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THE USE OF BACTERIOPHAGE IN
DETECTING FOODBORNE BACTERIAL PATHOGENS
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University o f Guelph
by
STACY JANE FAVRIN
In partial fdfihent of requirements
for the degree
Master of Science
May, 1998
OStacy Jane Favrin, 1998
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THE USE OF BACTERIOPHAGE IN DETECTING FOODBORNE BACTERIAL PATHOGENS
S t acy Jane Favrin University of Guelph, 1998
Advisor: Dr. M.W. GrifEths
Salmonella is the second leading cause of foodbome illness in most developed counties
causing diarrhea, crampg vomiting and often fever. Poultry, eggs and rnilk are fiequently
implicated as vehicles of infection. Simple, rapid, and specific methods are required for the
detection of Salmonella in foods. A bacteriophage assay combining the use of
irnmunornagnetic separation WS) with fluorescence or absorbance measurements, was
developed for the detection of Salmonella Enteritidis. The sensitivity of the assay in pure
suspensions, skimmed milk powder, ground beef and liquid whole egg was determined. The
MS-bactenophage assay was able to detect 3 CFU/g or ml present initidy in each of these
food samples. The assay was generdy speciflc for serogroup D Sulmonel(a; with Salmonellu
Typhimurium also testing positive. The IMS-bacteriophage assay was adapted to the
detection of E- col2 0157:H7. The assay was able to detect 2.5 CFU/g E. coli 01 57:H7
present initially in ground beef.
Thanks to God, for the perspective that lmowing Him provides, and for His strength and faithfidness which helped me to persevere. This work has been completed for His glory.
1 would Like to express my sincere thanks to my advisory cornmittee. Special thanks to my advisor, Dr. Mansel Gritnths, for his support, guidance and great suggestions. Thanks to Dr. Heidi Schraft for her support and the opporhinity to be a teaching assistant in Food Microbiology 42-323. It was a great learning experience, and a highlight of my program. The basic concepts of this study were introduced to me by Dr. Sabah Jassim and for that, and his help and guidance early in rny program, 1 am grateful.
Thanks to Dr. Joseph Odurneru and Dr. Massimo Marcone for being a part of my examination committee.
Thanks to Dr. Ted Heying and GEM Biomedical for fùnding this project, and for the opportunity to be involved in such exciting and relevant research.
Special thanks to my husband, Steve, whose patience and support, especially at the end, wiil always be appreciated. Thanks also to my parents for their interest and support throughout my Master's studies.
1 am also grateful to Jinni, Larry, Doug and Thomas, for the good laughs and the help they each have provided me in reaching this goal.
TABLE OF CONTENTS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Salmonella Z . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1 Background: 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.2. Classification: 1
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Salmonellosis 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1. Clinical signs: 2
1.2.2. Infective dose: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 .2.3. Incidence and costs: 4
. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.4. Trends in Salmonella infection: 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.5. Sources of infection: 7
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 .3 Ecology and control: 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Detection of Salmonella 16
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 .4.1. Conventional methods: 16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 .4.2. Rapid methods: 17
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 .4.3. Immunomagnetic separation: 23 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.Bacteriophage 24
. . . . . . . . . . . . . . . . . . . . . . . . . 1 5 1 . Lytic and temperate bacteriophage: 24 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 S.2. Phage-typing: 24
. . . . . . . . . . . . . . . . . . . . 1 .5.3. Bacteriophage based detection methods: 26 . . . . . . . . . . . . . . . . . . 1.5.3.1. Luminescence and reporter genes: 26 . . . . . . . . . . . . . . . . . . 1 5 3 .2 . Fluorescent bacteriop hage assay: 28
. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.3.3. Metabolic inhibition: 28 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.3.4. BIND assay: 29
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6. Fluorescence 29 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6.1. Principles: 29
1.6.2. Fluorescence as a means of determining ce11 viability: . . . . . . . . . 30 . . . . . . . . . . . . . . . . . . . 1.6.3. Direct Epifluorescence Filter Technique: 32
1.7. Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2 . Development and characterization of a bacteriophage assay for the detection of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Salmonella Enteritidis 35
2- 1 . Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Introduction 35
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Materials and methods 38 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Bacterial strains: 38
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2.Bacteriophageandhost.. 41
LIST OF FIGURES
Figure 1.1.
Figure 1.2.
Figure 1 -3.
Figure 1.4.
Figure 2.1.
Figure 2.2.
Figure 2.3.
Figure 2.4.
Figure 2.5.
Figure 2.6.
Figure 2.7.
Figure 2.8.
Figure 2.9.
Figure 2.10.
Figure 2.1 1.
Number of human cases of illness fiom total Salmonella, S. Typhimurium, and S. Enteritidis reported to the National Laboratory for Bacteriology and Enteric Pathogens fiom 1985 to 1995. . . . . . . . . . . . . . . . . . . . . . . . . . . . - 5
Infection cycle of lytic bacteriophage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Biochemistry of the bioluminescence reaction . . . . . . . . . . . . . . . . . . . . . 26
Energy level schematic diagram illustrating energy changes involved in absorption and fluorescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1
Overview of the IMS-bacteriophage protoco t for the detection of Salmonella Enteritidis in enriched samples. . . . . . . . . . . . . . . . . . . . . . . . 47
Details of the IMS-bacteriophage protocol for the detection of Salmonella Enteritidis, . . . . . . . . . . . . . , , , , , . . . . . . . . . . - . . . . . . . . . . . . . . . . . . . - 48
Transmission electron micrograph of bacteriophage SJ2. . . . . . . . . . . . . 58
Scanning electron micrograph of bacteriophage SJ2 attached to S. Enteritidis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Resulu of one-step gowth experiment for phage SJ2. . . . . . . . . . . . . . . . 60
Average clump counts following rnicroscopy protocol for four initial populations of S. Enteritidis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Relationship between plate count and DEFT count for different dilutions (1 o-'~ IO5, 1 O', 10J) of S. Ententidis in buffer. . . . . . . . . . . . . . . . . . . . . 63
Epifluorescent micrograph of S. Enteritidis stained with Molecular Probes LIVE/DEAD@ BacLight" bacterial viability stain. . . . . . . . . . . . . . . . . . . 64
Percent of negative control value for five populations of S. Enteritidis in broth following IMS-bactenophage assay . . . . . i . . . . . . . . . . . . . . . . . . . 66
Sensitivity of the Pharmacia spectrophotometer as indicated by mean optical density values for various populations of S. Enteritidis. . . . . . . . 68
Sensitivity of the MGM Fluorometer as indicated by mean fluorescence values for variou populations of S. Ententidis. . . . . . . . . . . . . . . . . . . . . 68
Figure 2.12.
Figure 2.13.
Figure 2.14.
Figure 2.15
Figure 3.1.
Figure 3.2.
Figure 3.3.
Figure 3.4.
Figure 4.1
Figure 4.2
Relationship between plate count, and plate count following IMS for different dilutions of S. ~nteritidis in buffer. . . . . . . . . . . . . . . . . . . . . . . 69
Scanning electron rnicrographs of S. Enteritidis attached to magnetic beads. . . . . . . . . . . . .- . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Cornparison of L-broth, lambda buffer and three dextrose concentrations (0.2%, 0.5% and 1 .O%) for the reduction of S. Enteritidis by phage SJ2 at 37OC . . . . . . . . . . . , . . . . . . . . . . . . . . - ~ ~ . . . . . - . . * . ~ . . . . . . . . 73
Cornparison of L-broth, lambda buffer and three dextrose concentrations (0.2%, 0.5% and 2.0%) as media for growth of S. Enteritidis at 37°C. . . 73
Percent of negative control value for skimmed milk powder samples inoculated with S. Enteritidis at four levels (0, loO, 1 ol,andl O' CFUig). . 93
Percent of negative control value for ground chicken samples inoculated with S. Enteritidis at four levels (0, 10°, 1 ol,andl 0' CFU/g). . . . . . . . . . . 93
Percent of negative control value for ground chicken samples inoculated with S. Enteritidis at four levels (0, loO, 10',and 10' CFU/g). . . . . . . . . . . 97
Percent of negative control value for liquid whole egg samples inoculated with S. Enteritidis at four levels (0, 1 0°, 10',and 10' CFU/ml). . . . . . . . . . 97
Percent of negative control value for four populations of E. coli 0 157:H7 following IMS protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 13
Percent of negative control value for ground beef samples inoculated with E. coli0157:H7atthreelevels(O, loO, 10'CFU/g). . . . . . . . . . . . . . . . 113
LIST OF T B L E S
Table 1.1.
Table 1.2.
TabIe 1.3.
Table 2.1.
Table 2.2.
Table 2.3.
Tabfe 2.4.
Table 2.5.
TabIe 2.6.
Table 2.7.
Table 2.8.
TabIe 3.1.
Prevalence of Salmonella in foods of animal orïgin. produce and h i ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Cornparison of several existing tests for Salmonella spp . . . . . . . . . . . . . 20
Cornparison of five methods for recovery of Sa/monella fiom processed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . raw broiler carcasses 22
Non- Enteritidis SaZrnonella strains used for the specificity study . . . . . . 39
Salmonella Enteritidis strains used for the specificity study . . . . . . . . . . . 40
Non-Salmonella strains used for the specificity study ................ 40
Schedule for one-step growth experirnent- . . . . . . . . . . . . . . . . . . . . . . . . 44
Schedule for plating growth tubes (GT- 1 and GT.2) . . . . . . . . . . . . . . . . . 44
Bacteriophage assay and plaque assay specificity results for non- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Salmonella strains 76
Bacteriophage assay and plaque assay specificity results for Salmonella Enteritidis strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Bacteriophage assay and plaque assay spec i f ic i~ results for non- Salmonellastrains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Tally of Mse positive and false negative results obtained fiom skimmed rnilk powder. ground chicken and liquid whole egg samples when various
. . . . . . . . . numerical cut-offvalues are used to detemine a positive test 92
1. Introduction
1.1. Salmonella
1.1.1 Background:
Salmoneliae are small, Gram negative, non-spo~g rods that are widely distributed in
nature, with humans and animals being their p r i m q reservoirs. Salmonella was first
identined as a human pathogen in 1888 following a German outbreak caused by an
organism subsequently known as SaZrnonella Typhimurium (Tauxe, 199 1). Food
po isoning from Salmonella results when significant numbers of the appropriate strains are
ingested.
1.1.2, Classification:
The genus Salmonelh consists of around 2400 serotypes (Andrews, 1996). Antigenic
classification of Sahonellae is generally based on the Kaufham-White scheme and uses
somatic (O), capsular (Vi), and flagellar 0 antigens (Bergey and Holt, 1994). When
classification is made by this scheme, species and serovars are placed in serogroups
designated A, B, C, and D etc. according to similarities in content of one or more O
antigens. Narning of a Salmonella serovar is based on the place where it was first
isolated, for example S. London (Jay 1992).
For epidemiological purposes, Salmonella cm be divided into three groups (WHO, 1988):
1) Those that infect hurnans only, which include S. Typhi, Paratyphi 4 and S. Paratyphi
2) Host adapted serovars, some of which are human pathogens and may be conrracted
fi-om foods.
3) Unadapted serovars (no host preference). These cm be pathogenic for both humans
and animais, and include most foodborne serovars.
1 -2. Salmoneilosis
1 -2.1. Clinical signs:
With the exception of typhoid fever caused by S. Typhi, which is not normally foodbome,
there exists four main clinical manifestations of SaZmuneLIa infection (Andrews, 1996).
The most cornmon is gastroenteritis, characterized by diarrhea, cramps, vomiting and
often fever. Recovety generally occurs in two to three days. Second, the organisms
invade the bloodstrearn and settle in the liver, kidneys, gallbladder, heart and joints where
abscesses or other complications may occur. Third is a typhoid-like fever that is milder
and shorter in duration than the two to three week recovery from typhoid fever caused by
S. Typhi. Lastly there exists a carrier condition, whereby certain individuals harbour the
organism asymptomaticaily, and can spread the organism to others.
Large plasrnids have been demonstrated to be a prerequisite for the virulence and
invasiveness of various Salmonella serotypes, including S. Enteritidis (Helmuth et al.,
1993). Mouse studies indicate that virulence plasrnids enable strains to colonize and
multiply withîn animal organs (Gulig, 1990), and the fiequency of vinilence plasrnids is
almost 100% when strains are isolated from animal organs or human blood (Helmuth et
al., 1993). Although exceptions have been observed, virulence plasmids seem to play a
signïficant roIe in systemic infections of livestock and humans (Hehuth et al. 1993). It is
also thought that enterotoxins, cytotoxins, and lipopoiysaccharides are involved in the
pathogenesis of Salmonella. contributing to the local damage of the intestinal mucosa that
results in enteric syrnptoms (Suruki, 1994).
1.2.2. Infective dose:
Whether infection follows exposure to Salmonella depends on the numbers of organisms
ingested and the ability of those organisms to overcome local defences. Factors influencing
infective dose include differences in virulence among organisrns, variation in susceptibility
among hosts and conditions that might alter the number of organisms reaching the
intestine (Blaser and Newman, 1982). Early human feeding studies suggested that
salmonellosis occurred only after ingestion of large numbers of organisms but these
studies were found to be flawed for one or more reasons. Blaser and Newman (1982)
reviewed 11 outbreaks for which approximations of the infective dose were calcuiated. In
six of the 1 1 outbreaks the doses ingested were calculated to be <1 O3 organisms.
The low infective dose was confirmed more recently during a 1994 US outbreak of
Salmonella Enteritidis involkg contaminated ice cream. Using a three dilution Most
Probable Number procedure, the infective dose appeared to be no more than 28 cells
(Vought and Tatini, 1998).
1.2.3. Incidence and costs:
In 1995, a total of 6389 human cases of salmonellosis were reported in Canada through
provincial laboratones to the National Laboratory for Bacteriology and Enteric Pathogens
(NLBEP) (Health Canada, 1998). Typhirnunum and S. Ententidis were the most
frequent serortypes reported, with 1366 and 964 cases respectively. There were a
reported 244 hospital inpatient visits, 38 outpatient visits and ten deaths associated with
Salmoneh cases. Fifty two outbreaks were reported for 1995, most of which were
associated with comrnunity events, restaurants and nursing homes.
Over the past decade, the total number of Salmonella cases reported £tom the NLBEP, as
well as reported S. Typhimurium cases, has decreased (Figure 1.1). The number of annual
cases associated with S. Enteritidis has slightly increased over the same time penod. A
moderate seasonal trend was observed for all serotypes, with increased rates of
salmonellosis in the surnmertime mealth Canada, 1998). In cornpanson, there were
13,680 human cases of C m p y Z o b a c ~ infection and 1,493 human cases of pathogenic
1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 Year
Total
S. Ententidis
- - S. Typhimurium
Figure 1.1. Number of human cases of illness fiom total Suhonelltz, S. Typhimurium, and S. Ententidis reported in Canada to the National Laboratory for Bacteriology and Enteric Pathogens fiom 1985 to 1995 (Health Canada, 1998)
Escherichia coli infections reported for 1995 in the National Notifiable Diseases
databases (Heaith Canada, 1998).
The actual number of amual cases of salmoneilosis would greatly exceed the reported
numbers for several reasons including under-reporting and the large number of infected
individuals who do not seek medical attention. An estimated two million cases of
salmonellosis occur each year in the United States (USDA, 1998). It is estimated that the
average case of salmonellosis costs the US national economy $700 - $5000 (Mason and
Eebel, 1991). Total estimated costs of Salmonella infections in the United States are
about $ 4 billion annually (USDA, 1998).
1.2.4. Trends in Salmonella infection:
Tauxe (1 99 1) identifies four trends of Salmonella infection in the 1990s suggesting that
salmonellosis will present an ever increasing challenge to public health in the future. The
first is the increasing resistance of Salmonella to one or more antimicrobial agents.
Antimicrobial therapy may be compromised if the Salmonella is resistant to the agent of
choice. Infection can actually be promoted by such therapy as cornpeting flora, known to
have a protective effect against pathogen colonization, may be reduced. This reduction in
cornpetitive organisms may result in a smaller dose of Salmonella being infective.
The second trend is the exacerbation of salmonellosis symptoms in imrnuno-compromised
and HTV patients. S. Typhimurium, S. Dublin, and S. Enteritidis are particularly invasive
and can lead to bacteremia in such persons. Recurrent Salmonella bacteremia has been
included as an indicator disease of A l D S since 1987. Prophylactic use of antimicrobials
may fbrther increase nsk of salrnonellosis in this population.
The third trend is the association of S. Enteritidis with eggs. Investigations of S.
Enteritidis outbreaks have repeatedly shown that the most comrnon source is the grade A
sheIl egg, usuaily consumed raw or improperly cooked.
The fourth trend has been the occurrence of large and dispersed outbreaks linked to large-
scale food processing and widespread distribution. A massive outbreak in the United
States in 1985 was linked to contaminated pasteurized milk (Ryan et al., 1987). A strain
of S. Typhimurium, resistant to multiple antibiotics, was isolated from 17,000 people
during the course of the outbreak, with the estimated number of cases exceeding 180,000.
Such widespread outbreaks oAen require collaboration of public health authorities in many
jurîsdictions and even across national boundanes, illustrating the need for a global
approach to food safety.
1 -2.5. Sources of infection:
Sahonellae are ubiquitous and primarily reside in the intestinal tract of birds, reptiles,
farm animals and humans. Since salmonellae are intestinal organisms they can be
introduced into the environment through feces, poliuting water and foods. Transmission
to man is usudy food-borne firom eating undercooked rneat, milk or eggs, or by cross-
contamination to other foods which are eaten without further cooking. Salrnonellae grow
rapidly in foods at room temperature and cm survive refigeration and freezing but are
kdled by heat over 60°C.
Table 1.1. illustrates the results of various surveys in regards to the prevaience of
Salmonella in foods. Poultry is recognized as a major source of foodborne Salmonella.
The problem starts on the farm, where contaminated feed, water and the environment can
lead to Salmonella colonization of young chicks. Processing of poultry carcasses cm lead
to a decrease in the numbers of Salmonella on carcasses, but may also lead to an increase
in the total number of contarninated carcasses because of cross-contamination during
processing. The reported prevalence of salmonellae on poultry products varies nom 2 to
100%, with the median being about 30% positive (Bryan and Doyle, 1995).
Factors that contribute to outbreaks of poultry borne disease include irnproper cooling
(48%), foods prepared a day or more in advance of serving (34%), and inadequate
cooking (27%) @ryan, 1980). Cross-contamination occurred in 27% of the 26 poultry-
associated outbreaks investigated.
