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Antibiotic Resistance of Gram Negatives isolates from loggerhead sea turtles (Carettacaretta) in the central Mediterranean Sea
Marine Pollution Bulletin 58 (2009) 1363–1366
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Antibiotic Resistance of Gram Negatives isolates from loggerhead sea turtles (Caretta caretta) in the central Mediterranean Sea M. Foti a, C. Giacopello a, Teresa Bottari b,*, V. Fisichella a, D. Rinaldo a, C. Mammina c a
Dipartimento di Sanità Pubblica Veterinaria, Università degli Studi di Messina, Polo Universitario SS Annunziata, 98167 Messina, Italy Istituto per l’Ambiente Marino Costiero, CNR – Spianata S. Raineri, 86 – 98122 Messina, Italy c Dipartimento di Scienze per la Promozione della Salute ‘‘G. D’Alessandro”, Università degli Studi di Palermo, Via del Vespro 133, I-90127 Palermo, Italy b
a r t i c l e
i n f o
Keywords: Antibiotic resistance Antimicrobials Loggerhead sea turtle Caretta caretta Cloacal bacteria Mediterranean sea
a b s t r a c t Previous studies on fish and marine mammals support the hypothesis that marine species harbor antibiotic 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 carbenicillin (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, Providencia rettgeri and Pseudomonas aeruginosa. In this study, antibiotic resistant bacteria reflect marine contamination by polluted effluents and C. caretta is considered a bioindicator which can be used as a monitor for pollution. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction 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, 2001). 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; Mudryk and Skórczewski, 1998). Antibiotic resistance is also considered to be an ecological problem. Bacteria showing antibiotic resistance are an index of marine pollution.
* Corresponding author. Tel.: +39 90 711263; fax: +39 90 669007. E-mail address: [email protected] (T. Bottari). 0025-326X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2009.04.020
Marine turtles have been proposed as sentinel species useful as environmental health indicators for coastal marine habitats (Aguirre and Lutz, 2004; Owens et al., 2005). Ecological and physiological characteristics, such as their long life-span, long period of time to reach sexual maturity, and high site fidelity to near-coastal feeding 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 threatened. The aim of this study was to determine the resistance to antibiotics of Gram negative bacteria isolated from oral and cloacal swabs of loggerhead sea turtle from the central Mediterranean region.
2. Materials and methods 2.1. Sampling A total of 19 loggerhead sea turtles, five of them from the Sicilian channel, one from the South Tyrrhenian sea and 13 from the Ionian 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
M. Foti et al. / Marine Pollution Bulletin 58 (2009) 1363–1366
2.2. Isolation of bacteria The swabs were then plated onto Mc Conkey agar and Trypticase 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 samples 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 morphology of colonies (colour, size, etc.). The cultures were then transferred to Kligler Iron agar slants (Oxoid, Basingstoke, Hampshire – England). The following tests were performed on all isolated strains: Gram-staining, motility, catalase and oxidase activity. All isolates were identified by API 20 E and API 20 NE systems (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 optical 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 measured and recorded. Each bacterial species was classified as Resistant (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 parentheses, were tested amikacin (30 lg), ampicillin (10 lg), amoxicillin (30 lg), amoxicillin–clavulanic acid (30 lg), ampicillin– sulbactam (20 lg), aztreonam (30 lg), carbenicillin (100 lg), cefepime (30 lg), cephalothin (30 lg), cefoperazone (75 lg), cefotaxceftazidime (30 lg), ceftriaxone (30 lg), ime (30 lg), chloramphenicol (30 lg), ciprofloxacin (5 lg), colistin (25 lg), enrofloxacin (5 lg), gentamicin (10 lg), imipenem (10 lg), kanamycin (30 lg), lomefloxacine (10 lg), nalidixic acid (30 lg), neomycin (30 lg), netilmicin (30 lg), oxytetracycline (30 lg), piperacillin (100 lg), streptomycin (25 lg), tetracycline (30 lg), ticarcillin–clavulanic acid (85 lg), tobramycin (10 lg) and trimethoprim–sulphamethoxazole (25 lg). Screening for production of extended-spectrum-beta-lactamases 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.
