Polar Science 10 (2016) 123e131

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Polar Science
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Antibiotic resistance in Escherichia coli strains isolated from Antarctic

bird feces, water from inside a wastewater treatment plant, and
seawater samples collected in the Antarctic Treaty area
Virginia Rabbia a, Helia Bello-Toledo a, *, Sebastia

n Jime

nez b, Mario Quezada a,
a c
Mariana Domínguez , Luis Vergara , Claudio Go
mez-Fuentes d, Nancy Calisto-Ulloa d,
Daniel Gonza
lez-Acun~ a , Juana Lo
e
pez , Gerardo Gonza
f
lez-Rocha a
a
n en Agentes Antibacterianos, Facultad de Ciencias Biolo
Laboratorio de Investigacio
gicas, Universidad de Concepcio

n, Concepcio

n, Chile
b
Facultad de Ciencias Naturales y Oceanogra
ficas, Universidad de Concepcio
n, Concepcio
n, Chile
c
n, Concepcio
Facultad de Ciencia, Universidad San Sebastia
n, Chile
d
Departamento de Ingeniería Química, Universidad de Magallanes, Punta Arenas, Chile
e
Departamento de Ciencias Pecuarias, Facultad de Ciencias Veterinarias, Universidad de Concepcio
n, Chile
f
Departamento de Patología y Medicina Preventiva, Facultad de Ciencias Veterinarias, Universidad de Concepcio
n, Chile

a r t i c l e i n f o a b s t r a c t

Article history: Antibiotic resistance is a problem of global concern and is frequently associated with human activity.
Received 19 August 2015 Studying antibiotic resistance in bacteria isolated from pristine environments, such as Antarctica, ex-
Received in revised form tends our understanding of these fragile ecosystems. Escherichia coli strains, important fecal indicator
2 March 2016
bacteria, were isolated on the Fildes Peninsula (which has the strongest human influence in Antarctica),
Accepted 7 April 2016
Available online 8 April 2016
from seawater, bird droppings, and water samples from inside a local wastewater treatment plant. The
strains were subjected to molecular typing with pulsed-field gel electrophoresis to determine their
genetic relationships, and tested for antibiotic susceptibility with disk diffusion tests for several antibiotic
Keywords:
Antibiotic resistance
families: b-lactams, quinolones, aminoglycosides, tetracyclines, phenicols, and trimethoprim
Antarctica esulfonamide. The highest E. coli count in seawater samples was 2400 cfu/100 mL. Only strains isolated
Fildes Peninsula from seawater and the wastewater treatment plant showed any genetic relatedness between groups.
Escherichia coli Strains of both these groups were resistant to b-lactams, aminoglycosides, tetracycline, and trimethoprim
esulfonamide.In contrast, strains from bird feces were susceptible to all the antibiotics tested. We
conclude that naturally occurring antibiotic resistance in E. coli strains isolated from Antarctic bird feces
is rare and the bacterial antibiotic resistance found in seawater is probably associated with discharged
treated wastewater originating from Fildes Peninsula treatment plants.
© 2016 Elsevier B.V. and NIPR. All rights reserved.

1. Introduction increasing dissemination of antibiotic-resistant bacteria has two

Antibiotic resistance was reported before the widespread use of
antibiotics in hospitals (Abraham and Chain, 1940), indicating that
it is a natural phenomenon and has its origins in environmental
microbiota (Martínez, 2012). However, a study carried out from
1940 to 2008 showed a worldwide increase in antibiotic-resistant
bacteria, attributed to the misuse of antibiotics (Dodd, 2012). The
* Corresponding author. Facultad de Ciencias Biolo
gicas, Departamento de
Microbiología, Arco Universidad de Concepcio
n, Barrio Universitario S/N, POB: 160-
C, Concepcio
n, Chile.
E-mail address: hbello@udec.cl (H. Bello-Toledo).
major consequences. First, pathogenic bacteria can acquire
antibiotic-resistance genes and become multidrug resistant (MDR),
leaving few treatment options for infected patients. Second, the
introduction and dissemination of antibiotic-resistance genes into
the Antarctic bacterial community changes the original resistome
of the native microbial community, leading to genetic homogeni-
zation (Cowan et al., 2011).
The extent of antibiotic resistance in the bacterial communities
of a given environment has been frequently linked to anthropo-
genic activities. Therefore, in areas in which human activity is
limited, bacteria are expected to contain few antibiotic-resistance
genes. An example of this is the Antarctic continent, where