Eggs were identified in the early 1960s as the source of large outbreaks of salmone1Iosis.
As a result of egg related illnesses, it is recommended that pasteurized eggs be substituted
in recipes that require raw eggs (Morse et al.. 1994). Pasteurization of buik egg products
have become cornmon practice and the numbers of outbreaks have decreased but localized
Food Country Prevalence Reference
Melons Importedb 1 1 / 1440 (0.8%) Madden, 1992 SE = S. Enteritidis
Chicken
Unpasteurized egg
Unpasteurized egg (SEa)
SEa in eggs fiom infected flocks
Bulk tank raw milk
Pork
Lamb carcasses
Beef sarnples
Smoked fish
Japan
us us UK us
Ireland us US
Japan
Spain
Japan
US
Spain
(O, 16%) 26/292 (8.9%)
69/225 (3 1 %) 3/94 (3 -2%)
a
Tokumaru et al., 199 1
Ebel et al., 1993
EbeI et al., 1993
Humphrey et al., 199 1 Henzler et al., 1994
Rea et al., 1992 Rohrbach et al., 1992
Epling et al., 1993 Tokumani et al., 199 I
Sierra et al., 1995
Tokurnam et al., 199 1
Heinitz and Johnson, 1998
Garcia-Villanova Ruiz et al., 1987
Table 1.1 - Prevalence of Salmonella in foods of animal origin, produce and fniits
I
Imported into US fiom Central Amerka, Mexico and the Carribean
outbreaks have still been traced to the use of ungraded eggs. Eggs have been found to be
responsible for the majority of S. Enteritidis outbreaks for which a food vehicle has been
identified (Hogue et al., 1997; Gast and Beard, 1993). Eggs become contarninated
internally with S. Ententidis as a result of infection of the hens ovarïes and oviducts
(Stephenson et al., 1991). A survey by Bamhart et al. (199 1) found an overall mean of
26.6% of pooled ovary sarnples within infected flocks to be positive for Salmonella
Enteritidis, with a range of 0400%. Humphrey (1994) reported a prevalence of 0.6% of
S. Enteritidis in eggs originating nom naturally infected poultry laying flocks. Subsequent
growth of these bactena is govemed by temperature and length of storage and it appears
that high levels of contamination occur only when the yolk is invaded (Humphrey, 1994).
A cornparison of 1991 and 1995 survey results in the United States suggests there has
been no decline in S. Ententidis occurrence in the commercial egg industry between these
years .
Although Salmonella is most frequently associated with eggs and poultry, outbreaks of
Salmonella are not tirnited to these food sources, An outbreak of Salmonella Infantis
involving 500 confirmed cases in Denmark was traced to a single pig slaughterhouse
(Wegener and Baggesen, 1996). A ready to eat savoury snack produced in Israel was
responsible for an outbreak in that country as weil as numerous cases in England, Wales,
Canada and the United States (Killalea et al., 1996; Shohat et al., 1996). The municipal
water supply in a rural Missouri township was responsible for a 1993 S. Typhimurium
outbreak involving more than 650 persons with seven deaths (hgu lo et al., 1997).
Salmonella outbreaks have also been linked to goats' mik cheese (Desenclos et al., 1996),
pasteurized milk (Sun, 198 5), cured ham (Gonzilez-Hevia, 1 W6), and cheddar cheese
(D'Aoust et al., 1985; Bezanson et al., 1985). Most recently, a March 1998 outbreak in
Canada implicated cheddar cheese fiom V ~ ~ O U S prepared lunch snacks as a source of
Salmonella Ententidis infection. At the tirne of this report, over 500 children had become
il1 (Food Safety Net, 1998). In addition to foods of animal origin, outbreaks have been
linked to unpasteurized orange juice (Ekish, 1998), tomatoes (Hedberg et al., 1994), and
watermelon (Larson et al., 1979). arnong others. Fruits and vegetables may become
contaminated while growing in orchards and fields, during harvesting and post-harvest
handling, processing and distribution (Beauchat, 1996). According to D' Aoust (1994) the
prevalence of Salmonella in fiesh h i t s and vegetables remains quite low (O to 8%).
Between 1983 and 1987, the percentage of outbreaks of gastrointestinal illness caused by
fruits and vegetables in the US averaged 5.5% Wadden, 1992). This value includes al1
reported foodborne illness, not just outbreaks associated with Salmonella.
In some instances, outbreaks are the result of cross-contamination. A 1994 US outbreak
of Salmonella Enteritidis in ice cream was linked to tanker trucks which carried
unpasteurized liquid egg pnor to transporting ice cream premix (Hemessy et al., 1996)-
Ninety percent of party-goers at a catered party in Toronto, Canada became ill,
presumably following consumption of curry chicken and fish cutlets (PHERO, 1994). An
inspection of the catering premises revealed several serious violations, and cross-
contamination of ready to eat food with dirty utensils and food contact surfaces was
thought to be a contributing factor.
1 -3. Ecology and control:
It is evident that the control of foodborne illness due to infection with Salmonella is of
great national and world-wide importance. The current increase in Salmonella infection
associated with poultry suggests that reducing infection in, or contamination of poultry
could significantly decrease human illness (Sockett, 1995). The control of salmonellosis
requires action at d levels of the food chah which may be addressed by the application of
the Hazard Analysis Critical Control Point system (HACCP). Reliable testhg and
sensitive analytical methods are still an important part of the process.
Feed ingredients are recognized as being fiequent sources of Salmonelln, but the Iink
between S. Enteritidis infections in laying flocks and the consumption of contaminated
feed is not well established (Gast and Beard, 1993). Mice are highly susceptible to S.
Enteritidis infection and may serve as amplifiers by shedding large numbers of the
organism. Mice have been observed to persist through the cleaning and dissection
process in poultry houses and rnay be the main vehicle in the spread of S. Enteritidis fiom
house to house, and between flocks (Gast and Beard, 1993; Mason, 1994). Surface
water, insects, crops, pet animals, humans and other livestock contribute to the prevalence
of Salmonella in poultry (Noordhuizen and Frankena, 1994). The most important source
of contamination appears to be the resident Salmonella of the flock (Lahellec et al., 1986).
A Canada-wide survey of 295 randornly selected layer flocks found that environmental
(faecal and eggbelt) samples fiom 52.9% of the flocks were contaminated with
salmonellae (Poppe, 199 1).
Experimental infections of young chickens 16th SaZrnonelh Enteritidis leads to extensive
intestinal colonization which is sometimes followed by invasion of the interna1 organs
(Gast, 1994). Young chicks reared in disinfected production units were found to be
particularly susceptible to Salmonella colonization, as the development of protective
intestinal microflora was severely delayed. It was found, however, that adult-level
resistance could be conferred in young broilers when a bacterial preparation, given orally,
established an adult Iike intestinal ffora in these young birds (Hirn et al., 1992). This
concept later became known as the Numi concept or cornpetitive exclusion (CE).
Bacterial cornpetition in the gut for nutrients and surface receptor sites, as well as
chemical inhibition of pathogens (for exarnple by production of volatile fatty acids and
bacteriocins) are thought to be involved in the protective eEect of these cultures (Nurmi et
al., 1992; Schneitz et al., 1992).
Until recently, there has been only one cornmercially available CE product. ~roilact"
(Orion Corportaion, Farmos, Finland) is an undefined culture, usually administered in the
first drinking water. A recently developed product, preernptm (MS Biosciences Lnc.
Dundee, Illinois), is applied as a spray on newly hatched chicks.
Competitive exclusion has been used in Sweden since 1981 as part of their national
control program for SaZmoneZZa. A 1990 nationwide study in Sweden demonstrated that
less than 1% of broiler chickens were contaminated with Sahonelia after slaughter
(Wierup et al., 1992). Competitive exclusion is not seen as a panacea for the control of
Salmonella, but in conjunction with good hygiene and rearing practice it has been
demonstrated to have a profound effect on the incidence of Salmo~ella infection in treated
flocks.
Once the broilers leave the farm, adequate hygiene must be rnaintained to prevent cross-
contamination and proliferation dunng transport and processing. A study by Jones et al.
(1991), concluded that catching, loading and live haul procedures appear to be major
contributors to the contamination rates seen in ready-to-cook broilers. A significant factor
affecting the persistence of Salmonella throughout processing is that of attachment and
entrapment. Bacteria that become attached to the skin or muscle cannot be easily
removed by subsequent processing steps, and enteric bactena are sometirnes M y
attached to the skin before the birds arrive at the plant (Lillard, 1989; Bryan and Doyle,
1995).
Hygiene problems associated with poultry processing are similar to that of other meat
animals. There are certain features, however, that make microbial control with poultry
more dificult. First is the rapid rate of processing; up to 6000 birds per hour. Secondly,
the carcass remains whole during processing therefore it is difficult to remove the
intestines without breakage through a reiatively small hole in the abdomen (Mead, 1989).
The main problem in poultry processing is limiting the spread of surface and faecal
bacteria. In positive birds, Salmonella can be found fiom the crop to caeca, but most
frequently in the caecal content (Riemann, 1993). Cross-contamination can occur at any
point in the process. Scalding, plucking, evisceration, washing, and chilling are cntical
control points in the process (Mead, 1980). The number of Salmonel/a positive carcasses
can Vary at dflerent stages throughout processing. Percentage of positive carcasses pre-
evisceration, pre-CM, post-chi11 and post-automatic cut were 58%, 48%, 72% and 77%
respectively (James et al., 1992). The use of biocides in the washing and chilling water
have been investigated as a means of reducing the microbial Ioad on carcasses. Agents
such as chlorine, hydrogen peroxide and lactic acid have been demonstrated to reduce the
prevalence of Salmonella on carcasses, but are often associated with adverse quality
effects (Izat et al., 1989; Lillard, 1989). No processing step completely destroys
Salmonella and no definitive solution to poultry carcass contamination is available unless
the flocks are Salmonella fkee (Lahellec et al., 1986).
A more direct approach would be decontamination of processed carcasses prior to retail
distribution. Irradiation is the only suit able treatment currently available which will
eliminate Salmonella fiom carcasses and retain the desired characteristics of raw poultry
(Simonsen et al.. 1987). A cost-benefit analysis of poultry irradiation (costs of
salmonellosis iess costs of irradiation) performed in England and Wales indicates that only
under the most severe assumptions regarding irradiation costs and effectiveness was the
net benefit negative (Sockett, 1995). The mode1 presented indicated that even for a
modest 25% reduction in human salmonellosis, the minimum benefits wouId be substantiai
(£261 million - £540 million over 15 years).
Epidemiological information can also be used to aid in prevention. If the contribution of
the major components to infection occurrence could be quantified, risk factors could be
ranked and preventative measures prioritized (Noordhuizen and Frankena, 1994). A
monitoring and surveillance system (MOSS) can provide accurate and reliable information
about infection and disease occurrence over time and thus provide a means of predicting
infection and evaluating the effectiveness of control measures. The sensitivity and
specîfïcity of diagnostic tests applied would be of great importance here.
A remaining option is to increase public awareness of food poisoning hazards by providing
relevant Somation and instruction regarding the proper handling and preparation of
foods. This may ensure the steps needed to make food safe could be applied more
fiequently and reliably both commercially and at home.
1.4. Detection of Salmonella
1.4.1. Conventional methods:
Detection of Salmonella by conventional methods consists of four phases. The first is a
non-seiective pre-enrichment which permits recovery of stressed organisms, and growth of
al1 organisms present. The second is a selective e ~ c h m e n t which allows for the survival
or growth of Salmonella while reducing the numbers of non-SaImonelZa in the media.
Next is isolation using selective agar to produce presumptive isolates. The last is a
confirmation step, often employing serological and biochemical tests to codirrn that the
isolate is Salmonella and to determine its serotype (Mansfield and Forsythe 1993; van der
Zee 1994). The total time for deiection is around 72 to 96 hours. Conventional methods
can provide a theoreticai sensitivity of one Salmonella cell per 25 g of food, however the
presence of cornpetitive flora can prevent detection. van der Zee (1994) notes that fdse
negative results may be obtained when non-salmonellae outgrow salmoneilae in pre-
e~chment , seiective enrichment or plating agars.
Regarding the specinc isolation of S. Enteritidis, conventional methods are scarce. This is
mainiy attributed to the minor differences that exist between S. Enteritidis and other
serovars. Such differences are not detectable by cultural methods (van der Zee 1994).
1.4.2. Rapid methods:
The tirne required for Salmonella detection by conventional methods can cause prolonged
and expensive storage of foods prior to distribution. Ideaiiy, the goal is to detect smdl
numbers of target bacteria in the presence of large numbers of non-target cells, after they
have been injured by manufacturing processes or disinfectants, rapidly enough to prevent
the manufacture or sale of contaminated product (Wolber and Green, 1990). The genus
Salmonella is a 'zero tolerance' contaminant and detection methods should be able to
detect one viable bacterium in 25g of food. A need exists for methods that provide results
quicker and with equal or greater sensitivity than conventional methods. Such rapid
methods should be robust, reliable, cost-effective and specific, minimizing false positive
results (Blackburn, 1993). Rapid methods could also facilitate routine monitoring (Mossel
et al., 1994).
Shortening non-selective or selective ennchment times from 16- 18 hours to 6-8 hours for
recovery of Salmonella ftom foodstuffs has been considered as a means of simplifving
methodology for tirnely identification of contarninated foods (D'Aoust et al., 1992). In
some cases this strategy was found to be as reliable as standard methods, but in others it
has led to a high number of false negative resuks (Allen et al., 1991; D'Aoust et ai., 1992;
Patel and Williams, 1994). Results of a 16 to 24 hour selective enrichment were found to
consistently exceed that obtained with 6 hour selective enrichment cultures for recovery of
foodbome SalmoneZZa (D' Aoust et al., 1992). Because resuscitation of injured celis does
not occur in selective media, the benefit of direct selective enrichment is also questionable
(Andrews, 1 986).
New media and modifications of existing media have improved recovery of Salmonella.
In a three-way study selenite cystine broth, Tetrathionate broth and Rappaport-Vassiliadis
(FW) medium were evaluated for the recovery of Salmonella from highly contaminated
foods (June et al., 1996). RV medium was found to be supenor for al1 food types tested.
SaZrnonella can be also isolated by their motility in a semi-solid version of RV medium.
In regards to rapidity, Mossel et al. (1994) noted three traits of microorganisrns that
hinder attempts to speed up detection methods. First is the sublethally stressed state of
microorganisms which may be an inevitable consequence of production and distribution
chains. This forces at least a short penod of enrichment for recovery. Secondly, target
organisms are often present in very low numbers. Lastly, the organism itself, rather than a
signal indicating its presence, is often required as proof in commercial or legal disputes.
This necessitates some confinnatory procedures.
Table 1.2. outhes some existing rapid methods for the detection of SuZmonelZu in food.
Enzyme-linked immunosorbent assays (ELISA) tests typically have a sensitivity of 1 o4 to
106 CFW/ml and often have very good agreement with conventional cuitUral methods
(Patel and Wiuiams, 1994), but they of€en suffer £?om a lack of specificity (Blackburn,
1993). Manual ELISA'S often require a high level of technical ski1 but automated
systems are available. A study by Juune et al. (1992) compared two AOAC-approved
enzyme irnmunoassays, S almonella-TekTM and ReportTM, wit h the standard culture
method of the AOAC and the Food and Dmg Administrations Bacteriological Analytical
Manual @AM), for the recovery of Salmonella spp. fiom four low-moisture foods. Of
the 300 inoculated food samples, 199 were contirrned positive by Salmonella-TekN, 193
by R e p o r P , and 206 by the AOACDAM method. When preenrichments were
inoculated after incubation, the lowest concentration identified by Salmonella-TekTM was
2.0~10' ceils per ml, and 2.0 x108 for Reportm. The TECRA LNnue,capture ELISA
uses an antibody coated dipstick to recover Salmonella fiom the food pree~chment.
This is followed by a five hour e ~ c h r n e n t to increase the number of Salmonella in the
sample. Cornparison of the TECRA ELISA with the FDA culture method indicated close
agreement over 176 tests (Flint and
CFU/ml of S. Typhimunum after the
Hartley, 1993). These authors detennined that 30
16 h preenrichrnent was sufficient for a positive test.
The TECRA procedure incorportates an additional 5 h incubation followhg
preenrichrnent.
'abIe 1.2: Cornparison of several existing tests for Salmonella spp (Wolber and Green, 1990).
Test Manufacturer Assay Method Approximate Time cost
(hours) (US$/test)
Gene-Trak SalmonelIa
Salmonella-Tek
Salmonella 1-2
Tecra Salmonella
Q-Trol Salmonel f a
Oxoid Salmonella Ra~id Test
M a y
Gene-Trak
Organon Teknika
BioControl Systems
Dynatech La bs
Oxoid
Standard Microbiology 2.00
DNA probe; 8.75 Radioisotope or colorimetric detection
ELISA: Co lorimetric 5.00 detection
Immunodifision and 20.00 irnmobilization; fluorescence
ELISA; 6.25 Colorimetric detection
Enzyme immunoassay; 3 -00 Fluorescence
Standard Microbiology; 6.00 Latex agglutination
" BAM-AOAC: based on the US Food and Drug Administration Bacterial AnaZysis Man& and approved by the Association of Officia1 Analytical Chemists
Latex agglutination tests are usually used to confirm suspect colonies on solid media and
require high levels (10' to lo9 CFU) to give a positive agglutination reaction (Patel and
Williams, 1994). They are very rapid, being perfomed in about 3 minutes. Feng (1992)
notes that the sensitivity and specifkity of the antibodies used to prepare the latex reagents
needs improvement.
Nucleic acid-based tests can be highly specific, but are often labour intensive and require a
skilled technician to perform the assays. Commercial test kits such as the GENE-TRAK@
DNA hybndization test are applied after 48 hour incubation and Save approximately 18-24
hours relative to conventional methods (PateI and Williams, 1994). An evaluation of
GENE-TRAKB with 600 artificially contaminated, and 404 naturally contaminated food
samples showed excellent agreement with the BAMIAOAC culture method (Wilson et al,
1990).
Polymerase chah reaction (PCR) is a technique whereby a small amount of target DNA is
amplified, giving a theoretical detection lirnit of one molecule of target DNA
(Swaminathan and Feng, 1994). PCR is highly sensitive but generally requires a pure
culture and considerable laboratory tirne and expertise (Yu and Stopa, 1996), thus
offsetting PCR's high sensitivity advantage. PCR also has a disadvantage in that it cannot
dserentiate viable and non-viable bacteria (Mossel et al., 1994).
