Mediated Colistin Resistance Research Articles

First report of detection of mcr-1 and virulence genes in avian pathogenic Escherichia coli in the center of Algeria.

Antimicrobial resistance in avian pathogenic Escherichia coli (APEC) represents a major concern in the avian industry worldwide and limited studies have investigated Colistin resistance among APEC in Algeria. Investigate antibiotic resistance, in particular, Colistin, and mediated-Colistin resistance (mcr) genes, as well as the virulence genes in APEC. One hundred E. coli were isolated from poultry suspected of colibacillosis. Antimicrobial susceptibility testing was done on 14 antibiotics by the disk diffusion method. Colistin minimum inhibitory concentration (MIC) was assessed by the broth microdilution method. Using multiplex PCR, mcr genes (mcr-1 to 5) and 7 virulence-related genes were investigated in Colistin-resistant isolates. Results showed high resistance to Tetracycline (99%), Nalidixic acid (92%), Doxycycline (90%), Ampicillin (89%), Ofloxacin (74%), Sulfamethoxazole-Trimethoprim (72%), and Amoxicillin-Clavulanic acid (57%); in addition, 92% of isolates were multidrug resistant. The rate of resistance to Colistin was 27% (27/100) of which 96.3% (26/27) of isolates carried the mcr-1 gene. Twenty-five of the Colistin-resistant isolates (92.59%) had at least three virulence genes. The most frequently isolated virulence genes were: fim H (96.3%) followed by hlyF, iroN, and iss (77.7%, each), iutA and ompT were found in 59.25% and 55.5% of isolates, respectively. The most prevalent combination of virulence factors was hlyF-iss-iroN-iutA-ompT-fimH. This is the first report which highlighted Colistin resistance with the detection of mcr-1 in APEC isolates in the area of study. Colistin resistance and carriage of mcr-1 in virulent and multidrug-resistant isolates of E. coli are alarming and a surveillance program to limit the spread of these pathogens is mandatory.

Molecular epidemiological characteristics of E. coli strains carrying the mcr-1 gene isolatedfrom healthy human feces, domestic animal feces,food and water in Thanh Ha commune, Thanh Liem district, Ha Nam province

Colistin is a critically important antimicrobial for the treatment of carbapenem-resistant gram-negative intestinal infections. E. coli strains carrying the colistin-resistant mediated colistin resistance (mcr-1)gene have been globally distributed, found in humans, animals and the environment with diverse resistance mechanisms, namely intrinsic, mutation, and horizontal gene transfer [1]. Evaluation of the epidemiological characteristics and transmission status of E. coli strains to carry the mcr-1 gene is urgent. Sequencing results of 87 community E. coli strains isolated from healthy human feces and environment showed that the mcr-1 gene of these strains in the study was found on both chromosomes and plasmids, the ratio of mcr-1 plasmid-mediated is much higher than on the chromosome (71.3% vs 26.4%, p<0.001). 100% of E. coli strains that carry the mcr-1 gene were subtype mcr-1.1. The plasmids diversified in size (from <50 kb to >300 kb). There were 7 types of single replicon carrying mcr-1 gene detected from 29 strains, including IncI2 (n=7, 10.9%), IncP (n=7, 10.9%), IncX4 (n=5, 7.8%), IncFIA (n=3, 4.7%), IncHI1B (n=3, 4.7%), IncN (n=1, 1.6%) and IncX1 (n=1, 1.6%), were found in healthy humans feces, animals and household water. There were also 21 different multi-replicons with combinations of IncFIA, IncFIB, IncHI1B, IncHI2, IncN, IncY, IncX1 and IncI2 plasmids, found in 31 strains of E.coli in humans, animals and water. Strains were classified into 56 different sequence types (STs). ST10 (n=10, 11%), ST48 (n=9, 9%) and ST206 (n=8, 8%) were common STs.

Enhancement by pyrazolones of colistin efficacy against mcr-1-expressing E. coli: an in silico and in vitro investigation.

