(Gekko gecko) destined for the pet trade

Science of the Total Environment 505 (2015) 299–305

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The carriage of antibiotic resistance by enteric bacteria from imported tokay geckos (Gekko gecko) destined for the pet trade Christine L. Casey a, Sonia M. Hernandez a,b,⁎, Michael J. Yabsley a,b, Katherine F. Smith c, Susan Sanchez d,e a

Southeastern Cooperative Wildlife Disease Study, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, United States Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602, United States Department of Ecology and Evolutionary Biology, Brown University, Providence, RI 02912, United States d The Athens Veterinary Diagnostic Laboratory, Athens, GA 30602, United States e The Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, United States b c

H I G H L I G H T S • • • •

Tokay geckos for the pet trade are wild-caught and imported from Indonesia. After imported into USA, their enteric bacteria were resistant to several antibiotics. When housed at high densities, resistance to some increased, but not significantly. Import of resistant bacteria in wildlife is an understudied public health concern.

a r t i c l e

i n f o

Article history: Received 25 July 2014 Received in revised form 26 September 2014 Accepted 29 September 2014 Available online 16 October 2014 Editor: F. Riget Keywords: Antibiotic resistance Enterobacteriaceae Pets Reptile Tokay gecko

a b s t r a c t The emergence of antibiotic-resistant bacteria is a growing public health concern and has serious implications for both human and veterinary medicine. The nature of the global economy encourages the movement of humans, livestock, produce, and wildlife, as well as their potentially antibiotic-resistant bacteria, across international borders. Humans and livestock can be reservoirs for antibiotic-resistant bacteria; however, little is known about the prevalence of antibiotic-resistant bacteria harbored by wildlife and, to our knowledge, limited data has been reported for wild-caught reptiles that were specifically collected for the pet trade. In the current study, we examined the antibiotic resistance of lactose-positive Enterobacteriaceae isolates from wild-caught Tokay geckos (Gekko gecko) imported from Indonesia for use in the pet trade. In addition, we proposed that the conditions under which wild animals are captured, transported, and handled might affect the shedding or fecal prevalence of antibiotic resistance. In particular we were interested in the effects of density; to address this, we experimentally modified densities of geckos after import and documented changes in antibiotic resistance patterns. The commensal enteric bacteria from Tokay geckos (G. gecko) imported for the pet trade displayed resistance against some antibiotics including: ampicillin, amoxicillin/clavulanic acid, cefoxitin, chloramphenicol, kanamycin and tetracycline. There was no significant difference in the prevalence of antibiotic-resistant bacteria after experimentally mimicking potentially stressful transportation conditions reptiles experience prior to purchase. There were, however, some interesting trends observed when comparing Tokay geckos housed individually and those housed in groups. Understanding the prevalence of antibiotic resistant commensal enteric flora from common pet reptiles is paramount because of the potential for humans exposed to these animals to acquire antibiotic-resistant bacteria and the potential for released pets to disseminate these bacteria to native wildlife. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Since the introduction of antibiotics in the 1950s, there has been a steady and significant increase in the number of resistant strains of ⁎ Corresponding author at: 180 Green Street, University of Georgia, Athens, GA 30602, United States. Tel.: +1 706 542 9727; fax: +1 706 542 8356. E-mail address: [email protected] (S.M. Hernandez).

http://dx.doi.org/10.1016/j.scitotenv.2014.09.102 0048-9697/© 2014 Elsevier B.V. All rights reserved.

bacteria (Hawkey, 2008). Partly because the development of new antibiotics has been relatively stagnant, antibiotic resistance has become a major public health concern (Cohen, 1992). The dissemination of antimicrobial resistance is exacerbated by the global dispersion of people, animals, and goods (Okeke and Edelman, 2001). An effort has been made to understand the epidemiology of antibiotic resistance by increasing surveillance and through the use of predictive mathematical models (Gould, 2008). Additionally, governmental agencies of