Rapid biochemical profile systems, such as Analytical Profile Index (AH) strips
(Biomerieu, St. Laurent, Quebec) are available but require isolated colonies on solid
media. These tests generdy provide information to the genus level for Salmonella.
TabIe 1.3 outlines the results of a study by Bailey et al. (1991) comparing confirmed
culture with enzyme immunoassay (Salmonella-Tekm), DNA Hybndkation (GENE-
TRAK"), antibody immobilization (1-2 Testm) and the Food Safety Inspection Service
(FSIS) culture rnethod for the recovery of Salmonella fiom naturally contaminated
processed broiler carcasses.
Table 1.3. Cornparison of five rnethods for recovery of salmonellae fiorn processed raw
Test Positive (%) False + (Yo) False - (%) - - -- - - - - -- - -
Confirrned culture 71
FSIS Culture 65.1
Salmonella-Tekm 75.6
GENE-TRAP 71.3
1-2 Test- 66- 1
broiler carcasses @ d e y et al., 199 1) r -
-
Table 1.3. illustrates the difficulty in comparing methods. Feng (1992) noted that the
complexity of food matrices make cornparisons between test kits difficult. Test sensitivity
and the incidence of false positive and negative results are infiuenced by the food types
analysed. Feng reviewed various comparative studies of commercial assay kits for
Salmonella detection and found that some rapid rnethods performed better than other
methods in some instances but not in others. This highlights the importance of choosing a
rnethod appropriate to the application, and validating that method. He noted that despite
22
the advantages and limitations of each method, most assays exarnined were found to be
rapid, simple and at least as sensitive as conventional methods.
1.4.3. Imrnunomagnetic separation:
Irnrnunomagnetic separation (IMS) has been used as an alternative to selective enrichment
broths for a variety of bacteria including Salmonella. Paramagnetic beads are coated with
polycional antibodies which can target and separate Salmonella fiorn a rnixed suspension
without loss of viability, producing a normal isolate for further confirmation. IMS can
eliminate the need for selective enrichment and reduce the time required for conventional
methods by one day. Mansfield and Forsythe (1993) compared IMS as an enrichment
method with approved selective enrichment protocols for Sulmorrella using selenite
cystine, Rappaport-Vassiliadis and Muller-Kaufham tetrathionate broths. The results of
the 120 food samples tested indicated that Dynabeadsa gave comparable results after a 10
minute capture compared with 24 and 48 hour exuichrnent periods with the approved
media. The authors concluded that MS could be a reliable alternative to standard
methods for Salmonella screening. A collaborative ring-trial compared the use of IMS
with conventional enrichment broths for the recovery of stressed Salmonella fiom herbs
and spices (Mansfield and Forsythe, 1996). IMS was found to be a suitable alternative to
conventional enrichment and reduced the time required for detection by 24 hours.
IMS has also been used in conjunction with other rapid detection methods. Dziadkowiec
et al. (1995) found that Salmonella was successfùlly isolated and enumerated in skimmed
milk powder by IMS and indirect conductance despite the 1000-fold greater number of
non-target cells. IMS has been successfuiiy used in conjunction with ELISA'S,
conductance rnicrobiology, electrochemilumlnescence, and PCR (Cudjoe et al., 1995; Holt
et al., 1995; Parmar et al., 1992; Yu and Stopa, 1996; FIuit et ai, 1993).
1 S. Bacteriophage
1 S. 1. Lytic and temperate bactenophage:
Bacteriophage can generally be divided into two categories, temperate and Iytic. Infection
by lytic phage, such as Felix-O1 of Salmonella spp., always results in the imrnediate
production of progeny phage. The infection cycle of lytic phages is illustrated in Figure
1.2. The total infection cycle can take as little as 25 minutes with the number of progeny
phage being highly variable. Temperate phages, such as the P22 phage of Salmonella
Typhimurium have two possible outcornes (Poteete, 1994). It can grow lytically, as
above, or form a lysogen. In the latter case, instead of unrestrained DNA replication and
phage assembly, a stable relationship is established with the host ceii which is maintained
over many generations. In this temperate state, the viral DNA replicates at the same rate
as host ce11 DNA and is distributed to daughter celis at each ce11 division (Birge, 198 1).
Phage DNA Phage head Mature phage proteins Tailandfibre particie
proteins
l nfection Phage DNA Repl. Setf assembly
Eariy mRNA Late rnRNA
!
v Early proteins l Late proteins Lysis
Time (minutes)
Figure 1.2 Infection cycle of lytic bactenophage. During infection, the viral DNA is injected into the host cell. The early proteins produced are enzymes necessary for the replication of the viral nucleic acid. Late proteins include proteins required for the virus coat.
Bactenophages have been used in the identification and classification of bacteria since
1925. The specificity of bacteriophage for their hosts have made them ideal agents for
what has been cailed 'phage-typing'. Phage-typing schemes have significance in medicine,
epidemiological studies, as well as industrial fermentations and cheese formation (DuBow,
1994).
1.5.3. Bacteriophage based detection rnethods:
1 -5 -3.1. Luminescence and reporter genes:
Many marine organisms, including Vibrio and Photobacteritrm, are biolurninescent, that is
they are capable of emitting light. The light reaction requires oxygen, a source of energy,
a luciferase enzyme and a long chain fatty aldehyde. The reaction is illustrated in Figure
1.3.
E;MN-H2 + O, + RCOH -+ luFife- + FMN + RCOOH + H,O + Light
Figure 1.3 Biochemistry of the bioluminescence reaction.
Bacterial luciferase is encoded by the lm A and lzrx B genes. In bacterial cells, detection
based on bioluminescence has the advantage of being non-invasive, non-destnictive and
can offer high sensitivity in real-time analysis (Stewart and Williams, 1992).
Bacterial luciferase has been used in reporter gene expression, particularly for Gram
negative organisms whose light output is typicafly one hundred-fold that of Gram positive
organisms. It has also been used as a reporter of cellular viability. Since light output
would be dependent on a functional intracellular biochemistry, it was reasoned that
compromised cells with impaired biochemical activity would elicit a reduction in light
emission (Stewart and Williams, 1992). This may have application in the field of
predictive Mcrobiology, where light output in geneticdy engineered bioluminescent
bacteria can be monitored as cells are exposed to diffierent stresses (Baker et al., 1992).
Chen and Griffiths (1996a) successfùliy used luminescent SaZmoneZZu as real tirne
reporters of growth and recovery from sublethal injury in foods.
In addition to these applications, bacterial luciferase has been used as a reporter for
bacterial detection and enurneration. In this case, lux genes are introduced into the
genome of bactenophage. Bactenophage lack the intracellular mechanisms necessary for
light production and thus remain dark. Upon host infection the phage genes, including the
additional lux genes, are expressed and within an hour of infection the host bacteria are
bioluminescent (Stewart, 1990). This method exploits bacteno phage-host specificity.
Recombinant lux+ bactenophage can detect target bacteria in a food matrix without
enrichment provided they are present at levels greater than 103 CF'U/ml (Stewart, 1990).
Kodikara et al. (1991) has used ZZJ& recombinant phage for near on-line detection of
enteric indicator organisms. If the target is present at levels greater than IO" C N per g or
cmZ, it can be detected without enrichment in less than an hour. A 4 h o u enrichment was
found to be suscient for detection when counts were 10 CFU per g or cm2. Chen and
Griffiths (1996b) used lm+ recombinant bacteriophage for the detection of Salmorrelia in
eggs. Recombinant transducing phage were introduced into artificially inoculated eggs.
Infection of target Salmonella by the phage resulted in luminescence, which could be
detected through the egg shell using a BIQ Bioview Image Quantifier. A 6 hour
preenrichrnent was sufficient for the detection of as few as 10 Salmonella cells per ml
present in the original sample
An advantage of these luminescent applications is the sensitivity of instrumentation
availabte to detect and quant* light output. A detection limit of less than 10
biolurninescent bacteria per millilitre of sample has been reported (Stewart and Williams,
1992), with inexpensive luminorneters detecting as few as 102 luminescent bacteria per ml
(Stewart, 1990). A limitation of these methods is the genetic manipulation required to
prepare the bacteriophage for the test.
1 -5.3 -2. Fluorescent bacteriophage assay:
In addition to using genes as reporter molecules in phage, Goodndge (1997) developed a
fluorescent bactenophage assay based on a method described by Hennes et al. (1995),
whei-e the genetic material of phage LGl of Escherichia coli O1 57:H7 was labelled with a
fluorescent probe. Attachent of the labelled phage to the surface of the target celk
could be visualized by epinuorescent rnicroscopy or altematively quantified by flow
cytornetry. The assay was successfûliy applied to artificially inoculated ground beef and
raw milk samples.
1.5 -3 -3. Metabolic inhibition:
Bacteriophage has been used in conjunction with turbidity and colonmetric rnethods as
well as conductance rnicrobiology (McIntyre, unpublished data). Listeria, Sahonella,
and E. coli were prepared for detection in semi-selective media according to their
respective protocols; with and without bacteriophage added. If the target organism was
present, the time for detection of sarnples containhg phage increased, if it occurred at d,
relative to the sarnples without phage. In this case, the lytic activity of bactenophage was
exploited as a means of confhming the target organism of interest was indeed responsible
for the positive test.
1.5.3.4. B W assay:
The only commercially available bacteriophage assay for Salmonella is the Bactenal Ice
Nucleation Diagnostic (BIND) test. The Salmonella-specific bacteriophage P22 has been
engineered with the bacterial gene responsible for an ice-nucleating protein. When
infected, the bactena produce ice crystals in supercooled water (Worthy, 1990).
Detection of fkeezing is aided by addition of a dye that fluoresces in supercooled water,
but is quenched and changes colour when the water freezes. The test can be completed in
2-6 hours and has a sensitivity of 10 CFU/rnl. No false positives were observed with non- . salmoneiiae and food material did not interfere with the assay (Wolber and Green, 1990).
1.6. Fluorescence
1.6.1. Principles:
Fluorescence has been used for decades in the field of microbiology, and more recently in
food microbiology. The term fluorescence refers to the light (luminescence) emitted when
a substance retums f?om an excited or higher energy state to its normal or lower energy
state. A fluorescent molecule is raised to a higher energy state by the absorption of
radiant energy such as W rays. Some of the absorbed energy is dissipated in collisions
with other molecules. When the molecule drops back to it's normal ground state, much of
the absorbed energy is emitted at a Iower energy (longer wavelength) than the absorbed
energy (White and Argauer, 1970). The change in energy states results in fluorescence
and is illustrated in Figure 1.4. Instruments for fluorescence analysis consist of two
essential units; a source of exciting energy, and a means of observing or measuring the
intensity of the fluorescence emission.
1.6.2. Fluorescence as a means of determining cell viability:
Fluorescence has been used as a means of determining ce11 viability. To this end, several
fluorescent stains have been investigated. Rhodamine 123, which stains viable cells based
on transmembrane potential, was a moderate predictor of ce11 viability (Matsuyama,
1984). Dead ceUs or cells with a dissipated transmembrane potential showed markedly
diminished fluorescence. Gram positive bacteria stained well with rhodamiie 123, but half
of the 14 Gram negative strains tested stained sparsely (Matsuyama, 1984). Kasprelants
and Kell (1992) concluded that flow cytometry using rhodamine 123 was an effective
method for the rapid assessrnent of bacterial viability. In another study rhodamine 123 and
carbocyanine dyes were found to exhibit only small changes in fluorescence between
GROUND
Figure 1.4. Energy level schematic diagram illusmting energy changes involved in absorption and fluorescence. A photon of energy hv,, is supplied by an extemal source such as an incandescent lamp or a laser and is absorbed by the fluorophore, creathg an excited singlet state (S,, S2, S3). The excited state exists for only a fraction of a second, during which time some of the energy is dissipated. The dotted lines indicate energy dissipation without producing fluorescence. A photon of light, hv,, is emitted, rehiming the fluorophore to its ground state, S,. Due to energy dissipation, the fluorescence ernission is at a longer (less energy) wavelength than the absorbed energy (Adapted fiom White and Argauer, 1970; Johnson, 1996).
viable and non-viable populations of bacteria using flow cytometry (Mason et al., 1995).
In the same study, calcafluor white and oxonol dye (bis 1,3-dibutylbarbituric acid
trimethine oxonol) were much more usefùl (Mason et al., 1995). Ethidium bromide, a
nucleic acid stain, was investigated to assess the viability of Pseudomonas after fkeeze
thawing (Puchkov and Melkozemov, 1995). Ethidium fluorescence increased with
decreased viability, as disruption of the ce11 led to interaction of the fiuorochrome with
intracellular nucleic acids. Ethidium bromide was determined to be a simple, low cost and
rapid means of comparative bacterial viability assessment. A novel viability stain has been
developed by Molecular Probes that involves two fluorescent cornponents, a live stain,
SYTO 9, and a dead stain, propidium iodide (Molecular Probes Product Information).
The live and dead staining is based on the permeability of the ce11 membrane. #en used
together, live cells fluoresce green and dead cells fluoresce red when excited by blue light.
1 -6.3. Direct Epifluorescence Filter Technique:
One of the earliest applications of fluorescence in food microbiology was the direct
epinuorescence filter technique (DEFT). It was onginally developed for counting bactena
in milk and was suitable for milk containing between 5x10~ to 5x10~ bacteria per mi
(Pettipher et al., 1980). DEFT has subsequently been used for a vanety of other
applications, including estimating bacterial counts on meat, poultry and food contact
surfaces (Shaw and Farr, 1989; Holah et al., 1988). Microorganisms are recovered kom
the sample by the use of membrane filtration, and stained with the fluorescent nucleic acid
stain, acridine orange. Cells are then counted by means of a fluorescent microscope.
Acridine orange has been used as a vital stain, based on the colour of fluorescence
reflecting the RNA to DNA ratio in the cell, but this has not always been proven reliable
(Pettipher, 1983).
In this present study, the advantages of IMS as an alternative to selective enrichment, the
speciscity of bacteriophage and the sirnplicity of the novel Molecular Probes fluorescent
viability stain were exploited in the development of a rapid assay for the detection of
Salmonella Enteritidis in food. The assay exploits the normal infection cycle of a lytic
bacteriophage, thus requiring no genetic manipulation. The endpoint of the assay is
evaluated by monitoring phage infection. When bacterial cells are lysed by phage, they
are no longer viable, and the amount of green fluorescence in the sample decreases. This
decrease in fluorescence can be quantified, either by microscopie count by a modified
DEFT count, or by fluorescence measurements of cells in suspension.
1.7. Objectives
The objectives of this study were to:
Develop a bacteriophage-based assay to detect Salmonella Enteritidis in a timely,
inexpensive and technically simple manner without the need for manipulation,
genetic or othenuise, of the biological species involved.
Determine the endpoint of the assay by simple, standard, and inexpensive
laboratory instrumentation.
Evaluate the specificity of the assay for SaZrnonelia Enteritidis, other Salmonella
serotypes as well as other bacteria.
Investigate the use of the assay for the detection of Salmonella Enteritidis in
skimmed milk powder, gound chicken, and liquid whole egg.
Investigate the use of the assay with a different phagehost systern, namely phage
LGl and host Escherichia coli 0 157:H7.
The assay would capitalize on the inherent specificity of bacteriophage for its target
bacterium, and be based on the normal infection cycle of bacteriophage in its host. As
such the assay should be easily applicable to other bacteriophage and host combinations.
In addition, the assay would detect only viable bacteria, leaving those target bactena
killed by disinfectants or other bacteriocidal activities undetected.
2. Development and characteruation of a bacteriophage assay for the detection of Salmonella Enteritidis.
2.1. Abstract
Salmonella is the second most common source of foodborne illness in Canada and the
United States, perhaps responsible for millions of cases of gastroenteritis a year. Children,
the elderly and the immunocompromised are particularly susceptible. Many rapid methods
are available for the detection of SnIrnoneIla in foods but are often insensitive, expensive
or require a high degree of technical ability to perform. This study describes the
development and characterization of a novel assay that utilizes the normal infection cycle
of bacteriophage SJ2 for the detection of Salmonella Enteritidis in broth. Initial
experiments with a modified direct epifluorescent filter technique (DEFT) demonstrated
that the bioiogy of the bacteriophage could be exploited for the detection of S. Enteritidis.
To make the assay more applicable to food samples, subsequent experiments were
performed using irnrnunomagnetic beads as a separation method, and fluorescence and
optical density as the endpoint. The lower detection limit in broth was calculated to about
104 CFU/ml. The results of this study demonstrate that the bacteriophage assay is a rapid,
simple and sensitive technique for the detection of Salmottella Enteritidis in broth culture.
2.2. Introduction
Salmonella was first identified as a human pathogen in 1888 following a German outbreak
caused by an organism subsequently known as Salmonella Tyhimurhm (Tauxe, 199 1).
The most cornmon clinical manifestation of salmonellosis is gastroententis, characterized
by diarrhea, crarnps, vomiting and often fever. Salmonellae are ubiquitous in the
environment and primarily reside in the intestinal tract of birds, reptiles, f m anirnals and
humans. Transmission to man is usually foodbome from eating undercooked meat, miik
or eggs, or by cross-contamination to other foods which are eaten without cooking.
Four trends in Salmonella infection have been identified in the 1990s suggesting
Salmonella will continue to be a challenge to public health (Tauxe, 1991). These trends
include increased antirnicrobial resistance of SuZmoneIZa to one or more rnicrobial agents,
the exacerbation of salmonellosis in irnmunocompromised individuals, the association of S.
Enteritidis with grade A shell eggs, and lastly the occurrence of large and dispersed
outbreaks linked to large-scale production and distribution of foods. A multiple-antibiotic
resistant strain of S. Typhimurium found in pasteurized milk was responsible for a large
outbreak in the U.S. involving an estimated 180,000 cases (Ryan et al. 1987).
SalmonelIu is most fiequently associated with eggs and poultry, but outbreaks have been
linked to other sources such as cheese, pork products, water, and citrus juices (D'Aoust et
al. 1985, Wegener and Baggersen, 1996, Angulo et al,, 1997, Parish, 1998). In many
cases, improper handling and preparation of food in homes and institutions are responsible
for salmonellosis outbreaks (Bryan, 1980).
Conventional cultural methods for the enurneration of SalmonelZu require 72 to 96 hours.
This cm cause prolonged and expensive storage of foods prior to distribution. Advances
have been made in shortening the time required for Salmonella detection in food.
