Antibiotic Resistance of Gram Negatives isolates from loggerhead sea turtles
(Caretta caretta) in the central Mediterranean Sea
M. Fotia, C. Giacopelloa, Teresa Bottarib,*, V. Fisichellaa, D. Rinaldoa, C. Mamminac
aDipartimento di Sanità Pubblica Veterinaria, Università degli Studi di Messina, Polo Universitario SS Annunziata, 98167 Messina, Italy
bIstituto per l’Ambiente Marino Costiero, CNR – Spianata S. Raineri, 86 – 98122 Messina, Italy
cDipartimento di Scienze per la Promozione della Salute ‘‘G. D’Alessandro”, Università degli Studi di Palermo, Via del Vespro 133, I-90127 Palermo, Italy
a r t i c l ei n f o
Loggerhead sea turtle
a b s t r a c t
Previous studies on fish and marine mammals support the hypothesis that marine species harbor antibi-
otic resistance and therefore may serve as reservoirs for antibiotic-resistance genetic determinants. The
aim of this study was to assess the resistance to antimicrobial agents of Gram negative strains isolated
from loggerhead sea turtles (Caretta caretta). Oral and cloacal swabs from 19 live-stranded loggerhead
sea turtles, with hooks fixed into the gut, were analyzed. The antimicrobial resistance of the isolates to
31 antibiotics was assessed using the disk-diffusion method. Conventional biochemical tests identified
Citrobacter spp., Proteus spp., Enterobacter spp., Escherichia spp., Providencia spp., Morganella spp., Pantoea
spp., Pseudomonas spp. and Shewanella spp. Highest prevalences of resistance was detected to carbenicil-
lin (100%), cephalothin (92.6%), oxytetracycline (81.3%) and amoxicillin (77.8%). The isolates showing
resistance to the widest range of antibiotics were identified as Citrobacter freundii, Proteus vulgaris, Prov-
idencia rettgeri and Pseudomonas aeruginosa. In this study, antibiotic resistant bacteria reflect marine con-
tamination by polluted effluents and C. caretta is considered a bioindicator which can be used as a
monitor for pollution.
? 2009 Elsevier Ltd. All rights reserved.
Antibiotic resistance (ABR) in bacteria is a growing problem in
human and veterinary medicine worldwide. Emergence of bacteria
resistant to antibiotics is predictable in any environment where
antibiotics are being used, but occurrence of antibiotic resistant
bacteria is also increasing in aquatic environments (Al-Bahry
et al., 2009; Lima-Bittercourt et al., 2007) Studies have been carried
out to identify environmental reservoirs of bacterial antibiotic
resistance in wild animal populations. Research was conducted
on fish and marine mammals support the hypothesis that marine
species harbor resistant microbial species and therefore may serve
as reservoirs for ABR (Johson et al., 1998; Miranda and Zemelman,
Studies indicated the distribution of antibiotic resistant bacteria
in freshwater basins, estuaries and marine waters (Nemi et al.,
1983; Herwig et al., 1997; Ash et al., 2002; Nair et al., 1992; Mu-
dryk and Skórczewski, 1998).
Antibiotic resistance is also considered to be an ecological prob-
lem. Bacteria showing antibiotic resistance are an index of marine
Marine turtles have been proposed as sentinel species useful as
environmental health indicators for coastal marine habitats (Agu-
irre and Lutz, 2004; Owens et al., 2005). Ecological and physiolog-
ical characteristics, such as their long life-span, long period of time
to reach sexual maturity, and high site fidelity to near-coastal feed-
ing habitats, make them very reliable bio-indicators. Moreover,
marine turtles appear highly susceptible to biological and chemical
insults (Lutcavage et al., 1997). C. caretta is included in the Red List
of the world conservation union (IUNC/SSC, 2002) and is highly
The aim of this study was to determine the resistance to antibi-
otics of Gram negative bacteria isolated from oral and cloacal
swabs of loggerhead sea turtle from the central Mediterranean
2. Materials and methods
A total of 19 loggerhead sea turtles, five of them from the Sicil-
ian channel, one from the South Tyrrhenian sea and 13 from the Io-
nian sea, were captured or found stranded alive between July 2006
and June 2007. All the loggerhead sea turtles examined were
immature individuals and their weight ranged between 2 and
0025-326X/$ - see front matter ? 2009 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +39 90 711263; fax: +39 90 669007.
E-mail address: email@example.com (T. Bottari).
Marine Pollution Bulletin 58 (2009) 1363–1366
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20 kg. The presence of hooks and lines in various portions of the
alimentary tract was confirmed in all of them. Sixteen cloacal
swabs and nine oral swabs were collected. The swabs were placed
in Stuart’s media (Meus, Piove di Sacco – Italy) and then sent to the
laboratory for bacteriological investigation.
