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FEMS Microbiology Ecology 48 (2004) 157–167
www.fems-microbiology.org
Antarctic bacteria inhibit growth of food-borne microorganismsat low temperatures
Andrea O’Brien a, Richard Sharp a, Nicholas J. Russell b, Sibel Roller a,*
a Microbiology Research Group, Faculty of Health and Human Sciences, Thames Valley University, 32–38 Uxbridge Road Ealing, London W5 2BS, UKb Department of Agricultural Sciences, Imperial College London, Wye Campus, Ashford, Kent TN25 5AH, UK
Received 22 September 2003; received in revised form 8 January 2004; accepted 8 January 2004
First published online 11 February 2004
Abstract
The aim of this study was to identify Antarctic microorganisms with the ability to produce cold-active antimicrobial compounds
with potential for use in chilled food preservation. Colonies (4496) were isolated from 12 Antarctic soil samples and tested against
Listeria innocua, Pseudomonas fragi and Brochothrix thermosphacta. Thirteen bacteria were confirmed as being growth-inhibitor
producers (detection rate 0.29%). When tested against a wider spectrum of eight target organisms, some of the isolates also inhibited
the growth of L. monocytogenes and Staphylococcus aureus. Six inhibitor producers were psychrotrophic (growth optima between 18
and 24 �C), halotolerant (up to 10% NaCl) and catalase-positive; all but one were Gram-positive and oxidase-positive. The inhibitors
produced by four bacteria were sensitive to proteases, suggesting a proteinaceous nature. Four of the inhibitor–producers were shown
to be species of Arthrobacter, Planococcus and Pseudomonas on the basis of their 16S rRNA gene sequences and fatty acid com-
positions. It was concluded that Antarctic soils represent an untapped reservoir of novel, cold-active antimicrobial-producers.
� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Antarctica; Psychrotrophic; Food-borne; Bacterial growth inhibition; Bacterial diversity
1. Introduction
The production of antimicrobial compounds such as
bacteriocins by lactic acid bacteria is widely recognized
and there is much interest in developing novel applica-
tions for these natural agents in the food, cosmetic andpharmaceutical industries [1]. However, the antagonistic
properties of cold-loving organisms have not been in-
vestigated as extensively as those of the mesophiles.
Unlike the inhibitors produced by mesophiles, the an-
timicrobials produced in cold environments need to
function at low temperatures for the organisms to gain a
competitive advantage during their growth cycle. Such
cold-active antimicrobial compounds may be exploit-able in industrial applications including chilled-food
preservation.
* Corresponding author. Tel.: +44-20-8280-5108; fax: +44-20-8280-
5289.
E-mail address: sibel.roller@tvu.ac.uk (S. Roller).
0168-6496/$22.00 � 2004 Federation of European Microbiological Societies
doi:10.1016/j.femsec.2004.01.001
Antarctica is a rocky continent almost completely
covered by a massive ice sheet, with regions of cold
desert soils that have little free water and temperatures
that rarely rise above freezing. The marine soils are free
of snow and ice in the summer months and receive in-
puts from the ocean and animal life [2]. In coastal areasseal and penguin rookeries may contribute significant
quantities of organic material to soils, known as or-
nithogenic soils; although they are high in nutrients,
such soils are marked by rapid freeze–thaw cycles, which
are more lethal than a permanently cold environment
and hence restrict life. Microbial survival and growth in
Antarctic soils are limited not only by low temperatures
but also by low aw and osmotic stress [3]. The transitionfrom winter to summer involves a freeze–thaw phase,
which is critical for the onset of microbial activity. The
microorganisms need to be efficient at rapidly switching
their metabolism on and off according to prevailing
conditions. In view of the severe environmental
conditions, it could be argued that the production of
. Published by Elsevier B.V. All rights reserved.
158 A. O’Brien et al. / FEMS Microbiology Ecology 48 (2004) 157–167
extracellular antimicrobial compounds would be a par-
ticular advantage in order to reduce inter-species com-
petition.
The aim of the present study was to identify Antarctic
microorganisms with the ability to produce cold-activeantimicrobial compounds with potential for use in
chilled-food preservation. Both classical and molecular
methods were used to characterize and identify a small
number of inhibitor producers with potential for use at
low temperatures. Since the extent of microbial species
and diversity in soil varies with the location and is in-
fluenced by salinity, pH and the availability of moisture
and nutrients, a number of soils from different Antarcticsites have been screened.
2. Materials and methods
2.1. Bacteria and their cultivation
Antarctic bacteria were maintained in dilute tryptonesoya broth/agar (DTSB/DTSA, one-tenth the strength
of TSB/TSA); marine broth/agar (MB/MA); and Luria–
Bertani broth/agar (LBB/LBA) containing tryptone (10
g l�1), yeast extract (5 g l�1) and NaCl (5 g l�1) with agar
(15 g l�1) in solid media. Indicator bacteria used for
detecting antimicrobial activity were maintained on
tryptone soya broth/agar (TSB/TSA) and nutrient
broth/agar (NB/NA). Overnight cultures for antimicro-bial testing were prepared using Listeria innocua NCTC
10528 and NCRZ 4202, L. monocytogenesNCTC 11984,
Brochothrix thermosphacta NCTC 2304 and Pseudomo-
nas fragi (Leatherhead Food International, UK), all
Table 1
Numbers of bacteria cultured from Antarctic soils incubated on dilute trypto
at 10 �C for 5 days
Location Description
Nutrient-rich, ornithogenic
Cape Hallett Disused penguin rookery
Edmonson Point (a) Penguin rookery
Kay Island Entrance to snow petrel nesting site
Cape Russell Exposed guano-covered ridge
Low altitude (<500 m)
Edmonson Point (b) Maritime soil
Lake Hoare Polygon-crack silt in dry valley
Harrow Peaks Light gravel
Crater Circe Raised lake beach/pond
High altitude (>500 m)
Battleship Promontory Scree beneath rockface with endoliths
Mount McGee Coarse gravel soil
Mount Rittmann Moist clay silt/soil
Mount Melbourne North flank, exposed volcanic soil
NG, no growth.a Incubated for 8 weeks.
grown at 30 �C; and Escherichia coli O157:H7 (attenu-
ated strain, Unilever Research, UK), Salmonella enterica
sv. Enteritidis (13244WT University of Bath, UK) and
Staphylococcus aureus NCTC 10788 grown at 37 �C. All
microbiological media were from Oxoid (Basingstoke,England) except MB/MA, which was from Difco (De-
troit, USA).