Antibody-based tests, such as ELISA tests, have decreased detection times. Non-specific
binding of competing organisms can lead to a lack of specificity, however, and long
enrichment steps may be required to reach detection limits (Patel and Williams, 1994).
Nucleic acid-based tests, although they can be highly specific, are often labour intensive
and require a high degree of technical skill. As such they may not be suitable for routine
analysis of food samples.
Irnmunomagnetic separation uses paramagnetic beads coated with covalently bound
afitibodies against specific surface markers on the target rnicroorganism. With the aid of a
magnetic particle separator, target cells can be pulled out of a food pree~chment,
eliminating the need for selective enrichment and shortening assay time by as much as 24
hours (Mansfield and Forsythe, 1993).
In addition to these immunological and genetic applications, bacteriophage assays have
been developed, where reporter genes, such as the lux genes responsible for bacterial
luminesence, are introduced into the phage DNA, producing light when these genes are
expressed in the target cells. Stewart (1990) reports that target bactena present at levels
greater than IO3 per mi, can be detected by recombinant lm+ bacteriophage in a food
matrix without enrichment.
The BIND assay is the only comrnercially available bacteriophage assay for the detection
of Salmonella in food, and is based on the production of ice nuclei upon infection by
phage. (Worthy, 1990). A sensitivity of 10 CFU/ml have been reported, and food
material was found not to interfere with the assay (Wolber and Green, IWO). The major
limitation of these bacteriophage assays is the genetic manipulation required to produce
the detecting agent.
This study describes the development and characterization of a bacteriophage assay for the
rapid detection of Salmoizella Enteritidis in broth cultures. IMS was employed to separate
and concentrate target Salmonella. Additional assay specificity was inherent with the use
of bacteriophage. The end point of the assay was detected either by optical density at 600
nm or by fluorescence using Molecular Probes LIVE/DEAD' BacLight" bactenal viability
stain.
2.3. Materials and methods
2.3.1. Bacterial strains:
Forty-one strains were used for enurneration, recovery and specificity studies. Tables 2.1.
2.2 and 2.3 outline the 30 strains of non-Enteritidis Salmonella, 5 strains of Salmonella
Enteritidis and 6 strains of non-Salmonella bacteria used in this study. Cultures were
obtained from Health Canada, the Center for Disease Control and the University of
Guelph, Department of Food Science culture collection.
Stock cultures were maintained fiozen at -20°C in 15% glycerol. Fresh bacterial cultures
for use in experiments were produced by inoculation of frozen stock cultures ont0 L-agar
able 2.1. Non-Enteritidis SaZrnonella strains used for the specincity shidy.
Serogroup Bacterial Species Strain Ongin
Group B
Group C
S. Typhimurium
S. Typhimurium
S. Heidelberg
S. Saintpaul
S. Bredeney
S. Schwartzengmnd
S. Schwartzengnuid
S. Agona
S. Indiana
S. Brandenburg
S. Reading
S. Infântis
S. Thompson
S. Mbandaka
S. Braenderup
S. Montevideo
S. Ohio
S. Oranienburg
S. Tenenssee
S. Johannesburg
S. Urbana
S. Rubislaw
S. Hadar
S. Kentucb
Hedth Canada
LCDCa
Heaith Canada
Heaith Canada
Heaith Canada
LCDCa
Heal th Canada
Health Canada
Health Canada
Health Canada
Health Canada
Heaith Canada
Health Canada
Health Canada
Health Canada
Health Canada
Health Canada
Health Canada
Health Canada
Health Canada
Health Canada
Health Canada
Health Canada
Health Canada
Health Canada " Laboratory, Center for Disease Control.
39
Table 2.1. continued. Non-Enteriticlis SuZmonelh strains used for the specificity study.
Serogroup Bacterial Species Strain Ongin
Group C S. Haardt SA 9 14074 HeaIth Canada
I S. Choleraesuis S-870 LCDCa
1 Group D S. Berta SA 941 123 HeaIth Canada
I S. Panama SA 93 1592 Health Canada
S. Sendai S-5 13 LCDCa " Laboratory, Center for Disease Control.
Table 2.2 Bacteriophage assay and plaque assay specificity results for Salmonella Enteritidis strains.
Bacterial species S train Ongin
S. Enteritidis SA 932451 Health Canada
1 S. Enteritidis Laboratory strain Egg
1 S. Enteritidis ATCC 13076 ATCCa
1 S. Enteritidis SA 94245 1 Health Canada
S. Enteritidis EN 2588 LCDC~ I " Amencan Type Culture Collection
Laboratory, Center for Disease Control
Table 2.3. Bacteriophage assay and plaque assay specificity results for non-Salmonella strains.
Bacterial species S train Origin
Serratia marsescens ATCC 8 100 ATCCa
Klebsiella pneumoniae ATCC 1388 ATCC
Citrobacter fieundii Laboratory S train University of ~ u e l p h ~
Shigella flemeri Laboratory Stmh University of ~ u e l p h ~
Escherichia coli ATCC 25922 ATCC
Escherichia coli 015 7:H7 EC 920333 Health Canada " Amencan Type Culture Collection
Department of Food Science
plates (1% tryptone (Difco Laboratories, Detroit, MI), 0.5% sodium chloride @$CO),
0.5% yeast extract (Difco) and 1% agar (Difco)). Plates were incubated overnight at
37T. Broth cultures were prepared by inoculating L-broth with cells from an L-agar
plate and incubating them ovenllght, with shaking, at 37OC.
2.3 -2. Bacteriophage and host:
The bacteriophage and host used in this study, SJ2 and S. Enteritidis respectively, were
obtained £tom Dr. Sabah Jassim, Department of Food Science, University of Guelph.
Both the bacteriophage and the host had been isolated from egg. Phage SJ2 was amplified
in its host by the plate method. Ten-fold serial dilutions of undiluted phage stock (10''
PFU/ml) were prepared in lambda buffer (0.25% MgSOJHfl, 0.0005% gelatin, and 0.6%
1 M TRIS pH 7.2 in distilled water, adjusted to pH 7.2). From each dilution tube, 0.1 ml
of phage suspension was added in duplicate to 0.1 ml of an overnight culture of host.
Tubes containing phage and host were incubated for 10 minutes at 37°C to allow
attachment. Samples were then added to 2.5 ml of melted top layer agar (1% tryptone,
0.5% yeast extract, 0.5% NaCI, and 0.4% agar) cooled to 45°C. The mixture of agar,
phage and cells was poured quickly onto a Petri plate containing dried L-Agar and the
plate was swirled so that the mixture covered the entire plate. When the top layer had
hardened, plates were incubated overnight at 3 P C . Phage was recovered fiom the plates
by adding 8-10 ml of lambda buffer to a plate containing the highest dilution of phage.
Phage was harvested with a glass hockey stick and the resulting lysate (bufer, phage, host
cells, and media residue) was transferred to the next highest dilution. Plates were washed
sequentially, transferring lysate to sterile tubes and using ffesh buffer as required.
Following phage recovery, chloroform was added to the lysate tube at a 1:3
chloroform:lysate ratio. Lysate was put on ice for 10 minutes to facilitate recovery of
phage from unlysed cells. The lysate suspension was centrifuged at 5000x g for 15
minutes. The supernatant was withdrawn and filtered through 0.2 prn pore size syringe
filters (Nalgene, Rochester, New York).
Phage was enumerated by the plaque assay method. Ten-fold serial dilutions of phage
were prepared in lambda buffer. From appropriate dilution tubes, 0.1 ml of phage
suspension was added in duplicate to 0.1 ml of an ovemight culture of host S. Enteritidis.
Tubes containing phage and host were incubated for IO minutes at 37°C to ailow
attachment. SampIes were then added to 2.5 ml of melted top layer agar cooled to 4S°C.
The mixture of agar, phage and cells was poured quickly ont0 a Petri plate containing
dned L-Agar and the plate was swirled so that the mixture covered the entire p!ate. When
the top layer had hardened, plates were incubated overnight at 37OC. Plaques were
counted, and the phage titre in the original tube was calculated.
2.3.2.1- One-step growth experiment:
In order to charactenze phage SJ2, a one-step growth experiment was performed to
determine a) the 'burst tirne' (the period between adsorption and lysis) and b) the 'burst
size' (the average number of phage particles released per infected host cell). The method
used was adapted from Maramorosh and Koprowski (1967). Forty microlitres of an
overnight culture of S. Ententidis was inoculated into 40 ml of L-broth. The culture was
incubated at 37OC, shaking, for 2.5 hours. The tube was centrifuged at 5000x g for ten
minutes in a Sorvall RT6000 refngerated centrifuge (DuPont Inc., Mississauga, Ontario).
The pellet was resuspended in 2 ml of L-broth. The phage population used was 5x10'
PEZl/ml. Three tubes containing 10 ml of L-broth were labeled D L , GT-1, and GT-2
(dilution tube, first growth tube, and second growth tube).
The schedule of the growth experiment is outlined in Tables 2.4 and 2.5. Plating occurred
by adding 0.1 ml of sarnple from GT-1 or GT-2 to 2.5 ml of top layer agar containing 0.1
ml of host culture. Samples were immediately poured ont0 solidified L-agar plates and
incubated ovemight at 37OC. Following counting of plaques, the relative titre of each
plate was divided by the titre of plate 1 and ptotted linearly as a function of time. The
percentage of adsorption during the first four minutes was calculated by comparing the
count on plate 13 with the count on plate 1.
2.3.3. Procedure for analysis - Microscopy protocol:
Ten microlitres of bacteriophage SJ2 (2x10~ PFUI10 pl) was added to a microfuge tube
containing 100 pl of stationary phase culture of S. Enteritidis. Tubes were incubated for
15 minutes at 37OC to allow for phage at tachent and were then centrifùged at 14,000
rpm for 10 minutes to remove unattached phage. The pellet was resuspended in 100 pl
stationary phase host (approxùnately 2x10~ CFU/lOOpl) to act as a 'helper'. Samples
were incubated for 1 hour at 37°C. Dunng this incubation penod, progeny phage fiom
Time (minutes)
Table 2.4. Schedule for one-step growth experiment (adapted from Maramorosch and Koprowski, 1967).
O 0.1 ml phage was added to 0.9rnl of cells in Eppendorf tube, incubated at 3 7OC, shaking
4 0.1 ml fiom Eppendorf to DIL (1 : 100 dilution)
4.5 0.1 ml fiom DIL to GT-1 (1 : 10,000 dilution)
" DL, dilution tube; GT-1, first growth tube; GT-2, second growth tube.
5 2 ml sample fiom GT-1 was cenûifbged for 8 minutes. Supernatant was titrated for phage
7 0.1 ml fiom GT- 1 to GT-2 (1 : 1,000,000). Sarnples (O. 1 ml) are taken in sequence fiom GT-1 and GT-2 and plated for plaque forrning units according to the schedule in Table 2.5.
Tabte 2.5. Schedute for plating growth tubes (GT-I and GT-2) (adapted fiom Maramorosch and Koprowski, 1967).
Tirne" (minutes) Frorn GT-1 From GT-2
Plate I
-
Plate 3
-
Plate 5
-
Plate 7
-
Plate 9
-
Plate 1 1
-
-
PIate 2
-
Plate 4
-
Plate 6
-
Plate 8
-
Plate 10
-
Plate 12
a Note: Between minutes 12 and 20, the centrifuged supernatant sample was ready, and 0.1 ml was assayed on Plate 13.
the original cell population would be released and the helper population would be
influenced based on the number of target cells present initially. Following incubation, 900
pl of lambda buffer and 10 pl of Molecular Probes LIVE/DEAD@ Baclight- bacterial
viability stain solution were added. Each 10 pl of dye solution contained 1.5 pl dye A
(live stain) and 1.5 pl dye B (dead stain) in lambda buEer, which was consistent with
Molecular Probes recornmendation of 3 pl of dye AB per millilitre of sample.
2.3.3.1. Epifiuorescent microscopy:
One millilitre samples were filtered using a lOcc syringe (Becton Dickinson & Co.
Franklin Lakes, NJ) ont0 13 mm diameter, 0.2 pl black polycarbonate membrane filters
(Millipore Corporation, Bedford, MA) housed in Swimex filtration devices (Millipore).
Foliowing filtration, the membrane was removed fiom the filtration device and placed on a
glas slide, a drop of Baclight Mounting oil (Molecular Probes, Eugene, OR) was added,
and the filter was covered with a cover slip. The samples were viewed with a Nikon
Labophot Microscope with epduorescence equipment (Nikon Corporation, Mississauga,
ON) with an excitation range of 450-490 nm. Clumps were counted according to a
modified DEFT procedure.
2.3 -3.2. Bacterial counts:
The micoscopy protocol relied on the ability to accurately enurnerate fluorescently stained
bacterial cells. The epifluorescent technique (DEFT) described by Pettipher (1983), was
modified to enumerate Live and dead cells using the Molecular Probes LIVE/DEAD@
BaclightTM bacterial viability stain. The resulting DEFT counts were CO rrelated to
standard plate counts. Ten-fold dilutions of an overnight culture of S. Enteritidis were
prepared in lambda buffer. CeUs were stained according to the procedure outlined in 2.3.3
and lei? in the dark for 15 min. Ten randomly selected fields of view were counted and
averaged. The DEFT count per ml was calculated by multiplying the average clump count
by a microscope factor. The microscope factor was calculated as folows:
Microscope factor = Area of membrane throueh which sample is filtered !mm2) Microscopy field area (mm2) x Sarnple volume (ml)
The filter area was calculated from the interna1 radius of the filter membrane (n x radius2)).
The area of the microscope field of view was calculated from the radius of the field of
view determined with a hemocytometer.
2.3.4. Procedure for analysis - IMS protocol:
2.3 -4.1. IMS, phage attachent and amplification:
Flowcharts for the IMS protocol are illustrated in Figures 2.1 and 2.2. Twenty microlitre
volumes of Anti-Salmonella ~ ~ n a b e a d s @ @ynal Inc., Lake Success, NY) were added to
rnicrofbge tubes containhg 1 ml of stationary-phase culture of S. Enteritidis. Samples
were rotated at 30rpm for 30 min at room temperature on an Orbitron Rotator II (Fisher
Scientific, Mssissauga, ON). Sampies were placed in a magnetic rack @pal Inc., Lake
Success, NY) in order to separate the magnetic beads from the broth. The beads were
washed two times in lambda buffer, and resuspended in 500 pl L-broth. One hundred
microlitres of phage were added and samples were incubated for 15 min at 37OC to allow
for attachent. Magnetic beads were separated and washed twice in lambda buffer to
remove unbound phage. Beads were resuspended in 100 pl of lambda buffer and
incubated for 30 min at 37OC to allow for release of progeny phage. Magnetic beads were
captured and the progeny phage recovered in the supernatant was added to a 1 ml volume
of helper S. Enteritidis ceUs in L-broth, adjusted to an optical density of 0.100
(approximately 1x10~ CN/rnl). Helper population and progeny phage were incubated at
37°C for one hour.
2.3 -4.2. Optical density:
Following incubation of progeny phage with the helper cells, the samples were transferred
to 1.5 ml disposable plastic cuvettes (Fisher Scientific, Mississauga, ON). Absorbance
was read at 600nrn in a Pharmacia Novaspec II Spectrohotometer (Arnersham Pharmacia
Biotech Inc, Uppsala, Sweden).
2.3 -4.3. Fluorescence staining:
Alternatively, samples were centrifùged at 14,000 rpm for 10 minutes in an Eppendorf
54 15 C bench top centrifuge (Brinkmann Instruments Inc., Westbury, NY). Supernatant
was discarded and the pellet was resuspended in 900 pl lambda buffer. Samples were
stained wit h 1 00 pl Molecular Probes LIVE/DEAD@ ~ a c ~ i g h t " bacterial viability stain
solution. Dye was prepared such that a 100 pl portion of dye solution contained 1.5 pl
each of the [ive stain (component A) and the dead stain (component B) in lambda buffer.
Sarnples were left at room temperature in the dark for 15 minutes.
2.3 -4.4. FLSOO fluorometer:
Four 200pl portions of each lm1 sample were distributed into Coming clear 96 well
microtitre plates (Fisher scientific, Mississauga, ON). Samples were read in the FL500
fluorescence plate reader (Bio-Tek Instruments, Winooski, VT). Excitation was 485 nm
and emission was 530 nm, with band-widths of 20 and 25 nm respectively.
2.3.4.5, MGM fluorometer:
Samples were distributed into 6 x 50 mm glass tubes (Fisher ScientSc, Mississauga,
Ontario) and read immediately in the MGM fluorometer (MGM Instruments, Hamden,
Connecticut). Excitation wavelength was 460 nm and emission wavelength was 5 10 nm.
2.3 -4.6. Efficiency of immunomagnetic separation:
An ovemight culture of S. Ententidis was serially diluted IO-fold in L-Broth to a dilution
of 10". The cell concentrations in the six dilutions were estimated to be 1 08, IO', 106, los,
1 04, and lo3 CFU/ml. Irnrnediately after dilution, the efficiency of magnetic capture was
tested on each dilution in duplicate. To determine the ce11 populations in the initial
dilutions, duplicate spread plate counts were performed. Following magnetic capture, the
beads were resuspended in 1 ml of lambda buEer and duplicate counts of each sample
were performed. Dilutions were made as required to enumerate sarnples containing high
numbers of ceus. Ail plates were incubated ovemight at 37°C. colonies were counted and
the resulting counts were compared to the number ofCFU/rnl in the original dilutions.
2.3.5 Assay parameters:
2.3 -5.1. Temperature:
The IMS assay was tested at three temperatures (30°C, 37OC, and 42°C) to determine the
optimum temperature for the assay. Each incubation period. attachent, amplification,
and helper incubation were performed at one of the three experimental temperatures.
Positive and negative samples were tested in duplicate at each experimental temperature.
2.3.5 -2. Phage population:
The IMS assay was performed using three phage populations (Io6, 10'. and 108 PFU/100
pl) to determine the optimum phage concentration for the assay. Positive and negative
samples were tested in duplicate at each phage population. The experirnent was
conducted twice.
