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Dogs and goats as sentinels for environmental lead burden in Caribbean basin West Indian Veterinary Journal 2009, 9 (2) 21-26 Original Article Bacterial Contamination of Leatherback Turtle (Dermochelys coriacea) eggs and sand in nesting chambers at Levera Beach, Grenada, West Indies - a preliminary Study

Ulrike Zieger*¹, Heather Trelease¹, Nicolas Winkler², Vanessa Mathew³, Ravindra N. Sharma³ ¹Anatomy, Physiology & Pharmacology Program, School of Veterinary Medicine, St. George’s University, Grenada, W.I. ²Ocean Spirits Inc., P.O. Box 1373, St. George’s, Grenada, W.I. ³Pathobiology Program, School of Veterinary Medicine, St. George’s University, Grenada, W.I.

*Corresponding author: Tel: 1-473-444-4175 ext 3328 Email: [email protected]

Abstract The pilot study determines the potential role of bacterial contamination of egg surfaces at the time of oviposition and in the sand of the nesting chambers in lowering hatchability. A total of 15 of were isolated from 20 eggs and 17 sand samples of egg nests, with little overlap in the species spectrum between eggs and sand. The most frequent bacteria found on the egg surface were Pseudomonas spp. followed by Citrobacter spp., Enterobacter cloacae and Morganella morganii, whereas from sand samples most frequent isolates were spp. followed by Enterobacter spp. and Pseudomonas spp. All 15 isolated species are considered opportunistic , and could be potential causes for the reported lower hatchability. These pathogens also constitute a public health risk when eggs are consumed by humans. The majority of isolates showed drug resistance, indicating environmental pollution.

Key Words: Leatherback Turtle, turtle eggs, sand, nesting chamber, bacterial isolates, microbial resistance, Grenada

Introduction oil, incidental capture in oceanic fisheries and oceanic pollution1. evera Beach in the north of Grenada, W.I., is a major Lnesting site for leatherback turtles (Dermochelys Levera Beach in the north of the island of Grenada is coriacea) in the Caribbean. Hatching success for of particular significance as a nesting site receiving 200 leatherback turtles is generally expected to be around to 900 nesting activities annually4. Generally, hatching 50%, but Levera Beach has seen a decline to 30-35% success for sea turtle eggs is estimated to be 80%5, but hatching success during the years 2000 to 2008. One for Leatherback Turtles it is typically lower with an potential cause for this decline could be bacterial average of 50%6. Levera Beach has seen a decline in contamination. hatching success during the years 2000 to 20087, and hatching success rates are currently estimated to be The Leatherback Turtle (Dermochelys coriacea) is below 30-35% at Levera Beach (Winkler, unpublished listed by the International Union for Conservation data). Causes for the low rates of hatching success are of Nature (IUCN) Red List (2010)1 as a critically not clear, but degradation of the nesting habitat due to endangered species. It has a worldwide distribution that sand mining and beach front development has been has experienced drastic declines in the Pacific during reported as a potential reason4. Furthermore, changes in the past two decades². The Atlantic has been reported water run-off pattern caused by the nearby construction to hold approximately 70% of the Leatherback Turtle sites could have increased bacterial contamination of population³. Nesting populations in the Caribbean Levera Beach, which may be a contributing factor to islands are comparatively small and stable, and their the low hatching success rate. Bacterial contamination protection becomes the more important as the species was incriminated in Georgia, USA, with egg failure in continues to be threatened by legal and illegal egg eggs8, 9. To date no data exist in harvesting, killing of nesting females for meat and

West Indian Veterinary Journal, 9 (2) December 2009 • 21 Bacterial Contamination of Leatherback Turtle Eggs and Sand in Nesting Chamber

