Original Contribution INTRODUCTION. Adam M. Schaefer, 1 Juli D. Goldstein, 1 John S. Reif, 2 Patricia A. Fair, 3 and Gregory D.

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1 EcoHealth DOI: /s Ó 2009 International Association for Ecology and Health Original Contribution Antibiotic-Resistant Organisms Cultured from Atlantic Bottlenose Dolphins (Tursiops truncatus) Inhabiting Estuarine Waters of Charleston, SC and Indian River Lagoon, FL Adam M. Schaefer, 1 Juli D. Goldstein, 1 John S. Reif, 2 Patricia A. Fair, 3 and Gregory D. Bossart 1,4 1 Harbor Branch Oceanographic Institute at Florida Atlantic University, 5600 US 1, North, Ft. Pierce, FL Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 3 Center for Coastal Environmental Health and Biomolecular Research, NOAA, NOS, Charleston, SC 4 University of Miami, Miller School of Medicine, Miami, FL Abstract: Bottlenose dolphins (Tursiops truncatus) from estuarine waters of Indian River Lagoon, FL (IRL) and Charleston, SC (CHS) were cultured to screen for microorganism colonization and to assess antibiotic sensitivity. Swabs (n = 909) were collected from the blowhole, gastric fluid, and feces of 171 individual dolphins The most frequently cultured organisms were Plesiomonas shigelloides (n = 161), Aeromonas hydrophila (n = 144), Escherichia coli (n = 85), and Pseudomonas fluorescens (n = 82). In descending frequency, organisms demonstrated resistance to erythromycin, ampicillin, and cephalothin. Human and animal pathogens resistant to antibiotics used in human and veterinary medicine were cultured. Escherichia coli (E. coli) more often was resistant in IRL dolphins. Three cases of methicillin-resistant Staphylococcus aureus (MRSA) were found at CHS. Emergence of antibiotic resistance is not confined to humans. Bottlenose dolphins may serve as sentinels for transfer of resistance from humans and animals or indicate that antibiotics are reaching the marine environment and causing resistance to emerge through selective pressure and genetic adaptation. Keywords: Atlantic bottlenose dolphin (Tursiops truncatus), Antibiotic-resistant organisms, Indian River Lagoon, Charleston Harbor INTRODUCTION The increasing prevalence of antibiotic-resistant strains of bacteria in both animals and humans is an emerging public health issue. Of particular concern is bacterial resistance in wild animal populations due to antibiotics commonly used in human and veterinary medicine, aquaculture, and agriculture. The proliferation of antibiotic-resistant organisms (ARO) in the environment reflects extension of resistance Correspondence to: Adam M. Schaefer, aschaefer@hboi.fau.edu beyond the hospital setting and likely indicates multiple sources of exposure and transfer of resistant strains to nontarget populations. Current evidence suggests that the widespread use of antimicrobial agents for therapy is directly linked to the increase in the prevalence of AROs (Nue, 1999; Prescott et al., 2000; Phillips et al., 2004). Once AROs become persistent in the environment, their genetic material can be passed to other bacteria through a variety of mechanisms, thus increasing the occurrence of antimicrobial resistant bacteria in nature (Kruse and Sorum, 1994). As a result, the

2 Adam M. Schaefer et al. potential for infection of aquatic animals with AROs becomes increasingly likely. Previous environmental research has shown that antibiotic resistant bacteria, including Escherichia coli can be isolated from a variety of aquatic sources (Webster et al., 2004). The frequent presence of resistant bacteria in aquatic environments has served as a useful tool to track and identify sources of fecal contamination (Wiggin, 1996; Hagedorn et al., 1999; Harwood et al., 2000). An additional concern is environmental persistence of resistant strains of bacteria in the face of unused antimicrobial agents, which potentially increase levels and change resistance patterns of bacteria (Kummerer, 2001). Thus, contamination of coastal environments with discarded antibiotics through effluents or runoffs could lead to further selective pressure and the emergence of antibiotic resistance microbes in marine mammal populations inhabiting coastal waters. As the prevalence of AROs in the environment increases, so does the importance of assessing the ecological effects of such microbes. The occurrence of AROs has been reported in a variety of wild animals, including trout, tree frogs, and Canada geese (Gonzalez et al., 