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C.L. Casey et al. / Science of the Total Environment 505 (2015) 299–305

developed nations have implemented surveillance programs of antibiotic usage and resistance, primarily focusing on humans and food animals (Vlieghe et al., 2010). In contrast, very limited information is available on antibiotic use and resistant bacteria from wildlife, including those transported around the globe through the pet trade (Blackburn et al., 2010; Leonard et al., 2012; Lloyd, 2007; Weese, 2008). There is an urgent need for information regarding the role of these animals in the dissemination of antibiotic resistant bacteria because of the intimate contact owners and handlers often have with these animals and the ability of pets to act as reservoirs of resistant organisms (Allen et al., 2010; Lloyd, 2007). Reptile-associated salmonellosis in humans and problems with antibiotic-resistant Salmonella spp. have been well documented and remain an important public health concern; however, far less is known about the antibiotic resistance patterns of other enteric organisms commonly shed by reptiles (CDC, 2006; Mermin et al., 2004). Herein we describe the resistance patterns observed in these less frequently described genera as a way to address the potential for imported reptiles to disseminate antibiotic resistant genes to both the biosphere and anthrosphere (Stull et al., 2013). The United States imported over 1.4 billion live animals between 2000 and 2006, of which, the majority were designated for commercial uses like the pet trade and approximately 80% of these animals originated from wild populations (Smith et al., 2009). Of the wild animals imported into the United States during this six year period, 69% originated from Southeast Asia, an area specifically known for high rates of antibiotic resistance in bacterial isolates cultured from humans, where antibiotics are readily available, often misused, and the antibiotics used are typically older generations of drugs no longer used in developed nations because of resistance potential (Simanjuntak et al., 2004). Wild animals imported to the United States from Southeast Asia often endure stressful transport conditions. Transport conditions such as overcrowding, thermal extremes, poor diets, and poor ventilation have been shown to be stressful to livestock and are possibly more stressful to wild animals because, unlike domestic animals, they are not acclimated to novel stimuli (Grandin, 1997; Hartung, 2003; Schumacher, 2006; Weston and Memon, 2009). These conditions often translate into physiological changes within the host, most of which have been linked to immunosuppression (Jacobson, 1993), increased mutations and exchange of antibiotic resistance among their enteric bacteria (Blázquez, 2003; Silbergeld et al., 2008), and an increased rate of shedding of pathogens (Molitoris et al., 1987; Sorum and Sunde, 2001). In contrast to many exotic pets which are bred in captivity, the majority of Tokay geckos (G. gecko; Fa: Gekkonidae) currently sold in the United States are wild caught in their native range (Nijman et al., 2012; Okeke and Edelman, 2001). The Tokay gecko is a relatively long-lived nocturnal, arboreal, small lizard and males are territorial and aggressive (Yu et al., 2011). Tokay geckos inhabit tropical rainforests of the Indo-Australian Archipelago, where they are typically solitary. In their native range and under natural conditions, they are found 30–100 m apart from each other and display aggressive behaviors towards one another if they come closer (Tang et al., 2001; Yu et al., 2011). Housing solitary reptiles together, a practice common in the wildlife trade, can lead to stress. For example, tree skinks (Egernia striolata), which under natural conditions are observed singly or in breeding pairs several meters apart, experienced social stress indicated by elevated corticosterone levels at relatively modest densities in captive settings (four individuals housed together) (Lancaster et al., 2011). Additionally, Martinez-Silvestre suggested that stress from either social dynamics or overcrowding would result in release of corticosterone after just 10–14 days (Martínez-Silvestre, 2014). Geckos are increasingly found in peridomestic settings in Indonesia, such as rural dwellings or barns (Aowphol et al., 2006; Sanchez et al., 2012). Many imported wild-caught geckos are captured from these peridomestic settings, where they might have acquired antibiotic-

