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496 Journal of Basic Microbiology 2007, 47, 496 – 505

Research Paper
Characterization of Antarctic psychrotrophic bacteria
with antibacterial activities against terrestrial microorganisms

Angelina Lo Giudice, Vivia Bruni, Luigi Michaud

Department of Animal Biology and Marine Ecology (DBAEM-CIBAN), University of Messina, Messina, Italy

Five-hundreds and eighty bacterial strains, isolated from various Antarctic marine sources and
locations, were screened for antimicrobial activity against terrestrial microorganisms. Twenty-
two Antarctic isolates (3.8%), mainly retrieved from the water column at Terra Nova Bay (Ross
Sea), expressed antagonistic activity against one to three indicator organisms. Escherichia coli
and Proteus mirabilis resulted as the more susceptible, followed by Micrococcus luteus and Bacillus
subtilis. None of the isolates inhibited the growth of Pseudomonas aeruginosa, Staphylococcus aureus,
Salmonella enterica and the eukaryotic fungus Candida albicans.
Active Antarctic isolates, identified by 16S rDNA sequencing and phenotypically characteriz-
ed by classical methods, were phylogenetically affiliated to the Actinobacteria (16 strains) and
the γ-Proteobacteria (6 strains). Inhibition patterns, as well as phenotypic characteristics,
highly vary for different isolates, even though they were affiliated to the same genus or closely
related to the identical microorganism retrieved from the database, suggesting that these
features were more likely strain-rather than species-specific.
Results obtained from the present study confirm previous observations and highlight the
potentiality of Antarctic marine bacteria as novel source of antibacterial substances.

Keywords: Antarctic marine bacteria / Inhibitory activity / Seawater / Sediment

Received: July 12, 2007; accepted: August 09, 2007

DOI 10.1002/jobm.200700227

*
Introduction some cases, bactericidal substances with novel chemical
structures were purified and characterized [1, 10-12].
Terrestrial microorganisms have been the major source Nevertheless, in recent decades there has been a dearth
of the most important antibiotics in human history for of new classes of discovered antibiotics [5, 13]. The in-
decades, while the inhibitory effects of marine bacteria creasing global resistance to existing antibiotics has
against human pathogens have been only recently in- became a public health problem and, in attempts to
vestigated, becoming a focal point of interest for natu- overcome it, research efforts are now addressed to the
ral product chemistry. In fact, it has been demon- discovery of novel and efficient antibacterial com-
strated that marine bacteria are able to produce pounds [14]. In this context, unusual sources, such as
interesting bioactive compounds which could integrate microorganisms from extreme environments, have
those recovered from microorganisms of terrestrial begun to capture the attention of scientists.
origin [1, 2]. In the last two decades, research on antimi- Bacteria inhabiting Antarctica, characterized by ad-
crobial activity has mainly regarded particle-attached verse environmental conditions, adopt peculiar survival
or epibiotic marine bacteria. In particular, special at- strategies to achieve a competitive advantage. In par-
tention has been directed to microorganisms isolated ticular, antagonistic activity may contribute to the
from invertebrates, such as sponges and algae [3 – 9]. In adaptation of Antarctic bacteria to permanently low
temperatures by reducing the presence of competitive
Correspondence: Angelina Lo Giudice, Dipartimento di Biologia Ani-
male ed Ecologia Marina (DBAEM), Università di Messina, Salita
microorganisms. In a previous investigation [15], we
Sperone 31, 98166 Messina, Italy demonstrated the occurrence of inter-specific antago-
E-mail: alogiudice@unime.it nistic interactions among marine Antarctic isolates,
Tel.: +39 090 6765533
Fax: +39 090 393409 whereas no reports exist regarding their inhibition

