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Evaluation of five susceptibility test methods for detection of tobramycin resistance in a cluster of epidemiologically related Acinetobacter baumannii isolates.

ABSTRACT: Acinetobacter baumannii is a major nosocomial pathogen causing infections in critically ill patients. This organism has acquired the propensity to rapidly develop resistance to most antibiotics. At several hospitals within Cape Town, South Africa, tobramycin and colistin are frequently the only therapeutic options. Vitek2 automated susceptibility testing (AST) is used in the clinical laboratory to determine selected susceptibility profiles. The suspicion of a possible AST-related technical error when testing for susceptibility to tobramycin in A. baumannii precipitated this study. Thirty-nine A. baumannii strains isolated from clinical specimens (June to December 2006) were included in this prospective study. Tobramycin susceptibility testing results obtained by AST, disc diffusion, the epsilometer test (Etest), and agar dilution were compared to those for broth microdilution (BMD), the reference method. The tobramycin susceptibility results revealed errors in 25/39 (64%) isolates (10 very major and 15 minor errors) when AST was compared to BMD, 12/39 (31%) (2 very major and 10 minor errors) when Etest was compared to BMD, 16/39 (41%) (3 very major and 13 minor errors) when disc diffusion was compared to BMD, and 21/39 (54%) (10 very major and 11 minor errors) when agar dilution was compared to BMD. Using PCR, we detected aac(3)-IIa, which is associated with tobramycin resistance, in 21/25 of the discrepant isolates. Molecular typing (using pulsed-field gel electrophoresis and repetitive sequence-based PCR [rep-PCR]) showed that these isolates were genetically related. Clinical laboratories that routinely use the Vitek2 system should consider an alternative testing method for determining susceptibility to tobramycin.

SUBMITTER: Moodley VM

PROVIDER: S-EPMC3719664 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Evaluation of five susceptibility test methods for detection of tobramycin resistance in a cluster of epidemiologically related Acinetobacter baumannii isolates.

Moodley V Mischka VM Oliver Stephen P SP Shankland Iva I Elisha B Gay BG

Journal of clinical microbiology 20130522 8

Acinetobacter baumannii is a major nosocomial pathogen causing infections in critically ill patients. This organism has acquired the propensity to rapidly develop resistance to most antibiotics. At several hospitals within Cape Town, South Africa, tobramycin and colistin are frequently the only therapeutic options. Vitek2 automated susceptibility testing (AST) is used in the clinical laboratory to determine selected susceptibility profiles. The suspicion of a possible AST-related technical error ...[more]

PMID: 23698528

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Project description:Active efflux is regarded as a common mechanism for antibiotic and biocide resistance. However, the role of many drug efflux pumps in biocide resistance in Acinetobacter baumannii remains unknown. Using biocide-resistant A. baumannii clinical isolates, we investigated the incidence of 11 known/putative antimicrobial resistance efflux pump genes (adeB, adeG, adeJ, adeT1, adeT2, amvA, abeD, abeM, qacE, qacEΔ1, and aceI) and triclosan target gene fabI through PCR and DNA sequencing. Reverse transcriptase quantitative PCR was conducted to assess the correlation between the efflux pump gene expression and the reduced susceptibility to triclosan or chlorhexidine. The A. baumannii isolates displayed high levels of reduced susceptibility to triclosan, chlorhexidine, benzalkonium, hydrogen peroxide, and ethanol. Most tested isolates were resistant to multiple antibiotics. Efflux resistance genes were widely distributed and generally expressed in A. baumannii. Although no clear relation was established between efflux pump gene expression and antibiotic resistance or reduced biocide susceptibility, triclosan non-susceptible isolates displayed relatively increased expression of adeB and adeJ whereas chlorhexidine non-susceptible isolates had increased abeM and fabI gene expression. Increased expression of adeJ and abeM was also demonstrated in multiple antibiotic resistant isolates. Exposure of isolates to subinhibitory concentrations of triclosan or chlorhexidine induced gene expression of adeB, adeG, adeJ and fabI, and adeB, respectively. A point mutation in FabI, Gly95Ser, was observed in only one triclosan-resistant isolate. Multiple sequence types with the major clone complex, CC92, were identified in high level triclosan-resistant isolates. Overall, this study showed the high prevalence of antibiotic and biocide resistance as well as the complexity of intertwined resistance mechanisms in clinical isolates of A. baumannii, which highlights the importance of antimicrobial stewardship and resistance surveillance in clinics.

Project description:Gene amplification is believed to play an important role in antibiotic resistance but has been rarely documented in clinical settings because of its unstable nature. We report a rise in MICs from 0.5 to 16 ?g/ml in successive Acinetobacter baumannii isolated over 4 days from a patient being treated with tobramycin for an infection by multidrug-resistant A. baumannii, resulting in therapeutic failure. Isolates were characterized by whole-genome sequencing, real-time and reverse transcriptase PCR, and growth assays to determine the mechanism of tobramycin resistance and its fitness cost. Tobramycin resistance was associated with two amplification events of different chromosomal fragments containing the aphA1 aminoglycoside resistance gene part of transposon Tn6020. The first amplification event involved low amplification (6 to 10 copies) of a large DNA fragment that was unstable and conferred tobramycin MICs of ? 8 ?g/ml. The second event involved moderate (10 to 30 copies) or high (40 to 110 copies) amplification of Tn6020. High copy numbers were associated with tobramycin MICs of 16 ?g/ml, impaired fitness, and genetic instability, whereas lower copy numbers resulted in tobramycin MICs of ?8 ?g/ml and no fitness cost and were stably maintained in vitro. Exposure in vitro to tobramycin of the initial susceptible isolate and of the A. baumannii AB0057 reference strain led to similar aphA1 amplifications and elevated tobramycin MICs. To the best of our knowledge, this is the first report of in vivo development of antibiotic resistance secondary to gene amplifications resulting in therapy failure. IMPORTANCE A combination of whole-genome sequencing and mapping were used to detect an antibiotic resistance mechanism, gene amplification, which has been presumed for a long time to be of major importance but has rarely been reported in clinical settings because of its unstable nature. Two gene amplification events in a patient with an Acinetobacter baumannii infection treated with tobramycin were identified. One gene amplification event led to high levels of resistance and was rapidly reversible, while the second event led to low and more stable resistance since it incurred low fitness cost on the host. Gene amplification, with an associated rise in tobramycin MICs, could be readily reproduced in vitro from initially susceptible strains exposed to increasing concentrations of tobramycin, suggesting that gene amplification in A. baumannii may be a more common mechanism than currently believed. This report underscores the importance of rapid molecular techniques for surveillance of drug resistance.

Dataset Information

Evaluation of five susceptibility test methods for detection of tobramycin resistance in a cluster of epidemiologically related Acinetobacter baumannii isolates.

Publications

Evaluation of five susceptibility test methods for detection of tobramycin resistance in a cluster of epidemiologically related Acinetobacter baumannii isolates.

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