resistant bacteria from human or livestock waste. Often perceived as attractive display animals because of their bright coloration, Tokay geckos are common pet reptiles (Cavendish, 2001; Tapley et al., 2011). Yet, like other beautiful but surprisingly aggressive exotic pets, Tokay geckos are often released by their owners into the environment, and they are now considered an established, breeding, invasive species in several US states (Hawaii, Florida, Texas) and countries including Belize, and Martinique (Norval et al., 2011; Witmer et al., 2007), creating a unique potential method for the introduction of resistant bacteria from Southeast Asia into new regions and hosts. To our knowledge, limited reports exist on the antibiotic susceptibility of the enteric bacteria of reptiles from Indonesia (Graves et al., 1988; Smith et al., 2012). In this study, we examined the antimicrobial resistance of the commensal enteric flora of wild-caught Tokay geckos imported for use in the pet trade. Enterobacteriaceae was selected specifically because of their status as common commensal bacteria of reptile gastrointestinal tracts and their potential to be pathogens for humans (Jacobson, 2007; Rosenthal and Mader, 2006). We propose that the pet trade can influence the movement and prevalence of antibiotic resistant bacteria. To test this, we measured 1) the antimicrobial resistance patterns of culturable lactose fermenting Enterobacteriaceae isolates from individuals immediately upon arrival and, 2) how antibiotic resistance patterns might change after manipulating housing density—mimicking stressful transport and distribution practices. We predicted that wild-caught Tokay geckos are imported with antibiotic-resistant commensal enteric flora, and that the detection resistance from individually-housed Tokay geckos would become more frequent as animal density was increased. 2. Materials and methods 2.1. Animal collection, shipping, and housing Geckos were captured from rural locations outside Jakarta on the island of Java by a reptile distributor, following standard business practices but modified for the purpose of this study, and shipped as airfreight, via Miami, to the University of Georgia's (UGA) College of Veterinary Medicine (Smith et al., 2012). Briefly, geckos were handcaptured from locations where they are typically captured for the reptile trade, including homes, barns and other anthropogenic structures, placed in clean plastic containers and individually housed for shipping. The reptile distributor was instructed to keep all geckos in individual, clean containers from the moment of capture through shipping and to practice basic hygiene practices (hand washing) to minimize transfer of fecal bacteria among individuals. One hundred and fifty geckos were shipped to UGA in two batches, 60 in March 2009 (Batch 1) and 90 in June 2009 (Batch 2). A total of 50 geckos from Batch 1 and 60 geckos from Batch 2 were used in this study. Animals were initially housed for 10 days individually in sterile, clear propylene boxes to allow animals to acclimate to their new environment. Fecal samples were collected within the first 48 h of the acclimation period. Their specific husbandry requirements were met and monitored as previously described (Bartlett and Bartlett, 1995). Briefly, they were fed a standard diet of crickets and mealworms daily. The temperature and humidity were maintained at 26.6° ± 4 °C and 50–70%, respectively, and the lighting was maintained on a 12 hour cycle. After the 10 day acclimatization period, individual geckos were randomly assigned to three groups: low density (n = 5), medium density (n = 15), and high density (n = 30) and then transferred to their respective enclosures measuring 61 cm wide × 91 cm deep × 183 cm high. Batch 2 was imported in June and housed individually as above. As aforementioned, fecal samples were collected from animals while they were housed individually within the first 48 hrs, after which, 15 geckos from Batch 2 were assigned to each of the pre-existing density groups (low, medium, and high) and a fourth, Temporal group, was created (n = 15). The Temporal group was comprised of only geckos from Batch 2 and was created to determine if there was any difference in genera shed from the second,


