Response of bacterial isolates from Antarctic shallow sediments (2013)

Response of bacterial isolates from Antarctic shallow sedimentstowards heavy metals, antibiotics and polychlorinated biphenyls

Angelina Lo Giudice • Patrizia Casella •

Vivia Bruni • Luigi Michaud

Accepted: 15 November 2012 / Published online: 27 November 2012

� Springer Science+Business Media New York 2012

Abstract The response of bacterial isolates from Antarctic

sediments to polychlorinated biphenyls (Aroclor 1242

mixture), heavy metal salts (cadmium, copper, mercury and

zinc) and antibiotics (ampicillin, chloramphenicol, kana-

mycin and streptomycin) was investigated. Overall, the

ability to growth in the presence of Aroclor 1242 as a sole

carbon source was observed for 22 isolates that mainly

belonged to Psychrobacter spp. Tolerance to the heavy

metals assayed in this study was in the order of Cd [Cu [ Zn [ Hg and appeared to be strictly related to the

metal concentrations, as determined during previous

chemical surveys in the same area. With regards to anti-

biotic assays, the response of the isolates to the tested

antibiotics ranged from complete resistance to total sus-

ceptibility. In particular, resistances to ampicillin and

chloramphenicol were very pronounced in the majority of

isolates. Our isolates differently responded to the presence

of toxic compounds primarily based on their phylogenetic

affiliation and secondarily at strain level. Moreover, the

high incidence of resistance either to metal or antibiotics,

in addition to the capability to grow on PCBs, confirm that

bacteria are able to cope and/or adapt to the occurrence

pollutants even in low human-impacted environments.

Keywords Heavy metal tolerance � Antibiotic resistance �PCB oxidation

Introduction

In spite of the precautionary measures taken to avoid pol-

lution, Antarctica is not exempt from a moderate anthro-

pogenic contamination. Toxic compounds can reach the

Antarctic environment through long-range transport by

mass flow in the atmosphere and water, contamination due

to improper disposal practices and/or incineration of waste

produced at research bases. This is the case, for example,

of heavy metals, antibiotics and polychlorinated biphenyls

(PCBs) (Montone et al. 2001, 2005; De Souza et al. 2006).

These latter are long-term persistent compounds synthe-

sized as mixtures during the mid-twentieth century and

sold for industrial application. Their use has been regulated

or prohibited since the 1970 s in western nations. Con-

versely, antibiotics and heavy metals are also introduced

through natural processes into the environment.

The low seawater temperature is responsible for reduc-

ing both metabolic (which makes the excretion process less

efficient) and growth rates of organisms, thus promoting

high concentrations of contaminants in the Antarctic biota

and accumulation throughout the food chain. Such con-

centrations are often comparable or even higher than those

from the Northern Hemisphere (Grotti et al. 2008). Con-

trary to heavy metals and PCBs that can have a negative

effect on all forms of life, antimicrobials mainly alter the

microbiosphere by inhibiting microbial activity and disturb

the biogeochemical circulation of biogenesis elements

(Martinez 2009).

Once in the marine environment, all these problematic

contaminants cannot be easily dispersed, thus becom-

ing localized and accumulated in sediments which are the

natural collectors of pollutants (Andrade et al. 2001;

Caroppo et al. 2006). Their fate is then strictly linked to

bacteria, as the most abundant sediment organisms, which

A. Lo Giudice (&) � P. Casella � V. Bruni � L. Michaud

Department of Biological and Environmental Sciences,

University of Messina, Viale Ferdinando Stagno d’Alcontres 31,

98166 Messina, Italy

e-mail: [email protected]

123

Ecotoxicology (2013) 22:240–250

DOI 10.1007/s10646-012-1020-2

represent the first step in the transfer of toxic compounds to

higher trophic levels (Gillan et al. 2005). Bacteria possess

genetic and biochemical capacities for remediation of

heavy metal and PCB pollution. PCBs can be transformed,

despite their recalcitrant nature, into chemical substances

by different microbial metabolic pathways, both aerobic

and anaerobic, facilitating further metabolization. The

biodegradation of certain, usually less chlorinated, PCB

congeners has been reported for several microorganisms

elsewhere in the world (Bedard et al. 1987; Kohler et al.

