Detection of genes mediating beta-lactamase production in isolates of enterobacteria recovered from wild pets in Saudi Arabia

Aim: To determine the genetic basis and types of beta-lactamase encountered among enterobacterial isolates of wild pets from the animal exhibit. Materials and Methods: A total of 17 beta-lactamase-producing enterobacteria recovered from fecal samples of wild pet animals were analyzed for a selected beta-lactamase gene by polymerase chain reaction. Results: Molecular analysis identified one or more β-lactamase-encoding genes in 14 enterobacterial isolates as a single or gene combination. The most frequent extended-spectrum β-lactamases types were TEM and CTX-M, and the most common AmpC enzymes were CMY-2 and DHA types. Conclusions: The study is the first in Saudi Arabia, have established the presence of β-lactamase-encoding genes in the fecal isolates of wild pets.


Introduction
Antibiotic-resistant bacteria are extremely important to human health. The production of ß-lactamases is the major mechanism of bacterial resistance to β-lactam antibiotics which are considered the most widely used class of antibiotics against both Gramnegative and Gram-positive bacteria. Resistance to this class of antimicrobial agents is therefore of immense clinical significance.
A major reason for resistance of Enterobacteriaceae to beta-lactam antibiotics is the production of extended-spectrum β-lactamases (ESBLs) and AmpC beta-lactamases, capable of inactivating the effects of broad-spectrum cephalosporins and penicillins [1]. Exposure to ESBL/ AmpC-producing microorganisms can occur through any means, but the hospital has always been thought to be the greatest risk [2]. The occurrence of ESBL/AmpC-producing microorganisms is on the rise globally, with prevalence varying from country to country and within a country from institution to institution [3]. The genes that encode for these enzymes may be plasmid-borne or chromosomally located.
Data from the Arabian Peninsula, including Saudi Arabia, suggested that extended-spectrum and AmpC beta-lactam-resistant bacteria constitute a major problem in nosocomial and community-acquired infections [15,16]. However, there is scarce information on the occurrence and genetic characteristics of β-lactamase-producing bacteria in wild pet animals. Therefore, this study was carried to investigate the occurrence and distribution of beta-lactamase encoding genes within enterobacteria derived from wild pet animals in Saudi Arabia.

Ethical approval
The fecal samples were collected aseptically with adequate precautionary measures to minimize pain and/or discomfort to the animals and carried out in accordance with the Saudi animal welfare laws.

Molecular investigation
Rapid DNA preparation was performed by a boiling technique that includes heating at boiling of an overnight bacterial culture (200 μl) mixed with 800 μl of distilled water, followed by cooling, centrifugation and the supernatant was used as the DNA template for the polymerase chain reaction (PCR).
The presence of genes encoding TEM, SHV, OXA, CTX-M, CMY-2, and DHA type β-lactamases was studied by multiplex PCR using universal primers and conditions previously reported [17,18]. The PCR was conducted in a Thermal Cycler PXE-0.5 (THERMO; Electron Corporation) and the resulting PCR products were subjected to electrophoretic separation in 1.5% agarose gel. Visualization of amplicons was completed by staining with ethidium bromide (Sigma-Aldrich) (1 μg/ml) under UV transluminator and photographed. DNA bands of each amplicon were compared with 100-bp DNA mass marker ( Figure-1a-c). Primers sequence and PCR condition are presented in Table-1.

PCR detection of ß-lactamase encoding genes
A total of 17 beta-lactamase positive enterobacterial strains recovered from the feces of wild pet animals were screened for beta-lactamase (bla)-encoding genes. The PCR screening identified the presence of the beta-lactamase genes encoding TEM, CTX-M, CMY-2, and DHA in 14 of them (Figure-1a-c). None of the isolates were reacted positively for bla OXA and bla SHV . No beta-lactamase genes were identified in the remaining three isolates.
Overall, variety of beta-lactamase genes were found within nine bacterial species isolated from various wild pets species. TEM enzyme was detected in nine isolates of beta-lactamase-producing, respectively, which included 4 isolates of E. coli and single isolate of E. aerogenes, P. mirabilis, C. youngae, and P. vulgaris (Table-2). The CTX-M enzyme was identified in five strains among of beta-lactamase-producing isolates, as a single isolate of E. coli, K. pneumonia, E. cloacae, K. oxytoca and C. freundii (Table-2). Both of CMY-2 and DHA, a plasmid-mediated AmpC beta-lactamases were detected in two different isolate of E. coli (Table-2).

