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Inactivation of Listeria monocytogenes by Disinfectants andBacteriophages in Suspension and Stainless Steel Carrier Tests
N CHAITIEMWONG W C HAZELEGER AND R R BEUMER
Laboratory of Food Microbiology Wageningen University PO Box 17 6700 AA Wageningen The Netherlands
MS 14-151 Received 31 March 2014Accepted 16 July 2014
ABSTRACT
To simulate food contact surfaces with pits or cracks stainless steel plates with grooves (depths between 02 and 5 mm)
were constructed These plates were artificially contaminated with Listeria monocytogenes in clean conditions with organic
soiling or after 14 days of biofilm formation after which inactivation of the pathogen by Suma Tab D4 (sodium
dichloroisocyanurate 240 and 300 mgliter) Suma Bac D10 (quaternary ammonium compound 740 mgliter) and bacteriophage
suspension (Listex P100) was determined Both chemical disinfectants performed well in suspension tests and in clean carrier
tests according to the European standard with a reduction of more than 5 and 4 log units respectively of Listeria cells after 5 min
of contact time However for the plates with grooves the reduction could not meet the standard requirement although a higher
reduction of L monocytogenes was observed in the shallow grooves compared with the deeper grooves Furthermore presence of
food residues and biofilm reduced the effect of the disinfectants especially in the deep grooves which was dependent on type of
food substrate Bacteriophages showed the best antimicrobial effect compared with the chemical disinfectants (sodium
dichloroisocyanurate and quaternary ammonium compound) in most cases in the shallow grooves but not in the deep grooves
The chlorine based disinfectants were usually less effective than quaternary ammonium compound The results clearly
demonstrate that surfaces with grooves influenced the antimicrobial effect of the chemical disinfectants and bacteriophages
because the pathogen is protected in the deep grooves The use of bacteriophages to inactivate pathogens on surfaces could be
helpful in limited cases however use of large quantities in practice may be costly and phage-resistant strains may develop
Listeria monocytogenes a ubiquitous pathogenic bac-
terium is widely found in the natural environment including
plants soil sewage water and also in human and animal
feces (27 29 67) Due to this widespread occurrence in
nature the pathogen can enter food processing plants via
several vectors such as personnel equipment raw materi-
als pest animals and process waste (67) The pathogen is
able to adhere to and form biofilms on several surface
materials such as stainless steel rubber and plastic which
are used in food processing facilities (11 14 49)Furthermore the bacterium has been frequently isolated
from wet and cold spots (23) as well as from surfaces and
equipment that are difficult to clean (18 33 48)In food processing plants cleaning and disinfection are
the most important steps to eliminate dirt and microorgan-
isms that accumulate on food-contact surfaces equipment
walls and floors Improperly cleaned and disinfected
surfaces can lead to cross-contamination when food
products come into contact with the surfaces or when dirt
and microorganisms are dispersed during processing
Additionally biofilms can be formed on surfaces particu-
larly in pits or cracks that are difficult to clean Once the
biofilm is formed it is problematic to remove the pathogens
by normal cleaning and disinfecting procedures (8 54)because abraded surfaces can provide protection to firmly
attached bacteria Moreover microorganisms inside soil are
protected against disinfectants (2 9)Various disinfectants such as peracetic acid quaternary
ammonium compounds (Qacs) hypochloride sodium
dichloroisocyanurate (SDC) and iodofores have demon-
strated their efficacy against L monocytogenes in both
suspension (12 24 59 69) and surface tests (2 10 41)where the antimicrobial activity of the disinfectants was
more effective in suspension than in surface tests In a
suspension the disinfectants come into contact with free-
floating cells whereas on a surface the disinfectants can
reach the organisms only from one side (28 36 63)Moreover the effectiveness of the disinfectants will be
reduced when the disinfectants are inactivated by or bind to
organic matter present on the surface resulting in less
disinfectant coming into contact with the microorganism
(32 68)Apart from chemical disinfectants bacteriophages
(viruses that infect bacteria) have been used to control
bacteria in all stages of food production from preharvest to
finished products (22 44 47 52 57) Although some
studies on the efficiency of bacteriophages in removing
bacterial biofilm cells have been performed with Esche-richia coli (26) L monocytogenes (38) and Pseudomonas
Author for correspondence Tel z317-484982 E-mail
wilmahazelegerwurnl
2012
Journal of Food Protection Vol 77 No 12 2014 Pages 2012ndash2020doi1043150362-028XJFP-14-151Copyright G International Association for Food Protection
fluorescens (60) still little information is available on the
efficiency of bacteriophages on the inactivation of patho-
gens in biofilms (43 65 66)The effectiveness of disinfectants against bacteria on
smooth surfaces such as stainless steel coupons or chips (17 12 32 34) buna-N rubber (58 64) Teflon (55)polyester fabrics and polyurethane surfaces of conveyor
belt (64) has been well documented however only few
studies have been performed on surfaces with pits or
grooves The purpose of this study was to simulate the
practical situation where a Listeria biofilm is formed in
cracks in the equipment and to investigate the effect of two
commercial disinfectants based on chlorine and Qacs and a
Listeria bacteriophage product against L monocytogenes in
suspension and in carrier tests with flat and rutted (grooved)
surfaces
MATERIALS AND METHODS
Bacterial strains L monocytogenes LF 38 (serotype 12a
isolated from cooked ham) LF 36 (serotype 12b isolated from
cooked sausage) and LF 29 (serotype 4e isolated from cooked
sliced sausage) were obtained from The Netherlands Food and
Consumer Product Safety Authority (Wageningen The Nether-
lands) Stationary-phase cultures of all strains (24 h 30uC) were
stored in 1-ml cryo vials (Greiner Bio-one Frickenhausen
Germany) with 25 (volvol) glycerol (Fluka-Chemica Buchs
Switzerland) in brain heart infusion (BHI) broth (Difco BD
Sparks MD) and 2-mm glass beads (Emergo Landsmeer The
Netherlands) at 220uC For each experiment one glass bead of
each strain was inoculated to a separate tryptone soy agar plate
(Oxoid Basingstoke England) and then incubated at 30uC for
24 h A single colony was picked inoculated into BHI broth and
incubated at 30uC for 24 h Test suspensions were prepared by
mixing cultures of the three L monocytogenes strains where 2 ml
of overnight cultures of each strain (BHI broth 30uC for 24 h)
were combined to give a 6-ml mixed culture of L monocytogeneswhich was serially diluted in peptone saline solution (PSS 85 g
liter NaCl Merck Darmstadt Germany) and 1 gliter Neutralised
Bacteriological Peptone (Oxoid) to obtain appropriate dilutions for
inoculation
Bacteriophages Listeria bacteriophage Listex P100 was
obtained from Micreos Food Safety BV (Wageningen The
Netherlands) as a suspension with a concentration of 1011 PFU
ml Listex P100 was serially diluted with 9 ml of PSS to obtain
109 PFUml The phage suspensions were freshly prepared
before use to prevent deterioration during storage The
concentration of the phages was determined by a double agar
overlay method according to Carey-Smith et al (19) using BHI
agar (BHI plus Agar technical Agar no 3 12 and 4 gliter for
bottom and top layer respectively Oxoid) and Listeria innocua(serovar 5 Micreos Food Safety BV) as the indicator organism
The plates were incubated at 30uC for 24 h and plaques were
counted
Test surfaces Two types of surfaces were used in this study
stainless steel coupons (AISI type 304 standard ODS Bare-
ndrecht The Netherlands) with a total surface (both sides) of
38 cm2 and stainless steel plates (AISI type 304 standard ODS)
measuring 12 by 10 by 1 cm in which two identical sets of grooves
(5- 2- 1- 05- 02-mm depth 1-mm width and 10-cm length)
were cut (Fig 1)
Disinfectants Suma Tab D4 (SDC Diversey Inc Utrecht
The Netherlands) and Suma Bac D10 (Qac Diversey Inc) were
used in this experiment at concentrations of 240 mgliter for SDC
(pH 6) and 740 mgliter for Qac (pH 101 to 103) according to the
manufacturerrsquos recommendations Sterile water was used as a
control in the experiments The disinfectants were freshly prepared
shortly before use on the day of the experiments to prevent
deterioration during storage
Neutralizers For the chemical disinfectants inactivation
liquid was used as a neutralizer (6) containing 1 gliter L-histidine
(Merck) 3 gliter lecithin (VWR International Poole England)
5 gliter sodium thiosulfate pentahydrate (Na2S2O3-5H2O VWR
International) 30 mlliter Tween 80 (Merck) and 10 mlliter
phosphate buffer The phosphate buffer was prepared by dissolving
34 g of potassium dihydrogen phosphate (KH2PO4 Merck) in
500 ml of demi water after adjustment of the pH to 72 with 1 M
sodium hydroxide (NaOH VWR Prolabo Lutterworth England)
the volume was filled with 1 liter of demi water There were 9 ml
of inactivation liquid that were dispensed into test tubes and
sterilized at 121uC for 15 minutes
For Listeria bacteriophages a dead cell suspension of Linnocua (serovar 5) was used as a neutralizer (37) which was
prepared by adding 1 ml of an overnight culture (BHI broth 30uC
24 h) of L innocua to 9 ml of PSS which were subsequently
sterilized for 15 min at 121uC
Preparation of food residues Modified atmospherendashpacked
cooked ham vacuum-packed smoked salmon and chopped
endives were bought from a local supermarket kept in a
refrigerator at 4 to 7uC and used within 24 h To prepare a 10
FIGURE 1 Pictures of the stainless steel plate (12 by 10 by 1 cm)with 2 sets of 5 grooves (5- 2- 1- 05- 02-mm depth 1-mmwidth 10-cm length) used in carrier tests top (a) and side view (b)
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2013
suspension of the food residue 10 g of each food was aseptically
placed in a stomacher bag with 90 ml PSS and subsequently
homogenized in a stomacher (Stomacher 400 Circulator Seward
England) for 1 min at 260 rpm kept at 4 to 7uC and used within 4 h
One milliliter of ultrahigh-temperature-processed milk was asep-
tically added to 9 ml PSS vortexed and used immediately Prior to
the test all food residues were tested for the presence of Lmonocytogenes according to ISO 11290-11996Amd 12004 (5) to
verify if the food matrix was free of L monocytogenes
Suspension tests (case 1) The tests were performed
according to the European standard EN 10402005 (6) An
overnight culture of L monocytogenes with or without 10 food
residue was added to each disinfectant at recommended concen-
trations at room temperature for 5 min of contact time The tests
were performed in triplicate for each disinfectant on different days
Figure 2 shows a diagram of all tests that were performed in this
study
Carrier tests The tests were performed on smooth surfaces
(case 2) and surfaces with grooves (case 3) Before the experiments
started the stainless steel coupons and plates with grooves were
soaked overnight in anionic-active detergent (Dreft Procter amp
Gamble Netherlands BV Rotterdam The Netherlands) Thereafter
both surfaces were rinsed with hot water (70 to 80uC) and air dried
The coupons were put in a glass container covered with aluminum
foil while the plates were wrapped in paper and then all were
sterilized at 121uC for 15 min
Carrier tests on smooth surfaces (case 2) (4) To inoculate
the stainless steel coupons 10 ml of the test suspensions (109
CFUml resulting in 64 log CFUcm2) were put on one side of
the coupon and allowed to air dry for 1 h in a biological safety
cabinet After inoculation of the stainless steel coupons these were
placed in a 12-well plate (Greiner Bio-One BV Alphen ad Rijn
The Netherlands) containing 1 ml of SDC (240 mgliter) or Qac
(740 mgliter) and left at 1- 2- and 5-min contact time at room
temperature The experiment was repeated three times on different
days for each disinfectant
Carrier tests on surfaces with grooves (case 31) To
inoculate the surface with grooves (case 31) 10 ml of test
suspension (107 CFUml resulting in 6 log CFUcm2) with or
without 10 food suspension were put on the plates and evenly
spread over the surfaces to fully fill the grooves by using a sterile
sponge The plates were then kept at room temperature for 15 min
and 24 h to simulate wet and dry conditions respectively After
inoculation of the plates 240 mgliter SDC or 740 mgliter Qac
were sprayed on the plates (5 ml) by using a sterile spray bottle
(perfume spray bottle Etos Wageningen The Netherlands) and
left to achieve 5- 10- 15- or 20-min contact time at room
temperature The experiment was repeated two times on different
days for each disinfectant
Carrier tests on surfaces with grooves in presence of abiofilm (case 32) To mimic the situation in a food processing
plant with biofilm formation in cracked equipment a biofilm was
grown by daily inoculating a diluted overnight culture of the three
L monocytogenes strains with each food residue (3 ml 106 CFU
ml) in the grooves for 7 consecutive days Thereafter only food
suspension (3 ml) was added daily to the grooves for another 7 days
to allow further development of the biofilm During this period the
plates were stored at 15uC in a closed plastic box to maintain
stable humidity (50 relative humidity measured by a digital
hygrothermometer VWR International BV Amsterdam The
Netherlands) The disinfectant tests were carried out as described
in the previous paragraph for 20-min contact time
For the bacteriophage test 5 ml of a 109 PFUml phage
suspension were sprayed over the biofilm plates Thereafter the
plates were put in sterile plastic bags (Aseptic vacuum bags 400
by 510 mm Hevel Zaandam The Netherlands) to maintain
humidity and left for 30-min contact time
The tests on surfaces with grooves were performed in
duplicate on different days for each disinfectant and for
bacteriophages
Effect of disinfectants on suspended biofilm cells The 2-
week biofilm cells were taken out of each groove by scraping the
grooves with sterile extra-thin toothpicks (Jordan Intec BV
Utrecht The Netherlands) The cells were mixed with 9 ml PSS
and tested with disinfectants as described in the suspension test
Microbiological analysis In the suspension test (case 1) the
reaction was terminated after the required contact time by
FIGURE 2 Diagram shows all tests per-formed in this study The suspension andcarriers tests on smooth surface wereperformed with or without food residues(w or wo) according to the Europeanstandard EN 10402005 Tests on thesurface with grooves were performed withL monocytogenes cells on wet and dryconditions in presence and absence of foodresidues and 2-week food biofilm Thedisinfectants used were SDC (240 mgliter)and Qac (740 mgliter) while sterile waterwas used as a control
2014 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
transferring 1 ml of the suspensions to 9 ml of neutralizer for 1 min
Of these suspensions 100 ml was then plated on Agar Listeriaaccording to Ottaviani and Agosti (ALOA BioMerieux SA Marcy
lrsquoEtoile France) using a spiral plater (Eddy Jet IUL Instrument
SA Barcelona Spain) For the carrier tests on flat surfaces (case
2) the disinfectant reaction was terminated by transferring the
coupons plus disinfectant to a 9-ml tube containing neutralizer and
45-g glass beads (1 mm Emergo Landsmeer The Netherlands)
As a blank inoculated coupons were transferred to 10 ml of PSS
plus glass beads The cells were removed from the coupons by
vortexing the tubes for 30 s Thereafter 1 ml of the suspension was
pour plated in tryptone soy agar
For the carrier tests on surfaces with grooves (case 31)
samples were taken using cotton swabs and cotton threads after the
required contact time Cotton swabs made with extra-thin toothpicks
and cotton wool (01 g 100 Hygienic cotton Etos) and cotton
threads (27-cm length 075-mm diameter [01 g] Super Durable
Glanskatoen no 8 HWS Markoma BV Ninove Belgium) were wet
sterilized at 121uC for 15 min The sterile cotton swabs were used to
take the samples from the 2- 1- 05- and 02-mm grooves by
swabbing three times through the grooves The sterile cotton threads
were used to take samples from the 5-mm groove by flossing and
scraping the groove for 30 s After sampling the cotton swabs and
cotton threads were put in 9-ml neutralizer vortexed and 100 ml
were plated on ALOA plates using a spiral plater
For the carrier tests with bacteriophages (case 32) the cotton
threads and cotton swabs were soaked for 1 min in a heat killed
suspension of L innocua (109 CFUml) to bind free phage particles
as neutralizer Subsequent sampling and plating were similar as
described above
During the 14 days of growing biofilms the numbers of
Listeria aerobic plate count and lactic acid bacteria were
determined at different time intervals on separate plates using
ALOA agar tryptone soy agar and De Man Rogosa and Sharpe
Agar (Lactobacillus broth Merck and 12 [wtvol] Agar
technical Agar no 3 Oxoid) respectively
Data and statistical analysis The volume of each groove
(cubic millimeters) was calculated and transformed to milliliters
The amount of organisms in a groove was then divided by this
volume to obtain a concentration in log CFU per milliliter The
value was calculated as log CFU per milliliter of the grooves in
order to equally compare the results obtained from the grooves that
have different depths
For statistical analysis the results of duplicate experiments
were combined The significance of differences (P 005) in
numbers of L monocytogenes that were reduced by different types
of disinfectants food residues and types of surface were
determined with analysis of the variance and a general linear
model using PASW Statistics 17 (SPSS Inc Chicago IL)
RESULTS AND DISCUSSION
Suspension tests (case 1) Reduction of more than 5
log units of Listeria planktonic cells and biofilm cells in
suspension was observed after 5-min contact time with
240 mgliter SDC and 740 mgliter Qac in all test
conditions where there was no significant difference (P
005) in reduction of the cells between the two disinfectants
in absence of food residues In contrast the effectiveness of
SDC (1-log reduction in 5 min with 3 ultrahigh-
temperature-processed milk) was less than that of Qac (5-
log reduction in 1 min with 10 ultrahigh-temperature-
processed milk) in presence of food residues as expected
(21 25 46) The effectiveness of SDC and Qac in absence
of food residues was higher than in presence of food
residues (data not shown) However these results comply
with the European standard EN1040 (6) which indicates a
reduction of 5 log units should be obtained after 5-min
contact time for an effective disinfectant
Carrier tests on smooth surfaces (case 2) The results
from the tests on the stainless steel coupons showed more
than a 5-log reduction after 1- 2- and 5-min contact time
with no significant difference (P 005) between SDC and
Qac (data not shown) These results complied with the
European standard EN13697 requirement (4) for a reduction
of 4 log units on clean surfaces indicating an effective
disinfectant in the surface test
Carrier tests on surfaces with grooves (case 3) The
shape of the 1-mm wide grooves with depths of 02 05 1
2 and 5 mm used as test surfaces in this study was beyond
the capability of standard sampling methods such as surface
contact plates and swabbing techniques Therefore several
methods eg the use of filter paper cotton swabs and
flossing threads were evaluated for their effectiveness to
recover Listeria cells from the grooves The results showed
that the flossing technique with cotton thread led to 70
recovery for the 5-mm groove For the other grooves this
method could recover only 20 With cotton swabs
prepared from extra-thin toothpicks and cotton wool a
recovery of 70 could be obtained for the remaining
grooves (data not shown) Therefore flossing and swab
methods were chosen as sampling methods for the 5-mm
groove and the other grooves respectively
In these tests the contact times of disinfectant on carriers
were 5 10 15 and 20 min Since the inactivation obtained for
the different contact times was not significantly different (P 005) which may be due to exhaustion of the disinfectants
(3 51) only the results from the contact time of 5 min are
shown Furthermore reduction of L monocytogenes was
dependent on the groove depth the deeper the groove the
lower the reduction This indicates that the activity of the
disinfectants stopped due to the inability to reach the cells in
the deeper areas Only the results of the extremes the 5- and
02-mm grooves are presented in Figures 3 and 4
All food residues were tested negative for L monocy-togenes In absence of food residues on a wet surface after
exposure to water (control) and the disinfectants a high
reduction of the cells was observed in the 02-mm grooves
whereas a similar trend but less reduction was observed in
the 5-mm grooves On the dry surfaces treatment of the
cells in the 02-mm groove resulted in levels below the
detection limit which can be partly explained by a high
reduction observed already as a result of the drying of the
plates for 24 h For the 5-mm grooves this initial reduction
was much lower and not all listerias were inactivated by the
treatment In most cases there was no significant difference
(P 005) between the control and disinfectants The
results demonstrated that drying of surfaces alone could
already largely reduce the cells on the surfaces especially in
the shallow grooves where deeper grooves provided suitable
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2015
FIGURE 3 Numbers of L monocytogenes (log CFU per milliliter) in wet conditions for the 02- (a) and 5-mm (b) grooves and dryconditions for the 02- (c) and 5-mm (d) grooves in presence and absence of food residues at initial inoculation ( T ~ 0) before (amp15 min or 24 h for wet and dry conditions respectively) and after exposure to sterile water ( control) SDC ( 240 mgliter) and Qac( 740 mgliter) for 5 min Data are means iexcl standard deviations of duplicate experiments for each disinfectant (n ~ 2) Dashed linesand vertical arrows (Q) indicate values lower than the detection limit DL detection limit of the method
FIGURE 4 Numbers of L monocytogenes biofilm cells (log CFU per milliliter) in presence of food residues in 02- (a) and 5-mm (b)grooves at initial inoculation ( T ~ 0) before (amp D 14) and after exposure to sterile water ( control) SDC ( 240 mgliter) Qac( 740 mgliter) and bacteriophages ( 109 PFUml) Data are means iexcl standard deviations of duplicate experiments for eachdisinfectant (n ~ 2) Dashed lines and vertical arrows (Q) indicate values lower than the detection limit
2016 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
conditions to prevent complete drying protecting some of
the cells from the disinfectants Drying of surfaces has been
considered as a significant bactericidal method to maintain
low numbers of bacteria in any environment and is
dependent on type of organism temperature relative
humidity and soiling (39 45 53) Furthermore type of
surface and topography have been reported to play an
important role on efficiency of disinfectants (30 40) in
particular surfaces with scrapes or defects where soil can
accumulate and protect bacteria against cleaning and
disinfecting agents (16 17 51)In presence of food residues in the wet 02-mm
grooves Listeria was reduced 2 to 3 log dependent on
types of food residues (Fig 3a) After exposure to the
disinfectants a reduction to below the detection limit was
observed for all food residues with no significant difference
(P 005) between SDC and Qac In the 5-mm grooves
before treatment less reduction (2 log CFUml) was
observed SDC was significantly (P 005) less effective
(2 log) than Qac ($2 log CFUml) for all food residues
except fish In the dry 02-mm grooves already a reduction
of 3 log CFUml was observed 24 h after inoculation
without treatment After exposure to the disinfectants the
numbers were further reduced to below the detection limit
In the 5-mm groove only a small (05 log CFUml ie
vegetable fish and ham residues) or no reduction (milk
residue) was observed immediately before treatment
probably because the cells were protected from the drying
in the deep grooves Treatment with SDC and Qac did not
inactivate all listerias with no significant difference (P
005) between the two disinfectants except in the case of
ham and vegetable residues where Qac performed signif-
icantly better than SDC in the wet grooves and for
vegetables in the dry grooves Previous studies have
reported an inactivation of the antimicrobial activity of
disinfectants by organic matter on surfaces showing 1- to 2-
log reductions on soiled surfaces compared with 3- to 4-
log reductions on clean surfaces (2 34) This could be due
to the chemical compositions such as protein and fat which
can bind with the disinfectants or reduction of penetration
into the bacterial layer (32 68) The chlorine-based
disinfectants have been found to be easily inactivated by
organic materials (25 46) whereas Qac was reported to be
less inactivated by organic matters (21) which was
confirmed in most cases in this study In general the test
on surfaces is dependent on the surface materials used and
viability of the cells dried on the surfaces resulting in
inconsistent results when compared with the test in
suspensions (15) Under practical conditions high amounts
of organic materials may be present resulting in less or no
activity of the disinfectant When the concentration of SDC
was increased from 240 to 300 mgliter a comparable
reduction of the cells to that of Qac was obtained however
there was no effect on reduction in the deep grooves (data
not shown) The results indicated that the disinfectant could
not reach all cells in the bottom of the groove while such
grooves (5-mm depth 1-mm width) might be present in
practice (eg small spaces between connection parts of
slicers) The effectiveness of the disinfectants in the deep
areas could be increased by using high pressure spraying
(32) however this method might have adverse effects since
it could also spread the bacterial cells to other areas and
environments in particular when high numbers of bacterial
cells are accumulated (42)
Carrier tests on surfaces with grooves in presence ofa biofilm (case 32) After 14 days of biofilm growing in
02-mm grooves (Fig 4) the number of cells remained
more or less stable with a decrease of the cells in vegetable
and ham biofilms (15 and 18 log CFUml respectively) In
5-mm grooves however growth of biofilm cells was
observed in all food residues A previous study reported that
L monocytogenes has the ability to form biofilms at
numbers of 104 to 107 CFUcm2 (34) and adheres to a
variety of food contact surfaces such as plastic glass
rubber and stainless steel particularly in pits or crevices
that are difficult to clean (31 61)To check if the biofilm cells were more resistant to the
disinfectants than the planktonic cells the biofilm cells were
brought into suspension and then tested The obtained