compared to the first batch of imported geckos due to the amount of extra time Batch 1 spent together. There was no statistical difference in the number/diversity of genera shed by the animals in the combined groups formed from the Temporal group, when compared to the combined groups that consistent of animals from Batch 1 (Casey et al., in press). At the completion of the study (September; 6 mos and 3 mos after Batch 1 animals were placed in their various combined groups), fecal samples were collected from the groups of combined geckos living at varying densities in the same manner as described above. Therefore, individuals from Batch 1 were sampled in March when they arrived, individuals from Batch 2 were sampled in June, and combined animals were all sampled in September. All animal handling and care procedures were approved by the University of Georgia's Institutional Animal Use Care Committee (#A2008 12-051). 2.2. Isolation and identification Fecal samples were homogenized in 1 μl of sterile water and submitted to the laboratory within 12 h from collection. A 10 μl loop was used to streak the diluted fecal solution on MacConkey media plates and the plates were incubated at 37 °C ± 2° for 24 h. One sample of each morphologically distinct lactose positive colony was collected. If the colonies were later identified as the same genus and species, only one of the isolates was used. Pure isolates were suspended in freezer media consisting of 1% peptone and 15% glycerol and frozen at − 80 °C until further analysis. Biochemical tests as recommended in the Manual of Clinical Microbiology were used to determine the identity of isolated organisms (Murray et al., 2003). Rapid Analytical Profile Index strips® (API20E)1 were used to definitively determine the identification of the isolate, if results were conflicting. 2.3. Minimum inhibitory concentrations To determine antibiotic resistance, minimum inhibitory concentrations (MIC) were performed by using broth microdilution methods and a National Antimicrobial Resistance Monitoring System (NARMS) panel for Gram-negative enteric bacteria (CMV1AGNF). 2 The antibiotics included on the plate were four aminoglycosides (amikacin: 0.5–32 μg/ml, streptomycin: 32–64 μg/ml, gentamicin: 0.25–16 μg/ml, kanamycin: 8–64 μg/ml), two penicillins (ampicillin: 1–32 μg/ml, amoxicillin/clavulanic acid: 1/0.5–32/16 μg/ml), three cephalosporins (cefoxitin: 0.5–32 μg/ml, ceftriaxone: 0.25–64 μg/ml, ceftiofur: 0.12–8 μg/ml), two quinolones (ciprofloxacin: 0.015– 4 μg/ml, nalidixic acid: 0.5–32 μg/ml), two sulfa drugs (sulfisoxazole: 16–256 μg/ml, trimethoprim/sulfamethoxazole: 0.12/2.38–4/76 μg/ml), as well as chloramphenicol (2–32 μg/ml) and tetracycline (4–32 μg/ml) at various concentrations and used according to the Clinical Laboratory Standards Institute guidelines (CLSI, 2008). Aseptic technique was used to transfer colonies into sterile water to create a 0.5 Mcfarland standard. Ten microliters of the 0.5 Mcfarland solution was added to 10 ml of cation-adjusted Mueller–Hinton broth and vortexed. The MIC plate was inoculated with 50 μl of the broth. The plates were incubated at 37 °C ± 2° for 18 h. Escherichia coli ATCC 25922 was routinely included as a quality control. Interpretations of susceptibility for all the antibiotics tested were made by comparison with CLSI criteria for Enterobacteriaceae (CLSI, 2010). 2.4. Data analysis The MIC results were assigned numerical values of 0 if the bacterial was completely susceptible, 0.5 if it displayed intermediate resistance, and 1 if resistant. Logistic regression was used to look at the prevalence of resistance between the combined groups Low, Medium, High and 1 2

Biomerieux, Durham, NC, USA. Sensititre; TREK Diagnostics, Cleveland, OH, USA.

301

Table 1 The prevalence of culturable lactose positive Enterobacteriaceae cultured from individually housed Tokay geckos (n = 137) destined for the pet trade and from geckos combined in groups (n = 52). There was a total of 189 isolates recovered in this study. Bacteria

Individually-housed geckosa

Geckos combined in groupsb

Citrobacter spp. Klebsiella spp. Enterobacter spp. Kluyvera spp. Echerichia coli Serratia spp. Pantoea spp. S. arizonae

55% (n 20% (n 10% (n 7% (n 5% (n 3% (n 1% (n 1% (n

75% (n = 39) 4% (n = 2) 4% (n = 2) 4% (n = 2) – – 2% (n = 1) 12% (n = 6)

= = = = = = = =

75) 27) 13) 9) 7) 4) 1) 1)

a Percentage and the number of isolates recovered from all individually housed geckos (n = 137). b Percentage and the number of isolates recovered from all the geckos combined in varying density groups (n = 52).