1988; Dercova et al. 2009). However, to date PCB-

degraders have been rarely isolated from the Antarctic

environment and they derive exclusively from seawater

(Yakimov et al. 1999; De Domenico et al. 2004; Michaud

et al. 2007). Conversely, a single work exists about the

abundance of PCB-degrading bacteria in Antarctic sedi-

ments, but no information on the isolation and identifica-

tion of these PCB-degrading microorganisms was furnished

(Tumeo and Guinn 1997).

Bacteria can adsorb, accumulate (by a passive metabo-

lism-independent or an active metabolism-dependent pro-

cess) and transform heavy metals in most food chains

(De Souza et al. 2006; Haferburg and Kothe 2007). Heavy-

metal resistance has been reported for a number of different

bacterial genera and in some cases it is present together

with antibiotic resistances. Both kinds of resistance are

becoming a global phenomenon, with resistant bacteria that

are ubiquitous and have been also detected in Antarctic

seawater (De Souza et al. 2006).

In this context, the present work was mainly aimed at

analysing the response of bacteria from Antarctic sedi-

ments to heavy metals, antibiotics and PCBs, in order to

provide further insights toward this under-investigated

topic in cold-systems.

Materials and methods

Study area and sample collection

Shallow marine sediment samples were aseptically col-

lected by scuba from two stations in the Terra Nova Bay

(Ross Sea), i.e. Tethys Bay (THS; coordinates: 74�41.

6980S–164�04021400E; depth: 21 m) and Road Bay (ROB;

coordinates: 74�41080.300S–164�07080.300E; depth: 15 m),

which are both close to the Italian Base ‘‘Mario Zucchelli’’.

Bacterial isolation

Sediment samples for bacterial isolation were processed

within approximately 2 h after sampling. Serial dilutions

were prepared (1:10 and 1:100, using filter-sterilized

seawater) and 100 ll of each dilution was spread-plated in

three replicates on Marine Agar 2216 (MA, Difco). Inoc-

ulated plates were incubated in the dark at 4 �C for

1 month. Colonies were randomly isolated from agar

plates, picked and subcultured almost three times under the

same conditions.

16S rDNA PCR amplification of bacterial isolates

PCR-amplification of 16S rDNA from bacterial isolates

was carried out under the conditions described earlier

(Michaud et al. 2004). Briefly, a single colony of each

strain was lysed by heating at 95 �C for 10 min. Amplifi-

cation of 16S rDNA was performed with an ABI 9600

thermocycler (PE, Applied Biosystems) using the domain

Bacteria-specific primers 27F (50-AGAGTTTGATCCTGG

CTCAG-30) (spanning positions from 8 to 27 in E. coli

rRNA coordinates) and 1492R (50-CTACGGCTACCTTGT

TACGA-30) (spanning positions from 1492 to 1513 in

E. coli). The reaction mixtures were assembled at 0 �C and

contained 1–10 ng DNA, 10X buffer, 1.5 mM MgCl2,

150 ng of each forward and reverse primer (MWG, Ger-

many), 250 lM dNTP (Polymed, Italy), 0.5 units of

PolyTaq polymerase (Polymed, Italy) and sterile distilled

water to a final volume of 20 ll. The PCR program was as

follows: 3 min at 95 �C, followed by 30 cycles of 1 min at

94 �C, 1 min at 50 �C, 2 min at 72 �C and a final extension

step of 10 min at 72 �C. The expected size of the PCR

product was approximately 1.4 kb. The results of the

amplification reactions were analyzed by agarose gel

electrophoresis (1 %, w/v) in TAE buffer (0.04 M Tris–

acetate, 0.02 M acetic acid, 0.001 M EDTA), containing

1 lg/ml of ethidium bromide.

Sequencing and analysis of 16S rRNA gene

Automated sequencing of 16S rRNA gene from isolates

was carried out by cycle sequencing using the dye termi-

nator method. Sequencing was carried out at the Sequenc-

ing Service of the Macrogen Laboratory (Korea). Next

relatives of isolates were determined by comparison to 16S

rRNA gene sequences in the NCBI GenBank and the

EMBL databases using BLAST, and the ‘‘Seqmatch’’ and

‘‘Classifier’’ programs of the Ribosomal Database Project

II (http://rdp.cme.msu.edu/). All sequences with similarity

C97 % were considered to represent one phylogenetic

group or phylotype and grouped. Selected sequences were

further aligned using the program Clustal W (Thompson

et al. 1994) to the most similar orthologous sequences

retrieved from database. Each alignment was checked

manually, corrected and then analyzed using the neigh-

bour-joining method (Saitou and Nei 1987) according to

the model of Jukes-Cantor distances. Phylogenetic tree was

Response of bacterial isolates 241

123

http://rdp.cme.msu.edu/

constructed using the MEGA 5 (Molecular Evolutionary

Genetics Analysis) software (Kumar et al. 1993). The

robustness of the inferred trees was evaluated by 500

bootstrap re-samplings.