results a 5-log reduction in all conditions after 5-min
exposure to the disinfectants indicated no difference in
reduction of biofilm cells (case 32) compared with
planktonic cells (case 1) These results complied with a
previous study where reduction of the suspended biofilm
cells was higher than that of biofilm cells attached to
surfaces after exposure to disinfectants (50) It has been
described that a 10 to 100 times higher concentration of
disinfectants may be required to obtain the same reduction
of biofilm cells compared with planktonic cells (40 56)indicating the importance of preventing biofilm formation
For the bacteriophages an efficacy test was previously
performed with L monocytogenes strains in presence and
absence of food residues on flat stainless steel surfaces The
results demonstrated that rapid inactivation of Listeria cells
(2 to 3 log CFUcm2) within 30 min of contact time was
obtained (data not shown) which was similar with the
results of Soni and Nannapaneni (65) where a reduction of
35 log CFUcm2 was obtained in the same time with a
phage treatment on a 1-week-old L monocytogenes biofilm
on stainless steel coupons
In the 02-mm grooves (Fig 4a) Qac showed better
reduction (2 to 3 log) than SDC (12 to 27 log) with
inactivation below the detection limit in vegetable biofilms
The phages showed higher reduction (3 log CFUml P
005) than that of water or the chemical disinfectants in three
food biofilms except for the vegetable biofilms where all
cells were inactivated by every disinfectant In the 5-mm
grooves SDC and Qac did not reduce Listeria better than
water alone However the phages reduced approximately
14 log CFUml in ham and fish biofilms and approxi-
mately 08 log CFUml in milk and vegetable biofilms
which was not significantly different (P 005) from that of
the disinfectants except for ham biofilms The results
indicated that only in the 02-mm grooves the phages were
likely to be more effective in the biofilms with food matrix
than the chemical disinfectants This was probably due to
the fact that the disinfectants and phages did not penetrate
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2017
the foodbacterial matrix completely in the deep groove (2041) Furthermore the effectiveness of the disinfectants on Lmonocytogenes biofilms may be reduced by presence of
other bacteria (6 to 7 log CFUml) including lactic acid
bacteria (4 log CFUml) in the grooves as well (data not
shown) Phage efficacy depends on susceptibility of the
bacterial cells to the phages and availability of receptor sites
between phages and the bacterial cells (13) which could be
blocked by the food matrix (35) In this study the phage
efficacy was enhanced by keeping the biofilm plates in
plastic bags after application of the phages to maintain a
high humidity that would enhance the diffusion and the
chance of contact between Listeria cells and phage particles
(66)Strikingly the biofilm removal by sterile water
(control) resulted in a similar reduction of the biofilm cells
compared with disinfectants In the present study the tests
were performed to simulate survival of bacteria in presence
or absence of organic matter in pits or cracks on food
contact surfaces and to test the effect of disinfection
therefore cleaning of the surfaces before using disinfectants
was excluded So in this case plates and grooves were
dirtier than soiled surfaces in reality which explains the
observed low efficacy of the disinfectants and the water
washed away the biofilm cells from the grooves Rinsing the
biofilm on surfaces by water has been reported to reduce the
biofilm cells at about 1 log unit of cells (34)The results in this study clearly demonstrated that
presence of grooves or spaces on surfaces humidity and
presence of food substrates influenced the antimicrobial
effect of disinfectants and bacteriophages Bacteriophages
showed a better antimicrobial effect than the chemical
disinfectants (ie SDC and Qac) in the shallow grooves but
not in the deep grooves Use of bacteriophages as a
biocontrol could only be a promising method in limited
cases however use of large quantities in practice may be
costly and phage-resistant strains may occur (35 62)
ACKNOWLEDGMENTS
The authors thank Diversey Inc (Utrecht The Netherlands) who
provided the chemical disinfectants for this study We also thank Micreos
Food Safety BV (Wageningen The Netherlands) for Listeria bacteriophage
Listex P100 and L innocua (serovar 5) Our great appreciation goes to
Feifei Gao for her technical assistance in the phage experiments and Hilda
Nyati for her work on smooth surfaces
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1 Aarnisalo K J Lunden H Korkeala and G Wirtanen 2007
Susceptibility of Listeria monocytogenes strains to disinfectants and
chlorinated alkaline cleaners at cold temperatures LWT - Food SciTechnol 401041ndash1048
2 Aarnisalo K S Salo H Miettinen M Suihko G Wirtanen T
Autio J Lunden H Korkeala and A Sjoberg 2000 Bactericidal
efficiencies of commercial disinfectants against Listeria monocyto-
genes on surfaces J Food Saf 20237ndash250
3 Adams M R A D Hartley and L J Cox 1989 Factors affecting
the efficacy of washing procedures used in the production of prepared
salads Food Microbiol 669ndash77
4 Anonymous 2001 European Standard EN 136972001 Chemical
disinfectants and antisepticsmdashqualitative non-porous surface test for
the evaluation of bacteriocidal andor fungicidal activity of chemical
disinfectants used in food industrial domestic and institutional
areasmdashtest method and requirements without mechanical action (phase
2 step 2) Available at httpwwwceneucenSectorsTechnical
CommitteesWorkshopsCENTechnicalCommitteesPagesStandards
aspxparam~6197amptitle~Chemical20disinfectants20and20
antiseptics Accessed February 2014
5 Anonymous 2004 Microbiology of food and animal feeding stuffs mdash
horizontal method for the detection and enumeration of Listeria
monocytogenesmdashpart 1 detection method ISO 11290-11996Amd
12004 International Organization for Standardization Geneva
Available at httpswwwisoorgobpuiisostdiso11290-1ed-1
v1enamd1amd1 Accessed January 2014
6 Anonymous 2005 European Standard EN 10402005 Chemical
disinfectants and antiseptics Quantitative suspension test for the
evaluation of basic bactericidal activity of chemical disinfectants
and antisepticsmdashtest method and requirements (phase 1) Available
at httpwwwceneucenSectorsTechnicalCommitteesWorkshops
CENTechnicalCommitteesPagesStandardsaspxparam~6197amptitle~
Chemical20disinfectants20and20antiseptics Accessed February
2014
7 Arachchi G J G A G Cridge B M Dias-Wanigasekera C D
Cruz L McIntyre R Liu S H Flint and A N Mutukumira 2013
Effectiveness of phages in the decontamination of Listeria monocy-
togenes adhered to clean stainless steel stainless steel coated with
fish protein and as a biofilm J Ind Microbiol Biotechnol 401105ndash
1116
8 Autio T R Keto-Timonen J Bjorkroth and H Korkeala 2003
Characterization of persistent and sporadic Listeria monocytogenes
strains by pulsed-field gel electrophoresis (PFGE) and amplified
fragment length polymorphism (AFLP) Syst Appl Microbiol 26
539ndash545
9 Bagge-Ravn D Y Ng M Hjelm J N Christiansen C Johansen
and L Gram 2003 The microbial ecology of processing equipment
in different fish industriesmdashanalysis of the microflora during
processing and following cleaning and disinfection Int J Food
Microbiol 87239ndash250
10 Belessi C A A S Gounadaki A N Psomas and P N Skandamis
2011 Efficiency of different sanitation methods on Listeria
monocytogenes biofilms formed under various environmental condi-
tions Int J Food Microbiol 145S46ndashS52
11 Beresford M R P W Andrew and G Shama 2001 Listeria
monocytogenes adheres to many materials found in food-processing
environments J Appl Microbiol 901000ndash1005
12 Best M M M M E Kennedy and F F Coates 1990 Efficacy of a
variety of disinfectants against Listeria spp Appl Environ Micro-
biol 56377ndash380
13 Bigwood T J A Hudson and C Billington 2009 Influence of host
and bacteriophage concentrations on the inactivation of food-borne
pathogenic bacteria by two phages FEMS Microbiol Lett 29159ndash
64
14 Blackman I C and J F Frank 1996 Growth of Listeria
monocytogenes as a biofilm on various food-processing surfaces J
Food Prot 59827ndash831
15 Bloomfield S F M Arthur B Klingeren W van Pullen J T
Holah and R Elton 1994 An evaluation of the repeatability and
reproducibility of a surface test for the activity of disinfectants J
Appl Bacteriol 7686ndash94
16 Boulange-Petermann L J Rault and M-N Bellon-Fontaine
1997 Adhesion of Streptococcus thermophilus to stainless steel
with different surface topography and roughness Biofouling 11
201ndash216
17 Bower C K J McGuire and M A Daeschel 1996 The adhesion
and detachment of bacteria and spores on food contact surfaces
Trends Food Sci Technol 7152ndash157
18 Bryan F L 2002 Where we are in the retail food safety how we got
to where we are and how do we get there J Environ Health 6529ndash
36
19 Carey-Smith G V C Billington A J Cornelius J A Hudson and
J A Heinemann 2006 Isolation and characterization of bacterio-
phages infecting Salmonella spp FEMS Microbiol Lett 258182ndash
186
2018 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
20 Carpentier B and O Cerf 1993 Biofilms and their consequences
with particular reference to hygiene in the food industry J Appl
Bacteriol 75499ndash511
21 Carsberg H 1996 Selecting your sanitizers Food Qual 235ndash36
61ndash62
22 Chibeu A L Agius A Gao P M Sabour A M Kropinski and S
Balamurugan 2013 Efficacy of bacteriophage LISTEXTMP100
combined with chemical antimicrobials in reducing Listeria mono-
cytogenes in cooked turkey and roast beef Int J Food Microbiol
167208ndash214
23 Cox L J T Kleiss J L Cordier C Cordellana P Konkel C
Pedrazzini R Beumer and A Siebenga 1989 Listeria spp in food
processing non-food and domestic environments Food Microbiol 6
49ndash61
24 Cruz C D and G C Fletcher 2012 Assessing manufacturersrsquo
recommended concentrations of commercial sanitizers on inactivation
of Listeria monocytogenes Food Control 26194ndash199
25 De Beer D R Srinivasan and P S Stewart 1994 Direct
measurement of chlorine penetration into biofilms during disinfec-
tion Appl Environ Microbiol 604339ndash4344
26 Doolittle M M J J Cooney and D E Caldwell 1995 Lytic
infection of Escherichia coli biofilms by bacteriophage T4 Can J
Microbiol 4112ndash18
27 Environmental Protection Agency (EPA) Office of Wastewater
Enforcement and Compliance 2014 Guide to field storage of
biosolids and other organic by-products used in agriculture and for
soil resource management Available at httpwaterepagovscitech
wastetechbiosolidsguidecfm Accessed January 2014
28 Fatemi P and J F Frank 1999 Inactivation of Listeria
monocytogenesPseudomonas biofilms by peracid sanitizer J Food
Prot 62761ndash765
29 Fenlon D R J Wilson and W Donachie 1996 The incidence and
level of Listeria monocytogenes contamination of food sources at
primary production and initial processing J Appl Bacteriol 81641ndash
650
30 Frank J F and R A N Chmielewski 1997 Effectiveness of
sanitation with quaternary ammonium compound or chlorine on
stainless steel and other domestic food-preparation surfaces J Food
Prot 6043ndash47
31 Frank J F and R A Koffi 1990 Surface-adherent growth of
Listeria monocytogenes is associated with increased resistance of
surfactant sanitizers and heat J Food Prot 53550ndash554
32 Gibson H J H Taylor K E Hall and J T Holah 1999
Effectiveness of cleaning techniques used in the food industry in terms
of the removal of bacterial biofilms J Appl Microbiol 8741ndash48
33 Gombas D E Y Chen R S Clavaro and V N Scott 2003 Survey
of Listeria monocytogenes in foods J Food Prot 66559ndash569
34 Gram L D Bagge-Ravn Y Y Ng P Gymoese and B F Vogel
2007 Influence of food soiling matrix on cleaning and disinfection
efficiency on surface attached Listeria monocytogenes Food Control
181165ndash1171
35 Greer G G 2005 Bacteriophage control of foodborne bacteria J
Food Prot 681102ndash1111
36 Gronholm L G Wirtanen K Ahlgren K Nordstrom and A
Sjoberg 1999 Screening of antimicrobial activities of disinfectants
and cleaning agents against foodborne spoilage microbes Eur Food
Res Technol 208289ndash298
37 Hagens S 2014 Personal communication
38 Hibma A M S A Jassim and M W Griffiths 1997 Infection and
removal of L-forms of Listeria monocytogenes with bred bacterio-
phage Int J Food Microbiol 34197ndash207
39 Hirai Y 1991 Survival of bacteria under dry conditions from a
viewpoint of nosocomial infection J Hosp Infect 19191ndash200
40 Holah J T 1992 Industrial monitoring hygiene in food processing
p 645ndash659A In L F Melo T R Bott M Fletcher and B
Capdeville (ed) Biofilmsmdashscience and technology Kluwer Aca-
demic Publishers Dordrecht
41 Holah J T C Higgs S Robinson D Worthington and H
Spenceley 1990 conductance-based surface disinfection test for food
hygiene Lett Appl Microbiol 11255ndash259
42 Holah J T J H Taylor and J S Holder 1993 The spread of
Listeria by cleaning systems part II Technical Memorandum no 673
Campden and Chorleywood Food Research Association Chipping
Campden UK
43 Hughes K A I W Sutherland and M V Jones 1998 Biofilm
susceptibility to bacteriophage attack the role of phage-borne
polysaccharide depolymerase Microbiology 1443039ndash3047
44 Joerger R D 2003 Alternatives to antibiotics bacteriocins
antimicrobial peptides and bacteriophages Poult Sci 82640ndash647
45 Kusumaningrum H D G Riboldi W C Hazeleger and R R
Beumer 2003 Survival of foodborne pathogens on stainless steel
surfaces and cross-contamination to foods Int J Food Microbiol 85
227ndash236
46 Lee S-H and J Frank 1991 Inactivation of surface adherent
Listeria monocytogenes hypochlorite and heat J Food Prot 544ndash
6
47 Leverentz B W S Conway M J Camp W J Janisiewicz T
Abuladze M Yang R Saftner and A Sulakvelidze 2003
Biocontrol of Listeria monocytogenes on fresh-cut produce by
treatment with lytic bacteriophages and a bacteriocin Appl Environ
Microbiol 694519ndash4526
48 Lin C M K Takeuchi L Zhang C B Dohm J D Meyer P A
Hall and M P Doyle 2006 Cross-contamination between
processing equipment and deli meats by Listeria monocytogenes J
Food Prot 6971ndash79
49 Lunden J M T J Autio and H J Korkeala 2002 Transfer of
persistent Listeria monocytogenes contamination between food-
processing plants associated with a dicing machine J Food Prot
651129ndash1133
50 Luppens S B I M W Reij R W L van der Heijden F M
Rombouts and T Abee 2002 Development of a standard test to
assess the resistance of Staphylococcus aureus biofilm cells to
disinfectants Appl Environ Microbiol 684194ndash4200
51 Mafu A A D Roy J Goulet L Savoie and R Roy 1990
Efficiency of sanitizing agents for destroying Listeria monocytogenes
on contaminated surfaces J Dairy Sci 733428ndash3432
52 Mahony J O McAuliffe R Paul and D van Sinderen 2011
Bacteriophages as biocontrol agents of food pathogens Curr Opin
Biotechnol 22157ndash163
53 McEldowney S and M Fletcher 1988 Effect of temperature and
humidity on survival of bacteria attached to dry solid surfaces Lett
Appl Microbiol 783ndash86
54 Miettinen L K J Bjorkroth and H Korkeala 1999 Characterisa-
tion of Listeria monocytogenes from an ice-cream plant by serotyping
and pulsed-field gel electrophoresis Int J Food Microbiol 46187ndash
192
55 Mosteller T M and J R Bishop 1993 Sanitizer efficacy against
attached bacteria in a milk biofilm J Food Prot 5634ndash41
56 Norwood D E and A Gilmour 2000 The growth and resistance to
sodium hypochlorite of Listeria monocytogenes in a steady-state
multispecies biofilm J Appl Microbiol 88512ndash520
57 Oliveira M I Vinas P Colas M Anguera J Usall and M
Abadias 2014 Effectiveness of a bacteriophage in reducing Listeria
monocytogenes on fresh-cut fruits and fruit juices Food Microbiol
38137ndash142
58 Ronner A B and A C L Wong 1993 Biofilm development and
sanitizer inactivation of Listeria monocytogenes and Salmonella
typhimurium on stainless steel and buna-n rubber J Food Prot 56
750ndash758
59 Sallam S S and C W Donnelly 1992 Destruction injury and
repair of Listeria species exposed to sanitizing compounds J Food
Prot 55771ndash1033
60 Sillankorva S D R Oliveira M J Vieira I W Sutherland and J
Azeredo 2004 Bacteriophage V S1 infection of Pseudomonas
fluorescens planktonic cells versus biofilms Biofouling 20133ndash138
61 Silva S P Teixeira R Olieveira and J Azeredo 2008 Adhesion to
and viability of Listeria monocytogenes on food contact surfaces J
Food Prot 711379ndash1385
62 Skurnik M and E Strauch 2006 Phage therapy facts and fiction
Int J Food Microbiol 2965ndash14 (Review)
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2019
63 Somers E B J L Schoeni and A C L Wong 1994 Effect of
trisodium phosphate on biofilm and planktonic cells of Campylo-
bacter jejuni Escherichia coli O157 H7 Listeria monocytogenes
and Salmonella typhimurium Int J Food Microbiol 22269ndash
276
64 Somers E B and A C L Wong 2004 Efficacy of two cleaning
and sanitizing combination on Listeria monocytogenes biofilm
formed at low temperature on a variety of materials in the presence
of ready-to-eat meat residue J Food Prot 672218ndash2229
65 Soni K A and R Nannapaneni 2010 Removal of Listeria
monocytogenes biofilms with bacteriophages P100 J Food Prot 73
1519ndash1524
66 Sutherland I W K A Hughes L C Skillman and K Tait 2004
The interaction of phage and biofilm FEMS Microbiol Lett 2321ndash6
(MiniReview)
67 Swaminathan B 2001 Listeria monocytogenes p 383ndash409 In L R
Beuchat M P Doyle and T J Montville (ed) Food microbiology
fundamentals and frontiers 2nd ed ASM Press Washington DC
68 Thevenot D A Dernburg and C Vernozy-Rozand 2006 An
updated review of Listeria monocytogenes in the pork meat industry
and its products J Appl Microbiol 1017ndash17
69 Tuncan E U 1993 Effect of cold temperature on germicidal efficacy
of quaternary ammonium compound iodophor and chlorine on
Listeria J Food Prot 531029ndash1033
2020 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
fluorescens (60) still little information is available on the
efficiency of bacteriophages on the inactivation of patho-
gens in biofilms (43 65 66)The effectiveness of disinfectants against bacteria on
smooth surfaces such as stainless steel coupons or chips (17 12 32 34) buna-N rubber (58 64) Teflon (55)polyester fabrics and polyurethane surfaces of conveyor
belt (64) has been well documented however only few
studies have been performed on surfaces with pits or
grooves The purpose of this study was to simulate the
practical situation where a Listeria biofilm is formed in
cracks in the equipment and to investigate the effect of two
commercial disinfectants based on chlorine and Qacs and a
Listeria bacteriophage product against L monocytogenes in
suspension and in carrier tests with flat and rutted (grooved)
surfaces
MATERIALS AND METHODS
Bacterial strains L monocytogenes LF 38 (serotype 12a
isolated from cooked ham) LF 36 (serotype 12b isolated from
cooked sausage) and LF 29 (serotype 4e isolated from cooked
sliced sausage) were obtained from The Netherlands Food and
Consumer Product Safety Authority (Wageningen The Nether-
lands) Stationary-phase cultures of all strains (24 h 30uC) were
stored in 1-ml cryo vials (Greiner Bio-one Frickenhausen
Germany) with 25 (volvol) glycerol (Fluka-Chemica Buchs
Switzerland) in brain heart infusion (BHI) broth (Difco BD
Sparks MD) and 2-mm glass beads (Emergo Landsmeer The
Netherlands) at 220uC For each experiment one glass bead of
each strain was inoculated to a separate tryptone soy agar plate
(Oxoid Basingstoke England) and then incubated at 30uC for
24 h A single colony was picked inoculated into BHI broth and
incubated at 30uC for 24 h Test suspensions were prepared by
mixing cultures of the three L monocytogenes strains where 2 ml
of overnight cultures of each strain (BHI broth 30uC for 24 h)
were combined to give a 6-ml mixed culture of L monocytogeneswhich was serially diluted in peptone saline solution (PSS 85 g
liter NaCl Merck Darmstadt Germany) and 1 gliter Neutralised
Bacteriological Peptone (Oxoid) to obtain appropriate dilutions for
inoculation
Bacteriophages Listeria bacteriophage Listex P100 was
obtained from Micreos Food Safety BV (Wageningen The
Netherlands) as a suspension with a concentration of 1011 PFU
ml Listex P100 was serially diluted with 9 ml of PSS to obtain
109 PFUml The phage suspensions were freshly prepared
before use to prevent deterioration during storage The
concentration of the phages was determined by a double agar
overlay method according to Carey-Smith et al (19) using BHI
agar (BHI plus Agar technical Agar no 3 12 and 4 gliter for
bottom and top layer respectively Oxoid) and Listeria innocua(serovar 5 Micreos Food Safety BV) as the indicator organism
The plates were incubated at 30uC for 24 h and plaques were
counted
Test surfaces Two types of surfaces were used in this study
stainless steel coupons (AISI type 304 standard ODS Bare-
ndrecht The Netherlands) with a total surface (both sides) of
38 cm2 and stainless steel plates (AISI type 304 standard ODS)
measuring 12 by 10 by 1 cm in which two identical sets of grooves
(5- 2- 1- 05- 02-mm depth 1-mm width and 10-cm length)
were cut (Fig 1)
Disinfectants Suma Tab D4 (SDC Diversey Inc Utrecht
The Netherlands) and Suma Bac D10 (Qac Diversey Inc) were
used in this experiment at concentrations of 240 mgliter for SDC
(pH 6) and 740 mgliter for Qac (pH 101 to 103) according to the
manufacturerrsquos recommendations Sterile water was used as a
control in the experiments The disinfectants were freshly prepared
shortly before use on the day of the experiments to prevent
deterioration during storage
Neutralizers For the chemical disinfectants inactivation
liquid was used as a neutralizer (6) containing 1 gliter L-histidine
(Merck) 3 gliter lecithin (VWR International Poole England)
5 gliter sodium thiosulfate pentahydrate (Na2S2O3-5H2O VWR
International) 30 mlliter Tween 80 (Merck) and 10 mlliter
phosphate buffer The phosphate buffer was prepared by dissolving
34 g of potassium dihydrogen phosphate (KH2PO4 Merck) in
500 ml of demi water after adjustment of the pH to 72 with 1 M
sodium hydroxide (NaOH VWR Prolabo Lutterworth England)
the volume was filled with 1 liter of demi water There were 9 ml
of inactivation liquid that were dispensed into test tubes and
sterilized at 121uC for 15 minutes
For Listeria bacteriophages a dead cell suspension of Linnocua (serovar 5) was used as a neutralizer (37) which was
prepared by adding 1 ml of an overnight culture (BHI broth 30uC
24 h) of L innocua to 9 ml of PSS which were subsequently
sterilized for 15 min at 121uC
Preparation of food residues Modified atmospherendashpacked
cooked ham vacuum-packed smoked salmon and chopped
endives were bought from a local supermarket kept in a
refrigerator at 4 to 7uC and used within 24 h To prepare a 10
FIGURE 1 Pictures of the stainless steel plate (12 by 10 by 1 cm)with 2 sets of 5 grooves (5- 2- 1- 05- 02-mm depth 1-mmwidth 10-cm length) used in carrier tests top (a) and side view (b)
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2013
suspension of the food residue 10 g of each food was aseptically
placed in a stomacher bag with 90 ml PSS and subsequently
homogenized in a stomacher (Stomacher 400 Circulator Seward
England) for 1 min at 260 rpm kept at 4 to 7uC and used within 4 h
One milliliter of ultrahigh-temperature-processed milk was asep-
tically added to 9 ml PSS vortexed and used immediately Prior to
the test all food residues were tested for the presence of Lmonocytogenes according to ISO 11290-11996Amd 12004 (5) to
verify if the food matrix was free of L monocytogenes
Suspension tests (case 1) The tests were performed
according to the European standard EN 10402005 (6) An
overnight culture of L monocytogenes with or without 10 food
residue was added to each disinfectant at recommended concen-
trations at room temperature for 5 min of contact time The tests
were performed in triplicate for each disinfectant on different days
Figure 2 shows a diagram of all tests that were performed in this
study
Carrier tests The tests were performed on smooth surfaces
(case 2) and surfaces with grooves (case 3) Before the experiments
started the stainless steel coupons and plates with grooves were
soaked overnight in anionic-active detergent (Dreft Procter amp
Gamble Netherlands BV Rotterdam The Netherlands) Thereafter
both surfaces were rinsed with hot water (70 to 80uC) and air dried
The coupons were put in a glass container covered with aluminum
foil while the plates were wrapped in paper and then all were
sterilized at 121uC for 15 min
Carrier tests on smooth surfaces (case 2) (4) To inoculate
the stainless steel coupons 10 ml of the test suspensions (109
CFUml resulting in 64 log CFUcm2) were put on one side of
the coupon and allowed to air dry for 1 h in a biological safety
cabinet After inoculation of the stainless steel coupons these were
placed in a 12-well plate (Greiner Bio-One BV Alphen ad Rijn
The Netherlands) containing 1 ml of SDC (240 mgliter) or Qac
(740 mgliter) and left at 1- 2- and 5-min contact time at room
temperature The experiment was repeated three times on different
days for each disinfectant
Carrier tests on surfaces with grooves (case 31) To
inoculate the surface with grooves (case 31) 10 ml of test
suspension (107 CFUml resulting in 6 log CFUcm2) with or
without 10 food suspension were put on the plates and evenly
spread over the surfaces to fully fill the grooves by using a sterile
sponge The plates were then kept at room temperature for 15 min
and 24 h to simulate wet and dry conditions respectively After
inoculation of the plates 240 mgliter SDC or 740 mgliter Qac
were sprayed on the plates (5 ml) by using a sterile spray bottle
(perfume spray bottle Etos Wageningen The Netherlands) and
left to achieve 5- 10- 15- or 20-min contact time at room
temperature The experiment was repeated two times on different
days for each disinfectant
Carrier tests on surfaces with grooves in presence of abiofilm (case 32) To mimic the situation in a food processing
plant with biofilm formation in cracked equipment a biofilm was
grown by daily inoculating a diluted overnight culture of the three
L monocytogenes strains with each food residue (3 ml 106 CFU
ml) in the grooves for 7 consecutive days Thereafter only food
suspension (3 ml) was added daily to the grooves for another 7 days
to allow further development of the biofilm During this period the
plates were stored at 15uC in a closed plastic box to maintain
stable humidity (50 relative humidity measured by a digital
hygrothermometer VWR International BV Amsterdam The
Netherlands) The disinfectant tests were carried out as described
in the previous paragraph for 20-min contact time
For the bacteriophage test 5 ml of a 109 PFUml phage
suspension were sprayed over the biofilm plates Thereafter the
plates were put in sterile plastic bags (Aseptic vacuum bags 400
by 510 mm Hevel Zaandam The Netherlands) to maintain
humidity and left for 30-min contact time
The tests on surfaces with grooves were performed in
duplicate on different days for each disinfectant and for
bacteriophages
Effect of disinfectants on suspended biofilm cells The 2-
week biofilm cells were taken out of each groove by scraping the
grooves with sterile extra-thin toothpicks (Jordan Intec BV
Utrecht The Netherlands) The cells were mixed with 9 ml PSS