1999; Slaughter et al., 2001; Middleton and Ambrose, 2005). Among marine animals, resistance to multiple antibiotics has been observed in cultures from demersal and pelagic fish, sharks, and pinnipeds (Johnson et al., 1998; Miranda and Zemelman, 2001; Blackburn, 2003; Lockwood et al., 2006). However, this issue warrants further examination in cetaceans and other marine mammals. The objective of the current study was to evaluate the prevalence and distribution of antibiotic-resistant organisms cultured from free-ranging Atlantic bottlenose dolphins (Tursiops truncatus) in two locations on the Atlantic coast of the United States. Our goals were to describe the patterns of antibiotic resistance amongst organisms cultured from dolphins at both sites and to determine whether differences existed between the study areas. METHODS Dolphin HERA Project Data were collected as a part of the Bottlenose Dolphin Health and Risk Assessment Project (HERA), which is a collaborative multidisciplinary effort to assess health in two estuarine regions along the eastern coast of the United States. Research was conducted by the Marine Mammal Research and Conservation Program/Harbor Branch Oceanographic Institute at Florida Atlantic University, Fort Pierce, Florida, and National Ocean Service (NOS) Center for Coastal Environmental Health and Bimolecular Research (CCEHBR), Charleston, South Carolina. All research was approved by the Harbor Branch Animal Care and Use Committee. Bottlenose dolphins were sampled from the Indian River Lagoon, Florida (IRL; Figure 1) and the estuarine waters of Charleston, South Carolina (CHS; Figure 2; Fair et al., 2006). The goal of HERA was to examine potential associations between health status and Figure 1. Indian River Lagoon, Florida.

3 Antibiotic-Resistant Organisms Cultured from Dolphins Figure 2. Estuarine waters of Charleston, South Carolina. environmental conditions (Fair and Bossart, 2005; Fair et al., 2006). The assessment of free-ranging dolphins included physical examination and collection of blood, tissue samples, and cultures for microbiologic investigation from a total of 171 individual dolphins (Reif et al., 2008). Clinicopathologic parameters, including hematology and serum chemistry, also were used to assess individual animal health. Study Sites Dolphins were examined in the IRL and at CHS during the months of June and August, respectively between 2003 and The IRL extends 260 km along the east coast of Florida from the Ponce De Leon Inlet in the north to the Jupiter Inlet in the south and is an aggregate of three estuaries. The CHS study site includes Charleston Harbor and its adjacent estuarine waterways, which encompass 1,200 square miles. Sample Collection Microbiological samples were collected from the blowhole, gastric fluid, and feces from each dolphin for aerobic/ anaerobic bacterial culture, fungal culture, as well as antibiotic sensitivity testing. Two sterile Aimes culturettes (Fischer Scientific, Pittsburg, PA) were inserted into the blowhole during a breath, gently rotated along the wall of the nares, and removed during the subsequent breath. Gastric fluid was collected by inserting a well-lubricated soft, flexible, plastic stomach tube past the oropharynx to the first stomach, followed by gentle aspiration and placement of fluid into a 15-ml conical vial. A sterile Aimes culturette was then inserted in the gastric fluid and placed in culture media. Fecal samples were collected in sterile 15- ml conical vials opportunistically as the animal voluntarily defecated, or by catheterization under sterile conditions. A saline-filled 20-ml syringe with a 14-French self-catheter (Best Buy Medical/Healthcare, Santa Clarita, CA) was used to collect the fecal specimen by gentle aspiration. All swabs were placed in culture media and held on ice packs for storage and overnight shipment to Micrim Laboratories, Ft. Lauderdale, FL. Organism Identification Microbiologic testing was conducted in a commercial laboratory (Micrim Laboratories). Standard methods were used for organism identification, including growth on selective and differential media, macroscopic appearance, gram stain morphology, and biochemical reaction according to the National Committee for Clinical Laboratory