2.3 -5.3. Media:
The L-broth used for the finai step in the assay interfered with the fluorescent dye,
necessitating a centrifugation step to wash cells and remove media residue pnor to
fluorescent staining. An alternative to L-broth, whkh would not interfere with fluorescent
staining, was sought in order to eliminate the washing step. The media would have to
support both the growth of helper cells in the absence of phage, and the reduction of
helper by phage. Prelirninary studies suggested that lambda buffer was not a suitable
alternative- It was thought that the addition of sugars to the buffer rnay increase the
performance of the buffer. Two sugars were tested (dextrose and maltose), at three
concentrations (02.%, OS%, and 1.0%) in duplicate. These samples were compared with
L-broth and lambda buffer, also tested in duplicate. Optical density readings at 600 nm
were taken of al1 sarnples at time O, 1 h, 1.5 h and 2 h.
2.3 -5 -4 Freeze-dried helper:
Because of the potential problems with maintainhg and manipulatirtg iive SaImortella
stock cultures in a food lab, the use of a fieeze dried helper population was investigated as
an alternative to an overnight culture. An ovemight culture of S. Enteritidis was diluted 1
in 10 in L-broth and 1 ml samples were distributed into stenle Eppendorf tubes. Samples
were fiozen ovemight at -20°C on a slant, and then transferred to a -80°C freezer for 30
min. Small holes were pierced in the top of the tubes and they were placed in a Lyph-
~ o c k @ flask (Labconco Corp., Kansas City, MO) and freeze-dried for 21 h at 5 . 7 ~ 1 0 ~ ~
mBar vacuum pressure in a Labconco ~ ~ ~ h - ~ o c k @ Freeze Dry System (Labconco Corp.).
The titre of the cell population in two samples was determined before and aîter freeze-
drying by duplicate plate counts on L-agar. Samples were reconstituted in 1 ml of lambda
buffer immediately prior to use.
The feasibility of freeze-dried helper cultures for use with the bacteriophage assay was
determined. At the start of the protocol, four freeze-dried samples were reconstituted in 1
ml of lambda buffer and incubated at 37°C until ready for use. The IMS assay was
conducted with four negative sarnples (broth only) and four positive samples (1 in 10
dilution of an overnight S. Enteritidis culture) as outlined in section 2.3.4.1. Following the
amplification step, the average optical density (600 nrn) of the fieeze-drïed helper ce11
samples was determined. An ovemight culture of S. Enteritidis was adjusted to the same
optical density. The progeny phage recovered h m the assay samples were divided such
that there were duplicate positive and negative sarnples for each helper type. The
overnight helper population and the fieeze-dned helper population were compared for
their ability to support the growth of helper bactena in negative samples, and the reduction
in helper ceil numbers in Salmonella positive sarnples.
2.3 -6. Scanning electron rnicroscopy :
Four samples containing 1 ml of ovemight S. Ententidis culture and 20 pl
immunomagnetic beads were incubated at room temperature on an Orbitron Rotator II at
30 rpm for 30 minutes. The samples were washed twice in lambda buffer and resuspended
in 0.5 ml of lambda buffer. To two of the samples, 100 pl of bacteriophage SJ2 were
added, and sarnples were incubated at 30 rpm on an Orbitron Rotator II for 10 minutes at
room temperature. Samples were washed once and beads were resuspended in 0.5 ml
lambda buffer. The samples were filtered with the use ofa Buchner vacuum filtration unit
ont0 a 0.2 pm polycarbonate membrane filter (Poretics Corp. Livermore, California)
situated in a 13 mm syringe apparatus (MiIlipore, Bedford, Massecheusses). The filter
was transferred to 2% glutaraldehyde in Sorensen's phosphate buffer (SPB, 1: 1 ratio of
0.07 M Na2B0, 7H20 and 0.07 M KHJ'O, pH 6 . 9 for one hour a t room temperature
to fix proteins. Filters were rinsed three times in SPB, flooded with 2% osmium
tetraoxide and left in the dark for one hour at room temperature for lipid fixation.
Samples were rinsed three times in SPB and gradually dehydrated in a graded senes (50%.
70%, 80%, 90%, 95%, 100% (three times)) of ethanol for 10 minutes at each level. The
sarnples were cntical point dried with CO, in a LADD cntical point dryer (LADD
Research Industries, Burlmgton, Vermont). The samples were rnounted on a specimen
stubb, sputter coated with goldlpalladium in a Polaron SC500 sputter coater ( Soquelec,
Montreal, Quebec), and scamed in a Hitachi S-4500 Scanning electron microscope
@tachi, Tokyo, Japan) at a 7 and 25 keV accelerating voltage.
2.3.7. Transmission electron microscopy:
Bacteriophage SJ2 was prepared for transmission electron microscopy by negative
staining. A drop of the phage suspension was placed on parafilm and a formvar/carbon
coated copper grid (200 mesh) (Marivac Ltd., Halifax, Nova Scotia) was floated on top of
the sample for 2 minutes. The copper grid was blotted dry, and was floated, sample side
down, on a drop of 1% wt/vol aqueous uranyl acetate (Fisher Scientific, Nepean, ON) for
2 minutes and blotted dry. The sample was viewed in a Phillips EM300 transmission
electron microscope (Phillips Electrical Corp. New York, NY) operating at 60 keV with a
liquid nitrogen cold trap.
2.3 -8. Bacteriophage specificity:
Bacteriophage SJ2 was tested against 37 bacterial stains (Table 2.1-2.3) to determine its
specificity. Plaque assays were performed, based on a modified method for the
quantitative assay of phage outlined by Maramomsch and Koprowski (1967). Phage SJ2
suspension (0.1 ml) and 0.1 ml of an ovemight culture to be tested were added to top
layer agar cooled to 45OC. The mixture of phage, cells and agar was quickly poured onto
Petri plates containing hardened L-agar and swirled to cover the plate. When the top
layer agar had hardened, plates were incubated overnight at 37OC. Plates were observed
for the presence of plaques. The IMS bacteriophage protocol was performed on overnight
cultures of al1 41 strains, diluted 1 in1 O in L-broth.
2.3.9. Statistical analysis:
Linear regression comparing plate counts with DEFT counts and IMS plate counts was
performed using Quattro-Pro (Corel Corporation, Ottawa, Ontario). Unless otherwise
indicated, all statistical analysis involving treatment cornparisons was performed using a
one way analysis of variance (ANOVA) at a signincance level of a = 0.05 in Quattro-Pro
(Corel). Where statisticdly significant ciifferences existed, post hoc analysis was
performed using Scheffe' s contrast test (Scheffe, 1959).
2.4. Results
2.4.1. Bacteriophage and host:
A transmission electron rnicrograph of bactenophage SJ2 showed a head and a long tail
(Figure 2.3). The base plate was visible but tail fibres, if they exist, were not. Scanning
electron rnicrographs of bactenophage SJ2 attached to S. Enteritidis are shown in Figure
2.4.
2.4.2. One-step growth experiment:
The propagation curve of phage SJ2 (Figure 2.5) shows a burst time of approximately 30
minutes with a burst size of approximately 100. Companng the plaque count of plate 13
with plate 1 indicates that approximately 97% of the phage had adsorbed to the host celis
during the first four minutes.
2.4.3. Microscopy protocol:
Ten-fold dilutions of S. Enteritidis were evaluated by the centrifugation and epifluorescent
rnicroscopy protocol. The average counts obtained fiom Salmonella positive samples
were compared with samples containing no Sa/moneZZa initially. Live (green) celis were
counted, as prelirninary studies (not shown) indicated dead (red) ce11 counts were a poor
representation of what was occurring in the assay. DEFT counting methodology requires
Figure 2.3. Transmission electron micrograph of bactenophage SJ2. Bar =IO0 nrn. Ma@cation, x 207,850.
Figure 2.4. Scanning electron microgaphs of bacteriophage SJ2 attached to S. Enteritidis. A) Bar = 900 nm. Magnification, x 20,000. B) Bar = 360 nrn. Magnification, x 50,000.
20 25 30 35 Time (minutes)
Figure 2.5. Resdts of one-step growth experirnent for phage SJ2. Lysis occurs d e r approximately 3 0 minutes, with a burst size of about 1 00, observed within 42 minutes.
that any cell, or group of cells, separated by a distance less than twice the diameter of the
cells nearest each other, be counted as one clump. Theoreticaiiy, one clump would be
responsible for one colony forming unit on solid agar. This convention was adopted in
the assay. The results of one of the trials is illustrated in Figure 2.6. At initial ce11
populations of 1o3, Io4, and 10' CFU/ml, the expected decrease in clump counts was
observed relative to the Salmonella negative sample. A one way analysis of variance
(ANOVA) showed a statistically significant difference between samples. The calcuiated
F-value was 154, giving a p value <0.001 (F,, = 2.9). Post hoc analysis using Scheffe's
contrast test demonstrates there was a significant difference between the negative samples
and the positive samples at the 95% confidence level. Likewise, the 103 CFU sample was
significantly different from the IO4 and 10' CFU levels. The latter two were not
signifïcantly dEerent from each other. These results were confirmed in two additional
trials.
2.4.3-1. Bacterial counts:
Figure 2.7 illustrates the relationship between standard plate counts and DEFT counts. In
al1 instances, live (green) cells were counted, as dead (red) ce11 counts were not a good
indicator of phage activity. DEFT counts generally correlated well with plate counts (i =
0.89). Figure 2.8 is an epinuorescent rnicrograph of a stationary phase S. Enteritidis
culture stained with the BacLightm bacterial viability stain and filtered on a 13mrn black
polycarbonate filter.
O 7x1 O= 7x1 o4 7x1 o5 Initial Population (CFUI100pf)
Figure 2.6. Average clump counts following microscopy protocol for four initial populations of S. Enteritidis. Bar represents one standard deviation.
4 5 6 Log,, , plate countlrnl
Figure 2.7. Relationship between plate count and DEFT count for different dilutions (1 O-', 1 05, 1 O-4, 1 O") of S. Enteritidis in buffer, tested in triplicate. Line represents fitted regression line (y = 0 . 8 3 ~ + 0.52, ? = 0.95)
Figure 2.8. Epifluorescent micrograph of S. Ententidis stained with Molecular Probes LISEDEAD@ BacLight" bacterial viability srain. Live ceiis fluoresce green, dead ceUs fluoresce red.
2.4.4. IMS protocol:
2.4.4.1. Optical density:
Ten-fold serial dilutions of S. Enteritidis were prepared in L-broth. Dilutions representing
plate counts of 102, 10" 104, los, and 106 CFUIml, were tested in duplicate. Three trials
were performed. For each sample, the absorbance value was calculated as a percentage of
the mean of the negative control values for that trial. The results over al1 three trials were
pooled and are illustrated in Figure 2.9. The sensitivity of the assay with pure cultures in
broth is approximately 104 CFU/rnl. If a value of 70% of the mean of the negative control
value is used as the cut-off for a positive sample (determined in section 3.4.1), then none
of the samples tested in the 1o3 CFU/ml range were positive. Two of the 10' CFU/ml
samples were determined to be 1.5xl0'CFU/ml by plate count. These samples were both
exactly 70% of the negative control after two hours incubation. The other four samples in
the 104 range were determined by plate count to be above 5x10' CN/ml and had values
55% or lower. Samples above IO4 CFülrnl were al1 clearly positive as weli.
2.4.4.2. Fluorescence:
The assay was performed as indicated in section 2.4.3.1. Following optical density
readings, cells were stained and read in the FL-500 fluororneter. As expected, the
resulting distributions were very similar to those obtained by the absorbance readings.
The sensitivity of the assay with pure cultures in broth is comparable to the optical density
6x10' 6x10' 6 x 1 0 ~ 6x1 0' 6 x 1 0 ~
Mean Initial Population (CF Ufrnl)
Figure 2.9. Percent of negative control value for five populations of S. Enteritidis in broth following TMS-bacteriophage assay. Values s h o w are averages of three trials. Endpoints were determined by optical density. Bar represents one standard deviation.
results of approximately104 CFU/ml. The lowest population for which a positive test was
observed was 8xid CFU/ml. When the MGM tluorometer was used for one of the trials,
it was noted that the detection Limit was closer to 1x10' CFU/ml, even though absorbance
readings taken before fluorescent staining indicated a lower detection S i t by more than
two log cycles. A second trial showed a difFerence in detection limit by more than one log
cycle in favor of the spectrophotometer.
The sensitivity of the MGM Fluorometer and the spectrophotometer were compared to
determine their ability to detect low numbers of cells. An ovemight culture of S.
Enteritidis was washed in lambda buffer and was serially diluted. Each 10-fold dilution
was then further diluted 2:3, and 1:3 in buffer to represent populations between full log
cycles. Tnplicate sarnples of each dilution were analysed in each machine according to
sections 2.3.4.2. and 2.3.4.4. The results are illustrated in Figure 2.10. and Figure 2.1 1.
The spectrophotometer was able to detect a bacterial population of approximately 6.35
log,, CN/ml . The MGM Fluorometer was more sensitive by approximately one log
cycle. At the lowest dilution tested (5.35 log,, CFU/ml), the MGM fluororneter signal
was more than one standard deviation over the blank.
2.4.4.3. Efficiency of irnrnunomagnetic separation:
A cornparison of plate counts following IMS was compared to spread plate counts for six
dilutions of S. Enteritidis. The results are illustrated in Figure 2.12. There was a good
correlation between plate counts and IMS plate counts (? = 0.99). The efficiency of IMS
Figure 2.10. Sensitivity of the Pharmacia spectrophotometer as indicated by mean optical density values for various populations of S. Enteritidis. Bar represents one standard deviation.
Figure 2.1 1. Sensitivity of the MGM Fluorometer as indicated by mean fluorescence values for various populations of S. Enteritidis. Bar represents one standard deviation.
I L I 1 1
3 4 5 6 7 8 9 Log,, plate countlml
Figure 2.12. Relationship between plate count, and plate count following MS for different dilutions of S. Enteritidis in buffer. Two replicates from each 10- fold dilution (1 03, 1 04, 1 os, 1 06, 10' and 1 o8 CFU/ml) were counted. Line represents fitted regression line (y = 0 . 8 9 ~ + 0.39, $ = 0.99)
was reduced at higher celi populations, presumably because of the larger ce11 to bead ratio.
Figure 2.13 shows scanning electron micrographs of S. Ententidis attached to
irnrnonumagnetic beads.
2.4.5. Assay parameters:
2.4.5.1- Temperature:
The bactenophage assay was tested at 30°C, 37OC and 42T. There was no significant
difference in helper reduction between 37°C and 42T, but these temperatures were
significantly better than 30°C (px0.05). Growth of helper appeared to be reduced at 30°C
relative to the other temperatures, but this was not significant at the 95% confidence level
(p = 0.15). As a result, 37°C was used for the assay, as this is a common incubation
temperature and there was not a 42OC incubator routinely available in the laboratory.
2.4.5.2. Phage population:
Three phage populations (Io6, IO7 and 108 PFU/100 pl) were tested in duplicate to
determine the optimum phage population for the assay. There was no si,-cant
difference in helper reduction when 10' and 10' PFU were used, but these phage
populations performed signincantly better than 106 PFU @< 0.05). This was confirmed in
a second trial. Efficient removal of unbound phage after the attachrnent step is very
important to the success of the protocol. As a result 10' PFU was considered optimal for
Figure 2.13. Scanning electron rnicrographs of S. Ententidis attached to immmunomagnetic beads A) Bar = 2.0 Pm. Magnification, x 9,000. B) Bar = 4.5 Pm- Madcat ion, x 4,000.
the assay, as removal of phage would presumably be more complete if a lower initial
population was added.
2.4.5.3- Media:
The L-broth used for the helper incubation step in the assay interfered with the fluorescent
dye, necessitating a centrifugation step to wash cells and remove media residue prior to
fluorescent staining. An alternative to L-broth, which would not interfere with fluorescent
staining, was sought in order to eliminate the washing step. In addition to cornparhg L-
broth and lambda buEer, two sugars were tested (dextrose and maltose), at three
concentrations (0.2%, OS%, and 1.0%). Results for dextrose are shown in Figure 2.14
and 2.15. Regarding reduction of helper by phage, an ANOVA perfomed on results at
1.5 hours resulted in an F value of 424 with a calculated p value of < 0.00 1 (F,, = 5.19).
Post hoc analysis using Scheffe's test indicated that at 1.5 hours. L-broth perfomed better
than al1 the other media, lambda buffer was the least acceptable, and there was no
significant difference between the rhree dextrose concentrations (p = 0.05). Results
obtained with the maltose samples were very similar to dextrose.
Regarding growth of helper, L-broth was superior to the other treatments at ail time
intervals, with the dextrose solutions perforrning only marginally better than the buffer
afone (Figure 2.15). Again, distributions for the maltose samples were similar to those
for dextrose.
- L-Broth --F
g 0.04 ' Buffer Cu -- -5- e g 0.03 - 6,
-'\ 0.2% Dextrose
2 - - - - -----,< - 0.02 - 0.5% Dextrose
- -t
0.01 O 0.5 1 1-5 2 1,0% Dextrose
Time (Hours)
Figure 2.14. Cornparison of L-broth, lambda buffer, and three dextrose concentrations (0.2%, 0.5% and 1.0%) as media for the reduction of S. Ententidis by phage S J2 at 3 7OC.
- O ' ,
- 0 0.5 1 1.5 2
Time (HOUE)
-e
L-Broth
3-
Buffer - 0.2% Dextrose
-E-
0.5% Dextrose
-t
1 .O% Dextrose
Figure 2.15. Comparison of L-broth, lambda buffer, and three dernose concenirations (0.2%, 0.5% and 1 -0%) as media for the growth of S. Enteriticlis at 37OC.
In another study, ammonium chlonde was added as a nitrogen source to lambda buffer
containhg 0.2% dextrose. When compared to 0.2% dextrose in buffer, there was no
significant dflerence for both growth of helper population and reduction of helper by
phage (p <O.OS).
None of the media treatments tested compared well with the L-broth, particularly for
facilitating growth of the helper population. A washing step will continue to be necessary
when fluorescence is used as an endpoint.
2.4.5.4. Freeze-dried helper:
One millilitre samples of helper bacteria were fieeze-dried and tested with the IMS assay,
in order to possibly eliminate the need for overnight growth of Salmonella cultures for the
completion of the protocol. A cornparison of the titre of samples before and after fieeze-
drying indicated a one log reduction in ce11 numbers following freeze-drying.
The recovery penod of fieeze-dried samples was investigated. Freeze-dried samples were
reconstituted in 1 ml of lambda buffer and incubated at 37T. When phage was added
following reconstitution, a decrease in optical density (600 nrn) was observed within one
hour. Growth, as measured by opticaI density, was observed within two hours.
Freeze-dried helper was compared with an overnight helper culture in the IMS assay.