3. Results A total of 57 bacterial isolates (38 from Ionian sea, 10 from Sicilian channel and nine from south Tyrrhenian sea) were identified 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), Enterobacter 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, Pseudomonas 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 antimicrobial. 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%), oxytetracycline (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 antibiotics 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. 4. Discussion This study describes resistance to antimicrobial agents within Gram negative isolates from loggerhead sea turtle in the Mediterranean sea. Determining microbial antibiotic resistance from marine 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 antimicrobials tested. Only imipenem proved to be effective against all isolates. Carbenicillin resistance was present in 100% of the iso-
% frequency of isolation
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.
30 25 20 15 10 5 0 A
B
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F
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Isolates 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.
Number of Isolates
1364
10 9 8 7 6 5 4 3 2 1 0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Number of Antibiotics Fig. 2. Frequency of resistant isolates to antibiotics.
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100 90
% frequency ofresistant bacteria
80 70 60 50 40 30 20 10
30 31
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15 16
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Antibiotics 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) chloramphenicol; (15) ciprofloxacin; (16) colistin; (17) enrofloxacin; (18) gentamicin; (19) imipenem; (20) kanamycin; (21) lomefloxacine; (22) nalidixic acid; (23) neomycin; (24) netilmicin; (25) oxytetracycline; (26) piperacillin; (27) streptomycin; (28) tetracycline; (29) ticarcillin–clavulanic acid; (30) tobramycin; (31) trimethoprim– sulphamethoxazole.
lates. High resistance rates were recorded as follows: cephalosporins (cephalothin 92.6%; ceftriaxone 45.5%), tetracyclines (oxytetracycline 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 resistances in Gram negative isolates from C. caretta from North Carolina (USA) were to lincomycin, clindamycin, cephalexin, erythromycin, cephalothin and penicillin. Little or no resistance was observed in the same isolates to gentamicin, amikacin, enrofloxacin, 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 cephalothin 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 interesting 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 antibiotic 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 antimicrobial agents. Acknowledgements Sincere thanks to Annalisa Liotta and to the staff of the ‘‘Centro Recupero Tartarughe Brancaleone – CTS”. References Aguirre, A.A., Lutz, P.L., 2004. Marine turtles as sentinels of ecosystem health: is fibropapillomatosis an indicator? EcoHealth 1, 275–283. Al-Bahry, S., Mahmoud, I., Elshfie, A., Al-Harthy, A., Al-Ghafri, S., Al-Amri, I., Alkindi, A., 2009. Bacterial flora and antibiotic resistance from eggs of green turtles Chelonia mydas: An indication of polluted effluents. Marine Pollution Bulletin 58, 720–725. Ash, R.J., Mauck, B., Morgan, M., 2002. Antibiotic resistance of Gramnegative bacteria in rivers, Unites States. Emerging Infectious Diseases 8, 713–716. Giraud, E., Douet, D.G., Le Bris, H., Bouju-Albert, A., Donnay_Moreno, C., Thorin, C., Pouliquen, H., 2006. Survey of antibiotic resistance in an integrated marine aquaculture system under oxolinic acid treatment. FEMS Microbiology Ecology 55 (3), 439–448. Gordon, L., Giraud, E., Ganiere, J.P., Armend, F., Bouju_Albert, A., de la Cotte, N., Mangion, C., Le Bris, H., 2007. Antimicrobial resistance survey in a river receiving effluents from freshwater fish farms. Journal of Applied Microbiology 102 (4), 1167–1176. Guglielmetti, E., Korhonen, J.M., Heikkinen, J., Morelli, L., von Wright, A., 2009. Transfer of plasmid-mediated resistance to tetracycline in pathogenic bacteria from fish and aquaculture environments. FEMS Microbiology Letters 293, 28– 34. Harms, C.A., Mihnovets, A.N., Braun-McNeill, J., Kelly, T.R., Avens, L., Goodman, M.A., Goshe, L.R., Godfrey, M.H., Hohn, A.A., 2006. Cloacal bacterial isolates and antimicrobial resistance patterns in juvenile loggerhead turtles in North Carolina, USA. In: Proceedings of Annual Symposium on Sea Turtle Conservation and Biology, p. 58. Herwig, R.P., Gray, J.P., Weston, D.P., 1997. Antibacterial resistant bacteria in surficial sediments near salmon net-cage farms in Puget Sound, Washington. Aquaculture 149, 263–283. IUNC/SSC (International Union for Conservation of Nature and Natural Resources/ Specie Survival Commission), 2002. 2002 IUCN Red List of Threatened Species. IUCN, Balmar, Arlington, VA. Johson, S.P., Nolan, S., Gulland, F.M., 1998. Antimicrobial susceptibility of bacteria isolated from pinnipeds stranded in central and northern California. Journal of Zoo and Wildlife Medicine 29 (3), 288–294.