Temporal for the 15 different antibiotics tested, by bacterial genera. This method was also used to compare the prevalence of resistance of various bacterial genera from individual animals against the resistance of bacteria from each combined group. Due to the low number of observations, not all genera or antibiotics were compared for every group (Table 4). Statistical analyses were performed using SAS V 9.2.3 All hypothesis tests were 2-sided and the significance level was set at α = 0.05.

3. Results & dicussion A total of 189 lactose positive Enterobacteriaceae isolates were recovered from both individuals and combined groups (Table 1). We obtained 137 isolates of seven genera from the 110 individually-housed animals. There was no statistical significant difference in diversity of genera cultured between individually housed animals or combined into groups. The three most frequently isolated genera were Citrobacter (55%), Klebsiella (20%), and Enterobacter (10%) among individually housed geckos. E. coli and Serratia spp. were only cultured from individually housed geckos. Twenty nine animals died during the experiment and five animals were not used at the end of the study because we could not collect a fecal sample. Of the remaining 76 geckos housed in combined groups, 52 lactose positive isolates were cultured (Table 1). Overall there was no statistical difference in the prevalence of resistance among the combined groups or when isolates from individually-housed geckos were compared to their respective combined groups. Additionally, although Salmonella arizonae, a lactose positive Salmonella sp. was one of two most common genera cultured from all the combined groups (12%; Table 1), its analysis of antibiotic resistance is not included here because it was incorporated with the data from non-lactose fermenting Salmonella spp. in another report (Smith et al., 2012). Our data support the first prediction that commensal bacteria from wild-caught Tokay geckos imported into the United States display resistance against antibiotics that are commonly used in Southeast Asia, and specifically Indonesia (chloramphenicol, aminopenicillins and tetracyclines) (Hadi et al., 2008). We found relatively high rates of resistance among Citrobacter spp., Enterobacter spp., Klebsiella spp., and Serratia spp. against both these and other commonly-used antibiotics in the United States, such as cephalosporins. Isolates of Klebsiella spp. and E. coli from individually housed geckos were resistant against cefoxitin, a cephalosporin. These isolates also expressed some level of decreased susceptibility against amoxicillin/ clavulanic acid. In particular, the prevalence of Klebsiella spp. resistant against cefoxitin was much lower in individually housed geckos (4%) compared to animals in combined groups (50%). Resistance to cefoxitin and amoxicillin/clavulanic acid is a typical pattern observed when 3

SAS Institute Inc. Cary, NC, USA.

302 Table 2 Prevalence (%) of antibiotic resistance (R) and intermediate resistance (I.R.) among bacterial isolates from individually housed Tokay geckos and in geckos combined in groups arranged by bacterial genera. Cells that are left blank indicate a 0% prevalence. Bacteria & resistance

Antibiotic

Citrobacter spp. R I. R. Klebsiella spp. R I. R. Enterobacter spp. R I. R. Kluyvera spp. R I. R. Pantoea spp. R I. R. E. coli R I. R. Serratia spp. R I. R.

CHL

TET

Individual

Combined

Individual

Combined

Individual

81 11

85 5

1 33

18

4

50 4

50

15 23

100

85 8

100

AMP

KAN

Individual

Combined

Individual

Combined

Individual

1 3

7 13

3 10

47 27

36 20

1 1

15

70 15

50

4

23 8

Combined

AUG

50

22

STR Combined

Individual

FIS Combined

Individual

Combined

1

17

13

4

52

50

77

100

67

50

50

46 23

50

92

11 55

50

100

8

100

50

100 100 14

100

100

—a — — —

— —

50

— —

75

— —

14

— —

— —

25 25

— —

— — 100

— —

14

— —

— —

14

— —

— —

— —

100

— —

FOX (cefoxitin), CHL (chloramphenicol), TET (tetracycline), AMP (ampicillin), AUG (amoxicillin/clavulanic acid), KAN (kanamycin), STR (streptomycin) which has no intermediate resistance value, FIS (sulfisoxazole) which has no intermediate resistance value. a The dash indicates that no isolates of E. coli and Serratia were recovered from any of the combined geckos.