and tested with disinfectants as described in the suspension test
Microbiological analysis In the suspension test (case 1) the
reaction was terminated after the required contact time by
FIGURE 2 Diagram shows all tests per-formed in this study The suspension andcarriers tests on smooth surface wereperformed with or without food residues(w or wo) according to the Europeanstandard EN 10402005 Tests on thesurface with grooves were performed withL monocytogenes cells on wet and dryconditions in presence and absence of foodresidues and 2-week food biofilm Thedisinfectants used were SDC (240 mgliter)and Qac (740 mgliter) while sterile waterwas used as a control
2014 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
transferring 1 ml of the suspensions to 9 ml of neutralizer for 1 min
Of these suspensions 100 ml was then plated on Agar Listeriaaccording to Ottaviani and Agosti (ALOA BioMerieux SA Marcy
lrsquoEtoile France) using a spiral plater (Eddy Jet IUL Instrument
SA Barcelona Spain) For the carrier tests on flat surfaces (case
2) the disinfectant reaction was terminated by transferring the
coupons plus disinfectant to a 9-ml tube containing neutralizer and
45-g glass beads (1 mm Emergo Landsmeer The Netherlands)
As a blank inoculated coupons were transferred to 10 ml of PSS
plus glass beads The cells were removed from the coupons by
vortexing the tubes for 30 s Thereafter 1 ml of the suspension was
pour plated in tryptone soy agar
For the carrier tests on surfaces with grooves (case 31)
samples were taken using cotton swabs and cotton threads after the
required contact time Cotton swabs made with extra-thin toothpicks
and cotton wool (01 g 100 Hygienic cotton Etos) and cotton
threads (27-cm length 075-mm diameter [01 g] Super Durable
Glanskatoen no 8 HWS Markoma BV Ninove Belgium) were wet
sterilized at 121uC for 15 min The sterile cotton swabs were used to
take the samples from the 2- 1- 05- and 02-mm grooves by
swabbing three times through the grooves The sterile cotton threads
were used to take samples from the 5-mm groove by flossing and
scraping the groove for 30 s After sampling the cotton swabs and
cotton threads were put in 9-ml neutralizer vortexed and 100 ml
were plated on ALOA plates using a spiral plater
For the carrier tests with bacteriophages (case 32) the cotton
threads and cotton swabs were soaked for 1 min in a heat killed
suspension of L innocua (109 CFUml) to bind free phage particles
as neutralizer Subsequent sampling and plating were similar as
described above
During the 14 days of growing biofilms the numbers of
Listeria aerobic plate count and lactic acid bacteria were
determined at different time intervals on separate plates using
ALOA agar tryptone soy agar and De Man Rogosa and Sharpe
Agar (Lactobacillus broth Merck and 12 [wtvol] Agar
technical Agar no 3 Oxoid) respectively
Data and statistical analysis The volume of each groove
(cubic millimeters) was calculated and transformed to milliliters
The amount of organisms in a groove was then divided by this
volume to obtain a concentration in log CFU per milliliter The
value was calculated as log CFU per milliliter of the grooves in
order to equally compare the results obtained from the grooves that
have different depths
For statistical analysis the results of duplicate experiments
were combined The significance of differences (P 005) in
numbers of L monocytogenes that were reduced by different types
of disinfectants food residues and types of surface were
determined with analysis of the variance and a general linear
model using PASW Statistics 17 (SPSS Inc Chicago IL)
RESULTS AND DISCUSSION
Suspension tests (case 1) Reduction of more than 5
log units of Listeria planktonic cells and biofilm cells in
suspension was observed after 5-min contact time with
240 mgliter SDC and 740 mgliter Qac in all test
conditions where there was no significant difference (P
005) in reduction of the cells between the two disinfectants
in absence of food residues In contrast the effectiveness of
SDC (1-log reduction in 5 min with 3 ultrahigh-
temperature-processed milk) was less than that of Qac (5-
log reduction in 1 min with 10 ultrahigh-temperature-
processed milk) in presence of food residues as expected
(21 25 46) The effectiveness of SDC and Qac in absence
of food residues was higher than in presence of food
residues (data not shown) However these results comply
with the European standard EN1040 (6) which indicates a
reduction of 5 log units should be obtained after 5-min
contact time for an effective disinfectant
Carrier tests on smooth surfaces (case 2) The results
from the tests on the stainless steel coupons showed more
than a 5-log reduction after 1- 2- and 5-min contact time
with no significant difference (P 005) between SDC and
Qac (data not shown) These results complied with the
European standard EN13697 requirement (4) for a reduction
of 4 log units on clean surfaces indicating an effective
disinfectant in the surface test
Carrier tests on surfaces with grooves (case 3) The
shape of the 1-mm wide grooves with depths of 02 05 1
2 and 5 mm used as test surfaces in this study was beyond
the capability of standard sampling methods such as surface
contact plates and swabbing techniques Therefore several
methods eg the use of filter paper cotton swabs and
flossing threads were evaluated for their effectiveness to
recover Listeria cells from the grooves The results showed
that the flossing technique with cotton thread led to 70
recovery for the 5-mm groove For the other grooves this
method could recover only 20 With cotton swabs
prepared from extra-thin toothpicks and cotton wool a
recovery of 70 could be obtained for the remaining
grooves (data not shown) Therefore flossing and swab
methods were chosen as sampling methods for the 5-mm
groove and the other grooves respectively
In these tests the contact times of disinfectant on carriers
were 5 10 15 and 20 min Since the inactivation obtained for
the different contact times was not significantly different (P 005) which may be due to exhaustion of the disinfectants
(3 51) only the results from the contact time of 5 min are
shown Furthermore reduction of L monocytogenes was
dependent on the groove depth the deeper the groove the
lower the reduction This indicates that the activity of the
disinfectants stopped due to the inability to reach the cells in
the deeper areas Only the results of the extremes the 5- and
02-mm grooves are presented in Figures 3 and 4
All food residues were tested negative for L monocy-togenes In absence of food residues on a wet surface after
exposure to water (control) and the disinfectants a high
reduction of the cells was observed in the 02-mm grooves
whereas a similar trend but less reduction was observed in
the 5-mm grooves On the dry surfaces treatment of the
cells in the 02-mm groove resulted in levels below the
detection limit which can be partly explained by a high
reduction observed already as a result of the drying of the
plates for 24 h For the 5-mm grooves this initial reduction
was much lower and not all listerias were inactivated by the
treatment In most cases there was no significant difference
(P 005) between the control and disinfectants The
results demonstrated that drying of surfaces alone could
already largely reduce the cells on the surfaces especially in
the shallow grooves where deeper grooves provided suitable
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2015
FIGURE 3 Numbers of L monocytogenes (log CFU per milliliter) in wet conditions for the 02- (a) and 5-mm (b) grooves and dryconditions for the 02- (c) and 5-mm (d) grooves in presence and absence of food residues at initial inoculation ( T ~ 0) before (amp15 min or 24 h for wet and dry conditions respectively) and after exposure to sterile water ( control) SDC ( 240 mgliter) and Qac( 740 mgliter) for 5 min Data are means iexcl standard deviations of duplicate experiments for each disinfectant (n ~ 2) Dashed linesand vertical arrows (Q) indicate values lower than the detection limit DL detection limit of the method
FIGURE 4 Numbers of L monocytogenes biofilm cells (log CFU per milliliter) in presence of food residues in 02- (a) and 5-mm (b)grooves at initial inoculation ( T ~ 0) before (amp D 14) and after exposure to sterile water ( control) SDC ( 240 mgliter) Qac( 740 mgliter) and bacteriophages ( 109 PFUml) Data are means iexcl standard deviations of duplicate experiments for eachdisinfectant (n ~ 2) Dashed lines and vertical arrows (Q) indicate values lower than the detection limit
2016 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
conditions to prevent complete drying protecting some of
the cells from the disinfectants Drying of surfaces has been
considered as a significant bactericidal method to maintain
low numbers of bacteria in any environment and is
dependent on type of organism temperature relative
humidity and soiling (39 45 53) Furthermore type of
surface and topography have been reported to play an
important role on efficiency of disinfectants (30 40) in
particular surfaces with scrapes or defects where soil can
accumulate and protect bacteria against cleaning and
disinfecting agents (16 17 51)In presence of food residues in the wet 02-mm
grooves Listeria was reduced 2 to 3 log dependent on
types of food residues (Fig 3a) After exposure to the
disinfectants a reduction to below the detection limit was
observed for all food residues with no significant difference
(P 005) between SDC and Qac In the 5-mm grooves
before treatment less reduction (2 log CFUml) was
observed SDC was significantly (P 005) less effective
(2 log) than Qac ($2 log CFUml) for all food residues
except fish In the dry 02-mm grooves already a reduction
of 3 log CFUml was observed 24 h after inoculation
without treatment After exposure to the disinfectants the
numbers were further reduced to below the detection limit
In the 5-mm groove only a small (05 log CFUml ie
vegetable fish and ham residues) or no reduction (milk
residue) was observed immediately before treatment
probably because the cells were protected from the drying
in the deep grooves Treatment with SDC and Qac did not
inactivate all listerias with no significant difference (P
005) between the two disinfectants except in the case of
ham and vegetable residues where Qac performed signif-
icantly better than SDC in the wet grooves and for
vegetables in the dry grooves Previous studies have
reported an inactivation of the antimicrobial activity of
disinfectants by organic matter on surfaces showing 1- to 2-
log reductions on soiled surfaces compared with 3- to 4-
log reductions on clean surfaces (2 34) This could be due
to the chemical compositions such as protein and fat which
can bind with the disinfectants or reduction of penetration
into the bacterial layer (32 68) The chlorine-based
disinfectants have been found to be easily inactivated by
organic materials (25 46) whereas Qac was reported to be
less inactivated by organic matters (21) which was
confirmed in most cases in this study In general the test
on surfaces is dependent on the surface materials used and
viability of the cells dried on the surfaces resulting in
inconsistent results when compared with the test in
suspensions (15) Under practical conditions high amounts
of organic materials may be present resulting in less or no
activity of the disinfectant When the concentration of SDC
was increased from 240 to 300 mgliter a comparable
reduction of the cells to that of Qac was obtained however
there was no effect on reduction in the deep grooves (data
not shown) The results indicated that the disinfectant could
not reach all cells in the bottom of the groove while such
grooves (5-mm depth 1-mm width) might be present in
practice (eg small spaces between connection parts of
slicers) The effectiveness of the disinfectants in the deep
areas could be increased by using high pressure spraying
(32) however this method might have adverse effects since
it could also spread the bacterial cells to other areas and
environments in particular when high numbers of bacterial
cells are accumulated (42)
Carrier tests on surfaces with grooves in presence ofa biofilm (case 32) After 14 days of biofilm growing in
02-mm grooves (Fig 4) the number of cells remained
more or less stable with a decrease of the cells in vegetable
and ham biofilms (15 and 18 log CFUml respectively) In
5-mm grooves however growth of biofilm cells was
observed in all food residues A previous study reported that
L monocytogenes has the ability to form biofilms at
numbers of 104 to 107 CFUcm2 (34) and adheres to a
variety of food contact surfaces such as plastic glass
rubber and stainless steel particularly in pits or crevices
that are difficult to clean (31 61)To check if the biofilm cells were more resistant to the
disinfectants than the planktonic cells the biofilm cells were
brought into suspension and then tested The obtained
results a 5-log reduction in all conditions after 5-min
exposure to the disinfectants indicated no difference in
reduction of biofilm cells (case 32) compared with
planktonic cells (case 1) These results complied with a
previous study where reduction of the suspended biofilm
cells was higher than that of biofilm cells attached to
surfaces after exposure to disinfectants (50) It has been
described that a 10 to 100 times higher concentration of
disinfectants may be required to obtain the same reduction
of biofilm cells compared with planktonic cells (40 56)indicating the importance of preventing biofilm formation
For the bacteriophages an efficacy test was previously
performed with L monocytogenes strains in presence and
absence of food residues on flat stainless steel surfaces The
results demonstrated that rapid inactivation of Listeria cells
(2 to 3 log CFUcm2) within 30 min of contact time was
obtained (data not shown) which was similar with the
results of Soni and Nannapaneni (65) where a reduction of
35 log CFUcm2 was obtained in the same time with a
phage treatment on a 1-week-old L monocytogenes biofilm
on stainless steel coupons
In the 02-mm grooves (Fig 4a) Qac showed better
reduction (2 to 3 log) than SDC (12 to 27 log) with
inactivation below the detection limit in vegetable biofilms
The phages showed higher reduction (3 log CFUml P
005) than that of water or the chemical disinfectants in three
food biofilms except for the vegetable biofilms where all
cells were inactivated by every disinfectant In the 5-mm
grooves SDC and Qac did not reduce Listeria better than
water alone However the phages reduced approximately
14 log CFUml in ham and fish biofilms and approxi-
mately 08 log CFUml in milk and vegetable biofilms
which was not significantly different (P 005) from that of
the disinfectants except for ham biofilms The results
indicated that only in the 02-mm grooves the phages were
likely to be more effective in the biofilms with food matrix
than the chemical disinfectants This was probably due to
the fact that the disinfectants and phages did not penetrate
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2017
the foodbacterial matrix completely in the deep groove (2041) Furthermore the effectiveness of the disinfectants on Lmonocytogenes biofilms may be reduced by presence of
other bacteria (6 to 7 log CFUml) including lactic acid
bacteria (4 log CFUml) in the grooves as well (data not
shown) Phage efficacy depends on susceptibility of the
bacterial cells to the phages and availability of receptor sites
between phages and the bacterial cells (13) which could be
blocked by the food matrix (35) In this study the phage
efficacy was enhanced by keeping the biofilm plates in
plastic bags after application of the phages to maintain a
high humidity that would enhance the diffusion and the
chance of contact between Listeria cells and phage particles
(66)Strikingly the biofilm removal by sterile water
(control) resulted in a similar reduction of the biofilm cells
compared with disinfectants In the present study the tests
were performed to simulate survival of bacteria in presence
or absence of organic matter in pits or cracks on food
contact surfaces and to test the effect of disinfection
therefore cleaning of the surfaces before using disinfectants
was excluded So in this case plates and grooves were
dirtier than soiled surfaces in reality which explains the
observed low efficacy of the disinfectants and the water
washed away the biofilm cells from the grooves Rinsing the
biofilm on surfaces by water has been reported to reduce the
biofilm cells at about 1 log unit of cells (34)The results in this study clearly demonstrated that
presence of grooves or spaces on surfaces humidity and
presence of food substrates influenced the antimicrobial
effect of disinfectants and bacteriophages Bacteriophages
showed a better antimicrobial effect than the chemical
disinfectants (ie SDC and Qac) in the shallow grooves but
not in the deep grooves Use of bacteriophages as a
biocontrol could only be a promising method in limited
cases however use of large quantities in practice may be
costly and phage-resistant strains may occur (35 62)
ACKNOWLEDGMENTS
The authors thank Diversey Inc (Utrecht The Netherlands) who
provided the chemical disinfectants for this study We also thank Micreos
Food Safety BV (Wageningen The Netherlands) for Listeria bacteriophage
Listex P100 and L innocua (serovar 5) Our great appreciation goes to
Feifei Gao for her technical assistance in the phage experiments and Hilda
Nyati for her work on smooth surfaces
REFERENCES
1 Aarnisalo K J Lunden H Korkeala and G Wirtanen 2007
Susceptibility of Listeria monocytogenes strains to disinfectants and
chlorinated alkaline cleaners at cold temperatures LWT - Food SciTechnol 401041ndash1048
2 Aarnisalo K S Salo H Miettinen M Suihko G Wirtanen T
Autio J Lunden H Korkeala and A Sjoberg 2000 Bactericidal
efficiencies of commercial disinfectants against Listeria monocyto-
genes on surfaces J Food Saf 20237ndash250
3 Adams M R A D Hartley and L J Cox 1989 Factors affecting
the efficacy of washing procedures used in the production of prepared
salads Food Microbiol 669ndash77
4 Anonymous 2001 European Standard EN 136972001 Chemical
disinfectants and antisepticsmdashqualitative non-porous surface test for
the evaluation of bacteriocidal andor fungicidal activity of chemical
disinfectants used in food industrial domestic and institutional
areasmdashtest method and requirements without mechanical action (phase
2 step 2) Available at httpwwwceneucenSectorsTechnical
CommitteesWorkshopsCENTechnicalCommitteesPagesStandards
aspxparam~6197amptitle~Chemical20disinfectants20and20
antiseptics Accessed February 2014
5 Anonymous 2004 Microbiology of food and animal feeding stuffs mdash
horizontal method for the detection and enumeration of Listeria
monocytogenesmdashpart 1 detection method ISO 11290-11996Amd
12004 International Organization for Standardization Geneva
Available at httpswwwisoorgobpuiisostdiso11290-1ed-1
v1enamd1amd1 Accessed January 2014
6 Anonymous 2005 European Standard EN 10402005 Chemical
disinfectants and antiseptics Quantitative suspension test for the
evaluation of basic bactericidal activity of chemical disinfectants
and antisepticsmdashtest method and requirements (phase 1) Available
at httpwwwceneucenSectorsTechnicalCommitteesWorkshops
CENTechnicalCommitteesPagesStandardsaspxparam~6197amptitle~
Chemical20disinfectants20and20antiseptics Accessed February
2014
7 Arachchi G J G A G Cridge B M Dias-Wanigasekera C D
Cruz L McIntyre R Liu S H Flint and A N Mutukumira 2013
Effectiveness of phages in the decontamination of Listeria monocy-
togenes adhered to clean stainless steel stainless steel coated with
fish protein and as a biofilm J Ind Microbiol Biotechnol 401105ndash
1116
8 Autio T R Keto-Timonen J Bjorkroth and H Korkeala 2003
Characterization of persistent and sporadic Listeria monocytogenes
strains by pulsed-field gel electrophoresis (PFGE) and amplified
fragment length polymorphism (AFLP) Syst Appl Microbiol 26
539ndash545
9 Bagge-Ravn D Y Ng M Hjelm J N Christiansen C Johansen
and L Gram 2003 The microbial ecology of processing equipment
in different fish industriesmdashanalysis of the microflora during
processing and following cleaning and disinfection Int J Food
Microbiol 87239ndash250
10 Belessi C A A S Gounadaki A N Psomas and P N Skandamis
2011 Efficiency of different sanitation methods on Listeria
monocytogenes biofilms formed under various environmental condi-
tions Int J Food Microbiol 145S46ndashS52
11 Beresford M R P W Andrew and G Shama 2001 Listeria
monocytogenes adheres to many materials found in food-processing
environments J Appl Microbiol 901000ndash1005
12 Best M M M M E Kennedy and F F Coates 1990 Efficacy of a
variety of disinfectants against Listeria spp Appl Environ Micro-
biol 56377ndash380
13 Bigwood T J A Hudson and C Billington 2009 Influence of host
and bacteriophage concentrations on the inactivation of food-borne
pathogenic bacteria by two phages FEMS Microbiol Lett 29159ndash
64
14 Blackman I C and J F Frank 1996 Growth of Listeria
monocytogenes as a biofilm on various food-processing surfaces J
Food Prot 59827ndash831
15 Bloomfield S F M Arthur B Klingeren W van Pullen J T
Holah and R Elton 1994 An evaluation of the repeatability and
reproducibility of a surface test for the activity of disinfectants J
Appl Bacteriol 7686ndash94
16 Boulange-Petermann L J Rault and M-N Bellon-Fontaine
1997 Adhesion of Streptococcus thermophilus to stainless steel
with different surface topography and roughness Biofouling 11
201ndash216
17 Bower C K J McGuire and M A Daeschel 1996 The adhesion
and detachment of bacteria and spores on food contact surfaces
Trends Food Sci Technol 7152ndash157
18 Bryan F L 2002 Where we are in the retail food safety how we got
to where we are and how do we get there J Environ Health 6529ndash
36
19 Carey-Smith G V C Billington A J Cornelius J A Hudson and
J A Heinemann 2006 Isolation and characterization of bacterio-
phages infecting Salmonella spp FEMS Microbiol Lett 258182ndash
186
2018 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
20 Carpentier B and O Cerf 1993 Biofilms and their consequences
with particular reference to hygiene in the food industry J Appl
Bacteriol 75499ndash511
21 Carsberg H 1996 Selecting your sanitizers Food Qual 235ndash36
61ndash62
22 Chibeu A L Agius A Gao P M Sabour A M Kropinski and S
Balamurugan 2013 Efficacy of bacteriophage LISTEXTMP100
combined with chemical antimicrobials in reducing Listeria mono-
cytogenes in cooked turkey and roast beef Int J Food Microbiol
167208ndash214
23 Cox L J T Kleiss J L Cordier C Cordellana P Konkel C
Pedrazzini R Beumer and A Siebenga 1989 Listeria spp in food
processing non-food and domestic environments Food Microbiol 6
49ndash61
24 Cruz C D and G C Fletcher 2012 Assessing manufacturersrsquo
recommended concentrations of commercial sanitizers on inactivation
of Listeria monocytogenes Food Control 26194ndash199
25 De Beer D R Srinivasan and P S Stewart 1994 Direct
measurement of chlorine penetration into biofilms during disinfec-
tion Appl Environ Microbiol 604339ndash4344
26 Doolittle M M J J Cooney and D E Caldwell 1995 Lytic
infection of Escherichia coli biofilms by bacteriophage T4 Can J
Microbiol 4112ndash18
27 Environmental Protection Agency (EPA) Office of Wastewater
Enforcement and Compliance 2014 Guide to field storage of
biosolids and other organic by-products used in agriculture and for
soil resource management Available at httpwaterepagovscitech
wastetechbiosolidsguidecfm Accessed January 2014
28 Fatemi P and J F Frank 1999 Inactivation of Listeria
monocytogenesPseudomonas biofilms by peracid sanitizer J Food
Prot 62761ndash765
29 Fenlon D R J Wilson and W Donachie 1996 The incidence and
level of Listeria monocytogenes contamination of food sources at
primary production and initial processing J Appl Bacteriol 81641ndash
650
30 Frank J F and R A N Chmielewski 1997 Effectiveness of
sanitation with quaternary ammonium compound or chlorine on
stainless steel and other domestic food-preparation surfaces J Food
Prot 6043ndash47
31 Frank J F and R A Koffi 1990 Surface-adherent growth of
Listeria monocytogenes is associated with increased resistance of
surfactant sanitizers and heat J Food Prot 53550ndash554
32 Gibson H J H Taylor K E Hall and J T Holah 1999
Effectiveness of cleaning techniques used in the food industry in terms
of the removal of bacterial biofilms J Appl Microbiol 8741ndash48
33 Gombas D E Y Chen R S Clavaro and V N Scott 2003 Survey
of Listeria monocytogenes in foods J Food Prot 66559ndash569
34 Gram L D Bagge-Ravn Y Y Ng P Gymoese and B F Vogel
2007 Influence of food soiling matrix on cleaning and disinfection
efficiency on surface attached Listeria monocytogenes Food Control
181165ndash1171
35 Greer G G 2005 Bacteriophage control of foodborne bacteria J
Food Prot 681102ndash1111
36 Gronholm L G Wirtanen K Ahlgren K Nordstrom and A
Sjoberg 1999 Screening of antimicrobial activities of disinfectants
and cleaning agents against foodborne spoilage microbes Eur Food
Res Technol 208289ndash298
37 Hagens S 2014 Personal communication
38 Hibma A M S A Jassim and M W Griffiths 1997 Infection and
removal of L-forms of Listeria monocytogenes with bred bacterio-
phage Int J Food Microbiol 34197ndash207
39 Hirai Y 1991 Survival of bacteria under dry conditions from a
viewpoint of nosocomial infection J Hosp Infect 19191ndash200
40 Holah J T 1992 Industrial monitoring hygiene in food processing
p 645ndash659A In L F Melo T R Bott M Fletcher and B
Capdeville (ed) Biofilmsmdashscience and technology Kluwer Aca-
demic Publishers Dordrecht
41 Holah J T C Higgs S Robinson D Worthington and H
Spenceley 1990 conductance-based surface disinfection test for food
hygiene Lett Appl Microbiol 11255ndash259
42 Holah J T J H Taylor and J S Holder 1993 The spread of
Listeria by cleaning systems part II Technical Memorandum no 673
Campden and Chorleywood Food Research Association Chipping
Campden UK
43 Hughes K A I W Sutherland and M V Jones 1998 Biofilm
susceptibility to bacteriophage attack the role of phage-borne
polysaccharide depolymerase Microbiology 1443039ndash3047
44 Joerger R D 2003 Alternatives to antibiotics bacteriocins
antimicrobial peptides and bacteriophages Poult Sci 82640ndash647
45 Kusumaningrum H D G Riboldi W C Hazeleger and R R
Beumer 2003 Survival of foodborne pathogens on stainless steel
surfaces and cross-contamination to foods Int J Food Microbiol 85
227ndash236
46 Lee S-H and J Frank 1991 Inactivation of surface adherent
Listeria monocytogenes hypochlorite and heat J Food Prot 544ndash
6
47 Leverentz B W S Conway M J Camp W J Janisiewicz T
Abuladze M Yang R Saftner and A Sulakvelidze 2003
Biocontrol of Listeria monocytogenes on fresh-cut produce by
treatment with lytic bacteriophages and a bacteriocin Appl Environ
Microbiol 694519ndash4526
48 Lin C M K Takeuchi L Zhang C B Dohm J D Meyer P A
Hall and M P Doyle 2006 Cross-contamination between
processing equipment and deli meats by Listeria monocytogenes J
Food Prot 6971ndash79
49 Lunden J M T J Autio and H J Korkeala 2002 Transfer of
persistent Listeria monocytogenes contamination between food-
processing plants associated with a dicing machine J Food Prot
651129ndash1133
50 Luppens S B I M W Reij R W L van der Heijden F M
Rombouts and T Abee 2002 Development of a standard test to
