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

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

Journal of Basic Microbiology 2007, 47, 496 – 505
properties against terrestrial microorganisms. To our
knowledge, only one study on the production of antimi-
crobial compounds, acting against food-borne microor-
ganisms, by Antarctic bacteria was performed, but these
active strains were isolated exclusively from soils [16].
In this context, the aim of the present work was to
characterize, both phenotypically and phylogenetically,
Antarctic bacteria from the Ross Sea able to inhibit the
growth of microorganisms of terrestrial origin and/or
pathogenic for man.
Materials and methods
Sampling and bacterial isolation
The 580 bacterial strains used in this study were iso-
lated from samples of different origins collected in
Antarctica (coordinates: 72°19′S to 74°53′S – 163°48′E to
70°16′E) during four oceanographic campaigns: i) a
total of 253 strains were isolated from seawater sam-
ples collected along the water column at the three fixed
stations Mergellina (MER, 0 – 25 m depth), Santa Maria
Novella (SMN, 0 – 200 m depth) and Tiburtina (TIB, 0 –
150 m depth) during the 1989 – 90 (MER and SMN) and
1994 – 95 (TIB) austral summers [17, 18]. Sampling along
the water column was performed by using 10 l Niskin
bottles, pre-treated with a HCl 10 N solution; ii) 43 ad-
ditional strains were isolates from samples of sea-
surface microlayer (SSM, 20 µm), collected by submerg-
ing a microscope glass, and intestinal content of two
Antarctic fish (Trematomus bernacchii – TRB, and Cygno-
draco mawsoni – CIM) during the 1997 – 98 season. In the
latter case, the surface of each fish was firstly pre-
treated with absolute ethanol, the intestines were asep-
tically removed and their content squeezed in pre-
sterilized seawater; iii) 150 strains were isolated from
surface seawater samples manually collected by using
pre-sterilized polycarbonate bottles during the 2002 – 03
season at nine stations located at Road Bay (ROB, st. 1 to
4), Gerlache Inlet (GIN, st. 6), Evans Cove (EVC, st. 11),
Inexpressible Island (INI, st. 12) and Cape Hallet (CPH,
st. 13 and 14). Finally, 134 strains were isolated from
sediment samples collected by scuba at two of the
above stations (st. 1 and 3) plus at Tethys Bay (THS, st.
7 to 9).
All samples were processed within 4 h after sam-
pling. Serial dilutions of each sample were prepared
(1 : 10 and 1 : 100, using filter-sterilized seawater) and
100 µl of each dilution was plated on 2 replicate plates
of Marine Agar 2216 (MA, Difco). Inoculated plates were
incubated in the dark for 21 days at 4 °C. Colonies were
selected at random from the cultures on MA and iso-
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Inhibitory activity of Antarctic bacteria 497
lates were streaked at least three times before being
considered pure. Isolation procedure was always carried
out on Marine Agar 2216 (MA; Difco).
All the isolates belong to the Italian Collection of
Antarctic Bacteria (CIBAN) of the National Antarctic
Museum (MNA) “Felice Ippolito” kept in our laboratory
at the University of Messina. They are maintained on
MA slopes at 4 °C and routinely streaked on agar plates
from tubes every six months to control purity and vi-
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.
Indicator microorganisms
Indicator microorganisms used throughout this study
were as follow: Escherichia coli ATCC 25922, Pseudomonas
aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538,
Micrococcus luteus ATTC 4698, Bacillus subtilis ATCC 6633,
Proteus mirabilis ATCC 12453, Salmonella enterica ATCC
14028 and the eukaryotic fungus Candida albicans ATCC
10231, all maintained at + 37 °C on Nutrient Agar (NA;
Oxoid).
Screening for inhibitory activity against indicator
organisms
Antarctic isolates were screened for their ability to
inhibit the growth of the eight indicator organisms
mentioned in the above section. Experiments were
performed on both MA and the solid medium proposed
by Ivanova et al. [4], containing (w/v): 0.2% Bacto-
peptone, 0.2% casein hydrolysate, 0.2% yeast extract,
0.1% glucose, 0.02% KH2PO4, 0.005% MgSO4 × 7 H2O,
0.1% CaCl2, 0.01% KBr and 1.5% Bacto-agar. The latter
medium was prepared in a mixture of 75% (v/v) natural
seawater and 25% (v/v) distilled water (pH 7.8). Indica-
tor organisms grew well on the two media mentioned
above when streaked alone. Antibacterial activity was
detected by the cross-streak method as previously de-
scribed by Lo Giudice et al. [15]. Briefly, Antarctic bacte-
ria were streaked across one-third of an agar plate and
incubated at 15 °C (due to the psychrotrophic nature of
the isolates). After good growth was obtained (generally
in 7 – 10 days, depending on growth of the isolates),
indicator organisms were streaked perpendicular to the
initial streak and plates were further incubated at 15 °C
for 72/120 h and at 37 °C overnight and checked after-
wards for inhibition zones. The antagonistic effect was
indicated by the failure of the target strain to grow in
the confluence area. Inhibition had to be observed at
least twice to be considered positive. If the first two
assays showed ambiguous results, an additional assay
was performed to re-assess inhibitory activity.
www.jbm-journal.com
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