2.2. Isolation of Antarctic bacteria and detection of
inhibitor production
Soil samples were collected aseptically in Antarctica
from the locations given in Table 1 (72� 190S to 77� 830S;160� 550E to 170� 160E) by one of the authors (NJR)
during the 1995/1996 field season and were stored at )80�C. For isolations the soil samples were defrosted and 1
g of each was inoculated into 50 ml DTSB, MB and LB.
The samples were incubated at 10 �C with shaking for 24
h. Those turbid samples, indicating growth, were di-
luted, plated on their respective agars and incubated at
10 �C for 5 days or longer as necessary. Colonies werecounted and their morphological characteristics re-
corded. Master plates were made by randomly picking
over 4000 colonies from the three types of agar. Five of
the soil samples were also incubated at 5 �C for up to a
month using the same method.
Inhibitor production was determined using the de-
ferred antagonism procedure of Kekessy and Piguet [4].
Replica plates were made from the master plates using aMultipoint Inoculator (Sigma Chemical Co., UK).
Replica plates were prepared using the same agar as that
used for isolating the organisms on the master plates
(e.g. Marine Agar was used to prepare replica plates for
ne soy agar (DTSA), marine agar (MA) and Luria–Bertani agar (LBA)
Altitude (m) Bacterial numbers (log cfu g�1)
DTSA MA LBA
Sea level >9.0 6.8 >9.0
50 7.3 7.5 6.7
�80 9.3 6.0 6.3
�600 8.3 8.3 8.3
Sea level NGa 7.0 NGa
�150 4.3 3.7a 8.9
200 6.5 7.9 8.0
500 6.0 6.0 5.8
1000 NGa 9.0 >9.0
1410 4.8 4.8 5.0
2000 NGa >9.0 8.3
2650 4.0a <3.0a 6.3
A. O’Brien et al. / FEMS Microbiology Ecology 48 (2004) 157–167 159
organisms isolated on Marine Agar master plates). The
replica plates were overlaid with soft TSA containing
one of eight indicator organisms. Zones of clearance
surrounding the producer colony following incubation
at 10 �C (or 15 �C when Staph. aureus, E. coli O157:H7and S. Enteritidis were used) for 4/5 days indicated the
presence of an antagonistic agent.
2.3. Sensitivity of inhibitors to enzymes
The enzymes trypsin (EC 3.4.21.4) in 67 mM sodium
phosphate buffer (pH 7.6) and pronase E (EC 3.4.24.31)
in 50 mM phosphate buffer (pH 7.5) were used to de-termine whether the inhibitory substances produced by
the Antarctic bacteria were proteinaceous. Catalase (EC
1.11.1.6) in 50 mM phosphate buffer (pH 7.0) was used
to eliminate inhibition by hydrogen peroxide. Lipase
(EC 3.1.1.3) in 50 mM phosphate buffer (pH 7.7) and a-amylase (EC 3.2.1.1) in phosphate buffer (pH 6.9) were
used to determine the presence of lipid or glycogen
components in the inhibitory substances. Final concen-tration of all enzymes was 25 mg ml�1. Deferred an-
tagonism assays were performed by placing a 5 llaliquot of the enzyme solution next to the fully grown
producer-cell spot on an agar plate and incubating at 37
�C (or 20 �C for a-amylase) for 1 h before overlaying the
agar with the target organism. Sensitivity of the inhibi-
tor to the enzyme was indicated when the zone of inhi-
bition around the producer colony was altered, giving a‘‘half-moon’’ appearance.
2.4. Characterisation and identification of Antarctic
bacteria
Bacteria were identified initially on the basis of their
Gram reaction, morphology (by light microscopy), plus
their catalase and oxidase reactions tested according toRoberts [5]. The cardinal growth temperatures (i.e.
minimum, optimum and maximum) were determined on
DTSA or MA according to the method of White et al.
[6]. Briefly, a purpose-built vertical temperature gradient
incubator (range 3.3–40 �C) with 2.5 �C increments be-
tween the shelves was used. The time taken for the ap-
pearance of colonies visible to the naked eye was used as
the measure of growth. Agar plates were monitoreddaily for signs of growth. Results were plotted as growth
rate in days�1 versus temperature of growth. Salt gra-
dient plates (1–10% NaCl) were made in 25 cm� 25 cm
square Petri dishes according to Venables et al. [7].
Bacterial isolates were streaked from high to low salt
concentrations on the plates and incubated at 4, 10, 22,
30 and 37 �C for 7–10 days. Growth was scored as the
distance in cm reached by visible colonies across thegradient.
The fatty acid composition of selected Antarctic
bacteria was determined by gas chromatography ac-
cording to the method of Kates et al. [8]. Cultures were
grown on DTSA or MA for 5–7 days at 10 �C. Fattyacid methyl esters (FAME) were prepared with 2.5% (v/
v) sulphuric acid in dry methanol and analysed using a
Hewlett–Packard 5880A gas chromatograph fitted witha Supelco 2380 polar fused silica capillary column and a
Spectraphysics SP420 integrator. The carrier gas was
nitrogen (flow rate 1 mlmin�1) with detection using
flame ionisation. Detection and injection temperatures
were 250 �C. Oven temperatures were set initially at 130
�C for 10 min and then programmed to rise at 4
�Cmin�1 to 250 �C, which was held for a further 15 min.
Individual peaks were identified by comparison of re-tention times with standards (14:0, 16:0, 18:0, 16:1 and
18:1) and confirmed by sequential hydrogenation with
platinum dioxide/H2 gas (to reveal unsaturated com-
ponents) and bromination with bromide/N2 gas (to
identify cyclopropane fatty acids).
