Mobile Colistin Resistance Enzyme MCR-3 Facilitates Bacterial Evasion of Host Phagocytosis.
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ABSTRACT:
SUBMITTER: Yin W
PROVIDER: S-EPMC8456205 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Objectives:To characterize the mobile colistin resistance gene mcr-5 in Aeromonas hydrophila from backyard pigs in rural areas of China. Methods:Pig faecal samples from 194 households were directly tested for the presence of mcr-5 by PCR assay and the phenotypic antimicrobial susceptibility profiles of the mcr-5-positive isolates were determined using the broth dilution method. The genomic location and transferability of mcr-5 were analysed by S1-PFGE with Southern blotting and DNA hybridization, and natural transformation, respectively. One strain isolated from an mcr-5-positive sample was subjected to WGS and the stability of the mcr-5-harbouring plasmid over successive generations was examined by subculturing. Results:One mcr-5-positive A. hydrophila isolate showing resistance, with a colistin MIC of 4?mg/L, was isolated from a backyard pig faecal sample. mcr-5 was located on a 7915?bp plasmid designated pI064-2, which could naturally transform into a colistin-susceptible A. hydrophila strain of porcine origin and mediated colistin resistance in both the original isolate and its transformants. The plasmid backbone (3790?bp) of pI064-2 showed 81% nucleotide sequence identity to the corresponding region of the ColE2-type plasmid pAsa1 from Aeromonas salmonicida, while similar replication primases are widely distributed among aeromonads, Enterobacteriaceae and Pseudomonas species. Conclusions:To the best of our knowledge, this is the first identification of the novel colistin resistance gene mcr-5 in an A. hydrophila isolate from the faeces of a backyard pig. mcr-5 is expected to be able to disseminate among different bacterial species and genera.
| S-EPMC6005042 | biostudies-literature
Project description:Mobile colistin resistance (mcr) genes represent an emerging challenge. Here we describe a novel mcr gene, mcr-10, on an IncFIA plasmid of an Enterobacter roggenkampii clinical strain. mcr-10 has the highest nucleotide identity (79.69%) with mcr-9 and encodes MCR-10 with 82.93% amino acids identical to MCR-9. mcr-10 confers 4-fold increase in colistin MIC (from 1 to 4 mg/L) when cloned into a colistin-susceptible E. roggenkampii strain. By screening GenBank, mcr-10 was found in various Enterobacteriaceae species of countries in four continents, suggesting that this gene has widely spread. MCR-10 shows 79.04% to 83.67% amino acid identity and highly conserved predicted protein structures with chromosomally encoded MCR-like phosphoethanolamine transferases (designated MCR-B here) of various Buttiauxella species. MCR-10, MCR-9 and MCR-B proteins may, therefore, originate from a common ancestor. mcr-10 was adjacent to a site-specific recombinase-encoding gene and was bracketed by IS903 and may be mobilized by site-specific recombination or composite transposon. Our results indicate that mcr-10 is a novel plasmid-borne colistin resistance gene and warrants immediate monitoring and further studies.
| S-EPMC7067168 | biostudies-literature
Project description:Colistin is the last-resort antibiotic against lethal infections with multidrug-resistant bacterial pathogens. A rainbow coalition of mobile colistin resistance (mcr) genes raises global health concerns. Here, we describe the action and mechanism of colistin resistance imparted by MCR-4, a recently-identified member from the broader MCR family. We found that MCR-4 originates from the silenced variant of Shewanella frigidimarina via progressive evolution and forms a phylogenetically-distinct group from the well-studied MCR-1/2 family. Domain-swapping experiments further confirmed that MCR-1 and MCR-4 transmembrane and catalytic domains are not functionally-interchangeable. However, structural and functional analyses demonstrated that MCR-4 possesses a similar PE lipid substrate-recognizable cavity and exploits an almost-identical ping-pong catalysis mechanism. MCR-4 also can alleviate colistin-triggered accumulation of reactive oxygen species (ROS). Taken together, this finding constitutes a functional proof that MCR-4 proceeds in a distinct evolutionary path to fulfill a consistent molecular mechanism, resulting in phenotypic colistin resistance.
| S-EPMC6347640 | biostudies-literature
Project description:The initial report of the mcr-1 (mobile colistin resistance) gene has led to many reports of mcr-1 variants and other mcr genes from different bacterial species originating from human, animal and environmental samples in different geographical locations. Resistance gene nomenclature is complex and unfortunately problems such as different names being used for the same gene/protein or the same name being used for different genes/proteins are not uncommon. Registries exist for some families, such as bla (?-lactamase) genes, but there is as yet no agreed nomenclature scheme for mcr genes. The National Center for Biotechnology Information (NCBI) recently took over assigning bla allele numbers from the longstanding Lahey ?-lactamase website and has agreed to do the same for mcr genes. Here, we propose a nomenclature scheme that we hope will be acceptable to researchers in this area and that will reduce future confusion.