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

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

 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 + – + + + + + + + + + + + + + + – – – – – +

www.jbm-journal.com
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 – – – – – – – – – – – + + – – – – – – – – +

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

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

www.jbm-journal.com
501
502 A. Lo Giudice et al.
and 11%, respectively. A narrower NaCl range for
growth was observed for three members of the Arthro-
bacter genus, and for the unique Pseudomonas isolates
(range 0 to 5%).
All Gram-positive isolates were catalase positive (ex-
cept Arthrobacter sp. F40) and oxidase negative (except
Janibacter sp. G4) while the contrary was observed for
Gram-negative isolates which were generally catalase
negative and oxidase positive. Lysine and ornithine
decarboxylases were never detected. Tryptophane
deaminase was present only in Arthrobacter members.
The majority of the isolates were arginine dihydrolase
and urease positive (17 and 18 strains, respectively).
β-galactosidase was detected only in seven isolates. Only
Janibacter members were able to produce indole. Many
isolates resulted positive to the Voges-Proskauer reac-
tion and were able to reduce nitrate. The patterns of
assimilation and acidification of different carbon
sources strongly varied between the bacterial isolates.
Acids were never produced from mannitol, inositol,
sorbitol, rhamnose and amygdaline.
Macromolecule hydrolysis was particularly observed
for Pseudoalteromonas members. Agar hydrolysis was
never observed. All strains belonging to the Nester-
enkonia and Pseudomonas genera, together with Rhodococ-
cus sp. G77, did not degrade the macromolecules tested.
Isolates were sensitive to at least two of the antibiot-
ics tested. All strains were susceptible to chloram-
phenicol (except for Arthrobacter sp. F40) and ampicillin
(except for Janibacter spp. F39 and G4), followed by peni-
cillin G (18 sensitive isolates) and polymixyn B (14 sen-
sitive isolates). Only six and eight of them were inhib-
ited by tobramycin and tetracycline, respectively.
Nalidixic acid almost exclusively inhibited the growth
of γ-Proteobacteria, as well as the vibriostatic agent
O/129 negatively acted on Actinobacteria only.
Discussion
Each of the 580 Antarctic isolates analyzed in this work
was examined for its ability to inhibit the growth of
E. coli, Ps. aeruginosa, S. aureus, M. luteus, B. subtilis, P. mir-
abilis, Salm. typhimurium and C. albicans. The 22 active
strains inhibited Gram negative bacteria much more
readily than Gram positive ones. This finding could be
explained by the predominance of Gram negative bac-
teria in these marine environments [17, 18], so that
antagonistic activities work better with them.
In our experiments, media composition did not affect
the inhibition process. Furthermore, identical results
were obtained after incubation at 15 °C or 37 °C, sug-
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Journal of Basic Microbiology 2007, 47, 496 – 505
gesting that the antibacterial compounds eventually
produced by Antarctic bacteria could be thermostable.
In comparison with other inhibitory screening test-
ing marine-deriving bacteria against indicator organ-
isms, the detection rate of 3.8% here reported for Ant-
arctic bacterial strains was generally lower. In a study
carried out on 238 bacteria associated with two Medi-
terranean sponges, Hentschel et al. [6] reported a detec-
tion rate of 11.3%; Nair and Simidu [3] found that the