Selected bacterial isolates were also subjected to
partial sequencing of their 16S rRNA gene. Briefly,
DNA was extracted from exponential phase culturesusing phenol–chloroform extraction according to the
method of Sambrook and Maniatis [9]. A set of primers
was designed based on probe sequences made from en-
vironmentally derived and taxon-specific 16SrRNA se-
quences [10,11]. PCR reactions were performed in a
Techne Progene PCR thermocycler as follows: 94 �C for
2 min, denaturation at 94 �C for 30 s, annealing at 45 �Cfor 45 s and extension at 72 �C for 2 min, for 30 cycles.The PCR product was ligated into the pGem-Teasy
vector and transformed in CaCl2-competent E. coli
(Promega, Madison, WI, USA). Plasmid DNA of six
clones was isolated and sequenced (MWG Biotech) us-
ing an Automated Biosystems Sequencer. The 16S
rRNA gene sequences obtained ranged in size from 920
to 940 bp. Sequences were identified using the Ribo-
somal Database Project (RDP) 16S rRNA gene se-quence database and BLAST searching of the NCBI
database. To further analyse the relationships of these
organisms, the percent sequence similarities of the 16S
rRNA genes were calculated using the CLUSTAL V
programme of the MegAlign package from DNA Star
Software, Editseq. (DNA Star Inc., USA).
3. Results
3.1. Isolation of bacteria from Antarctic soils
Three types of media were used for the isolation of
bacteria from Antarctic soils: dilute tryptone soya broth
and agar to isolate bacteria from nutrient-deficient soils,
marine broth and agar to isolate bacteria with high saltrequirements, and Luria–Bertani broth and agar to
isolate fastidious bacteria. Two temperatures were used,
10 and 5 �C, in order to isolate both psychrotrophic and
160 A. O’Brien et al. / FEMS Microbiology Ecology 48 (2004) 157–167
psychrophilic organisms. Table 1 shows the viable bac-
terial counts obtained from twelve soil samples on the
three different media after 5 days at 10 �C. Ornithogenic
soils are rich in nitrogen and phosphorus due to the
deposition of guano by penguins: one would anticipate,therefore, that these soils would be capable of support-
ing good microbial growth. As shown in Table 1, all
four ornithogenic soil samples in this study (penguin
rookery at Edmonson Point, Cape Russell, Kay Island
and Cape Hallet) yielded relatively high bacterial counts
(6–9 log cfu g�1) on all media. Very high counts (10
log cfu g�1) determined in ornithogenic soil by direct
microscopic counting have been reported previously butonly about 20% of these could be cultured [10]. Total
counts obtained by microscopy may be misleadingly
high due to the presence of dead cells and other debris.
Maritime soil samples from Edmonson Point gave no
growth on DTSA or LBA, whereas on MA counts of
more than 7 log cfu g�1 were obtained. This indicated
that a halotolerant group of organisms dominated in
this region and is a reflection of the maritime nature ofthe soil, which had been collected at sea level. Maritime
soils often have additional nutrients available from
coastal macrofauna, penguins and other seabirds and
seals. Lake Hoare samples gave low counts on both
DTSA and MA (3–4 log cfu g�1); however, a high count
of 8 log cfu g�1 was obtained on LB agar suggesting that
the microflora was nutritionally fastidious. The Harrow
Peaks and Crater Circe samples produced medium-level(6–8 log cfu g�1) counts on all three agars (Table 1).
The Battleship Promontory sample supported the
growth of lichens beneath the rock face, hence providing
nutrients for a microbial population. In this sample, no
bacterial growth was detected on DTSA but very high
numbers (9 log cfu g�1) were observed on MA and LBA.
The Mount Rittmann sample, consisting of moist clay/
silt, also yielded no growth on DTSA but high numbers(8–9 log cfu g�1) onMA and LBA. It is interesting to note
that the apparently halophilic populations from these two
samples were from sites that were 1000–2000 m above sea
level. It is often the case in Antarctic soils that with
freezing of water, there is an increase in solute concen-
tration in the soil resulting in creation of a hypersaline
micro-environment [11]. High altitude soils in the Trans-
Antarctic mountains provide the least hospitable envi-ronment for microbial growth since temperatures rise
above freezing for only brief periods each year. Mount
McGee soil at over 1000 m above sea level contained low
levels of organisms (4–5 log cfu g�1) on all three media.
The numbers of viable bacteria isolated from the
Antarctic soils after incubation at 5 �C for one month
were much lower (3–4 log cfu g�1 and often <3
log cfu g�1) than at 10 �C. It has been observed previ-ously that it is difficult to isolate true psychrophiles even
at lower temperatures [6]. This is probably because of
the occasional high ground temperature and the fact
that the samples in this study were all collected in the
summer months. True psychrophiles are more often
found in consistently cold environments such as the
Southern Ocean [12].
3.2. Antimicrobial screening
As shown in Table 2, 4496 Antarctic colonies were
picked at random and screened for inhibitor production
against the psychrotrophic P. fragi, B. thermosphacta
and L. innocua. The former two species are common
causes of food spoilage and L. innocua was chosen as a
model for the food-borne pathogen L. monocytogenes.B. thermosphacta was sensitive to 51 of the inhibitors
produced, whereas P. fragi was sensitive to only one
inhibitor. Notably, soil samples from sites at an altitude
of over 1400 m (Mt. McGee, Mt. Melbourne and Mt.
Rittmann) yielded no inhibitor producers. Crater Circe
seemed to be the best source of inhibitor producers,
yielding 42 strains, whereas Lake Hoare provided 14
producers. When the 74 putative producers obtained at10 �C were re-tested against the three target organisms
to confirm inhibition, 13 strains were positive. The loss
of inhibitor production following the second screening
may have been due to an overcrowding of colonies on
the plates in the primary screening, making it difficult, at
times, to assess true zones of inhibition. Furthermore,
the genes for inhibitors may be plasmid encoded and
their loss on subculturing could account for the loss ofability to produce the inhibitor compound on re-
screening [13].
A total of 576 colonies isolated at 5 �C were also
tested for antimicrobial production; however, only four
inhibitor producers were detected and all four strains
were active against B. thermosphacta only. On re-
screening, none of the strains produced zones of inhi-
bition against the same target organisms. The dearth ofinhibitor producers at 5 �C suggests that psychrophilic
organisms may have developed other survival strategies,
e.g. dormancy at very cold temperatures.
