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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    • "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.
    BMC Microbiology 05/2014; 14(1):119. DOI:10.1186/1471-2180-14-119 · 2.73 Impact Factor
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    • "When adeIJK is inactivated, the MICs of β-lactams, fluoroquinolones, tetracyclines, tigecycline, lincosamides, rifampicin, chloramphenicol, co-trimoxazole, novobiocin and fusidic acid are decreased, while susceptibility to aminoglycosides is unaffected.8,9 The AdeFGH RND system, also identified in BM4454 by the Courvalin team, confers MDR when overexpressed, but did not contribute to innate resistance to the antibiotics tested.9,10 Inactivation of AdeFGH decreased the MICs of fluoroquinolones, chloramphenicol, trimethoprim, clindamycin, tetracyclines, tigecycline and sulfamethoxazole, but not of aminoglycosides or β-lactams.10 "
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    ABSTRACT: Objectives Overexpression of efflux pumps in Acinetobacter baumannii is a common mechanism of multidrug resistance in this nosocomial pathogen. Increased efflux pump expression is often assumed from MICs of antibiotics and dyes, without measurement of efflux levels. This study describes a safe, rapid and simple 96-well plate assay that measures the accumulation of a fluorescent dye, Hoechst (H) 33342. Methods The growth kinetics of three resistant and three susceptible Singaporean clinical isolates of A. baumannii in the presence of carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and phenylalanine-arginine-β-naphthylamide (PAβN) were studied to determine non-inhibitory concentrations for use in the assay. Accumulation of H33342 was measured in these clinical isolates with and without efflux inhibitors. Accumulation was also measured in an adeB efflux pump deletion mutant and its parental strain to assess the ability of the assay to identify altered efflux in strains lacking efflux pumps. Results were compared with data from accumulation assays with ethidium bromide and norfloxacin. Results Increased accumulation, indicative of reduced efflux, was observed in AB211ΔadeB compared with parental strain AB211. Clinical isolates demonstrated different levels of accumulation of H33342. The addition of both CCCP and PAβN significantly increased the accumulation of H33342. The pattern of norfloxacin accumulation broadly reflected H33342 accumulation. Ethidium bromide showed a different pattern of accumulation in clinical isolates. Conclusions The measurement of the intracellular accumulation of H33342 in real time allowed a comparison of efflux activity between strains of A. baumannii.
    Journal of Antimicrobial Chemotherapy 03/2013; 68(7). DOI:10.1093/jac/dkt052 · 5.31 Impact Factor
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    • "Previous studies including ours have indicated that the sequence of AdeR and AdeS are widely variable in clinical isolates, but several mutations are still recommended for causing overexpression of the AdeABC efflux pump [10], [41], [42]. Most of the publications describe point mutations in the sensor domain (Asp30Gly), in the linker domain (Aln94Val) between the recognition domain and the histidine box, and in the histidine box (Tro153Met) of the AdeS [14], [44]. A mutation (Pro116Leu) in AdeR is also reported to cause adeABC operon overexpression likely due to altering the structural conformation of the regulator [14]. "
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    ABSTRACT: Over-expression of AdeABC efflux pump stimulated continuously by the mutated AdeRS two component system has been found to result in antimicrobial resistance, even tigecycline (TGC) resistance, in multidrug-resistant Acinetobacter baumannii (MRAB). Although the insertion sequence, ISAba1, contributes to one of the AdeRS mutations, the detail mechanism remains unclear. In the present study we collected 130 TGC-resistant isolates from 317 carbapenem resistant MRAB (MRAB-C) isolates, and 38 of them were characterized with ISAba1 insertion in the adeS gene. The relationship between the expression of AdeABC efflux pump and TGC resistant was verified indirectly by successfully reducing TGC resistance with NMP, an efflux pump inhibitor. Further analysis showed that the remaining gene following the ISAba1 insertion was still transcribed to generate a truncated AdeS protein by the Pout promoter on ISAba1 instead of frame shift or pre-termination. Through introducing a series of recombinant adeRS constructs into a adeRS knockout strain, we demonstrated the truncated AdeS protein was constitutively produced and stimulating the expression of AdeABC efflux pump via interaction with AdeR. Our findings suggest a mechanism of antimicrobial resistance induced by an aberrant cytoplasmic sensor derived from an insertion element.
    PLoS ONE 11/2012; 7(11):e49534. DOI:10.1371/journal.pone.0049534 · 3.23 Impact Factor
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