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Lima-Bittercourt, C.I., Cursino, L., Goncalves, H., Pontes, D.S., Nardi, R.M.D., Callisto, M., Chartone-Souza, E., Nascimento, A.M.A., 2007. Multiple antimicrobial resistance in Enterobacteriaceae isolates from pristine freshwater. Genetics and Molecular Research 6 (3), 510–521. Lutcavage, M., Plotkin, P., Witherington, B., Lutz, P.L., 1997. Human impacts on sea turtle survival. In: Lutz, P.L., Musik, J.A. (Eds.), The Biology of Sea Turtles. CRC Press, Boca Raton, FL, pp. 387–410. Miranda, C.D., Zemelman, R., 2001. Antibiotic resistant bacteria in fish from the Concepcion Bay, Chile. Marine Pollution Bulletin 42, 1096–1102. Mudryk, Z., Skórczewski, P., 1998. Antibiotic resistance in marine neustonic and planktonic bacteria isolated from the Gdan´sk Deep. Oceanologia 40, 125– 136.
Nair, S., Chandramohan, D., Bharathi, L., 1992. Differential sensitivity of pigmented and non-pigmented marine bacteria to metals and antibiotics. Water Research 26, 431–434. Nemi, M., Sibakov, M., Niemela, S., 1983. Antibiotic resistance among different species of fecal coliforms isolated from water samples. Applied and Environmental Microbiology 45, 79–83. Owens, D.W., Day, R.D., Blanvillain, G.J., Schwenter, J.A., Christopher, S.J., Roumillat W.A., 2005. Turtles as physiological models for environmental stress: can they be ‘‘used” and is it ethical. In: 25th Annual Symposium on Sea Turtle Biology and Conservation. San Jose, Costarica. Pinera-Pasquino, L., 2006. Patterns of Antibiotic Resistance in Bacteria Isolated from Marine Turtles. Master Thesis.
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Antibiotic Resistance of Gram Negatives isolates from loggerhead sea turtles (Caretta caretta) in the central Mediterranean Sea M. Foti a, C. Giacopello a, Teresa Bottari b,*, V. Fisichella a, D. Rinaldo a, C. Mammina c a
Dipartimento di Sanità Pubblica Veterinaria, Università degli Studi di Messina, Polo Universitario SS Annunziata, 98167 Messina, Italy Istituto per l’Ambiente Marino Costiero, CNR – Spianata S. Raineri, 86 – 98122 Messina, Italy c Dipartimento di Scienze per la Promozione della Salute ‘‘G. D’Alessandro”, Università degli Studi di Palermo, Via del Vespro 133, I-90127 Palermo, Italy b
a r t i c l e
i n f o
Keywords: Antibiotic resistance Antimicrobials Loggerhead sea turtle Caretta caretta Cloacal bacteria Mediterranean sea
a b s t r a c t Previous studies on fish and marine mammals support the hypothesis that marine species harbor antibiotic 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 carbenicillin (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, Providencia rettgeri and Pseudomonas aeruginosa. In this study, antibiotic resistant bacteria reflect marine contamination by polluted effluents and C. caretta is considered a bioindicator which can be used as a monitor for pollution. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction 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, 2001). 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; Mudryk and Skórczewski, 1998). Antibiotic resistance is also considered to be an ecological problem. Bacteria showing antibiotic resistance are an index of marine pollution.
* Corresponding author. Tel.: +39 90 711263; fax: +39 90 669007. E-mail address: [email protected] (T. Bottari). 0025-326X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2009.04.020
Marine turtles have been proposed as sentinel species useful as environmental health indicators for coastal marine habitats (Aguirre and Lutz, 2004; Owens et al., 2005). Ecological and physiological characteristics, such as their long life-span, long period of time to reach sexual maturity, and high site fidelity to near-coastal feeding 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 threatened. The aim of this study was to determine the resistance to antibiotics of Gram negative bacteria isolated from oral and cloacal swabs of loggerhead sea turtle from the central Mediterranean region.