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Journal of Basic Microbiology 2007, 47, 496 – 505 Inhibitory activity of Antarctic bacteria 497

properties against terrestrial microorganisms. To our lates were streaked at least three times before being
knowledge, only one study on the production of antimi- considered pure. Isolation procedure was always carried
crobial compounds, acting against food-borne microor- out on Marine Agar 2216 (MA; Difco).
ganisms, by Antarctic bacteria was performed, but these All the isolates belong to the Italian Collection of
active strains were isolated exclusively from soils [16]. Antarctic Bacteria (CIBAN) of the National Antarctic
In this context, the aim of the present work was to Museum (MNA) “Felice Ippolito” kept in our laboratory
characterize, both phenotypically and phylogenetically, at the University of Messina. They are maintained on
Antarctic bacteria from the Ross Sea able to inhibit the MA slopes at 4 °C and routinely streaked on agar plates
growth of microorganisms of terrestrial origin and/or from tubes every six months to control purity and vi-
pathogenic for man. ability. Antarctic strains are also preserved by freezing
cell suspensions at – 80 °C in Marine Broth (MB; Difco)
to which 20% (v/v) glycerol is added.
Materials and methods
Indicator microorganisms
Sampling and bacterial isolation Indicator microorganisms used throughout this study
The 580 bacterial strains used in this study were iso- were as follow: Escherichia coli ATCC 25922, Pseudomonas
lated from samples of different origins collected in aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538,
Antarctica (coordinates: 72°19′S to 74°53′S – 163°48′E to Micrococcus luteus ATTC 4698, Bacillus subtilis ATCC 6633,
70°16′E) during four oceanographic campaigns: i) a Proteus mirabilis ATCC 12453, Salmonella enterica ATCC
total of 253 strains were isolated from seawater sam- 14028 and the eukaryotic fungus Candida albicans ATCC
ples collected along the water column at the three fixed 10231, all maintained at + 37 °C on Nutrient Agar (NA;
stations Mergellina (MER, 0 – 25 m depth), Santa Maria Oxoid).
Novella (SMN, 0 – 200 m depth) and Tiburtina (TIB, 0 –
150 m depth) during the 1989 – 90 (MER and SMN) and Screening for inhibitory activity against indicator
1994 – 95 (TIB) austral summers [17, 18]. Sampling along organisms
the water column was performed by using 10 l Niskin Antarctic isolates were screened for their ability to
bottles, pre-treated with a HCl 10 N solution; ii) 43 ad- inhibit the growth of the eight indicator organisms
ditional strains were isolates from samples of sea- mentioned in the above section. Experiments were
surface microlayer (SSM, 20 µm), collected by submerg- performed on both MA and the solid medium proposed
ing a microscope glass, and intestinal content of two by Ivanova et al. [4], containing (w/v): 0.2% Bacto-
Antarctic fish (Trematomus bernacchii – TRB, and Cygno- peptone, 0.2% casein hydrolysate, 0.2% yeast extract,
draco mawsoni – CIM) during the 1997 – 98 season. In the 0.1% glucose, 0.02% KH2PO4, 0.005% MgSO4 × 7 H2O,
latter case, the surface of each fish was firstly pre- 0.1% CaCl2, 0.01% KBr and 1.5% Bacto-agar. The latter
treated with absolute ethanol, the intestines were asep- medium was prepared in a mixture of 75% (v/v) natural
tically removed and their content squeezed in pre- seawater and 25% (v/v) distilled water (pH 7.8). Indica-
sterilized seawater; iii) 150 strains were isolated from tor organisms grew well on the two media mentioned
surface seawater samples manually collected by using above when streaked alone. Antibacterial activity was
pre-sterilized polycarbonate bottles during the 2002 – 03 detected by the cross-streak method as previously de-
season at nine stations located at Road Bay (ROB, st. 1 to scribed by Lo Giudice et al. [15]. Briefly, Antarctic bacte-
4), Gerlache Inlet (GIN, st. 6), Evans Cove (EVC, st. 11), ria were streaked across one-third of an agar plate and
Inexpressible Island (INI, st. 12) and Cape Hallet (CPH, incubated at 15 °C (due to the psychrotrophic nature of
st. 13 and 14). Finally, 134 strains were isolated from the isolates). After good growth was obtained (generally
sediment samples collected by scuba at two of the in 7 – 10 days, depending on growth of the isolates),
above stations (st. 1 and 3) plus at Tethys Bay (THS, st. indicator organisms were streaked perpendicular to the
7 to 9). initial streak and plates were further incubated at 15 °C
All samples were processed within 4 h after sam- for 72/120 h and at 37 °C overnight and checked after-
pling. Serial dilutions of each sample were prepared wards for inhibition zones. The antagonistic effect was
(1 : 10 and 1 : 100, using filter-sterilized seawater) and indicated by the failure of the target strain to grow in
100 µl of each dilution was plated on 2 replicate plates the confluence area. Inhibition had to be observed at
of Marine Agar 2216 (MA, Difco). Inoculated plates were least twice to be considered positive. If the first two
incubated in the dark for 21 days at 4 °C. Colonies were assays showed ambiguous results, an additional assay
selected at random from the cultures on MA and iso- was performed to re-assess inhibitory activity.