FOX


an AmpC beta-lactamase is present (Bradford, 2001; Livermore, 1987). β-Lactamase activity is one mechanism by which cephalosporins display resistance. Without further testing, the mechanisms responsible for this observed resistance pattern is unknown; however, currently Klebsiella spp. is not known to contain a chromosomal ampC β-lactamase gene (Black et al., 2005; Philippon et al., 2002). Recent descriptions of plasmid-mediated AmpC β-lactamases, which allow for horizontal transfer of resistance, have been identified and reported in human Klebsiella spp. isolates at varying rates (3.3–8.5%) (Alvarez et al., 2004; Coudron et al., 2000; Moland et al., 2006). Citrobacter spp. cultured from individuals and combined groups was consistently resistant to cefoxitin (81 and 85% respectively). This is in accordance with a study that reported 63% resistance to cefoxitin by the enteric flora of Bothrops jararaca, a venomous snake from Brazil (Bastos et al., 2008). The prevalence of resistance against cefoxitin and amoxicillin/clavulanic acid remained high among Enterobacter spp. isolates from both individuals and combined animals. Additionally, all four isolates of Serratia spp. were resistant against amoxicillin/ clavulanic acid and expressed intermediate resistance against cefoxitin. The resistance to cefoxitin and amoxicillin/clavulanic acid by Citrobacter spp., Enterobacter spp., and Serratia spp. was expected because these genera commonly express β-lactamase activity (Hawser et al., 2009; Janda and Abbott, 2006; Lewis, 2002). However, it is important to document this resistance because there is potential for horizontal transfer of mobile genetic elements disseminating resistance genes to other bacterial genera within the gastrointestinal tract, and facilitating the development of resistance against other extended spectrum beta-lactams (Livermore, 1987; Nordmann, 1998; Paterson, 2006). There was also a low prevalence of resistance against kanamycin, streptomycin, nalidixic acid, trimethoprim/sulfamethoxazole, and tetracycline among Citrobacter spp. (Table 2). Two isolates of Enterobacter sp. were both resistant against gentamicin and one isolate expressed intermediate resistance against kanamycin. An isolate of E. coli was resistant against kanamycin. This resistance pattern, although low, is of paramount significance because aminoglycosides and fluoroquinolones are often used to treat severe Gram-negative bacterial infections in the United States (Galimand et al., 2003; Neuhauser et al., 2003).

303

Specifically, kanamycin is an aminoglycoside commonly used to treat Citrobacter spp. infections and multidrug resistant tuberculosis (Krüüner et al., 2003). Previous studies have demonstrated that plasmid-mediated resistance mechanisms against aminoglycosides can also confer resistance against other classes of antibiotics such as quinolones, like ciprofloxacin (Périchon et al., 2007; Robicsek et al., 2005). We observed a low prevalence of resistance against quinolones yet, again, they are important because of the high prevalence of quinolone and extend-spectrum beta lactam resistance reported from clinical isolates of Enterobacteriaceae in Southeast Asia and the potential for geckos living in close proximity to humans to acquire these resistant genes and disseminate them across international borders (Hawser et al., 2009; Nordmann and Poirel, 2005; Okeke et al., 2005; Sheng et al., 2002). In recent years, due to the rise in resistance against aminoglycosides and fluoroquinolones, there has been a growing concern and a call for more judicious use of these antibiotics, especially against suspected multidrug-resistant bacterial infections in the United States (Paterson, 2006; Pitout and Laupland, 2008). Bacterial infections acquired from imported geckos have the potential to be unrecognized sources of resistant bacteria leading to complications with antibiotic therapy. The intermediate resistance we documented by Citrobacter spp., Klebsiella spp., Enterobacter spp., and Serratia spp. against chloramphenicol (Table 2) is consistent with other studies of wild reptiles in Indonesia (Graves et al., 1988; Okeke et al., 2005; Schwarz et al., 2010). In contrast, Kluyvera spp. expressed intermediate resistance to chloramphenicol (22%), which has been previously reported to be very effective against Kluyvera spp. (Sarria et al., 2001). Lastly, one isolate of Enterobacter spp. cultured from an individually housed gecko and one from the combined animals were the only isolates to express intermediate resistance against third generation cephalosporins (Table 3). Table 3 represents the infrequent prevalence of resistance against several classes of antibiotics among all the genera isolated. Intermediate resistance is reported because it represents the decreased (or waning) susceptibility of a bacterium to an antibiotic (Levy and Marshall, 2004). The prevalence of low-level resistance in a population of microbes is also important because they have the potential to

