Dataset Information

Rough-type and loss of the LPS due to lpx genes deletions are associated with colistin resistance in multidrug-resistant clinical Escherichia coli isolates not harbouring mcr genes.

ABSTRACT: The emergence of multidrug-resistant Escherichia coli has become a great challenge in treating nosocomial infections. The polymyxin antibiotic colistin is used as a 'last-line' therapy for such strains, but resistance to colistin is increasingly emerging all over the world. In this study, we investigated lipopolysaccharides (LPS) of colistin-resistant isolates and examined mutations in lpx genes in strains not harbouring mcr genes. We examined 351 clinical E. coli isolates with 38 showing reduced susceptibility to colistin. These isolates were collected from different clinical specimens including blood, urine, and wounds, but no stool. After confirmation of the isolates via a BD Phoenix-100 system (Becton Dickinson, USA), we performed antimicrobial susceptibility tests to characterize the resistance pattern of these isolates to different classes of antibiotics, using the disk diffusion test. The Minimum Inhibitory Concentration (MIC) of colistin was determined using E-test strips. The presence of mobile colistin resistance (mcr-1 and mcr-2) genes was tested for all isolates. LPS (including lipid A) were extracted from all isolates and associated lpx genes analyzed by PCR and sequencing. Among the 38 clinical E. coli isolates with reduced susceptibility to colistin, 52% were resistant to colistin. The MICs of colistin ranged from 0.5 ?g/ml to ?256 ?g/ml. Within the 20 colistin-resistant strains, six isolates carried the mcr-1 gene, but not mcr-2. Heterologous expression of the mcr-1 gene in susceptible E. coli DH5? increased the MIC of colistin by eight-fold. The remaining 14 isolates, were negative for both mcr genes. Six isolates were further negative for LPS production and five showed rough LPS phenotypes. Here we present evidence that loss of LPS or lipid A-deficiency can lead to colistin-resistance in clinical E. coli isolates not harbouring mcr genes.

SUBMITTER: Moosavian M

PROVIDER: S-EPMC7239443 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Rough-type and loss of the LPS due to lpx genes deletions are associated with colistin resistance in multidrug-resistant clinical Escherichia coli isolates not harbouring mcr genes.

Moosavian Mojtaba M Emam Nasrin N Pletzer Daniel D Savari Mohammad M

PloS one 20200520 5

The emergence of multidrug-resistant Escherichia coli has become a great challenge in treating nosocomial infections. The polymyxin antibiotic colistin is used as a 'last-line' therapy for such strains, but resistance to colistin is increasingly emerging all over the world. In this study, we investigated lipopolysaccharides (LPS) of colistin-resistant isolates and examined mutations in lpx genes in strains not harbouring mcr genes. We examined 351 clinical E. coli isolates with 38 showing reduce ...[more]

PMID: 32433662

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Project description:Mobilized colistin resistance (mcr) genes are plasmid-borne genes that confer resistance to colistin, an antibiotic used to treat severe bacterial infections. To date, eight known mcr homologues have been described (mcr-1 to -8). Here, we describe mcr-9, a novel mcr homologue detected during routine in silico screening of sequenced Salmonella genomes for antimicrobial resistance genes. The amino acid sequence of mcr-9, detected in a multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium (S Typhimurium) strain isolated from a human patient in Washington State in 2010, most closely resembled mcr-3, aligning with 64.5% amino acid identity and 99.5% coverage using Translated Nucleotide BLAST (tblastn). The S. Typhimurium strain was tested for phenotypic resistance to colistin and was found to be sensitive at the 2-mg/liter European Committee on Antimicrobial Susceptibility Testing breakpoint under the tested conditions. mcr-9 was cloned in colistin-susceptible Escherichia coli NEB5α under an IPTG (isopropyl-β-d-thiogalactopyranoside)-induced promoter to determine whether it was capable of conferring resistance to colistin when expressed in a heterologous host. Expression of mcr-9 conferred resistance to colistin in E. coli NEB5α at 1, 2, and 2.5 mg/liter colistin, albeit at a lower level than mcr-3 Pairwise comparisons of the predicted protein structures associated with all nine mcr homologues (Mcr-1 to -9) revealed that Mcr-9, Mcr-3, Mcr-4, and Mcr-7 share a high degree of similarity at the structural level. Our results indicate that mcr-9 is capable of conferring phenotypic resistance to colistin in Enterobacteriaceae and should be immediately considered when monitoring plasmid-mediated colistin resistance.IMPORTANCE Colistin is a last-resort antibiotic that is used to treat severe infections caused by MDR and extensively drug-resistant (XDR) bacteria. The World Health Organization (WHO) has designated colistin as a "highest priority critically important antimicrobial for human medicine" (WHO, Critically Important Antimicrobials for Human Medicine, 5th revision, 2017, https://www.who.int/foodsafety/publications/antimicrobials-fifth/en/), as it is often one of the only therapies available for treating serious bacterial infections in critically ill patients. Plasmid-borne mcr genes that confer resistance to colistin pose a threat to public health at an international scale, as they can be transmitted via horizontal gene transfer and have the potential to spread globally. Therefore, the establishment of a complete reference of mcr genes that can be used to screen for plasmid-mediated colistin resistance is essential for developing effective control strategies.

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.

Dataset Information

Rough-type and loss of the LPS due to lpx genes deletions are associated with colistin resistance in multidrug-resistant clinical Escherichia coli isolates not harbouring mcr genes.

Publications

Rough-type and loss of the LPS due to lpx genes deletions are associated with colistin resistance in multidrug-resistant clinical Escherichia coli isolates not harbouring mcr genes.

Similar Datasets

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