Isolates are part of the Italian Collection of Antarctic

Bacteria (CIBAN) of the National Antarctic Museum

(MNA, www.mna.it) ‘‘Felice Ippolito’’ kept in our labo-

ratory at the University of Messina. They are currently

maintained on MA slopes at 4 �C and routinely streaked on

agar plates from tubes every 6 months to control purity and

viability. 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.

The nucleotide sequences of the representative isolates

sequenced in this study have been deposited in the Gen-

Bank database under the accession numbers JX103222–

JX103234.

Bacterial growth in the presence of polychlorinated

biphenyls (PCBs)

Aerobic PCB degradation was screened into the mineral

liquid medium Bushnell Haas (BH; Difco) supplemented

with NaCl (3 %, w/v) and an Aroclor 1242 solution

(100 ppm in dichloromethane) as sole carbon and energy

source (final concentration 0.1 %, v/v) (De Domenico et al.

2004; Michaud et al. 2007). Aroclor 1242 is a mixture of

PCB congeners (ranging from dichloro- to hexachlorobi-

phenyls) made of twelve carbon atoms in the biphenyl

molecule and containing 42 % chlorine by weight.

Before adding the bacterial inoculum, 10 ll of Aroclor

solution were transferred to 100 ml flasks containing

9.5 ml of BH and left in a cabinet until CH2Cl2 had

completely evaporated. The medium was inoculated with

0.5 ml of a freshly prepared bacterial suspension (5 %, v/v)

in 3 % (w/v) NaCl supplemented filter-sterilized distilled

water. The optical density (OD) of the suspension was

adjusted to 1.0 at 600 nm. Cultures were incubated at 4 �C

for 3 weeks on a rotary shaker operated at 100 rpm. The

ability to use PCBs as growth substrates was evaluated

according to the degree of turbidity or the appearance of

cellular flocs in the test tubes. Uninoculated medium was

incubated in parallel as a negative control.

Heavy metal tolerance

Heavy metal tolerance was tested by modifying the method

reported by Chandy (1999). Four metals [i.e. cadmium

(Cd), zinc (Zn), mercury (Hg) and copper (Cu)] were tes-

ted. Metal salt solutions were prepared in filter-sterilized

phosphate buffer saline (PBS) and stored at 4 �C until use

(Selvin et al. 2009). The following concentrations (ppm)

were used: 10, 50, 100, 500, 1,000, 2,500, 5,000, 7,500 and

10,000 ppm. The metals were added as CdCl2 9 2.5 H2O,

HgCl2, CuCl2 9 2H2O and ZnCl2.

Isolates were grown in MB at 4 �C until the OD600nm

was approximately 0.500. Aliquots (100 ll) of each cell

suspension were spread-plated in duplicate on MA plates.

The inoculated plates were considered ready for the

assessment of heavy metal tolerance when the inoculum

was completely absorbed (after 10–15 min). Ten microli-

ters of each metal solution were then spotted on the inoc-

ulated agar plates. Sterile PBS was used as negative

control. Plates were further incubated at 4 �C and exam-

ined for the appearance of well-defined inhibition zone

after 96 h.

Antibiotic resistance

The susceptibility tests to antibiotics were carried out on

MA plates supplemented with chloramphenicol (25 and

50 ppm), ampicillin (range 50 and 100 ppm), streptomycin

(25, 250, 350 and 500 ppm) and kanamycin (25, 150 and

250 ppm). Inoculated plates were incubated at 4 �C for

96 h.

Data elaboration

Results were converted to a binary matrix and used to

calculate a Jaccard similarity matrix. The matrix was then

analyzed by non-metric multidimensional scaling (nMDS)

by using the software Primer-6 (Plymouth Marine Labo-

ratory, Roborough, United Kingdom). nMDS is a multi-

variate ordination method transforming complex data into a

new format by constructing a new set of variables.

This procedure condenses data to one point in a two-

dimensional plane so that highly similar data are plotted

close together.

Results

Phylogenetic affiliation of Antarctic isolates

A total of 98 strains were isolated from MA plates: 72 were

from the site ROB and 26 from THS, respectively. The

phylogenetic affiliation of isolates is reported in Fig. 1.