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498 A. Lo Giudice et al. Journal of Basic Microbiology 2007, 47, 496 – 505

PCR amplification and analysis of 16S rDNA Table 1. Number of isolates showing inhibitory activity against
sensitive indicator organisms, with respect to the marine source
PCR-amplification, sequencing and phylogenetic analy-
and location. The table reports only the sources from which
sis of 16S rDNA from active bacterial isolates were car- active bacteria were isolated. Negative results obtained for Ps.
ried out as previously described by Michaud et al. [19]. aeruginosa, S. aureus, Salm. enterica and C. albicans are not
The first half (about 700 nucleotides) of each amplifica- showed.
tion product was sequenced by using the primer 27f. Sourcea n° of n° of isolate active Total n°
Each sequence was then used as a query in a BLASTn isolates againstb of active
search [20] and further aligned using the program isolates
Clustal W [21] to the most similar orthologous se- Ec Ml Bs Pm
quences retrieved from database. Each alignment
Water column
was checked manually and corrected. The 16S rRNA MER 81 3 2 1 3 5
gene sequences were submitted to GenBank and as- SMN 60 8 5 – 9 10
signed to the following accession numbers: EF513616- TIB 112 3 – – 2 3
EF513622. Superficial seawater
ROB 93 1 – 1 – 1
Morphological, biochemical and physiological Sediment
ROB 83 2 – – – 2
characterization
Fish intestinal con-
Isolates were characterized by means of a combination tent
of phenotypic tests as previously described in Bruni TRB 10 – – – 1 1
et al. [18] and Lo Giudice et al. [22]. All media used in this Total 17 7 2 15 22
study were sterilized by autoclaving at 121 °C for a)
Water Column: MER, Mergellina; SMN, Santa Maria Novella;
15 min and the plates were incubated at 15 °C for TIB, Tiburtina. Superficial seawater: ROB, Road Bay. Sediment:
14 days. The analyses were performed at least twice. ROB, Road Bay. Intestinal Content: TRB, Trematomus bernacchii.
b)
Ec: E. coli; Ml: M. luteus; Bs: B. subtilis; Pm: P. mirabilis.

Results Identification of Antarctic isolates with inhibitory


activity
Inhibitory activity against indicator organisms Active Antarctic isolates were placed within two differ-
No differences were recorded by using the two different ent phylogenetic groups: 16 fell in the Actinobacteria
media or incubating plates at 15 °C and 37 °C. As shown (Gram-positive bacteria with high GC content), while
in Table 1, 22 Antarctic isolates (detection rate of 3.8%) the remaining six were affiliated to the γ-Proteobacteria
expressed antagonistic activity against E. coli, M. luteus, subclass (Table 2), clustering into four and two different
B. subtilis and P. mirabilis, whereas Ps. aeruginosa, families, respectively. The highest diversity of isolates
S. aureus, Salm. enterica and C. albicans were never inhib- was found within the Actinobacteria (with Janibacter
ited. All except five and seven isolates had a negative members as the dominant representatives), whereas the
effect on the growth of E. coli and P. mirabilis, respec- γ-Proteobacteria were represented by only two genera
tively. Only seven isolates showed an inhibitory effect (Pseudoalteromonas and Pseudomonas).
against M. luteus, while B. subtilis resulted as the more
resistant indicator organism, being inhibited by only Inhibitory activity in relation to affiliation
two Antarctic strains. Members of Actinobacteria inhibited one to three indi-
The marine water column at SMN, MER and TIB re- cator organisms, whereas a slightly lower activity was
sulted as the main source of bacteria with inhibitory observed for the γ-Proteobacteria tested (Table 3). The
activity, yielding eighteen strains (10, five and three, latter had a negative effect exclusively on the growth of
respectively). Among the 150 isolates retrieved from the E. coli and P. mirabilis; on the contrary, Actinobacteria
surface seawater, only one (from the st. 4 located at inhibited also M. luteus and B. subtilis, although at a less
ROB) showed an antagonistic feature. The sediment extent than the other two sensitive indicator strains.
collected at ROB (st. 1) and the intestinal content of Inhibition patterns highly vary for different isolates,
the fish T. bernacchii yielded two (out of 134) and one even though they were affiliated to the same genus or
(out of 10) active strains, respectively. Finally, none closely related to the identical microorganism retrieved
of the strains isolated from the sea-surface microlayer from the database. For example, differences were re-
was able to inhibit the growth of the indicator organ- corded between the isolates B7 and G77 (both strongly
isms. related to Rhodococcus fascians, AY771765) or among