assess the resistance of Staphylococcus aureus biofilm cells to
disinfectants Appl Environ Microbiol 684194ndash4200
51 Mafu A A D Roy J Goulet L Savoie and R Roy 1990
Efficiency of sanitizing agents for destroying Listeria monocytogenes
on contaminated surfaces J Dairy Sci 733428ndash3432
52 Mahony J O McAuliffe R Paul and D van Sinderen 2011
Bacteriophages as biocontrol agents of food pathogens Curr Opin
Biotechnol 22157ndash163
53 McEldowney S and M Fletcher 1988 Effect of temperature and
humidity on survival of bacteria attached to dry solid surfaces Lett
Appl Microbiol 783ndash86
54 Miettinen L K J Bjorkroth and H Korkeala 1999 Characterisa-
tion of Listeria monocytogenes from an ice-cream plant by serotyping
and pulsed-field gel electrophoresis Int J Food Microbiol 46187ndash
192
55 Mosteller T M and J R Bishop 1993 Sanitizer efficacy against
attached bacteria in a milk biofilm J Food Prot 5634ndash41
56 Norwood D E and A Gilmour 2000 The growth and resistance to
sodium hypochlorite of Listeria monocytogenes in a steady-state
multispecies biofilm J Appl Microbiol 88512ndash520
57 Oliveira M I Vinas P Colas M Anguera J Usall and M
Abadias 2014 Effectiveness of a bacteriophage in reducing Listeria
monocytogenes on fresh-cut fruits and fruit juices Food Microbiol
38137ndash142
58 Ronner A B and A C L Wong 1993 Biofilm development and
sanitizer inactivation of Listeria monocytogenes and Salmonella
typhimurium on stainless steel and buna-n rubber J Food Prot 56
750ndash758
59 Sallam S S and C W Donnelly 1992 Destruction injury and
repair of Listeria species exposed to sanitizing compounds J Food
Prot 55771ndash1033
60 Sillankorva S D R Oliveira M J Vieira I W Sutherland and J
Azeredo 2004 Bacteriophage V S1 infection of Pseudomonas
fluorescens planktonic cells versus biofilms Biofouling 20133ndash138
61 Silva S P Teixeira R Olieveira and J Azeredo 2008 Adhesion to
and viability of Listeria monocytogenes on food contact surfaces J
Food Prot 711379ndash1385
62 Skurnik M and E Strauch 2006 Phage therapy facts and fiction
Int J Food Microbiol 2965ndash14 (Review)
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2019
63 Somers E B J L Schoeni and A C L Wong 1994 Effect of
trisodium phosphate on biofilm and planktonic cells of Campylo-
bacter jejuni Escherichia coli O157 H7 Listeria monocytogenes
and Salmonella typhimurium Int J Food Microbiol 22269ndash
276
64 Somers E B and A C L Wong 2004 Efficacy of two cleaning
and sanitizing combination on Listeria monocytogenes biofilm
formed at low temperature on a variety of materials in the presence
of ready-to-eat meat residue J Food Prot 672218ndash2229
65 Soni K A and R Nannapaneni 2010 Removal of Listeria
monocytogenes biofilms with bacteriophages P100 J Food Prot 73
1519ndash1524
66 Sutherland I W K A Hughes L C Skillman and K Tait 2004
The interaction of phage and biofilm FEMS Microbiol Lett 2321ndash6
(MiniReview)
67 Swaminathan B 2001 Listeria monocytogenes p 383ndash409 In L R
Beuchat M P Doyle and T J Montville (ed) Food microbiology
fundamentals and frontiers 2nd ed ASM Press Washington DC
68 Thevenot D A Dernburg and C Vernozy-Rozand 2006 An
updated review of Listeria monocytogenes in the pork meat industry
and its products J Appl Microbiol 1017ndash17
69 Tuncan E U 1993 Effect of cold temperature on germicidal efficacy
of quaternary ammonium compound iodophor and chlorine on
Listeria J Food Prot 531029ndash1033
2020 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
suspension of the food residue 10 g of each food was aseptically
placed in a stomacher bag with 90 ml PSS and subsequently
homogenized in a stomacher (Stomacher 400 Circulator Seward
England) for 1 min at 260 rpm kept at 4 to 7uC and used within 4 h
One milliliter of ultrahigh-temperature-processed milk was asep-
tically added to 9 ml PSS vortexed and used immediately Prior to
the test all food residues were tested for the presence of Lmonocytogenes according to ISO 11290-11996Amd 12004 (5) to
verify if the food matrix was free of L monocytogenes
Suspension tests (case 1) The tests were performed
according to the European standard EN 10402005 (6) An
overnight culture of L monocytogenes with or without 10 food
residue was added to each disinfectant at recommended concen-
trations at room temperature for 5 min of contact time The tests
were performed in triplicate for each disinfectant on different days
Figure 2 shows a diagram of all tests that were performed in this
study
Carrier tests The tests were performed on smooth surfaces
(case 2) and surfaces with grooves (case 3) Before the experiments
started the stainless steel coupons and plates with grooves were
soaked overnight in anionic-active detergent (Dreft Procter amp
Gamble Netherlands BV Rotterdam The Netherlands) Thereafter
both surfaces were rinsed with hot water (70 to 80uC) and air dried
The coupons were put in a glass container covered with aluminum
foil while the plates were wrapped in paper and then all were
sterilized at 121uC for 15 min
Carrier tests on smooth surfaces (case 2) (4) To inoculate
the stainless steel coupons 10 ml of the test suspensions (109
CFUml resulting in 64 log CFUcm2) were put on one side of
the coupon and allowed to air dry for 1 h in a biological safety
cabinet After inoculation of the stainless steel coupons these were
placed in a 12-well plate (Greiner Bio-One BV Alphen ad Rijn
The Netherlands) containing 1 ml of SDC (240 mgliter) or Qac
(740 mgliter) and left at 1- 2- and 5-min contact time at room
temperature The experiment was repeated three times on different
days for each disinfectant
Carrier tests on surfaces with grooves (case 31) To
inoculate the surface with grooves (case 31) 10 ml of test
suspension (107 CFUml resulting in 6 log CFUcm2) with or
without 10 food suspension were put on the plates and evenly
spread over the surfaces to fully fill the grooves by using a sterile
sponge The plates were then kept at room temperature for 15 min
and 24 h to simulate wet and dry conditions respectively After
inoculation of the plates 240 mgliter SDC or 740 mgliter Qac
were sprayed on the plates (5 ml) by using a sterile spray bottle
(perfume spray bottle Etos Wageningen The Netherlands) and
left to achieve 5- 10- 15- or 20-min contact time at room
temperature The experiment was repeated two times on different
days for each disinfectant
Carrier tests on surfaces with grooves in presence of abiofilm (case 32) To mimic the situation in a food processing
plant with biofilm formation in cracked equipment a biofilm was
grown by daily inoculating a diluted overnight culture of the three
L monocytogenes strains with each food residue (3 ml 106 CFU
ml) in the grooves for 7 consecutive days Thereafter only food
suspension (3 ml) was added daily to the grooves for another 7 days
to allow further development of the biofilm During this period the
plates were stored at 15uC in a closed plastic box to maintain
stable humidity (50 relative humidity measured by a digital
hygrothermometer VWR International BV Amsterdam The
Netherlands) The disinfectant tests were carried out as described
in the previous paragraph for 20-min contact time
For the bacteriophage test 5 ml of a 109 PFUml phage
suspension were sprayed over the biofilm plates Thereafter the
plates were put in sterile plastic bags (Aseptic vacuum bags 400
by 510 mm Hevel Zaandam The Netherlands) to maintain
humidity and left for 30-min contact time
The tests on surfaces with grooves were performed in
duplicate on different days for each disinfectant and for
bacteriophages
Effect of disinfectants on suspended biofilm cells The 2-
week biofilm cells were taken out of each groove by scraping the
grooves with sterile extra-thin toothpicks (Jordan Intec BV
Utrecht The Netherlands) The cells were mixed with 9 ml PSS
and tested with disinfectants as described in the suspension test
Microbiological analysis In the suspension test (case 1) the
reaction was terminated after the required contact time by
FIGURE 2 Diagram shows all tests per-formed in this study The suspension andcarriers tests on smooth surface wereperformed with or without food residues(w or wo) according to the Europeanstandard EN 10402005 Tests on thesurface with grooves were performed withL monocytogenes cells on wet and dryconditions in presence and absence of foodresidues and 2-week food biofilm Thedisinfectants used were SDC (240 mgliter)and Qac (740 mgliter) while sterile waterwas used as a control
2014 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
transferring 1 ml of the suspensions to 9 ml of neutralizer for 1 min
Of these suspensions 100 ml was then plated on Agar Listeriaaccording to Ottaviani and Agosti (ALOA BioMerieux SA Marcy
lrsquoEtoile France) using a spiral plater (Eddy Jet IUL Instrument
SA Barcelona Spain) For the carrier tests on flat surfaces (case
2) the disinfectant reaction was terminated by transferring the
coupons plus disinfectant to a 9-ml tube containing neutralizer and
45-g glass beads (1 mm Emergo Landsmeer The Netherlands)
As a blank inoculated coupons were transferred to 10 ml of PSS
plus glass beads The cells were removed from the coupons by
vortexing the tubes for 30 s Thereafter 1 ml of the suspension was
pour plated in tryptone soy agar
For the carrier tests on surfaces with grooves (case 31)
samples were taken using cotton swabs and cotton threads after the
required contact time Cotton swabs made with extra-thin toothpicks
and cotton wool (01 g 100 Hygienic cotton Etos) and cotton
threads (27-cm length 075-mm diameter [01 g] Super Durable
Glanskatoen no 8 HWS Markoma BV Ninove Belgium) were wet
sterilized at 121uC for 15 min The sterile cotton swabs were used to
take the samples from the 2- 1- 05- and 02-mm grooves by
swabbing three times through the grooves The sterile cotton threads
were used to take samples from the 5-mm groove by flossing and
scraping the groove for 30 s After sampling the cotton swabs and
cotton threads were put in 9-ml neutralizer vortexed and 100 ml
were plated on ALOA plates using a spiral plater
For the carrier tests with bacteriophages (case 32) the cotton
threads and cotton swabs were soaked for 1 min in a heat killed
suspension of L innocua (109 CFUml) to bind free phage particles
as neutralizer Subsequent sampling and plating were similar as
described above
During the 14 days of growing biofilms the numbers of
Listeria aerobic plate count and lactic acid bacteria were
determined at different time intervals on separate plates using
ALOA agar tryptone soy agar and De Man Rogosa and Sharpe
Agar (Lactobacillus broth Merck and 12 [wtvol] Agar
technical Agar no 3 Oxoid) respectively
Data and statistical analysis The volume of each groove
(cubic millimeters) was calculated and transformed to milliliters
The amount of organisms in a groove was then divided by this
volume to obtain a concentration in log CFU per milliliter The
value was calculated as log CFU per milliliter of the grooves in
order to equally compare the results obtained from the grooves that
have different depths
For statistical analysis the results of duplicate experiments
were combined The significance of differences (P 005) in
numbers of L monocytogenes that were reduced by different types
of disinfectants food residues and types of surface were
determined with analysis of the variance and a general linear
model using PASW Statistics 17 (SPSS Inc Chicago IL)
RESULTS AND DISCUSSION
Suspension tests (case 1) Reduction of more than 5
log units of Listeria planktonic cells and biofilm cells in
suspension was observed after 5-min contact time with
240 mgliter SDC and 740 mgliter Qac in all test
conditions where there was no significant difference (P
005) in reduction of the cells between the two disinfectants
in absence of food residues In contrast the effectiveness of
SDC (1-log reduction in 5 min with 3 ultrahigh-
temperature-processed milk) was less than that of Qac (5-
log reduction in 1 min with 10 ultrahigh-temperature-
processed milk) in presence of food residues as expected
(21 25 46) The effectiveness of SDC and Qac in absence
of food residues was higher than in presence of food
residues (data not shown) However these results comply
with the European standard EN1040 (6) which indicates a
reduction of 5 log units should be obtained after 5-min
contact time for an effective disinfectant
Carrier tests on smooth surfaces (case 2) The results
from the tests on the stainless steel coupons showed more
than a 5-log reduction after 1- 2- and 5-min contact time
with no significant difference (P 005) between SDC and
Qac (data not shown) These results complied with the
European standard EN13697 requirement (4) for a reduction
of 4 log units on clean surfaces indicating an effective
disinfectant in the surface test
Carrier tests on surfaces with grooves (case 3) The
shape of the 1-mm wide grooves with depths of 02 05 1
2 and 5 mm used as test surfaces in this study was beyond
the capability of standard sampling methods such as surface
contact plates and swabbing techniques Therefore several
methods eg the use of filter paper cotton swabs and
flossing threads were evaluated for their effectiveness to
recover Listeria cells from the grooves The results showed
that the flossing technique with cotton thread led to 70
recovery for the 5-mm groove For the other grooves this
method could recover only 20 With cotton swabs
prepared from extra-thin toothpicks and cotton wool a
recovery of 70 could be obtained for the remaining
grooves (data not shown) Therefore flossing and swab
methods were chosen as sampling methods for the 5-mm
groove and the other grooves respectively
In these tests the contact times of disinfectant on carriers
were 5 10 15 and 20 min Since the inactivation obtained for
the different contact times was not significantly different (P 005) which may be due to exhaustion of the disinfectants
(3 51) only the results from the contact time of 5 min are
shown Furthermore reduction of L monocytogenes was
dependent on the groove depth the deeper the groove the
lower the reduction This indicates that the activity of the
disinfectants stopped due to the inability to reach the cells in
the deeper areas Only the results of the extremes the 5- and
02-mm grooves are presented in Figures 3 and 4
All food residues were tested negative for L monocy-togenes In absence of food residues on a wet surface after
exposure to water (control) and the disinfectants a high
reduction of the cells was observed in the 02-mm grooves
whereas a similar trend but less reduction was observed in
the 5-mm grooves On the dry surfaces treatment of the
cells in the 02-mm groove resulted in levels below the
detection limit which can be partly explained by a high
reduction observed already as a result of the drying of the
plates for 24 h For the 5-mm grooves this initial reduction
was much lower and not all listerias were inactivated by the
treatment In most cases there was no significant difference
(P 005) between the control and disinfectants The
results demonstrated that drying of surfaces alone could
already largely reduce the cells on the surfaces especially in
the shallow grooves where deeper grooves provided suitable
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2015
FIGURE 3 Numbers of L monocytogenes (log CFU per milliliter) in wet conditions for the 02- (a) and 5-mm (b) grooves and dryconditions for the 02- (c) and 5-mm (d) grooves in presence and absence of food residues at initial inoculation ( T ~ 0) before (amp15 min or 24 h for wet and dry conditions respectively) and after exposure to sterile water ( control) SDC ( 240 mgliter) and Qac( 740 mgliter) for 5 min Data are means iexcl standard deviations of duplicate experiments for each disinfectant (n ~ 2) Dashed linesand vertical arrows (Q) indicate values lower than the detection limit DL detection limit of the method
FIGURE 4 Numbers of L monocytogenes biofilm cells (log CFU per milliliter) in presence of food residues in 02- (a) and 5-mm (b)grooves at initial inoculation ( T ~ 0) before (amp D 14) and after exposure to sterile water ( control) SDC ( 240 mgliter) Qac( 740 mgliter) and bacteriophages ( 109 PFUml) Data are means iexcl standard deviations of duplicate experiments for eachdisinfectant (n ~ 2) Dashed lines and vertical arrows (Q) indicate values lower than the detection limit
2016 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
conditions to prevent complete drying protecting some of
the cells from the disinfectants Drying of surfaces has been
considered as a significant bactericidal method to maintain
low numbers of bacteria in any environment and is
dependent on type of organism temperature relative
humidity and soiling (39 45 53) Furthermore type of
surface and topography have been reported to play an
important role on efficiency of disinfectants (30 40) in
particular surfaces with scrapes or defects where soil can
accumulate and protect bacteria against cleaning and
disinfecting agents (16 17 51)In presence of food residues in the wet 02-mm
grooves Listeria was reduced 2 to 3 log dependent on
types of food residues (Fig 3a) After exposure to the
disinfectants a reduction to below the detection limit was
observed for all food residues with no significant difference
(P 005) between SDC and Qac In the 5-mm grooves
before treatment less reduction (2 log CFUml) was
observed SDC was significantly (P 005) less effective
(2 log) than Qac ($2 log CFUml) for all food residues
except fish In the dry 02-mm grooves already a reduction
of 3 log CFUml was observed 24 h after inoculation
without treatment After exposure to the disinfectants the
numbers were further reduced to below the detection limit
In the 5-mm groove only a small (05 log CFUml ie
vegetable fish and ham residues) or no reduction (milk
residue) was observed immediately before treatment
probably because the cells were protected from the drying
in the deep grooves Treatment with SDC and Qac did not
inactivate all listerias with no significant difference (P
005) between the two disinfectants except in the case of
ham and vegetable residues where Qac performed signif-
icantly better than SDC in the wet grooves and for
vegetables in the dry grooves Previous studies have
reported an inactivation of the antimicrobial activity of
disinfectants by organic matter on surfaces showing 1- to 2-
log reductions on soiled surfaces compared with 3- to 4-
log reductions on clean surfaces (2 34) This could be due
to the chemical compositions such as protein and fat which
can bind with the disinfectants or reduction of penetration
into the bacterial layer (32 68) The chlorine-based
disinfectants have been found to be easily inactivated by
organic materials (25 46) whereas Qac was reported to be
less inactivated by organic matters (21) which was
confirmed in most cases in this study In general the test
on surfaces is dependent on the surface materials used and
viability of the cells dried on the surfaces resulting in
inconsistent results when compared with the test in
suspensions (15) Under practical conditions high amounts
of organic materials may be present resulting in less or no
activity of the disinfectant When the concentration of SDC
was increased from 240 to 300 mgliter a comparable
reduction of the cells to that of Qac was obtained however
there was no effect on reduction in the deep grooves (data
not shown) The results indicated that the disinfectant could
not reach all cells in the bottom of the groove while such
grooves (5-mm depth 1-mm width) might be present in
practice (eg small spaces between connection parts of
slicers) The effectiveness of the disinfectants in the deep
areas could be increased by using high pressure spraying
(32) however this method might have adverse effects since
it could also spread the bacterial cells to other areas and
environments in particular when high numbers of bacterial
cells are accumulated (42)
Carrier tests on surfaces with grooves in presence ofa biofilm (case 32) After 14 days of biofilm growing in
02-mm grooves (Fig 4) the number of cells remained
more or less stable with a decrease of the cells in vegetable
and ham biofilms (15 and 18 log CFUml respectively) In
5-mm grooves however growth of biofilm cells was
observed in all food residues A previous study reported that
L monocytogenes has the ability to form biofilms at
numbers of 104 to 107 CFUcm2 (34) and adheres to a
variety of food contact surfaces such as plastic glass
rubber and stainless steel particularly in pits or crevices
that are difficult to clean (31 61)To check if the biofilm cells were more resistant to the
disinfectants than the planktonic cells the biofilm cells were
brought into suspension and then tested The obtained
results a 5-log reduction in all conditions after 5-min
exposure to the disinfectants indicated no difference in
reduction of biofilm cells (case 32) compared with
planktonic cells (case 1) These results complied with a
previous study where reduction of the suspended biofilm
cells was higher than that of biofilm cells attached to
surfaces after exposure to disinfectants (50) It has been
described that a 10 to 100 times higher concentration of
disinfectants may be required to obtain the same reduction
of biofilm cells compared with planktonic cells (40 56)indicating the importance of preventing biofilm formation
For the bacteriophages an efficacy test was previously
performed with L monocytogenes strains in presence and
absence of food residues on flat stainless steel surfaces The
results demonstrated that rapid inactivation of Listeria cells
(2 to 3 log CFUcm2) within 30 min of contact time was
obtained (data not shown) which was similar with the
results of Soni and Nannapaneni (65) where a reduction of
35 log CFUcm2 was obtained in the same time with a
phage treatment on a 1-week-old L monocytogenes biofilm
on stainless steel coupons
In the 02-mm grooves (Fig 4a) Qac showed better
reduction (2 to 3 log) than SDC (12 to 27 log) with
inactivation below the detection limit in vegetable biofilms
The phages showed higher reduction (3 log CFUml P
005) than that of water or the chemical disinfectants in three
food biofilms except for the vegetable biofilms where all
cells were inactivated by every disinfectant In the 5-mm
grooves SDC and Qac did not reduce Listeria better than
water alone However the phages reduced approximately
14 log CFUml in ham and fish biofilms and approxi-
mately 08 log CFUml in milk and vegetable biofilms
which was not significantly different (P 005) from that of
the disinfectants except for ham biofilms The results
indicated that only in the 02-mm grooves the phages were
likely to be more effective in the biofilms with food matrix
than the chemical disinfectants This was probably due to
the fact that the disinfectants and phages did not penetrate
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2017
the foodbacterial matrix completely in the deep groove (2041) Furthermore the effectiveness of the disinfectants on Lmonocytogenes biofilms may be reduced by presence of
other bacteria (6 to 7 log CFUml) including lactic acid
bacteria (4 log CFUml) in the grooves as well (data not
shown) Phage efficacy depends on susceptibility of the
bacterial cells to the phages and availability of receptor sites
between phages and the bacterial cells (13) which could be
blocked by the food matrix (35) In this study the phage
efficacy was enhanced by keeping the biofilm plates in
plastic bags after application of the phages to maintain a
high humidity that would enhance the diffusion and the
chance of contact between Listeria cells and phage particles
(66)Strikingly the biofilm removal by sterile water
(control) resulted in a similar reduction of the biofilm cells
compared with disinfectants In the present study the tests
were performed to simulate survival of bacteria in presence
or absence of organic matter in pits or cracks on food
contact surfaces and to test the effect of disinfection
therefore cleaning of the surfaces before using disinfectants
was excluded So in this case plates and grooves were
dirtier than soiled surfaces in reality which explains the
observed low efficacy of the disinfectants and the water
washed away the biofilm cells from the grooves Rinsing the
biofilm on surfaces by water has been reported to reduce the
biofilm cells at about 1 log unit of cells (34)The results in this study clearly demonstrated that
presence of grooves or spaces on surfaces humidity and
presence of food substrates influenced the antimicrobial
effect of disinfectants and bacteriophages Bacteriophages
showed a better antimicrobial effect than the chemical
disinfectants (ie SDC and Qac) in the shallow grooves but
not in the deep grooves Use of bacteriophages as a
biocontrol could only be a promising method in limited
cases however use of large quantities in practice may be
costly and phage-resistant strains may occur (35 62)
ACKNOWLEDGMENTS
The authors thank Diversey Inc (Utrecht The Netherlands) who
provided the chemical disinfectants for this study We also thank Micreos
Food Safety BV (Wageningen The Netherlands) for Listeria bacteriophage
Listex P100 and L innocua (serovar 5) Our great appreciation goes to
Feifei Gao for her technical assistance in the phage experiments and Hilda
Nyati for her work on smooth surfaces
REFERENCES
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Susceptibility of Listeria monocytogenes strains to disinfectants and
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2 Aarnisalo K S Salo H Miettinen M Suihko G Wirtanen T
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disinfectants used in food industrial domestic and institutional
areasmdashtest method and requirements without mechanical action (phase
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CommitteesWorkshopsCENTechnicalCommitteesPagesStandards
aspxparam~6197amptitle~Chemical20disinfectants20and20
antiseptics Accessed February 2014
5 Anonymous 2004 Microbiology of food and animal feeding stuffs mdash
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6 Anonymous 2005 European Standard EN 10402005 Chemical
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and antisepticsmdashtest method and requirements (phase 1) Available
at httpwwwceneucenSectorsTechnicalCommitteesWorkshops
CENTechnicalCommitteesPagesStandardsaspxparam~6197amptitle~
Chemical20disinfectants20and20antiseptics Accessed February
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7 Arachchi G J G A G Cridge B M Dias-Wanigasekera C D
Cruz L McIntyre R Liu S H Flint and A N Mutukumira 2013
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togenes adhered to clean stainless steel stainless steel coated with
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1116
8 Autio T R Keto-Timonen J Bjorkroth and H Korkeala 2003
Characterization of persistent and sporadic Listeria monocytogenes
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539ndash545
9 Bagge-Ravn D Y Ng M Hjelm J N Christiansen C Johansen
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10 Belessi C A A S Gounadaki A N Psomas and P N Skandamis
2011 Efficiency of different sanitation methods on Listeria
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12 Best M M M M E Kennedy and F F Coates 1990 Efficacy of a
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biol 56377ndash380
13 Bigwood T J A Hudson and C Billington 2009 Influence of host