Only one representative isolate per phylotype is reported.

Based on the results of the 16S rDNA sequencing isolates

mainly belonged to the Gammaproteobacteria (65.3 % of

total isolates), followed by the CF group of Bacteroidetes

(24.5 %), Actinobacteria (9.2 %) and Alphaproteobacteria

(1.0 %). Comparative sequence analysis indicated that

isolates were closely related to already known and/or pre-

viously isolated bacteria (16S rRNA similarity, C97 %).

242 A. Lo Giudice et al.

123

http://www.mna.it

Fig. 1 Rooted phylogenetic

tree calculated by Jukes-Cantor

distance estimation algorithm

showing affiliation of Antarctic

isolates to closest-related

sequences from either cultivated

or cloned bacteria. Percentages

of 400 bootstrap resampling that

supported the branching orders

in each analysis are shown

above or near the relevant

nodes. The tree was outgrouped

with 16S rRNA gene sequence

of Methanocaldococcusjannaschii DSM 2661


123

The Gammaproteobacteria clustered in two main groups

(Fig. 1). The first one branched in two sub-clusters formed

by Pseudoalteromonas (strain S8-16 representing 12

isolates) and Shewanella (strain S1-58 representing three

isolates) affiliates, respectively. The larger group included

isolates that were strongly related to the genus Psychrob-

acter (strains S8-7, S1-47 and S8-3 representing 49 isolates).

The next abundant group, the CF group of Bacteroidetes,

formed a single cluster that included representatives of

the genera Bizionia (nine isolates represented by the strains

S1-28 and S1-75), Pibocella (three isolates represented by

the strain S1-16) and Psychroserpens (12 isolates repre-

sented by the strain S1-61).

The Actinobacteria clustered in two groups. In particu-

lar, the first one included exclusively isolates that were

strongly related to the genus Rhodococcus (strain S3-8).

Isolates in the second group branched in two clusters

formed by Agreia (five isolates represented by the strain

S1-15) and Arthrobacter/Nesterenkonia (one and two

isolates represented by the strains S8-47 and S1-40,

respectively). Finally, the sole affiliate (strain S1-74) to the

Alphaproteobacteria shared the highest degree of sequence

identity with a Sulphitobacter sp.

Bacterial growth in the presence of polychlorinated

biphenyls (PCBs)

A total of 22 isolates (18 Gammaproteobacteria, three

Actinobacteria and one Bacteroidetes) were able to grow

into the mineral salt medium amended with Aroclor 1242. In

particular, PCB-oxidizing bacteria were mainly affiliated to

Psychrobacter spp. (16 isolates, namely S1-6, S1-30, S1-33,

Fig. 2 Percentage of resistant

isolates. a Resistance to heavymetals. Cu-1000 and Cu-2500:

isolates that resisted to up to

1,000 or 2,500 ppm of CuCl2,

respectively; Cd-50, Cd-100,

Cd-500, Cd-1000, Cd-2500,

Cd-5000 and Cd-10000: isolates

that resisted to up to 100, 500,

1,000, 2,500, 5,000 or

10,000 ppm of CdCl2,

respectively; Zn-500, Zn-1000

and Zn-2500: isolates that

resisted to up to 500, 1,000 or

2,500 ppm of ZnCl2,

respectively. b Resistance toantibiotics. chl-25: isolates that

resisted to up to 25 ppm of

chloramphenicol; amp-50:

isolates that resisted to up to

50 ppm of ampicillin; str-25,

str-250 or str-350: isolates that

resisted to up to 25, 250 or

350 ppm of streptomycin,

respectively; kan-25 and kan-

150: isolates that resisted to up

to 25 or 150 ppm of kanamycin


123

S1-41, S1-47, S1-60, S3-2, S3-4, S8-2, S8-3, S8-5, S8-7,

S8-12, S8-14, S8-42, S8-43) with Pseudoalteromonas,

Shewanella, Bizionia, Nesterenkonia, Arthrobacter and Rho-

dococcus that were represented by a single isolate each (namely

S8-16, S1-58, S1-67, S1-21, S8-47 and S3-8, respectively).