13.3% of the free-living bacteria tested inhibited the
growth of S. aureus; Ivanova et al. [4] screened 491 bacte-
ria isolated from different marine sources, detecting
the 26% of active isolates; finally, Zheng et al. [9] se-
lected eight among the twenty-nine strains (28%) they
tested due to their ability to inhibit at least one of the
target microorganisms. Moreover, in the present study
the percentage of marine bacteria showing inhibitory
activity was higher than that recorded by O’Brien et al.
[16; 0.29%], who screened 4496 strains isolated from soil
samples collected at different locations in Antarctica.
However, it could be noted that the differences
among the detection rates reported in literature
strongly depend on both the isolation and assay proce-
dures (including the organisms used as a target and the
media employed), as well as on the sources of bacterial
isolates. In fact, antimicrobial activity is generally con-
sidered to be more common for bacteria inhabiting
living surfaces or particles, than for free-living ones [3,
23]. In our study, the majority of bacteria (18 out of 22)
with inhibitory activity were retrieved from the water
column (below 10 m depth), while little contributions
were given by sediments (two isolates), surface seawater
(one isolate) and intestinal content of Antarctic fish
(one isolate). The lack of antimicrobial activity in the
isolates obtained from the sea surface microlayer, gen-
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-
tat of a bacterial community that probably has devel-
oped other kinds of survival strategies [24].
The 16S rRNA gene fragment sequencing of the
twenty-two Antarctic bacteria revealed their affiliation
to the Actinobacteria and γ-Proteobacteria, both well
known as producers of biologically active compounds.
To our knowledge, members of the genera Nesterenkonia
and Rhodococcus among Actinobacteria have been never
indicated as being able to inhibit the growth of terres-
trial microorganisms; thus, it could represent a novel
source of antimicrobial compounds. Janibacter have
been rarely reported as inhibitors of indicator organ-
isms. As an example, Asolkar et al. [25] characterized a
new antibiotic produced by J. limosus isolated from the
www.jbm-journal.com
Journal of Basic Microbiology 2007, 47, 496 – 505
North Sea. Also Arthrobacter spp. are not generally
known as producers of bioactive molecules. Neverthe-
less, Hentschel et al. [6] reported about a marine sponge-
associated representative of this genus showing an
inhibitory effect against S. aureus. Other two Arthrobac-
ter members with antimicrobial activity were isolated
from soils by Kamigiri et al. [26] and O’Brien et al. [16].
However, in a previous investigation on the antagonism
among Antarctic bacteria, Arthrobacter spp. F40 and
G75, Janibacter spp. B20 and I44, and Rhodococcus sp. B7
were among the active producers detected [15], con-
firming their inhibitory potential.
Among γ-Proteobacteria, members of the Pseudoal-
teromonas genus are recognized as antibiotic producers.
Sobolevskaya et al. [12] and Zheng et al. [9] reported
about the antimicrobial compounds extracted from
Pseudoalteromonas strains isolated from marine sponges,
while Isnansetyo and Kamei [27] deeply characterized
the newly discovered P. phenolica, a bacterium produc-
ing anti-methicillin-resistant S. aureus substances. Fi-
nally, antimicrobial activity has been often associated
with Pseudomonas species [16]. Kamei and Isnansetyo [28]
reported about the activity of Pseudomonas sp. AMSN,
isolated from a marine alga, against methicillin-
resistant S. aureus.
As previously observed by Grossart et al. [29] for bac-