3.3. Host spectrum of inhibitor-producing bacteria
Table 3 shows the effect of the 13 inhibitor–producers
obtained at 10 �C against the three initial target bacteria,plus another five spoilage/pathogenic bacteria: E. coli
O157:H7, L. innocua 4202, L. monocytogenes, S. Ente-
ritidis and Staph. aureus. None of the Antarctic bacteria
inhibited E. coli O157:H7 or S. Enteritidis. The latter
two are Gram-negative bacteria that are often more
inherently resistant to microbiocidal substances, in-
cluding most bacteriocins, which is due to the protective
outer membrane [14]. Only one isolate (HPG8) inhibitedthe Gram-negative P. fragi, whereas all except one
(HPF5) inhibited B. thermosphacta with a zone radius of
up to 22 mm.
Table 3
Zones of inhibition formed by 13 inhibitor-producing bacteria isolated from Antarctic soils against eight food-borne bacteria at 10 �C (unless
otherwise indicated)
Soil source Isolate no. Radius of zone of inhibition (mm) against
B. thermos-phacta L. innocua 10528 L. innocua 4202 L. monocy-togenes P. fragi Staph. Aureus
(at 15 �C)
Nutrient-rich, ornithogenic
Cape Hallett CHF8 12 5 20 5 0 0
Low altitude (<500 m)
Lake Hoare LHA8 5 3 5 0 0 0
LHC14 5 0 0 0 0 0
LHE9 2 2 0 0 0 0
Harrow Peaks HPC22 8 0 0 0 0 2
HPF5 0 3 2 2 0 0
HPG8 10 12 8 6 2 5
HPH17 3 5 8 0 0 3
HPH22 5 0 4 0 0 2
Crater Circe CrCB23 20 4 0 0 0 10
CrCD21 22 5 5 0 0 12
CrCE1 5 0 10 5 0 4
CrCF15 8 0 0 0 0 5
Note. No zones of inhibition were formed against Escherichia coli O157:H7 or Salmonella enterica sv. Enteritidis by any of the Antarctic isolates
at 15 �C. Isolates shown in boldface were selected for further study.
Table 2
Identification of inhibitor–producers at 10 �C against three food-borne bacteria (Brochothrix thermosphacta, Listeria innocua and Pseudomonas fragi
Location No. of colonies tested for
inhibitor productionNo. of inhibitor–producers identified against Total no. of inhibitor–
producers identifiedB. thermosaphacta L. innocua P. fragi
Nutrient-rich, ornithogenic
Cape Hallett 436 1 0 0 1
Edmonson Point (a) 288 0 0 0 0
Kay Island 576 2 0 0 2
Cape Russell 408 0 0 0 0
Subtotal 1708 3 0 0 3
Low altitude (<500 m)
Edmonson Point (b) 216 0 1 0 1
Lake Hoare 412 8 6 0 14
Harrow Peaks 528 3 2 1 6
Crater Circe 576 29 13 0 42
Subtotal 1732 40 22 1 63
High altitude (>500 m)
Battleship Promontory 312 8 0 0 8
Mount McGee 384 0 0 0 0
Mount Rittmann 192 0 0 0 0
Mount Melbourne 168 0 0 0 0
Subtotal 1056 8 0 0 8
Grand total 4496 51 22 1 74
A. O’Brien et al. / FEMS Microbiology Ecology 48 (2004) 157–167 161
3.4. Characterisation of inhibitor-producing bacteria
Six inhibitor producers were selected for further study
on the basis of their host spectrum and size of inhibition
zone. As shown in Table 4, all six inhibitor producers
except for the isolate from Crater Circe (CrCD21) were
Gram-positive. All of the isolates were catalase positive
and with the exception of HPG8, all were oxidase
Table 4
Morphology and growth characteristics of six inhibitor-producing bacteria from Antarctic soils
Soil
sourceIsolate Gram
stain
Oxidase
reaction
Catalase
reactionMorphologya Growth optimum Growth in salt (1–10% NaCl)c at
Temp (�C) Mediumb 4 �C 10 �C 22 �C 30 �C 37 �C
Cape
Hallett
CHF8 + + + Orange colonies,
single cocci
24 MA Y (10%) Y (10%) Y (10%) N N
Crater
Circe
CrCD21 ) + + Cream colonies,
short fat curved
rods
21 MA Y (5%) Y (5%) Y (10%) N N
CrCB23 + + + Cream colonies,
long thin rods
21 MA Y (5%) Y (10%) Y (10%) Y (5%) N
Harrow
Peaks
HPG8 + ) + Orange colonies,
cocci in pairs and
tetrads, slime
18 DTSA Y (5%) Y (10%) Y (10%) N N
HPH17 + + + Yellow colonies,
cocci in long
chains, slime
18 DTSA Y (5%) Y (5%) Y (5%) N N
HPC22 + + + Yellow colonies,
cocci in tetrads,
slime
18 DTSA Y (10%) Y (10%) Y (10%) N N
Y, yes; N, no. Growth was tested in concentrations of salt ranging from 1% to 10% .aWhen grown at 10 �C.b Three media tested: dilute tryptone soy agar (DTSA), marine agar (MA) and Luria–Bertani agar (LBA). No isolates producing antimicrobial
were obtained from LBA.cGrown in optimum medium (marine broth for Cape Hallett and Crater Circe isolates; dilute tryptone soy broth for Harrow Peaks isolates).
162 A. O’Brien et al. / FEMS Microbiology Ecology 48 (2004) 157–167
positive. All of the six inhibitor-producing bacteria were
pigmented, and it was noted that many of the original
4496 colonies isolated were pigmented, e.g. yellow, or-
ange, red and pink. The predominance of pigmented
isolates in Antarctic collections has been observed pre-
viously [15,16]. Armstrong [17] suggested that these
pigments might have a role in the regulation of mem-
brane fluidity at cold temperatures. Pigments may alsohave a role in protecting the organisms from UV radi-
ation that is very strong in snowy mountains even below
ice cover [3]. Many of the colonies produced extracel-
lular slime, which is often associated with psychro-
trophic organisms growing in cold habitats that are also
subjected to frequent or continuous desiccation.
The optimum temperatures for growth shown in
Table 4 were determined from Figs. 1(a) and (b). At 22�C, which was close to the optimum growth temperature
for the six inhibitor producers, all bacteria except HPH
17 tolerated up to 10% (w/v) salt. In Antarctica, salts are
derived from a variety of sources including rock
weathering, sea spray, seawater evaporation and atmo-
spheric aerosols. In this study, HPC22 and CHF8 were
the most salt-tolerant bacteria as they tolerated 10%
NaCl at temperatures as low as 4 �C. Isolate CHF8came from a disused penguin rookery, where the soils
have high levels of salts due to penguin nasal secretions
and ammonium salts derived from the decomposition of
penguin guano. The salt tolerance limits obtained in this
study need to be treated with some caution as the gra-
dient plate technique may not be as reliable after 3–4
days of incubation due to diffusion of salt through the
agar.