Distribution of bla genes
The ß-lactamase-producing isolates were distributed into two categories, the first harbored only one type of ß-lactamase encoding gene, the second harbored two types (Table-2). Twelve (12/17) of the total beta-lactamase-producing entrerobacteria were harboring only one beta-lactamase encoding gene, including five strains of E. coli and a single isolate of E. cloacae, K. oxytoca, C. youngae, P. vulgaris, C. freundii, P. mirabilis and E. aerogenes.
The bla TEM , a narrow-spectrum ß-lactamase was detected alone in 7 isolates; E. coli (3 isolates) and a single isolate of C. youngae, P. vulgaris, P. mirabilis and E. aerogenes. The bla CTX-M, an extended-spectrum ß-lactamase was detected alone in four isolates; single isolate of K. oxytoca from Yemen linnet feces, E. coli from common kestrel, E. cloacae from rock dove, and C. freundii from African gray parrot (Table-3). The plasmid-mediated ß-lactamases, bla CMY-2 and bla DHA were detected in two different E. coli isolates recovered from Arabian red fox and Hill Mynah, respectively.
A total of two (2/17) of the total beta-lactamase-producing isolates were harboring gene combinations of bla TEM and bla DHA in E. coli recovered from the feces of Hill Mynah and bla TEM and bla CTX-M in K. pneumonia delivered from the feces of baboon monkey.

Discussion
The resistance to beta-lactam and beta-lactamase inhibitors is of great clinical significance in several countries. Resistance to beta-lactam antibiotics is primarily mediated by beta-lactamases production. Many different β-lactamases have been described, but TEM, SHV, OXA, CMY-2, and CTX-M β-lactamases are currently regarded the most common among Enterobacteriaceae spp. [2].
Recently, many studies carried out in different countries describing the prevalence and characteristics of beta-lactamase gene harbored Enterobacteriaceae in wildlife free-living Canada geese in Georgia and North California [19], wild animals in Portugal [8,20], zoo animals in Japan [21], black-headed gulls in the Czech Republic [4] and wild birds and free-range poultry in Bangladesh [22]. Since there seem to be geographical variations in the occurrence of different ESBLs, we describe prevalence and characteristics of ESBL/AmpC-genotypes within enterobacterial isolates from wild pet animals presenting at live animal market in Taif, Western Saudi Arabia.

Prevalence of beta-lactamase genes
The beta-lactamase genes harboring enterobacterial isolates from wild pet animals were detected in     [8,20,21], whereas E. coli is the most prevalent and encountered bla genes among enterobacteria from wild animals.

Determination of the types of bla genes
In this study, PCR screening revealed detection of beta-lactamase encoding genes of TEM, CTX-M, CMY, and DHA. None of the isolates were positive for bla OXA and bla SHV . The remaining three isolates did not show any of the bla genes investigated. Similarly, previous studies also detected many β-lactamase-encoding genes in wild animals [12,14,20,21,22].
A TEM-β-lactamase is a narrow-spectrum beta-lactamase gene, which confers resistance against penicillin's and first-generation cephalosporins [23]. In this study, bla TEM being detected in 7 isolates out of 17 enterobacteria-producing beta-lactamase as a sole mechanism of resistance to beta-lactams and all these isolates showed an ampicillin, cephalothin and or cefuroxime resistance phenotypes. TEM-β-lactamase has been previously detected in fecal isolates from magpies and wild rabbits from West Wales [24], free-living Canada geese in Georgia and North Carolina [19], wild animals in Portugal [20], Zoo animals in Japan [21], black-headed gulls in the Czech Republic [4], yellow-legged gulls in France [5], imported flamingos in Japan [25], gulls population in Sweden [12], migratory and resident population of rooks in Austria [26], seagulls and crows in Bangladesh [27].
Recently, there has been worldwide increase in the incidence of ESBLs [3]. In this study, bla CTX-M , an ESBL-encoding gene, was detected in five isolates of enterobacteria from feces of wild animals. The bla CTX-M has been previously identified in fecal bacteria from wild animals in Portugal [20], masked palm civet in Japan [21], imported flamingos in Japan [25], gulls in Sweden [12], migrating and resident population of rooks in Austria [26].
Furthermore, the plasmid-mediated AmpC genes (bla CMY-2 and bla DHA ), were observed in two of strains of enterobacteria showed a typical AmpCbeta-lactamase resistance phenotype. The presence of AmpC β-lactamases have been found worldwide but are less common than ESBLs [28]. The information on the presence of AmpC producing Enterobacteriaceae in wildlife is scarce. Recently, the bla CMY has been reported previously from jaybird isolates of K. oxytoca in Japan [21], migrating and resident population of rooks in Austria [26]. The bla DHA was the first identified from clinical isolates of Salmonella enteritidis in Saudi Arabia [29]. Recently, in Magnolia, the bla DHA was detected in one E. coli from clinical sources [30].

Analysis of bla genes multiplicity among isolates
As in previous studies, bla-genes in this study were detected within enterobacteria from wild animals either as a single gene loci or as gene combination of two or more gene loci for beta-lactamases [13,21,26].

Conclusions
It is of interest the detection of ESBL/AmpCproducing bacteria in wild animals at pet market. This is the first study, to our knowledge, of enterobacteria harboring β-lactamase genes in wild animals in Saudi Arabia. The fact that these animals often live in close contact with their owners and other people in market make the occurrence of transmission between them even more likely. More studies should be carried out in the future in order to track the variants and evolution of β-lactamase genes compared to those from human isolates.

Authors' Contributions
SAH conceived, designed the study, drafted and revised the manuscript. MYS collected and analyzed samples. Both authors read and approved the final manuscript.