Freeze-dned samples were reconstituted at the start of the protocol and incubated at 37OC
until required (approximately a 2.5 hour recovery period). The desired trend was
observed with the fieeze-dned sarnples, but the ovemight helper population perfomed
significantly better for both growth of the helper, and reduction of the helper by phage (p
< 0.001)
2.4.6. Specificity of bacteriophage SJ2:
Bacteriophage SJ2 was tested against 41 strains of bacteria to detennine its host range.
The results are presented in Tables 2.6-2.8. Al1 six non-Salmonella strains tested were
negative by both plaque assay and phage assay. Phage SJ2 infected al1 five of the S.
Enteritidis strains tested as detemined by plaque assay, and resulted in a positive test
when these strains were tested in the IMS phage assay. Phage SJ2 is not specific to S.
Ententidis however. Strong positive results for both tests were observed for S.
Typhimurium SA 942256 and S. Sendai. Interestingly, S. Typhimunum 94-51 was not
positive by either method. Other serotypes belonging to group D,, namely S. Berta and S.
Panama, showed somewhat inconsistent results for both the plaque assay and the phage
assay. Weak lysis was observed by the plaque method on two of four trials for both of
these organisms. In addition, these strains were positive by the phage assay the fïrst time
they were tested, but this was not confirmed in a second trial. It folIows then, that these
two strains would not be reliably detected by the IMS assay.
Table 2.6. Bactenophage assay and plaque assay specificity results for non-Enteritidis
Serogroup Bacterial Species Strain Plaque assay / Phage assay
Group B
Group C
S. Typhimurium
S. Typhimurium
S. Heideiberg
S. Saintpaul
S. Bredeney
S. Schwartzengnuid
S. Schwartzengnrnd
S. Agona
S. Indiana
S. Bmdenburg
S. Reading
S. Infantis
S. Thornpsoa
S. Mbandaka
S. Braenderup
S. Montevideo
S. Ohio
S. Oranienburg
S. Tenenssee
S. JO hannes burg
S. Ubana
S. Rubislaw
S. Hadar
S. Kentucky
S. Newport
Table 2.6. continued. Bacteriophage assay and plaque assay specificity results for non- Ententidis Salmonella stiains. I
Serogroup Bacterial Species S train Plaque assay I Phage assay
Group C S. Haardt SA 914074
S. Choleraesuis S-870
Group D S. Berta SA 941 123
S. Panama SA 93 1592
S. Sendai S-5 13 -- -- -
a Weak lysishot consistently positive by bacteriophage assay.
Table 2.7. Bactenophage assay and plaque assay specificity resdts for SolmoneZZa Enteritidis strains.
Bacterial species S train Plaque assay/Phage assay
S. Enteritidis SA 932451 +/+ S. Enteritidis Laboratory Strain +/+
S. Enteritidis ATCC 13076 +/+ S. Enteritidis SA 94245 1 +/+
S. Enteritidis EN 2588 +/+
Table 2.8. Bacteriophage assay and plaque assay specificity results for non-Salmonella strains.
Bacterial species Sîrain Plaque AssayPhage assay
Serratia marsescens ATCC 8 1 O0 -1-
Cino bacter fieundii -/-
Sh ige lla flexne ri -/-
Escherichia coli ATCC 25922 4-
Escherichia coli 0 1 5 7:H7 EC 920333 -1-
2.5. Discussion
2.5.1. Bacteriophage specificity:
It became clear early on that phage SJ2 was not specific for only Salmonella Ententidis
nor was it's host range sufficient for a generic SalmonefZa test. SpecScity testing was
nonetheless camied out to further characterize the phage. The phage was not infectious
for any of the non-Salmonella strains tested. AI1 five of the S. Enteritidis strains tested
showed strong positive reactions by both methods. Strong positive results were also
observed for S. Typhirnurium SA 942256 and S. Sendai. S. Typhimunum 94-5 1 was not
positive by either method. This particula. strain of S. Typhimunum is highly resistant to
multiple antibiotics. Perhaps the mutations responsible for antibiotic resistance also
imparts some resistance to infection by phage. Other serotypes belonging to group D,,
namely S. Berta and S. Panama, showed somewhat inconsistent results for both the plaque
assay and the phage assay. There are no diagnostically important somatic or flagellar
antigens among cornrnon among the strains testing positive that were not also present in
strains that were not sensitive to phage SJ2. Somatic (O) antigen 09 was cornmon to the
serogroup D Salmonella tested (which includes S. Enteritidis), but this antigen is not
present on S. Typhirnurium. O-antigen 01, and 12 are common to al1 the strains testing
positive, but are also found on other group C strains tested (Bergey et al., 1984). Since
phage SJ2 was isolated fiom the environment, and has not been characterized, it is
unknown what constitutes a receptor. A more suitable choice for a genenc Salmonella
test may be bactenophage Felix-01, which has been shown to uifect 98-99.5% of the
greater than 5000 SaIrnonella strains tested (Bergey et al., 1984)
2.5 -2. One-step growth experiment:
The one step growth experiment was important in that it dictated some of the incubation
tirnes required for the assay. Since lysis began to occur around 30 minutes, the attachent
and washing steps could not exceed this time, ~îherwïse progeny phage rnay be lost. As a
result, 15 minutes was permitted for attachment of phage, followed by washing. The
results of the growth experiment suggest the 30 minute incubation penod for amplification
would be sufficient for one complete round of infection.
It is important to note that results of this procedure are estimates only. Samples taken
more frequently would give greater precision for determining the latent period. Also, if
adsorption was not swifi and very high, burst-size calculation may not be very accurate
(Mararnorosch and Koprowski, 1967). The 97% adsorption rate caiculated in this
experirnent indicated that adsorption occurred very rapidly. The plates representing times
greater than 42 minutes were too numerous too count, so it is difficult to estimate how
much higher the relative plaque titre would be. If this increase is due to late-bursting cells,
the burst size may be underestimated.
2.5.3. Microscopy protocol:
This protocol demonstrated the feasibility of the basic principles of the assay. That is,
progeny phage from Salmonella present initially in the sample could be indirectly detected
by its effect on a helper population. When this effect was compared to samples containing
no Salmonella initially, it became clear which sarnples were positive. Furthemore, the
greater the initiai Salmonella population, the greater the effect on the helper population.
There are several reasons the centrifugation protocol would not be suitable for testing
food samples. First, there is no means of dealing with the background microflora that
would be in a food sarnple. At the very least, a selective ennchrnent step would be
necessary. Second, centrifugation is not desirable as it would require additional
instrumentation to perform the assay. Lastly, direct microscopie counts are labour
intensive, time-consuming and not suitable for routine analysis, not to mention the capital
costs of an epifluorescent microscope. If the bactenal population of the sample exceeded
the upper lirnit for the filter used, accurate counts could not be obtained and there would
be no opportunity to diiute the samples for additional counting. Each sample would also
require some mathematical manipulation to calculate averages which would become
cumbersorne with large numbers of samples.
2.5.3.1. Bacterial counts:
Molecular Probes LIVE/DEAD' BacLightTM bacterial viability stain for microscopy and
quantitative anaiysis was employed in the assay. The stain i s a two-colour fluorescence
assay consisting of SYTO 9 green fluorescent nucleic acid stain, and the red fluorescent
nucleic acid stain propidiurn iodide (Molecular Probes, Product Information). The stains
ditfer in their spectral characteristics and their ability to penetrate healthy bacterial cells.
SYTO 9 is able to penetrate healthy bacterial cells and thus will label al1 cells in a sample,
those with and without intact membranes. Propidium iodide is unable to penetrate healthy
cells and cornpetes for binding sites with SYTO 9 only in cells with damaged membranes.
As a result, the appropriate mixture of the two stains results in Iive cells staining
fluorescent green and the dead cells staining fluorescent red. Both stains excite in the blue
range (470nrn). The live stain emits between 510 and 540 nrn, and the dead stain emits
between 620 and 650 nrn.
There was good correlation between DEFT counts and plate counts (3 = 0.95) in this
present study. Pettipher et al. (1980) reported a correlation of r = 0.83, when comparing
plate counts with acridine orange clump counts of bacteria in raw milk. It is reasonable to
assume that food samples would have a lower correlation coefficient given the greater
dieculty in recovering bacteria from food samples relative to cells in broth. For example,
Shaw and Farr (1989) used DEFT for the analysis of meat and poultry. They reported
that orangelred debris on the DEFT filter overestimated the actual count by 5 to 1000-
fold.. The use of 1% vlv Tween 80 has been found to improve the correlation to more
acceptable values however. This indicates another potential drawback of the present
microscopy/centrifugation protocol in that pretreatment of food samples would be
necessary to rernove fat and debris.
2.5.4. IMS protocol:
The IMS protocol was developed to make the bacteriophage assay suitable for the analysis
of food sarnples. The use of IMS eliminates the need for selective e~chment , thereby
reducing the total assay tirne by as much as 24 hours. Many authors have reported the
utility of IMS in increasing the sensitivity and specincity of Solmonella detection, as well
as significantly reducing total assay time (Mansfield and Forsythe, 1993; Holt et al., 1995;
Parmar et al., 1992). The application of IMS as a separation technique in this assay also
eliminates the need for centrifugation. Magnetic particle separatioii is important, not only
for initial recovery of target cells, but for permitting subsequent washing steps (phage
removal), and for imrnobolizing the beads and cells for recoveiy of progeny phage in the
supernatant.
Regarding endpoint detection, a Buororneter is considerably easier to use than obtaining
microsco pic counts and requires less sample manipulation. In addition, results obtained
require only minimal mathematical manipulation.
Optical density was evaluated as an endpoint when it was observed that, in many cases,
positive and negative samples could be visually differentiated by differences in the turbidity
of the samples. Using optical density has sorne further advantages. First. a
spectrophotometer is a relatively inexpensive and common piece of laboratory equipment.
Second, sarnple manipulation and total costs are reduced as there is no need for
fluorescent staining. There is no need for a washing step to remove media, as samples can
be read directly fiom the incubator. The assay also becomes more flexible, since samples
can be re-incubated after initial readings, if necessary, to further distinguish between
positive and negative samples. The average incubation tirne was about 1.5 h.
Optimal parameters for the assay were determined to be an assay temperature of 37"C, a
phage population of 10' P m , with L-broth being the most suitable media for helper
incubation. Freeze-dried helper cells show some promise as an alternative to overnight
cultures, but a longer recovery period (>2.5 hours) may be required in order to approach
the performance of the ovemight helper population. Andrews (1986) reviewed the
resuscitation of injured Salmonella spp. and coliforms from foods. He reported that slow
(drop-wise) rehydration of dried cultures result in higher viable counts when compared to
rehydration by rapid addition of water. This is Iikely attributed to ce11 wall damage
occuning dunng rapid rehydration. Temperature was also found to be an important
factor. Spray-dried culture recovered best when rehydrated at 50°C, while freeze-dried
cultures were maximally recovered at a rehydration temperature of 20 to 25°C. These
factors should be taken into consideration when contemplating the use of a fkeeze-dned
helper population in the present assay.
2.5.5 Efficiency of immunomagnetic separation:
The eficiency of IMS was evaluated. There was a good correlation between JMS and
plate counts over the range of ce11 populations tested (r = 0.99). At the 104 dilution
(1 .4x103 CFU/ml), 86% of the bacteria in the sarnple were recovered. This dropped to
15.7% for the 10-' dilution (1 .4x108 CFU/ml), presumably because of the increased ce11 to
bead ratio in the sample. There are approximately 1.3 x10' beads added per 1 ml sample
(Dynal Inc., personal communication). At the highest dilution tested, tbis meant a cell to
bead ratio of about 10 to 1. Vermunt et al. (1992) reported a recovery of 5 1% * 7.8%
following IMS. The etticiency of IMS in this experiment was better than the 10%
recovery reported by Hanai et al. (1997).
One of the problems cornmon to antibody based tests is non-specific binding. Parmar et
al. (1992) evaluated an IMS-conductance method for the detection of Salmonella in milk
powders. In evaluating the specificity of IMS, they noted that exposure to Cipobacfer
freundii resulted in a Salmonella-type conductance curve and concluded the magnetic
beads were only partially specific for the Salmonella strains tested. Non-specific binding
of Citrobacter has been reported by other authors (Cudjoe et al., 1995). . ~ i t h the use of
bactenophage providing additional specificity, non-specific binding should not interfere
with the assay provided an appropriate number of Salmonella can be recovered on the
beads. The Citrobacferfrerndii tested as part of the phage specificity test in this study
did not result in a positive test. Factors affecting irnmunologicai and non-specific binding
of bactena to magnetic particles include the culture medium, the bacterial strain and the
particlekell incubation time and temperature (Blackburn, 1993).
2.5.6. Assay sensitivity:
Optical density was chosen as the means of determining assay sensitivity, since incubation
time was flexible. The assay sensitivity is approximately 104 CFU/rnl, as
indicated in Figure 2.9.
Another Salmonella detection assay based on the principle of assaying progeny phage was
reported by Hirsch and Martin (1983a; 1983b). They describe a method for the detection
of SuZmoneIZu using Felix-O 1 bacteriop hage and high performance liquid chromatograp hy
(HPLC). The interaction of bacteriophage Felix-O1 with S. Typhimurium resulted in
increased numbers of bactenophage which were subsequently detected by HPLC. It was
determined that 106 Salmonella per ml need to be present to be detected. The detection
limit of the present assay is about two log cycles lower. The lower detection lirnit of
ELISA'S have been reported to range frorn 104 CFU/ml up to 10' CFU/d (Patel and
Williams, 1994; Cudjoe et al. 1995; June et al., 1992). This present assay has a detection
limit comparable to or better than other available detection methodg excluding gene
amplification methods..
2.5 -6 . Conclusion:
The bacteriophage assay described here is based on the cornbined specificity of
immunomagnetic beads and bacteriophage SJ2 for SalmoneIIa spp. The study
demonstrates that the normal biology of bacteriophage SJ2 in its host bacterium can be
exploited, with progeny phage subsequently assayed indirecty by its eEect on a helper
population of healthy S. Ententidis cells. The assay endpoint can be determined either by
optical density or fluorescence measurements. The assay is not specific to S. Ententidis,
nor is it suitable for a genenc Salmonella test, but provides a good working mode1 for the
future development with other host/phage systems. The assay is simple to perform, can br:
completed in under 4 hours, and has a sensitivity in the range of 1 O' CFU/rnl.
3. The use of the IMS-bacteriophage assay for the detection of Salmonella Enteritidis in skimmed mük powder, ground chicken and liquid whole egg.
3.1 Abstract
Salmonella infection is the second most prevalent cause of foodborne illness in most
developed countries. Poultry products, eggs and milk are ftequently implicated in
outbreaks. The objective of this study was to appty an Unmunomagnetic separation @MS)
bacteriophage assay to the detection of S. Enteritidis in artificially inoculated skimmed
milk powder, ground chicken, and liquid whole egg. In al1 food types tested, the IMS
assay was able to detect S. Enteritidis at the lowest initial inoculation level tested, 10"
CFU/g, following a preenrichment ranging fiom 10 to 18 hours. These results indicate
that the IMS assay is a rapid and sensitive means of detecting S. Ententidis in these foods.
3.2 Introduction
Salmonella is widely recognized as an important cause of foodborne illness. Poultry
products, eggs and milk are frequently implicated as vehicles of infection in cases and
outbreaks of Sa~monelZu infection (Bryan and Doyle, 1995; Henzler et al., 1994; Lin et al.,
1988; Ryan et al., 1987). Detection methods can play a role in stopping the transmission
of disease by preventing the sale of contaminated product. There are many available
methods for the detection of Salmonella in foods. Conventional cultural methods are very
slow, and require expensive storage of foods before results are available. The largest
group of rapid methods for Salinonellu detection are antibody-based tests such as enzyme
linked immunosorbent assays (ELISAs), latex agglutination, imrnunodifision and
methods incorporating M S . Drawbacks of these methods include non-specific binding
leading to false positive results, the high degree of technical skill required, and in some
cases, high detection Limits. Nucleic acid-based tests such as PCR can be very sensitive
and specific but can be expensive and labor intensive-
The specificity of bacteriophage has been exploited in the detection or typing of several
bacterial species (Stewart, 1990; Wobler and Green, 1990; Hirsch and Martin, 1983a;
Chen and Grifliths, 1996; Dubow, 1994).
This study describes the use of a novel IMS-bactenophage assay for the detection of S.
Ententidis in skimmed milk powder, ground chicken, and liquid whole egg.
3.3 Materiais and Methods
3 3.1 Bacteriophage and host:
The bacteriophage and host used in this study, SJ2 and S. enterifidis respectively, were
obtained fkom Dr. Sabah Jassim, Department of Food Science, University of Guelph.
Both the bacteriophage and the host were isolated fiorn egg. The method of isolation is
unknown. Bacteriophage SJ2 and host S. Ententidis were maintained as previously
descnbed in sections 2.3.1 and 2-3 -2.
3 -3 -2. Food enrichment preparation:
Ail food samples were purchased from a local supermarket. Overnight cultures of S.
Enteritidis were serially diluted IO-fold in lambda buffer. The population of S. Enteritidis
in the dilution tubes was determined by triplicate plate counts. Tluee replicates of
skimmed milk powder, six replicates of ground chicken, and three replicates of Liquid
whole egg were inoculated to give initial ce11 concentrations of 102, 10' and 10' C m / g or
ml. In each case, 1 millilitre of the appropriate dilutions of S. Enteritidis were added to 25
g or ml samples to give the required final ce11 concentrations. A negative control for ali
experirnents included uninoculated food. For each experiment, the results of food samples
were compared to a negative control consisting of 1 ml of L-broth, tested in triplicate.
For skimmed milk powder, spiked samples and unspiked skirnrned milk powder samples
were added to 225 ml of L-broth in a 500 ml Erlenmeyer flask and incubated, with
shaking, for 10 hours at 37OC. On the day of testing, each inoculation level was tested in
duplicate.
For ground chicken, controls, spiked samples and unspiked samples were added to a 500
ml Erlenrneyer flask containing 225 ml of L-broth. Samples were incubated for 16 to 18
hours at 37OC. For trial one and two, each inoculation level was tested in triplicate. In
trial three and four, samples were tested uninoculated and inoculated at the 10' CFU/g
level only. Each inoculation level was tested in tnplicate. A second package of ground
chicken was used for trials five and six. Sarnples were incubated for 10 hours in a
stomacher bag with a filter at 37OC and each of the four inoculation levels were tested in
duplicate. Negative controls @roth ody) were tested in triplicate.