teria isolated from organic aggregates, and by Lo
Giudice et al. [15] for Antarctic marine bacteria, closely
related isolates (sharing a degree of 16rDNA sequence
similarity > 99%) showed some differences in inhibitory
activities. This was observed, for example, in the case of
isolates identified as Janibacter thuringensis (97 to 99%)
and Rhodococcus fascians (99%). These findings suggest
that inhibitory activity was more likely strain-rather
than species-specific and that single species could
probably produce multiple compounds acting on dif-
ferent targets.
Each phenotypic feature was not generally peculiar
of a single strain or a distinct phylogenetic group. In
fact, a given characteristic was rarely observed only in
few isolates, e.g. the chitin hydrolysis by Pseudoalteromo-
nas spp. G24 and F26, and the exclusive production of
tryptophane deaminase by Arthrobacter isolates. More-
over, as stated above for antimicrobial patterns, different
phenotypic features were often detected among closely
related strains, suggesting again a strain- rather than
species-specificity. Similar observations were reported by
O’Brien et al. [16] for soil Antarctic bacteria.
Particular attention must be paid to the temperature
range for growth and to the salt tolerance of the Ant-
arctic isolates tested. All of them were psychrotrophic
according to the definition given by Morita [30]. It is
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Inhibitory activity of Antarctic bacteria 503
well known that the isolation of true psychrophiles is
very difficult and that the predominance of psychro-
trophs is a common feature of permanently cold habi-
tats, such as the Southern Ocean seawater [16, 31, 32].
All isolates (except for three Arthrobacter members
and Pseudomonas sp. 65/3) tolerated up to 9% (w/v) NaCl,
and in some cases even up to 13% (Nesterenkonia iso-
lates). In particular, Actinobacteria were able to growth
in the absence of NaCl, suggesting their probable ter-
restrial origin. Nevertheless, as previously reported by
Grossart et al. [29], their tolerance to high salt concen-
trations suggests their strong adaptation to marine
environment.
The wide range of salt tolerance observed for our
Antarctic isolates could be explained taking into con-
sideration the variability in seawater salinity, which
strongly depends on the freezing and melting cycles of
Antarctic sea-ice. In fact, brine rejection during the ice
formation significantly contributes to increase the Ant-
arctic water salinity, whereas ice-melting dilutes the
underlying water column [33, 34].
In conclusion, the isolation and characterization of
Antarctic isolates inhibiting the growth of terrestrial
microorganisms, some of which are pathogenic for
man, highlight the potential exploitation of cold-
adapted bacteria as a new source of antibiotics of
pharmaceutical interest. In accordance with O’Brien
et al. [16], we think that Antarctica could represent an
untapped environment for the discovery of biotechno-
logically exploitable microorganisms. Nevertheless,
further studies are needed in order to clarify the nature
of the inhibitory activities observed which could be
linked to the synthesis of antimicrobial compounds, as
well as to alterations of the culture medium features
(in terms of nutrient availability or pH).
Acknowledgements
This research was supported by grants from PNRA (Pro-
gramma Nazionale di Ricerche in Antartide), Italian
Ministry of Education and Research (Research Project
PNRA 2004/1.6) and from the PhD Course in Environ-
mental Sciences (Scienze Ambientali: Ambiente Marino
e Risorse) of the University of Messina. The experiments
