Prevalence of Klebsiella oxytoca in Anolis carolensis of Louisiana

Introduction

K lebsiella spp. are gram-negative bacteria in the family Enterobacteriaceae and are found in the environment where they have been identified in water, soil, and on plants (Podschun and Ullman 1998). These bacteria have also been found to colonize mucosal surfaces of both healthy and diseased mammals including humans (Darby et al. 2014). In humans, K. oxytoca is a nosocomial infection responsible for outbreaks in hospitals throughout the world and is responsible for many types of human infections including pneumonia and colitis (Leitner et al. 2015; Vergara-Lopez et al. 2015).

K. oxytoca has also been identified in reptiles. Three percent of wild tuatara (Sphenodon punctatus) in New Zealand harbor this bacterium in their digestive tracts (Gartrell et al. 2007). In addition, ∼20% of garter snakes were positive for K. oxytoca in their oral flora (Goldstein et al. 1981). This bacterium was also identified in 31% of green turtles (Chelonia mydas) from Costa Rica (Santoro et al. 2006).

Reptiles are known to be a potential source for human bacterial infections. Research has shown that in England, 27% of Salmonella cases in children below 5 years of age are reptile associated (Murphy and Oshin 2015). Research in Germany showed that 15 of 19 households with a child suffering from gastroenteritis because of Salmonella had a reptile that tested positive for the same Salmonella serovar (Pees et al. 2013). Green anoles (Anolis carolensis) are a common lizard found throughout the southeastern part of the United States. As they live in proximity to humans, green anoles could be a potential source for exposure to zoonotic bacteria. For this reason, fecal samples were collected from green anoles living in Southwest Louisiana and were analyzed for K. oxytoca by PCR.

Materials and Methods

To test the specificity of the PCR primers, one strain of K.oxytoca and nine strains of non-K. oxytoca (Remel; PML Microbiologicals) were grown overnight in broth and T-streaked onto agar plates. A single colony was isolated from each strain and cultured overnight in LB broth at 37°C.

Forty-two green anoles were captured in Calcasieu Parish located in Southwest Louisiana. Animals that appeared healthy were captured by hand and were housed overnight with minimal human interaction. Fecal samples were collected in the morning and were placed in a microcentrifuge tube at −20°C. All animals were immediately released back into the environment after fecal collection.

Bacteria cultures or whole fecal samples were used for DNA isolation using the E.Z.N.A. Bacterial DNA Isolation Kit (Omega). DNA concentrations were measured using a UV-Spectrophotometer (Model UV-1800; Shimadzu). DNA from bacteria cultures was diluted in distilled water to a concentration of 1 μg/μL, whereas DNA from fecal samples was diluted to a concentration of 2 μg/μL.

PCR primers used in this study recognized the polygalacturonase pehX gene of K.oxytoca and are as follows: forward primer 5′-GATACGGAGTATGCCTTTACGGTG-3′ and reverse primers 5′-TAGCCTTTATCAAGCGGATACTGG-3′ (Chander et al. 2011). PCR amplifications were performed on 1 μg DNA from bacteria cultures and 2 μg DNA from fecal samples in a reaction mixture consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.1% triton X-100, 1 μM of each primer, 0.4 mM dNTPs (Invitrogen), and 1 U Taq Polymerase (GenScript) in a total of 50 μL. Amplifications were performed using a Mastercycler Thermocycler (Eppendorf) with an initial step of 94°C for 1 min followed by 40 cycles of 94°C for 15 s, 55°C for 15 s, 72°C for 30 s, and a final extension step of 72°C for 5 min. Bands were observed by gel electrophoresis using ethidium bromide.

Results

Specificity of the K. oxytoca primers used in this study was shown by Chander et al. (2011) and was further verified on one strain of K. oxytoca and nine strains of non-K. oxytoca. Only the K. oxytoca strain amplified a product of 343 bp, validating the use of these primers for this study.

Sensitivity of the PCR assay was examined on K. oxytoca DNA at decreasing concentrations of 1 ng, 500 pg, 200 pg, and 100 pg. The minimum amount of DNA detectable in this assay was 100 pg. Based on the amount of DNA found in a single K. oxytoca cell, 15,500 cells is the minimum number needed for detection. Of the 42 fecal samples isolated from green anoles, 7 (17%) were positive for K. oxytoca.

Discussion

At present, there is little information available in the literature on the prevalence of K. oxytoca in free-living reptiles. To our knowledge, this is the first study describing the prevalence of K. oxytoca in wild green anoles. In this study, 17% of green anoles were positive for this bacterium. These results are in close agreement with previous studies that reported 20% of garter snakes testing positive for Klebsiella oxytoca (Goldstein et al. 1981).

For this study, DNA was isolated from whole fecal samples rather than culturing the samples overnight to allow for exponential growth of bacteria as seen in most studies of this nature. The fecal samples collected for this study were used to analyze three different strains of pathogenic bacteria. Therefore, samples were not cultured before DNA isolation to assure that growth of one bacteria strain did not prevent the identification of other bacteria strains. Therefore, this assay required a large number of bacteria to be present in fecal samples for detection. Since K. oxytoca was identified in fecal samples of 17% of the anoles tested, this suggests that these animals are harboring this bacterium in large numbers.

K. oxytoca is responsible for infections in multiple regions of the human body, including the respiratory, digestive, and urinary tracts as well as the skin. In addition, some strains of K. oxytoca are resistant to antibiotics, making this a potentially dangerous bacterium for people with weakened immune systems such as the very young and the elderly. Therefore, it is important to have a better understanding of the possible sources of exposure to this bacterium. K. oxytoca is known to be present in soil and water samples, but in this study we have shown that green anoles harbor this bacterium in their digestive tracts, and it is shed in their feces. In Southwest Louisiana, green anoles are ubiquitous, and human exposure to feces from these animals is relatively common. These data prove that free-living green anoles are a potential source for human exposure to K.oxytoca and may be a source for human infections by this bacterium.