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A novel plasmid-encoded mcr-4.3 gene in a colistin-resistant Acinetobacter baumannii clinical strain.

ABSTRACT: OBJECTIVES:To identify the molecular mechanism of colistin resistance in an MDR Acinetobacter baumannii clinical strain isolated in 2008 from a meningitis case in Brazil. METHODS:Long- and short-read WGS was used to identify colistin resistance genes in A. baumannii strain 597A with a colistin MIC of 64?mg/L. MS was used to analyse lipid A content. mcr was cloned into pET-26b (+) and transformed into Escherichia coli BL21(?DE3)pLysS for analysis. RESULTS:A novel plasmid (pAb-MCR4.3) harbouring mcr-4.3 within a Tn3-like transposon was identified. The A. baumannii 597A lipid A MS spectra showed a main molecular ion peak at m/z=2034, which indicated the addition of phosphoethanolamine to the lipid A structure. E. coli BL21 transformed with pET-26b-mcr-4.3 gained colistin resistance with a colistin MIC of 8?mg/L. CONCLUSIONS:Colistin resistance in A. baumannii 597A was correlated with the presence of a novel plasmid-encoded mcr-4.3 gene.

SUBMITTER: Martins-Sorenson N

PROVIDER: S-EPMC6910164 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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A novel plasmid-encoded mcr-4.3 gene in a colistin-resistant Acinetobacter baumannii clinical strain.

Martins-Sorenson Natacha N Snesrud Erik E Xavier Danilo Elias DE Cacci Luciana Camila LC Iavarone Anthony T AT McGann Patrick P Riley Lee W LW Moreira Beatriz Meurer BM

The Journal of antimicrobial chemotherapy 20200101 1

<h4>Objectives</h4>To identify the molecular mechanism of colistin resistance in an MDR Acinetobacter baumannii clinical strain isolated in 2008 from a meningitis case in Brazil.<h4>Methods</h4>Long- and short-read WGS was used to identify colistin resistance genes in A. baumannii strain 597A with a colistin MIC of 64 mg/L. MS was used to analyse lipid A content. mcr was cloned into pET-26b (+) and transformed into Escherichia coli BL21(λDE3)pLysS for analysis.<h4>Results</h4>A novel plasmid (pA ...[more]

PMID: 31578567

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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.

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:Shewanella species have been identified as progenitors of several clinically important antibiotic resistance genes. The aim of our study was to analyze Shewanella baltica strains isolated from the gut contents of wild Atlantic mackerel (Scomber scombrus) for the presence of both known and novel variants of antibiotic resistance genes (ARGs), using Illumina-based whole-genome sequencing (WGS). Thirty-three S. baltica strains were isolated from Atlantic mackerel collected in the northern North Sea. WGS revealed the presence of several new variants of class C and class D beta-lactamases. Nearly 42% (14/33) of the strains carried the mobile colistin resistance gene mcr-4.3. To understand the genetic context of mcr-4.3, we determined the complete genome sequence of strain 11FHM2, using a combination of Oxford Nanopore- and Illumina-based sequencing. The complete genome sequence is 5,406,724 bp long, with one contig representing a chromosome of 5,068,880 bp and three contigs representing novel plasmids (pSBP1, 194,145 bp; pSBP2_mcr4, 86,727 bp; and pSBP3, 56,972 bp). Plasmid pSBP2_mcr4 contains the mobile colistin resistance gene mcr-4.3, as well as the mercury resistance operon merRPAT. Plasmid pSBP1 carries genes encoding resistance against copper, zinc, chromium, and arsenic. Plasmid pSBP3 does not carry any antibiotic or heavy metal resistance genes. Analysis of the flanking region of mcr-4.3 suggests that a phage integrase may be involved in the mobilization of mcr-4.3 in Shewanella spp. Our results provide insights into the mobile mcr-4.3 present in Shewanella spp. and highlight the importance of the marine environment in the emergence and dissemination of clinically important resistance genes. IMPORTANCE We identified two new plasmids in Shewanella baltica isolated from wild Atlantic mackerel (Scomber scombrus) collected from the northern North Sea, one plasmid carrying the mcr-4.3 gene for colistin resistance and the operon merRPAT for mercury resistance and the other carrying multiple heavy metal resistance genes. The marine environment has been recognized as a source of new resistance genes that are found in human pathogens. Selection pressure from heavy metals is seen in the marine environment, especially associated with human activities, such as waste discharge, mining, and in aquaculture settings. This would help maintain and disseminate these plasmids in the environment. Our study provides insights into the mobilization of colistin resistance genes in Shewanella spp. and highlights the importance of the marine environment in the emergence and dissemination of clinically important antibiotic resistance genes.

Dataset Information

A novel plasmid-encoded mcr-4.3 gene in a colistin-resistant Acinetobacter baumannii clinical strain.

Publications

A novel plasmid-encoded mcr-4.3 gene in a colistin-resistant Acinetobacter baumannii clinical strain.

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