Of the six inhibitor–producers studied, only one
(CrCB23) grew above 30 �C (Fig. 1(b)). The lowest
temperature that could be achieved in the incubator
used in this study was 3.3 �C, so temperatures below this
value could not be tested. The maximum growth tem-
perature for HPG8 was 24 �C, whereas for the remain-
der of the strains it was 28 �C. The optimum growth
temperature for the three Harrow Peaks bacteria was 18�C. At 10 �C (temperature at which antimicrobial assays
were performed), HPG8 and HPC22 had the fastest
growth rate of the six bacteria. The optimum growth
temperature for CrCB23 and CrCD21 (both from Cra-
ter Circe) was 22 �C, although the latter isolate did not
grow above 28 �C. All of the strains fitted the descrip-
tion by Morita (1975) for psychrotrophic bacteria, in
that their maximum temperature of growth was above20 �C but they could also grow at temperatures close to
0 �C.Following growth at 10 �C for 7 days in shake-flask
cultures, only one of the six bacteria (HPG8) secreted its
inhibitor into cell-free supernatant (CFS). The cell-free
supernatants of the remaining five isolates failed to in-
hibit B. thermosphacta in the deferred antagonism assay.
3.5. Sensitivity of inhibitors to enzymes
The sensitivity of the inhibitors to enzymes was tested
in order to gain insight into their chemical structure.
None of the inhibitors produced by the six strains shown
in Table 4 were sensitive to treatment with catalase, li-
pase or a-amylase for 1 h, indicating that the active
moiety was not hydrogen peroxide, a lipid or a glycan,
Temperature (˚C)
10 20 30 4000
1
2
HPG8HPH17HPC22
0 5 10 15 20 25 30 35 40
Gro
wth
rat
e (d
ays-1
)
0
1
2
CrCB23CrCD21CHF8
(b)
(a)
Fig. 1. Growth temperature profiles of Antarctic inhibitor-producing
soil isolates cultivated in dilute tryptone soy agar (HPC22, HPH17 and
HPG8) and marine agar (CrCB23, CrCD21 and CHF8).
A. O’Brien et al. / FEMS Microbiology Ecology 48 (2004) 157–167 163
respectively. The inhibitors from CHF8, HPC22 and
HPH17 were sensitive to both trypsin and pronase;
those from CrCB23 and CrCD21 were resistant to
trypsin but sensitive to pronase; and that from HPG8
was resistant to both trypsin and pronase. These resultssuggested that five of the six inhibitors may have been
proteinaceous in nature.
3.6. Identification of inhibitor producers
Fatty acid profiles of microorganisms are species-
specific and can help in the identification of a particular
strain. The fatty acid composition of five Antarctic in-hibitor producers is shown in Table 5. The fatty acids of
the three Harrow Peaks bacteria were similar and typical
of Gram-positive soil bacteria such as Arthrobacter [6].
The major fatty acid for these three bacteria was aC15:0
but there were large differences in the amount of aC15:1
present: 20% in HPG8 and less than 1% in HPH 17 and
HPC22. The fatty acid composition of CHF8 was also
typical of Gram-positive cocci of the family Micrococ-
caceae that includes the genus Planococcus [18]. In
contrast, the fatty acid composition of CrCD21 was
typical of many Gram-negative bacteria, including the
family Pseudomonadaceae that contains the genusPseudomonas [19].
Sequence analyses of 16S rRNA from five of the in-
hibitor producers revealed that four bacteria could be
assigned to known genera: HPG8 and HPH17 to Arth-
robacter, CHF8 to Planococcus and CrCD21 to Pseu-
domonas. Alignments were carried out using MegAlign
(DNA Star) software and phylogenetic trees were con-
structed using the Clustal V program (Fig. 2). The se-quence from one of the strains, HPC22, had less than
50% similarity to any of the sequences in the database
and so is not shown in Fig. 2. The phylogenetic analyses
of the Harrow Peaks isolates HPH17 and HPG8 showed
that they had a 99.2% similar identity. This would sug-
gest that they belonged to the same species; however,
they had different phenotypic characteristics. For ex-
ample, their maximum growth temperatures differed by4 �C and HPG8 was more halotolerant than HPH 17,
which did not grow in 10% NaCl. Furthermore, HPG8
produced an antimicrobial different to that of HPC22.
However, because most species descriptions of Arthro-
bacter are based on single strains, the range of variation
within species is not known [20,21]. It is possible,
therefore, that some of the variation in the phenotypic
features of these two bacteria may be strain-, rather thanspecies-specific.
4. Discussion and conclusions
In this study of bacteria isolated from Antarctic soils,
we found that no single growth medium yielded more
organisms than another, and counts varied greatly be-tween the origin of soil and the type of agar medium
used. The predominance of psychrotolerant isolates is a
common finding in permanently cold environments.
Although the temperature of most Antarctic soils sel-
dom rises above 10 �C, exposed soils may reach tem-
peratures as high as 20–25 �C during the summer.
Therefore, psychrotolerant bacteria with growth optima
at 18–24 �C are able to take full advantage of the shorttime available for rapid growth when the soil is warm.
The soil samples in this study were collected during the
summer, which could account for the predominance of
psychrotolerant bacteria isolated.