http://dx.doi.org/10.1016/j.polar.2016.04.002
1873-9652/© 2016 Elsevier B.V. and NIPR. All rights reserved.
124 V. Rabbia et al. / Polar Science 10 (2016) 123e131
bacteria have negligible or no antibiotic resistance, although the
anthropogenic impact has increased in recent years with tourist
and scientific activities (Bonnedahl et al., 2008; Cowan et al., 2011;
Skurnik et al., 2006). Escherichia coli is found in the feces of warm-
blooded animals, so its presence in a given environment strongly
indicates (99% certainty) the presence of animals, like mammals
and birds, or humans in the area (Stevens et al., 2003).
The main vectors of the foreign microorganisms introduced into
Antarctica are the people who inhabit research stations and Ant-
arctic animals, mainly birds (Cowan et al., 2011; Hughes, 2003).
Several studies have reported the presence of coliforms, as well as
E. coli, near sewage outfalls in different locations in Antarctica,
suggesting that sewage is an extrinsic source of nonnative micro-
organisms in the Antarctic Treaty area (Delille and Delille, 2000;
Delille and Gleizon, 2003). The E. coli strains derived from
humans and birds usually differ in their antibiotic-resistance pat-
terns. In previous studies, the presence of E. coli in Antarctic ani-
mals or birds that had no direct human contact was rare
(Bonnedahl et al., 2008; Gordon and Cowling, 2003), and when
E. coli strains were found, they showed little or no antibiotic
resistance (Bonnedahl et al., 2008). However, human-associated
E. coli strains showed higher antibiotic resistance, correlating
with antibiotic use (Davies and Davies, 2010).
The Fildes Peninsula, located on King George Island (Fig. 1),
contains the greatest number of research stations in the Antarctic
Treaty area (Peter et al., 2013). The only study of antibiotic resis-
tance among E. coli isolates in the Fildes Peninsula was performed
by Herna
ndez et al. (2012). In their study, eight E. coli strains
resistant to tetracycline, ampicillin, nalidixic acid, trimetho-
primesulfonamide, and/or streptomycin were isolated from
seawater samples. Based on their antibiotic-resistance patterns, the
researchers concluded that the strains were of human origin.
The aim of this work was to evaluate and compare the antibiotic
resistance in three different groups of E. coli strains isolated on the
Fildes Peninsula from samples of Antarctic bird droppings,
seawater, and water collected from inside a wastewater treatment
Fig. 1. Overall view of the sampling area in Antarctica. Left: the Antarctic Peninsula showing Gabriel Gonz
alez Videla station (GGVS), General Bernardo O'Higgins station (GBOS),
and Fildes Peninsula (FP). Right: an inset of Fildes Peninsula, on which the North Fildes Peninsula (NFP), South Fildes Peninsula (SFB), and Ardley Island (AI) sampling sites are
shown.
plant (WWTP), to assess the contribution of these strains to the
antibiotic-resistant gene pool.
2. Materials and methods
2.1. Sampling
Samples were collected from the Antarctic Treaty area during
expeditions in 2011 and 2013, and were divided into four groups
according to the sampling site: North Fildes Peninsula (NFP), South
Fildes Peninsula (SFP), Antarctic birds, and WWTP (Fig. 1). Thirty-
nine sea water samples were collected in NFP from the sea sur-
face, on- and offshore at distances of 0, 10, 15, 25, 50, 150, 350, and
650 m from the sewage outfalls of two Chilean scientific bases: 1)
Fuerza Ae
rea de Chile (FACH; 62
1201.6500 S, 58
570 36.9600 W); and 2)
Instituto Anta
rtico Chileno (INACH) and Estacio
n Marítima Bahia
Fildes (62
120 5.0700 S, 58
570 39.5800 W). Samples were also collected
in the same manner from the Kitiesh River mouth (62
11059.3700 S,
58
570 31.1600 W), where the Russian Bellinghausen station dis-
charges its treated wastewater (Fig. 2A). Seven seawater samples
were also taken on the coastline at the Chinese Great Wall station
located in SFP (62
120 58.6500 S, 58
570 35.2100 W) (Fig. 2B). A single
control sample was taken 2000 m off shore from NFP, which we
assumed was unaffected by any WWTP discharge.
The “Antarctic birds” group included 25 E. coli strains collected
in 2011 by Gonz
alez-Acun~ a et al. (2013) from cloacal swabs or
environmental stool samples from Pygoscelis papua (Gentoo pen-
guin), Larus dominicanus (kelp gull), and Chionis alba (snowy
sheathbill). These samples were collected at three locations within
the Antarctic Treaty area (Fig. 1): General Bernardo O'Higgins sta-
tion (GBOS; 63
190 16.4400 S, 57
540 5.0900 W), Gabriel Gonza
lez Videla
station (GGVS; 64
490 26.0300 S, 62
51026.1000 W), and Ardley Island
(AI; 62
120 47.4400 S, 58
550 58.7000 W). The “wastewater treatment
plant” group consisted of strains isolated in 2013 from one water
sample taken inside of the INACH WWTP located on the Fildes
Peninsula (Fig. 1).
 V. Rabbia et al. / Polar Science 10 (2016) 123e131 125

