Microarray-Based Detection of 90 Antibiotic Resistance Genes of Gram-Positive Bacteria

Abstract

A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broadhost-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.

Identification of an African Bacillus anthracis Lineage That Lacks Expression of the Spore Surface-Associated Anthrose-Containing Oligosaccharide

“…Thirty-microliter reaction mixtures were used with a GeneAmp 9700 PCR system (Applied Biosystems). Template DNA for PCR was obtained by cell lysis followed by filtration (15) and was denatured at 94°C for 7 min. Thirty-three amplification cycles of 30 s of denaturation at 94°C, 30 s of annealing at 55°C, and 100 s of extension at 72°C were performed, followed by one cycle of 10 min at 72°C.…”

Genotypic relatedness and characterization of Staphylococcus pseudintermedius associated with post-operative surgical infections in dogs

“…PCRs to detect the mecA gene and the virulence genes SIET, lukS, lukF and Panton-Valentine leukocidin (PVL) were carried out as described previously (Futagawa-Saito et al, 2006;Lina et al, 1999;Perreten et al, 2005;Poulsen et al, 2003). For detection of the mecA gene, only the mecA-specific primer set was used.…”

Outbreak of encephalitic listeriosis in red-legged partridges (Alectoris rufa)

“…Resistance gene testing was performed by using an antimicrobial resistance gene microarray for Gram positive organisms that is available through Alere Technologies (Jena, Germany). Genomic DNA was isolated as described by Perreten et al (2005). Approximately 100 ng of DNA was labelled by a randomly primed polymerization reaction using Sequenase, version 2.0 (USB Corporation, Cleveland, OH, USA) and consisted of three cycles of enzymatic reactions (Bohlander et al, 1992).…”

“…Approximately 100 ng of DNA was labelled by a randomly primed polymerization reaction using Sequenase, version 2.0 (USB Corporation, Cleveland, OH, USA) and consisted of three cycles of enzymatic reactions (Bohlander et al, 1992). This was followed by hybridization of the labelled fragments to the array following the protocol of Perreten et al (2005). The arrays were subsequently scanned and the IconoClust software (Alere TM ) used to identify positive signals.…”