Grenada for Leatherback Turtles to support a similar Antimicrobial susceptibility test claim. The bacterial isolates were tested for antimicrobial The aim of this study was to collect preliminary data susceptibility by the Kirby-Bauer disk diffusion 10 on bacterial contamination of unhatched Leatherback method on Mueller-Hinton agar (Remel). Isolates Turtle eggs and of sand in the nesting chamber at the were subjected to the antimicrobial susceptibility test 11 time of oviposition as a potential cause for the reported and results interpreted as per NCCLS guidelines . high rate of egg loss. Antimicrobial agents used and their concentrations are as follows: (AM) 10 µg, amoxicillin- clavulanic acid (AMC) 30 µg, gentamycin (OM) Materials and Methods 10 µg, sulphamethaxasole-trimethoprim (SXT) 25 µg, enrofloxin (ENO) 30 µg and tetracycline 30 µg. Sampling and bacterial isolation Multidrug resistance was defined as resistance to three or more antimicrobial agents. Due to ethical restrictions when dealing with a critically endangered species, only non-invasive sampling Results techniques were employed. The types of samples collected were as follows: Bacterial isolation of egg surface swabs and sand:

Egg surface swabs Table 1 summarizes the bacterial isolates found in egg Egg surface swabs were collected from 20 turtles surface swabs and in sand samples. All 20 eggs examined between May and July 2009. During oviposition eggs were contaminated by bacteria with 15 samples (75%) were collected by sterile-gloved hand before falling revealing single isolates and 5 samples (25%) revealing into the nesting chamber and their surface swabbed by a mixed isolates. On the 20 eggs, a total of 25 isolates sterile swab. Care was taken to obtain swabs from eggs belonging to 4 genera with 9 species were identified. which were clearly covered in oviductal fluid and devoid The most frequent isolates were Pseudomonas spp (n=l1 of sand contamination. Swabs were placed in Cary Blair or 55%), followed by Citrobacter spp (n=l0 or 50%), transport medium (Becton-Dickinson, Maryland USA), Enterobacter cloacae (n=3 or 15%) and Morganella and kept on ice or 4°C until processing within 36 hours. morganii (n=1 or 5%). Each sample was streaked on blood agar (Columbia agar with 5% sheep blood, Remel, Lennexa, KS, USA) and A corresponding sand sample was taken from 17 of the McConkey agar plates (Remel) and incubated for 24 above 20 turtles’ nesting chambers. All sand samples hr at 37°C. For enrichment, swabs were also incubated were contaminated by bacteria with 14 (82%) revealing separately with Trypticase soy broth (Becton-Dickinson, single isolates and 3 (18%) mixed isolates. The overall Maryland, USA) at 37°C for 24 hour and then cultured 20 isolates belong to 7 genera with 9 species. The on blood agar and McConkey agar. Plates that did not most frequent isolates were Bacillus spp (n=6 or 35%) show any growth were incubated for a further 24 hour and Enterobacter cloacae (n=6 or 35%), followed by period. Bacterial colonies were further purified and Pseudomonas species (n=4 or 24%). identified using standard bacteriological methods10 and confirmed by using API strips (BioMerieux, Marcy- Comparing egg surface sample to sand sample isolates, I’Etoile, France). only 3 of the overall 15 species were isolated from both sources. Sand samples Sand was collected from 17 of the above 20 nesting chambers. A sterile wooden spatula (Fisher Scientific, Pittsburgh, USA) was used to collect a sand sample Table 2 summarizes the frequency of resistance to from the wall of the nesting chamber once the female antimicrobial agents for isolates from egg surface swabs had excavated it and was preparing to lay her eggs. and sand samples. Twelve of the 14 tested bacterial The sand was transferred to sterile containers (Fisher species exhibited resistance to at least 1 antimicrobial Scientific, Pittsburgh, USA) for transport on ice. One agent and 6 species were resistant to 2 antimicrobial gram of sand was mixed with 10 ml of Trypticase soy agents and 4 species showing multi-drug resistance. broth, incubated for 24 hour at 37°C and then cultured The highest frequency of resistance was to ampicillin aerobically on blood agar and McConkey agar. Bacterial and amoxicillin, followed by sulfamethoxazole- colonies were picked up and identified as described for trimethroprim. No resistance was found against egg swabs isolates. enrofloxacin and gentamycin. 22 • West Indian Veterinary Journal, 9 (2) December 2009 Bacterial Contamination of Leatherback Turtle Eggs and Sand in Nesting Chamber