Fig. 2. Map of the sampling sites in NFP (A) and SFP (B). Each sampling site is highlighted with a white dot and the total coliform count for the sample is shown next to it.

2.2. Bacterial strains
Total coliforms and E. coli were counted by filtering 0.1 mL, 1 mL,
and 134 mL seawater samples. The membrane filters were placed
on chromogenic selective agar (ChromoCult® Coliform Agar ES,
Merck, Darmstadt, Germany) and incubated at 37
C for 24 h. The
filters with countable salmon-red colonies (coliforms) and dark
blue to violet colonies (E. coli) were selected. The bacterial isolates
were purified by streaking a single colony on Levine agar (Merck)
and incubating it at 37
C for 18e24 h. Trypticase soy broth (Oxoid
Ltd., Basingstoke, Hampshire, England) was then inoculated with
the presumptive E. coli strains and incubated at 37
C overnight.
Aliquots were stored at 80
C in 50% (v/v) glycerol in Trypticase
soy broth.
Human-associated E. coli strains were isolated by spreading
100 mL of water from the WWTP on chromogenic selective agar
plates (ChromoCult® Coliform Agar ES), which were incubated at
37
C for 18e24 h. The strains were purified and stored as described
above. Ecoli was confirmed with conventional biochemical tests,
according to Nataro et al. (2011).
126 V. Rabbia et al. / Polar Science 10 (2016) 123e131

Fig. 3. Dendrogram of E. coli strains isolated from WWTP, NFP, and SFP. Dendrogram based on XbaI macrorestriction patterns of E. coli. The 80% cut-off for the determination of
clusters of genetic relatedness clusters is shown with a dashed line. : North Fildes Peninsula (NFP); : South Fildes Peninsula (SFP); : Wastewater treatment plant (WWTP).
Clonal strains are highlighted with a black box.
 V. Rabbia et al. / Polar Science 10 (2016) 123e131
Fig. 3. continued
2.3. Molecular typing
The genetic diversity of the E. coli strains was evaluated with
pulsed-field gel electrophoresis (PFGE) in a CHEF-DR II apparatus
(Bio-Rad, La Jolla, CA) under the following conditions: XbaI diges-
tion, 6 V/cm2, 6.8 s (initial pulse), 35.4 s (final pulse), and 19 h run at
14
C, according to the protocol of ISP (2007). Salmonella Braen-
derup H9812 was used as the control strain. A fingerprint analysis
was performed with the software BioNumerics v.6.611 based on the
Dice coefficient, and a dendrogram was constructed with UPGMA.
Strains sharing at least 80% similarity were considered genetically
related, and those sharing 100% similarity were classified as clones
(Chung et al., 2012; Kao et al., 2015).
2.4. Antibiotic susceptibility testing
Antibiotic susceptibility patterns were determined with the disk
diffusion method (Clinical and Laboratory Standards Institute, CLSI,
2012) using different groups of antibiotics: penicillins (ampicillin,
AMP), cephalosporins (cephalothin, CEP; cefuroxime, CXM; cefox-
itin, FOX; cefotaxime, CTX; ceftazidime, CAZ; cefepime, FEP), car-
bapenems (ertapenem, ETP; meropenem, MEM; imipenem, IMP),
aminoglycosides (streptomycin, STR; kanamycin, KAN; gentamicin,
GEN; amikacin, AMK), quinolones (nalidixic acid, NAL; ciprofloxa-
cin, CIP), tetracycline (TET), phenicols (chloramphenicol, CLO),
sulfonamide (SUL), and trimethoprim (TMP). Escherichia coli ATCC
25922 was used as the control for the susceptibility tests. The
127
results were interpreted according to the CLSI document M100-S24
(CLSI, 2014).
2.5. Statistical analysis
One-way ANOVA and Tukey's multiple-range test were used to
compare the mean total coliform and E. coli counts using IBM SPSS
Statistics version 23.0 (SPSS Inc.®, Chicago, IL). Statistical signifi-
cance was set at p < 0.05.
3. Results
3.1. Bacterial counts
The sampling sites and counts of E. coli (dark blue to violet
colonies) and total coliforms (salmon-red colonies) are summa-
rized in Table 1. In NFP (Fig. 2A), total coliforms and E. coli were
detected in 53% and 46% of samples, respectively, whereas in SFP
(Fig. 2B), total coliforms and E. coli were both detected in 85% of
samples. The highest bacterial counts were found at the sites of
treated wastewater discharge and decreased towards the open sea
(Fig. 2A). There were no statistically significant differences
(p > 0.05) between the maximum total coliform counts at any of the
sampling sites. In the samples taken at the Kitiesh River mouth and
0 m from the discharge outlets of the INACH, FACH, and Great Wall
WWTPs, E. coli constituted 0.35%,1.4%, 2.2%, and 24.5%, respectively,
of the total coliform counts. The only statistically significant
128 V. Rabbia et al. / Polar Science 10 (2016) 123e131