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com


Journal of Basic Microbiology 2007, 47, 496 – 505 Inhibitory activity of Antarctic bacteria 499

Table 2. 16S rDNA gene sequence affiliation of the twenty-two active Antarctic isolates to their closest phylogenetic neighbors.

Phylum or Strain Accession Next relative by GenBank alignment Homb Family Source
class number (ANa, organism) (%)
Actinobacteria B20 DQ646858 DQ172990, Arthrobacter sp. TSBY-20 99 Micrococcaceae [15]
F40 DQ667096 DQ366002, Arthrobacter sp. 20/4 99 Micrococcaceae [15]
G18 DQ667118 AF409000, Arthrobacter sp. Ellin158 98 Micrococcaceae [15]
G75 AY451331 AY383043, Arthrobacter sp. DY12-2 99 Micrococcaceae [19]
B8 AY444156 Y08540, Janibacter thuringensis 97 Intrasporangiaceae [19]
F21 DQ667087 Y08540, Janibacter thuringensis 99 Intrasporangiaceae [15]
F34 DQ667092 Y08540, Janibacter thuringensis 99 Intrasporangiaceae [15]
F39 DQ667095 Y08540, Janibacter thuringensis 99 Intrasporangiaceae [15]
G4 DQ667104 Y08540, Janibacter thuringensis 98 Intrasporangiaceae [15]
G5 DQ667105 Y08540, Janibacter thuringensis 99 Intrasporangiaceae [15]
I44 DQ831968 Y08540, Janibacter thuringensis 97 Intrasporangiaceae [15]
S1-21 EF513616 AJ717365, Nesterenkonia sp. AC84 99 Micrococcaceae This study
Actinobacteria S1-40 EF513617 AJ717365, Nesterenkonia sp. AC84 98 Micrococcaceae This study
B7 DQ646853 AY771765, Rhodococcus fascians clone SE59 99 Nocardiaceae [15]
G77 DQ667129 AY771765, Rhodococcus fascians clone SE59 99 Nocardiaceae [15]
W4-5 EF513618 AY730713, Rhodococcus fascians 99 Nocardiaceae This study
γ-Proteobacteria F26 DQ667088 AY664306, Pseudoalteromonas cl. JL-BS-K14 98 Pseudoalteromonadaceae [15]
G24 AY451336 AM110950, Pseudoalteromonas sp. 1002 99 Pseudoalteromonadaceae [19]
59 EF513619 DQ186668, Pseudoalteromonas sp. YAAJ-9 99 Pseudoalteromonadaceae This study
129 EF513620 DQ517879, Pseudoalteromonas elyakovii strain 98 Pseudoalteromonadaceae This study
BSi20670
131 EF513621 AY383040, Pseudoalteromonas sp. 2-3-6-2 100 Pseudoalteromonadaceae This study
65/3 EF513622 AY573030, Pseudomonas sp. ARCTIC-P14 99 Pseudomonadaceae This study
a)
AN: Accession Number
b)
Hom: sequence homology

