Screening and quantification of the expression of antibiotic resistance genes in Acinetobacter baumannii with a microarray

Abstract

An oligonucleotide-based DNA microarray was developed to evaluate expression of genes for efflux pumps in Acinetobacter baumannii and to detect acquired antibiotic resistance determinants. The microarray contained probes for 205 genes, including those for 47 efflux systems, 55 resistance determinants, and 35 housekeeping genes. The microarray was validated by comparative analysis of mutants overexpressing or deficient in the pumps relative to the parental strain. The performance of the microarray was also evaluated using in vitro single-step mutants obtained on various antibiotics. Overexpression, confirmed by quantitative reverse transcriptase PCR, of RND efflux pumps AdeABC, due to a G30D substitution in AdeS in a multidrug-resistant (MDR) strain obtained on gentamicin, and AdeIJK, in two mutants obtained on cefotaxime or tetracycline, was detected. A new efflux pump, AdeFGH, was found to be overexpressed in a mutant obtained on chloramphenicol. Study of MDR clinical isolates, including the AYE strain, whose entire sequence has been determined, indicated overexpression of AdeABC and of the chromosomally encoded cephalosporinase as well as the presence of several acquired resistance genes. The overexpressed and acquired determinants detected by the microarray could account for nearly the entire MDR phenotype of the isolates. The microarray is potentially useful for detection of resistance in A. baumannii and should allow detection of new efflux systems associated with antibiotic resistance.

FIG. 1.

Differential gene expression in the class 1 integron of the AbaR1 island from strain AYE. The ratio was determined by comparing the expression levels of every gene between strains AYE and CIP 70-10. 5′CS and 3′CS, 5′ and 3′ conserved sequences of class 1 integron; *, statistically significant; ND, not determined. Arrows indicate coding sequences and sense of transcription. Bent arrows represent the promoters for the gene cassettes, with IS1999 bringing a strong promoter. Decreased intensity in gray indicates decreased overexpression from bla_VEB-1 to bla_OXA-10.