Table 1
Total coliform and E. coli counts in seawater samples from the Fildes Peninsula.

Sample sites Distance from sewage outfall (m) Number of samples Number of total coliformsa Number of E. colia

North Fildes Peninsula (NFP) 0 6 2400e9940 14e140

10 18 96e8400 0e100
25 20 3e580 1e35
50 12 16e249 1e40
150 4 0e141 0e10
350 8 0 0
650 10 0 0
South Fildes Peninsula (SFP) 0 2 9600e9800 2300e2400
10 4 60e1400 4e780
150 2 90e101 0e1
200 2 20e31 1
240 2 0e20 0e3
350 2 0 0
b
Control sample 2 0 0
a
cfu/100 mL.
b
2000 m offshore.

difference was between the E. coli counts of the Great Wall and the
other stations (INACH and FACH) (p < 0.05). No coliforms were
detected in the control samples or in samples taken 350 m from a
sewage outfall. The total coliform counts in the samples taken from
the WWTP outfalls exceeded 20,000 cfu/100 mL.
3.2. E.coli identification
We randomly selected 70, 18, and 26 strains isolated from
samples collected in NFP, SFP, and WWTP, respectively, on chro-
mogenic selective agar plates and confirmed them as E. coli.
Twenty-five strains isolated from Antarctic birds in 2011 were
included, for a total of 139 E. coli strains.
3.3. Genetic diversity among strains
The genetic relatedness of the E. coli strains from the samples
collected in SFP, NFP, and WWTP and from Antarctic birds is shown
in Fig. 3. Of the 139 strains analyzed with PFGE, 10 strains from the
NFP and Antarctic bird groups were classified as non typeable.
Some strains within each group were genetically related. The
highest clonality rate was in the WWTP group, with 76% of the
strains considered clones. In both SFP and NFP, 67% of the strains
were genetically related. Five clusters of two clonal strains were
detected in the Antarctic bird group (data not shown), three of
them formed by strains isolated from Gentoo penguins at
geographic locations separated by a distance of approximately
130 km. Clonality and genetic relatedness between groups was only
found between strains of the WWTP and NFP groups (23%).
3.4. Antibiotic susceptibility testing
To avoid misinterpretation of the percentage of antibiotic-
resistant strains, only one strain with each different antibiotic-
resistance pattern was selected from each distinct clonal cluster.
Therefore, the results shown in Table 2 and Fig. 4 were obtained
from a total of 95 E. coli strains, including 53 strains from NFP, 11
strains from SFP, 20 strains from the Antarctic bird group, and 11

Table 2
Resistance to antibiotics in E. coli strains isolated on the Fildes Peninsula, Antarctica.