For liquid whole egg samples, eggs were dipped in 95% ethanol and left to dry in a
laminar flow hood under W light for 10 minutes to disinfect the shells. Eggs were
asepticaiiy cracked and the contents of four eggs were pooled. Twenty five millilitre
portions were spiked as indicated above, added to 225 ml of L-broth in a 500 ml
Erlenmeyer fiask and incubated, with shaking, for 16 hours at 37°C. On the day of
testing, each inoculation level was tested in duplicate.
3 -3 -3 . Testing of food samples with the IMS bacteriophage assay:
Enrichment cultures from the skimmed milk powder, ground chicken and liquid whole egg
were tested according to the IMS protocol as previously described in section 2.3.4. The
results of some experiments were determined using optical density as the endpoint, others
employed both optical density and fluorescence as the endpoint.
3.3 -4. Determination of the assay endpoint:
A quantitative means of distinguishing positive from negative samples was determined.
The results h m the testing of d three food types were pooled, and a numerical cut-off
was determined based on the value that gave the minimal number of false positive and
false negative results. The results of al1 food samples tested were compared to the mean
of the negative control samples in each experiment. Cut-offs based on 80%, 75%, 70%,
65%, and 60% of the mean of the negative control samples for each experiment were
calculated the number of false negative and false positive results at each cut-off were
ascertained.
3.4. Resdts
3 -4.1. Determination of the assay endpoint:
Table 3.1 shows the results when five cut-off values were used to determine the endpoint
of the assay. False positive results occur when an unspiked sample has a value less than or
equal to the calculated cut-off. False negatives occur when a spiked sample has a value
above the calculated cut-off. A cut-off of 70% was chosen as the cut-off for a test to be
considered positive. At this level, one of 18 uninoculated samples (5.5%) gave a false
positive result. Two of 69 of the inoculated food sarnples gave a false negative result. AU
three of these samples were fiom liquid whole egg.
3 -4.2. Skimrned rnilk powder sarnples:
Samples of skirnrned rnilk powder inonilated with four levels of S. Entedidis (0, loO, 1 01,
and IO* CFU/g) were evaluated by optical density and in trial two by fluorescence, in
duplicate over three experirnental trials. Figure 3.1 shows the pooled results from the three
trials. Al1 four inoculation levels are reported as a percentage of the mean negative
Table 3.1. Tally of false positive and false negative results obtained fiom skimmed milk powder, ground chicken and liquid whole egg sarnples when various numerical cut-off values are used to determine a positive test.
Cut-OP 60%
positive, it must be less than or equal to the cut-off value,
65% 70% 75% 80%
False positive (+) and False negative (-) results are reported as the number of false positive or false negatiie samples out of
Skimmed mitk powder False +b False -b 015(0%) 1115(7%)
the number of true negative and true positive samples tested. Corresponding percentages are indicated in brackets.
a Cut-off was calculated from the mean of the negative control values in each experiment. In order for an experimental result to be considered
O15 (0%) 1/15 (7%) 0/5(0°h) 0/15(0°h) 115 (20%) 011 5 (0%) 215 (40%) 01 15 (0%)
In trials 2 ,3 and 4, uninoculated ground chicken sampler gave a strong positive result. Chicken was confirnied to be naturally contaminated with Salmonella. Results from these uninoculated samples are not included in the table.
Ground chickenC False t False - 0/3(0%) 0124(0%)
Second set of hiais with ground chicken; new package.
O13 (0%) 0124 (0%) 0/3(0%) 0124(0°/o) 013 (0%) 0124 (0%) 013 (0%) 0124 (0%)
Ground chickend False -I- False - 014(0%) 3/12(25%) 014 (0%) 2112 (16.6%) OI4(0%) 0/12(0%) 014 (0%) O112 (0%) 014 (0%) O11 2 (0%)
Liquid w h o u False + False -
116(16.7%) 3118(16,7%)
Tota b False + False - 1/18(5.5%) 7/69(10%)
116 (16.7%) 311 8 (16.7%) 116(16.7%) 2118(ll.1°/o) 116 (16.7%) 111 8 (5,5%) 116 (1 6.7%) 011 8 (0%)
111 8 (5.5%) 6169 (8.7%) 1/18(5.5%)2169(2,9%: 211 8 (1 1,1%) 1169 (1.4%) 311 8 (1 6,6%) 0169 (0%)
O 3 35 350
Mean Initial lnoculum (CFUIg)
Figure 3.1. Percent of negative control value for skimmed milk powder samples inoculated with S. Enteritidis at four levels (O, 10'. 1 o', and 10' CFU/g). Values shown are averages of three trial S. Endpoints were determined using optical density. Bar represents one standard deviation.
O 1 6 60
Mean Initial InocuIurn (CFUIg)
Figure 3 -2. Percent of negative control value for ground chicken samples inoculated with S. Enteritidis at four levels (0, IO', IO', and 10' CFU/g). Values shown are averages of four trials. Endpoints were determined using the FL500 fluororneter. Bar represents one standard deviation.
93
control value. The assay was consistently positive at the lowest inoculation level tested
(10' CFU/g) and there were no fdse positive or false negative results in any of the three
trials as determined by optical density measurements. For trial two, fluorescence was
measured using the MGM fluorometer. Only one of the six inoculated samples (102
CFU/g) was positive at the 70% cut-off. At the 75% and 80% cut-off, the number of
positive sarnples increased to three and four respectively. Duplicate fluorescence samples
from each inocuiation level were pooled and transferred into cuvettes. Optical density
measurements were taken, resulting in all three inoculation levels being positive at the
70% cut-off level. The MGM fluorometer may be very sensitive to small amounts of
media residue that are not completely removed dunng the washing step, which may
account for the readings observed. No false positives were observed for optical density or
fluorescence.
3 -4.3. Ground chicken samples:
Samples of ground chicken inoculated with four levels of S. Enteritidis (0, loO, IO', and
102 CFU/g) were evaluated by fluorescence with the F'L500 fiuorometer in tnplicate. A
subsequent attempt to confirm these results showed a positive test at al1 inoculation levels,
including the uninoculated samples. Two subsequent trials were conducted with an
uninoculated sample tested in triplicate, and a sample inoculated at 10' CFUfg (confirmed
by plate count), also tested in tnplicate. Al1 six uninoculated samples, as well as the
spiked samples showed strong positive results, confhming the results of the second trial. It
was suspected that the ground chicken sample was naturally contarninated with
Salmonella, which may be responsible for the false positives observed. To test this
hypothesis, the IMS samples fiom the unspiked chicken were retained and analyzed
according to the Health Protection Branch method for the isolation and identification of
Salmonella fiom foods (Anon., 1989). Two hundred microlitres of the magnetic beads
were transferred in duplicate into selenite cystine broth (DIFCO, Detroit, Michigan) and
tetrathionate brilliant green broth @ECO) and incubated ovemight at 37°C. Samples
were streaked ont0 brilliant green sulfa agar @ECO) and bismuth sulfite agar
@IFCO)and incubated overnight at 37OC. Suspect colonies were streaked ont0
MacConkey agar @ECO) and incubated ovemight at 37OC. Suspect colonies were
streaked ont0 Tryptic Soy Agar (DIFCO) and incubated ovemight at 37°C. Two suspect
colonies were biochernically characterized by analytical profile index (API) 20E
(Biomerieux, St. Laurent, Quebec) and serology. The first API profile indicated an
excellent identification of Sulmonelh spp. Serology testing indicated positive
agglutination with Anti-Salmonella Factor 8 (DIFCO, Detroit, Michigan) confirming the
presence of a group C Salmonella. The result of the second API was Proleus mirdilis.
The results of this testing indicated the ground chicken was naturally contarninated with
Salmonella. The pooled results of these four trials are presented in Figure 3.2. The
uninoculated samples which were responsible for the positive test were not included in
Figure 3 -2, or Table 2.1.
A second package of ground chicken was purchased in an attempt to demonstrate the
utility of the bacteriophage assay on a ground chicken sarnple which was not naturally
contaminated. This would also elirninate the possibility that matrix eEects, perhaps via
the trapping of phage in unspiked sarnples, were responsible for the false positives
observed. Ground chicken was tested as indicated above with one exception. Samples
were incubated in stomacher bags with a filter in an attempt to reduce the fat and
particulate material introduced into the assay tubes. In addition, each inoculation level
was tested in duplicate, and compared to negative control tubes (broth only), tested in
triplicate. Results are shown in Figure 3.3. There were no fdse positives or fdse
negatives observed at the 70% cut-off level. These results suggest that the 'false-
positive' results obtained in the previous trials with unspiked ground chicken may be the
result of natural Salmonella contamination, and not matrix effects of the food sampie.
Regarding the instrumentation used for determinhg the assay endpoint, optical density
measurements were consistent and reliable. In addition, fluorescence results for ground
chicken trials one through four were obtained using the FL-500 fluorometer. These
results were also very predictable, and distributions corresponded well to those obtained
by the spectrophotometer. In trial five and six, results were obtained using optical density
and the MGM fluororneter. The optical density results were as expected. The
fluorescence results did not correspond to the optical density readings, with inoculated
chicken samples showing much higher readings than the negative control and uninoculated
chicken samples
3 -4.4. Liquid whole egg:
O 4 29 290
Mean Initial lnoculurn (CFU/g)
Figure 3 -3. Percent of negative control value for ground chicken samples inoculated with S. Ententidis at four levels (0, loO, IO', and 102CFU/g). Values shown are averages of two trials. Endpoints were determined using optical density. Bar represents one standard deviation.
Mean Initial lnoculum (CFUIml)
Figure 3.4. Percent of negative control value for liquid whole egg samples inoculated with S. Ententidis at four levels (0, 1 oO, 1 o', and 102 C F U / ~ ~ ) . Values shown are averages of three trials. Endpoints were detennined using optical density. Bar represents one standard deviation.
The pooled results of three trials with liquid whole egg are illustrated in Figure 3.4. Al1
three triais were evaluated by optical density. Three of the negative control samples had
values rnuch lower than expected. This is presumably because of insufficient removal of
phage after the attachrnent step, resulting in an inappropriate reduction in helper ce11
population. As a result, the uninoculated samples averaged over 120% of the negative
control values. There was one false positive sample in trial two (Table 3.1). This value
was quite low, again likely due to inefficient phage removai. This sample contributed
greatly to the large standard deviation shown in Figure 3.4 for the unspiked samples. Two
false negative samples were obsenred in trial three at the 70% cut-off level (Table 3.1).
Interestingly, these false negative sarnples were observed at the rniddle inoculation level
(10' CFU/ml). Had one of the negative control samples been closer to the expected value,
these samples would Wcely have been positive. This highlights the importance of carefùl
and efficient removal of unbound phage fiom the tubes following the attachent step.
3 -5. Discussion
3 -5.1. General comrnents:
The purpose of this study was to test artificially inoculated food samples using the IMS-
bacteriophage assay. A cut-off of 70% of the mean negative value for each trial was
arbitrarily chosen to indicate a positive sample. This value was determined based on the
cut-off that gave the most acceptable number of false positive and false negative results,
when al1 of the food sample results were pooled.
False positive results Likely occur when unattached phage are not efficiently removed
during the washing steps. The phage may then be recovered in the supernatant and added
to the helper population, resulting in an undesirable reduction in ce11 numbers. This was
observed with the egg samples, where unexpectedly low values for three of the negative
control samples were observed. This highlights the great importance of thÏs washing step,
and the care that must be taken to maximize the removal of unattached phage.
The food matrix may be responsible for inhibiting the efficient removal of phage.
Unspiked samples sometirnes have reduced endpoint values compared to the broth only
controls (Figure 3.2). This dflerence is seldom responsible for a false positive result, but
suggests that the food matrix may somehow inhibit removal of phage during the washing
steps. This phage may be recovered later and effect a small change in helper relative to
the negative control.
Experimental trials are not meant to be directly compared between days, but for the
purpose of this report, results were pooled. Variations in incubation times and dserences
in inoculation levels between experimental trials may be responsible for some of the larger
standard deviations depicted in Figures 3.1 through 3.4. Values of replicate samples
within trials are generally very consistent.
In most cases, a final incubation time of 1.5 h was sufficient to distinguish positive from
negative samples. An evaluation of the actual absorbance values following the IMS assay
indicates the progeny phage will not imrnediately reduce the helper population below it's
original reading of 0.100. In most cases, growth of helper will occur in both positive and
negative samples, but the rate of growth will be slower in positive samples. After a 70 to
80 minute incubation, two rounds of infection have occurred and the values for positive
samples will generally decline, while the helper fiom negative samples will continue to
grow. Incubation cannot occur indefinitely, however, as srnaIl numbers of phage nom
negative samples may eventually 'catch-up' to the growth and effect a negative change in
these samples.
Results of the IMS-bacteriophage assay are presumptive, and plating of the beads would
provide a viable isolate for fûrther confirmation.
3.5.2. Skirnmed d k powder:
Many outbreaks have been associated with rnilk products (El-Ganar and Martb 19921,
including milk powders (Rowe et al., 1987). Skimmed milk powder was chosen as an
opportunity to test the assay in a processed food. The IMS-bacteriophage assay worked
consistently with the skimmed milk powder samples. The assay was positive over al1 three
trials at the lowest inoculation level tested (10' CFU/g).
Parmar et al. (1992) investigated an IMS-conductance test for the rapid detection of S.
Enteritidis and S. Typhimuriurn in skirnrned milk powder. Low numbers of Salmonella
(<20 CFU/ml) were detected in a total time of 13.5 hours, consisting of a 6 hour
preenrichment followed by IMS and conductance rneasurements. For lower ce11 numbers
(one cell in 25g) the authors suggest that ovemight enrichment followed by IMS and same
day conductance may be necessary.
Dziadkowiec et al. (1995) compared conventional methods with IMS-conductance for the
detection of Salmonella in skimmed milk powder emichrnents. The IMS-conductance
assay was positive following a minimum 5 hour pree~chment, when the Salmondla in
the sample had reached 50 celldml. The initial inoculum was very low (20 CFU in 250 ml
broth). These authors noted that non-Salmonella skirnrned rnilk flora increased fiom
lx1 o3 to 3x 10' CFU/rnl after 2 and 12 hours incubation respectively, while the Salmonella
count did not exceed 3x106 CFU/ml throughout the 24 hour e ~ c h r m n t period. This
suggests that after the 10 hour incubation in this current study potentially large numbers of
background flora are present, dernonstrating the utility of the IMS-bactenophage in the
presence of high numbers of background flora. Detection limits of these studies ptior to
IMS cannot be compared directly with the present study, as conductance requires an
additional incubation period after IMS .
3 -5 -3. Ground chicken:
The IMS-bactenophage assay was successful at detecting S. Enteritidis at the lowest
inoculation level tested (10' CFWg). It also appears that the assay identified SalmonelIa
in a naturally contarninated product. Al1 of the group C Salmonella strains that were used
in the specificity testing of bactenophage SJ2 (section 2.5.1) were negative by both the
plaque assay and the phage assay. Another possibility is the presence of multiple
Salmonella serovars in the ground chicken sarnple. This is quite possible, since the
chicken tested is a comminuted meat product and particularly susceptible to
contamination. Individual flocks have been reported to harbor up to five Salmonella
serovars. (J3arnhart et al., 199 1)
Coleman et al. (1995a) evaluated IMS for the detection of salmonellae in raw chicken
carcasses. They found that IMS was superior to traditional culture methods in isolating
salmonellae frorn naturally contaminated poultry. In contrast, a study by Ripabelli et al.
(1997) found that selenite cysteine was slightly better than IMS for recovery of
Salmonella nom pork and chicken samples, giving false negative rates of 16.6% and
33.3% respectively. Studies with IMS indicate that high fat levels c m lead to loss of
organisms at the separation stage (Skjerve and Olsvik, 1991; Coleman et al., 1995a).
They suggested the use of stomacher bags with an inner filter may dirninish the amount of
fat and particulate matter in the homogenate. This was observed in the present study with
the ground chicken. Ground chicken samples which had been incubated in Erlenmeyer
flasks almost invarïably had food particulate matter in the sample tubes. The food
particulate persisted through washing steps, being separated with the magnetic bead
portion of the sarnple. This seemed to interfiere with the assay. It is reasonable to assume
the presence of such particulates would hinder the attachment of the phage to the
captured target cells. Efficiency of Salmonella capture rnay also be reduced, as beads
become trapped in the particulate material. In addition to food particulates, fat in the
sample made the walls of the tubes rather 'sticky' and it was difficult to wash beads f?om
the sides of the tubes. The use of stomacher bags with a filter in the second set of trials
considerably improved the problem caused by food particulate although the effects of fat
were still apparent. Skjerve and OlNik (1991) found that IMS was not suitable for al1
food products tested. For some food products (eg. yoghurt and chicken liver), a
substantial loss of beads was observed during initial separation and washing steps. Other
foods produced a visually impure pellet (soft cheese), although most beads were still
recovered. Preliminary studies with changes to broth and washing buffers using different
proteins and detergents were found to be ineffective.
When the IMS product fiom the unspiked ground chicken sample was tested by
conventional methods, a group C Salmonella was confirmed to be present. In addition,
Proteus mirubilis persisted throughout the isolation procedures as confirmed by API
identification. Several other isolates fiom the selective media showed negative
agglutination with polyvalent Salmonella anti-sera poly IA and Vi @ECO. Detroit, MI).
Other authors have reported problems of specificity with the magnetic beads, indicating
interference of salmonellae binding by coliforms, Citrobacterfrez~ndii, pseudomonads and
Proteus spp. (Coleman et al., 1995b; Cudjoe et al., 1995). The time and temperature of
incubation, the media used and the bactenal strain can affect irnmunological and non-
specitic binding to irnrnunomagnetic particles (Blackburn, 1993).
The combined effects of fat and particulate matter as well as the non-specific binding of
non-Salmonella organisms to the beads may have an effect on the magnitude of the
positive reaction seen in the second round of ground chicken samples by reducing the
efficiency of target ce11 capture and subsequent attachent of pnage. Conversely, the
trapping of phage by fat and food particulates may contribute to the reduction in the
helper population seen in uninoculated food samples, relative to the negative control.
Non-specific binding should not produce false positive results because of the specificity of
the bacteriophage employed.
3 5 4 . Liquid whole egg:
Grade A shell eggs have emerged as major sources Salmonella infection. Seventy-seven
percent of 35 S. Enteritidis outbreaks in the US between 1985 and 1987 were linked to
eggs alone or egg-containing foods (St. Louis et al., 1988).
