Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial resistance and virulence factor genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae

Stephen G. Smith,Fiona Walsh,Niamh M. Cooke,Gary P. Moran,Alasdair Ivens,Fiona J. Cooke,Thomas R. Rogers,John Wain

doi:10.1016/j.ijantimicag.2010.02.011

Abstract

A DNA microarray was developed to detect plasmid-mediated antimicrobial resistance (AR) and virulence factor (VF) genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. The array was validated with the following bacterial species: Escherichiacoli (n=17); Klebsiellapneumoniae (n=3); Enterobacter spp. (n=6); Acinetobacter genospecies 3 (n=1); Acinetobacterbaumannii (n=1); Pseudomonasaeruginosa (n=2); and Stenotrophomonasmaltophilia (n=2). The AR gene profiles of these isolates were identified by polymerase chain reaction (PCR). The DNA microarray consisted of 155 and 133 AR and VF gene probes, respectively. Results were compared with the commercially available Identibac AMR-ve Array Tube. Hybridisation results indicated that there was excellent correlation between PCR and array results for AR and VF genes. Genes conferring resistance to each antibiotic class were identified by the DNA array. Unusual resistance genes were also identified, such as blaSHV-5 in a blaOXA-23-positive carbapenem-resistant A. baumannii. The phylogenetic group of each E. coli isolate was verified by the array. These data demonstrate that it is possible to screen simultaneously for all important classes of mobile AR and VF genes in Enterobacteriaceae and non-Enterobacteriaceae whilst also assigning a correct phylogenetic group to E. coli isolates. Therefore, it is feasible to test clinical Gram-negative bacteria for all known AR genes and to provide important information regarding pathogenicity simultaneously.

Highlights

Levels of antimicrobial resistance in some developed countries are reaching crisis point, severely limiting treatment options for an increasing number of nosocomially acquired infections [1]
We studied two microarray systems compared with conventional multiplex polymerase chain reaction (PCR) methods, which rapidly provide comprehensive information about the genotypic profile of clinical bacterial isolates
Directed at E. coli bloodstream isolates, have highlighted the importance of phylogenetic group analyses in providing key information on mechanisms of pathogenesis and levels of antimicrobial resistance (AR), and in highlighting strains with enhanced invasive potentials as well as aiding epidemiological studies [10,11,12]

Summary

Introduction

Levels of antimicrobial resistance in some developed countries are reaching crisis point, severely limiting treatment options for an increasing number of nosocomially acquired infections [1]. Identification and characterisation of the genes responsible for AR and virulence in Gram-negative pathogens have been typically limited to gene-specific multiplex polymerase chain reaction (PCR) and sequencing. A commercial microarray has become available, the Identibac AMR-veTM Array Tube (Veterinary Laboratories Agency, Addlestone, UK) This is a microtube DNA array that detects up to 58 AR genes of clinical importance in Salmonella spp. and Escherichia coli. The capacity of the Identibac AMR-ve Array Tube to detect all AR genes in these strains was investigated for comparison Results of both microarray systems were validated by PCR amplification and, where necessary, by direct sequencing

Objectives

Methods

Results

Conclusion