Antibiotics (disc potency) Percentage of resistant strains isolated from:

Antarctic birdsa(20)* WWTP* Seawater samples

(11)b SFPc(11)* NFPd(53)*

Ampicillin (10 mg) 0 9.1 45.5 9.4

Cephalothin (30 mg) 0 9.1 18.2 0
Cefuroxime (30 mg) 0 0 0 0
Cefoxitin (30 mg) 0 0 18.2 0
Cefotaxime (30 mg) 0 0 18.2 0
Ceftazidime (30 mg) 0 0 9.1 0
Cefepime (30 mg) 0 0 18.2 0
Ertapenem (10 mg) 0 0 0 0
Meropenem (10 mg) 0 0 0 0
Imipenem (10 mg) 0 0 0 0
Streptomycin (10 mg) 0 18.2 9.1 3.8
Kanamycin (30 mg) 0 9.1 0 0
Gentamicin (10 mg) 0 0 9.1 0
Amikacin (30 mg) 0 0 0 0
Nalidixic acid (30 mg) 0 0 81.8 0
Ciprofloxacin (5 mg) 0 0 9.1 0
Tetracycline (30 mg) 0 9.1 81.8 17
Chloramphenicol (30 mg) 0 0 0 0
Sulfonamide (300 mg) 0 18.2 27.3 11.3
Trimethoprim (5 mg) 0 9.1 9.1 7.5

()* number of isolates.