Table 3. Inhibitory activity of Antarctic isolates against indicator those identified as Janibacter thuringensis (Y08540). The
organisms (negative results obtained for Ps. aeruginosa, S. au-
growth of E. coli was weakly inhibited by Nesterenkonia
reus, Salm. enterica and C. albicans are not reported).
spp. S1 – 21 and S1 – 40.
Affiliation Strain Indicator organism
Phenotypic characterization of Antarctic isolates
E. coli M. lu- B. sub- P. mi- with inhibitory activity
teus tilis rabilis Isolates showing inhibitory activity were further char-
Actino Arthrobacter B20 – + – + acterized by using a combination of classical pheno-
bacteria F40 – – + – typical tests. Results are summarized in Table 4. Only
G18 + + – – members of the genera Nesterenkonia (yellow pigment),
G75 + + – +
Janibacter B8 + + – +
Arthrobacter (strain F40, yellow) and Rhodococcus were
F21 – – – + pigmented (orange). All Gram-negatives were motile by
F34 + + – + means of polar flagella. Endospores were detected in
F39 – – – + any isolates.
G4 + – – +
G5 + – – + None of the isolates grew above 30 °C. Temperatures
I44 + + – + below 4 °C could not be tested. The pH range for
Nesterenkonia S1-21 + – – – growth was generally between 4 – 5 and 9, with the
S1-40 + – – – exception for the isolates Arthrobacter sp. B20 and Jani-
Rhodococcus G77 + – – –
B7 + + – + bacter sp. G5, growing in the range 6 – 9 and 7 – 9,
W4-5 + – + – respectively. All Gram-positive isolates grew in the
γ-Proteo Pseudoaltero F26 + – – + absence of NaCl, whereas among Gram-negatives Pseu-
bacteria monas G24 + – – +
doalteromonas members needed NaCl concentration of
59 + – – –
129 + – – + almost 1% (w/v). The majority of isolates tolerated up to
131 + – – + 9% (w/v) NaCl; the two Nesterenkonia members and four
Pseudomonas 65/3 – – – + Pseudoalteromonas isolates were able to grow up to 13

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Table 4. Phenotypic features of Antarctic isolates able to inhibit the growth of terrestrial microorganisms.

500
Arthrobacter Janibacter Nesteren- Rhodococcus Pseudoalteromonas Pseudo-
konia monas
Characteristica B20 F40 G18 G75 B8 F21 F34 F39 G4 G5 I44 S1-21 S1-40 B7 G77 W4-5 F26 G24 59 129 131 65/3
Gram reaction + + + + + + + + + + + + + + + + – – – – – –
Motility – – – – – – – – – – – – – – – – + + + + + +
b
Cell morphology c r c r c c c c c c c c c c c c r r r r r r
A. Lo Giudice et al.

Polar flagellum – – – – – – – – – – – – – – – – + + + + + +
Colony pigmentation – + – – – – – – – – – + + + + + – – – – – –
pH range for growth 6–9 5–9 5–9 5–9 5–9 4–9 5–9 5–9 5–9 7–9 5–9 5–9 5–9 5–9 5–9 5–9 4–9 4–9 4–9 4–9 4–9 5–9
NaCl conc. for growth (%)
minimum 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 0
maximum 5 9 5 5 9 9 9 9 9 9 9 13 13 9 9 9 11 11 9 11 11 5
Growth in the absence of + + + + + + + + + + + + + + + + – – – – – +

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


NaCl
Utilization as a carbon
source:
Glucose + + + + + + + – + + + + – + – + – + + + + +
Arabinose – – + – – – – – – – – + – – – + – – – – – +
Mannose + + + + – – – – – + + – – – – + – – + + – –
Mannitol + + + + – – – – – + + + + – – + – – + + + –
N-acetyl-glucosa- – + – – – – – – + + + – – – – + + + – – – –
mine
Maltose + – + + + – – – – + + + + + – + + + + + – –
Gluconate + + + + + – – – + + + + – + – + + – – + – +
Caprate – – – – – – – – – + + – – – – – – – – – – +
Adipate – – – – + + + + + + + – – + – – – + – – – +
Malate + + + + + + + – + + + + + + – + – + – – – +
Citrate + + + + – – – – – – + – – – – + – – + – – +
Phenyl-acetate + – + + – – – – – – + + – – – + – – + – – –
Biochemical tests:
Urease + + + + + + + + – + + + – + – + + + + + + –
β-galactosidase + + + + – – + – – – – – – – – – – – + + + –
Arginine dihydro- + + + – + + + + – + + + – + – – + + + + + +
lase
Tryptophane + + + + – – – – – – – – – – – – – – – – – –
deaminase
Oxidase – – – – – – – – + – – – – – – – + + – + – +
Catalase + – + + + + + + + + + + + + + + – – – – – +