and bacteriophage concentrations on the inactivation of food-borne
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64
14 Blackman I C and J F Frank 1996 Growth of Listeria
monocytogenes as a biofilm on various food-processing surfaces J
Food Prot 59827ndash831
15 Bloomfield S F M Arthur B Klingeren W van Pullen J T
Holah and R Elton 1994 An evaluation of the repeatability and
reproducibility of a surface test for the activity of disinfectants J
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16 Boulange-Petermann L J Rault and M-N Bellon-Fontaine
1997 Adhesion of Streptococcus thermophilus to stainless steel
with different surface topography and roughness Biofouling 11
201ndash216
17 Bower C K J McGuire and M A Daeschel 1996 The adhesion
and detachment of bacteria and spores on food contact surfaces
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18 Bryan F L 2002 Where we are in the retail food safety how we got
to where we are and how do we get there J Environ Health 6529ndash
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19 Carey-Smith G V C Billington A J Cornelius J A Hudson and
J A Heinemann 2006 Isolation and characterization of bacterio-
phages infecting Salmonella spp FEMS Microbiol Lett 258182ndash
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20 Carpentier B and O Cerf 1993 Biofilms and their consequences
with particular reference to hygiene in the food industry J Appl
Bacteriol 75499ndash511
21 Carsberg H 1996 Selecting your sanitizers Food Qual 235ndash36
61ndash62
22 Chibeu A L Agius A Gao P M Sabour A M Kropinski and S
Balamurugan 2013 Efficacy of bacteriophage LISTEXTMP100
combined with chemical antimicrobials in reducing Listeria mono-
cytogenes in cooked turkey and roast beef Int J Food Microbiol
167208ndash214
23 Cox L J T Kleiss J L Cordier C Cordellana P Konkel C
Pedrazzini R Beumer and A Siebenga 1989 Listeria spp in food
processing non-food and domestic environments Food Microbiol 6
49ndash61
24 Cruz C D and G C Fletcher 2012 Assessing manufacturersrsquo
recommended concentrations of commercial sanitizers on inactivation
of Listeria monocytogenes Food Control 26194ndash199
25 De Beer D R Srinivasan and P S Stewart 1994 Direct
measurement of chlorine penetration into biofilms during disinfec-
tion Appl Environ Microbiol 604339ndash4344
26 Doolittle M M J J Cooney and D E Caldwell 1995 Lytic
infection of Escherichia coli biofilms by bacteriophage T4 Can J
Microbiol 4112ndash18
27 Environmental Protection Agency (EPA) Office of Wastewater
Enforcement and Compliance 2014 Guide to field storage of
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soil resource management Available at httpwaterepagovscitech
wastetechbiosolidsguidecfm Accessed January 2014
28 Fatemi P and J F Frank 1999 Inactivation of Listeria
monocytogenesPseudomonas biofilms by peracid sanitizer J Food
Prot 62761ndash765
29 Fenlon D R J Wilson and W Donachie 1996 The incidence and
level of Listeria monocytogenes contamination of food sources at
primary production and initial processing J Appl Bacteriol 81641ndash
650
30 Frank J F and R A N Chmielewski 1997 Effectiveness of
sanitation with quaternary ammonium compound or chlorine on
stainless steel and other domestic food-preparation surfaces J Food
Prot 6043ndash47
31 Frank J F and R A Koffi 1990 Surface-adherent growth of
Listeria monocytogenes is associated with increased resistance of
surfactant sanitizers and heat J Food Prot 53550ndash554
32 Gibson H J H Taylor K E Hall and J T Holah 1999
Effectiveness of cleaning techniques used in the food industry in terms
of the removal of bacterial biofilms J Appl Microbiol 8741ndash48
33 Gombas D E Y Chen R S Clavaro and V N Scott 2003 Survey
of Listeria monocytogenes in foods J Food Prot 66559ndash569
34 Gram L D Bagge-Ravn Y Y Ng P Gymoese and B F Vogel
2007 Influence of food soiling matrix on cleaning and disinfection
efficiency on surface attached Listeria monocytogenes Food Control
181165ndash1171
35 Greer G G 2005 Bacteriophage control of foodborne bacteria J
Food Prot 681102ndash1111
36 Gronholm L G Wirtanen K Ahlgren K Nordstrom and A
Sjoberg 1999 Screening of antimicrobial activities of disinfectants
and cleaning agents against foodborne spoilage microbes Eur Food
Res Technol 208289ndash298
37 Hagens S 2014 Personal communication
38 Hibma A M S A Jassim and M W Griffiths 1997 Infection and
removal of L-forms of Listeria monocytogenes with bred bacterio-
phage Int J Food Microbiol 34197ndash207
39 Hirai Y 1991 Survival of bacteria under dry conditions from a
viewpoint of nosocomial infection J Hosp Infect 19191ndash200
40 Holah J T 1992 Industrial monitoring hygiene in food processing
p 645ndash659A In L F Melo T R Bott M Fletcher and B
Capdeville (ed) Biofilmsmdashscience and technology Kluwer Aca-
demic Publishers Dordrecht
41 Holah J T C Higgs S Robinson D Worthington and H
Spenceley 1990 conductance-based surface disinfection test for food
hygiene Lett Appl Microbiol 11255ndash259
42 Holah J T J H Taylor and J S Holder 1993 The spread of
Listeria by cleaning systems part II Technical Memorandum no 673
Campden and Chorleywood Food Research Association Chipping
Campden UK
43 Hughes K A I W Sutherland and M V Jones 1998 Biofilm
susceptibility to bacteriophage attack the role of phage-borne
polysaccharide depolymerase Microbiology 1443039ndash3047
44 Joerger R D 2003 Alternatives to antibiotics bacteriocins
antimicrobial peptides and bacteriophages Poult Sci 82640ndash647
45 Kusumaningrum H D G Riboldi W C Hazeleger and R R
Beumer 2003 Survival of foodborne pathogens on stainless steel
surfaces and cross-contamination to foods Int J Food Microbiol 85
227ndash236
46 Lee S-H and J Frank 1991 Inactivation of surface adherent
Listeria monocytogenes hypochlorite and heat J Food Prot 544ndash
6
47 Leverentz B W S Conway M J Camp W J Janisiewicz T
Abuladze M Yang R Saftner and A Sulakvelidze 2003
Biocontrol of Listeria monocytogenes on fresh-cut produce by
treatment with lytic bacteriophages and a bacteriocin Appl Environ
Microbiol 694519ndash4526
48 Lin C M K Takeuchi L Zhang C B Dohm J D Meyer P A
Hall and M P Doyle 2006 Cross-contamination between
processing equipment and deli meats by Listeria monocytogenes J
Food Prot 6971ndash79
49 Lunden J M T J Autio and H J Korkeala 2002 Transfer of
persistent Listeria monocytogenes contamination between food-
processing plants associated with a dicing machine J Food Prot
651129ndash1133
50 Luppens S B I M W Reij R W L van der Heijden F M
Rombouts and T Abee 2002 Development of a standard test to
assess the resistance of Staphylococcus aureus biofilm cells to
disinfectants Appl Environ Microbiol 684194ndash4200
51 Mafu A A D Roy J Goulet L Savoie and R Roy 1990
Efficiency of sanitizing agents for destroying Listeria monocytogenes
on contaminated surfaces J Dairy Sci 733428ndash3432
52 Mahony J O McAuliffe R Paul and D van Sinderen 2011
Bacteriophages as biocontrol agents of food pathogens Curr Opin
Biotechnol 22157ndash163
53 McEldowney S and M Fletcher 1988 Effect of temperature and
humidity on survival of bacteria attached to dry solid surfaces Lett
Appl Microbiol 783ndash86
54 Miettinen L K J Bjorkroth and H Korkeala 1999 Characterisa-
tion of Listeria monocytogenes from an ice-cream plant by serotyping
and pulsed-field gel electrophoresis Int J Food Microbiol 46187ndash
192
55 Mosteller T M and J R Bishop 1993 Sanitizer efficacy against
attached bacteria in a milk biofilm J Food Prot 5634ndash41
56 Norwood D E and A Gilmour 2000 The growth and resistance to
sodium hypochlorite of Listeria monocytogenes in a steady-state
multispecies biofilm J Appl Microbiol 88512ndash520
57 Oliveira M I Vinas P Colas M Anguera J Usall and M
Abadias 2014 Effectiveness of a bacteriophage in reducing Listeria
monocytogenes on fresh-cut fruits and fruit juices Food Microbiol
38137ndash142
58 Ronner A B and A C L Wong 1993 Biofilm development and
sanitizer inactivation of Listeria monocytogenes and Salmonella
typhimurium on stainless steel and buna-n rubber J Food Prot 56
750ndash758
59 Sallam S S and C W Donnelly 1992 Destruction injury and
repair of Listeria species exposed to sanitizing compounds J Food
Prot 55771ndash1033
60 Sillankorva S D R Oliveira M J Vieira I W Sutherland and J
Azeredo 2004 Bacteriophage V S1 infection of Pseudomonas
fluorescens planktonic cells versus biofilms Biofouling 20133ndash138
61 Silva S P Teixeira R Olieveira and J Azeredo 2008 Adhesion to
and viability of Listeria monocytogenes on food contact surfaces J
Food Prot 711379ndash1385
62 Skurnik M and E Strauch 2006 Phage therapy facts and fiction
Int J Food Microbiol 2965ndash14 (Review)
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63 Somers E B J L Schoeni and A C L Wong 1994 Effect of
trisodium phosphate on biofilm and planktonic cells of Campylo-
bacter jejuni Escherichia coli O157 H7 Listeria monocytogenes
and Salmonella typhimurium Int J Food Microbiol 22269ndash
276
64 Somers E B and A C L Wong 2004 Efficacy of two cleaning
and sanitizing combination on Listeria monocytogenes biofilm
formed at low temperature on a variety of materials in the presence
of ready-to-eat meat residue J Food Prot 672218ndash2229
65 Soni K A and R Nannapaneni 2010 Removal of Listeria
monocytogenes biofilms with bacteriophages P100 J Food Prot 73
1519ndash1524
66 Sutherland I W K A Hughes L C Skillman and K Tait 2004
The interaction of phage and biofilm FEMS Microbiol Lett 2321ndash6
(MiniReview)
67 Swaminathan B 2001 Listeria monocytogenes p 383ndash409 In L R
Beuchat M P Doyle and T J Montville (ed) Food microbiology
fundamentals and frontiers 2nd ed ASM Press Washington DC
68 Thevenot D A Dernburg and C Vernozy-Rozand 2006 An
updated review of Listeria monocytogenes in the pork meat industry
and its products J Appl Microbiol 1017ndash17
69 Tuncan E U 1993 Effect of cold temperature on germicidal efficacy
of quaternary ammonium compound iodophor and chlorine on
Listeria J Food Prot 531029ndash1033
2020 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
transferring 1 ml of the suspensions to 9 ml of neutralizer for 1 min
Of these suspensions 100 ml was then plated on Agar Listeriaaccording to Ottaviani and Agosti (ALOA BioMerieux SA Marcy
lrsquoEtoile France) using a spiral plater (Eddy Jet IUL Instrument
SA Barcelona Spain) For the carrier tests on flat surfaces (case
2) the disinfectant reaction was terminated by transferring the
coupons plus disinfectant to a 9-ml tube containing neutralizer and
45-g glass beads (1 mm Emergo Landsmeer The Netherlands)
As a blank inoculated coupons were transferred to 10 ml of PSS
plus glass beads The cells were removed from the coupons by
vortexing the tubes for 30 s Thereafter 1 ml of the suspension was
pour plated in tryptone soy agar
For the carrier tests on surfaces with grooves (case 31)
samples were taken using cotton swabs and cotton threads after the
required contact time Cotton swabs made with extra-thin toothpicks
and cotton wool (01 g 100 Hygienic cotton Etos) and cotton
threads (27-cm length 075-mm diameter [01 g] Super Durable
Glanskatoen no 8 HWS Markoma BV Ninove Belgium) were wet
sterilized at 121uC for 15 min The sterile cotton swabs were used to
take the samples from the 2- 1- 05- and 02-mm grooves by
swabbing three times through the grooves The sterile cotton threads
were used to take samples from the 5-mm groove by flossing and
scraping the groove for 30 s After sampling the cotton swabs and
cotton threads were put in 9-ml neutralizer vortexed and 100 ml
were plated on ALOA plates using a spiral plater
For the carrier tests with bacteriophages (case 32) the cotton
threads and cotton swabs were soaked for 1 min in a heat killed
suspension of L innocua (109 CFUml) to bind free phage particles
as neutralizer Subsequent sampling and plating were similar as
described above
During the 14 days of growing biofilms the numbers of
Listeria aerobic plate count and lactic acid bacteria were
determined at different time intervals on separate plates using
ALOA agar tryptone soy agar and De Man Rogosa and Sharpe
Agar (Lactobacillus broth Merck and 12 [wtvol] Agar
technical Agar no 3 Oxoid) respectively
Data and statistical analysis The volume of each groove
(cubic millimeters) was calculated and transformed to milliliters
The amount of organisms in a groove was then divided by this
volume to obtain a concentration in log CFU per milliliter The
value was calculated as log CFU per milliliter of the grooves in
order to equally compare the results obtained from the grooves that
have different depths
For statistical analysis the results of duplicate experiments
were combined The significance of differences (P 005) in
numbers of L monocytogenes that were reduced by different types
of disinfectants food residues and types of surface were
determined with analysis of the variance and a general linear
model using PASW Statistics 17 (SPSS Inc Chicago IL)
RESULTS AND DISCUSSION
Suspension tests (case 1) Reduction of more than 5
log units of Listeria planktonic cells and biofilm cells in
suspension was observed after 5-min contact time with
240 mgliter SDC and 740 mgliter Qac in all test
conditions where there was no significant difference (P
005) in reduction of the cells between the two disinfectants
in absence of food residues In contrast the effectiveness of
SDC (1-log reduction in 5 min with 3 ultrahigh-
temperature-processed milk) was less than that of Qac (5-
log reduction in 1 min with 10 ultrahigh-temperature-
processed milk) in presence of food residues as expected
(21 25 46) The effectiveness of SDC and Qac in absence
of food residues was higher than in presence of food
residues (data not shown) However these results comply
with the European standard EN1040 (6) which indicates a
reduction of 5 log units should be obtained after 5-min
contact time for an effective disinfectant
Carrier tests on smooth surfaces (case 2) The results
from the tests on the stainless steel coupons showed more
than a 5-log reduction after 1- 2- and 5-min contact time
with no significant difference (P 005) between SDC and
Qac (data not shown) These results complied with the
European standard EN13697 requirement (4) for a reduction
of 4 log units on clean surfaces indicating an effective
disinfectant in the surface test
Carrier tests on surfaces with grooves (case 3) The
shape of the 1-mm wide grooves with depths of 02 05 1
2 and 5 mm used as test surfaces in this study was beyond
the capability of standard sampling methods such as surface
contact plates and swabbing techniques Therefore several
methods eg the use of filter paper cotton swabs and
flossing threads were evaluated for their effectiveness to
recover Listeria cells from the grooves The results showed
that the flossing technique with cotton thread led to 70
recovery for the 5-mm groove For the other grooves this
method could recover only 20 With cotton swabs
prepared from extra-thin toothpicks and cotton wool a
recovery of 70 could be obtained for the remaining
grooves (data not shown) Therefore flossing and swab
methods were chosen as sampling methods for the 5-mm
groove and the other grooves respectively
In these tests the contact times of disinfectant on carriers
were 5 10 15 and 20 min Since the inactivation obtained for
the different contact times was not significantly different (P 005) which may be due to exhaustion of the disinfectants
(3 51) only the results from the contact time of 5 min are
shown Furthermore reduction of L monocytogenes was
dependent on the groove depth the deeper the groove the
lower the reduction This indicates that the activity of the
disinfectants stopped due to the inability to reach the cells in
the deeper areas Only the results of the extremes the 5- and
02-mm grooves are presented in Figures 3 and 4
All food residues were tested negative for L monocy-togenes In absence of food residues on a wet surface after
exposure to water (control) and the disinfectants a high
reduction of the cells was observed in the 02-mm grooves
whereas a similar trend but less reduction was observed in
the 5-mm grooves On the dry surfaces treatment of the
cells in the 02-mm groove resulted in levels below the
detection limit which can be partly explained by a high
reduction observed already as a result of the drying of the
plates for 24 h For the 5-mm grooves this initial reduction
was much lower and not all listerias were inactivated by the
treatment In most cases there was no significant difference
(P 005) between the control and disinfectants The
results demonstrated that drying of surfaces alone could
already largely reduce the cells on the surfaces especially in
the shallow grooves where deeper grooves provided suitable
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2015
FIGURE 3 Numbers of L monocytogenes (log CFU per milliliter) in wet conditions for the 02- (a) and 5-mm (b) grooves and dryconditions for the 02- (c) and 5-mm (d) grooves in presence and absence of food residues at initial inoculation ( T ~ 0) before (amp15 min or 24 h for wet and dry conditions respectively) and after exposure to sterile water ( control) SDC ( 240 mgliter) and Qac( 740 mgliter) for 5 min Data are means iexcl standard deviations of duplicate experiments for each disinfectant (n ~ 2) Dashed linesand vertical arrows (Q) indicate values lower than the detection limit DL detection limit of the method
FIGURE 4 Numbers of L monocytogenes biofilm cells (log CFU per milliliter) in presence of food residues in 02- (a) and 5-mm (b)grooves at initial inoculation ( T ~ 0) before (amp D 14) and after exposure to sterile water ( control) SDC ( 240 mgliter) Qac( 740 mgliter) and bacteriophages ( 109 PFUml) Data are means iexcl standard deviations of duplicate experiments for eachdisinfectant (n ~ 2) Dashed lines and vertical arrows (Q) indicate values lower than the detection limit
2016 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
conditions to prevent complete drying protecting some of
the cells from the disinfectants Drying of surfaces has been
considered as a significant bactericidal method to maintain
low numbers of bacteria in any environment and is
dependent on type of organism temperature relative
humidity and soiling (39 45 53) Furthermore type of
surface and topography have been reported to play an
important role on efficiency of disinfectants (30 40) in
particular surfaces with scrapes or defects where soil can
accumulate and protect bacteria against cleaning and
disinfecting agents (16 17 51)In presence of food residues in the wet 02-mm
grooves Listeria was reduced 2 to 3 log dependent on
types of food residues (Fig 3a) After exposure to the
disinfectants a reduction to below the detection limit was
observed for all food residues with no significant difference
(P 005) between SDC and Qac In the 5-mm grooves
before treatment less reduction (2 log CFUml) was
observed SDC was significantly (P 005) less effective
(2 log) than Qac ($2 log CFUml) for all food residues
except fish In the dry 02-mm grooves already a reduction
of 3 log CFUml was observed 24 h after inoculation
without treatment After exposure to the disinfectants the
numbers were further reduced to below the detection limit
In the 5-mm groove only a small (05 log CFUml ie
vegetable fish and ham residues) or no reduction (milk
residue) was observed immediately before treatment
probably because the cells were protected from the drying
in the deep grooves Treatment with SDC and Qac did not
inactivate all listerias with no significant difference (P
005) between the two disinfectants except in the case of
ham and vegetable residues where Qac performed signif-
icantly better than SDC in the wet grooves and for
vegetables in the dry grooves Previous studies have
reported an inactivation of the antimicrobial activity of
disinfectants by organic matter on surfaces showing 1- to 2-
log reductions on soiled surfaces compared with 3- to 4-
log reductions on clean surfaces (2 34) This could be due
to the chemical compositions such as protein and fat which
can bind with the disinfectants or reduction of penetration
into the bacterial layer (32 68) The chlorine-based
disinfectants have been found to be easily inactivated by
organic materials (25 46) whereas Qac was reported to be
less inactivated by organic matters (21) which was
confirmed in most cases in this study In general the test
on surfaces is dependent on the surface materials used and
viability of the cells dried on the surfaces resulting in
inconsistent results when compared with the test in
suspensions (15) Under practical conditions high amounts
of organic materials may be present resulting in less or no
activity of the disinfectant When the concentration of SDC
was increased from 240 to 300 mgliter a comparable
reduction of the cells to that of Qac was obtained however
there was no effect on reduction in the deep grooves (data
not shown) The results indicated that the disinfectant could
not reach all cells in the bottom of the groove while such
grooves (5-mm depth 1-mm width) might be present in
practice (eg small spaces between connection parts of
slicers) The effectiveness of the disinfectants in the deep
areas could be increased by using high pressure spraying
(32) however this method might have adverse effects since
it could also spread the bacterial cells to other areas and
environments in particular when high numbers of bacterial
cells are accumulated (42)
Carrier tests on surfaces with grooves in presence ofa biofilm (case 32) After 14 days of biofilm growing in
02-mm grooves (Fig 4) the number of cells remained
more or less stable with a decrease of the cells in vegetable
and ham biofilms (15 and 18 log CFUml respectively) In
5-mm grooves however growth of biofilm cells was
observed in all food residues A previous study reported that
L monocytogenes has the ability to form biofilms at
numbers of 104 to 107 CFUcm2 (34) and adheres to a
variety of food contact surfaces such as plastic glass
rubber and stainless steel particularly in pits or crevices
that are difficult to clean (31 61)To check if the biofilm cells were more resistant to the
disinfectants than the planktonic cells the biofilm cells were
brought into suspension and then tested The obtained
results a 5-log reduction in all conditions after 5-min
exposure to the disinfectants indicated no difference in
reduction of biofilm cells (case 32) compared with
planktonic cells (case 1) These results complied with a
previous study where reduction of the suspended biofilm
cells was higher than that of biofilm cells attached to
surfaces after exposure to disinfectants (50) It has been
described that a 10 to 100 times higher concentration of
disinfectants may be required to obtain the same reduction
of biofilm cells compared with planktonic cells (40 56)indicating the importance of preventing biofilm formation
For the bacteriophages an efficacy test was previously
performed with L monocytogenes strains in presence and
absence of food residues on flat stainless steel surfaces The
results demonstrated that rapid inactivation of Listeria cells
(2 to 3 log CFUcm2) within 30 min of contact time was
obtained (data not shown) which was similar with the
results of Soni and Nannapaneni (65) where a reduction of
35 log CFUcm2 was obtained in the same time with a
phage treatment on a 1-week-old L monocytogenes biofilm
on stainless steel coupons
In the 02-mm grooves (Fig 4a) Qac showed better
reduction (2 to 3 log) than SDC (12 to 27 log) with
inactivation below the detection limit in vegetable biofilms
The phages showed higher reduction (3 log CFUml P
005) than that of water or the chemical disinfectants in three
food biofilms except for the vegetable biofilms where all
cells were inactivated by every disinfectant In the 5-mm
grooves SDC and Qac did not reduce Listeria better than
water alone However the phages reduced approximately
14 log CFUml in ham and fish biofilms and approxi-
mately 08 log CFUml in milk and vegetable biofilms
which was not significantly different (P 005) from that of
the disinfectants except for ham biofilms The results
indicated that only in the 02-mm grooves the phages were
likely to be more effective in the biofilms with food matrix
than the chemical disinfectants This was probably due to
the fact that the disinfectants and phages did not penetrate
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2017
the foodbacterial matrix completely in the deep groove (2041) Furthermore the effectiveness of the disinfectants on Lmonocytogenes biofilms may be reduced by presence of
other bacteria (6 to 7 log CFUml) including lactic acid
bacteria (4 log CFUml) in the grooves as well (data not
shown) Phage efficacy depends on susceptibility of the
bacterial cells to the phages and availability of receptor sites
between phages and the bacterial cells (13) which could be
blocked by the food matrix (35) In this study the phage
efficacy was enhanced by keeping the biofilm plates in
plastic bags after application of the phages to maintain a
high humidity that would enhance the diffusion and the
chance of contact between Listeria cells and phage particles
(66)Strikingly the biofilm removal by sterile water
(control) resulted in a similar reduction of the biofilm cells
compared with disinfectants In the present study the tests
were performed to simulate survival of bacteria in presence
or absence of organic matter in pits or cracks on food
contact surfaces and to test the effect of disinfection
therefore cleaning of the surfaces before using disinfectants
was excluded So in this case plates and grooves were
dirtier than soiled surfaces in reality which explains the
observed low efficacy of the disinfectants and the water
washed away the biofilm cells from the grooves Rinsing the
biofilm on surfaces by water has been reported to reduce the
biofilm cells at about 1 log unit of cells (34)The results in this study clearly demonstrated that
presence of grooves or spaces on surfaces humidity and
presence of food substrates influenced the antimicrobial
effect of disinfectants and bacteriophages Bacteriophages
showed a better antimicrobial effect than the chemical
disinfectants (ie SDC and Qac) in the shallow grooves but
not in the deep grooves Use of bacteriophages as a
biocontrol could only be a promising method in limited
cases however use of large quantities in practice may be
costly and phage-resistant strains may occur (35 62)
ACKNOWLEDGMENTS
The authors thank Diversey Inc (Utrecht The Netherlands) who
provided the chemical disinfectants for this study We also thank Micreos
Food Safety BV (Wageningen The Netherlands) for Listeria bacteriophage
Listex P100 and L innocua (serovar 5) Our great appreciation goes to
Feifei Gao for her technical assistance in the phage experiments and Hilda
Nyati for her work on smooth surfaces
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Susceptibility of Listeria monocytogenes strains to disinfectants and
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2 Aarnisalo K S Salo H Miettinen M Suihko G Wirtanen T
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disinfectants used in food industrial domestic and institutional
areasmdashtest method and requirements without mechanical action (phase
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CommitteesWorkshopsCENTechnicalCommitteesPagesStandards