a
Cloacal swabs and stool samples.
b
Water samples from INACH wastewater treatment plant.
c
South Fildes Peninsula.
d
North Fildes Peninsula.
 V. Rabbia et al. / Polar Science 10 (2016) 123e131
Fig. 4. Distribution of E. coli strains according to the number of antibiotic groups to which they are resistant.
strains from WWTP.
The strains isolated from water samples and Antarctic bird feces
displayed different antibiotic-resistance patterns. The isolates from
the Antarctic bird group showed no resistance to any of the 20
antibiotics tested (Table 2). However, strains with only intermedi-
ate susceptibility to aminoglycosides were found: more than 20%
were slightly resistant to GEN or KAN and up to 80% were slightly
resistant to STR. These strains also showed intermediate suscepti-
bility to AMP and CEP (data not shown).
Among the strains isolated from the WWTP, 18% (2/11 strains)
were resistant to antibiotics from at least three different groups
(KANeTETeSUL and AMPeSTReSULeTMP; Fig. 4), and can
therefore be classified as MDR strains, according to the criteria of
Magiorakos et al. (2012). Two strains from the WWTP group
showed susceptibility to every compound tested, whereas the
other six strains showed intermediate susceptibility to at least
one antibiotic, with resistance to STR most common.
The strains isolated from sea water also showed diverse
antibiotic-resistance patterns. The highest percentage of antibi-
otic resistance strains was found in SFP (Table 2). These strains
were resistant to 13 antibiotics, whereas the strains from NFP
were resistant to only five antibiotics. All but two strains from
SFP showed resistance to NAL and TET. This was also the only
group in which strains resistant to CTX, CAZ, GEN, NAL, and CIP
were found. Only 2/11 strains (18%) were susceptible to every
antibiotic tested, whereas 6/11 strains (55%) displayed an MDR
phenotype (Table 2; Fig. 4). All the MDR strains were resistant to
AMP, NAL, and TET, and another two strains were resistant to 8
and 10 of the 20 antibiotics tested. Molecular typing revealed
clusters of closely related strains (strain CHþ15-3 and strain
CHMVþ-25-3) and clonal strains (CHþ15-1, CHþ15-4, and
CHþ15-5) in the SFP group (Fig. 3), which shared the same
antibiotic-resistance phenotype (AMP, NAL, and TET). Strain
CHþ15-3 was also resistant to GEN, SUL, and TMP.
The strains from NFP showed minimal antibiotic resistance,
mainly to TET, AMP, SUL, TMP, and STR (Table 2). Twenty-eight of
the 53 strains (52%) were susceptible to all the antibiotics tested
and 13 (25%) showed susceptibility and intermediate susceptibility,
mainly to CEP. Only 12 strains (23%) were resistant to at least one
antibiotic, and five strains (9%) were MDR, all of which were
resistant to TET.
4. Discussion
Because bacterial antibiotic resistance is frequently related to
129
human activity, antibiotic-resistant bacteria are rarely isolated from
Antarctica (Bonnedahl et al., 2008). However, humans and Ant-
arctic animals may introduce and disseminate antibiotic-resistant
bacteria in Antarctica (Delille and Delille, 2000; Delille and
Gleizon, 2003), which could potentially transfer resistance genes
to the endemic bacteria, thereby disturbing the native resistome.
The E. coli:total coliform ratio in the outfall seawater samples in
this study was significantly higher in SFP (Great Wall station) than
in NFP (INACH, FACH, and Bellinghausen stations). All four WWTPs
declare that they treat wastewater both mechanically and biologi-
cally, but only the NFP stations use a physicochemical treatment to
disinfect the water bodies before discharging them into the sea
(Peter et al., 2013). UV light is used for this purpose at all three
stations (ATIPR, 2012; Gomez-Fuentes and Calisto-Ulloa, 2013;
Peter et al., 2013). Chang et al. (1985) studied the influence of
different doses of UV light on E. coli and total coliforms in water and
demonstrated that E. coli is more susceptible to UV light than are
other coliforms. Therefore, because the total coliform and E. coli
counts at all four sampling sites were high, the wastewater treat-
ments were not working as they should. However, the E. coli load
discharged into the sea was lower at those WWTPs at which UV
light was used to treat the wastewater (FACH, INACH, and
Bellinghausen).
The Bellinghausen station discharges onto the Kitiesh River, so
the water samples were obtained from the mouth of the river.
Therefore, the recorded bacterial counts corresponded to water
exposed to natural UV light and diluted along the river. However,
interestingly, there was no statistically significant difference be-
tween the total coliform and E. coli counts in the FACH, INACH, and
Bellinghausen groups.
At the moment, no limits have been established for coliforms or
E. coli in wastewater discharged into the sea or any kind of waters of
Antarctica, because the protocol for environmental protection in
the Antarctic Treaty does not address the issue. However, because
all the samples taken in the course of this study were seawater,
rather than treated wastewater, we cannot use the guidelines for
the limits of E. coli in wastewater discharged at sea. Therefore,
because scuba diving activities for scientific purposes take place in
́
the bay, we used the guidelines for recreational water as the
benchmark against which to discuss our results. The European
Union allows a maximum of 500 E. coli cfu/100 mL in seawater for
recreational use (European Union, 2007), and the Environmental
Protection Agency (US EPA) allows a maximum of 200 E. coli cfu/
100 mL in water bodies used for recreational purposes (EPA, 1986).
However, the Chilean guidelines are less strict, allowing a
130 V. Rabbia et al. / Polar Science 10 (2016) 123e131
maximum of 1000 fecal coliform cfu/100 mL in recreational water
with direct human contact (NCh 1333 Of. 78). The results of this
study indicate that the NFP samples meet the Chilean standard (and
the US EPA and EU criteria) because the maximum E. coli count in
this area was 140 cfu/100 mL water. However, the SFP samples did
not meet these standards, because counts as high as 2400 E. coli cfu/
100 mL water were detected, exceeding all three guidelines
mentioned above. Because the Antarctic is an especially pristine
environment that warrants conservation, it is disturbing that no