Table 3 Infrequent prevalence (%) of resistance (R) and intermediate resistance (I.R.) among bacterial isolates from both individually housed Tokay geckos and geckos in combined groups against selected antibiotics. Ceftriaxone and amikacin were excluded from this table because no resistance was detected from any isolate. Cells that are left blank indicate a 0% prevalence. Bacteria & resistance

Antibiotic SXT Individual

Citrobacter spp. R. I. R. Klebsiella spp. R. I. R. Enterobacter spp. R. I. R. Kluyvera spp. R. I. R. Pantoea spp. R. I. R. E. coli R. I. R. Serratia spp. R. I. R.

NAL Combined

1

Individual

CIP Combined

Individual

GEN Combined

Individual

TIO Combined

Individual

Combined

1

100

100

100 50

8

—a —

— —

— —

— —

— —

— —

— —

— —

— —

— —

SXT (trimethoprim/sulfamethoxazole) which has no intermediate resistance value, NAL (nalidixic acid) which has no intermediate resistance value, CIP (ciprofloxacin), GEN (gentamicin), TIO (ceftiofur). a The dash indicates that no isolates of E. coli and Serratia were recovered from any of the combined geckos.

304

C.L. Casey et al. / Science of the Total Environment 505 (2015) 299–305 100

Table 4 Results of the logistic regression utilized to compare the antibiotic resistance against Citrobacter spp. cultured from individual and combined groups of Tokay geckos for selected antibiotics. Anitibiotics tested against Citrobacter spp

Low:

FOX 0.8928a FOX 0.5235 FOX 0.6829 CHL 0.7348

Medium: High: Temporal:

AUG 0.8928 AUG 0.4055 CHL 0.3651 AUG 0.3857

FIS 0.4637 AUG 0.30 AMP 0.6755

AMP 0.4252 FIS 0.20

Prevelance

Density

80

AMP 0.3209

FOX (cefoxitin), AUG (amoxicillin/clavulanic acid), FIS (sulfisoxazole), AMP (ampicillin), CHL (chloramphenicol). a P-value.

