Screening and Quantification of the Expression of Antibiotic Resistance Genes in Acinetobacter baumannii with a Microarray

Institut Pasteur, Unité des Agents Antibactériens, 75724 Paris, Cedex 15, France.
Antimicrobial Agents and Chemotherapy (Impact Factor: 4.48). 11/2009; 54(1):333-40. DOI: 10.1128/AAC.01037-09
Source: PubMed


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.

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Available from: Sébastien Coyne
    • "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 . "
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    ABSTRACT: Integrons are genetic elements that are common in bacteria and are hotspots for genome evolution. They facilitate the acquisition and reassembly of gene cassettes encoding a variety of functions, including drug resistance. Despite their importance in clinical settings, the selective forces responsible for the evolution and maintenance of integrons are poorly understood. We present a mathematical model of integron evolution within bacterial populations subject to fluctuating antibiotic exposures. Bacteria carrying a functional integrase that mediates reshuffling of cassette genes and thereby modulates gene expression patterns compete with bacteria without a functional integrase. Our results indicate that for a wide range of parameters, the functional integrase can be stably maintained in the population despite substantial fitness costs. This selective advantage arises because gene-cassette shuffling generates genetic diversity, thus enabling the population to respond rapidly to changing selective pressures. We also show that horizontal gene transfer promotes stable maintenance of the integrase and can also lead to de novo assembly of integrons. Our model generates testable predictions for integron evolution, including loss of functional integrases in stable environments and selection for intermediate gene-shuffling rates in changing environments. Our results highlight the need for experimental studies of integron population biology.The ISME Journal advance online publication, 5 February 2016; doi:10.1038/ismej.2015.222.
    No preview · Article · Feb 2016 · The ISME Journal
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    • "Polymorphisms of the AdeN reported are His111Pro, Ile112Phe, Pro16Lys (Rumbo et al. 2013). Studies have shown that overexpression of AdeFGH is associated with tigecycline resistance (Coyne et al. 2010a). AdeL point mutations, Val139Gly, Thr319Lys, insertion at position 981 of a thymidine leading to 300-and 200-increase in adeG have been described (Coyne et al. 2010b). "
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    ABSTRACT: Acinetobacter baumannii is a pathogen of increasing concern, commonly causing outbreaks in the hospital environment. Of particular concern, A. baumannii strains exhibiting resistance to carbapenems, which were previously considered the treatment of choice for infected patients, have dramatically increased worldwide, leaving a few antibacterial choices. Tigecycline, a broad-spectrum modified minocycline derivative, isconsidered as a last resort drug against multidrug-resistant A. baumannii. Though, resistance to tigecycline has emerged and is growing notably following increasing tigecycline usage. Comparative evaluation of the tigecycline resistance rates reported worldwide is challenging due to the absence of official interpretative criteria for in vitro susceptibility testing and the discrepancies among the different susceptibility methodologies used, with broth microdilution being considered the reference method. Tigecycline resistance is mainly associated with resistance-nodulation-cell division (RND)-type transporters, mainly the AdeABC, AdeFGH and AdeIJK efflux pumps, but other resistance mechanisms have also been implicated. Tigecycline is still an attractive choice for A. baumannii , but further investigations are warranted so that treatment of MDR Α. baumannii could be guided by validated in vitro data.
    Full-text · Article · Nov 2015 · Advances in Experimental Medicine and Biology
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    • "Previous studies that investigated the regulation of AdeABC efflux pumps in A. baumannii primarily focused on the AdeRS TCS, which is located upstream of the adeABC operon and is transcribed in the opposite direction [15]. Several point mutations in adeR or adeS have been proposed as the major cause of AdeABC efflux pump overexpression, including a threonine-to-methionine substitution at position 153 [15], a glycine-to-aspartate mutation at position 30 [24], an alanine-to-valine substitution at position 94 of AdeS [25], or a proline-to-leucine substitution at position 116 of AdeR [15]. However, the effect of AdeR or AdeS mutations on the expression of AdeABC is not always consistent. "
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    ABSTRACT: BackgroundTigecycline resistance in Acinetobacter baumannii is primarily acquired through overexpression of the AdeABC efflux pump. Besides AdeRS, other two-component regulatory systems (TCSs) involving the regulation of this transporter have not been clarified.ResultsIn this study, we found that the TCS genes baeR and baeS are co-transcribed and function as stress responders under high osmotic conditions. The baeSR and adeAB genes showed increased transcription in both the laboratory-induced and clinical tigecycline-resistant strains compared with the wild-type strain. The deletion of baeR in the ATCC 17978 strain led to 67–73% and 68% reduction in adeA and adeB expression, respectively, with a resultant 2-fold decrease in the tigecycline minimal inhibition concentration (MIC). In contrast, the overexpression of baeR resulted in a doubled tigecycline MIC, with a more than 2-fold increase in adeA and adeB expression. The influence of baeR knockout on adeAB gene expression can also be observed in the laboratory-induced tigecycline-resistant strain. A time-kill assay showed that the baeR deletion mutant showed an approximate 1-log10 reduction in colony forming units (CFUs) relative to the wild-type strain when the tigecycline concentration was 0.25 μg/mL throughout the assay period. The wild-type phenotype could be restored by trans-complementation with pWH1266-kan r -baeR. Increasing the tigecycline concentration to 0.5 μg/mL produced an even more marked 4.7-log10 reduction in CFUs of the baeR deletion mutant at 8 h, while only a 2.1-log10 reduction was observed for the wild-type strain.ConclusionsTaken together, these data show for the first time that the BaeSR TCS influences the tigecycline susceptibility of A. baumannii through the positive regulation of the resistance-nodulation-division efflux pump genes adeA and adeB.
    Full-text · Article · May 2014 · BMC Microbiology
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