Dataset Information

An Evaluation of BfmR-Regulated Antimicrobial Resistance in the Extensively Drug Resistant (XDR) Acinetobacter baumannii Strain HUMC1.

ABSTRACT: Acinetobacter baumannii is a problematic pathogen due to its common expression of extensive drug resistance (XDR) and ability to survive in the healthcare environment. These characteristics are mediated, in part, by the signal transduction system BfmR/BfmS. We previously demonstrated, in antimicrobial sensitive clinical isolates, that BfmR conferred increased resistance to meropenem and polymyxin E. In this study, potential mechanisms were informed, in part, by a prior transcriptome analysis of the antimicrobial sensitive isolate AB307-0294, which identified the porins OprB and aquaporin (Omp33-36, MapA) as plausible mediators for resistance to hydrophilic antimicrobials such as meropenem. Studies were then performed in the XDR isolate HUMC1, since delineating resistance mechanisms in this genomic background would be more translationally relevant. In HUMC1 BfmR likewise increased meropenem and polymyxin E resistance and upregulated gene expression of OprB and aquaporin. However, the comparison of HUMC1 with isogenic mutant constructs demonstrated that neither OprB nor aquaporin affected meropenem resistance; polymyxin E susceptibility was also unaffected. Next, we determined whether BfmR-mediated biofilm production affected either meropenem or polymyxin E susceptibilities. Interestingly, biofilm formation increased resistance to polymyxin E, but had little, if any effect on meropenem activity. Additionally, BfmR mediated meropenem resistance, and perhaps polymyxin E resistance, was due to BfmR regulated factors that do not affect biofilm formation. These findings increase our understanding of the mechanisms by which BfmR mediates intrinsic antimicrobial resistance in a clinically relevant XDR isolate and suggest that the efficacy of different classes of antimicrobials may vary under biofilm inducing conditions.

SUBMITTER: Marr CM

PROVIDER: S-EPMC7658413 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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An Evaluation of BfmR-Regulated Antimicrobial Resistance in the Extensively Drug Resistant (XDR) <i>Acinetobacter baumannii</i> Strain HUMC1.

Marr Candace M CM MacDonald Ulrike U Trivedi Grishma G Chakravorty Somnath S Russo Thomas A TA

Frontiers in microbiology 20201029

<i>Acinetobacter baumannii</i> is a problematic pathogen due to its common expression of extensive drug resistance (XDR) and ability to survive in the healthcare environment. These characteristics are mediated, in part, by the signal transduction system BfmR/BfmS. We previously demonstrated, in antimicrobial sensitive clinical isolates, that BfmR conferred increased resistance to meropenem and polymyxin E. In this study, potential mechanisms were informed, in part, by a prior transcriptome analy ...[more]

PMID: 33193275

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Project description:Nosocomial infections caused by extensively drug-resistant (XDR) or Pan-Drug resistant (PDR) Acinetobacter (A.) baumannii have recently increased dramatically creating a medical challenge as therapeutic options became very limited. The aim of our study was to investigate the antibiotic-resistance profiles and evaluate the various combinations of ciprofloxacin (CIP) or levofloxacin (LEV) with antimicrobial agents and non-antimicrobial agents to combat antimicrobial resistance of XDR A. baumannii. A total of 100 (6.25%) A. baumannii clinical isolates were recovered from 1600 clinical specimens collected from hospitalized patients of two major university hospitals in Upper Egypt. Antimicrobial susceptibility tests were carried out according to CLSI guidelines. Antimicrobial susceptibility testing of the respective isolates showed a high percentage of bacterial resistance to 19 antimicrobial agents ranging from 76 to99%. However, a lower percentage of resistance was observed for only colistin (5%) and doxycycline (57%). The isolates were categorized as PDR (2; 2%), XDR (68; 68%), and multi-drug resistant (MDR) (30; 30%). Genotypic analysis using ERIC-PCR on 2 PDR and 32 selected XDR isolates showed that they were not clonal. Combinations of CIP or LEV with antibiotics (including, ampicillin, ceftriaxone, amikacin, or doxycycline) were tested on these A. baumannii non-clonal isolates using standard protocols where fractional inhibitory concentrations (-FICs) were calculated. Results of the respective combinations showed synergism in 23.5%, 17.65%, 32.35%, 17.65% and 26.47%, 8.28%, 14.71%, 26.47%, of the tested isolates, respectively. CIP or LEV combinations with either chlorpromazine (CPZ) 200 μg/ml, propranolol (PR) in two concentrations, 0.5 mg/ml and 1.0 mg/ml or diclofenac (DIC) 4 mg/ml were carried out and the MIC decrease factor (MDF) of each isolate was calculated and results showed synergism in 44%, 50%, 100%, 100% and 94%, 85%, 100%, 100%, of the tested isolates, respectively. In conclusion, combinations of CIP or LEV with CPZ, PR, or DIC showed synergism in most of the selected PDR and XDR A. baumannii clinical isolates. However, these combinations have to be re-evaluated in vivo using appropriate animal models infected by XDR- or PDR- A. baumannii.

