Development of a DNA Microarray for Enterococcal Species, Virulence, and Antibiotic Resistance Gene Determinations among Isolates from Poultry

Abstract

A DNA microarray (Enteroarray) was designed with probes targeting four species-specific taxonomic identifiers to discriminate among 18 different enterococcal species, while other probes were designed to identify 18 virulence factors and 174 antibiotic resistance genes. In total, 262 genes were utilized for rapid species identification of enterococcal isolates, while characterizing their virulence potential through the simultaneous identification of endogenous antibiotic resistance and virulence genes. Enterococcal isolates from broiler chicken farms were initially identified by using the API 20 Strep system, and the results were compared to those obtained with the taxonomic genes atpA, recA, pheS, and ddl represented on our microarray. Among the 171 isolates studied, five different enterococcal species were identified by using the API 20 Strep system: Enterococcus faecium, E. faecalis, E. durans, E. gallinarum, and E. avium. The Enteroarray detected the same species as API 20 Strep, as well as two more: E. casseliflavus and E. hirae. Species comparisons resulted in 15% (27 isolates) disagreement between the two methods among the five API 20 Strep identifiable species and 24% (42 isolates) disagreement when considering the seven Enteroarray identified species. The species specificity of key antibiotic and virulence genes identified by the Enteroarray were consistent with the literature adding further robustness to the redundant taxonomic probe data. Sequencing of the cpn60 gene further confirmed the complete accuracy of the microarray results. The new Enteroarray should prove to be a useful tool to accurately genotype strains of enterococci and assess their virulence potential.Enterococci are commensal bacteria found in the gastrointestinal tracts of humans, animals, and birds, as well as in soil and water. Sequenced Enterococcus faecalis and E. faecium genomes revealed the presence of virulence factors and mobile and/or exogenously acquired DNA giving some insight on how these bacteria made the transition from a commensal organism to a nosocomial pathogen (21,46,53). Enterococci can infect the endocardium, abdomen, urinary tract, burn or surgical wounds, and numerous other tissues, especially in immunocompromised patients (24, 59). E. faecalis causes the majority of infections, followed by E. faecium (53). Other enterococci such as E.

Characterization of hospital-associated lineages of ampicillin-resistant Enterococcus faecium from clinical cases in dogs and humans

“…Microarray hybridization experiments were performed as previously described (Champagne et al, 2011). This enterococcal virulence microarray was developed (Diarra et al, 2010) at the National Research Council in Montreal and carries 70 taxonomic probes for species identification as well as 15 virulence factors, and 173 antibiotic resistance probes for a total of 262 probes.…”

Aquaculture Can Promote the Presence and Spread of Antibiotic-Resistant Enterococci in Marine Sediments

“…They have been recognized as major agents of nosocomial infections [13], [14] whose treatment is often complicated by antibiotic resistance (AR), either intrinsic and acquired [15]. Acquired AR is mainly due to integration of external genetic material mediated by transposon or plasmid transfer [16], [17].…”

Characterization of Antimicrobial Resistance and Virulence Genotypes of Enterococcus faecalis Recovered from a Pork Processing Plant

“…A custom microarray was used to provide information on the taxonomy and virulence and antimi crobial resistance genes in individual enterococcal isolates. The array containing 70 taxonomic probes (50-mer oligonucleotides), 17 virulence gene probes (70-mer oligonucleotides), and 171 antimicrobial resistance gene probes (70-mer oligonucleotides), for a total of 258 probes, was constructed and validated in previous studies (7,10).…”

“…This bacterium can survive and adapt to environm ental stresses and can serve as a vector for the spread o f virulence and resistance genes to other pathogens. T he results o f several studies indicate the possibility for transfer o f antim icrobial resistance genes to other bacteria in the food environm ent (7,15,17).…”