Dataset Information

Methods to Evaluate Colistin Heteroresistance in Acinetobacter baumannii.

ABSTRACT: The nosocomial pathogen Acinetobacter baumannii is a growing threat to public health due to its increasing resistance to antibiotics including the last-line polymyxin, colistin. Heteroresistance to colistin has been described in A. baumannii, wherein a resistant subpopulation of cells coexisting with a majority susceptible subpopulation actively grows in the presence of antibiotic and can cause treatment failure. The shortcomings of diagnostic tests in detecting colistin heteroresistance are especially worrisome as they may lead to clinicians unknowingly prescribing an ineffective antibiotic, leading to increased patient morbidity and mortality.Several techniques can be used to detect heteroresistance, and the purpose of this chapter is to outline effective methods for identifying, quantifying, and analyzing heteroresistance to colistin in A. baumannii. We will highlight the advantages and disadvantages of techniques including population analysis profile (PAP), Etest, and disc diffusion, as well as additional methods to distinguish heteroresistance from other forms of resistance. While the scope of this chapter will focus on colistin heteroresistance in A. baumannii, these techniques can be adapted for the study of heteroresistance to other antibiotics and in other bacteria with slight modifications.

SUBMITTER: Sherman EX

PROVIDER: S-EPMC6637766 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Methods to Evaluate Colistin Heteroresistance in Acinetobacter baumannii.

Sherman Edgar X EX Wozniak Jessie E JE Weiss David S DS

Methods in molecular biology (Clifton, N.J.) 20190101

The nosocomial pathogen Acinetobacter baumannii is a growing threat to public health due to its increasing resistance to antibiotics including the last-line polymyxin, colistin. Heteroresistance to colistin has been described in A. baumannii, wherein a resistant subpopulation of cells coexisting with a majority susceptible subpopulation actively grows in the presence of antibiotic and can cause treatment failure. The shortcomings of diagnostic tests in detecting colistin heteroresistance are esp ...[more]

PMID: 30798542

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Project description:Heteroresistance is a phenomenon where a subpopulation of cells exhibits higher levels of antibiotic resistance than the general population. Analysis of tobramycin resistance in Acinetobacter baumannii AB5075 using Etest strips demonstrated that colonies with increased resistance arose at high frequency within the zone of growth inhibition. The presence of a resistant subpopulation was confirmed by population analysis profiling (PAP). The tobramycin-resistant subpopulation was cross resistant to gentamicin but not amikacin. The increased tobramycin resistance phenotype was highly unstable, and cells reverted to a less resistant population at frequencies of 60 to 90% after growth on nonselective media. Furthermore, the frequency of the resistant subpopulation was not increased by preincubation with subinhibitory concentrations of tobramycin. The tobramycin-resistant subpopulation was shown to replicate during the course of antibiotic treatment, demonstrating that these were not persister cells. In A. baumannii AB5075, a large plasmid (p1AB5075) carries aadB, a 2?-nucleotidyltransferase that confers resistance to both tobramycin and gentamicin but not amikacin. The aadB gene is part of an integron and is carried adjacent to four additional resistance genes that are all flanked by copies of an integrase gene. In isolates with increased resistance, this region was highly amplified in a RecA-dependent manner. However, in a recA mutant, colonies with unstable tobramycin resistance arose by a mechanism that did not involve amplification of this region. These data indicate that tobramycin heteroresistance occurs by at least two mechanisms in A. baumannii, and future studies to determine its effect on patient outcomes are warranted.IMPORTANCEAcinetobacter baumannii has become an important pathogen in hospitals worldwide, where the incidence of these infections has been increasing. A. baumannii infections have become exceedingly difficult to treat due to a rapid increase in the frequency of multidrug- and pan-resistant isolates. This has prompted the World Health Organization to list A. baumannii as the top priority for the research and development of new antibiotics. This study reports for the first time a detailed analysis of aminoglycoside heteroresistance in A. baumannii We define the mechanistic basis for heteroresistance, where the aadB(ant2?)Ia gene encoding an aminoglycoside adenylyltransferase becomes highly amplified in a RecA-dependent manner. Remarkably, this amplification of 20 to 40 copies occurs stochastically in 1/200 cells in the absence of antibiotic selection. In addition, we provide evidence for a second RecA-independent mechanism for aminoglycoside heteroresistance. This study reveals that aminoglycoside resistance in A. baumannii is far more complex than previously realized and has important implications for the use of aminoglycosides in treating A. baumannii infections.

Project description:BackgroundWith an increase in the use of colistin methansulfonate (CMS) to treat carbapenem-resistant Acinetobacter baumannii infections, colistin resistance is emerging.MethodsPatients with infection or colonization due to colistin-resistant A. baumannii were identified at a hospital system in Pennsylvania. Clinical data were collected from electronic medical records. Susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were performed. To investigate the mechanism of colistin resistance, lipid A was subjected to matrix-assisted laser desorption/ionization mass spectrometry.ResultsTwenty patients with colistin-resistant A. baumannii were identified. Ventilator-associated pneumonia was the most common type of infection. Nineteen patients had received intravenous and/or inhaled CMS for treatment of carbapenem-resistant, colistin-susceptible A. baumannii infection prior to identification of colistin-resistant isolates. The 30-day all-cause mortality rate was 30%. The treatment regimen for colistin-resistant A. baumannii infection associated with the lowest mortality rate was a combination of CMS, a carbapenem, and ampicillin-sulbactam. The colistin-susceptible and -resistant isolates from the same patients were highly related by PFGE, but isolates from different patients were not, suggesting evolution of resistance during CMS therapy. By MLST, all isolates belonged to the international clone II, the lineage that is epidemic worldwide. Phosphoethanolamine modification of lipid A was present in all colistin-resistant A. baumannii isolates.ConclusionsColistin-resistant A. baumannii occurred almost exclusively among patients who had received CMS for treatment of carbapenem-resistant, colistin-susceptible A. baumannii infection. Lipid A modification by the addition of phosphoethanolamine accounted for colistin resistance. Susceptibility testing for colistin should be considered for A. baumannii identified from CMS-experienced patients.

Dataset Information

Methods to Evaluate Colistin Heteroresistance in Acinetobacter baumannii.

Publications

Methods to Evaluate Colistin Heteroresistance in Acinetobacter baumannii.

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