Table 1: Comparison of bacterial isolates from egg surface and sand samples

No. (%) of samples position for : Isolates Egg surface Sand (n=20) (n=17) 7 1 Pseudomonas oryzihabitans 3 2 Pseudomonas species 1 - - 1 Citrobacter youngae 1 - Citrobacter Koserilamalonticus 5 - Citrobacter freudii 3 - Citrobacter species 1 - Enterobacter cloacae 3 6 Morganella morganii 1 - baumanii - 1 Proteus mirabilis - 1 Klebsiella pneumoniae - 1 Salmonella spp. (nonmotile) - 1 Bacillus spp. - 6

Total number of isolates 25 20

Single contamination 15 14

Mixed contamination 5 3

Total number of specimen 20 17

Species of Bacteria No.(%) of samples positive for bacteria from: Egg surface a Egg surface b (n = 20) (n = 17)

Pseudomonas aeruginosa 7(35.0) 1 (5.9)

Bascillus spp. 0(0.0) 6 (35.3 )

a For egg surface swab samples, 15 (75.0%) were contaminated by a single species of bacteria while 5(25.0%) had mixed contamination.

b For sand samples, 14 (70%) and 3 (17.6%) had single and mixed contamination respectively

West Indian Veterinary Journal, 9 (2) December 2009 • 23 Bacterial Contamination of Leatherback Turtle Eggs and Sand in Nesting Chamber

Table 2: Frequency of resistance to antimicrobial agents amongst bacterial isolates from egg surface and sand

Bacterial spp Frequency (%) of resistance to antimicrobial agents amongs isolates from Egg surface Sand Isolates AM AMC TE ENO GM SXT AM AMC TE ENO GM SXT

Pseudomonas aeruginosa 86 86 14 0 0 14 100 100 0 0 0 100 Pseudomonas oryzihabitans 100 100 0 0 0 100 0 50 0 0 0 50 Pseudomonas species 100 100 0 0 0 100 ------Pseudomonas luteola ------100 100 0 0 0 0 Citrobacter youngae 100 100 0 0 0 0 ------Citrobacter koseri/ amalonticus 60 20 0 0 0 20 ------Citrobacter freudii 100 67 0 0 0 0 ------Citrobacter species 0 0 0 0 0 0 ------Enterobacter cloacae 100 100 0 0 0 0 66 66 0 0 0 0 Morganella morganii 100 100 0 0 0 0 ------Acinetobacter baumanii ------100 100 0 0 0 0 Proteus mirabilis ------0 0 100 0 0 0 Klebsiella pneumoniae ------100 0 0 0 0 0 Salmonella sp.(nonmotile) ------0 0 0 0 0 0

Bacillus spp.

Percentage frequency of 84 72 4 0 0 24 50 57 7 0 0 14 resistance to

(AM: Ampicillin; AMC: Amoxicillin with clavulanic acid; TE: Tetracycline; ENO: Enrofloxacin; GM: ; SXT: Sulfamethoxazole- Trimethoprim)

Discussion Furthermore, potentially have been suggested as a possible cause of embryonic death in In this study, the bacterial contamination of Leatherback loggerhead sea turtles8, 9. Thus, the presence of bacteria Turtle eggs and of sand from nesting chambers was isolated from eggs and nesting chambers in our investigated as a potential factor contributing to the present study could be contributing cause of the low surprisingly low hatching success rate reported from hatchability in leatherback turtle eggs at Levera Beach, Levera beach, Grenada. Fifteen species of bacteria Grenada. However, further studies such as the isolation were isolated with little overlap in the species spectrum of bacteria from eggs that failed to hatch are warranted between eggs and sand. to elucidate this hypothesis.

The surfaces of all 20 eggs and 17 sand samples from All bacteria identified in this study, on egg surfaces as nests were contaminated with potentially pathogenic well as in sand samples, can be considered potentially bacteria. It is interesting to note that the bacterial pathogenic. The 15 isolated species have been spectrum differed between egg surfaces and sand associated with stomatitis, pharyngitis, gastrointestinal with only three of the 15 species being isolated from disorders, pneumonia, dermatitis, wound infections both sources. Leatherback turtles therefore introduce and sepsis in a variety of chelonians13. Furthermore, new bacteria into the nesting environment during the same isolates are associated in humans with oviposition. The bacterial contamination from either urinary tract infections, respiratory and gastrointestinal source could possibly have fatal consequences for the disease, wound infections, sepsis, and meningitisl4. In developing embryo, as it has recently been shown that addition, Acinetobacter spp. are known to cause serious bacteria are able to penetrate the eggshell of green nosocomial infections and can cause life-threatening turtle eggs and contaminate both albumen and yolk12. infections in hospital patients14.