aspxparam~6197amptitle~Chemical20disinfectants20and20
antiseptics Accessed February 2014
5 Anonymous 2004 Microbiology of food and animal feeding stuffs mdash
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6 Anonymous 2005 European Standard EN 10402005 Chemical
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evaluation of basic bactericidal activity of chemical disinfectants
and antisepticsmdashtest method and requirements (phase 1) Available
at httpwwwceneucenSectorsTechnicalCommitteesWorkshops
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Chemical20disinfectants20and20antiseptics Accessed February
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7 Arachchi G J G A G Cridge B M Dias-Wanigasekera C D
Cruz L McIntyre R Liu S H Flint and A N Mutukumira 2013
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1116
8 Autio T R Keto-Timonen J Bjorkroth and H Korkeala 2003
Characterization of persistent and sporadic Listeria monocytogenes
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9 Bagge-Ravn D Y Ng M Hjelm J N Christiansen C Johansen
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2011 Efficiency of different sanitation methods on Listeria
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12 Best M M M M E Kennedy and F F Coates 1990 Efficacy of a
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13 Bigwood T J A Hudson and C Billington 2009 Influence of host
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64
14 Blackman I C and J F Frank 1996 Growth of Listeria
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15 Bloomfield S F M Arthur B Klingeren W van Pullen J T
Holah and R Elton 1994 An evaluation of the repeatability and
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1997 Adhesion of Streptococcus thermophilus to stainless steel
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201ndash216
17 Bower C K J McGuire and M A Daeschel 1996 The adhesion
and detachment of bacteria and spores on food contact surfaces
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19 Carey-Smith G V C Billington A J Cornelius J A Hudson and
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20 Carpentier B and O Cerf 1993 Biofilms and their consequences
with particular reference to hygiene in the food industry J Appl
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21 Carsberg H 1996 Selecting your sanitizers Food Qual 235ndash36
61ndash62
22 Chibeu A L Agius A Gao P M Sabour A M Kropinski and S
Balamurugan 2013 Efficacy of bacteriophage LISTEXTMP100
combined with chemical antimicrobials in reducing Listeria mono-
cytogenes in cooked turkey and roast beef Int J Food Microbiol
167208ndash214
23 Cox L J T Kleiss J L Cordier C Cordellana P Konkel C
Pedrazzini R Beumer and A Siebenga 1989 Listeria spp in food
processing non-food and domestic environments Food Microbiol 6
49ndash61
24 Cruz C D and G C Fletcher 2012 Assessing manufacturersrsquo
recommended concentrations of commercial sanitizers on inactivation
of Listeria monocytogenes Food Control 26194ndash199
25 De Beer D R Srinivasan and P S Stewart 1994 Direct
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26 Doolittle M M J J Cooney and D E Caldwell 1995 Lytic
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27 Environmental Protection Agency (EPA) Office of Wastewater
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28 Fatemi P and J F Frank 1999 Inactivation of Listeria
monocytogenesPseudomonas biofilms by peracid sanitizer J Food
Prot 62761ndash765
29 Fenlon D R J Wilson and W Donachie 1996 The incidence and
level of Listeria monocytogenes contamination of food sources at
primary production and initial processing J Appl Bacteriol 81641ndash
650
30 Frank J F and R A N Chmielewski 1997 Effectiveness of
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stainless steel and other domestic food-preparation surfaces J Food
Prot 6043ndash47
31 Frank J F and R A Koffi 1990 Surface-adherent growth of
Listeria monocytogenes is associated with increased resistance of
surfactant sanitizers and heat J Food Prot 53550ndash554
32 Gibson H J H Taylor K E Hall and J T Holah 1999
Effectiveness of cleaning techniques used in the food industry in terms
of the removal of bacterial biofilms J Appl Microbiol 8741ndash48
33 Gombas D E Y Chen R S Clavaro and V N Scott 2003 Survey
of Listeria monocytogenes in foods J Food Prot 66559ndash569
34 Gram L D Bagge-Ravn Y Y Ng P Gymoese and B F Vogel
2007 Influence of food soiling matrix on cleaning and disinfection
efficiency on surface attached Listeria monocytogenes Food Control
181165ndash1171
35 Greer G G 2005 Bacteriophage control of foodborne bacteria J
Food Prot 681102ndash1111
36 Gronholm L G Wirtanen K Ahlgren K Nordstrom and A
Sjoberg 1999 Screening of antimicrobial activities of disinfectants
and cleaning agents against foodborne spoilage microbes Eur Food
Res Technol 208289ndash298
37 Hagens S 2014 Personal communication
38 Hibma A M S A Jassim and M W Griffiths 1997 Infection and
removal of L-forms of Listeria monocytogenes with bred bacterio-
phage Int J Food Microbiol 34197ndash207
39 Hirai Y 1991 Survival of bacteria under dry conditions from a
viewpoint of nosocomial infection J Hosp Infect 19191ndash200
40 Holah J T 1992 Industrial monitoring hygiene in food processing
p 645ndash659A In L F Melo T R Bott M Fletcher and B
Capdeville (ed) Biofilmsmdashscience and technology Kluwer Aca-
demic Publishers Dordrecht
41 Holah J T C Higgs S Robinson D Worthington and H
Spenceley 1990 conductance-based surface disinfection test for food
hygiene Lett Appl Microbiol 11255ndash259
42 Holah J T J H Taylor and J S Holder 1993 The spread of
Listeria by cleaning systems part II Technical Memorandum no 673
Campden and Chorleywood Food Research Association Chipping
Campden UK
43 Hughes K A I W Sutherland and M V Jones 1998 Biofilm
susceptibility to bacteriophage attack the role of phage-borne
polysaccharide depolymerase Microbiology 1443039ndash3047
44 Joerger R D 2003 Alternatives to antibiotics bacteriocins
antimicrobial peptides and bacteriophages Poult Sci 82640ndash647
45 Kusumaningrum H D G Riboldi W C Hazeleger and R R
Beumer 2003 Survival of foodborne pathogens on stainless steel
surfaces and cross-contamination to foods Int J Food Microbiol 85
227ndash236
46 Lee S-H and J Frank 1991 Inactivation of surface adherent
Listeria monocytogenes hypochlorite and heat J Food Prot 544ndash
6
47 Leverentz B W S Conway M J Camp W J Janisiewicz T
Abuladze M Yang R Saftner and A Sulakvelidze 2003
Biocontrol of Listeria monocytogenes on fresh-cut produce by
treatment with lytic bacteriophages and a bacteriocin Appl Environ
Microbiol 694519ndash4526
48 Lin C M K Takeuchi L Zhang C B Dohm J D Meyer P A
Hall and M P Doyle 2006 Cross-contamination between
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Food Prot 6971ndash79
49 Lunden J M T J Autio and H J Korkeala 2002 Transfer of
persistent Listeria monocytogenes contamination between food-
processing plants associated with a dicing machine J Food Prot
651129ndash1133
50 Luppens S B I M W Reij R W L van der Heijden F M
Rombouts and T Abee 2002 Development of a standard test to
assess the resistance of Staphylococcus aureus biofilm cells to
disinfectants Appl Environ Microbiol 684194ndash4200
51 Mafu A A D Roy J Goulet L Savoie and R Roy 1990
Efficiency of sanitizing agents for destroying Listeria monocytogenes
on contaminated surfaces J Dairy Sci 733428ndash3432
52 Mahony J O McAuliffe R Paul and D van Sinderen 2011
Bacteriophages as biocontrol agents of food pathogens Curr Opin
Biotechnol 22157ndash163
53 McEldowney S and M Fletcher 1988 Effect of temperature and
humidity on survival of bacteria attached to dry solid surfaces Lett
Appl Microbiol 783ndash86
54 Miettinen L K J Bjorkroth and H Korkeala 1999 Characterisa-
tion of Listeria monocytogenes from an ice-cream plant by serotyping
and pulsed-field gel electrophoresis Int J Food Microbiol 46187ndash
192
55 Mosteller T M and J R Bishop 1993 Sanitizer efficacy against
attached bacteria in a milk biofilm J Food Prot 5634ndash41
56 Norwood D E and A Gilmour 2000 The growth and resistance to
sodium hypochlorite of Listeria monocytogenes in a steady-state
multispecies biofilm J Appl Microbiol 88512ndash520
57 Oliveira M I Vinas P Colas M Anguera J Usall and M
Abadias 2014 Effectiveness of a bacteriophage in reducing Listeria
monocytogenes on fresh-cut fruits and fruit juices Food Microbiol
38137ndash142
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sanitizer inactivation of Listeria monocytogenes and Salmonella
typhimurium on stainless steel and buna-n rubber J Food Prot 56
750ndash758
59 Sallam S S and C W Donnelly 1992 Destruction injury and
repair of Listeria species exposed to sanitizing compounds J Food
Prot 55771ndash1033
60 Sillankorva S D R Oliveira M J Vieira I W Sutherland and J
Azeredo 2004 Bacteriophage V S1 infection of Pseudomonas
fluorescens planktonic cells versus biofilms Biofouling 20133ndash138
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and viability of Listeria monocytogenes on food contact surfaces J
Food Prot 711379ndash1385
62 Skurnik M and E Strauch 2006 Phage therapy facts and fiction
Int J Food Microbiol 2965ndash14 (Review)
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63 Somers E B J L Schoeni and A C L Wong 1994 Effect of
trisodium phosphate on biofilm and planktonic cells of Campylo-
bacter jejuni Escherichia coli O157 H7 Listeria monocytogenes
and Salmonella typhimurium Int J Food Microbiol 22269ndash
276
64 Somers E B and A C L Wong 2004 Efficacy of two cleaning
and sanitizing combination on Listeria monocytogenes biofilm
formed at low temperature on a variety of materials in the presence
of ready-to-eat meat residue J Food Prot 672218ndash2229
65 Soni K A and R Nannapaneni 2010 Removal of Listeria
monocytogenes biofilms with bacteriophages P100 J Food Prot 73
1519ndash1524
66 Sutherland I W K A Hughes L C Skillman and K Tait 2004
The interaction of phage and biofilm FEMS Microbiol Lett 2321ndash6
(MiniReview)
67 Swaminathan B 2001 Listeria monocytogenes p 383ndash409 In L R
Beuchat M P Doyle and T J Montville (ed) Food microbiology
fundamentals and frontiers 2nd ed ASM Press Washington DC
68 Thevenot D A Dernburg and C Vernozy-Rozand 2006 An
updated review of Listeria monocytogenes in the pork meat industry
and its products J Appl Microbiol 1017ndash17
69 Tuncan E U 1993 Effect of cold temperature on germicidal efficacy
of quaternary ammonium compound iodophor and chlorine on
Listeria J Food Prot 531029ndash1033
2020 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
FIGURE 3 Numbers of L monocytogenes (log CFU per milliliter) in wet conditions for the 02- (a) and 5-mm (b) grooves and dryconditions for the 02- (c) and 5-mm (d) grooves in presence and absence of food residues at initial inoculation ( T ~ 0) before (amp15 min or 24 h for wet and dry conditions respectively) and after exposure to sterile water ( control) SDC ( 240 mgliter) and Qac( 740 mgliter) for 5 min Data are means iexcl standard deviations of duplicate experiments for each disinfectant (n ~ 2) Dashed linesand vertical arrows (Q) indicate values lower than the detection limit DL detection limit of the method
FIGURE 4 Numbers of L monocytogenes biofilm cells (log CFU per milliliter) in presence of food residues in 02- (a) and 5-mm (b)grooves at initial inoculation ( T ~ 0) before (amp D 14) and after exposure to sterile water ( control) SDC ( 240 mgliter) Qac( 740 mgliter) and bacteriophages ( 109 PFUml) Data are means iexcl standard deviations of duplicate experiments for eachdisinfectant (n ~ 2) Dashed lines and vertical arrows (Q) indicate values lower than the detection limit
2016 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
conditions to prevent complete drying protecting some of
the cells from the disinfectants Drying of surfaces has been
considered as a significant bactericidal method to maintain
low numbers of bacteria in any environment and is
dependent on type of organism temperature relative
humidity and soiling (39 45 53) Furthermore type of
surface and topography have been reported to play an
important role on efficiency of disinfectants (30 40) in
particular surfaces with scrapes or defects where soil can
accumulate and protect bacteria against cleaning and
disinfecting agents (16 17 51)In presence of food residues in the wet 02-mm
grooves Listeria was reduced 2 to 3 log dependent on
types of food residues (Fig 3a) After exposure to the
disinfectants a reduction to below the detection limit was
observed for all food residues with no significant difference
(P 005) between SDC and Qac In the 5-mm grooves
before treatment less reduction (2 log CFUml) was
observed SDC was significantly (P 005) less effective
(2 log) than Qac ($2 log CFUml) for all food residues
except fish In the dry 02-mm grooves already a reduction
of 3 log CFUml was observed 24 h after inoculation
without treatment After exposure to the disinfectants the
numbers were further reduced to below the detection limit
In the 5-mm groove only a small (05 log CFUml ie
vegetable fish and ham residues) or no reduction (milk
residue) was observed immediately before treatment
probably because the cells were protected from the drying
in the deep grooves Treatment with SDC and Qac did not
inactivate all listerias with no significant difference (P
005) between the two disinfectants except in the case of
ham and vegetable residues where Qac performed signif-
icantly better than SDC in the wet grooves and for
vegetables in the dry grooves Previous studies have
reported an inactivation of the antimicrobial activity of
disinfectants by organic matter on surfaces showing 1- to 2-
log reductions on soiled surfaces compared with 3- to 4-
log reductions on clean surfaces (2 34) This could be due
to the chemical compositions such as protein and fat which
can bind with the disinfectants or reduction of penetration
into the bacterial layer (32 68) The chlorine-based
disinfectants have been found to be easily inactivated by
organic materials (25 46) whereas Qac was reported to be
less inactivated by organic matters (21) which was
confirmed in most cases in this study In general the test
on surfaces is dependent on the surface materials used and
viability of the cells dried on the surfaces resulting in
inconsistent results when compared with the test in
suspensions (15) Under practical conditions high amounts
of organic materials may be present resulting in less or no
activity of the disinfectant When the concentration of SDC
was increased from 240 to 300 mgliter a comparable
reduction of the cells to that of Qac was obtained however
there was no effect on reduction in the deep grooves (data
not shown) The results indicated that the disinfectant could
not reach all cells in the bottom of the groove while such
grooves (5-mm depth 1-mm width) might be present in
practice (eg small spaces between connection parts of
slicers) The effectiveness of the disinfectants in the deep
areas could be increased by using high pressure spraying
(32) however this method might have adverse effects since
it could also spread the bacterial cells to other areas and
environments in particular when high numbers of bacterial
cells are accumulated (42)
Carrier tests on surfaces with grooves in presence ofa biofilm (case 32) After 14 days of biofilm growing in
02-mm grooves (Fig 4) the number of cells remained
more or less stable with a decrease of the cells in vegetable
and ham biofilms (15 and 18 log CFUml respectively) In
5-mm grooves however growth of biofilm cells was
observed in all food residues A previous study reported that
L monocytogenes has the ability to form biofilms at
numbers of 104 to 107 CFUcm2 (34) and adheres to a
variety of food contact surfaces such as plastic glass
rubber and stainless steel particularly in pits or crevices
that are difficult to clean (31 61)To check if the biofilm cells were more resistant to the
disinfectants than the planktonic cells the biofilm cells were
brought into suspension and then tested The obtained
results a 5-log reduction in all conditions after 5-min
exposure to the disinfectants indicated no difference in
reduction of biofilm cells (case 32) compared with
planktonic cells (case 1) These results complied with a
previous study where reduction of the suspended biofilm
cells was higher than that of biofilm cells attached to
surfaces after exposure to disinfectants (50) It has been
described that a 10 to 100 times higher concentration of
disinfectants may be required to obtain the same reduction
of biofilm cells compared with planktonic cells (40 56)indicating the importance of preventing biofilm formation
For the bacteriophages an efficacy test was previously
performed with L monocytogenes strains in presence and
absence of food residues on flat stainless steel surfaces The
results demonstrated that rapid inactivation of Listeria cells
(2 to 3 log CFUcm2) within 30 min of contact time was
obtained (data not shown) which was similar with the
results of Soni and Nannapaneni (65) where a reduction of
35 log CFUcm2 was obtained in the same time with a
phage treatment on a 1-week-old L monocytogenes biofilm
on stainless steel coupons
In the 02-mm grooves (Fig 4a) Qac showed better
reduction (2 to 3 log) than SDC (12 to 27 log) with
inactivation below the detection limit in vegetable biofilms
The phages showed higher reduction (3 log CFUml P
005) than that of water or the chemical disinfectants in three
food biofilms except for the vegetable biofilms where all
cells were inactivated by every disinfectant In the 5-mm
grooves SDC and Qac did not reduce Listeria better than
water alone However the phages reduced approximately
14 log CFUml in ham and fish biofilms and approxi-
mately 08 log CFUml in milk and vegetable biofilms
which was not significantly different (P 005) from that of
the disinfectants except for ham biofilms The results
indicated that only in the 02-mm grooves the phages were
likely to be more effective in the biofilms with food matrix
than the chemical disinfectants This was probably due to
the fact that the disinfectants and phages did not penetrate
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2017
the foodbacterial matrix completely in the deep groove (2041) Furthermore the effectiveness of the disinfectants on Lmonocytogenes biofilms may be reduced by presence of
other bacteria (6 to 7 log CFUml) including lactic acid
bacteria (4 log CFUml) in the grooves as well (data not
shown) Phage efficacy depends on susceptibility of the
bacterial cells to the phages and availability of receptor sites
between phages and the bacterial cells (13) which could be
blocked by the food matrix (35) In this study the phage
efficacy was enhanced by keeping the biofilm plates in
plastic bags after application of the phages to maintain a
high humidity that would enhance the diffusion and the
chance of contact between Listeria cells and phage particles
(66)Strikingly the biofilm removal by sterile water
(control) resulted in a similar reduction of the biofilm cells
compared with disinfectants In the present study the tests
were performed to simulate survival of bacteria in presence
or absence of organic matter in pits or cracks on food
contact surfaces and to test the effect of disinfection
therefore cleaning of the surfaces before using disinfectants
was excluded So in this case plates and grooves were
dirtier than soiled surfaces in reality which explains the
observed low efficacy of the disinfectants and the water
washed away the biofilm cells from the grooves Rinsing the
biofilm on surfaces by water has been reported to reduce the
biofilm cells at about 1 log unit of cells (34)The results in this study clearly demonstrated that
presence of grooves or spaces on surfaces humidity and
presence of food substrates influenced the antimicrobial
effect of disinfectants and bacteriophages Bacteriophages
showed a better antimicrobial effect than the chemical
disinfectants (ie SDC and Qac) in the shallow grooves but
not in the deep grooves Use of bacteriophages as a
biocontrol could only be a promising method in limited
cases however use of large quantities in practice may be
costly and phage-resistant strains may occur (35 62)
ACKNOWLEDGMENTS
The authors thank Diversey Inc (Utrecht The Netherlands) who
provided the chemical disinfectants for this study We also thank Micreos
Food Safety BV (Wageningen The Netherlands) for Listeria bacteriophage
Listex P100 and L innocua (serovar 5) Our great appreciation goes to
Feifei Gao for her technical assistance in the phage experiments and Hilda
Nyati for her work on smooth surfaces
REFERENCES
1 Aarnisalo K J Lunden H Korkeala and G Wirtanen 2007
Susceptibility of Listeria monocytogenes strains to disinfectants and
chlorinated alkaline cleaners at cold temperatures LWT - Food SciTechnol 401041ndash1048
2 Aarnisalo K S Salo H Miettinen M Suihko G Wirtanen T
Autio J Lunden H Korkeala and A Sjoberg 2000 Bactericidal
efficiencies of commercial disinfectants against Listeria monocyto-
genes on surfaces J Food Saf 20237ndash250
3 Adams M R A D Hartley and L J Cox 1989 Factors affecting
the efficacy of washing procedures used in the production of prepared
salads Food Microbiol 669ndash77
4 Anonymous 2001 European Standard EN 136972001 Chemical
disinfectants and antisepticsmdashqualitative non-porous surface test for
the evaluation of bacteriocidal andor fungicidal activity of chemical
disinfectants used in food industrial domestic and institutional
areasmdashtest method and requirements without mechanical action (phase
2 step 2) Available at httpwwwceneucenSectorsTechnical
CommitteesWorkshopsCENTechnicalCommitteesPagesStandards
aspxparam~6197amptitle~Chemical20disinfectants20and20
antiseptics Accessed February 2014
5 Anonymous 2004 Microbiology of food and animal feeding stuffs mdash
horizontal method for the detection and enumeration of Listeria
monocytogenesmdashpart 1 detection method ISO 11290-11996Amd
12004 International Organization for Standardization Geneva
Available at httpswwwisoorgobpuiisostdiso11290-1ed-1
v1enamd1amd1 Accessed January 2014
6 Anonymous 2005 European Standard EN 10402005 Chemical
disinfectants and antiseptics Quantitative suspension test for the
evaluation of basic bactericidal activity of chemical disinfectants
and antisepticsmdashtest method and requirements (phase 1) Available
at httpwwwceneucenSectorsTechnicalCommitteesWorkshops
CENTechnicalCommitteesPagesStandardsaspxparam~6197amptitle~
Chemical20disinfectants20and20antiseptics Accessed February
2014
7 Arachchi G J G A G Cridge B M Dias-Wanigasekera C D
Cruz L McIntyre R Liu S H Flint and A N Mutukumira 2013
Effectiveness of phages in the decontamination of Listeria monocy-
togenes adhered to clean stainless steel stainless steel coated with
fish protein and as a biofilm J Ind Microbiol Biotechnol 401105ndash
1116
8 Autio T R Keto-Timonen J Bjorkroth and H Korkeala 2003
Characterization of persistent and sporadic Listeria monocytogenes
strains by pulsed-field gel electrophoresis (PFGE) and amplified
fragment length polymorphism (AFLP) Syst Appl Microbiol 26
539ndash545
9 Bagge-Ravn D Y Ng M Hjelm J N Christiansen C Johansen
and L Gram 2003 The microbial ecology of processing equipment
in different fish industriesmdashanalysis of the microflora during
processing and following cleaning and disinfection Int J Food
Microbiol 87239ndash250
10 Belessi C A A S Gounadaki A N Psomas and P N Skandamis
2011 Efficiency of different sanitation methods on Listeria
monocytogenes biofilms formed under various environmental condi-
tions Int J Food Microbiol 145S46ndashS52
11 Beresford M R P W Andrew and G Shama 2001 Listeria
monocytogenes adheres to many materials found in food-processing
environments J Appl Microbiol 901000ndash1005
12 Best M M M M E Kennedy and F F Coates 1990 Efficacy of a
variety of disinfectants against Listeria spp Appl Environ Micro-
biol 56377ndash380
13 Bigwood T J A Hudson and C Billington 2009 Influence of host
and bacteriophage concentrations on the inactivation of food-borne
pathogenic bacteria by two phages FEMS Microbiol Lett 29159ndash
64
14 Blackman I C and J F Frank 1996 Growth of Listeria
monocytogenes as a biofilm on various food-processing surfaces J
Food Prot 59827ndash831
15 Bloomfield S F M Arthur B Klingeren W van Pullen J T
Holah and R Elton 1994 An evaluation of the repeatability and
reproducibility of a surface test for the activity of disinfectants J
Appl Bacteriol 7686ndash94
16 Boulange-Petermann L J Rault and M-N Bellon-Fontaine
1997 Adhesion of Streptococcus thermophilus to stainless steel
with different surface topography and roughness Biofouling 11
201ndash216
17 Bower C K J McGuire and M A Daeschel 1996 The adhesion
and detachment of bacteria and spores on food contact surfaces
Trends Food Sci Technol 7152ndash157
18 Bryan F L 2002 Where we are in the retail food safety how we got
to where we are and how do we get there J Environ Health 6529ndash
36
19 Carey-Smith G V C Billington A J Cornelius J A Hudson and
J A Heinemann 2006 Isolation and characterization of bacterio-
phages infecting Salmonella spp FEMS Microbiol Lett 258182ndash
186
2018 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
20 Carpentier B and O Cerf 1993 Biofilms and their consequences
with particular reference to hygiene in the food industry J Appl
Bacteriol 75499ndash511
21 Carsberg H 1996 Selecting your sanitizers Food Qual 235ndash36
61ndash62
22 Chibeu A L Agius A Gao P M Sabour A M Kropinski and S
Balamurugan 2013 Efficacy of bacteriophage LISTEXTMP100
combined with chemical antimicrobials in reducing Listeria mono-
cytogenes in cooked turkey and roast beef Int J Food Microbiol
167208ndash214
23 Cox L J T Kleiss J L Cordier C Cordellana P Konkel C
Pedrazzini R Beumer and A Siebenga 1989 Listeria spp in food
processing non-food and domestic environments Food Microbiol 6
49ndash61
24 Cruz C D and G C Fletcher 2012 Assessing manufacturersrsquo
recommended concentrations of commercial sanitizers on inactivation
of Listeria monocytogenes Food Control 26194ndash199
25 De Beer D R Srinivasan and P S Stewart 1994 Direct
measurement of chlorine penetration into biofilms during disinfec-
tion Appl Environ Microbiol 604339ndash4344
26 Doolittle M M J J Cooney and D E Caldwell 1995 Lytic
infection of Escherichia coli biofilms by bacteriophage T4 Can J
Microbiol 4112ndash18
27 Environmental Protection Agency (EPA) Office of Wastewater
Enforcement and Compliance 2014 Guide to field storage of
biosolids and other organic by-products used in agriculture and for
soil resource management Available at httpwaterepagovscitech
wastetechbiosolidsguidecfm Accessed January 2014
28 Fatemi P and J F Frank 1999 Inactivation of Listeria
monocytogenesPseudomonas biofilms by peracid sanitizer J Food
Prot 62761ndash765
29 Fenlon D R J Wilson and W Donachie 1996 The incidence and
level of Listeria monocytogenes contamination of food sources at
primary production and initial processing J Appl Bacteriol 81641ndash
650
30 Frank J F and R A N Chmielewski 1997 Effectiveness of
sanitation with quaternary ammonium compound or chlorine on
stainless steel and other domestic food-preparation surfaces J Food
Prot 6043ndash47
31 Frank J F and R A Koffi 1990 Surface-adherent growth of
Listeria monocytogenes is associated with increased resistance of