Owing to the emergence of antibiotic resistance, the polymyxin colistin has been recently revived to treat acute, multidrug-resistant Gram-negative bacterial infections. Positively charged colistin binds to negatively charged lipids and damages the outer membrane of Gram-negative bacteria. However, the MCR-1 protein, encoded by the mobile colistin resistance (mcr) gene, is involved in bacterial colistin resistance by catalysing phosphoethanolamine (PEA) transfer onto lipid A, neutralising its negative charge, and thereby reducing its interaction with colistin. Our preliminary results showed that treatment with a reference pyrazolone compound significantly reduced colistin minimal inhibitory concentrations in Escherichia coli expressing mcr-1 mediated colistin resistance (Hanpaibool et al. in ACS Omega, 2023). A docking-MD combination was used in an ensemble-based docking approach to identify further pyrazolone compounds as candidate MCR-1 inhibitors. Docking simulations revealed that 13/28 of the pyrazolone compounds tested are predicted to have lower binding free energies than the reference compound. Four of these were chosen for in vitro testing, with the results demonstrating that all the compounds tested could lower colistin MICs in an E. coli strain carrying the mcr-1 gene. Docking of pyrazolones into the MCR-1 active site reveals residues that are implicated in ligand-protein interactions, particularly E246, T285, H395, H466, and H478, which are located in the MCR-1 active site and which participate in interactions with MCR-1 in ≥ 8/10 of the lowest energy complexes. This study establishes pyrazolone-induced colistin susceptibility in E. coli carrying the mcr-1 gene, providing a method for the development of novel treatments against colistin-resistant bacteria.

Pyrazolones Potentiate Colistin Activity against MCR-1-Producing Resistant Bacteria: Computational and Microbiological Study.

The polymyxin colistin is a last line antibiotic for extensively resistant Gram-negative bacteria. Colistin binding to lipid A disrupts the Gram-negative outer membrane, but mobile colistin resistance (mcr) gene family members confer resistance by catalyzing phosphoethanolamine (PEA) transfer onto lipid A, neutralizing its negative charge to reduce colistin interactions. Multiple mcr isoforms have been identified in clinical and environmental isolates, with mcr-1 being the most widespread and mcr-3 being common in South and East Asia. Preliminary screening revealed that treatment with pyrazolones significantly reduced mcr-1, but not mcr-3, mediated colistin resistance. Molecular dynamics (MD) simulations of the catalytic domains of MCR-1 and a homology model of MCR-3, in different protonation states of active site residues H395/H380 and H478/H463, indicate that the MCR-1 active site has greater water accessibility than MCR-3, but that this is less influenced by changes in protonation. MD-optimized structures of MCR-1 and MCR-3 were used in virtual screening of 20 pyrazolone derivatives. Docking of these into the MCR-1/MCR-3 active sites identifies common residues likely to be involved in protein-ligand interactions, specifically the catalytic threonine (MCR-1 T285, MCR-3 T277) site of PEA addition, as well as differential interactions with adjacent amino acids. Minimal inhibitory concentration assays showed that the pyrazolone with the lowest predicted binding energy (ST3f) restores colistin susceptibility of mcr-1, but not mcr-3, expressing Escherichia coli. Thus, simulations indicate differences in the active site structure between MCR-1 and MCR-3 that may give rise to differences in pyrazolone binding and so relate to differential effects upon producer E. coli. This work identifies pyrazolones as able to restore colistin susceptibility of mcr-1-producing bacteria, laying the foundation for further investigations of their activity as phosphoethanolamine transferase inhibitors as well as of their differential activity toward mcr isoforms.

Evaluation of Plasmid Mediated Colistin Resistance in Colistin Resistant Gram-negative Bacilli

Background: Infections caused by carbapenem-resistant gram-negative bacteria are treated with colonistin as a last resort. However, the increased resistance in recent years is significant for treating multiple drug-resistant infections. Objectives: This study aimed to determine the resistance genes mobile colistin resistance (mcr)-1 and mcr-2 in the colistin-resistant isolates, understand the resistance mechanism, and help with treatment. Methods: Isolates were identified in the Vitek MS automated system. The antibiotic susceptibility was tested with the Vitek 2 Compact automated system. Enteric gram-negative bacilli isolates were stored at -20°C until the molecular study. DNA extraction of colistin-resistant isolates was performed by boiling method. Then, polymerase chain reaction (PCR) optimization was performed using the specific primers, and mcr-1 and mcr-2 genes were investigated by the multiplex PCR method. Results: About 170 enteric gram-negative bacilli isolate were mainly sent from internal medicine (44.7%) and neurology (13.5%) services. According to the species identification, 37.6% of the isolates were Klebsiella pneumonia, and 31.7% were Serratia marcescens. Based on PCR results, mcr-1 and mcr-2 genes were not detected in the isolates. Conclusions: Increased colistin resistance and the worrying discovery of mcr genes require urgent precautions, even though the study did not detect mcr-1 and mcr-2. New studies investigating mcr genes in new isolates are needed to understand the mechanism of resistance and identify resistant isolates.