water quality standards for wastewater disposal into the Antarctic
sea have been instituted and that E. coli counts exceeding estab-
lished guidelines are found in its waters. Therefore, developing a
good environmental protection protocol is vital for the protection
of the Antarctic Treaty area.
Self-relatedness was only observed in the bacterial strains
within the seawater groups, indicating that all the strains in each
group had a common origin and that strains were not exchanged
between groups, probably because the North and South Fildes
Peninsula are physically isolated. The high clonality among the
strains inside the INACH WWTP might be attributable to the sam-
pling procedure (only one sample was taken) or to the wastewater
treatment system used at this facility, because the extended aera-
tion of activated sludge stimulates bacterial growth to reduce the
organic load of the water body, and therefore also stimulates clonal
growth of the bacterial strains entering the bioreactor (Van Veen,
1973).
The Antarctic bird group included strains that were clonal,
although physically separated by as much as 130 km. Most of the
clonal strains were isolated from Ppapua, which are known to live
close to their feces when breeding (Meyer-Rochow and Gal, 2003).
Therefore, one explanation of this phenomenon is that these birds
share strains through contact with feces, leading to a certain degree
of homogeneity among the E. coli strains in the bird population.
Only intermediate antibiotic resistance was detected in the
strains from the Antarctic bird group. This result is consistent with
studies suggesting that antibiotic resistance is rare among the
bacteria isolated from wild birds (Bonnedahl et al., 2008;
Herna
ndez et al., 2012; Skurnik et al., 2006). It has been sug-
gested that antibiotic-resistance patterns can be used as biological
indicators of environmental pollution (Radhouani et al., 2012). The
bacterial strains isolated from Gentoo penguins by Bonnedahl et al.
(2008) mainly belonged to the genus Edwardsiella, with only one
E. coli strain, which was susceptible to all 17 antibiotics tested.
The strains isolated from WWTPs may show widely varying
antibiotic-resistance patterns, depending on the characteristics of
the inflow, the wastewater treatment process, the sampling date,
and antibiotic use, among other factors (Novo and Manaia, 2010).
Therefore, no values for the antibiotic resistance of bacteria isolated
from WWTPs have been established with which to compare our
data. However, Novo and Manaia (2010) reported that one of the
major driving forces contributing to the dissemination of antibiotic
resistance by WWTPs is the input of antibiotic-resistant bacteria
with the raw inflow. Therefore, the antibiotic-resistant bacteria
detected should be compared with those in the human populations
inhabiting the research stations. Because the INACH station ac-
commodates predominantly Chilean scientists, our results were
compared with those of a study of E. coli strains isolated from a
Chilean WWTP located in Antofagasta (Silva et al., 2005). In that
study, 74% of strains showed resistance to AMP, 15% to NAL, 43% to
CLO, 44% to STR, 4% to GEN, 17% to KAN, and 67% to TET. These
antibiotic-resistance patterns are similar to those detected in the
INACH WWTP in our study.
The E. coli strains isolated from the seawater samples differed in
their antibiotic resistance depending on the sampling site. The SFP
group showed resistance to a greater number of antibiotics than the
isolates from the NFP group. It would be interesting to clarify the
factors responsible for this difference. Strain CHþ15-3 was the only
one of the five strains forming a closely related cluster that showed
resistance to GEN, SUL, and TMP, suggesting that the genetic de-
terminants of resistance to these three antibiotics are located in the
same plasmid, present only in this strain.
Only two unrelated strains, both isolated from SFP, showed
resistance to third-generation cephalosporins. Only strains from
the NFP group were related to the strains sampled from the WWTP
because these strains were collected from inside the Chilean
WWTP, which is the only plant discharging water into the NFP area
sampled, and not into SFP. The presence of antibiotic resistance in
E. coli strains isolated from the seawater around the Fildes Penin-
sula is associated with the treated wastewater discharged in the
area, because coliforms were only found in the vicinity of the
outfall, but did not occur beyond 50 m off the coast.
Herna
ndez et al. (2012) reported that strains with similar
resistance patterns were isolated from the same area in January
2011, so the results of this study demonstrate the persistence of
antibiotic resistance in the seawater around the Fildes Peninsula.
The permanence of antibiotic-resistant bacteria in the Fildes
Peninsula seawater is more likely attributable to the periodicity of
wastewater discharge than to the persistence and survival of the
strains in Antarctic seawater, where the environmental conditions
are harsh, making the survival of non indigenous microorganisms
unlikely (Hughes, 2005; Cowan et al., 2011).
During preliminary work, we confirmed that some antibiotic-
resistance genes are transferred by conjugation to other E. coli
(data not shown), so the presence of antibiotic-resistant E. coli
strains in Antarctic seawater must be addressed in future studies.
5. Conclusions
The antibiotic-resistance profiles of E. coli isolated from the
seawater around the Fildes Peninsula are a cause for concern,
especially considering the high E. coli counts in the southern area
and the MDR in the majority of strains. When the antibiotic-
resistance patterns are compared, it is highly probable that the
strains isolated from seawater originated from the WWTPs that
discharge into the area.
This work contributes to the field of bacterial antibiotic resis-
tance in Antarctica. Further research is required to establish
whether the horizontal transfer of genetic resistance determinants
to autochthonous Antarctic bacteria is possible.
Acknowledgments
The contribution of INACH projects RT_06-12, RT_12-13 and
MA_01-12 to the funding of this study is greatly appreciated. We
also thank Dr. Celia Fernandes de Lima and Dr. Diane Haughney for
their help with English grammar and editing.
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