Methods to Evaluate Colistin Heteroresistance in Acinetobacter baumannii.
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SUBMITTER: Sherman EX
PROVIDER: S-EPMC6637766 | biostudies-literature | 2019
REPOSITORIES: biostudies-literature
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Project description:Multidrug-resistant Acinetobacter baumannii infection has recently emerged as a worldwide clinical problem, and colistin is increasingly being used as a last-resort therapy. Despite its favorable bacterial killing, resistance and heteroresistance (HR) to colistin have been described. The purpose of the present study was to investigate the role of the PmrAB regulatory pathway in laboratory-selected mutants representative of global epidemic strains. From three unrelated A. baumannii clinical strains (sequence types 2, 3, and 20), eight colistin-resistant mutants were selected. Half of the mutants showed HR to colistin according to the reference method (population analysis profiling), whereas the other half exhibited stable resistance. M12I mutation within pmrA and M308R, S144KLAGS, and P170L mutations for pmrB were associated with HR to colistin, while T235I, A226T, and P233S mutations within pmrB were associated with stable resistance. The transcript levels of the pmrCAB operon were upregulated in all the mutants. Compensatory mutations were explored for some mutants. A single mutant (T235I mutant) displayed a compensatory mutation through ISAba1 mobilization within the pmrB gene that was associated with the loss of colistin resistance. The mutant resistance phenotype associated with T235I was partially restored in a trans-complementation assay turning to HR. The level of colistin resistance was correlated with the level of expression of pmrC in the trans-complemented strains. This report shows the role of different mutations in the PmrAB regulatory pathway and warns of the development of colistin HR that could be present but not easily detected through routine testing.
| S-EPMC6125516 | biostudies-other
Project description:In this study, we investigated the colistin heteroresistance patterns in Acinetobacter baumannii isolates. To identify colistin heteroresistance, population analysis profiling was performed for six in vitro colistin-susceptible A. baumannii isolates. Survival rates with and without prior exposure to colistin (at concentrations between 0 and 32 mg/L) were measured in media with and without colistin. Amino acid substitutions were also detected in colonies that survived in media with 4 mg/L colistin without further antibiotic treatment in six A. baumannii isolates. A stability test was also performed to investigate whether colistin resistance is maintained without antibiotic treatment. Although only three isolates showed typical colistin heteroresistance pattern, colistin-resistant populations were identified even without prior exposure to colistin in all A. baumannii isolates. Nearly all colonies of typical colistin-heteroresistant isolates (Type I heteroresistance) that survived after exposure to high colistin concentrations were found to be colistin-resistant, whereas no resistant colonies were identified in the other isolates (Type II heteroresistance). Stability tests showed that most of the surviving populations in media with 4 mg/L colistin without further antibiotic exposure failed to preserve resistance to colistin. Colistin-resistant populations also showed either no change in amino acid sequences, or diverse amino acid substitutions. We identified two types of colistin heteroresistance in A. baumannii isolates. Because Type I colistin-heteroresistant A. baumannii isolates could not be eradicated in vitro by high concentrations of colistin, differentiating two colistin heteroresistance types would be important for the treatment of A. baumannii infections using colistin.
| S-EPMC7534296 | biostudies-literature
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.
| S-EPMC6094062 | biostudies-literature
Project description:Background We investigated the presence of heteroresistance against both tigecycline and colistin in Acinetobacter baumannii and then evaluated the effectiveness of combined antibiotic treatment given the existence of discrete tigecycline- and colistin-resistant subpopulations. Methods We performed population analysis profiling (PAP) to evaluate the degree of composite heteroresistance in A. baumannii isolates, with the extent of this resistance quantified using subsequent antibiotic susceptibility testing. We then evaluated the amino acid sequence of PmrBAC and the relative mRNA expression levels of pmrB. Finally, we investigated the combined antibiotic efficacy of tigecycline and colistin in multiple-heteroresistant isolates using dual PAP and in vitro time-killing assays. Results All tigecycline-heteroresistant A. baumannii isolates, with the exception of one colistin-resistant isolate, were also heteroresistant to colistin. Evaluations of the colistin-resistant subpopulations revealed amino acid alterations in PmrA and PmrB and increased expression of pmrB. All tigecycline-resistant subpopulations were susceptible to colistin, and all colistin-resistant subpopulations were susceptible to tigecycline. Dual PAP analysis using tigecycline and colistin showed no heteroresistance, and in vitro time-killing assays revealed that a combination of these two antibiotics effectively eliminated the bacterial cells. Conclusion Our results suggest that multiple heteroresistance to tigecycline and colistin is highly prevalent among A. baumannii clinical isolates and that these resistant subpopulations exist independently in single multiple heteroresistant isolates. Therefore, our findings may explain the success of combined antibiotic therapies in these infections. Supplementary Information The online version contains supplementary material available at 10.1186/s12929-023-00914-6.
