Occurrence of Antibiotic Resistant Escherichia Coli in Green Iguanas (Iguana Iguana) in Grenada, West Indies

International Journal of Veterinary Medicine: Research & Reports

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W. R. B. Sylvester1, V. Amadi2, C. Hegamin-Younger3, R. Pinckney2, C. N. L. Macpherson4, J. S. McKibben5, R. Bruhl-Day1, K. D. John-Sylvester6 and H. Hariharan2

1Small Animal Medicine and Surgery Academic Program, School of Veterinary Medicine, St. George’s University, Grenada, West Indies

2Pathobiology Academic Program, School of Veterinary Medicine, St. George’s University, Grenada, West Indies

3Department of Public Health and Preventative Medicine, School of Medicine, St. George’s University, Grenada, West Indies

4Department of Microbiology, School of Medicine and Windward Islands Research and Education Foundation, St. George’s University, Grenada, West Indies

5Anatomy and Physiology Academic Program, School of Veterinary Medicine, St. George’s University, Grenada, West Indies

6Graduate Studies Program, School of Veterinary Medicine, St George’s University, Grenada, West Indies

Volume 2014 (2014), Article ID 260412, International Journal of Veterinary Medicine: Research & Reports, 8 pages, DOI: 10.5171/2014.260412

Received date : 3 February 2014; Accepted date : 11 March 2014; Published date : 23 April 2014

Academic editor: Modestas Ružauskas

Cite this Article as: W. R. B. Sylvester, V. Amadi, C. Hegamin-Younger, R. Pinckney, C. N. L. Macpherson, J. S. McKibben, R. Bruhl-Day, K.D. John-Sylvester and H. Hariharan (2014), "Occurrence of Antibiotic Resistant Escherichia Coli in Green Iguanas (Iguana Iguana) in Grenada, West Indies," International Journal of Veterinary Medicine: Research & Reports, Vol. 2014 (2014), Article ID 260412, DOI: 10.5171/2014.260412

Copyright © 2014 W. R. B. Sylvester, V. Amadi, C. Hegamin-Younger, R. Pinckney, C. N. L. Macpherson, J. S. McKibben, R. Bruhl-Day, K.D. John-Sylvester and H. Hariharan. Distributed under Creative Commons CC-BY 3.0

Abstract

Cloacal swabs from 62 green iguanas (Iguana iguana) from five parishes of Grenada were sampled during the period January to April 2013, and examined by culture for presence of Escherichia coli. Forty percent of the green iguanas were positive for E. coli. This organism is documented to cause health problems in wildlife species, but it is rare. Isolates were further tested for the presence of E. coli O157:H7, a serotype known to cause severe zoonotic illnesses globally. None of the isolates tested positive for this serotype. The results of this study indicate that green iguanas are not important reservoirs of E. coli O157:H7.  Antimicrobial susceptibility tests conducted by a disk diffusion method against amoxicillin-clavulanic acid, ampicillin, cefotaxime, ceftazidime, ciprofloxacin, enrofloxacin, gentamicin, nalidixic acid, streptomycin, tetracycline and trimethoprim-sulfamethoxazole showed that the most frequent resistance was detected t to amoxicillin-clavulanic acid and streptomycin. Isolates resistant to ampicillin, cefotaxime, tetracycline, and trimethoprim-sulfa were also detected. Overall, drug resistance was found for seven of the twelve antibiotics used in this study. Multiple drug resistance was found in 3 isolates. The antibiotic resistance patterns of E. coli found in this study is of public health concern since there are ample opportunities for transmission of enteric bacteria from iguanas to humans in Grenada. This is the first report of isolation of E. coli and antimicrobial resistance profiles from green iguanas in Grenada, West Indies.

Keywords: Iguana, Escherichia coli, drug resistance, Grenada

Introduction

Non-pathogenic Escherichia coli constitute an important part of the normal intestinal microbiota of healthy mammals and birds whereas pathogenic strains may cause zoonotic infections in humans (Quinn et al., 2011).  E. coli has been responsible for both intestinal and extra-intestinal infections in humans (Santos et al., 2013). In recent years, E. coliO157:H7 and other entero-hemorrhagic serotypes have emerged as major food-borne, zoonotic pathogens in humans (Perna et al., 2001, Quinn et al., 2011).