4 Adam M. Schaefer et al. Standards (1997). After identification of each organism, subsequent screening for antibiotic resistance was performed under aerobic and anaerobic culture conditions. Antibiotic Resistance Screening Antibiograms were constructed using standardized concentrations of each antibiotic. Antibiotic resistance was determined by a disk diffusion test (National Committee for Clinical Laboratory Standards, 1997). The protocol included standardized inoculums of bacteria swabbed on Mueller-Hinton agar plates. Impregnated antimicrobial discs were placed and the samples were incubated for approximately 24 to 48 hours. Zones of inhibition were measured and the Clinical and Laboratory Standards Institute (CLSI) antimicrobial susceptibility testing standards M2-A9 and M7-A7 were applied. Statistical Analysis Descriptive frequencies were calculated for isolation of each aerobic and anaerobic organism from all three collection sites. The frequency of resistance to each antibiotic for each organism was analyzed by culture site. Chi-square tests were used to compare resistance to each antibiotic by organism and to analyze resistance patterns by collection site (blowhole, gastric, feces). The proportions of resistance for each organism and for all organisms pooled were compared between the IRL and CHS samples with the Mantel-Haenszel procedure. Odds ratios with their 95 percent confidence limits were calculated to estimate risk of antibiotic resistance between the IRL and CHS. Fisher s exact test was used when >20% of the cells had an expected count of less than five or any cells had an expected count of less than one. Results were considered statistically significant when p < All analyses were completed by using SPSS 15.0 for Windows (SPSS Inc., Chicago, IL). frequency, these organisms demonstrated resistance to erythromycin (96%), ampicillin (88%), and cephalothin (57%). The patterns of antibiotic resistance in organisms cultured from the IRL (Table 1) and CHS (Table 2) were similar. However, all organisms pooled together resulted in significant differences for four antibiotics between capture sites. A significantly higher proportion of organisms isolated from dolphins in the IRL were resistant to amoxicillin clavulanate (odds ratio (OR), 1.77; 95% confidence interval (CI), ), chloramphenicol (OR, 2.23; 95% CI, ), and furadantin (OR, 2.2; 95% CI, ). There also was a significant increase in the proportion of resistance to clindamycin (OR, 2.46; 95% CI, ) for organisms cultured from CHS. For all organisms isolated, resistance among all isolates was found most commonly for erythromycin (91.4%), followed by clindamycin (87%), ampicillin (77.2%), cephalothin (53.3%), piperacillin (51.1%), and amoxicillin clavulanate (46.1%). E. coli isolates from dolphins in the IRL showed greater resistance to piperacillin, tetracycline, and trimethoprim/sulfamethoxazole compared with isolates from CHS (p = 0.046). P. fluorescens showed a marginal difference in resistance to amikacin with CHS having a slightly higher proportion of resistant cultures; however, these findings were not statistically significant (p = 0.051). Resistance by culture site was statistically different for 11 antibiotics (Table 3). Resistance to ampicillin, amoxicillin clavulanate, cephalothin, erythromycin, and furadantin all occurred most often in organisms cultured from the blowhole. However, resistance to septra/bacrim, tetracycline, marbofloxacin, ciprofloxacin, and enrofloxacin occurred most often in gastric samples. Importantly, three positive cultures demonstrated resistance patterns consistent with methicillin-resistant Staphylococcus aureus (MRSA) were found. All three cultures grew from separate swabs take from three individual dolphins in CHS. Two cultures were taken from blowholes, whereas the other grew from a gastric swab. RESULTS A total of 556 isolates from IRL dolphins and 353 isolates from CHS dolphins collected from gastric, fecal, and blowhole swabs demonstrated identifiable organism growth and subsequent sensitivity testing was conducted. The most frequently cultured organisms were Plesiomonas shigelloides (17.7%), Aeromonas hydrophila (15.8%), Escherichia coli (9.3%), and Pseudomonas fluorescens (9%). In descending DISCUSSION Most of the most commonly cultured organisms from dolphins at both study sites also are human pathogens. P. shigelloides, A. hydrophila A. baumannii, and P. fluorescens are opportunistic in nature and may cause secondary infections in immunocompromised marine mammals and humans. Therefore, the resistance patterns that they exhibit