surfactant sanitizers and heat J Food Prot 53550ndash554
32 Gibson H J H Taylor K E Hall and J T Holah 1999
Effectiveness of cleaning techniques used in the food industry in terms
of the removal of bacterial biofilms J Appl Microbiol 8741ndash48
33 Gombas D E Y Chen R S Clavaro and V N Scott 2003 Survey
of Listeria monocytogenes in foods J Food Prot 66559ndash569
34 Gram L D Bagge-Ravn Y Y Ng P Gymoese and B F Vogel
2007 Influence of food soiling matrix on cleaning and disinfection
efficiency on surface attached Listeria monocytogenes Food Control
181165ndash1171
35 Greer G G 2005 Bacteriophage control of foodborne bacteria J
Food Prot 681102ndash1111
36 Gronholm L G Wirtanen K Ahlgren K Nordstrom and A
Sjoberg 1999 Screening of antimicrobial activities of disinfectants
and cleaning agents against foodborne spoilage microbes Eur Food
Res Technol 208289ndash298
37 Hagens S 2014 Personal communication
38 Hibma A M S A Jassim and M W Griffiths 1997 Infection and
removal of L-forms of Listeria monocytogenes with bred bacterio-
phage Int J Food Microbiol 34197ndash207
39 Hirai Y 1991 Survival of bacteria under dry conditions from a
viewpoint of nosocomial infection J Hosp Infect 19191ndash200
40 Holah J T 1992 Industrial monitoring hygiene in food processing
p 645ndash659A In L F Melo T R Bott M Fletcher and B
Capdeville (ed) Biofilmsmdashscience and technology Kluwer Aca-
demic Publishers Dordrecht
41 Holah J T C Higgs S Robinson D Worthington and H
Spenceley 1990 conductance-based surface disinfection test for food
hygiene Lett Appl Microbiol 11255ndash259
42 Holah J T J H Taylor and J S Holder 1993 The spread of
Listeria by cleaning systems part II Technical Memorandum no 673
Campden and Chorleywood Food Research Association Chipping
Campden UK
43 Hughes K A I W Sutherland and M V Jones 1998 Biofilm
susceptibility to bacteriophage attack the role of phage-borne
polysaccharide depolymerase Microbiology 1443039ndash3047
44 Joerger R D 2003 Alternatives to antibiotics bacteriocins
antimicrobial peptides and bacteriophages Poult Sci 82640ndash647
45 Kusumaningrum H D G Riboldi W C Hazeleger and R R
Beumer 2003 Survival of foodborne pathogens on stainless steel
surfaces and cross-contamination to foods Int J Food Microbiol 85
227ndash236
46 Lee S-H and J Frank 1991 Inactivation of surface adherent
Listeria monocytogenes hypochlorite and heat J Food Prot 544ndash
6
47 Leverentz B W S Conway M J Camp W J Janisiewicz T
Abuladze M Yang R Saftner and A Sulakvelidze 2003
Biocontrol of Listeria monocytogenes on fresh-cut produce by
treatment with lytic bacteriophages and a bacteriocin Appl Environ
Microbiol 694519ndash4526
48 Lin C M K Takeuchi L Zhang C B Dohm J D Meyer P A
Hall and M P Doyle 2006 Cross-contamination between
processing equipment and deli meats by Listeria monocytogenes J
Food Prot 6971ndash79
49 Lunden J M T J Autio and H J Korkeala 2002 Transfer of
persistent Listeria monocytogenes contamination between food-
processing plants associated with a dicing machine J Food Prot
651129ndash1133
50 Luppens S B I M W Reij R W L van der Heijden F M
Rombouts and T Abee 2002 Development of a standard test to
assess the resistance of Staphylococcus aureus biofilm cells to
disinfectants Appl Environ Microbiol 684194ndash4200
51 Mafu A A D Roy J Goulet L Savoie and R Roy 1990
Efficiency of sanitizing agents for destroying Listeria monocytogenes
on contaminated surfaces J Dairy Sci 733428ndash3432
52 Mahony J O McAuliffe R Paul and D van Sinderen 2011
Bacteriophages as biocontrol agents of food pathogens Curr Opin
Biotechnol 22157ndash163
53 McEldowney S and M Fletcher 1988 Effect of temperature and
humidity on survival of bacteria attached to dry solid surfaces Lett
Appl Microbiol 783ndash86
54 Miettinen L K J Bjorkroth and H Korkeala 1999 Characterisa-
tion of Listeria monocytogenes from an ice-cream plant by serotyping
and pulsed-field gel electrophoresis Int J Food Microbiol 46187ndash
192
55 Mosteller T M and J R Bishop 1993 Sanitizer efficacy against
attached bacteria in a milk biofilm J Food Prot 5634ndash41
56 Norwood D E and A Gilmour 2000 The growth and resistance to
sodium hypochlorite of Listeria monocytogenes in a steady-state
multispecies biofilm J Appl Microbiol 88512ndash520
57 Oliveira M I Vinas P Colas M Anguera J Usall and M
Abadias 2014 Effectiveness of a bacteriophage in reducing Listeria
monocytogenes on fresh-cut fruits and fruit juices Food Microbiol
38137ndash142
58 Ronner A B and A C L Wong 1993 Biofilm development and
sanitizer inactivation of Listeria monocytogenes and Salmonella
typhimurium on stainless steel and buna-n rubber J Food Prot 56
750ndash758
59 Sallam S S and C W Donnelly 1992 Destruction injury and
repair of Listeria species exposed to sanitizing compounds J Food
Prot 55771ndash1033
60 Sillankorva S D R Oliveira M J Vieira I W Sutherland and J
Azeredo 2004 Bacteriophage V S1 infection of Pseudomonas
fluorescens planktonic cells versus biofilms Biofouling 20133ndash138
61 Silva S P Teixeira R Olieveira and J Azeredo 2008 Adhesion to
and viability of Listeria monocytogenes on food contact surfaces J
Food Prot 711379ndash1385
62 Skurnik M and E Strauch 2006 Phage therapy facts and fiction
Int J Food Microbiol 2965ndash14 (Review)
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2019
63 Somers E B J L Schoeni and A C L Wong 1994 Effect of
trisodium phosphate on biofilm and planktonic cells of Campylo-
bacter jejuni Escherichia coli O157 H7 Listeria monocytogenes
and Salmonella typhimurium Int J Food Microbiol 22269ndash
276
64 Somers E B and A C L Wong 2004 Efficacy of two cleaning
and sanitizing combination on Listeria monocytogenes biofilm
formed at low temperature on a variety of materials in the presence
of ready-to-eat meat residue J Food Prot 672218ndash2229
65 Soni K A and R Nannapaneni 2010 Removal of Listeria
monocytogenes biofilms with bacteriophages P100 J Food Prot 73
1519ndash1524
66 Sutherland I W K A Hughes L C Skillman and K Tait 2004
The interaction of phage and biofilm FEMS Microbiol Lett 2321ndash6
(MiniReview)
67 Swaminathan B 2001 Listeria monocytogenes p 383ndash409 In L R
Beuchat M P Doyle and T J Montville (ed) Food microbiology
fundamentals and frontiers 2nd ed ASM Press Washington DC
68 Thevenot D A Dernburg and C Vernozy-Rozand 2006 An
updated review of Listeria monocytogenes in the pork meat industry
and its products J Appl Microbiol 1017ndash17
69 Tuncan E U 1993 Effect of cold temperature on germicidal efficacy
of quaternary ammonium compound iodophor and chlorine on
Listeria J Food Prot 531029ndash1033
2020 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
conditions to prevent complete drying protecting some of
the cells from the disinfectants Drying of surfaces has been
considered as a significant bactericidal method to maintain
low numbers of bacteria in any environment and is
dependent on type of organism temperature relative
humidity and soiling (39 45 53) Furthermore type of
surface and topography have been reported to play an
important role on efficiency of disinfectants (30 40) in
particular surfaces with scrapes or defects where soil can
accumulate and protect bacteria against cleaning and
disinfecting agents (16 17 51)In presence of food residues in the wet 02-mm
grooves Listeria was reduced 2 to 3 log dependent on
types of food residues (Fig 3a) After exposure to the
disinfectants a reduction to below the detection limit was
observed for all food residues with no significant difference
(P 005) between SDC and Qac In the 5-mm grooves
before treatment less reduction (2 log CFUml) was
observed SDC was significantly (P 005) less effective
(2 log) than Qac ($2 log CFUml) for all food residues
except fish In the dry 02-mm grooves already a reduction
of 3 log CFUml was observed 24 h after inoculation
without treatment After exposure to the disinfectants the
numbers were further reduced to below the detection limit
In the 5-mm groove only a small (05 log CFUml ie
vegetable fish and ham residues) or no reduction (milk
residue) was observed immediately before treatment
probably because the cells were protected from the drying
in the deep grooves Treatment with SDC and Qac did not
inactivate all listerias with no significant difference (P
005) between the two disinfectants except in the case of
ham and vegetable residues where Qac performed signif-
icantly better than SDC in the wet grooves and for
vegetables in the dry grooves Previous studies have
reported an inactivation of the antimicrobial activity of
disinfectants by organic matter on surfaces showing 1- to 2-
log reductions on soiled surfaces compared with 3- to 4-
log reductions on clean surfaces (2 34) This could be due
to the chemical compositions such as protein and fat which
can bind with the disinfectants or reduction of penetration
into the bacterial layer (32 68) The chlorine-based
disinfectants have been found to be easily inactivated by
organic materials (25 46) whereas Qac was reported to be
less inactivated by organic matters (21) which was
confirmed in most cases in this study In general the test
on surfaces is dependent on the surface materials used and
viability of the cells dried on the surfaces resulting in
inconsistent results when compared with the test in
suspensions (15) Under practical conditions high amounts
of organic materials may be present resulting in less or no
activity of the disinfectant When the concentration of SDC
was increased from 240 to 300 mgliter a comparable
reduction of the cells to that of Qac was obtained however
there was no effect on reduction in the deep grooves (data
not shown) The results indicated that the disinfectant could
not reach all cells in the bottom of the groove while such
grooves (5-mm depth 1-mm width) might be present in
practice (eg small spaces between connection parts of
slicers) The effectiveness of the disinfectants in the deep
areas could be increased by using high pressure spraying
(32) however this method might have adverse effects since
it could also spread the bacterial cells to other areas and
environments in particular when high numbers of bacterial
cells are accumulated (42)
Carrier tests on surfaces with grooves in presence ofa biofilm (case 32) After 14 days of biofilm growing in
02-mm grooves (Fig 4) the number of cells remained
more or less stable with a decrease of the cells in vegetable
and ham biofilms (15 and 18 log CFUml respectively) In
5-mm grooves however growth of biofilm cells was
observed in all food residues A previous study reported that
L monocytogenes has the ability to form biofilms at
numbers of 104 to 107 CFUcm2 (34) and adheres to a
variety of food contact surfaces such as plastic glass
rubber and stainless steel particularly in pits or crevices
that are difficult to clean (31 61)To check if the biofilm cells were more resistant to the
disinfectants than the planktonic cells the biofilm cells were
brought into suspension and then tested The obtained
results a 5-log reduction in all conditions after 5-min
exposure to the disinfectants indicated no difference in
reduction of biofilm cells (case 32) compared with
planktonic cells (case 1) These results complied with a
previous study where reduction of the suspended biofilm
cells was higher than that of biofilm cells attached to
surfaces after exposure to disinfectants (50) It has been
described that a 10 to 100 times higher concentration of
disinfectants may be required to obtain the same reduction
of biofilm cells compared with planktonic cells (40 56)indicating the importance of preventing biofilm formation
For the bacteriophages an efficacy test was previously
performed with L monocytogenes strains in presence and
absence of food residues on flat stainless steel surfaces The
results demonstrated that rapid inactivation of Listeria cells
(2 to 3 log CFUcm2) within 30 min of contact time was
obtained (data not shown) which was similar with the
results of Soni and Nannapaneni (65) where a reduction of
35 log CFUcm2 was obtained in the same time with a
phage treatment on a 1-week-old L monocytogenes biofilm
on stainless steel coupons
In the 02-mm grooves (Fig 4a) Qac showed better
reduction (2 to 3 log) than SDC (12 to 27 log) with
inactivation below the detection limit in vegetable biofilms
The phages showed higher reduction (3 log CFUml P
005) than that of water or the chemical disinfectants in three
food biofilms except for the vegetable biofilms where all
cells were inactivated by every disinfectant In the 5-mm
grooves SDC and Qac did not reduce Listeria better than
water alone However the phages reduced approximately
14 log CFUml in ham and fish biofilms and approxi-
mately 08 log CFUml in milk and vegetable biofilms
which was not significantly different (P 005) from that of
the disinfectants except for ham biofilms The results
indicated that only in the 02-mm grooves the phages were
likely to be more effective in the biofilms with food matrix
than the chemical disinfectants This was probably due to
the fact that the disinfectants and phages did not penetrate
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2017
the foodbacterial matrix completely in the deep groove (2041) Furthermore the effectiveness of the disinfectants on Lmonocytogenes biofilms may be reduced by presence of
other bacteria (6 to 7 log CFUml) including lactic acid
bacteria (4 log CFUml) in the grooves as well (data not
shown) Phage efficacy depends on susceptibility of the
bacterial cells to the phages and availability of receptor sites
between phages and the bacterial cells (13) which could be
blocked by the food matrix (35) In this study the phage
efficacy was enhanced by keeping the biofilm plates in
plastic bags after application of the phages to maintain a
high humidity that would enhance the diffusion and the
chance of contact between Listeria cells and phage particles
(66)Strikingly the biofilm removal by sterile water
(control) resulted in a similar reduction of the biofilm cells
compared with disinfectants In the present study the tests
were performed to simulate survival of bacteria in presence
or absence of organic matter in pits or cracks on food
contact surfaces and to test the effect of disinfection
therefore cleaning of the surfaces before using disinfectants
was excluded So in this case plates and grooves were
dirtier than soiled surfaces in reality which explains the
observed low efficacy of the disinfectants and the water
washed away the biofilm cells from the grooves Rinsing the
biofilm on surfaces by water has been reported to reduce the
biofilm cells at about 1 log unit of cells (34)The results in this study clearly demonstrated that
presence of grooves or spaces on surfaces humidity and
presence of food substrates influenced the antimicrobial
effect of disinfectants and bacteriophages Bacteriophages
showed a better antimicrobial effect than the chemical
disinfectants (ie SDC and Qac) in the shallow grooves but
not in the deep grooves Use of bacteriophages as a
biocontrol could only be a promising method in limited
cases however use of large quantities in practice may be
costly and phage-resistant strains may occur (35 62)
ACKNOWLEDGMENTS
The authors thank Diversey Inc (Utrecht The Netherlands) who
provided the chemical disinfectants for this study We also thank Micreos
Food Safety BV (Wageningen The Netherlands) for Listeria bacteriophage
Listex P100 and L innocua (serovar 5) Our great appreciation goes to
Feifei Gao for her technical assistance in the phage experiments and Hilda
Nyati for her work on smooth surfaces
REFERENCES
1 Aarnisalo K J Lunden H Korkeala and G Wirtanen 2007
Susceptibility of Listeria monocytogenes strains to disinfectants and
chlorinated alkaline cleaners at cold temperatures LWT - Food SciTechnol 401041ndash1048
2 Aarnisalo K S Salo H Miettinen M Suihko G Wirtanen T
Autio J Lunden H Korkeala and A Sjoberg 2000 Bactericidal
efficiencies of commercial disinfectants against Listeria monocyto-
genes on surfaces J Food Saf 20237ndash250
3 Adams M R A D Hartley and L J Cox 1989 Factors affecting
the efficacy of washing procedures used in the production of prepared
salads Food Microbiol 669ndash77
4 Anonymous 2001 European Standard EN 136972001 Chemical
disinfectants and antisepticsmdashqualitative non-porous surface test for
the evaluation of bacteriocidal andor fungicidal activity of chemical
disinfectants used in food industrial domestic and institutional
areasmdashtest method and requirements without mechanical action (phase
2 step 2) Available at httpwwwceneucenSectorsTechnical
CommitteesWorkshopsCENTechnicalCommitteesPagesStandards
aspxparam~6197amptitle~Chemical20disinfectants20and20
antiseptics Accessed February 2014
5 Anonymous 2004 Microbiology of food and animal feeding stuffs mdash
horizontal method for the detection and enumeration of Listeria
monocytogenesmdashpart 1 detection method ISO 11290-11996Amd
12004 International Organization for Standardization Geneva
Available at httpswwwisoorgobpuiisostdiso11290-1ed-1
v1enamd1amd1 Accessed January 2014
6 Anonymous 2005 European Standard EN 10402005 Chemical
disinfectants and antiseptics Quantitative suspension test for the
evaluation of basic bactericidal activity of chemical disinfectants
and antisepticsmdashtest method and requirements (phase 1) Available
at httpwwwceneucenSectorsTechnicalCommitteesWorkshops
CENTechnicalCommitteesPagesStandardsaspxparam~6197amptitle~
Chemical20disinfectants20and20antiseptics Accessed February
2014
7 Arachchi G J G A G Cridge B M Dias-Wanigasekera C D
Cruz L McIntyre R Liu S H Flint and A N Mutukumira 2013
Effectiveness of phages in the decontamination of Listeria monocy-
togenes adhered to clean stainless steel stainless steel coated with
fish protein and as a biofilm J Ind Microbiol Biotechnol 401105ndash
1116
8 Autio T R Keto-Timonen J Bjorkroth and H Korkeala 2003
Characterization of persistent and sporadic Listeria monocytogenes
strains by pulsed-field gel electrophoresis (PFGE) and amplified
fragment length polymorphism (AFLP) Syst Appl Microbiol 26
539ndash545
9 Bagge-Ravn D Y Ng M Hjelm J N Christiansen C Johansen
and L Gram 2003 The microbial ecology of processing equipment
in different fish industriesmdashanalysis of the microflora during
processing and following cleaning and disinfection Int J Food
Microbiol 87239ndash250
10 Belessi C A A S Gounadaki A N Psomas and P N Skandamis
2011 Efficiency of different sanitation methods on Listeria
monocytogenes biofilms formed under various environmental condi-
tions Int J Food Microbiol 145S46ndashS52
11 Beresford M R P W Andrew and G Shama 2001 Listeria
monocytogenes adheres to many materials found in food-processing
environments J Appl Microbiol 901000ndash1005
12 Best M M M M E Kennedy and F F Coates 1990 Efficacy of a
variety of disinfectants against Listeria spp Appl Environ Micro-
biol 56377ndash380
13 Bigwood T J A Hudson and C Billington 2009 Influence of host
and bacteriophage concentrations on the inactivation of food-borne
pathogenic bacteria by two phages FEMS Microbiol Lett 29159ndash
64
14 Blackman I C and J F Frank 1996 Growth of Listeria
monocytogenes as a biofilm on various food-processing surfaces J
Food Prot 59827ndash831
15 Bloomfield S F M Arthur B Klingeren W van Pullen J T
Holah and R Elton 1994 An evaluation of the repeatability and
reproducibility of a surface test for the activity of disinfectants J
Appl Bacteriol 7686ndash94
16 Boulange-Petermann L J Rault and M-N Bellon-Fontaine
1997 Adhesion of Streptococcus thermophilus to stainless steel
with different surface topography and roughness Biofouling 11
201ndash216
17 Bower C K J McGuire and M A Daeschel 1996 The adhesion
and detachment of bacteria and spores on food contact surfaces
Trends Food Sci Technol 7152ndash157
18 Bryan F L 2002 Where we are in the retail food safety how we got
to where we are and how do we get there J Environ Health 6529ndash
36
19 Carey-Smith G V C Billington A J Cornelius J A Hudson and
J A Heinemann 2006 Isolation and characterization of bacterio-
phages infecting Salmonella spp FEMS Microbiol Lett 258182ndash
186
2018 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
20 Carpentier B and O Cerf 1993 Biofilms and their consequences
with particular reference to hygiene in the food industry J Appl
Bacteriol 75499ndash511
21 Carsberg H 1996 Selecting your sanitizers Food Qual 235ndash36
61ndash62
22 Chibeu A L Agius A Gao P M Sabour A M Kropinski and S
Balamurugan 2013 Efficacy of bacteriophage LISTEXTMP100
combined with chemical antimicrobials in reducing Listeria mono-
cytogenes in cooked turkey and roast beef Int J Food Microbiol
167208ndash214
23 Cox L J T Kleiss J L Cordier C Cordellana P Konkel C
Pedrazzini R Beumer and A Siebenga 1989 Listeria spp in food
processing non-food and domestic environments Food Microbiol 6
49ndash61
24 Cruz C D and G C Fletcher 2012 Assessing manufacturersrsquo
recommended concentrations of commercial sanitizers on inactivation
of Listeria monocytogenes Food Control 26194ndash199
25 De Beer D R Srinivasan and P S Stewart 1994 Direct
measurement of chlorine penetration into biofilms during disinfec-
tion Appl Environ Microbiol 604339ndash4344
26 Doolittle M M J J Cooney and D E Caldwell 1995 Lytic
infection of Escherichia coli biofilms by bacteriophage T4 Can J
Microbiol 4112ndash18
27 Environmental Protection Agency (EPA) Office of Wastewater
Enforcement and Compliance 2014 Guide to field storage of
biosolids and other organic by-products used in agriculture and for
soil resource management Available at httpwaterepagovscitech
wastetechbiosolidsguidecfm Accessed January 2014
28 Fatemi P and J F Frank 1999 Inactivation of Listeria
monocytogenesPseudomonas biofilms by peracid sanitizer J Food
Prot 62761ndash765
29 Fenlon D R J Wilson and W Donachie 1996 The incidence and
level of Listeria monocytogenes contamination of food sources at
primary production and initial processing J Appl Bacteriol 81641ndash
650
30 Frank J F and R A N Chmielewski 1997 Effectiveness of
sanitation with quaternary ammonium compound or chlorine on
stainless steel and other domestic food-preparation surfaces J Food
Prot 6043ndash47
31 Frank J F and R A Koffi 1990 Surface-adherent growth of
Listeria monocytogenes is associated with increased resistance of
surfactant sanitizers and heat J Food Prot 53550ndash554
32 Gibson H J H Taylor K E Hall and J T Holah 1999
Effectiveness of cleaning techniques used in the food industry in terms
of the removal of bacterial biofilms J Appl Microbiol 8741ndash48
33 Gombas D E Y Chen R S Clavaro and V N Scott 2003 Survey
of Listeria monocytogenes in foods J Food Prot 66559ndash569
34 Gram L D Bagge-Ravn Y Y Ng P Gymoese and B F Vogel
2007 Influence of food soiling matrix on cleaning and disinfection
efficiency on surface attached Listeria monocytogenes Food Control
181165ndash1171
35 Greer G G 2005 Bacteriophage control of foodborne bacteria J
Food Prot 681102ndash1111
36 Gronholm L G Wirtanen K Ahlgren K Nordstrom and A
Sjoberg 1999 Screening of antimicrobial activities of disinfectants
and cleaning agents against foodborne spoilage microbes Eur Food
Res Technol 208289ndash298
37 Hagens S 2014 Personal communication
38 Hibma A M S A Jassim and M W Griffiths 1997 Infection and
removal of L-forms of Listeria monocytogenes with bred bacterio-
phage Int J Food Microbiol 34197ndash207
39 Hirai Y 1991 Survival of bacteria under dry conditions from a
viewpoint of nosocomial infection J Hosp Infect 19191ndash200
40 Holah J T 1992 Industrial monitoring hygiene in food processing
p 645ndash659A In L F Melo T R Bott M Fletcher and B
Capdeville (ed) Biofilmsmdashscience and technology Kluwer Aca-
demic Publishers Dordrecht
41 Holah J T C Higgs S Robinson D Worthington and H
Spenceley 1990 conductance-based surface disinfection test for food
hygiene Lett Appl Microbiol 11255ndash259
42 Holah J T J H Taylor and J S Holder 1993 The spread of
Listeria by cleaning systems part II Technical Memorandum no 673
Campden and Chorleywood Food Research Association Chipping
Campden UK
43 Hughes K A I W Sutherland and M V Jones 1998 Biofilm
susceptibility to bacteriophage attack the role of phage-borne
polysaccharide depolymerase Microbiology 1443039ndash3047
44 Joerger R D 2003 Alternatives to antibiotics bacteriocins
antimicrobial peptides and bacteriophages Poult Sci 82640ndash647
45 Kusumaningrum H D G Riboldi W C Hazeleger and R R
Beumer 2003 Survival of foodborne pathogens on stainless steel
surfaces and cross-contamination to foods Int J Food Microbiol 85
227ndash236
46 Lee S-H and J Frank 1991 Inactivation of surface adherent
Listeria monocytogenes hypochlorite and heat J Food Prot 544ndash
6
47 Leverentz B W S Conway M J Camp W J Janisiewicz T
Abuladze M Yang R Saftner and A Sulakvelidze 2003
Biocontrol of Listeria monocytogenes on fresh-cut produce by
treatment with lytic bacteriophages and a bacteriocin Appl Environ
Microbiol 694519ndash4526
48 Lin C M K Takeuchi L Zhang C B Dohm J D Meyer P A
Hall and M P Doyle 2006 Cross-contamination between
processing equipment and deli meats by Listeria monocytogenes J
Food Prot 6971ndash79
49 Lunden J M T J Autio and H J Korkeala 2002 Transfer of
persistent Listeria monocytogenes contamination between food-
processing plants associated with a dicing machine J Food Prot
651129ndash1133
50 Luppens S B I M W Reij R W L van der Heijden F M
Rombouts and T Abee 2002 Development of a standard test to
assess the resistance of Staphylococcus aureus biofilm cells to
disinfectants Appl Environ Microbiol 684194ndash4200
51 Mafu A A D Roy J Goulet L Savoie and R Roy 1990
Efficiency of sanitizing agents for destroying Listeria monocytogenes
on contaminated surfaces J Dairy Sci 733428ndash3432
52 Mahony J O McAuliffe R Paul and D van Sinderen 2011
Bacteriophages as biocontrol agents of food pathogens Curr Opin
Biotechnol 22157ndash163
53 McEldowney S and M Fletcher 1988 Effect of temperature and
humidity on survival of bacteria attached to dry solid surfaces Lett
Appl Microbiol 783ndash86
54 Miettinen L K J Bjorkroth and H Korkeala 1999 Characterisa-
tion of Listeria monocytogenes from an ice-cream plant by serotyping
and pulsed-field gel electrophoresis Int J Food Microbiol 46187ndash
192
55 Mosteller T M and J R Bishop 1993 Sanitizer efficacy against
attached bacteria in a milk biofilm J Food Prot 5634ndash41
56 Norwood D E and A Gilmour 2000 The growth and resistance to
sodium hypochlorite of Listeria monocytogenes in a steady-state
multispecies biofilm J Appl Microbiol 88512ndash520
57 Oliveira M I Vinas P Colas M Anguera J Usall and M
Abadias 2014 Effectiveness of a bacteriophage in reducing Listeria
monocytogenes on fresh-cut fruits and fruit juices Food Microbiol
38137ndash142
58 Ronner A B and A C L Wong 1993 Biofilm development and
sanitizer inactivation of Listeria monocytogenes and Salmonella
typhimurium on stainless steel and buna-n rubber J Food Prot 56
750ndash758
59 Sallam S S and C W Donnelly 1992 Destruction injury and
repair of Listeria species exposed to sanitizing compounds J Food
Prot 55771ndash1033
60 Sillankorva S D R Oliveira M J Vieira I W Sutherland and J
Azeredo 2004 Bacteriophage V S1 infection of Pseudomonas
fluorescens planktonic cells versus biofilms Biofouling 20133ndash138
61 Silva S P Teixeira R Olieveira and J Azeredo 2008 Adhesion to
and viability of Listeria monocytogenes on food contact surfaces J
Food Prot 711379ndash1385