contribute to the dispersion of antibiotic-resistant strains (Andersson and Levin, 1999; Baquero, 2001). Using a conservative definition of multi-drug resistance (resistance against three or more classes of antibiotics) we found four multidrugresistant isolates of Enterobacter spp. (Magiorakos et al., 2012). Two of these isolates were resistant to four classes of drugs, including one quinolone and two aminoglycosides, which often utilize plasmidmediated resistance mechanisms and are commonly reported in SE Asia (Nordmann and Poirel, 2005). If our definition is expanded to include intermediate resistance to three or more classes of drugs, then one isolate of Citrobacter spp. and Serratia spp. should also be included. Both of these isolates expressed either resistance or intermediate resistance to a beta lactam, chloramphenicol, and tetracycline which are the three most commonly used antibiotics reported in Indonesia (Hadi et al., 2008). Multidrug resistance is a concern because of the potential to transfer mobile genetic elements, which can confer resistance against classes of drugs to other genera lacking those resistant genes, interfering with clinical treatment and possibly facilitating the selection of antibiotic-resistant bacteria. One possible mechanism by which geckos acquire antibioticresistant bacteria in their home range may be directly through exposure to human or livestock waste, or indirectly through consumption of prey which harbor resistant bacteria. In fact, their prey (e.g. cockroaches, flies) is abundant in peridomestic settings and is known to feed on human or domestic animal waste, which effectively combines the two mechanisms by which geckos could acquire these bacteria. The concept that anthropogenic forces have influenced the prevalence of antibiotic resistance among wildlife has been demonstrated in previous studies (Allen et al., 2010; Baquero et al., 2008; Skurnik et al., 2006). A positive relationship between an antibiotic resistance prevalence in reptiles and proximity to humans or livestock was also recently demonstrated in marine iguanas in the Galapagos (Wheeler et al., 2012). We additionally theorized that increased densities in the combined groups of geckos would influence the exchange of bacteria (through close contact), which may result in an increase in the prevalence of resistant bacteria. Our data did not support this hypothesis. Comparisons between the individual and combined geckos were hindered because the same genera of enteric bacteria were not represented in both groups equally, making it difficult to determine the effect, if any, that grouping geckos had on the antibiotic resistance patterns of specific genera. However, we did observe important trends through a comparison of the most frequently cultured isolates obtained from individual geckos and those combined into groups. The most frequently isolated genus, Citrobacter spp., displayed a decrease in susceptibility to cefoxitin as the density of the animals was increased such that we observed more isolates that expressed resistance and intermediate resistance (Fig. 1). The prevalence of resistance of Enterobacter spp. isolates against chloramphenicol, tetracycline, and ampicillin decreased in the combined groups, but intermediate resistance increased (Table 2). However, our small sample sizes precluded statistical significance.

60

R 32 40

I 16 S 8

20

0

Low -20

Med

High

Density Groups

Fig. 1. The distribution of susceptibility against cefoxitin for Citrobacter spp cultured from Tokay geckos across the three combined density groups (Low, Medium, High), illustrating a trend for a loss of susceptibility as density of geckos increased.

This study focused on lactose fermenting Enterobacteriaceae at the genus level because these bacteria are commonly considered commensal organisms of the gastrointestinal tract, yet, under the right conditions, these genera can also become important opportunistic pathogens of reptiles (Janda and Abbott, 2006). Commensal flora can also act as a reservoir of antibiotic resistance genes for pathogenic bacteria (Van den Bogaard and Stobberingh, 2000). In conclusion, commensal enteric bacteria from Tokay geckos imported for the pet trade display resistance against some common antibiotics for which the development of resistance is a concern both in Southeast Asia and in the United States. Specifically, the prevalence of phenotypic resistance against quinolones and aminoglycosides expressed in both Citrobacter spp. and Enterobacter spp. should be of importance to public health. The mechanisms of resistance, for example, of a plasmid-mediated AmpC beta-lactamase harbored by Klebsiella spp. should be investigated further because the pet trade could be a novel route for introduction for this resistance determinant. While there was no significant increase in the prevalence of antibiotic-resistant bacteria as geckos were grouped, other patterns emerged: a high prevalence of resistance against commonly used antibiotics in SE Asia; a decreased susceptibility to cefoxitin among Citrobacter spp. isolates as density increased; and a high prevalence of multidrug resistance among Enterobacter spp. isolates. Further work should follow to understand whether the close contact between pet owners and imported reptiles harboring antibiotic-resistant bacteria results in the exchange of these organisms, and if the movement of antibiotic resistance bacteria from imported Tokay geckos impacts the gastrointestinal flora of domestic animals or native wildlife, particularly in areas in the USA where the Tokay gecko has become established. Acknowledgments The authors thank S. Arnold and A. Rogers for assistance with gecko care and a professional wildlife distributor who will remain anonymous.

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