| S-EPMC6148208 | biostudies-literature
Project description:BackgroundDue to the paucity of novel antibiotics, colistin has become a last resort antibiotic for treating multidrug resistant bacteria. Colistin acts by binding the lipid A component of lipopolysaccharides and subsequently disrupting the bacterial membrane. The recently identified plasmid-encoded MCR-1 enzyme is the first transmissible colistin resistance determinant and is a cause for concern for the spread of this resistance trait. MCR-1 is a phosphoethanolamine transferase that catalyzes the addition of phosphoethanolamine to lipid A to decrease colistin affinity.ResultsThe structure of the catalytic domain of MCR-1 at 1.32 Å reveals the active site is similar to that of related phosphoethanolamine transferases.ConclusionsThe putative nucleophile for catalysis, threonine 285, is phosphorylated in cMCR-1 and a zinc is present at a conserved site in addition to three zincs more peripherally located in the active site. As noted for catalytic domains of other phosphoethanolamine transferases, binding sites for the lipid A and phosphatidylethanolamine substrates are not apparent in the cMCR-1 structure, suggesting that they are present in the membrane domain.
| S-EPMC5031297 | biostudies-literature
Project description:Antibiotic resistance is a prevalent problem in public health worldwide. In general, the carbapenem β-lactam antibiotics are considered a final resort against lethal infections by multidrug-resistant bacteria. Colistin is a cationic polypeptide antibiotic and acts as the last line of defense for treatment of carbapenem-resistant bacteria. Very recently, a new plasmid-borne colistin resistance gene, mcr-2, was revealed soon after the discovery of the paradigm gene mcr-1, which has disseminated globally. However, the molecular mechanisms for MCR-2 colistin resistance are poorly understood. Here we show a unique transposon unit that facilitates the acquisition and transfer of mcr-2 Evolutionary analyses suggested that both MCR-2 and MCR-1 might be traced to their cousin phosphoethanolamine (PEA) lipid A transferase from a known polymyxin producer, Paenibacillus Transcriptional analyses showed that the level of mcr-2 transcripts is relatively higher than that of mcr-1 Genetic deletions revealed that the transmembrane regions (TM1 and TM2) of both MCR-1 and MCR-2 are critical for their location and function in bacterial periplasm, and domain swapping indicated that the TM2 is more efficient than TM1. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) confirmed that all four MCR proteins (MCR-1, MCR-2, and two chimeric versions [TM1-MCR-2 and TM2-MCR-1]) can catalyze chemical modification of lipid A moiety anchored on lipopolysaccharide (LPS) with the addition of phosphoethanolamine to the phosphate group at the 4' position of the sugar. Structure-guided site-directed mutagenesis defined an essential 6-residue-requiring zinc-binding/catalytic motif for MCR-2 colistin resistance. The results further our mechanistic understanding of transferable colistin resistance, providing clues to improve clinical therapeutics targeting severe infections by MCR-2-containing pathogens.IMPORTANCE Carbapenem and colistin are the last line of refuge in fighting multidrug-resistant Gram-negative pathogens. MCR-2 is a newly emerging variant of the mobilized colistin resistance protein MCR-1, posing a potential challenge to public health. Here we report transfer of the mcr-2 gene by a unique transposal event and its possible origin. Distribution of MCR-2 in bacterial periplasm is proposed to be a prerequisite for its role in the context of biochemistry and the colistin resistance. We also define the genetic requirement of a zinc-binding/catalytic motif for MCR-2 colistin resistance. This represents a glimpse of transferable colistin resistance by MCR-2.
| S-EPMC5424208 | biostudies-literature
Project description:The rapid increase in carbapenem resistance among gram-negative bacteria has renewed focus on the importance of polymyxin antibiotics (colistin or polymyxin E). However, the recent emergence of plasmid-mediated colistin resistance determinants (mcr-1, -2, -3, -4, -5, -6, and -7), especially mcr-1, in carbapenem-resistant Enterobacteriaceae is a serious threat to global health. Here, we characterized a novel mobile colistin resistance gene, mcr-8, located on a transferrable 95,983-bp IncFII-type plasmid in Klebsiella pneumoniae. The deduced amino-acid sequence of MCR-8 showed 31.08%, 30.26%, 39.96%, 37.85%, 33.51%, 30.43%, and 37.46% identity to MCR-1, MCR-2, MCR-3, MCR-4, MCR-5, MCR-6, and MCR-7, respectively. Functional cloning indicated that the acquisition of the single mcr-8 gene significantly increased resistance to colistin in both Escherichia coli and K. pneumoniae. Notably, the coexistence of mcr-8 and the carbapenemase-encoding gene blaNDM was confirmed in K. pneumoniae isolates of livestock origin. Moreover, BLASTn analysis of mcr-8 revealed that this gene was present in a colistin- and carbapenem-resistant K. pneumoniae strain isolated from the sputum of a patient with pneumonia syndrome in the respiratory intensive care unit of a Chinese hospital in 2016. These findings indicated that mcr-8 has existed for some time and has disseminated among K. pneumoniae of both animal and human origin, further increasing the public health burden of antimicrobial resistance.