62 Skurnik M and E Strauch 2006 Phage therapy facts and fiction
Int J Food Microbiol 2965ndash14 (Review)
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2019
63 Somers E B J L Schoeni and A C L Wong 1994 Effect of
trisodium phosphate on biofilm and planktonic cells of Campylo-
bacter jejuni Escherichia coli O157 H7 Listeria monocytogenes
and Salmonella typhimurium Int J Food Microbiol 22269ndash
276
64 Somers E B and A C L Wong 2004 Efficacy of two cleaning
and sanitizing combination on Listeria monocytogenes biofilm
formed at low temperature on a variety of materials in the presence
of ready-to-eat meat residue J Food Prot 672218ndash2229
65 Soni K A and R Nannapaneni 2010 Removal of Listeria
monocytogenes biofilms with bacteriophages P100 J Food Prot 73
1519ndash1524
66 Sutherland I W K A Hughes L C Skillman and K Tait 2004
The interaction of phage and biofilm FEMS Microbiol Lett 2321ndash6
(MiniReview)
67 Swaminathan B 2001 Listeria monocytogenes p 383ndash409 In L R
Beuchat M P Doyle and T J Montville (ed) Food microbiology
fundamentals and frontiers 2nd ed ASM Press Washington DC
68 Thevenot D A Dernburg and C Vernozy-Rozand 2006 An
updated review of Listeria monocytogenes in the pork meat industry
and its products J Appl Microbiol 1017ndash17
69 Tuncan E U 1993 Effect of cold temperature on germicidal efficacy
of quaternary ammonium compound iodophor and chlorine on
Listeria J Food Prot 531029ndash1033
2020 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
the foodbacterial matrix completely in the deep groove (2041) Furthermore the effectiveness of the disinfectants on Lmonocytogenes biofilms may be reduced by presence of
other bacteria (6 to 7 log CFUml) including lactic acid
bacteria (4 log CFUml) in the grooves as well (data not
shown) Phage efficacy depends on susceptibility of the
bacterial cells to the phages and availability of receptor sites
between phages and the bacterial cells (13) which could be
blocked by the food matrix (35) In this study the phage
efficacy was enhanced by keeping the biofilm plates in
plastic bags after application of the phages to maintain a
high humidity that would enhance the diffusion and the
chance of contact between Listeria cells and phage particles
(66)Strikingly the biofilm removal by sterile water
(control) resulted in a similar reduction of the biofilm cells
compared with disinfectants In the present study the tests
were performed to simulate survival of bacteria in presence
or absence of organic matter in pits or cracks on food
contact surfaces and to test the effect of disinfection
therefore cleaning of the surfaces before using disinfectants
was excluded So in this case plates and grooves were
dirtier than soiled surfaces in reality which explains the
observed low efficacy of the disinfectants and the water
washed away the biofilm cells from the grooves Rinsing the
biofilm on surfaces by water has been reported to reduce the
biofilm cells at about 1 log unit of cells (34)The results in this study clearly demonstrated that
presence of grooves or spaces on surfaces humidity and
presence of food substrates influenced the antimicrobial
effect of disinfectants and bacteriophages Bacteriophages
showed a better antimicrobial effect than the chemical
disinfectants (ie SDC and Qac) in the shallow grooves but
not in the deep grooves Use of bacteriophages as a
biocontrol could only be a promising method in limited
cases however use of large quantities in practice may be
costly and phage-resistant strains may occur (35 62)
ACKNOWLEDGMENTS
The authors thank Diversey Inc (Utrecht The Netherlands) who
provided the chemical disinfectants for this study We also thank Micreos
Food Safety BV (Wageningen The Netherlands) for Listeria bacteriophage
Listex P100 and L innocua (serovar 5) Our great appreciation goes to
Feifei Gao for her technical assistance in the phage experiments and Hilda
Nyati for her work on smooth surfaces
REFERENCES
1 Aarnisalo K J Lunden H Korkeala and G Wirtanen 2007
Susceptibility of Listeria monocytogenes strains to disinfectants and
chlorinated alkaline cleaners at cold temperatures LWT - Food SciTechnol 401041ndash1048
2 Aarnisalo K S Salo H Miettinen M Suihko G Wirtanen T
Autio J Lunden H Korkeala and A Sjoberg 2000 Bactericidal
efficiencies of commercial disinfectants against Listeria monocyto-
genes on surfaces J Food Saf 20237ndash250
3 Adams M R A D Hartley and L J Cox 1989 Factors affecting
the efficacy of washing procedures used in the production of prepared
salads Food Microbiol 669ndash77
4 Anonymous 2001 European Standard EN 136972001 Chemical
disinfectants and antisepticsmdashqualitative non-porous surface test for
the evaluation of bacteriocidal andor fungicidal activity of chemical
disinfectants used in food industrial domestic and institutional
areasmdashtest method and requirements without mechanical action (phase
2 step 2) Available at httpwwwceneucenSectorsTechnical
CommitteesWorkshopsCENTechnicalCommitteesPagesStandards
aspxparam~6197amptitle~Chemical20disinfectants20and20
antiseptics Accessed February 2014
5 Anonymous 2004 Microbiology of food and animal feeding stuffs mdash
horizontal method for the detection and enumeration of Listeria
monocytogenesmdashpart 1 detection method ISO 11290-11996Amd
12004 International Organization for Standardization Geneva
Available at httpswwwisoorgobpuiisostdiso11290-1ed-1
v1enamd1amd1 Accessed January 2014
6 Anonymous 2005 European Standard EN 10402005 Chemical
disinfectants and antiseptics Quantitative suspension test for the
evaluation of basic bactericidal activity of chemical disinfectants
and antisepticsmdashtest method and requirements (phase 1) Available
at httpwwwceneucenSectorsTechnicalCommitteesWorkshops
CENTechnicalCommitteesPagesStandardsaspxparam~6197amptitle~
Chemical20disinfectants20and20antiseptics Accessed February
2014
7 Arachchi G J G A G Cridge B M Dias-Wanigasekera C D
Cruz L McIntyre R Liu S H Flint and A N Mutukumira 2013
Effectiveness of phages in the decontamination of Listeria monocy-
togenes adhered to clean stainless steel stainless steel coated with
fish protein and as a biofilm J Ind Microbiol Biotechnol 401105ndash
1116
8 Autio T R Keto-Timonen J Bjorkroth and H Korkeala 2003
Characterization of persistent and sporadic Listeria monocytogenes
strains by pulsed-field gel electrophoresis (PFGE) and amplified
fragment length polymorphism (AFLP) Syst Appl Microbiol 26
539ndash545
9 Bagge-Ravn D Y Ng M Hjelm J N Christiansen C Johansen
and L Gram 2003 The microbial ecology of processing equipment
in different fish industriesmdashanalysis of the microflora during
processing and following cleaning and disinfection Int J Food
Microbiol 87239ndash250
10 Belessi C A A S Gounadaki A N Psomas and P N Skandamis
2011 Efficiency of different sanitation methods on Listeria
monocytogenes biofilms formed under various environmental condi-
tions Int J Food Microbiol 145S46ndashS52
11 Beresford M R P W Andrew and G Shama 2001 Listeria
monocytogenes adheres to many materials found in food-processing
environments J Appl Microbiol 901000ndash1005
12 Best M M M M E Kennedy and F F Coates 1990 Efficacy of a
variety of disinfectants against Listeria spp Appl Environ Micro-
biol 56377ndash380
13 Bigwood T J A Hudson and C Billington 2009 Influence of host
and bacteriophage concentrations on the inactivation of food-borne
pathogenic bacteria by two phages FEMS Microbiol Lett 29159ndash
64
14 Blackman I C and J F Frank 1996 Growth of Listeria
monocytogenes as a biofilm on various food-processing surfaces J
Food Prot 59827ndash831
15 Bloomfield S F M Arthur B Klingeren W van Pullen J T
Holah and R Elton 1994 An evaluation of the repeatability and
reproducibility of a surface test for the activity of disinfectants J
Appl Bacteriol 7686ndash94
16 Boulange-Petermann L J Rault and M-N Bellon-Fontaine
1997 Adhesion of Streptococcus thermophilus to stainless steel
with different surface topography and roughness Biofouling 11
201ndash216
17 Bower C K J McGuire and M A Daeschel 1996 The adhesion
and detachment of bacteria and spores on food contact surfaces
Trends Food Sci Technol 7152ndash157
18 Bryan F L 2002 Where we are in the retail food safety how we got
to where we are and how do we get there J Environ Health 6529ndash
36
19 Carey-Smith G V C Billington A J Cornelius J A Hudson and
J A Heinemann 2006 Isolation and characterization of bacterio-
phages infecting Salmonella spp FEMS Microbiol Lett 258182ndash
186
2018 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
20 Carpentier B and O Cerf 1993 Biofilms and their consequences
with particular reference to hygiene in the food industry J Appl
Bacteriol 75499ndash511
21 Carsberg H 1996 Selecting your sanitizers Food Qual 235ndash36
61ndash62
22 Chibeu A L Agius A Gao P M Sabour A M Kropinski and S
Balamurugan 2013 Efficacy of bacteriophage LISTEXTMP100
combined with chemical antimicrobials in reducing Listeria mono-
cytogenes in cooked turkey and roast beef Int J Food Microbiol
167208ndash214
23 Cox L J T Kleiss J L Cordier C Cordellana P Konkel C
Pedrazzini R Beumer and A Siebenga 1989 Listeria spp in food
processing non-food and domestic environments Food Microbiol 6
49ndash61
24 Cruz C D and G C Fletcher 2012 Assessing manufacturersrsquo
recommended concentrations of commercial sanitizers on inactivation
of Listeria monocytogenes Food Control 26194ndash199
25 De Beer D R Srinivasan and P S Stewart 1994 Direct
measurement of chlorine penetration into biofilms during disinfec-
tion Appl Environ Microbiol 604339ndash4344
26 Doolittle M M J J Cooney and D E Caldwell 1995 Lytic
infection of Escherichia coli biofilms by bacteriophage T4 Can J
Microbiol 4112ndash18
27 Environmental Protection Agency (EPA) Office of Wastewater
Enforcement and Compliance 2014 Guide to field storage of
biosolids and other organic by-products used in agriculture and for
soil resource management Available at httpwaterepagovscitech
wastetechbiosolidsguidecfm Accessed January 2014
28 Fatemi P and J F Frank 1999 Inactivation of Listeria
monocytogenesPseudomonas biofilms by peracid sanitizer J Food
Prot 62761ndash765
29 Fenlon D R J Wilson and W Donachie 1996 The incidence and
level of Listeria monocytogenes contamination of food sources at
primary production and initial processing J Appl Bacteriol 81641ndash
650
30 Frank J F and R A N Chmielewski 1997 Effectiveness of
sanitation with quaternary ammonium compound or chlorine on
stainless steel and other domestic food-preparation surfaces J Food
Prot 6043ndash47
31 Frank J F and R A Koffi 1990 Surface-adherent growth of
Listeria monocytogenes is associated with increased resistance of
surfactant sanitizers and heat J Food Prot 53550ndash554
32 Gibson H J H Taylor K E Hall and J T Holah 1999
Effectiveness of cleaning techniques used in the food industry in terms
of the removal of bacterial biofilms J Appl Microbiol 8741ndash48
33 Gombas D E Y Chen R S Clavaro and V N Scott 2003 Survey
of Listeria monocytogenes in foods J Food Prot 66559ndash569
34 Gram L D Bagge-Ravn Y Y Ng P Gymoese and B F Vogel
2007 Influence of food soiling matrix on cleaning and disinfection
efficiency on surface attached Listeria monocytogenes Food Control
181165ndash1171
35 Greer G G 2005 Bacteriophage control of foodborne bacteria J
Food Prot 681102ndash1111
36 Gronholm L G Wirtanen K Ahlgren K Nordstrom and A
Sjoberg 1999 Screening of antimicrobial activities of disinfectants
and cleaning agents against foodborne spoilage microbes Eur Food
Res Technol 208289ndash298
37 Hagens S 2014 Personal communication
38 Hibma A M S A Jassim and M W Griffiths 1997 Infection and
removal of L-forms of Listeria monocytogenes with bred bacterio-
phage Int J Food Microbiol 34197ndash207
39 Hirai Y 1991 Survival of bacteria under dry conditions from a
viewpoint of nosocomial infection J Hosp Infect 19191ndash200
40 Holah J T 1992 Industrial monitoring hygiene in food processing
p 645ndash659A In L F Melo T R Bott M Fletcher and B
Capdeville (ed) Biofilmsmdashscience and technology Kluwer Aca-
demic Publishers Dordrecht
41 Holah J T C Higgs S Robinson D Worthington and H
Spenceley 1990 conductance-based surface disinfection test for food
hygiene Lett Appl Microbiol 11255ndash259
42 Holah J T J H Taylor and J S Holder 1993 The spread of
Listeria by cleaning systems part II Technical Memorandum no 673
Campden and Chorleywood Food Research Association Chipping
Campden UK
43 Hughes K A I W Sutherland and M V Jones 1998 Biofilm
susceptibility to bacteriophage attack the role of phage-borne
polysaccharide depolymerase Microbiology 1443039ndash3047
44 Joerger R D 2003 Alternatives to antibiotics bacteriocins
antimicrobial peptides and bacteriophages Poult Sci 82640ndash647
45 Kusumaningrum H D G Riboldi W C Hazeleger and R R
Beumer 2003 Survival of foodborne pathogens on stainless steel
surfaces and cross-contamination to foods Int J Food Microbiol 85
227ndash236
46 Lee S-H and J Frank 1991 Inactivation of surface adherent
Listeria monocytogenes hypochlorite and heat J Food Prot 544ndash
6
47 Leverentz B W S Conway M J Camp W J Janisiewicz T
Abuladze M Yang R Saftner and A Sulakvelidze 2003
Biocontrol of Listeria monocytogenes on fresh-cut produce by
treatment with lytic bacteriophages and a bacteriocin Appl Environ
Microbiol 694519ndash4526
48 Lin C M K Takeuchi L Zhang C B Dohm J D Meyer P A
Hall and M P Doyle 2006 Cross-contamination between
processing equipment and deli meats by Listeria monocytogenes J
Food Prot 6971ndash79
49 Lunden J M T J Autio and H J Korkeala 2002 Transfer of
persistent Listeria monocytogenes contamination between food-
processing plants associated with a dicing machine J Food Prot
651129ndash1133
50 Luppens S B I M W Reij R W L van der Heijden F M
Rombouts and T Abee 2002 Development of a standard test to
assess the resistance of Staphylococcus aureus biofilm cells to
disinfectants Appl Environ Microbiol 684194ndash4200
51 Mafu A A D Roy J Goulet L Savoie and R Roy 1990
Efficiency of sanitizing agents for destroying Listeria monocytogenes
on contaminated surfaces J Dairy Sci 733428ndash3432
52 Mahony J O McAuliffe R Paul and D van Sinderen 2011
Bacteriophages as biocontrol agents of food pathogens Curr Opin
Biotechnol 22157ndash163
53 McEldowney S and M Fletcher 1988 Effect of temperature and
humidity on survival of bacteria attached to dry solid surfaces Lett
Appl Microbiol 783ndash86
54 Miettinen L K J Bjorkroth and H Korkeala 1999 Characterisa-
tion of Listeria monocytogenes from an ice-cream plant by serotyping
and pulsed-field gel electrophoresis Int J Food Microbiol 46187ndash
192
55 Mosteller T M and J R Bishop 1993 Sanitizer efficacy against
attached bacteria in a milk biofilm J Food Prot 5634ndash41
56 Norwood D E and A Gilmour 2000 The growth and resistance to
sodium hypochlorite of Listeria monocytogenes in a steady-state
multispecies biofilm J Appl Microbiol 88512ndash520
57 Oliveira M I Vinas P Colas M Anguera J Usall and M
Abadias 2014 Effectiveness of a bacteriophage in reducing Listeria
monocytogenes on fresh-cut fruits and fruit juices Food Microbiol
38137ndash142
58 Ronner A B and A C L Wong 1993 Biofilm development and
sanitizer inactivation of Listeria monocytogenes and Salmonella
typhimurium on stainless steel and buna-n rubber J Food Prot 56
750ndash758
59 Sallam S S and C W Donnelly 1992 Destruction injury and
repair of Listeria species exposed to sanitizing compounds J Food
Prot 55771ndash1033
60 Sillankorva S D R Oliveira M J Vieira I W Sutherland and J
Azeredo 2004 Bacteriophage V S1 infection of Pseudomonas
fluorescens planktonic cells versus biofilms Biofouling 20133ndash138
61 Silva S P Teixeira R Olieveira and J Azeredo 2008 Adhesion to
and viability of Listeria monocytogenes on food contact surfaces J
Food Prot 711379ndash1385
62 Skurnik M and E Strauch 2006 Phage therapy facts and fiction
Int J Food Microbiol 2965ndash14 (Review)
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2019
63 Somers E B J L Schoeni and A C L Wong 1994 Effect of
trisodium phosphate on biofilm and planktonic cells of Campylo-
bacter jejuni Escherichia coli O157 H7 Listeria monocytogenes
and Salmonella typhimurium Int J Food Microbiol 22269ndash
276
64 Somers E B and A C L Wong 2004 Efficacy of two cleaning
and sanitizing combination on Listeria monocytogenes biofilm
formed at low temperature on a variety of materials in the presence
of ready-to-eat meat residue J Food Prot 672218ndash2229
65 Soni K A and R Nannapaneni 2010 Removal of Listeria
monocytogenes biofilms with bacteriophages P100 J Food Prot 73
1519ndash1524
66 Sutherland I W K A Hughes L C Skillman and K Tait 2004
The interaction of phage and biofilm FEMS Microbiol Lett 2321ndash6
(MiniReview)
67 Swaminathan B 2001 Listeria monocytogenes p 383ndash409 In L R
Beuchat M P Doyle and T J Montville (ed) Food microbiology
fundamentals and frontiers 2nd ed ASM Press Washington DC
68 Thevenot D A Dernburg and C Vernozy-Rozand 2006 An
updated review of Listeria monocytogenes in the pork meat industry
and its products J Appl Microbiol 1017ndash17
69 Tuncan E U 1993 Effect of cold temperature on germicidal efficacy
of quaternary ammonium compound iodophor and chlorine on
Listeria J Food Prot 531029ndash1033
2020 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
20 Carpentier B and O Cerf 1993 Biofilms and their consequences
with particular reference to hygiene in the food industry J Appl
Bacteriol 75499ndash511
21 Carsberg H 1996 Selecting your sanitizers Food Qual 235ndash36
61ndash62
22 Chibeu A L Agius A Gao P M Sabour A M Kropinski and S
Balamurugan 2013 Efficacy of bacteriophage LISTEXTMP100
combined with chemical antimicrobials in reducing Listeria mono-
cytogenes in cooked turkey and roast beef Int J Food Microbiol
167208ndash214
23 Cox L J T Kleiss J L Cordier C Cordellana P Konkel C
Pedrazzini R Beumer and A Siebenga 1989 Listeria spp in food
processing non-food and domestic environments Food Microbiol 6
49ndash61
24 Cruz C D and G C Fletcher 2012 Assessing manufacturersrsquo
recommended concentrations of commercial sanitizers on inactivation
of Listeria monocytogenes Food Control 26194ndash199
25 De Beer D R Srinivasan and P S Stewart 1994 Direct
measurement of chlorine penetration into biofilms during disinfec-
tion Appl Environ Microbiol 604339ndash4344
26 Doolittle M M J J Cooney and D E Caldwell 1995 Lytic
infection of Escherichia coli biofilms by bacteriophage T4 Can J
Microbiol 4112ndash18
27 Environmental Protection Agency (EPA) Office of Wastewater
Enforcement and Compliance 2014 Guide to field storage of
biosolids and other organic by-products used in agriculture and for
soil resource management Available at httpwaterepagovscitech
wastetechbiosolidsguidecfm Accessed January 2014
28 Fatemi P and J F Frank 1999 Inactivation of Listeria
monocytogenesPseudomonas biofilms by peracid sanitizer J Food
Prot 62761ndash765
29 Fenlon D R J Wilson and W Donachie 1996 The incidence and
level of Listeria monocytogenes contamination of food sources at
primary production and initial processing J Appl Bacteriol 81641ndash
650
30 Frank J F and R A N Chmielewski 1997 Effectiveness of
sanitation with quaternary ammonium compound or chlorine on
stainless steel and other domestic food-preparation surfaces J Food
Prot 6043ndash47
31 Frank J F and R A Koffi 1990 Surface-adherent growth of
Listeria monocytogenes is associated with increased resistance of
surfactant sanitizers and heat J Food Prot 53550ndash554
32 Gibson H J H Taylor K E Hall and J T Holah 1999
Effectiveness of cleaning techniques used in the food industry in terms
of the removal of bacterial biofilms J Appl Microbiol 8741ndash48
33 Gombas D E Y Chen R S Clavaro and V N Scott 2003 Survey
of Listeria monocytogenes in foods J Food Prot 66559ndash569
34 Gram L D Bagge-Ravn Y Y Ng P Gymoese and B F Vogel
2007 Influence of food soiling matrix on cleaning and disinfection
efficiency on surface attached Listeria monocytogenes Food Control
181165ndash1171
35 Greer G G 2005 Bacteriophage control of foodborne bacteria J
Food Prot 681102ndash1111
36 Gronholm L G Wirtanen K Ahlgren K Nordstrom and A
Sjoberg 1999 Screening of antimicrobial activities of disinfectants
and cleaning agents against foodborne spoilage microbes Eur Food
Res Technol 208289ndash298
37 Hagens S 2014 Personal communication
38 Hibma A M S A Jassim and M W Griffiths 1997 Infection and
removal of L-forms of Listeria monocytogenes with bred bacterio-
phage Int J Food Microbiol 34197ndash207
39 Hirai Y 1991 Survival of bacteria under dry conditions from a
viewpoint of nosocomial infection J Hosp Infect 19191ndash200
40 Holah J T 1992 Industrial monitoring hygiene in food processing
p 645ndash659A In L F Melo T R Bott M Fletcher and B
Capdeville (ed) Biofilmsmdashscience and technology Kluwer Aca-
demic Publishers Dordrecht
41 Holah J T C Higgs S Robinson D Worthington and H
Spenceley 1990 conductance-based surface disinfection test for food
hygiene Lett Appl Microbiol 11255ndash259
42 Holah J T J H Taylor and J S Holder 1993 The spread of
Listeria by cleaning systems part II Technical Memorandum no 673
Campden and Chorleywood Food Research Association Chipping
Campden UK
43 Hughes K A I W Sutherland and M V Jones 1998 Biofilm
susceptibility to bacteriophage attack the role of phage-borne
polysaccharide depolymerase Microbiology 1443039ndash3047
44 Joerger R D 2003 Alternatives to antibiotics bacteriocins
antimicrobial peptides and bacteriophages Poult Sci 82640ndash647
45 Kusumaningrum H D G Riboldi W C Hazeleger and R R
Beumer 2003 Survival of foodborne pathogens on stainless steel
surfaces and cross-contamination to foods Int J Food Microbiol 85
227ndash236
46 Lee S-H and J Frank 1991 Inactivation of surface adherent
Listeria monocytogenes hypochlorite and heat J Food Prot 544ndash
6
47 Leverentz B W S Conway M J Camp W J Janisiewicz T
Abuladze M Yang R Saftner and A Sulakvelidze 2003
Biocontrol of Listeria monocytogenes on fresh-cut produce by
treatment with lytic bacteriophages and a bacteriocin Appl Environ
Microbiol 694519ndash4526
48 Lin C M K Takeuchi L Zhang C B Dohm J D Meyer P A
Hall and M P Doyle 2006 Cross-contamination between
processing equipment and deli meats by Listeria monocytogenes J
Food Prot 6971ndash79
49 Lunden J M T J Autio and H J Korkeala 2002 Transfer of
persistent Listeria monocytogenes contamination between food-
processing plants associated with a dicing machine J Food Prot
651129ndash1133
50 Luppens S B I M W Reij R W L van der Heijden F M
Rombouts and T Abee 2002 Development of a standard test to
assess the resistance of Staphylococcus aureus biofilm cells to
disinfectants Appl Environ Microbiol 684194ndash4200
51 Mafu A A D Roy J Goulet L Savoie and R Roy 1990
Efficiency of sanitizing agents for destroying Listeria monocytogenes
on contaminated surfaces J Dairy Sci 733428ndash3432
52 Mahony J O McAuliffe R Paul and D van Sinderen 2011
Bacteriophages as biocontrol agents of food pathogens Curr Opin
Biotechnol 22157ndash163
53 McEldowney S and M Fletcher 1988 Effect of temperature and
humidity on survival of bacteria attached to dry solid surfaces Lett
Appl Microbiol 783ndash86
54 Miettinen L K J Bjorkroth and H Korkeala 1999 Characterisa-
tion of Listeria monocytogenes from an ice-cream plant by serotyping
and pulsed-field gel electrophoresis Int J Food Microbiol 46187ndash
192
55 Mosteller T M and J R Bishop 1993 Sanitizer efficacy against
attached bacteria in a milk biofilm J Food Prot 5634ndash41
56 Norwood D E and A Gilmour 2000 The growth and resistance to
sodium hypochlorite of Listeria monocytogenes in a steady-state
multispecies biofilm J Appl Microbiol 88512ndash520
57 Oliveira M I Vinas P Colas M Anguera J Usall and M
Abadias 2014 Effectiveness of a bacteriophage in reducing Listeria
monocytogenes on fresh-cut fruits and fruit juices Food Microbiol
38137ndash142
58 Ronner A B and A C L Wong 1993 Biofilm development and
sanitizer inactivation of Listeria monocytogenes and Salmonella
typhimurium on stainless steel and buna-n rubber J Food Prot 56
750ndash758
59 Sallam S S and C W Donnelly 1992 Destruction injury and
repair of Listeria species exposed to sanitizing compounds J Food
Prot 55771ndash1033
60 Sillankorva S D R Oliveira M J Vieira I W Sutherland and J
Azeredo 2004 Bacteriophage V S1 infection of Pseudomonas
fluorescens planktonic cells versus biofilms Biofouling 20133ndash138
61 Silva S P Teixeira R Olieveira and J Azeredo 2008 Adhesion to
and viability of Listeria monocytogenes on food contact surfaces J
Food Prot 711379ndash1385
62 Skurnik M and E Strauch 2006 Phage therapy facts and fiction
Int J Food Microbiol 2965ndash14 (Review)
J Food Prot Vol 77 No 12 EFFECT OF DISINFECTANTS AND BACTERIOPHAGES ON L MONOCYTOGENES 2019
63 Somers E B J L Schoeni and A C L Wong 1994 Effect of
trisodium phosphate on biofilm and planktonic cells of Campylo-
bacter jejuni Escherichia coli O157 H7 Listeria monocytogenes
and Salmonella typhimurium Int J Food Microbiol 22269ndash
276
64 Somers E B and A C L Wong 2004 Efficacy of two cleaning
and sanitizing combination on Listeria monocytogenes biofilm
formed at low temperature on a variety of materials in the presence
of ready-to-eat meat residue J Food Prot 672218ndash2229
65 Soni K A and R Nannapaneni 2010 Removal of Listeria
monocytogenes biofilms with bacteriophages P100 J Food Prot 73
1519ndash1524
66 Sutherland I W K A Hughes L C Skillman and K Tait 2004
The interaction of phage and biofilm FEMS Microbiol Lett 2321ndash6
(MiniReview)
67 Swaminathan B 2001 Listeria monocytogenes p 383ndash409 In L R
Beuchat M P Doyle and T J Montville (ed) Food microbiology
fundamentals and frontiers 2nd ed ASM Press Washington DC
68 Thevenot D A Dernburg and C Vernozy-Rozand 2006 An
updated review of Listeria monocytogenes in the pork meat industry
and its products J Appl Microbiol 1017ndash17
69 Tuncan E U 1993 Effect of cold temperature on germicidal efficacy
of quaternary ammonium compound iodophor and chlorine on
Listeria J Food Prot 531029ndash1033
2020 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12
63 Somers E B J L Schoeni and A C L Wong 1994 Effect of
trisodium phosphate on biofilm and planktonic cells of Campylo-
bacter jejuni Escherichia coli O157 H7 Listeria monocytogenes
and Salmonella typhimurium Int J Food Microbiol 22269ndash
276
64 Somers E B and A C L Wong 2004 Efficacy of two cleaning
and sanitizing combination on Listeria monocytogenes biofilm
formed at low temperature on a variety of materials in the presence
of ready-to-eat meat residue J Food Prot 672218ndash2229
65 Soni K A and R Nannapaneni 2010 Removal of Listeria
monocytogenes biofilms with bacteriophages P100 J Food Prot 73
1519ndash1524
66 Sutherland I W K A Hughes L C Skillman and K Tait 2004
The interaction of phage and biofilm FEMS Microbiol Lett 2321ndash6
(MiniReview)
67 Swaminathan B 2001 Listeria monocytogenes p 383ndash409 In L R
Beuchat M P Doyle and T J Montville (ed) Food microbiology
fundamentals and frontiers 2nd ed ASM Press Washington DC
68 Thevenot D A Dernburg and C Vernozy-Rozand 2006 An
updated review of Listeria monocytogenes in the pork meat industry
and its products J Appl Microbiol 1017ndash17
69 Tuncan E U 1993 Effect of cold temperature on germicidal efficacy
of quaternary ammonium compound iodophor and chlorine on
Listeria J Food Prot 531029ndash1033
2020 CHAITIEMWONG ET AL J Food Prot Vol 77 No 12