The detection rate for antimicrobial production (%
inhibitor producers) in this study was 0.29%. This is
comparable to detection rates reported in the literature,
although they depend partly on the criteria used for se-lection. In a study [22] based on a total of 663,533 col-
onies isolated from dairy and meat sources, a detection
rate of 0.20% was reported for bacteriocin producers
Table 5
Fatty acid composition of five inhibitor-producing bacteria from Antarctic soils
Fatty acid Composition (wt%)
CHF8 CrCD21 HPG8 HPH17 HPC22
n14:0 nd 2.4� 0.2 nd nd nd
i14:0 6.4� 0.1 nd Tr Tr 0.9� 0.1
i15:0 0.8� 0.1 nd 13.6� 3.2 7.3� 2.0 3.7� 0.6
a15:0 32.8� 3.6 nd 45.6� 1.5 53.0� 4.4 51.9� 2.5
n15:0 2.0� 3.3 nd nd nd nd
i15:1 nd nd 3.0� 2.2 nd nd
a15:1 nd nd 20.1� 0.9 0.9� 0.3 0.5� 0.7
n15:1 1.0� 0.2 nd nd nd nd
i16:0 nd nd 12.1� 2.6 15.7� 1.3 17.9� 1.2
n16:0 3.4� 1.5 27.4� 4.2 1.3� 0.8 1.9� 2.2 0.9� 1.4
i16:1 11.6� 0.1 nd nd nd nd
n16:1 nd 40.4� 3.2 nd nd nd
i17:0 1.3� 0.3 nd 1.5� 0.5 1.9� 0.4 1.5� 0.3
a17:0 3.3� 0.1 nd 5.5� 1.1 13.6� 1.1 11.3� 2.5
n17:0 8.3� 3.1 1.5� 0.5 nd nd nd
17cyc 7.2� 2.3 9.4� 0.9 nd nd nd
i18:0 6.2� 1.1 nd nd nd nd
n18:0 nd 0.9� 0.5 nd nd nd
n18:1 nd 17.5� 0.9 nd nd nd
19cyc nd 1.6� 1.1 nd nd nd
Data represent means of at least three replicate determinations.
Note. 0.5% as Tr; nd, not detected.
Fig. 2. Phylogenetic trees derived from 16S rRNA gene sequence data recovered from four Antarctic inhibitor-producing soil isolates: (a) CHF8; (b)
HPG8 and HPH17; and (c) CrCD21. Clostridium thermocellum was used to root the trees.
164 A. O’Brien et al. / FEMS Microbiology Ecology 48 (2004) 157–167
A. O’Brien et al. / FEMS Microbiology Ecology 48 (2004) 157–167 165
using direct plating methods. When a further 83,000
colonies from fish and vegetable sources were isolated
using enrichment procedures, a detection rate of 3.4%
was reported for bacteriocin producers. Others [13] have
reported detection rates of 0.35% for bacteriocin pro-ducers following screening of 48 000 lactic acid bacteria
from food sources. These studies were focused on the
isolation of antimicrobial peptides from mesophilic mi-
croorganisms with generally recognized as safe (GRAS)
status. In studies focused on obtaining bacteria with
general antimicrobial activities, the detection rates have
been much higher. For example, Hentschel et al. [23]
isolated over 200 bacteria from Mediterranean spongesand found that 11.3% had antimicrobial activity.
Maximum likelihood analyses of HPG8 and HPH17
16S rRNA gene sequences with those from Arthrobacter
spp. obtained from the RDP database placed the Ant-
arctic isolates within the Arthrobacter genus. A. psy-
chrolactophilus was identified as the nearest neighbour
with percentage similarities of 94.6% and 94.3% for
HPH17 and HPG8, respectively. Stackebrandt andGoebel [24] have proposed that organisms with < 97%
similarity could be considered different species. They
have also emphasised that DNA–DNA hybridisation
remains the optimal method for confirming extent of
relatedness. When 16S rRNA gene sequence homology
was below 97.5%, these authors found that the two or-
ganisms may have no more than 60% similarity in their
DNA. The 16S rRNA gene sequence similarity of HPG8and HPH17 with A. psychrolactophilus was less than this
proposed threshold, suggesting that HPH17 and HPG8
may be new species of the genus Arthrobacter. Fur-
thermore, the sequence similarities between these two
microorganisms and their nearest Arthrobacter species
(A. psychrolactophilus) was significantly less than seen
between many other known species of Arthrobacter
[25,26]. For example, the reported similarities betweenA. globiformis and A. polychromogenes and A. oxydans
are 96.2% and 96.0%, respectively, and the similarity
between A. flavus and A. agilis is 97.9%. DNA–DNA
hybridisation studies would be necessary to substantiate
the case for the designation of HPG8 and HPH17 as
new species.
Analysis of the 16S rRNA gene sequence from the
Cape Hallet isolate, CHF8, placed it within the Plano-
coccus genus. Sequence alignments showed that CHF8
had 93.3% sequence similarity with Pl. kocurii, signifi-
cantly lower than the 97.5% threshold suggested by
Stackebrandt and Goebel [24] discussed above. On this
basis, isolate CHF8 is not a strain of Pl. citreus (90.9%)
or Pl. kocurii (93.3%) but is a distinct species.
Maximum likelihood analyses of the 16S rRNA gene
sequence from strain CrCD21 and those of Pseudomo-
nas species placed the isolate from Crater Circe within
the Pseudomonas genus. A high % similarity of 99.4%
was shown with an unidentified ‘‘gamma proteobacte-
rium’’; however, the fact that only an incomplete se-
quence of the rRNA gene from the latter was available
in the database precluded confirmation of identity.
Comparison of CrCD21 with the full rRNA sequences
(>1400 bp) from identified Pseudomonas spp. showedthat this isolate had 96.4%, 96.2% and 96.1% similarities
with P. veronii, P. migulae and P. mandelii, respectively.
The latter strains, originally isolated from mineral wa-
ters and identified as fluorescent pseudomonads, were
grouped into three phenotypic subclusters, XIIIb, XVa
and XVc. They were subsequently characterised at the
genotypic level and identified as novel species of Pseu-
domonas [27,28]. High 16S rRNA gene similarities(>99%) were reported for these new species; however,
the results were not supported by high DNA–DNA
hybridisation levels, which were <36% [27,28].
The fatty acid composition of the Antarctic inhibi-
tor–producers in this study added further support to the
genus designations obtained above. The main fatty acid
of the Harrow Peaks bacteria was identified as aC15:0,
which has also been reported for other Arthrobacter spp.including A. psychrolactophilus [25]. The isolate CHF8
had a complex fatty acid profile and not all of the fatty
acids could be identified. The main fatty acid was
aC15:0, which is a major component of Gram-positive
cell membrane lipids, is indicative of psychrotolerance
and has been observed in psychrotolerant Listeria spp.
[29]. The major cellular fatty acids of planococci are
branched-iso and anteiso-branched fatty acids withaC15:0 being the predominant cellular fatty acid [18].