Heavy metal and antibiotic tolerance

All strains were strongly sensitive to HgCl2 as they did not

grow even at the lowest concentration that was tested for

such salt (10 ppm). Conversely, as it is shown in Fig. 2a,

isolates generally tolerated up to 1,000 ppm of the

remaining three metals. More in detail, the heavy metal

tolerance per genus is shown in Table 1. In particular,

members of the genera Psychroserpens (among the CFB

group of Bacteroidetes), Psychrobacter and Pseudoalte-

romonas (among the Gammaproteobacteria) were among

those strains that tolerated higher metal concentrations.

The concentration of 1,000 ppm was considered as a

threshold in order to individuate more resistant strains

(41; listed in Table 2). Tolerance patterns were generally

similar within the same species, though they often

Table 1 Heavy metal tolerance

per genus (the unidentified

alphaproteobacterium S1-74

was not included in the table)

Genus (no. of isolates) Heavy metal concentration (ppm)

50 100 500 1,000 2,500 5,000 10,000

CdCl2

Psychrobacter (49) 1 15 1 19 13 0 0

Pseudoalteromonas (12) 0 3 2 6 0 0 1

Shewanella (3) 0 1 0 2 0 0 0

Bizionia (9) 0 2 2 2 3 0 0

Pibocella (3) 0 1 0 1 0 1 0

Psychroserpens (12) 0 0 3 1 3 1 4

Clavibacter (5) 0 1 1 2 1 0 0

Arthrobacter (1) 0 0 0 0 1 0 0

Nesterenkonia (2) 0 1 0 1 0 0 0

Rhodococcus (1) 0 0 0 0 0 1 0

Total (97) 1 24 9 34 21 3 5

CuCl2

Psychrobacter (49) 0 0 0 41 8 0 0


Shewanella (3) 0 0 0 2 1 0 0

Bizionia (9) 0 0 0 8 1 0 0

Pibocella (3) 0 0 0 3 0 0 0


Clavibacter (5) 0 0 0 4 1 0 0



Rhodococcus (1) 0 0 0 0 1 0 0

Total (97) 0 0 0 81 16 0 0

ZnCl2

Psychrobacter (49) 0 0 12 37 0 0 0


Shewanella (3) 0 0 2 1 0 0 0

Bizionia (9) 0 0 4 5 0 0 0

Pibocella (3) 0 0 2 1 0 0 0


Clavibacter (5) 0 0 1 4 0 0 0



Rhodococcus (1) 0 0 0 1 0 0 0

Total (97) 0 0 25 70 2 0 0


123

differed among species in the same genus. The highest

tolerated concentration of CuCl2 and ZnCl2 generally

resulted 2,500 ppm (16 and two strains, respectively),

whereas CdCl2 was also tolerated up to 5,000 and

10,000 ppm, even if by few strains (that were mainly

affiliated to Psychroserpens sp.). Multiple metal-resis-

tance was exhibited by only six strains, i.e. Psychrobacter

spp. S8-3 and S8-25, Pseudoalteromonas sp. S1-17,

Clavibacter sp. S1-37, Rhodococcus sp. S3-8 and Nest-

erenkonia sp. S1-40.

Table 2 Strains that tolerated heavy metal salts over the threshold concentration of 1,000 ppm (A: 2,500 ppm; B: 5,000 ppm; C: 10,000 ppm)

Phylum or class Next relative by GenBank alignment (AN, organism) Strain Metal salt