comply with the current laws of the country in which
the experiments were performed.
References
[1] Isnansetyo, A. and Kamei, Y., 2003. MC21-A, a bactericidal
antibiotic produced by a new marine bacterium, Pseudoal-
www.jbm-journal.com
504 A. Lo Giudice et al.
teromonas phenolica sp. nov. O-BC30T, against methicillin-
resistant Staphylococcus aureus. Antimicrob. Ag. Chem., 47,
480 – 488.
[2] Miao, L. and Qian, P.Y., 2005. Antagonistic antimicrobial
activity of marine fungi and bacteria isolated from ma-
rine biofilm and seawaters of Hong Kong. Aq. Microb.
Ecol., 38, 231 – 238.
[3] Nair, S. and Simidu, U., 1987. Distribution and signifi-
cance of heterotrophic marine bacteria with antibacterial
activity. Appl. Environ. Microbiol., 53, 2957– 2962.
[4] Ivanova, E.P., Nicolau, D.V., Yumoto, N. and Taguchi, T.,
1998. Impact of conditions of cultivation and adsorption
on antimicrobial activity of marine bacteria. Mar. Biol.,
130, 545 – 551.
[5] Burgess, J.G., Jordan, E.M., Bregu, M., Mearns-Spragg, A.
and Boyd, K.G., 1999. Microbial antagonism: a neglected
avenue of natural product research. J. Biotechnol., 70,
27 – 32.
[6] Hentschel, U., Schmid, M., Wagner, M., Fieseler, L., Ger-
nert, C. and Hacker, J., 2001. Isolation and phylogenetic
analysis of bacteria with antimicrobial activities from the
Mediterranean sponges Aplysina aerophoba and Aplysina
cavernicola. FEMS Microbiol. Ecol., 35, 305 – 312.
[7] Thiel, V. and Imhoff, J.F., 2003. Phylogenetic identifica-
tion of bacteria with antimicrobial activities from Medi-
terranean sponges. Biomol. Eng., 20, 421 – 423.
[8] Chelossi, E., Milanese, M., Milano, A., Pronzato, R. and
Riccardi, G., 2004. Characterization and antimicrobial ac-
tivity of epibiotic bacteria from Petrosia ficiformis (Porifera,
Demospongiae). J. Exp. Mar. Biol. Ecol., 309, 21 – 33.
[9] Zheng, L., Chen, H., Han, X., Lin, W. and Yan, X., 2005.
Antimicrobial screening and active compound isolation
from marine bacterium NJ6-3-1 associated with the
sponge Hymeniacidon perleve. World J. Microbiol. Biotech-
nol., 21, 201 – 206.
[10] Long, R.A., Qureshi, A., Faulkner, D.J. and Azam, F., 2003.
2-n-pentyl-4-quinolinol produced by a marine Alteromonas
sp. and its potential ecological and biogeochemical roles.
Appl. Environ. Microbiol., 69, 568 – 576.
[11] Brinkhoff, T., Bach, G., Heidorn, T., Liang, L., Schlingloff,
A. and Simon, M., 2004. Antibiotic production by a Roseo-
bacter clade-affiliated species from the German Wadden
Sea and its antagonistic effect on indigenous isolates.
Appl. Environ. Microbiol., 70, 2560 – 2565.
[12] Sobolevskaya, MP., Smetanina, O.F., Speitlimg, M., Shev-
chenko, L.S. and Dmitrenok, P.S., et al., 2005. Controlling
production of brominated cyclic depsipeptides by Pseudo-
alteromonas maricaloris KMM 636T. Lett. Appl. Microbiol.,
40, 243 – 248.
[13] Barton, S., 2006. New antibiotic on the horizons? Nature
Rev., 5, 539.
[14] Bax, R., Mullan, N. and Verhoef, J., 2000. The millennium
bugs – the need for and development of new antibacteri-
als. Int. J. Antimicr. Ag., 16, 51 – 59.
[15] Lo Giudice, A., Brilli, M., Bruni, V., De Domenico, M., Fani,
R. and Michaud, L., 2007. Bacterium-bacterium inhibitory
interactions among psychrotrophic bacteria isolated from
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Journal of Basic Microbiology 2007, 47, 496 – 505
Antarctic seawater (Terra Nova Bay, Ross Sea). FEMS Mi-
crobiol. Ecol., 60, 383 – 396.
[16] O’Brien, A., Sharp, R., Russell, N. and Roller, S., 2004.
Antarctic bacteria inhibit growth of food-borne microor-
ganisms at low temperatures. FEMS Microbiol. Ecol., 48,
157 – 167.