The profile fitted well with the DNA sequence, which
placed this isolate within the genus Planococcus.
The Crater Circe isolate, CrCD21, had a high content
of C16:0, C16:1 and C18:1, typical of Pseudomonas
species [19], including those that grow at low tempera-
tures [6]. From the results of 16S rRNA gene analysis
this isolate was identified as Pseudomonas. No publishedfatty acid profiles are available for P. veronii, P. migulae
or P. mandelli, the species most closely matching the 16S
rRNA gene sequence of CrCD21. The fatty acid com-
position of CrCD21 closely matches that of another
psychrotrophic Pseudomonas sp. CL1-1 isolated by
White et al. [6] also from Antarctic soil. However, they
are not the same species because, unlike CrCD21, CL1-1
does not form cyclopropane fatty acids.In the natural environment, antibiotics and other
secondary metabolites serve multiple functions related
to the survival of the microorganism [30]. There are
three reports in the literature of arthrobacters producing
antimicrobial compounds. Kamigiri et al. [31] found
that an Arthrobacter sp. isolated from Indonesian soil
produced a quinolone antibiotic. This organism was
classified using phenotypic characteristics, including theidentification of lysine in the peptidoglycan cell wall.
The antibiotic was purified and characterised; however,
no subsequent work has been reported. Carnio et al. [32]
166 A. O’Brien et al. / FEMS Microbiology Ecology 48 (2004) 157–167
investigated the anti-listerial properties of microorgan-
isms isolated from French and German red-smear
cheeses and reported a single strain of Arthrobacter sp.
showing antagonism against listeriae by testing 53
physiological characteristics, including cellular fatty acidcomposition. Hentschel et al. [23] isolated Arthrobacter
spp. capable of antimicrobial production from the
Mediterranean sponges Aplysinia aerophoba and Aply-
sinia cavernicola. One of the antimicrobial-producing
strains, Arthrobacter sp. SB95, was identified using 16S
rRNA gene sequence analysis and found to belong to
the same species as a marine Arthrobacter isolate MB8-
13 that had been recovered from Antarctic sea ice. While16S rRNA gene sequence analysis did not place Arth-
robacter sp. HPH17 in the same species as the marine
isolate MB8-13, it has previously been reported that
different bacteria can produce the same bacteriocin [33].
In the present study, Arthrobacter sp. HPH17, Pla-
nococcus sp. CHF8 and Pseudomonas sp. CrCD21 were
found to produce proteinaceous compounds sensitive to
proteases; the latter two strains produced compoundsactive against Listeria spp. and B. thermosphacta. No
reports of bacteriocin or other antimicrobial production
by Planococcus sp. have been published to date. Anti-
microbial activity has often been associated with Pseu-
domonas spp., e.g. Sano et al. [34] have reported the
production of narrow-spectrum bacteriocins known as
pyocins by P. aeruginosa. Similarly, Laue et al. [35] and
Parret and DeMot [36] have identified bacteriocins fromP. fluorescens.
In conclusion, this study has shown that bacteria
isolated from the Antarctic environment are an un-
tapped source of novel antimicrobial compounds, which
may be exploitable in food, therapeutic and health ap-
plications in the future. Further work is needed to
characterize the chemical structure of the antimicrobial
compounds and to verify the activity of the inhibitors inreal applications such as chilled foods.
References
[1] Natural Antimicrobials for the Minimal Processing of Foods
(Roller, S., Ed.), p. 306. Woodhead Publishing and CRC Press,
Cambridge, UK and Boca Raton, FL, USA, 2003.
[2] Wynn-Williams, D.D. (1990) Ecological aspects of Antarctic
microbiology. Adv. Microbial Ecol. 11, 71–146.
[3] Vincent, W.F. (1988) In: Microbial Ecosystems of Antarctica, pp.
1–6. Cambridge University Press, Cambridge, MA. 153–176.
[4] Kekessy, A.M. and Piguet, D.B. (1970) New method for detecting
bacteriocin production. Appl. Microbiol. 20, 282–283.
[5] Roberts, D. (1995) In: Practical Food Microbiology (Roberts, D.,
Hooper, W. and Greenwood, M., Eds.), pp. 191–202. Public
Health Laboratory Service.
[6] White, L.P., Wynn-Williams, D.D. and Russell, N.J. (2000)
Diversity of thermal responses of lipid composition in the
membranes of the dominant culturable members of an Antarctic
fellfield soil bacterial community. Ant. Sci. 12, 386–393.
[7] Venables, W.A., Wimpenny, J.W., Ayres, A., Cook, S.M. and
Thomas, L.V. (1995) The use of two-dimensional gradient plates
to investigate the range of conditions under which conjugal
plasmid transfer occurs. Microbiology 141, 2713–2718.
[8] Kates, M., Pugh, E.L. and Ferrante, G. (1984) Regulation of
membrane fluidity by lipid desaturases. In: Membrane Fluidity,
Biomembranes (Dates, M. and Manson, A., Eds.), vol. 12, pp.
379–395. Plenum Press, New York.
[9] Sambrook, I., Fritsch, E.F. and Maniatis, T. (1989) Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory,
Cold Spring Harbor, New York.
[10] Bowman, J.P., Cavanagh, J., Austin, J.J. and Sanderson, K.
(1996) Novel Psychrobacter species from Antarctic ornithogenic
soils. J. Syst. Bacteriol. 46, 841–848.
[11] Vishniac, H.S. (1993) Soil microgeology. In: Antarctic Microbi-
ology (Friedmann, E.I., Ed.), pp. 297–341. Wiley–Liss, New York.
[12] Morita, Y. (1975) Psychrophilic bacteria. Bacteriol. Rev. 39, 144–
167.
[13] Vaughan, E.E., Caplice, E., Looney, R., O’Rourke, N., Coveny,
H., Daly, C. and Fitzgerald, G.F. (1994) Isolation from food
sources, of lactic acid bacteria that produced antimicrobials. J.
Appl. Bacteriol. 76, 118–123.
[14] Helander, I., von Wright, A. and Matilla-Sandholm, T-M. (1997)
Potential of lactic acid bacteria and novel antimicrobials against
Gram-negative bacteria. Trends Fd. Sci. Technol. 8, 146–150.