Green iguanas are arboreal lizards that are native to the territories extending from Southern Mexico through to Central Brazil, Paraguay, Bolivia and the Galapagos islands (Taddei et al., 2010). They can also be found in North America, Hawaii, Fiji and the Caribbean islands (Alberts et al., 2004), including Grenada.  Green iguanas constitute an important component of terrestrial and arboreal herpetofauna of many Caribbean islands (Alberts et al., 2004). Green iguanas may act as sources of zoonotic pathogenic E. coli. The green iguana has been previously implicated as an important reservoir in the transmission of zoonotic pathogens to humans through contact with iguana feces (Mermin et al., 1997). The increasing popularity of green iguanas as pets and as sources of meat for human consumption in Grenada, justifies the need to investigate their microbiological and zoonotic potential.

The research hypothesis was that green iguanas could be carriers of E. coli O157:H7, but drug resistance is unlikely. Based on this, the first objective of this study was to determine the occurrence of E. coli in the feces of green iguanas in Grenada. The second objective was to determine the prevalence of E. coli O157:H7 among the isolates.  Determination of the resistance profiles of the E. coli isolates against antimicrobial drugs, commonly used in treating enteric infections in humans was the third and final objective of this study.

Materials and Methods

During the period January to April 2013, a total of 62 cloacal swab samples were collected from wild and pet green iguanas and  were analyzed for the presence of E. coli. Green iguanas were sampled in five of the seven parishes of Grenada, including from St. George 18, St Andrew 10, St. David 10, St. Patrick 19, Carriacou and Petite Martinique 5. The wild iguanas were trapped and restrained using non-chemical humane traps and hand catching while the pet iguanas were safely retrieved from their respective cages with consent from their owners.

The trapped iguanas were examined by a veterinarian to ensure that they did not have any clinical signs of illness or injury. Only healthy green iguanas were included in this study. For each sampling event, a sterile transport swab (Starswab IITM, StarplexTM scientific inc, Etobicoke, Ontario, Canada) was inserted two (2) inches into their rectum via the cloaca and gently rotated five times so as to obtain an adequate fecal sample. After sampling, the trapped and sampled iguanas were tagged for identification purposes with regard to the location, time and date of sample collection using a permanent, non-toxic paint (VIBE Standard White, European Body Art Laboratory, Newport Beach, CA, USA). All the sampled iguanas were then returned to their respective natural habitats without subjection to transportation, diet change or excessive handling. The cloacal swabs were immediately stored in a cooler with ice packs and transported to the Bacteriology Laboratory, School of Veterinary Medicine, St. George’s University where all the laboratory analysis were performed. The approximate time between sample collection and culture was three hours.

For the identification and isolation of E. coli, the cloacal swabs were placed in 10 ml of tryptic soy broth (Remel, Lenexa, KS, USA) and incubated at 37o C for 24 hours. After incubation, the swabs were streaked onto MacConkey agar and incubated at 37o C for 24 hours. One to two (1 — 2) pink to red color colonies with or without a zone of precipitated bile morphologically resembling E. coli were subcultured via streaking onto individual MacConkey agar and incubated at 37o C for 24 hours. The pure colonies from second MacConkey agar were further tested by using API-20E® (Analytical profile Index; Bio-Merieux Inc., Durham, NC, USA) strips for confirmation as E. coli.

For identification of E. coli O157:H7 pure colonies were first plated on sorbitol-MacConkey agar and then tested using Remel Wellcolex* E. coli O157 Rapid Latex Test (Remel Europe Ltd; Clipper Boulevard West, Crossways, Dartford, Kent, DA2 6PT, UK) and ProlexTM E. coli O157  Latex Kit (Pro-lab Diagnostics, 20 Mural Street, Units 3 & 4, Richmond Hill, Toronto, Canada).

The antimicrobial susceptibility tests were carried out using the disk diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI) using Mueller Hinton agar, and the inhibition zone sizes were interpreted as per CLSI guidelines (Jorgensen and Turnidge, 2003). The antibiotic disks used were amoxillin/clavulanic acid, ampicillin, cefotaxime, ceftazidime, ciprofloxacin, enrofloxacin, gentamicin, imipenem, nalidixic acid, streptomycin, tetracycline and trimethoprim/sulfamethoxazole (Becton, Dickinson and Co., Sparks, MD, USA).