2. Materials and methods 2.1. Sampling A total of 19 loggerhead sea turtles, five of them from the Sicilian channel, one from the South Tyrrhenian sea and 13 from the Ionian 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
M. Foti et al. / Marine Pollution Bulletin 58 (2009) 1363–1366
2.2. Isolation of bacteria The swabs were then plated onto Mc Conkey agar and Trypticase 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 samples 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 morphology of colonies (colour, size, etc.). The cultures were then transferred to Kligler Iron agar slants (Oxoid, Basingstoke, Hampshire – England). The following tests were performed on all isolated strains: Gram-staining, motility, catalase and oxidase activity. All isolates were identified by API 20 E and API 20 NE systems (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 optical 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 measured and recorded. Each bacterial species was classified as Resistant (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 parentheses, were tested amikacin (30 lg), ampicillin (10 lg), amoxicillin (30 lg), amoxicillin–clavulanic acid (30 lg), ampicillin– sulbactam (20 lg), aztreonam (30 lg), carbenicillin (100 lg), cefepime (30 lg), cephalothin (30 lg), cefoperazone (75 lg), cefotaxceftazidime (30 lg), ceftriaxone (30 lg), ime (30 lg), chloramphenicol (30 lg), ciprofloxacin (5 lg), colistin (25 lg), enrofloxacin (5 lg), gentamicin (10 lg), imipenem (10 lg), kanamycin (30 lg), lomefloxacine (10 lg), nalidixic acid (30 lg), neomycin (30 lg), netilmicin (30 lg), oxytetracycline (30 lg), piperacillin (100 lg), streptomycin (25 lg), tetracycline (30 lg), ticarcillin–clavulanic acid (85 lg), tobramycin (10 lg) and trimethoprim–sulphamethoxazole (25 lg). Screening for production of extended-spectrum-beta-lactamases 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.
3. Results A total of 57 bacterial isolates (38 from Ionian sea, 10 from Sicilian channel and nine from south Tyrrhenian sea) were identified 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), Enterobacter 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, Pseudomonas 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 antimicrobial. 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%), oxytetracycline (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 antibiotics 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. 4. Discussion This study describes resistance to antimicrobial agents within Gram negative isolates from loggerhead sea turtle in the Mediterranean sea. Determining microbial antibiotic resistance from marine 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 antimicrobials tested. Only imipenem proved to be effective against all isolates. Carbenicillin resistance was present in 100% of the iso-
% frequency of isolation
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.
30 25 20 15 10 5 0 A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
Isolates 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.
Number of Isolates
1364
10 9 8 7 6 5 4 3 2 1 0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Number of Antibiotics Fig. 2. Frequency of resistant isolates to antibiotics.
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100 90
% frequency ofresistant bacteria
80 70 60 50 40 30 20 10
30 31
28 29
25 26 27
23 24
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8
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7
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0
Antibiotics 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) chloramphenicol; (15) ciprofloxacin; (16) colistin; (17) enrofloxacin; (18) gentamicin; (19) imipenem; (20) kanamycin; (21) lomefloxacine; (22) nalidixic acid; (23) neomycin; (24) netilmicin; (25) oxytetracycline; (26) piperacillin; (27) streptomycin; (28) tetracycline; (29) ticarcillin–clavulanic acid; (30) tobramycin; (31) trimethoprim– sulphamethoxazole.
lates. High resistance rates were recorded as follows: cephalosporins (cephalothin 92.6%; ceftriaxone 45.5%), tetracyclines (oxytetracycline 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 resistances in Gram negative isolates from C. caretta from North Carolina (USA) were to lincomycin, clindamycin, cephalexin, erythromycin, cephalothin and penicillin. Little or no resistance was observed in the same isolates to gentamicin, amikacin, enrofloxacin, 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 cephalothin 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 interesting 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 antibiotic resistant bacteria present in marine organisms especially from the Mediterranean Sea. Such information would be useful
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