Isolation of Salmonella fiom eggs presents a special problem because egg albumin
contains one or more factors that can interfere with detection (Andrews, 1996). The
growth of bacteria in egg albumin is limited by the presence of lysozyme, enqme
inhibitors such as ovomucoid, avidin, conalbumin and a high pH (Banwart, 1979). Such
stresses may necessitate a long preenrichment to allow recovery of injured cells.
Stephenson et al. (1991) investigated the recovery of S. Enteritidis fkom shell eggs and
noted that egg albumin seemed to inhibit survival, as numbers of Salmonella inoculated
irito egg albumen decreased during refngerated storage. Salmonella introduced into the
egg yolk, however, increased under the same storage conditions, indicating possible
growth of Salmonella in this rnatrix. While Stephenson and others (Hammack et al.,
1993) suggest testing yolk, others report the albumen to be more frequently contaminated
(Humphrey et al., 199 1).
Holt et al. (1995), investigated a magnetic bead-ELISA systern for the detection of
Salmonella Ententidis in pooled liquid egg samples. The IMS-ELISA was compared to
an IMS-direct protocol, where the lMS product was plated on brilliant green agar
supplemented with novobiocin. They found that 100% of pooled egg initially
contaminated with 10 cells per ml were positive following a 24 h enrichment at 37OC. In
pooled egg initially contaminated with 1 ce11 per ml, 61% and 72% were positive by IMS-
ELISA and IMS-direct respectively. A PCR procedure for detection of Salmonella in
whole shell egg by Burkhalter et al. (1995), indicated a detection Iirnit of 1-10 ceils per
egg when a 16-24 h preenrichment and selective enrichment were used. This present
assay compares favourably with these results.
3.6. Conclusion:
One false positive and two false negatives were observed out of the 18 negative, and 69
positive samples tested, al1 from liquid whole egg. The IMS-bacteriophage assay was
positive at an initial inoculation Ievel of 10' CFU/ml for al1 three food types tested.
Efficient removal of unbound phage was proven to be criticai to the success of the assay,
since the results of food samples were compared to negative controls tested with each
trial. Optical density is the preferred endpoint, given the flexibility in extending the
incubation period and the reduced sample manipulation required. The FL-500 fluorometer
provided consistent results that were veiy sirnilar to that obtained with a
spectrophotometer. The MGM fluorometer, while being the most sensitive o f the three
instruments in terms of lower detection lirnit, did not provide reliable results when applied
to the food samples. The IMS-bactenophage assay was demonstrated to be a rapid and
sensitive test for the deteetion of Salmonella Ententidis in skimmed milk powder, ground
chicken, and iiquid whole egg.
4. Application of the IMS-bactenophage assay to the detection of Escherichia coli 0157:H7 in ground beef.
Escherichia coli 0 157:H7 gained widespread attention as a human pathogen following a
large US outbreak in 1992 involving contaminated hamburgers. Ulness caused by E. coli
O I57:H7 can range Eom watery diarrhea, to life-threatening conditions such as hernolytic
uremic syndrome or thrombotic thrombocytopenic purpura. Ground beef is frequently
identified as the vehicle of transmission in E. coli 0157:H7 outbreaks. The purpose of
this study was to investigate the application of a recently developed immunomagnetic
separation ( M S ) bactenophage assay, originally developed for the detection of
Salmonel[a Enteritidis, to Escherichia coli 0 157:H7. After demonstrating the assay
could be successfully applied to pure cultures of E. coli 0157:H7 in broth, ground beef
was tested at two inoculation levels. The ground beef assay was positive for the lowest
inoculation level tested (2.5 CFU/g). The results of this study dernonstrate that the IMS-
bacteriophage assay can easily be applied to the detection of other pathogens in foods.
4.2. Introduction
Escherichia coli is considered part of the normal intestinal flora of humans and other
warm-blooded animals, generally residing as harrnless commensals. Some strains are
pathogenic and cause distinct diarrhed syndromes. Foodbome diarrheagenic strains are
divided into four groups baseà on virulence properties, clinical syndromes, and distinct
O:H serogroups (Padhye and Doyle, 1992). The first main category is enteropathogenic
E. coli (EPEC), which is mainly associated with neonatal and infantile diarrhea. Adult
carriers are usually asyrnptomatic. The second group are enteroinvasive E. coZi (EEC)
which invade epithelial cells resulting in bloody diarrhea. This group of organisms are
similar to shigellae. The third group of pathogenic E. coli is enterotoxigenic E. coli
ETEC), which is responsible for watery diarrhea caused by adherance to the intestinal
epithelial and production of one or more toxins. The fourth category is
enterohemorrhagic E. coli (EHEC), which includes E. coZi 0157:H7. A principal
manifestation of E. coli 0 157:H7 is hemorrhagic colitis, characterized by sudden onset of
abdominal cramps, and watery diarrhea which later becomes grossly bloody. Other
manifestations include hemolytic uremic syndrome (HUS), and thrombocytopenic purpura
(TTP). HUS is a combination of hemolytic anemia, thrombocytopenia, and renal failure
that can occur acutely on otherwise healthy individuals. TTP is similar to HUS except
that the central nervous system is involved (Padhye and Doyle, 1992). Although human
illness associated with E. coli 0 157:H7 is infiequent compared to other pathogens such as
Salmonella, the severity of clinical symptoms and the potential for serious complications
and death, makes it a noteworthy food safety issue (USDA, 1994).
Various types of foods have been implicated in 0157 associated illness. The rnajority
(71%) have been linked to bovine products including ground beet raw milk and roast beef
(USDA, 1994). Cross-contamination of other foods such as apple cider, vegetables and
mayonnaise have been confirmed or suspected in other outbreaks &JSDA, 1994).
Improvements to culture media have been successful in irnproving the recovery of E. cdi
0157:H7 from foods. Weagant et al. (1995) describe a revised e~chmen t and agar-
plating system involving a 6 h enrichrnent in EHEC enrichrnent broth (modified tryptic soy
broth supplemented with vancomycin, cefsulodin, and cefixime) foilowed by spread plating
on sorbitol-MacConkey agar supplemented with tellurite and cefixime (TCSMAC).
The use of immunomagnetic separation combined with electrocherninuminescence (ECL)
was investigated for the detection of E. cok 0157 in food (Yu and Bruno, 1996).
Artificially inoculated rnilk, juices, senim. and supernatant fluids from ground beef, minced
chicken and fish were evaluated by IMS-ECL, giving a detection lirnit of 1000 to 2000
bacteria per ml of food. Detection limits in buEer ranged from 100 to 1000 bacteria per
ml. IMS was found to be more sensitive than direct culture for isolation of E. coli O 157
from inoculated meat samples and artificially mixed cultures (Wright et al. 1994;
Fratamico et al. 1993).
Nucleic acid based tests are available for the detection of E coli 01 57:H7. Polymerase
chain reaction (PCR) is a sensitive and highly specific method based on the amplification
of specific DNA fragments. Meng et al. (1996) describes a PCR protocol that was
successful in detecting as few as 25 CFU of E. coli 0157:H7 in three hours.
These test suffer some of the same drawbacks as described for SaIrnonelln tests including
lack of specificity, sensitivity, and tirne and technical ski11 required.
Goodridge (1997) developed a fluorescent bacteriophage assay for the detection of E. coli
O L57:H7 in foods, based on the work of Hemes et al. (1995). Bacteriophage DNA was
labeled with the fluorescent dye YOYO-1. Attachment of bacteriophage to target celis
could be visualized by epifluorescent mîcroscopy or quantified by flow cytometry. An
initial inoculum of 2.2 CFU/g in ground beef and between 10' and 1 O2 CFU/rnl in raw milk
could be detected by flow cytometry.
This present study involves the application of an IMS-bacteriophage assay, originally
developed for the detection of Salmonella Enteritidis, to the detection of E. coli O 157:H7
in ground beef
4.3. Materials and methods
4.3.1. Bacteriophage and host:
Escherichia cok 0 157:H7 strain EC920333, obtained from Health Canada, was used in
this study. Culture was maintained as described in section 2.3.1. The bacteriophage used
in this study, LG1, was obtained from Lamy Goodridge, in the Department of Food
Science, University of Guelph. The phage was amplified in its host as descnbed in section
4.3.2. Procedure for analysis:
The detection protocol was carried out as described for the Salmonella assay described in
section 2.3.4 with a few exceptions. Immunomagnetic separation was achieved using
~ y n a b e a d s ~ anti-E. coZi 0 1 5 7 (Dynal Inc., Lake Success, New York). Preliminary tests
indicated that phage LGl did not appear to be as infectious as phage SJ2. Perhaps phage
LGI has a longer burst time, or progeny phage are released more slowly. In any case, the
phage amplification step during the assay occurred in broth and the incubation time was
extended to one hour. The phage population used was 4x10' PFU/lOOpl, and the helper
population was and ovemight culture of E. coli 0157:H7 EC920333 adjusted to an
optical density of 0.100. The endpoint of the assay was determined by absorbance at 600
nm. A negative control consisting of broth only, and four initial inoculation levels (106,
105, IO', and 1 O3 CFU/ml) in broth were tested in duplicate.
4.3 -3. Food enrichment preparation:
Extra lean ground beef was purchased fiom a local supermarket. An ovemight culture of
E. coli 0 157:H7 was diluted 10-fold .in lambda buffer. Twenty five gram samples of
ground beef were inoculated with 1 ml of E. coli 0157:H7 to give inoculation levels of
102 and 10' CFU/g, as determined by tripiicate plate counts. A negative (unspiked) beef
sample was tested as well. Samples were placed in a stomacher bag with a filter and 225
ml of L-broth was added. Samples were homogenized by hand and incubated for 12 hours
at 37°C. Each sample, including negative controls consisting of broth only, was tested in
duplicate.
4.4. Results
4.4.1. Sensitivity test:
The assay was performed using four levels of E. coli 0157:H7 in broth. Triplicate plate
counts codinned the initial populations to be between 8.5~10' and 8 . 5 ~ 1 0 ~ CFU/ml. The
results are iuostrated in Figure 4.1. Based on the 70% cut-off determined in section
3 -4.1 ., the 8 . 5 ~ 1 0 ~ CFU/ml sarnple was negative, but the next lowest dilution was positive,
indicating that the detection IKnit is about 10' CFU/ml.
4.4.2. Ground beef:
The results of the ground beef experiment are illustrated in Fi y r e 4.2. Based on the 70%
cut-off for positive samples established in Chapter 3, ground beef samples were positive at
both inoculation levels, with no fdse positives observed.
4.5. Discussion
The purpose of this study was to investigate the feasibility of applying the IMS-
bacteriophage assay developed for Salmonda Enteritidis to the detection of another
foodbome pathogen. The application of the assay for the E. coli 0 1 57:H7 host and phage
was quick and simple, requiring only a few minor modifications. Since the characteristics
of different bacteriophage and subsequently the dynarnics of the host/phage relationship
8.5~1 O' 8 S x l O' 8 .5~1 os 8Sxlo6
Mean Initial Population (CFUlml)
Figure 4.1. Percent of negative control value for four populations of Ecoli O157:H7 in broth following IMS-bacteriophage assay. Endpoints were deterrnined by optical density. Bar represents one standard deviati on.
O 2.5 220
Mean Initial lnocuturn (CFUIg)
Figure 4.2. Percent of negative control value for ground beef samples inoculated with E - d i 0157:H7 at three levels (0, 1 oO, and 10' CFU/g). Endpoints were determined by optical density. Bar represents one standard devi ati on.
would v q , assay parameters would have to be optùnized for each new system
investigated.
The sensitivity of the E. coli 0 157337 assay for pure cultures in broth (104 CFU/ml) was
very similar to the detection iimit determined for the S. Ententidis assay.
Wright et al. (1994) reported that an initial inoculum of 2 CFU/g of E. coli 0 157337 in
minced beef could be detected by an IMS-direct plating protocoi following a 24 hour
pree~chment. This present assay had a comparable detection limit (2.5 CFU/g) with only
a 12 hour e~chrnent. An initial inoculurn lower than 2.5 CFU/g was not tested. Perhaps
the detection lirnit would actually be lower. The sensitivity of an ELISA developed for the
detection of E. coii 0157:H7 was found to be 0.2 to 0.9 cells per g in ground beef
(Padhye and Doyle, 1995). A similar detection limit (0.76-2.64 CFU/g) was determined
by an evaluation of the TECRA irnmunoassay for E. coli 0 157:H7 in dairy products (Flint
and Hartley, 1995). Again, this assay compared favorably.
4.6. Conclusion
The IMS bacteriophage assay, developed for the detection of S. Ententidis, was applied to
the detection of E. coli 0157:H7 in ground beef. The assay, employing the use of phage
LG1, was successful at detecting 2.5 CFU/g of E. coli 0157:H7 present initially in ground
beef. The total detection tirne was under 17 hours. The results of this study ïndicate that
the IMS-bacteriophage assay can be easily adapted to other foodbome pathogens
provided an appropriate bacteriophage is available.
5. Conclusion
Recent trends in SulmoneIIa infection suggest it will continue to be a public health
challenge (Tauxe, 1991). Poulhy, eggs and milk have been fiequently identified as
vehicles of infection. IZapid, simple and sensitive detection rnethods can play a role in
reducing the incidence of salmonellosis by preventing the sale of contarninated product. A
combination of immunomagnetic separation (IMS), bactenophage, and two assay
endpoints were investigated for the detection of Salmonella Enteritidis; first in broth, then
in food samples. Experiments with pure cultures in broth indicated that the Iower
detection limit of the assay was approximately 10' CF'U/ml. The assay detected al1 of the
S. Enteritidis strains tested, one of the S. Typhimurium strains tested, S. Sendai, and
showed weak reactions with two other group D Salmonella (S. Berta, and S. Panama).
Optical density is the preferred endpoint because of the ease of use and the flexibility it
imparts.
Artificially inoculated skimmed milk powder, ground chicken, and liquid whole egg were
evaluated by the IMS bactenophage assay. Three CFU/g or ml present initially in the food
samples were successfully detected in al1 of these foods. One false positive, and two false
negative results were obsenred for the liquid whole egg samples.
Since no special manipulation of the bacteriophage is required, it was reasoned that the
assay could be easily and rapidly applicable to other phage and host combinations. This
was demonstrated with the LGI phage and its host E. coli 0157:H7. The detection limit
in broth appeared to be very similar to the lirnit determined for the S. Entedidis assay.
The E. coli IMS successfülly detected E. coli 0157:H7 in ground beef at the lowest
inoculation level tested (2.5 C M g ) following a 12 h e ~ c h m e n t . The total time for
detection was under 17 hours.
The IMS bacteriophage assay has several advantages. Bacteriophage are very inexpensive
to pro~ahn+a ,,., ,.d an maintzii.. There is no manipulation of the phage, genetic or otherwise,
required; the assay simply exploits the normal infection cycle of the bacteriophage in it's
host. This makes the assay easily and rapidly applicable to other phagehost systems as
indicated by the E. coli 0 157:H7 assay. The assay is technically simple to perfomi, and
can be compieted in about four hours.
5.2. Limitations of the assay
A potential stumbling block in the assay is the eficiency of phage removal. This step is
absolutely critical, as false positives have been observed when unattached phage is not
sufficiently removed. Since the food sampies are compared to the mean of the negative
control samples to determine a positive or negative test, any inappropriate decrease in the
helper population in negative samples will influence the cut-off for a positive test, perhaps
leading to false negative results. Great care m u a be taken to ensure maximum removal of
unattached phage, without the loss of beads.
The current assay is also not suitable for the processing of large numbers of samples.
Twelve samples is the highest number of samples that can be comfortably processed at one
time, as the magnetic rack used in this study can accornrnodate 12 tubes. In addition,
increasing the number of samples tested will require more tirne for the washing steps. This
becomes important when considering that progeny phage are released in about 30 minutes
following infection. A 15 minute incubation for phage attachent leaves only 15 minutes
for the washing steps. It would be difficult to wash more than 12 tubes in that time
period. The tirne for attachent could be reduced to accornrnodate this, however.
It has been suggested in the literature that IMS is not suitable for al1 food types. Food
particulate matter and high background flora can inhibit the binding of target organisms,
although this was not thought to be a significant problem with the food samples tested in
this study. The specificity irnparted by the bactenophage should overcome any problems
of non-target organisms binding non-specifically to the immunomagnetic beads.
The IMS bacteriophage assay should be considered a presumptive test, and culture
confirmation should accompany a positive test. The IMS product can be plated on
selective media, and suspect colonies confirmed by serology.
5 -3. Future research
The total detection time of the assay is about 14-20 hours, depending on the
pree~chment tirne used. Manipulation of the assay steps, and shortened enrichment
times could be investigated to determine the potential of the IMS bacteriophage assay as a
same-day test. The potential use of a fieeze-dried helper population should also be fùrther
investigated to eliminate the need for maintenance of Solmonella cultures, and the
potential problems that would pose in an industriai food laboratory.
A washing step is currently required pnor to fluorescent staining to remove the broth
media which interferes with the stain. An alternative medium, which facilitates the growth
of the helper population in negative samples, and the reduction in ce11 numbers in positive
samples without interferhg with the fluorescent dye should be investigated. Such a
medium would reduce sample manipulation time, and perhaps provide the flexibility of
extending the helper incubation times, which is the case when optical density is used as
the endpoint.
Testing bacteriophage SJ2 against additional Salmonella serotypes would further
characterize the host range of the phage. It is clear however that SJ2 is not suitable for a
genenc Salmonella test, nor is it specific for S. Ententidis. A bacteriophage with a more
suitable host range, such as Felix-01, should be considered for the assay. The utility of
the assay in other food types, such as carcass nnses and raw milk, should also be
investigated.
A modified capture step, using bactenophage îmmobilized on magnetic beads should be
investigated. Some success of passive irnmobilization of phage on solid surfaces for the
capture of Salmonella has aiready been reported in the literature. This would be a
significant improvement over the current protocol. If phage could be immobilized by the
head, leaving the tail fibres free for capture and subsequent infection, the problerns
associated with residual phage in the tubes would be elirninated. The total assay tirne
would aiso be reduced, as target ce11 capture and infection would occur simultaneously.
Other formats for the assay c m be evaluated, such as a microtitre format or the use of
antibody coated tubes.
The experiments with E. coli 0157337 indicate that the IMS bacteriophage assay can
easily be applied to other foodbome pathogens. The potential for the assay to be adapted
to other pathogens, such as Lisferia and Campylobacter, should be investigated. There
are potential diagnostic applications as well, such as the detection of antibiotic resistant
organisms.
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