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ARC Journal of Animal and Veterinary Sciences
Volume-2 Issue-4, 2016, Page No: 26-29

Investigation for the Colistin Resistance Genes mcr-1 and mcr-2 in Clinical Enterobacteriaceae Isolates from Cats and Dogs in Switzerland

Sabrina Simmen 1, Katrin Zurfluh 1, Magdalena Nüesch-Inderbinen 1*, Sarah Schmitt 2

1.Institute for Food Safety and Hygiene, University of Zürich, Winterthurerstrasse Zürich, Switzerland.
2.Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Zürich, Switzerland

Citation : Simmen S, Zurfluh K, Inderbinen MN, Schmitt S. Investigation for the Colistin Resistance Genes mcr-1 and mcr-2 in Clinical Enterobacteriaceae Isolates from Cats and Dogs in Switzerland. ARC Journal of Animal and Veterinary Sciences. 2016;2(4):26–29.DOI: dx.doi.org/10.20431/2455-2518.0204004

Copyright : © 2016 Inderbinen MN. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract

Colistin resistant Enterobacteriaceae have emerged in humans, food producing animals and food. The aim of this study was to assess the occurrence of MCR-1 and MCR-2 producers among Enterobacteriaceae isolated from companion animals admitted to the University of Zürich veterinary clinic. A total of 347 isolates (231 from dogs, 116 from cats) were analysed. Thereof, 274 (79%) were from urine and 73 (21%) from surgical sites, abscesses and other sources. All isolates were screened by polymerase chain reaction (PCR) for the presence of the plasmid-mediated colistin resistance genes mcr-1and mcr-2. Phenotypic colistin resistance was screened for by the antibiotic broth microdilution method. None of the isolates tested positive by PCR for the mcr-1 or the mcr-2 gene. Broth microdilution tests revealed MICs of 2mg/L–64 mg/L of colistin for four isolates (two Enterobacter spp., one Klebsiella spp. and one E. coli) from dogs (one from skin exudate, one from a surgical site and two isolates from urine). This study documents the presence of non-transmissible colistin resistance in clinical isolates from dogs and cats and confirms that so far, companion animals in Switzerland do not represent a reservoir for plasmid-mediated colistin resistant Enterobacteriaceae.

Keywords: Colistin; Resistance; Escherichia Coli; Companion Animals

Abbreviations : PCR: Polymerase Chain Reaction, LPS: Lipopolysaccharides, MDR: Multi Drug Resistant,E. coli: Escherichia coli, K. pneumonia: Klebsiella pneumoniae,E. cloacae: Enterobacter cloacae, EUCAST: European Committee on Antimicrobial Susceptibility Testing, MIC: Minimal Inhibitory Concentration , L-Ara4N: 4-Amino-4-deoxy-L-arabinose, PEtN: Phosphoethanolamine


1. Introduction

Polymyxins are cationic polypeptide antibiotics that interact with the lipopolysaccharides (LPS) and phospholipids in the outer membrane of Gram-negative bacteria. In veterinary medicine, colistin (polymyxin E) is used regularly for the treatment of gastrointestinal infections in livestock, whereas polymyxin B is topically applied for the treatment of otitis media and skin infections in small animals [1]. In human medicine, colistin has become one of the last-resort antimicrobials for treating life- threatening infections caused by multidrug resistant (MDR) bacteria, and the emergence of plasmid- mediated polymyxin resistance in Enterobacteriaceae severely compromises its use [2]. Since its first description in China in 2015 [3], the colistin resistance gene mcr-1 has been reported worldwide in isolates from humans, food producing animals and food [4]. Furthermore, a novel variant, mcr-2 was detected recently in livestock associated E. coli in Belgium [5].

A recent report from China shows that MCR-1 producing E. coli can colonize companion animals available in pet markets and can be transferred between companion animals and humans [6]. Hence, in addition to food animals and humans, companion animals can represent a reservoir of colistin- resistant E. coli. The aim of this study was to assess the prevalence rate of MCR-1 and MCR-2 producing Enterobacteriaceae in clinical strains obtained from companion animals admitted to the small animal veterinary clinic of the University of Zürich, Switzerland between February 2012 and July 2016. In addition, phenotypic resistance to colistin among the isolates was determined.