24 • West Indian Veterinary Journal, 9 (2) December 2009 Bacterial Contamination of Leatherback Turtle Eggs and Sand in Nesting Chamber

The general hazards of consuming turtle eggs have been Acknowledgements summarized recently15, 16. Turtle eggs are popular food items in Grenada and a high rate of poaching of turtle The financial grant received through the Small eggs (91%) from Levera beach has been reported7. This Research Grant Initiative from St. George’s University may indeed constitute a serious public health hazard is thankfully acknowledged. The authors also thank the if eggs are consumed uncooked, such as seen in the team members and volunteers of the NPO Ocean Spirits cholera-like outbreaks in Costa Rica caused by Vibrio for their assistance during the collection of samples. mimicus after consumption of raw turtle eggs17. Such outbreaks may be further complicated by the fact that the bacteria isolated in this study showed a high degree References of resistance against the tested antimicrobial agents. Eighty-four percent of all isolates from egg surfaces 1. 2010 ICUN. IUCN Red List of Threatened Species. were resistant against ampicillin, followed by amoxillin, Version 2010.4. International Union for Conservation of sulfamethoxazole-trimethroprim, and tetracycline. Nature and Natural Resources; [cited 2010 December]; Available from: www.iucnredlist.org. It is generally accepted that uncontrolled use of 2. Spotila JR, Reina RD, Steyermark AC, Plotkin PT, Paladino FV. Pacific leatherback turtles face extinction. antibiotics in the treatment of humans and animals, Nature. 2000;405:529-530. as well as the widespread use of antibiotics as growth enhancers in livestock has increased the occurrence of 3. Troeng S, Chacon D, Dick B. Possible decline in leatherback turtle Dermochelys coriacea nesting multi-drug resistant bacteria. Multi-drug-resistance is along the coast of Caribbean Central America. Oryx. commonly found in pathogenic and commensal bacteria 2004;38(4):395-403. 18 that inhabit humans and animals . Such bacteria, 4. Maison KA, King R, Lloyd C, Eckert S. Leatherback especially from the gastrointestinal tract then enter the nest distribution and beach erosion pattern at Levera environment via polluted effluents, such as sewerage19, Beach, Grenada, West-Indies. Marine Turtle Newsletter. animal manure or agricultural run-offs18 including waste 2010;127:9-12 from fish farms20. Resistant bacteria can thus enter soil, 5. Miller J. Reproduction in sea turtles. In: Lutz P, Musick fresh water sources as well as the marine environment21. J, editors. The biology of sea turtles. New York, CRC Levera Beach has seen increased human and livestock Press; 1997:51-82. traffic in recent years (Winkler, unpublished data) and 6. Bell BA, Spotila JR, Paladino FV, Reina RD. Low the adjacent beach development site has altered the reproductive success of leatherback turtles, Dermochelys coriacea, is due to high embryonic mortality. Biological water run-off pattern onto Levera Beach, leading to Conservation. 2003;115:131-138. measurable changes in its sand composition4. 7. King RS, Lloyd CB. The current status of leatherback turtles (Dermochelys coriacea) in Grenada. W.I. NOAA As wildlife is typically not treated with antibiotics, Technical Memorandum NMFS SEFSC 582, 2008: our finding of resistant bacterial strains in leatherback p.141. turtles can be interpreted as an indication that they 8. Wyneken J, Burke TJ, Salmon M, Pederson DK. may have been exposed to polluted effluents. Indeed, Egg failure in natural and relocated sea turtle nests. J this has been speculated in fish in Chile22 and in green Herpetology. 1988;22(1):88-96. 12 turtles in Oman in coastal areas. As Leatherback 9. Craven KS, Awong-Taylor J, Griffith L, Bass C, Turtles migrate widely between the Caribbean, Central Muscarella M. Identification of bacterial isolates from and North America, Europe and even to the Arctic unhatched loggerhead (Caretta caretta) sea turtle eggs in Circle23, the contaminating sources may be difficult to Georgia, USA. Marine Turtle Newsletter. 2007;115:9- 11. determine. 10. Quinn PJ, Carter ME, Markey B, Carter GR. Clinical veterinary . London, UK: Wolf/Mosby, In summary, this is the first report on bacterial 1994: p.95-101, p.118-126. contamination of Leatherback Turtle eggs in Grenada. The isolated bacteria may contribute to the reported 11. National Committee for Clinical Laboratory Standards. Performance standard for antimicrobial disk and dilution low hatching success rates in this critically endangered susceptibility tests for bacteria isolated from animals; species and may constitute a potential public health Approved standard. M31-A2. Wayne, PA : NCCLS, hazard. 2002. 12. AI-Bahry S, Mahmoud I, Elshafie A, AI-Harthy A, AI- Ghafri S, AI-Amri I, et al. Bacterial flora and antibiotic resistance from eggs of green turtles (Chelonia mydas): An indication of polluted effluents. Marine Pollution Bulletin. 2009;58 (5):720-725.