Project description:Nosocomial infections with Acinetobacter baumannii are a global problem in intensive care units with high mortality rates. Increasing resistance to first- and second-line antibiotics has forced the use of colistin as last-resort treatment, and increasing development of colistin resistance in A. baumannii has been reported. We evaluated the transcriptional regulator PmrA as potential drug target to restore colistin efficacy in A. baumannii Deletion of pmrA restored colistin susceptibility in 10 of the 12 extensively drug-resistant A. baumannii clinical isolates studied, indicating the importance of PmrA in the drug resistance phenotype. However, two strains remained highly resistant, indicating that PmrA-mediated overexpression of the phosphoethanolamine (PetN) transferase PmrC is not the exclusive colistin resistance mechanism in A. baumannii A detailed genetic characterization revealed a new colistin resistance mechanism mediated by genetic integration of the insertion element ISAbaI upstream of the PmrC homolog EptA (93% identity), leading to its overexpression. We found that eptA was ubiquitously present in clinical strains belonging to the international clone 2, and ISAbaI integration upstream of eptA was required to mediate the colistin-resistant phenotype. In addition, we found a duplicated ISAbaI-eptA cassette in one isolate, indicating that this colistin resistance determinant may be embedded in a mobile genetic element. Our data disprove PmrA as a drug target for adjuvant therapy but highlight the importance of PetN transferase-mediated colistin resistance in clinical strains. We suggest that direct targeting of the homologous PetN transferases PmrC/EptA may have the potential to overcome colistin resistance in A. baumanniiIMPORTANCE The discovery of antibiotics revolutionized modern medicine and enabled us to cure previously deadly bacterial infections. However, a progressive increase in antibiotic resistance rates is a major and global threat for our health care system. Colistin represents one of our last-resort antibiotics that is still active against most Gram-negative bacterial pathogens, but increasing resistance is reported worldwide, in particular due to the plasmid-encoded protein MCR-1 present in pathogens such as Escherichia coli and Klebsiella pneumoniae Here, we showed that colistin resistance in A. baumannii, a top-priority pathogen causing deadly nosocomial infections, is mediated through different avenues that result in increased activity of homologous phosphoethanolamine (PetN) transferases. Considering that MCR-1 is also a PetN transferase, our findings indicate that PetN transferases might be the Achilles heel of superbugs and that direct targeting of them may have the potential to preserve the activity of polymyxin antibiotics.

