Antarctic bacteria inhibit growth of food-borne microorganisms at low temperatures

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: [email protected] (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.

mail to: [email protected]

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


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)


Cape Hallett CHF8 12 5 20 5 0 0


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


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


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


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).


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).


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.



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]


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.

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