Investigation of Faecal Carriage of High-Level Gentamicin Resistant Enterococci in Dogs and Cats

Abstract

The emergence and spread of high-level gentamicin resistant (HLGR) enterococci have been a concern due to the eliminatation of bactericidal effect between aminoglycosides and cell-wall-active antimicrobials. Therefore, this study to determine the fecal carriage of (HLGR) enterococci in dogs and cats, to investigate the antimicrobial resistance, resistance mechanisms implicated and virulence genes of the isolates. A total of 465 rectal swab samples from dogs (n=226) and cats (n=239) from three different cities (Istanbul, Ankara and Mersin) of Turkey were used for the analysis of HLGR enterococci. The antimicrobial susceptibilities of HLGR isolates were searched by disc diffusion method, resistance and virulence genes by polymerase chain reaction (PCR) and minimum inhibitory concentration (MIC) values for gentamicin were determined by macrodilution method. HLGR enterococci were detected in 25 (11.1%) dogs and in 28 (11.7%) cats. Based on PCR results, 12 were identified as Enterococcus feacalis and 41 as Enterococcus faecium. All of the isolates showed a MIC value of ≥2048 μg/ml for gentamicin, and except one isolate, the rest of the isolates showed multidrug resistance (MDR) phenotype. None of the isolates displayed vancomycin resistance phenotype. The bifunctional enzyme encoded by aac(6)-Ie-aph(2)-Ia were detected in HLGR isolates as well as other aminoglycoside resistance genes at varying rates. Virulens genes were only detected in 11 (20.8%) E. faecalis isolates with different combinations. However, none of the isolates carried the hly gene. The results showed that both dogs and cats are a potential reservoir for MDR HLGR enterococci which may play an important role in the spread of these nosocomial pathogens.

Keywords: Cat; Dog; Enterococci; Faecal carriage; High Level Gentamicin Resistance; Virulence genes.

Introduction

Enterococci are part of the microbiota of the gastrointesti- nal tract of animals and humans. In the last three decades, enterococci have emerged as one of the leading causes of nosocomial infections due to their increased ability to develop resistance to various classes of antimicrobials and their virulence factors (1). Underlying mechanisms causing antimicrobial resistance in enterococci may be intrinsic to species or may be acquired through mutation of target genes or horizontal transfer of genetic material encoding resistance genes (2).

High level aminoglycoside resistance (HLGR) in enterococci is mediated by bi-functional aminoglycoside- modifying enzyme (AME) with both 6′-acetyltransferase and 2ʺ-phosphotransferase (AAC(6′)-Ie-APH(2′′)-Ia) activities. This enzyme is encoded by aac(6′)-Ie-aph(2′′)-Ia gene, which is part of a conjugative transposon called Tn5281, and frequent localization of this transposon on the plasmid also facilitates rapid spread of resistance gene from cell to cell (3).

Numerous virulence determinants have been identified that play an important role in the pathogenesis of infections caused by enterococci (4). Of these, cytolysin (Cyl), aggregation substance (asa1), enterococcal surface protein (esp), hyaluronidase (hyl), and gelatinase (gelE) have been reported to render enterococci more prone to cause disease and exacerbate disease symptoms (5). Cytolysin is a bacteriocine-type exotoxin with hemolytic activity on erythrocytes, leukocytes and macrophages (6). Gelatinase is an extracellular zinc endopeptidase/protease that is capable of hydrolyzing gelatin, collagen, casein, hemoglobin, and other peptides, which has also been reported to contribute to adhesion and biofilm formation (7). Aggregation substance (AS) is a multifunctional adhesin consisting of closely related surface proteins that promotes bacterial adherence to renal tubular cells (8) and internalization by intestinal cells (9). In addition to mediating adherence and invasion of eukaryotic cells, AS allows donor and recipient strains to form tight aggregates, which permits strains to maintain close contact over a period of sufficient time for conjugative transfer (10). The esp gene participates in the formation of biofilm, which plays an important role in the transfer of genetic material between cells and increase resistance to antibiotics (4).

Pet animals have long been known to play an important role as a reservoir of antimicrobial resistant bacteria. Considering the potential for resistant bacteria to pass from pet animals to humans due to close physical contact, this issue is becoming an even more important public health issue (2, 11).

In Turkey, there are a few studies examining the reservoir status of dogs and cats for the presence of antimicrobial resistant enterococci. These studies aimed mainly either to identify specific antimicrobial-resistant enterococci species (12, 13, 14), or to determine the antimicrobial susceptibilities of isolated enterococci (15, 16). Estimating the true rate of resistant strains to an antimicrobial is closely related to the isolation procedures used. Morever, there are no studies to identify HLGR enterococci in dogs and cats to date. Therefore, it was aimed to investigate the feacal carriage of enterococci positive for HLGR in cats and dogs in different provinces (İstanbul, Ankara and Mersin), to search for mechanisms mediating HLG, tetracycline and erythromycin resistance as well as virulence genes of the isolates.

Materials and methods

Ethical Statement

The study was approved by the Animal Ethical Committee of Hatay Mustafa Kemal University 2018/3-7

Sample collection: Between March 2018 and April 2018, rectal swab samples were collected from 226 dogs and 239 cats from three different provinces (İstanbul, Ankara and Mersin) of Turkey (Figure 1). During the collection of the samples, information related with location, age and gender of the animals was also recorded.  [...]