[17] Maugeri, T.L., Gugliandolo, C. and Bruni, V., 1996. Hete-
rotrophic bacteria in the Ross Sea (Terra Nova Bay, Antarc-
tica). Microbiologica, 19, 67 – 76.
[18] Bruni, V., Gugliandolo, C., Maugeri, T. and Allegra, A.,
1999. Psychrotrophic bacteria from a coastal station in
the Ross Sea (Terra Nova Bay, Antarctica). Microbiologica,
22, 357 – 363.
[19] Michaud, L., Di Cello, F., Brilli, M., Fani, R., Lo Giudice, A.
and Bruni, V., 2004. Biodiversity of cultivable psychrotro-
phic marine bacteria isolated from Terra Nova Bay (Ross
Sea, Antarctica). FEMS Microbiol. Lett., 230, 63 – 71.
[20] Altschul, S.F., Maden, T.L., Shäffer, A.A., Zhang, J., Miller,
W. and Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST:
a new generation of protein database search programs.
Nucl. Acids Res., 25, 3389 – 3402.
[21] Thompson, J.D., Higgins, D.G. and Gibson, T.J., 1994.
CLUSTAL W: improving the sensitivity of progressive
multiple sequence alignment through sequence weigh-
ting, position-specific gap penalties and weight matrix
choice. Nucl. Acids Res., 22, 4673 – 4680.
[22] Lo Giudice, A., Michaud, L., de Pascale, D., De Domenico,
M., di Prisco, G., Fani, R. and Bruni, V., 2006. Lipolytic
activity of Antarctic cold-adapted marine bacteria (Terra
Nova Bay, Ross Sea). J. Appl. Microbiol., 101, 1039 – 1048.
[23] Long, R.A. and Azam, F., 2001. Antagonistic interactions
among marine pelagic bacteria. Appl. Environ. Microbiol.,
67, 4975 – 4983.
[24] Agogué, H., Joux, F., Obernosterer, I. and Lebaron, P.,
2005. Resistance of marine bacterioneuston to solar radia-
tion. Appl. Environ. Microbiol., 71, 5282 – 5289.
[25] Asolkar, R.N., Schröder, D., Heckmann, R., Lang, S., Wag-
ner-Döbler, I. and Laatsch, H., 2004. Helquinoline, a new
tetrahydroquinoline antibiotic from Janibacter limosus Hel
1. J. Antibiot., 57, 17 – 23.
[26] Kamigiri, K., Tokunaga, T., Shibazaki, M., Setiavan, B.,
Rantiatmodjo, R.M., Morioka, M. and Suzuki, K.-I., 1996.
YM-3009, a novel quinolone antibiotic produced by Ar-
throbacter sp. J. Antibiot., 49, 823 – 825.
[27] Isnansetyo, A. and Kamei, Y., 2003. Pseudoalteromonas
phenolica sp. nov., a novel marine bacterium that produces
phenolic anti-methicillin-resistant Staphylococcus aureus
substances. Int. J. Syst. Evol. Microbiol., 53, 583 – 588.
[28] Kamei, Y. and Isnansetyo, A., 2003. Lysis of methicillin-
resistant Staphylococcus aureus by 2,4-diacetylphlorogluci-
nol produced by Pseudomonas sp. AMSN isolated from a
marine alga. Int. J. Antimicrob. Ag., 21, 71 – 74.
[29] Grossart, H.P., Schlingloff, A., Bernhard, M., Simon, M.
and Brinkhoff, T., 2004. Antagonistic activity of bacteria
isolated from organic aggregates of the German Wadden
Sea. FEMS Microbiol. Ecol., 47, 387 – 396.
www.jbm-journal.com
Journal of Basic Microbiology 2007, 47, 496 – 505 Inhibitory activity of Antarctic bacteria 505

[30] Morita, Y., 1975. Psychrophilic bacteria. Bacteriol. Rev.,
38, 144 – 167.
[31] Bowman, J.P., McCammon, S.A., Brown, M.V., Nichols,
D.S. and McMeekin, T.A., 1997. Diversity and association
of psychrophilic bacteria in Antarctic sea ice. Appl. Envi-
ron. Microbiol., 63, 3068 – 3078.
[32] Helmke, E. and Weyland, H., 2004. Psychrophilic versus
psychrotolerant bacteria-occurrence and significance in
polar and temperate marine habitats. Cell. Mol. Biol., 50,
553 – 561.
[33] Toggweiler, J.R. and Samuels, B., 1995. Effect of sea ice on
the salinity of Antarctic bottom waters. J. Phys. Ocean-
ogr., 25, 1980 – 1997.
[34] Jenkins, A., 1999. The impact of melting ice on ocean
waters. J. Phys. Oceanogr., 29, 2370 – 2381.

((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))

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