CuCl2 ZnCl2 CdCl2

Gammaproteobacteria PSU85875, Psychrobacter sp. IC008 S1-12 A

PSU85875, Psychrobacter sp. IC008 S1-47 A

HM771256, Psychrobacter sp. 4Dc S1-3 A










DQ521392, Psychrobacter sp. ice-oil-471 S1-4 A





DQ521392, Psychrobacter sp. ice-oil-471 S8-25 A A

DQ521392, Psychrobacter sp. ice-oil-471 S8-3 A A

AY657017, Pseudoalteromonas sp. 41 S1-17 A A

AY657017, Pseudoalteromonas sp. 41 S8-17 C

AY657017, Pseudoalteromonas sp. 41 S8-20 A

DQ533970, Shewanella sp. ice-oil-445 S1-49 A

Bacteroidetes FJ889663, Bizionia sp. NF1-21 S1-53 A

FJ889663, Bizionia sp. NF1-21 S1-75 A

AY694003, B. algoritergicola strain APA-1 S1-66 A

AY694003, B. algoritergicola strain APA-1 S1-67 A

AY771777, Pibocella ponti clone SE92 S1-10 B

GU644365, Psychroserpens sp. KLE1228 S1-54 C

GU644365, Psychroserpens sp. KLE1228 S1-55 A




GU644365, Psychroserpens sp. KLE1228 S1-52 B



Actinobacteria EU374910, Clavibacter sp. AP29 S1-37 A A

AF134184, Arthrobacter agilis strain LV7 S8-47 A

DQ108403, Nesterenkonia sp. Tibet-IBa2 S1-21 A

DQ108403, Nesterenkonia sp. Tibet-IBa2 S1-40 A A

GQ153645, Rhodococcus sp. ANT-2400 S9 S3-8 A B


123

Results from the antibiotic tolerance test are reported in

Fig. 2b. A total of 64 strains were resistant to at least one of

the tested antibiotics. Chloramphenicol and ampicillin

showed a strong inhibition effect against Antarctic isolates,

even at low concentrations (25–50 ppm), with resistant

strains that were 28 and 26, respectively. Overall, the

remaining two antibiotics were tolerated by a higher

number of isolates with the majority of them that were

resistant to kanamycin up to 150 ppm (37 out of 47

kanamycin-resistant strains) and streptomycin up to

250 ppm (29 out of 45 strains).

With regards to the phylogenetic affiliation, the unique

alphaproteobacterium (isolate S1-74) was susceptible to all

the tested antibiotics. Resistance to streptomycin and

kanamycin was predominantly observed for all bacteria

that belonged to the CF group of Bacteroidetes¸ in addition

to Pseudoalteromonas isolates among Gammaproteobac-

teria with Pseudoalteromonas sp. S1-46 that was resistant

to streptomycin up 350 ppm (Table 3). Multiple antibiotic-

resistance was exhibited by forty-five strains (Table 4).

Resistance patterns often differed within the same species.

Discussion

In the present study, we report for the first time on the

response of bacteria from Antarctic shallow sediments

towards heavy metals, antibiotics and PCBs, in order to

further contribute to the understanding of the role played

by microbial communities, as the initial step in most food

chains, in the transfer of toxic compounds to higher trophic

levels (De Souza et al. 2006). Such contaminants have been

detected in the Antarctic sedimentary compartment, also

in the Terra Nova Bay area (Fuoco et al. 1994, 1995;

Giordano et al. 1999; Dalla Riva et al. 2004). However, to

the best of our knowledge, their effect on indigenous

bacteria has been never investigated.

Interestingly, the nMDS analysis that was computed on

the entire data set from the screening showed that bacterial

isolates clustered according to their phylogenetic affiliation

(Fig. 3). In fact, only few Gammaproteobacteria isolates

were resistant to heavy metal concentrations higher than

1,000 ppm, whereas they were strongly represented among

antibiotic-resistant and/or PCB-oxidizing isolates. In con-

trast, the CF group of Bacteroidetes seemed to be more

resistant to the tested heavy metals. The comparison of the

antibiotic and heavy metal resistance patterns at genus

level revealed some distinctive features. For example,

members of the genus Psychroserpens (among the CF

group of Bacteroidetes) generally tolerated higher con-

centrations of the cadmium salt than the other genera,

whereas the genera Psychrobacter and Pseudoalteromonas

generally better tolerated copper and zinc. With respect to

the antibiotic resistance, the genera Psychrobacter,

Pseudoalteromonas, Shewanella and Bizionia showed the

widest range of resistance as they grew also in the presence

of ampicillin. Among Actinobacteria, all isolates that

belonged to the genera Nesterenkonia, Clavibacter and

Arthrobacter were resistant to kanamycin up to 150 ppm.

The observed differences in antibiotic and heavy metal

resistance/tolerance patterns can be dependent on the

ecology and physiology of the bacterial isolates, indicating

different modes and mechanisms of resistance/tolerance

acquisition, as it was suggested by Vaz-Moreira et al.

(2011). Additionally, such results indicate that distinctive

resistance/tolerance patterns could be related to the phy-

logenetic affiliation. Thus the dissemination of heavy metal

tolerance and antibiotic resistance may possess great rele-

vance for the population dynamics.

However, growth patterns often highly differed among

isolates in the same species, thus appearing to be more

likely strain- rather than species-specific. This finding

suggests a possible process of resistance/tolerance acqui-

sition or loss.

As it was stated in the introduction, information on

PCB-degrading cold-adapted microorganisms remain quite

scarce and, in particular, data regarding Antarctica are

limited to few investigations which demonstrated their occur-

rence in the water column at Terra Nova Bay (Yakimov

et al. 1999; De Domenico et al. 2004; Michaud et al. 2007).