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Journal of Basic Microbiology 2007, 47, 496 – 505
Biochemical tests:
Indole formation – – – – – + + – – + + – – – – – – – – – – –
Voges-Proskauer + – + – + + + + + + + – – + – – – – + – – –
reaction
Nitrate reduction + + + + + – + + + + + + + + – + + + – – – –
H2S formation – – – – – – – – – – – – – – – – – – – – – –
Macromolecule hydrolysis:
Aesculin + + + + – – + – – – – – – – – – + + + + + –
Gelatin + – + + + + + + + + + – – + – – + + + + + –
Chitin – – – – – – – – – – – – – – – – + + – – – –
Starch + – + – – + – + – + + – – – – + + + + + + –
Tween 80 – – – – – – – + + – + – – – – + + – + + + –
Growth on various solid
media
Journal of Basic Microbiology 2007, 47, 496 – 505

TSA + + + + + + + + + + + + + + + + + + + + + +
TSA + 3% NaCl + + + + + + + + + + + + + + + + + + + + + +
TCBS – – – – – – – – – – – + + – – – – – – – – +

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Acid produced from:
Glucose – – – – – – – – – – – – – – + – – – – – – –
Sucrose – – – – – – – – – – – – – – – – – + + + – –
Melibiose – – – – – – – – – – – – – – – – – – – – – +
Arabinose – – – – – – – – – – – – – – – – – – – – – +
Susceptibility to:
Nalidixic acid + – – – – – – – – – – – – – – – + + + + + +
Ampicillin + + + + + nd + – – + + + + + + + + + + + + +
Chloramphenicol + – + + + nd + + + + + + + + + + + + + + + +
O/129 + – + + + nd + + + + + + + + + + – – – – – –
Penicillin G + + + + – + + – + + + + + – + + + + + + + –
Polymixyn B + + – + + + – + – – – – – + – + + + + + + +
Tetracycline + – + + – nd – – – + + + + – – + – – – – – –
Tobramicin – – – + – – – + – – – – – – – – + + – + – +
a)
nd, not determined
b)
r, rod-shaped cells; c, coccoid cells.
Inhibitory activity of Antarctic bacteria

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501
502 A. Lo Giudice et al. Journal of Basic Microbiology 2007, 47, 496 – 505