West Indian Veterinary Journal, 9 (2) December 2009 • 25 Bacterial Contamination of Leatherback Turtle Eggs and Sand in Nesting Chamber

13. Jacobson ER. Bacterial diseases of reptiles. In: Jacobson ER, editor. Infectious diseases and pathology of reptiles - color atlas and text. Florida: Taylor & Francis Group; 2007:462-480. 14. Pathogenic bacteria database. Graduate School of Medicine and Faculty of Medicine, Kyoto University; 2010 [cited 2010 Dec 30]; Available from: http://bac. hs.med.kyoto-u.ac.ip/alphabete.html. 15. Aguirre AA, Gardner SC, Marsh JC, Delgado SG, Limpus CJ, Nichols WJ. Hazards associated with the consumption of sea turtle meat and eggs: a review for health care workers and the general public. EcoHealth. 2006;3:141-153. Doi: 10.1007/s10393-006-0032-x. 16. Magnino S, Colin P, Dei-Cas E, Madsen M, McLauchlin J, Noeckler K, et al. Biological risks associated with consumption of reptile products. I J Food Micro. 2009;134:163-175. 17. Campos E, Bolanos H, Acuna MT, Diaz G, Matamoros MC, Raventos H, et al. Vibrio mimicus diarrhea following ingestion of raw turtle eggs. Appl Environ Microbiol. 1996;62:1141-1144. 18. Rice EW, Messer JW, Johnson CH, Reasoner DJ. Occurrence of high-level amino glycoside resistance in environmental isolates of enterococci. Appl Environ Microbiol. 1995;61(1):374-376. 19. Al-Bahry SN, Elshafie AE, Al-Busaidy S, Al-Hinai J, Al- Shidi I. Antibiotic resistant Salmonella spp. from human and non-human sources in Oman. Mediterranean Health Journal. 2007;13:49-55. 20. Schmidt AS, Morten SB, Dalsgaard I, Pedersen K, Larsen JL. Occurrence of antimicrobial resistance in fish-pathogenic and environmental bacteria associated with four Danish rainbow trout farms. Appl Environ Microbio1. 2000;66(11):4908-4915. 21. Chee-Sandford GC, Aminov RI, Krapac 11, Guarriges- Jeanjean N, Mackie RI. Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities. Appl Environ Microbio1. 2001;67(4):1494-1502. 22. Miranda CD, Zemelman R. Antibiotic resistant bacteria in fish from the Conception Bay, Chile. Marine Pollution Bulletin. 2001;42(11):1096-1102. 23. Eckert SA, Bagley D, Kubis S, Ehrhart L, Johnson C, Stewart K, et al. Interesting and postnesting movements and foraging habitats of leatherback sea turtles (Dermochelys coriacea) nesting in Florida. Chelonian Conserv Biol. 2006;5:239-248.

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