Table 3 Antibiotic tolerance per genus (the unidentified alphapro-

teobacterium S1-74 was not included in the table)

Genus (no. of resistant

isolates)

Antibiotic concentration (ppm)

Chl

25

Amp

50

Str

25

Str

250

Str

350

Kan

25

Kan

150

Psychrobacter (18 out of

49)

4 15 4 2 0 6 1

Pseudoalteromonas (11

out of 12)

7 8 4 4 1 3 6

Shewanella (3 out of 3) 1 1 2 1 0 1 1

Bizionia (9 out of 9) 3 2 0 9 0 0 9

Pibocella (3out of 3) 3 0 0 3 0 0 3

Psychroserpens(11 out of 12)

11 0 0 11 0 0 11

Clavibacter

(5 out of 5)

0 0 1 0 0 0 5

Arthrobacter(1 out of 1)

0 0 0 0 0 0 1

Nesterenkonia(2 out of 2)

0 0 2 0 0 0 2

Rhodococcus(1 out of 1)

0 0 1 0 0 1 0

Total (64 out of 97) 29 26 14 30 1 11 39

Strains that were resistant to almost one antibiotic are considered


123

The isolation of PCB-oxidizing bacteria from Antarctic

sediments is here reported for the first time. Interestingly,

the percentage of bacteria that were able to grow into the

mineral salt medium amended with Aroclor 1242 was

consistently higher than that previously determined for

bacteria from seawater (21.4 vs 7.1 %; Michaud et al.

2007). This finding suggests that the sedimentary microbial

community may be more adapted to the presence of PCBs

than that inhabiting the water column, due to the accu-

mulation of PCBs in sediments.

The phylogenetic analysis revealed that the PCB-

oxidizers that were affiliated to Psychrobacter, Pseudoal-

teromonas, Arthrobacter and Rhodococcus species were

strongly related (97–99 % of similarity) to PCB-degrading

Table 4 Antibiotic resistance patterns of Antarctic isolates

Phylum or

class*

Strain (s) Next relative by GenBank alignment (AN,

organism)

Chl**

(ppm)

Amp**

(ppm)

Str** (ppm) Kan**

(ppm)