2. Materials and Methods

Strain Collection

A collection of 347 isolates recovered between 2012 and 2016 from 231 dogs and 116 cats presented to the University of Zürich veterinary clinic was available for analysis.

The isolates were obtained from urine (n=274), surgical sites (n=39), abscesses and wounds (n=27), and other sites (n=7). The isolates included E. coli (n=254), Klebsiella pneumoniae (n=32), Enterobacter cloacae (n=29), Proteus mirabilis (n=9), Klebsiellaspp. (n=8), Proteus spp. (n=8), Enterobacteraerogenes (n=2), Proteus vulgaris (n=2), Citrobacter freundii(n=1), Citrobacter koseri (n=1) and Enterobacter sp. (n=1).

Screening for mcr-1 and mcr-2 by PCR

DNA was extracted using a standard heat lysis protocol. All isolates were screened for the mcr-1 and mcr-2 genes using primers (custom synthesized by Microsynth, Balgach, Switzerland) and PCR conditions described previously [3,7]. Positive controls were included.

Antimicrobial Susceptibility Testing

Phenotypic colistin resistance was screened for using the broth microdilution method and a single selected screening concentration of 2mg/L of colistin, which represents the susceptibility breakpoint according to the guidelines of the European committee on antimicrobial susceptibility testing, EUCAST (www.eucast.org). Screening was applied to all isolates except Proteus spp., which possess intrinsic resistance to polymyxins. Isolates with growth in the screening medium were further tested for susceptibility to colistin by broth microdilution, and minimal inhibitory concentrations (MIC) were determined as described by EUCAST (www.eucast.org).


3. Results and Discussion

PCR results for all isolates remained negative. Based on this finding, no evidence for the occurrence of MCR-producing Enterobacteriaceae in companion animals in Switzerland need be established.

Screening for phenotypic resistance to colistin gave rise to seven isolates (2% of all isolates, all from dogs). Thereof, four isolates were resistant to colistin upon susceptibility testing by broth microdilution according to EUCAST. All four isolates originated from dogs (1.7% of the isolates from dogs): one E. cloacae (MIC 64 mg/L) isolated from a skin exudate, one Enterobacter spp. (MIC 64mg/L) from a surgical site (abdominal cavity), one Klebsiella spp. (MIC 64 mg/L) isolated from urine and one E. coli (MIC 2 mg/L) isolated from urine. While non-transmissible polymyxin resistance has been described for a number Gram-negative species, clinical E. coli isolates with this resistance mechanism are reported rarely [8,9], hence, the isolation of polymyxin resistant E. coli from a dog with UTI warrants attention. Although the mechanisms leading to colistin resistance were not investigated in this study, numerous complex mechanisms, predominantly those involving modifications of the outer cell membranes have been documented, including alterations of lipid A with 4-amino-4-deoxy-L-arabinose (L-Ara4N) and phosphoethanolamine (PEtN) [10]. The activation of such mechanisms is triggered by environmental stimuli and the development of polymyxin resistance under therapy with colistin has been observed in several instances of human infections with Klebsiella spp. [11,12]. Thus, it cannot be excluded that the occurrence of the colistin resistant isolates observed in this study is associated with polymyxin exposure. To avoid conditions that could promote the emergence of plasmid-mediated colistin resistance, judicious use of polymyxins in animals is of utmost importance.

Currently, the prevalence of MCR-producing enterobacterial clinical isolates from humans worldwide and in Switzerland is below 1% [13]. However, recent studies indicate a rising trend and point to an ongoing transmission of colistin resistance determinants and/or colistin resistant E. coli isolates from animals to humans [2]. Identification of reservoirs and transmission risks are warranted in order to prevent the spread of MCR producers. Strategies are needed to maintain the low prevalence of MCR producers in humans as well as animals, including screening of isolates from different sources.


4. Conclusion

This study represents the first screening for mcr genes in clinical isolates from companion animals in Switzerland and contributes to a better understanding of the epidemiology of mcr genes and the identification of risks to human and animal health. Although so far, dogs and cats in Switzerland do not represent a reservoir for plasmid-mediated colistin resistant Enterobacteriaceae, contact with companion animals from countries with a high prevalence of antimicrobial resistance, e.g. through international trade with pets or through travel, may be considered a risk with regard to the possible transmission of MCR producing bacteria.


5.Acknowledgements

This work was partly supported by the Swiss Federal Office of Public Health, Division Communicable Diseases.


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