| S-EPMC6030107 | biostudies-literature
Project description:The emergence and spread of mobile colistin (COL) resistance (mcr) genes jeopardize the efficacy of COL, a last resort antibiotic for treating deadly infections. COL has been used in livestock for decades globally. Bacteria have mobilized mcr genes (mcr-1 to mcr-9). Mcr-gene-containing bacteria (MGCB) have disseminated by horizontal/lateral transfer into diverse ecosystems, including aquatic, soil, botanical, wildlife, animal environment, and public places. The mcr-1, mcr-2, mcr-3, mcr-5, mcr-7, and mcr-8 have been detected in isolates from and/or directly in environmental samples. These genes are harboured by Escherichia coli, Enterobacter, Klebsiella, Proteus, Salmonella, Citrobacter, Pseudomonas, Acinetobacter, Kluyvera, Aeromonas, Providencia, and Raulotella isolates. Different conjugative and non-conjugative plasmids form the backbones for mcr in these isolates, but mcr have also been integrated into the chromosome of some strains. Insertion sequences (IS) (especially ISApl1) located upstream or downstream of mcr, class 1-3 integrons, and transposons are other drivers of mcr in the environment. Genes encoding multi-/extensive-drug resistance and virulence are often co-located with mcr on plasmids in environmental isolates. Transmission of mcr to/among environmental strains is clonally unrestricted. Contact with the mcr-containing reservoirs, consumption of contaminated animal-/plant-based foods or water, international animal-/plant-based food trades and travel, are routes for transmission of MGCB.
| S-EPMC7036836 | biostudies-literature
Project description:Colistin (polymyxin E) and polymyxin B have been used as last-resort agents for treating infections caused by multidrug-resistant Gram-negative bacteria. However, their efficacy has been challenged by the emergence of the mobile colistin resistance gene mcr-1, which encodes a transmembrane phosphoethanolamine (PEA) transferase enzyme, MCR-1. The enzyme catalyzes the transfer of the cationic PEA moiety of phosphatidylethanolamine (PE) to lipid A, thereby neutralizing the negative charge of lipid A and blocking the binding of positively charged polymyxins. This study aims to facilitate understanding of the mechanism of the MCR-1 enzyme by investigating its active-site sequence requirements. For this purpose, 23 active-site residues of MCR-1 protein were randomized by constructing single-codon randomization libraries. The libraries were individually selected for supporting Escherichia coli cell growth in the presence of colistin or polymyxin B. Deep sequencing of the polymyxin-resistant clones revealed that wild-type residues predominates at 17 active-site residue positions, indicating these residues play critical roles in MCR-1 function. These residues include Zn2+-chelating residues as well as residues that may form a hydrogen bond network with the PEA moiety or make hydrophobic interactions with the acyl chains of PE. Any mutations at these residues significantly decrease polymyxin resistance levels and the PEA transferase activity of the MCR-1 enzyme. Therefore, deep sequencing of the randomization libraries of MCR-1 enzyme identifies active-site residues that are essential for its polymyxin resistance function. Thus, these residues may be utilized as targets to develop inhibitors to circumvent MCR-1-mediated polymyxin resistance. IMPORTANCE Polymyxin antibiotics are used as last-line antibiotics in treating infections caused by multidrug-resistant pathogens. However, widespread use of polymyxins has led to the emergence of resistance. Although multiple mechanisms for resistance exist, that due to mcr-1 is a particular concern, as it can be readily transferred among bacterial pathogens. The mcr-1 gene encodes a transmembrane phosphoethanolamine (PEA) transferase that modifies lipid A to block the binding of polymyxin antibiotics. We utilized random mutagenesis coupled with next-generation sequencing to determine the amino acid sequence requirements of 23 residues in and near the active site of MCR-1. We show that the enzyme has stringent sequence requirements, with 75% of the residues examined being essential for function. Coupled with the finding that these residues are largely conserved among PEA enzymes, the results suggest inhibitors that bind near these sites will broadly inhibit MCR-1 and other enzymes of this class.
| S-EPMC8593676 | biostudies-literature
Project description:Plasmid-borne colistin resistance mediated by mcr-1 may contribute to the dissemination of pan-resistant Gram-negative bacteria. Here, we show that mcr-1 confers resistance to colistin-induced lysis and bacterial cell death, but provides minimal protection from the ability of colistin to disrupt the Gram-negative outer membrane. Indeed, for colistin-resistant strains of Enterobacteriaceae expressing plasmid-borne mcr-1, clinically relevant concentrations of colistin potentiate the action of antibiotics that, by themselves, are not active against Gram-negative bacteria. The result is that several antibiotics, in combination with colistin, display growth-inhibition at levels below their corresponding clinical breakpoints. Furthermore, colistin and clarithromycin combination therapy displays efficacy against mcr-1-positive Klebsiella pneumoniae in murine thigh and bacteremia infection models at clinically relevant doses. Altogether, these data suggest that the use of colistin in combination with antibiotics that are typically active against Gram-positive bacteria poses a viable therapeutic alternative for highly drug-resistant Gram-negative pathogens expressing mcr-1.
| S-EPMC5792607 | biostudies-literature