2.2. Isolation of bacteria
The swabs were then plated onto Mc Conkey agar and Trypti-
case soya agar with 5% sheep blood. The swabs were also dipped
in Selenite broth (Oxoid, Basingstoke, Hampshire – England). The
plates and broth were incubated at 37 ?C for 24–48 h. Those sam-
ples showing bacterial growth were subcultured using: Brilliant
green agar, Salmonella Shigella agar (Oxoid, Basingstoke, Hampshire
– England) and Hektoen enteric agar (Liofilchem, Teramo – Italy).
Fifty seven strains were selected from plates on the basis of mor-
phology of colonies (colour, size, etc.). The cultures were then
transferred to Kligler Iron agar slants (Oxoid, Basingstoke, Hamp-
shire – England). The following tests were performed on all iso-
lated strains: Gram-staining, motility, catalase and oxidase
activity. All isolates were identified by API 20 E and API 20 NE sys-
tems (Biomerieux, Marcy l’Etoile – France).
2.3. Determination of antibiotic resistance
Bacteria were tested for ABR using the Kirby-Bauer method.
Bacterial isolates stored on BHI agar (Oxoid) were inoculated on
5% blood agar (Oxoid) and incubated at 37 ?C for 24 h. Colonies
from the blood agar were then resuspended in saline until an opti-
cal density equal to a MacFarland 0.5 standard was achieved. The
bacterial suspensions were then plated onto Muller-Hinton agar
(Oxoid) and, then (Oxoid) antibiotic disks were placed. The plates
were incubated at 37 ?C for 18–24 h under aerobic conditions.
The diameter of the zone of inhibition around each disk was mea-
sured and recorded. Each bacterial species was classified as Resis-
tant (R), Intermediately Resistant (I), or Susceptible (S) according to
the guidelines of Clinical and Laboratory Standards Institute (CLSI).
The following antibiotics, with their concentrations given in paren-
theses, were tested amikacin (30 lg), ampicillin (10 lg), amoxicil-
sulbactam (20 lg), aztreonam (30 lg), carbenicillin (100 lg), cefe-
pime (30 lg), cephalothin (30 lg), cefoperazone (75 lg), cefotax-
ime (30 lg),ceftazidime(30 lg),
chloramphenicol (30 lg), ciprofloxacin (5 lg), colistin (25 lg),
enrofloxacin (5 lg), gentamicin (10 lg), imipenem (10 lg), kana-
mycin (30 lg), lomefloxacine (10 lg), nalidixic acid (30 lg), neo-
mycin(30 lg), netilmicin(30 lg),
piperacillin (100 lg), streptomycin (25 lg), tetracycline (30 lg),
ticarcillin–clavulanic acid (85 lg), tobramycin (10 lg) and tri-
methoprim–sulphamethoxazole (25 lg).
Screening for production of extended-spectrum-beta-lactamas-
es was conducted by using the double-disk synergy test (DDST) as
previously described. All isolated showed reduced susceptibility to
third generation cephalosporins and aztreonam. Escherichia coli
ATCC 25922 was used as quality control.
acid(30 lg), ampicillin–
A total of 57 bacterial isolates (38 from Ionian sea, 10 from
Sicilian channel and nine from south Tyrrhenian sea) were identi-
fied to at least the genus level. The predominant isolates in
descending order of frequency were: Proteus vulgaris (N = 14),
Citrobacter freundii (N = 10), Providencia rettgeri (N = 7), Enterobac-
ter cloacae (N = 7), Pantoea spp. (N = 4), Proteus mirabilis (N = 4),
Pseudomonas aeruginosa (N = 3), Citrobacter brakii, Enterobacter
sakazakii, E. coli, Morganella morgani, Providencia stuartii, Pseudomo-
nas mendocina, Pseudomonas luteola and Shewanella putrefaciens
(N = 1) (Fig. 1).
At least 50% of the strains displayed resistance to nine of the 31
antibiotic tested. All isolates showed resistance to at least one anti-
microbial. Moreover, as shown in Fig. 2, a prominent proportion of
bacterial strains exhibited simultaneous resistance to at least two
antibacterial drugs. The most frequently detected resistances were
to carbenicillin (100%), followed by cephalothin (92.6%), oxytetra-
cycline (81.3%) and amoxicillin (77.8%) (Fig. 3). Significant rates
of antibiotic resistance appeared to colistin (72.0%), tetracycline
(64.9), ampicillin (63.6%) ticarcillin–clavulanic acid (52.9%) and
lomefloxacine (51.9%). Lower rates of antibiotic resistance were
detected to amikacin (19%) and cefotaxime (9.1). No resistance
was observed to imipenem.