[15] Rotert, K.R., Toste, A.P. and Steiert, J.G. (1993) Membrane fatty
acid analysis of Antarctic bacteria. FEMS Microbiol. Lett. 114,
253–258.
[16] Pearce, D.A., van der Gast, C.J., Lawley, B. and Ellis-Evans, J.C.
(2003) Bacterioplankton community diversity in a maritime
Antarctic lake, determined by culture-dependent and culture-
independent techniques. FEMS Microbiol. Ecol. 45, 59–70.
[17] Armstrong, G.A. (1997) Genetics of eubacterial carotenoid
biosynthesis: a colourful tale. Annu. Rev. Microbiol. 51, 629–659.
[18] O’Leary, W.M. and Wilkinson, S.G. (1988) Gram-positive bac-
teria. In: Microbial Lipids (Ratledge, C. and Wilkinson, S.G.,
Eds.), vol. 1, pp. 117–201. Academic Press, London.
[19] Wilkinson, S.G. (1988) Gram-negative bacteria. In: Microbial
Lipids (Ratledge, C. and Wilkinson, S.G., Eds.), vol. 1, pp. 299–
488. Academic Press, London.
[20] Keddie, R.M., Collins, D. and Jones, D. (1986) Genus Arthro-
bacter. In: Bergey’s Manual of Systematic Bacteriology (Sneath,
P.H.A., Mair, N.S., Sharpe, M.E. and Holt, J.G., Eds.), vol. 2, pp.
1288–1301. Williams and Wilkins, Baltimore.
[21] Keddie, R.M., Jones, D. (2002) The Genus Arthrobacter. In: The
Prokaryotes (on line edition). Springer, New York, accessed 17/03/
02. Available from: <http://rizzo.springerny.com:6336/dynaweb/
verlagprok/prokbook/IDMAT>.
[22] Coventry, M.J., Gordon, J.B., Wilcock, A., Harmark, K.,
Davidson, B.E., Hickey, M.W., Hiller, A.J. and Wan, J. (1997)
Detection of bacteriocins of lactic acid bacteria isolated from
foods and comparison with pediocin and nisin. J. Appl. Micro-
biol. 83, 248–258.
[23] Hentschel, U., Schmid, M., Wagner, M., Fieseler, L., Gernert, C.
and Hacker, J. (2001) Isolation and phylogenetic analysis of
bacteria with antimicrobial activities from the Mediterranaen
sponges Aplysina aerophoba and Aplysina cavernicola. FEMS
Microbiol. Ecol. 35, 305–312.
[24] Stackebrandt, E. and Goebel, B.M. (1994) Taxonomic note: a
place for DNA–DNA reassociation and 16S rRNA gene sequence
analysis in the present species definition in bacteriology. Int. J.
Syst. Bacteriol. 44, 846–849.
[25] Loveland-Curtze, J., Sheridan, P.P., Gutshall, K.R. and Brench-
ley, J.E. (1999) Biochemical and phylogenetic analyses of psy-
chrophilic isolates belonging to the Arthrobacter subgroup and
description of Arthrobacter psychrolactophilus, sp. nov. Arch.
Microbiol. 171, 355–363.
A. O’Brien et al. / FEMS Microbiology Ecology 48 (2004) 157–167 167
[26] Crocker, F.H., Fredrickson, J.K., White, D.C., Ringelberg, D.B.
and Balkwill, D.L. (2000) Phylogenetic and physiological diversity
of Arthrobacter strains isolated from unconsolidated subsurface
sediments. Microbiology 146, 1295–1310.
[27] Elomari, M., Caroler, L., Hoste, B., Gillis, M., Izard, D. and
Leclerc, H. (1996) DNA relatedness among Pseudomonas strains
isolated from natural mineral waters and proposal of Pseudomo-
nas veronii sp. nov. Int. J. Sys. Bacteriol. 46, 1138–1144.
[28] Verhille, S., Baida, N., Dabboussi, F., Izard, D. and Leclerc, H.
(1999) Taxonomic study of bacteria isolated from natural mineral
waters: proposal of Pseudomonas jessenii sp. nov. and Pseudomo-
nas mandelii sp. nov. Syst. Appl. Microbiol. 22, 45–58.
[29] Annous, B.A., Becker, L.A., Bayles, D.O., Labeda, D.P. and
Wilkinson, B.J. (1997) Critical role of Anteiso-C15:0 fatty acid in
the growth of Listeria monocytogenes at low temperatures. Appl.
Environ. Microbiol. 63, 3887–3894.
[30] Vining, L.C. (1990) Functions of secondary metabolites. Annu.
Rev. Microbiol. 44, 395–427.
[31] Kamigiri, K., Tokunaga, T., Shibazaki, M., Setiavan, B., Rant-
iatmodjo, R.M., Morioka, M. and Suzuki, K-I. (1996) YM-30059,
a novel quinolone antibiotic produced by Arthrobacter sp. J.
Antibiot. 49, 823–825.
[32] Carnio, M.C., Eppert, I. and Scherer, S. (1999) Analysis of the
bacterial surface ripening flora of German and French smeared
cheeses with respect to their anti-listerial potential. Int. J. Food
Microbiol. 47, 89–97.
[33] Jack, R.W., Tagg, J.R. and Ray, B. (1995) Bacteriocins of Gram
positive bacteria. Microbiol. Rev. 59, 171–200.
[34] Sano, Y., Matsui, H. and Kobayashi, M. (1993) Molecular-
structures and functions of pyocin-S1 and pyocin-S2 in Pseudo-
monas aeruginosa. J. Bacteriol. 175, 2907–2916.
[35] Laue, R.E., Jiang, Y., Chabra, S.R., Jacob, S., Stewart, G.S.A.B.,
Hardman, A., Downie, J.A., O’Gara, F. and Williams, P. (2000)
The biocontrol strain Pseudomonas fluorescens F113 produces the
rhizobium small bacteriocin, N-(3-hydroxy-7-cis-tetradecenoyl)
homoserine lactone, via HdtS, a putative novel N-acylhomoserine
lactone synthase. Microbiology 146, 2469–2480.
[36] Parret, A.H.A. and De Mot, R. (2002) Bacteria killing their own
kind: novel bacteriocins of Pseudomonas and other gamma-
proteobacteria. Trends Microbiol. 10, 107–112.