5 Antibiotic-Resistant Organisms Cultured from Dolphins Table 1. Antibiotic resistance of most prevalent organisms cultured from dolphins in the IRL (n = 556) Organism # Isolates AM AU CE CH CI CL EN ER FU MA PI TE Aeromonas hydrophila % Resistant Plesiomonas shigelloides % Resistant Pseudomonas fluorescens % Resistant E. coli % Resistant Vibrio alginolyticus % Resistant Edwardsiella tarda % Resistant Acinetobacter baumannii % Resistant Shewanella putrefaciens % Resistant Clostridium perfringens % Resistant Pseudomonas aeruginosa % Resistant All organisms cultured % Resistant AM = ampicillin, AU = amoxicillin clavulanate, CE = cephalothin, CH = chloramphenicol, CI = ciprofloxacin, CL = clindamycin, EN = enrofloxacin, ER = erythomycin, FU = furadantin, MA = marbofloxacin, PI = piperacillin, TE tetracycline are of particular interest (Palleroni, 1984; Mathewson and Dupont, 1992; Gonzalez-Rey et al., 2004). P. shigelloides is most commonly resistant to piperacillin and intermediate in resistance to erythromycin, whereas ciprofloxacin and tetracycline are typically effective antibiotic treatment in humans (Reinhardt and George, 1985; Stock and Wiedmann, 2001). However, in this study erythromycin (99%) and piperacillin (69%) demonstrated extensive resistance, whereas ciprofloxacin and tetracycline resistance was not observed. Previous studies found A. hydrophila from human cultures to exhibit resistance to ampicillin with susceptibility to tetracycline and chloramphenicol; this pattern also was observed in the current study (Chang and Bolton, 1987). The pattern of resistance for P. aeruginosa differed slightly from that previously reported for human cultures; resistance to ciprofloxacin was not observed. In contrast resistance rates between 3% and 12% have been reported in Pseudomonas organisms isolated from humans (Reynolds et al., 2004; Raja and Singh, 2007). The pattern observed for E. coli isolates also differed slightly from previously reported human and avian isolates. There was no occurrence of E. coli resistance to ciprofloxacin, enrofloxacin, or tetracycline in dolphins, in contrast to previous reports of resistance between 9% and 82% for human and chicken isolates (Miles et al., 2006). The lack of concordance in resistance patterns between dolphin and human isolates was not an unexpected finding due to the differences in source environments and previous antibiotic exposure. The most alarming finding was the isolation of three positive cultures of methicillin-resistant Staphylococcus aureus (MRSA) from two blowhole samples and one gastric sample in three individual CHS dolphins. Methicillin resistance has been increasing among human S. aureus infections. Hospitalizations involving MRSA has increased 62% from 1999 to 2005 for both hospital and community acquired strains (Klein et al., 2007). Recently published data found 14% of MRSA cases to be acquired in the community rather than in a hospital setting (Klevens et al.,

6 Adam M. Schaefer et al. Table 2. Antibiotic resistance of most prevalent organisms cultured from dolphins in CHS (n = 353) Organism # Isolates AM AU CF CP CH CI CL EN ER FU PI S/B TE Plesiomonas shigelloides % Resistant Aeromonas hydrophila % Resistant E. coli % Resistant Clostridium perfringens % Resistant Pseudomonas fluorescens % Resistant Vibrio alginolyticus % Resistant Shewanella putrefaciens % Resistant Staph Coag. Neg % Resistant 7 7 Proteus mirabilis % Resistant Edwardsiella tarda 6 6 % Resistant 100 Total organisms Cultured % Resistant AM = ampicillin, AU = amoxicillin clavulanate, CF = cefotaxime, CE = cephalothin, CH = chloramyphenicol, CI = ciprofloxacin, CL = clindamycin, EN = enrofloxacin, ER = erythomycin, FU = furadantin, MA = marbofloxacin, PI = piperacillin, S/B = trimethoprim and sulfamethoxazole, TE = tetracycline Table 3. Antibiotics showing statistically significant differences in resistance between culture sites for IRL and CHS dolphins Blowhole Fecal Gastric p value Isolates % Resistant Isolates % Resistant Isolates % Resistant Ampicillin <0.001 Amoxicillin Clavulanate <0.001 Cephalothin <0.001 Chloramphenicol Erythomycin <0.001 Furadantin Septra/bactrim Tetracycline <0.001 Marbofloxacin <0.001 Ciprofloxacin <0.001 Enrofloxacin < ). The extent to which dolphins could be responsible for further transmission of MRSA to humans in the aquatic environment is unknown. However, both CHS and the IRL are used extensively for recreational purposes and human contact with MRSA is plausible if these strains become established in the dolphin population.