| S-EPMC10245395 | biostudies-literature
Project description:Colistin dependent (CD) isolates are dependent on colistin for optimal growth. Here we aimed to systematically determine the emergence of CD among colistin-heteroresistant carbapenem-resistant Acinetobacter baumannii (CRAB) isolates. We also examined the phenotypic characteristics of CD and the evolution of CD strains to overt resistance. Additionally, we examined whether detection of growth in blood cultures was impaired by CD. Heteroresistant isolates, as determined by population analysis profiling, were exposed to colistin; when the colony count with colistin was significantly higher than without, isolates were suspected to be CD. CD was confirmed by Etest and growth curves. CD strains with colistin minimum inhibitory concentrations > 2 mg/L after growth in colistin-free media were considered colistin-resistant. Of the 65 heteroresistant strains tested, eight became CD after colistin exposure. These strains attained higher colony counts and growth rates with colistin vs. without, and grew adjacent to the colistin Etest strip. CD strains exhibited increased susceptibilities to multiple antibiotics compared to their parent heteroresistant strains. All CD strains tested became colistin-resistant following growth without colistin. CD strains were detected in blood culture bottles, but time to detection was significantly prolonged compared with parent strains, suggesting that CD may lead to delay in detection of CRAB bacteremia.
| S-EPMC8780235 | biostudies-literature
Project description:These data are part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
2015-08-04 | E-ERAD-375 | biostudies-arrayexpress
Project description:Acinetobacter baumannii is an ESKAPE pathogen that rapidly develops resistance to antibiotics and persists for extended periods in the host or on abiotic surfaces. Survival in environmental stress such as phosphate scarcity, represents a clinically significant challenge for nosocomial pathogens. In the face of phosphate starvation, certain bacteria encode adaptive strategies, including the substitution of glycerophospholipids with phosphorus-free lipids. In bacteria, phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin are conserved glycerophospholipids that form lipid bilayers. Here, we demonstrate that in response to phosphate limitation, conserved regulatory mechanisms induce aminolipid production in A. baumannii. Specifically, phosphate limitation induces formation of three lipids, including amine-containing ornithine and lysine aminolipids. We show that phospahte limitation induced transcription of the olsB gene. Mutations that inactivate aminolipid biosynthesis exhibit fitness defects relative to wild type in colistin growth and killing assays.
2024-10-02 | GSE276010 | GEO
Project description:We recently demonstrated a high rate of colistin dependence in Acinetobacter baumannii isolates exposed to colistin in vitro. In the present study, we obtained a colistin-resistant (H08-391R) and colistin-dependent mutant (H08-391D) from a colistin-susceptible parental strain (H08-391). We found that the colistin-dependent mutant converted into a stable colistin-resistant mutant (H08-391D-R) in vitro after four serial passages without colistin. H08-391D and H08-391D-R were both found to harbor defective lipid A, as indicated by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis. Additionally, both contained an ISAba1 insertion in lpxC, which encodes a lipid A biosynthetic enzyme. Further, membrane potential measurements using the fluorescent dye 3,3'-diethyloxacarbocyanine iodide (DiOC2[3]) showed that the membrane potential of H08-391D and H08-391D-R was significantly decreased as compared to that of the parental strain, H08-391. Moreover, these mutant strains exhibited increased susceptibilities to antibiotics other than colistin, which may be attributed to their outer membrane fragility. Such phenomena were identified in other A. baumannii strains (H06-855 and its derivatives). Taken together, our study reveals that the colistin-dependent phenotype is a transient phenotype that allows A. baumannii to survive under colistin pressure, and can transition to the extremely resistant phenotype, even in an antibiotic-free environment.
| S-EPMC5660220 | biostudies-literature
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.
| S-EPMC4462660 | biostudies-literature
Project description:Persisters are phenotypic variants of normal susceptible bacterial populations that survive prolonged exposure to high doses of antibiotics and are responsible for pertinacious infections and post-treatment relapses. Out of the three antibiotics, Acinetobacter baumannii formed the highest percentage of persister cells against rifampicin followed by amikacin and the least against colistin. Colistin-treated cells formed the high levels of reactive oxygen species (ROS) whose quenching with bipyridyl and thiourea led to an increased persister population. Curcumin, a polyphenolic pro-oxidant, significantly decreased persistence against colistin. The quenching of ROS generated by curcumin-colistin combination and the use of resveratrol, an anti-oxidant, with colistin increased the persister population, supporting the significance of ROS in decreased persistence against this combination. The down-regulation of repair genes by this combination in comparison to colistin alone supported the modulation of gene expression in response to ROS and their importance in decreased persistence. Increased membrane permeability by colistin, facilitating the penetration of curcumin into cells and resulting in increased ROS and compromised repair compounded by the decreased efflux of colistin by the inhibition of efflux pumps, may be responsible for enhanced lethality and low persistence. Hence, the curcumin-colistin combination can be another option with anti-persister potential for the control of chronic A. baumannii infections.
| S-EPMC6056455 | biostudies-literature