Results

In this study, E. coli was isolated only from 25 (40%) of the 62 green iguanas (Table 1).  Based on gender, 43% (15 of 35) females and 36% (10 of 27) male iguanas tested positive for E. coli. The percentages of iguanas that tested positive for E. coli based on habitat, sex, life stage and parishes are summarized in Table 1. No statistical difference was found between male and female iguanas (p=0.64), between pet and wild iguanas (p=0.98) and between adult and young iguanas (p=0.71) in our study, however, significant statistical difference was found among the five parishes from which iguanas were sampled (p=0.003) using Chi-square test at a 95% confidence interval (Table 1). It may be noted that this may be a trend since the number of iguanas sampled from each parish varied as outlined in the methodology.  A total of 42 E. coli isolates were obtained from the 25 culture-positive iguanas, all confirmed by API-20E identification system.

Table 1: Statistical Analysis for E. coli Prevalence in Green Iguanas from Grenada.
 
260412-tab-1
With regard to identification of E. coli O157:H7 among the positive isolates; 12 of the 42 (29%) E. coli isolates tested negative for sorbitol fermentation on  sorbitol-MacConkey agar, however, none of the 42 E. coli isolates tested positive for E. coli O157:H7 using Remel Wellcolex* E. coli O157 Rapid Latex Test and ProlexTM E. coli O157 Latex.

Based on the result of the disk diffusion assays, five (12%) of the  isolates recovered from the positive iguanas were  resistant to amoxicillin/clavulanic acid and to streptomycin, 3 (7%) were resistant to ampicillin and 1(2%) each to nalidixic acid, cefotaxime, tetracycline and trimethoprim/sulfamethoxazole.  Seven (17%) showed intermediate resistance to ampicillin, 6 (14%) to streptomycin, 1 (2%) to tetracycline, cefotaxime, and ceftazidime. Three isolates showed multiple drug resistance. The antibiotic susceptibility profiles of the 42 E. coli isolates recovered from the positive iguanas are presented in Table 2.

Table 2: Antimicrobial Susceptibility Profiles of 42 E. coli Isolates from Green Iguanas in Grenada Using Disk Diffusion Method.
 
260412-tab-2
 
Discussion
 

The isolation rate for E. coli (40%) in the current study is somewhat similar to that reported in captive green iguanas in Trinidad and Tobago where 60% captive green iguanas tested positive for E. coli. That same study showed that 58% of free-ranging mammals yielded E. coli and 90% of free-flying birds were positive for E. coli (Adesiyun, 1999). In a study conducted in Australia on non-mammalian vertebrates, 33% of crocodiles, 4% of turtles, 2% of snakes and 10% of lizards tested positive for E. coli (Gordon and Cowling, 2003). E. coli is documented to cause health problems in wildlife species, but this is considered rare (Adesiyun, 1999). E. coli has been reported as a cause of diarrhea in a tortoise (Owuamanam et al. 2012). Free-flying birds and free-ranging mammals harbor more E. coli in their gastrointestinal tract than reptiles do (Adesiyun, 1999).

Furthermore, 29% of the E. coli isolates in the present study were negative for sorbitol fermentation on sorbitol-MacConkey agar. E. coli O157:H7 is typically a non-sorbitol fermenter, however other zoonotic pathogenic non-O157 strains of E. coli are also non-sorbitol fermenters. Some pathogenic non-O157 strains of E. coli including O26, O103 and O111, have been previously associated with infections in humans. There is a possibility that there are other pathogenic Shiga-toxin producing E. coli strains among the isolates, however this study focused on E. coli O157:H7 since it is of paramount public health importance (Adesiyun, 1999). Additionally, the most commonly isolated E. coliserotype implicated in hemorrhagic colitis disease outbreaks in humans globally is E. coli O157:H7 and it serves as a marker for virulent strains of E. coli in various species (Nataro and Kaper, 1998, Perna et al., 2001).