and 11%, respectively. A narrower NaCl range for gesting that the antibacterial compounds eventually
growth was observed for three members of the Arthro- produced by Antarctic bacteria could be thermostable.
bacter genus, and for the unique Pseudomonas isolates In comparison with other inhibitory screening test-
(range 0 to 5%). ing marine-deriving bacteria against indicator organ-
All Gram-positive isolates were catalase positive (ex- isms, the detection rate of 3.8% here reported for Ant-
cept Arthrobacter sp. F40) and oxidase negative (except arctic bacterial strains was generally lower. In a study
Janibacter sp. G4) while the contrary was observed for carried out on 238 bacteria associated with two Medi-
Gram-negative isolates which were generally catalase terranean sponges, Hentschel et al. [6] reported a detec-
negative and oxidase positive. Lysine and ornithine tion rate of 11.3%; Nair and Simidu [3] found that the
decarboxylases were never detected. Tryptophane 13.3% of the free-living bacteria tested inhibited the
deaminase was present only in Arthrobacter members. growth of S. aureus; Ivanova et al. [4] screened 491 bacte-
The majority of the isolates were arginine dihydrolase ria isolated from different marine sources, detecting
and urease positive (17 and 18 strains, respectively). the 26% of active isolates; finally, Zheng et al. [9] se-
β-galactosidase was detected only in seven isolates. Only lected eight among the twenty-nine strains (28%) they
Janibacter members were able to produce indole. Many tested due to their ability to inhibit at least one of the
isolates resulted positive to the Voges-Proskauer reac- target microorganisms. Moreover, in the present study
tion and were able to reduce nitrate. The patterns of the percentage of marine bacteria showing inhibitory
assimilation and acidification of different carbon activity was higher than that recorded by O’Brien et al.
sources strongly varied between the bacterial isolates. [16; 0.29%], who screened 4496 strains isolated from soil
Acids were never produced from mannitol, inositol, samples collected at different locations in Antarctica.
sorbitol, rhamnose and amygdaline. However, it could be noted that the differences
Macromolecule hydrolysis was particularly observed among the detection rates reported in literature
for Pseudoalteromonas members. Agar hydrolysis was strongly depend on both the isolation and assay proce-
never observed. All strains belonging to the Nester- dures (including the organisms used as a target and the
enkonia and Pseudomonas genera, together with Rhodococ- media employed), as well as on the sources of bacterial
cus sp. G77, did not degrade the macromolecules tested. isolates. In fact, antimicrobial activity is generally con-
Isolates were sensitive to at least two of the antibiot- sidered to be more common for bacteria inhabiting
ics tested. All strains were susceptible to chloram- living surfaces or particles, than for free-living ones [3,
phenicol (except for Arthrobacter sp. F40) and ampicillin 23]. In our study, the majority of bacteria (18 out of 22)
(except for Janibacter spp. F39 and G4), followed by peni- with inhibitory activity were retrieved from the water
cillin G (18 sensitive isolates) and polymixyn B (14 sen- column (below 10 m depth), while little contributions
sitive isolates). Only six and eight of them were inhib- were given by sediments (two isolates), surface seawater
ited by tobramycin and tetracycline, respectively. (one isolate) and intestinal content of Antarctic fish
Nalidixic acid almost exclusively inhibited the growth (one isolate). The lack of antimicrobial activity in the
of γ-Proteobacteria, as well as the vibriostatic agent isolates obtained from the sea surface microlayer, gen-
O/129 negatively acted on Actinobacteria only. erally characterized by high intensities of UV radiation
and high concentrations of organic toxic pollutants
could be ascribed to the presence in this peculiar habi-
Discussion tat of a bacterial community that probably has devel-
oped other kinds of survival strategies [24].
Each of the 580 Antarctic isolates analyzed in this work The 16S rRNA gene fragment sequencing of the
was examined for its ability to inhibit the growth of twenty-two Antarctic bacteria revealed their affiliation
E. coli, Ps. aeruginosa, S. aureus, M. luteus, B. subtilis, P. mir- to the Actinobacteria and γ-Proteobacteria, both well
abilis, Salm. typhimurium and C. albicans. The 22 active known as producers of biologically active compounds.
strains inhibited Gram negative bacteria much more To our knowledge, members of the genera Nesterenkonia
readily than Gram positive ones. This finding could be and Rhodococcus among Actinobacteria have been never
explained by the predominance of Gram negative bac- indicated as being able to inhibit the growth of terres-
teria in these marine environments [17, 18], so that trial microorganisms; thus, it could represent a novel
antagonistic activities work better with them. source of antimicrobial compounds. Janibacter have
In our experiments, media composition did not affect been rarely reported as inhibitors of indicator organ-
the inhibition process. Furthermore, identical results isms. As an example, Asolkar et al. [25] characterized a
were obtained after incubation at 15 °C or 37 °C, sug- new antibiotic produced by J. limosus isolated from the

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Journal of Basic Microbiology 2007, 47, 496 – 505 Inhibitory activity of Antarctic bacteria 503