25 50 25 250 350 25 150

GAM S1-29 PSU85875, Psychrobacter sp. IC008 X

S1-35 PSU85875, Psychrobacter sp. IC008 X

S1-45 HM771256, Psychrobacter sp. 4Dc X X X X

S8-7, S8-42, S8-46 HM771256, Psychrobacter sp. 4Dc X X X

S8-43 HM771256, Psychrobacter sp. 4Dc X X

S1-9 HM771256, Psychrobacter sp. 4Dc X X

S1-38, S8-44, S3-3,

S3-4, S8-2, S8-14

HM771256, Psychrobacter sp. 4Dc X

S3-2 HM771256, Psychrobacter sp. 4Dc X

S8-34 DQ521392, Psychrobacter sp. iceoil471 X X X X

S1-19 DQ521392, Psychrobacter sp. iceoil471 X

S8-29 DQ521392, Psychrobacter sp. iceoil471 X

S1-46 AY657017, Pseudoalteromonas sp. 41 X X X X

S8-13, S8-16,

S8-20, S8-24

AY657017, Pseudoalteromonas sp. 41 X X X X

S8-38 AY657017, Pseudoalteromonas sp. 41 X X X X

S8-17 AY657017, Pseudoalteromonas sp. 41 X X X

S1-14, S1-17 AY657017, Pseudoalteromonas sp. 41 X X

S1-22, S8-1 AY657017, Pseudoalteromonas sp. 41 X

S1-49 DQ533970, Shewanella sp. iceoil445 X X X X

S1-68 DQ533970, Shewanella sp. iceoil445 X X

S1-58 DQ533970, Shewanella sp. iceoil445 X

CFB S1-53 FJ889663, Bizionia sp. NF121 X X X X

S1-57, S1-75 FJ889663, Bizionia sp. NF121 X X

S1-44, S1-56 AY694003, B. algoritergicola strain APA1 X X X

S1-66 AY694003, B. algoritergicola strain APA1 X X X

S1-28, S1-67, S1-71 AY694003, B. algoritergicola strain APA1 X X

S1-10, S1-26, S1-16 AY771777, Pibocella ponti clone SE92 X X X

S1-13, S1-27, S1-54,

S1-55, S1-61, S1-63,

S1-11,

S1-52, S1-51, S1-72

GU644365, Psychroserpens sp. KLE1228 X X X

ACT S1-37 EU374910, Clavibacter sp. AP29 X X

S1-15, S3-7, S3-12 EU374910, Clavibacter sp. AP29 X

S1-21, S1-40 DQ108403, Nesterenkonia sp. TibetIBa2 X X

S8-47 AF134184, Arthrobacter agilis strain LV7 X

S3-8 GQ153645, Rhodococcus sp. ANT2400 S9 X X

* GAM, Gammaproteobacteria; CFB, CF group of Bacteroidetes; ACT, Actinobacteria

** Chl, chloramphenicol; Amp, ampicillin; Str, streptomycin; Kan, kanamycin


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bacteria that were isolated form Antarctic seawater during

previous expeditions at Terra Nova Bay (Yakimov et al.

1999; Michaud et al. 2007). Thus such bacteria could be

autochthonous and shared between the water column and

sediment compartments. Conversely, as far as we know, no

report exists about the ability of cold-adapted bacteria

belonging to the genera Shewanella, Bizionia and Nest-

erenkonia to utilize PCBs, as we observed through the

present study.

Microorganisms can develop resistance in the growing

presence of toxic compounds in the environment. The most

common resistance is to metals and antibiotics, which can

be a result of bio-essentiality or abuse of the metals, and/or

antibiotics. In the present study, multi-resistance either to

antibiotics or metals was seldom observed, thus confirming

results previous reported by De Souza et al. (2006) for

bacteria from Antarctic seawater. Tolerance to the four

heavy metals assayed in this study was in the order of

Cd [ Cu [ Zn [ Hg and appeared to be strictly related to

the metal concentrations, as determined during chemical

surveys in the same area. Concentrations of mercury in the

Terra Nova Bay sediments are among the lowest ever

reported for coastal marine environments (Bargagli et al.

1998). This feature could explain the very high suscepti-

bility to mercury of our Antarctic isolates which are

probably not adapted to the presence of mercury in their

surrounding environment. For the same reason, a strong

tolerance was recorded in the present study towards cad-

mium. In fact, cadmium content in sediments of the Terra

Nova Bay generally results considerably higher than

background typical concentrations due to the presence of

biogenic calcareous debris, where Cd tends to be enriched,

or to the Cd uptake by phytoplankton and the subsequent

discard with detritus and faecal pellets (Ianni et al. 2010).

Finally, a moderate tolerance was observed towards copper

and zinc whose concentrations in the Terra Nova Bay

generally resulted similar to those regarded as background

in most marine coastal areas (Bargagli et al. 1996).

With regards to antibiotic assays, the response of the

isolates to the tested antibiotics ranged from complete

resistance to total susceptibility. In particular, resistances to

ampicillin and chloramphenicol were very pronounced in

the majority of isolates, confirming previous results for

Antarctic marine bacteria (De Souza et al. 2006; Lo Giu-

dice et al. 2010, 2012) Antibiotic resistance was not

expected at so high frequency as tested bacteria were from

an environment where pollution from both chemicals and

drugs is quite scarce. So, it is possible that acquired anti-

biotic resistance traits could become widespread even in

environments where both the exposure to antibiotics and

the anthropic pressure are minimal.

In conclusion, physiological responses by bacteria from

Antarctic sediment towards PCBs, heavy metals and anti-

biotics were investigated. Antarctic bacteria responded

differently to the presence of such toxic compounds pri-

marily based on their phylogenetic affiliation and second-

arily at strain level. Moreover, the high incidence of

resistance either to metal or antibiotics, in addition to the

capability to grow on PCBs, confirm that bacteria are able

to cope and/or adapt to the occurrence pollutants even in

low human-impacted environments. Thus, the study of the

microbial responses to anthropogenic stress could furnish

useful information on the contaminant exposure of a cer-

tain ecosystem and, therefore, an overall picture of envi-

ronmental health. A genome-wide transcriptional level

analysis will be a focus of future research to reveal more

in-depth detail in the bacterial responses to such kind of

pollutants.

Acknowledgments This research was supported by grants from

PNRA (Programma Nazionale di Ricerche in Antartide), Italian

Ministry of Education and Research (Research Project PNRA

2004/1.6), and from MNA (Museo Nazionale dell’Antartide).

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Response of bacterial isolates from Antarctic shallow sediments (2013)

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