7 Antibiotic-Resistant Organisms Cultured from Dolphins To our knowledge, there are no published data to which our findings for antibiotic resistance to multiple bacterial species in free-ranging dolphins can be compared. Previously published research on antibiotic resistance in Tursiops truncatus focused solely on E. coli cultures from dolphin fecal samples collected during HERA in the IRL and CHS (Greig et al., 2007). Of 38 animals screened, 47% harbored E. coli resistant to one or more antibiotics, which is similar to the findings of this study. Wong (2002) found resistance to ciprofloxacin (2%) in captive Tursiops truncatus housed in open-water pens. However, captive animals are presumed to have different exposures to antibiotics compared with wild populations. Similar research on other marine species also is limited. A 2006 study by Lockwood et al., using otic, nares, and wound cultures from 46 stranded harbor seals, revealed a variety of microorganisms (Lockwood et al., 2006). The most frequently isolated organisms included E. coli, Streptococcus, Enterococcus, and P. aeruginosa. Additional bacteria cultured from multiple sites such as ear, nares, and wounds included Acinetobacter spp., Pseudomonas spp, and Lactobacillus spp. Gram-negative bacteria were isolated most frequently and exhibited resistance to multiple antibiotics more often than gram-positive organisms, although this could be attributed to the type of bacteria isolated. All isolates tested were least susceptible to ampicillin (26%). Cultures from multiple species of stranded pinnipeds along the California coast identified gram-positive and gramnegative bacteria from fecal swabs least susceptible to lincomycin (18%) and clindamycin (3%) (Johnson et al., 1998). Other marine species, such as demersal and pelagic fish, have shown high frequencies of resistance to ampicillin, streptomycin, and tetracycline (Miranda and Zemelman, 2001). Multidrug resistance to fluoroquinolones, such as ciprofloxacin and enrofloxacin, has been documented in another top-level predator, sharks, at multiple study sites (Blackburn, 2003). Although the current literature is limited, it demonstrates that a variety of organisms that colonize marine species exhibit antibiotic resistance. The extensive use of common antibiotics in both humans and livestock may create an environmental impact that leads to the development of resistance among bacteria found in or near coastal estuaries. The data from this study are inadequate to implicate point sources of pollution or discharges of residential or agricultural effluent into the IRL or CHS environments. However, the high occurrence of resistance to ampicillin, amoxicillin clavulanate, cephalosporin, and erythromycin seen in both the CHS and IRL dolphins suggests that agricultural runoff and discharge of human waste containing antibiotics or resistant organisms could be responsible. Two plausible mechanisms exist to explain the patterns of resistance to commonly used antibiotics among coastal dolphin populations. In one scenario, antibioticresistant organisms from human or animals may be transferred to the marine environment and colonize dolphins inhabiting coastal waters. Alternatively, discharge of pharmaceutical products into the marine environment may be responsible for the development of resistance followed by exposure of indigenous bacteria and selective genetic pressure. Further research is needed to explore potential pathways and mechanisms for acquisition of resistance. The current study was limited in its ability to determine the incidence of initial colonization due to data collection at a single point in time. However, the prevalence data exhibited alarming rates of resistance to commonly used antibiotics at both study sites. The analysis provides an overview of the microorganisms found in dolphins inhabiting the estuarine waters of CHS and the IRL and their resistance patterns. Due to the large geographic area of the IRL and variety of anthropogenic influences, further investigation of spatial variation in resistance patterns may be informative for both study sites. The findings from this study have significance not only for marine mammals but also for human health. Marine mammals are a sentinel for ecosystem health because of their long life span, high trophic level feeding, and bioaccumulation of anthropogenic toxins (Reddy et al., 2001; Wells et al., 2004; Bossart, 2006). Bottlenose dolphins are apex predators and their health reflects that of the ecosystem shared with humans. This is particularly important in the United States where more than half of the population lives along coastal freshwater or marine ecosystems (Bossart, 2007). Additionally, marine mammals have been known to carry zoonotic pathogens that pose a risk to human health (Bogomolni et al., 2008). Emerging infectious diseases in these animals may result from perturbations in the environment, both of anthropogenic and natural origins as sentinels. Therefore, the patterns of antibiotic resistance described in two coastal dolphin populations may indicate a potential public health risk. In Florida and South Carolina, this should raise concern for zoonotic and common source transmission of pathogenic microorganisms, because the IRL and CHS are used extensively for recreation and fishing. The environmental and public health impact of antibiotic resistance in the environment is significant and warrants further investigation.

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