North Sea. Also Arthrobacter spp. are not generally well known that the isolation of true psychrophiles is
known as producers of bioactive molecules. Neverthe- very difficult and that the predominance of psychro-
less, Hentschel et al. [6] reported about a marine sponge- trophs is a common feature of permanently cold habi-
associated representative of this genus showing an tats, such as the Southern Ocean seawater [16, 31, 32].
inhibitory effect against S. aureus. Other two Arthrobac- All isolates (except for three Arthrobacter members
ter members with antimicrobial activity were isolated and Pseudomonas sp. 65/3) tolerated up to 9% (w/v) NaCl,
from soils by Kamigiri et al. [26] and O’Brien et al. [16]. and in some cases even up to 13% (Nesterenkonia iso-
However, in a previous investigation on the antagonism lates). In particular, Actinobacteria were able to growth
among Antarctic bacteria, Arthrobacter spp. F40 and in the absence of NaCl, suggesting their probable ter-
G75, Janibacter spp. B20 and I44, and Rhodococcus sp. B7 restrial origin. Nevertheless, as previously reported by
were among the active producers detected [15], con- Grossart et al. [29], their tolerance to high salt concen-
firming their inhibitory potential. trations suggests their strong adaptation to marine
Among γ-Proteobacteria, members of the Pseudoal- environment.
teromonas genus are recognized as antibiotic producers. The wide range of salt tolerance observed for our
Sobolevskaya et al. [12] and Zheng et al. [9] reported Antarctic isolates could be explained taking into con-
about the antimicrobial compounds extracted from sideration the variability in seawater salinity, which
Pseudoalteromonas strains isolated from marine sponges, strongly depends on the freezing and melting cycles of
while Isnansetyo and Kamei [27] deeply characterized Antarctic sea-ice. In fact, brine rejection during the ice
the newly discovered P. phenolica, a bacterium produc- formation significantly contributes to increase the Ant-
ing anti-methicillin-resistant S. aureus substances. Fi- arctic water salinity, whereas ice-melting dilutes the
nally, antimicrobial activity has been often associated underlying water column [33, 34].
with Pseudomonas species [16]. Kamei and Isnansetyo [28] In conclusion, the isolation and characterization of
reported about the activity of Pseudomonas sp. AMSN, Antarctic isolates inhibiting the growth of terrestrial
isolated from a marine alga, against methicillin- microorganisms, some of which are pathogenic for
resistant S. aureus. man, highlight the potential exploitation of cold-
As previously observed by Grossart et al. [29] for bac- adapted bacteria as a new source of antibiotics of
teria isolated from organic aggregates, and by Lo pharmaceutical interest. In accordance with O’Brien
Giudice et al. [15] for Antarctic marine bacteria, closely et al. [16], we think that Antarctica could represent an
related isolates (sharing a degree of 16rDNA sequence untapped environment for the discovery of biotechno-
similarity > 99%) showed some differences in inhibitory logically exploitable microorganisms. Nevertheless,
activities. This was observed, for example, in the case of further studies are needed in order to clarify the nature
isolates identified as Janibacter thuringensis (97 to 99%) of the inhibitory activities observed which could be
and Rhodococcus fascians (99%). These findings suggest linked to the synthesis of antimicrobial compounds, as
that inhibitory activity was more likely strain-rather well as to alterations of the culture medium features
than species-specific and that single species could (in terms of nutrient availability or pH).
probably produce multiple compounds acting on dif-
ferent targets.
Each phenotypic feature was not generally peculiar Acknowledgements
of a single strain or a distinct phylogenetic group. In
fact, a given characteristic was rarely observed only in This research was supported by grants from PNRA (Pro-
few isolates, e.g. the chitin hydrolysis by Pseudoalteromo- gramma Nazionale di Ricerche in Antartide), Italian
nas spp. G24 and F26, and the exclusive production of Ministry of Education and Research (Research Project
tryptophane deaminase by Arthrobacter isolates. More- PNRA 2004/1.6) and from the PhD Course in Environ-
over, as stated above for antimicrobial patterns, different mental Sciences (Scienze Ambientali: Ambiente Marino
phenotypic features were often detected among closely e Risorse) of the University of Messina. The experiments
related strains, suggesting again a strain- rather than comply with the current laws of the country in which
species-specificity. Similar observations were reported by the experiments were performed.
O’Brien et al. [16] for soil Antarctic bacteria.
Particular attention must be paid to the temperature
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((Funded by:
● PNRA (Programma Nazionale di Ricerche in Antarti-
de)
● Italian Ministry of Education and Research; grant
number: PNRA 2004/1.6
● Course in Environmental Sciences (Scienze Ambien-
tali: Ambiente Marino e Risorse) of the University of
Messina))

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