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.Multidrug-resistant (MDR) strains of Acinetobacter baumannii have emerged in recent decades. This opportunistic pathogen is responsible for severe infections, particularly hospital-acquired pneumonia and bloodstream, urinary tract, and wound infections, and has become of worldwide concern (13). As in other bacterial species, multidrug resistance can be achieved by two mechanisms: (i) horizontal transfer of genetic information and (ii) mutation of endogenous genes. Acquired resistance determinants that are carried by plasmids (18, 28), transposons (23,29), and integrons (33, 43) have been described for Acinetobacter spp. Determination of the genomic sequence of several A. baumannii strains has improved our knowledge of the ways in which A. baumannii can develop antibiotic resistance (1,21,38,45). An 86-kb resistance island, AbaR1, found in strain AYE, contains as many as 25 antibiotic and 20 antiseptic and heavy metal resistance genes (16). Variants of this island are integrated at the same chromosomal locus in a significantly high proportion of MDR strains (37). In addition to these acquired resistance genetic elements, alterations in endogenous functions are involved in resistance, such as overexpression of chromosomally encoded ␤-lactamases ADC and OXA-51-like; loss of porins CarO and Omp33-36 contributing to carbapenem resistance; mutation in the GyrA and ParC fluoroquinolone targets; and overexpression of efflux systems (13).Efflux systems are components of the bacterial membrane that are thought to play a role in homeost...

The evolutionary dynamics of integrons in changing environments

“…With only few exceptions (Bissonnette et al, 1991;Stokes and Hall, 1991;Naas et al, 2001), the gene cassettes themselves are promoterless, so that their expression relies on P C (Cambray et al, 2010). Mechanistically, transcription initiated by P C or by other internal promoters can span several cassette genes, and generally expression of cassette genes decreases with distance from the promoter (Collis and Hall, 1995;Coyne et al, 2010). Consequently, integron gene-cassette dynamics modeled here is also relevant for internal genecassette promoters other than P C .…”

Integrons: Past, Present, and Future

“…sion drops off as cassettes become more distal to the promoter (5,87). This may be why the cassette arrays in clinical class 1 integrons rarely contain more than six cassettes, as any additional cassettes may not be expressed because of their distance from the promoter.…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…AdeFGH was initially identified through a microarray assessment of in vitro-selected multidrug-resistant mutants deficient in AdeABC and AdeIJK (546). This pump mediates acquired MDR and shows a broad substrate profile, including fluoroquinolones and tigecycline (184,547).…”