E. coli O157:H7 was not identified in this study using Latex Agglutination Test Kits. In Trinidad and Tobago, all samples from 5 green iguanas fermented sorbitol and also tested negative for E. coli O157:H7 on Latex Agglutination Test. Free-ranging and captive avian, reptile and mammalian wildlife species were not important reservoirs of E. coliO157:H7 in Trinidad and Tobago (Adesiyun, 1999). Similarly, it appears that green iguanas in Grenada do not harborE. coli O157:H7 and may therefore not pose a public health in this regard. However, it may be noted that other methods of detecting E. coli O157:H7, including PCR are available but they were not employed in this study.

Although there is no information on Shiga-toxin producing E. coli in green iguanas in England, wild rabbits appear to be significant reservoirs of E. coli O157:H7 in that country; 16.2% fecal samples from wild rabbits tested positive for Shiga-toxin producing E. coli  and 6.2% of said fecal samples were confirmed as E. coli O157:H7 (Sciafe and Crook, 2005).  In a study in the United States, using culture, latex agglutination and PCR, 521 fecal samples from wild life species including raccoons (230 samples), deer (141 samples), opossums (25 samples), birds (9 samples), and other species (16 samples) were analyzed; only one (0.19%) wild opossum of the 521 samples  tested positive for E. coli O157 (Renter et al., 2003). E. coli O157:H7 was isolated from 23.4% feral pigs in California, USA. This study confirmed that mammalian wild life species in California, USA may be implicated in cross contamination of vegetables destined for human consumption and therefore constitute an important public health source of pathogenic E. coli (Jay-Russell, 2010). In another study conducted in  three counties in California, USA, 730 samples from various wild life species including raccoons, opossums, striped skunk and tule elk tested negative for E. coliO157:H7 (Mandrell et al., 2010).  Obviously, wildlife species other than rabbits and feral pigs are not common reservoirs of this serotype of Shiga-toxin producing E. coli (Renter et al., 2003).

Green iguanas have however been shown to be significant sources of zoonotic pathogenic Salmonella in Grenada (Sylvester et al., 2013). It is possible that in the current study the prevalence of E. coli O157:H7 is underestimated since PCR and/or serotyping of E. coli were not performed. Due to the frequent human-wildlife interactions, the proximity of human and wildlife habitats as well as the tendency for Grenadians to consume wild meat, there is an urgent need to conduct continued research in wildlife species in Grenada. Future studies should include use of PCR and serotyping.

In this current study antibiotic resistance was detected for seven of the twelve antibiotics used. The highest resistance was 12%, recorded for  amoxicillin/clavalunic acid and streptomycin, followed by ampicillin and nalidixic acid, cefotaxime, tetracycline and trimethoprim/sulfamethoxazole.   It is interesting to note that resistance was found in multiple antimicrobial drug categories/classes including penicillins, tetracyclines, cephalosporins, aminoglycosides, sulfanomide-trimethoprim and quinolones (Table 2).  Additionally, it should be considered significant that one E. coliisolate showed intermediate resistance to two third generation cephalosporins (cefotaxime and ceftazidime) used in treating complicated systemic bacterial infections (Pournaras et al., 2004). This antibiotic resistance pattern differs to that reported in Salmonella isolates from green iguanas, blue land crabs and cane toads in Grenada where no resistance was reported (Sylvester et al., 2013; Peterson et al., 2013; Drake et al., 2012). In a study of 54 E. coliisolates from feral cats in Grenada, no resistance was found in 65% of the isolates tested against 6 drugs. Resistance to amoxicillin-clavulanic acid was found only in 2% of the isolates (Hariharan et al., 2011). The relatively high antibiotic resistance amongst E. coli isolates from green iguanas is of public health concern since these resistance genes may be transferred to other non-pathogenic and/or pathogenic E. coli and other members ofEnterobacteriaceae, including Salmonella in the environment or in the human gut. We propose that future studies onE. coli in wildlife should include determination of minimal inhibitory concentrations of antimicrobial drugs for critical analysis of drug resistance.

Conclusions

In this study E. coli O157:H7 was not detected in green iguanas in Grenada. The antibiotic resistance patterns found in this study are of public health concern since there are ample opportunities for transmission of pathogenic and/or commensal microorganisms from iguanas to humans, particularly because green iguanas are used for human consumption in Grenada.

Acknowledgements

This project was reviewed and approved by The St George’s University Institutional Animal Care and Use Committee (IACUC-10011-R).

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