The isolates showed resistance to the greatest number of antibi-
otics were 10 C. freundii strains (from 17% to 100% of antibiotic
tested), 14 P. vulgaris strains (from 17% to 100%); P. rettgeri (from
62.5% to 94.1%) and P. aeruginosa (94.1%).
No strains were found to produce ESBL.
This study describes resistance to antimicrobial agents within
Gram negative isolates from loggerhead sea turtle in the Mediter-
ranean sea. Determining microbial antibiotic resistance from mar-
ine animals is an important finding.
Furthermore, these results provide useful information for
researchers working in sea turtle rehabilitation facilities.
The isolates showed a high frequency of resistance to antimi-
crobials tested. Only imipenem proved to be effective against all
isolates. Carbenicillin resistance was present in 100% of the iso-
% frequency of isolation
Fig. 1. Percentage frequency of isolates from buccal cavity and cloacae of Caretta
caretta. A = Citrobacter brakii, B = Citrobacter freundii, C = Enterobacter cloacae,
D = Enterobacter sakazakii, E = Escherichia coli, F = Morganella morganii, G = Pantoea
spp., H = Proteus mirabilis, I = Proteus vulgaris, J = Providencia rettgeri, K = Providencia
stuartii, L = Pseudomonas aeruginosa, M = Pseudomonas luteola, N = Pseudomonas
mendocina, O = Shewanella putrefaciens.
23 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Number of Antibiotics
Number of Isolates
Fig. 2. Frequency of resistant isolates to antibiotics.
M. Foti et al./Marine Pollution Bulletin 58 (2009) 1363–1366
lates. High resistance rates were recorded as follows: cephalospo-
rins (cephalothin 92.6%; ceftriaxone 45.5%), tetracyclines (oxytet-
racycline 81.3%, tetracycline 64.9%), amoxicillin (77.8%), colistin
(72.0%) and quinolones (lomefloxacine 51.9%, enrofloxacin 48.1%).
Pinera-Pasquino (2006) found that the most frequent resis-
tances in Gram negative isolates from C. caretta from North Caro-
lina(USA) wereto lincomycin,
erythromycin, cephalothin and penicillin. Little or no resistance
was observed in the same isolates to gentamicin, amikacin, enro-
floxacin, ciprofloxacin, imipenem and neomycin. The isolates
showing resistance to the greatest number of antibiotics had been
identified in Pseudomonas strains. Significant levels of antibiotic
resistance had been found also for Morganella morganii, Citrobacter
freundii, and several E. coli strains. Harms et al. (2006) found that
among Gram negative rods coming from cloacal swabs of C. caretta
in North Carolina, ABR was most frequent to penicillin and cepha-
lothin and less to amikacin and gentamicin.
The present data induce deep concerns on the dissemination of
resistance to antimicrobial agents in the marine environment and
the mechanisms that drive its emergence. It would be very inter-
esting to investigate how specimens of C. caretta, which have never
been subjected to any antibiotic therapy, have acquired bacteria
that have developed resistances that habitually emerge because
of a selective pressure induced by the over use of antimicrobial
agents. It is possible that the source of multiple antibiotic resistant
bacteria could be from polluted effluents (Al-Bahry et al., 2009).
This issue has also been previously described by Gordon et al.
(2007) who found antibiotic resistant bacteria in a river receiving
effluents from fish farms. Furthermore, a role for marine culture
facilities and a possible sharing of pathogens among farmed and
wild fish cannot be excluded and should be carefully investigated
(Guglielmetti et al., 2009). This last hypothesis can also be also
supported by the results obtained by Giraud et al. (2006).
Bio-indicator used to monitor the spread of antibiotic resistant
bacteria in marine environment. More research is needed on anti-
biotic resistant bacteria present in marine organisms especially
from the Mediterranean Sea. Such information would be useful
to evaluate the degree of pollution caused by the overuse of anti-
Sincere thanks to Annalisa Liotta and to the staff of the ‘‘Centro
Recupero Tartarughe Brancaleone – CTS”.
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Fig. 3. Antibiotic resistance of different species of Gram negative isolated from Caretta caretta. (1) Amikacin; (2) amoxicillin; (3) amoxicillin–clavulanic acid; (4) ampicillin;
(5) ampicillin–sulbactam; (6) aztreonam; (7) carbenicillin; (8) cefepime; (9) cefoperazone; (10) cefotaxime; (11) ceftazidime; (12) ceftriaxone; (13) cephalothin; (14)
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