Project description:Carbapenem-resistant Acinetobacter baumannii (CRAB) is an important opportunistic pathogen linked to a variety of nosocomial infections and hospital outbreaks worldwide. This study aimed at investigating and characterizing a CRAB outbreak at a large tertiary hospital in Lebanon. A total of 41 isolates were collected and analyzed using pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing (WGS) was performed on all the isolates, and long-read PacBio sequencing was used to generate reference genomes. The multilocus sequence types (MLST), repertoire of resistance genes, and virulence factors were determined from the sequencing data. The plasmid content was analyzed both in silico and using the A. baumannii PCR-based replicon typing (AB-PBRT) method. Genome analysis initially revealed two clones, one carrying bla OXA-23 on Tn2006 (ST-1305, ST-195, and ST-218) and another carrying bla OXA-72 on pMAL-1 (ST-502 and ST-2059, a new ST), with the latter having two subclones, as revealed using the Bayesian transmission network. All isolates were extensively drug resistant (XDR). WGS analysis revealed the transmission pathways and demonstrated the diversity of CRAB isolates and mobile genetic elements in this health care setting. Outbreak detection using WGS and immediate implementation of infection control measures contribute to restraining the spread and decreasing mortality.IMPORTANCE Carbapenem-resistant Acinetobacter baumannii (CRAB) has been implicated in hospital outbreaks worldwide. Here, we present a whole-genome-based investigation of an extensively drug-resistant CRAB outbreak rapidly spreading and causing high incidences of mortality at numerous wards of a large tertiary hospital in Lebanon. This is the first study of its kind in the region. Two circulating clones were identified using a combination of molecular typing approaches, short- and long-read sequencing and Bayesian transmission network analysis. One clone carried bla OXA-23 on Tn2006 (ST-1305, ST-195, and ST-218), and another carried bla OXA-72 on a pMAL-1 plasmid (ST-502 and ST-2059, a new ST). A pMAL-2 plasmid was circulating between the two clones. The approaches implemented in this study and the obtained findings facilitate the tracking of outbreak scenarios in Lebanon and the region at large.

Project description:Identification and validation is the first phase of target-based antimicrobial development. BfmR (RstA), a response regulator in a two-component signal transduction system (TCS) in Acinetobacter baumannii, is an intriguing potential antimicrobial target. A unique characteristic of BfmR is that its inhibition would have the dual benefit of significantly decreasing in vivo survival and increasing sensitivity to selected antimicrobials. Studies on the clinically relevant strain AB307-0294 have shown BfmR to be essential in vivo. Here, we demonstrate that this phenotype in strains AB307-0294 and AB908 is mediated, in part, by enabling growth in human ascites fluid and serum. Further, BfmR conferred resistance to complement-mediated bactericidal activity that was independent of capsular polysaccharide. Importantly, BfmR also increased resistance to the clinically important antimicrobials meropenem and colistin. BfmR was highly conserved among A. baumannii strains. The crystal structure of the receiver domain of BfmR was determined, lending insight into putative ligand binding sites. This enabled an in silico ligand binding analysis and a blind docking strategy to assess use as a potential druggable target. Predicted binding hot spots exist at the homodimer interface and the phosphorylation site. These data support pursuing the next step in the development process, which includes determining the degree of inhibition needed to impact growth/survival and the development a BfmR activity assay amenable to high-throughput screening for the identification of inhibitors. Such agents would represent a new class of antimicrobials active against A. baumannii which could be active against other Gram-negative bacilli that possess a TCS with shared homology. IMPORTANCE Increasing antibiotic resistance in bacteria, particularly Gram-negative bacilli, has significantly affected the ability of physicians to treat infections, with resultant increased morbidity, mortality, and health care costs. In fact, some strains of bacteria are resistant to all available antibiotics, such as Acinetobacter baumannii, which is the focus of this report. Therefore, the development of new antibiotics active against these resistant strains is urgently needed. In this study, BfmR is further validated as an intriguing target for a novel class of antibiotics. Successful inactivation of BfmR would confer the multiple benefits of a decreased ability of A. baumannii to survive in human body fluids, increased sensitivity to complement-mediated bactericidal activity and, importantly, increased sensitivity to other antibiotics. Structural studies support the potential for this "druggable" target, as they identify the potential for small-molecule binding at functionally relevant sites. Next-phase high-throughput screening studies utilizing BfmR are warranted.

Dataset Information

An Evaluation of BfmR-Regulated Antimicrobial Resistance in the Extensively Drug Resistant (XDR) Acinetobacter baumannii Strain HUMC1.

Publications

An Evaluation of BfmR-Regulated Antimicrobial Resistance in the Extensively Drug Resistant (XDR) <i>Acinetobacter baumannii</i> Strain HUMC1.

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