Genomic characterization of colistin resistance in Klebsiella spp. under the pressure of colistin.

Introduction. Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a serious threat to global public health. Colistin is regarded as the last-resort antibiotic for CRKP infections, but colistin resistance among CRKP is increasingly being reported, making clinical treatment for CRKP infections more difficult.Hypothesis/Gap Statement. The molecular mechanisms of colistin resistance in Klebsiella spp. under the pressure of colistin have not been fully investigated.Aim. We aimed to investigate the phenotypic and genetic variation in two colistin-susceptible Klebsiella spp. strains under selective pressure of colistin.Methodology. One hundred microlitres of overnight cultures of the CRKP clinical strain CRKP12-130 and of ATCC 700603 was spread on five Mueller-Hinton Agar (MHA) plates with colistin concentrations of 2, 4, 8, 16 and 32 µg ml-1, and growth of colonies was observed for five consecutive days. Colonies collected from plates were passaged daily for 10 days on MHA plates without colistin and susceptibility testing of colistin was performed by broth microdilution. Thirty-four colistin-resistant strains randomly selected were submitted to whole genome sequencing (WGS). Transcriptional levels of genes involved in colistin resistance (mgrB, phoP, phoQ, pmrA, pmrB, pmrD, pmrE and pmrK) were measured by quantitative real-time PCR.Results. A total of 114 and 119 colistin-resistant colonies were obtained from CRKP12-130 and ATCC 700603 in this study, among which 16 and 18 colonies were submitted to WGS, respectively. Among these 34 sequenced isolates, mutation in phoQ (13/16, 81.25 %) was the main genetic factor mediating colistin resistance in strains from CRKP12-130, while for strains from ATCC 700603, mutation associated with mgrB (8/18, 44.44 %) was found to be the commonest. Mutation of mgrB led to a significant increase in the MIC for colistin (from 64 to >128 µg ml-1), and a novel mutation C28R in mgrB was first reported in this study.Conclusion. Colistin-resistant Klebsiella spp. could be easily selected under pressure of different concentrations of colistin. Mutations of mgrB, phoP, phoQ and pmrB genes were the main mechanisms leading to chromosomally mediated colistin resistance in Klebsiella spp.

Genomic Characterization of Colistin-Resistant Isolates from the King Fahad Medical City, Kingdom of Saudi Arabia.

Whole-genome sequencing is one of the best ways to investigate resistance mechanisms of clinical isolates as well as to detect and identify circulating multi-drug-resistant (MDR) clones or sub-clones in a given hospital setting. Here, we sequenced 37 isolates of Acinetobacter baumannii, 10 Klebsiella pneumoniae, and 5 Pseudomonas aeruginosa collected from the biobank of the hospital setting of the King Fahad Medical City. Complete phenotypic analyses were performed, including MALDI-TOF identification and antibiotic susceptibility testing. After the genome assembly of raw data, exhaustive genomic analysis was conducted including full resistome determination, genomic SNP (gSNP) analysis, and comparative genomics. Almost all isolates were highly resistant to all tested antibiotics, including carbapenems and colistin. Resistome analysis revealed many antibiotic resistance genes, including those with resistance to β-lactams, aminoglycosides, macrolides, tetracyclines, sulfamids, quinolones, and phenicols. In A. baumannii isolates, the endemic carbapenemase blaOXA-23 gene was detected in 36 of the 37 isolates. Non-synonymous mutations in pmrB were detected in almost all of the isolates and likely mediated colistin resistance. Interestingly, while classical analyses, such as MLST, revealed the predominance of an ST2 clone in A. baumannii isolates, the genomic analysis revealed the presence of five circulating sub-clones and identified several isolate transmissions between patients. In the 10 K. pneumoniae isolates, several resistance genes were identified, and the observed carbapenem resistance was likely mediated by overexpression of the detected extended-spectrum-β-lactamase (ESBL) genes associated with low membrane permeability as few carbapenemase genes were detected with just blaOXA-48 in three isolates. Colistin resistance was mediated either by non-synonymous mutations in the MgrB regulator, PmrA, PmrB, and PhoQ proteins or the presence of the MCR-1 protein. Here, gSNP analysis also revealed the existence of bacterial clones and cases of isolate transmissions between patients. The five analyzed P. aeruginosa isolates were highly resistant to all tested antibiotics, including carbapenems mediated by loss or truncated OprD porin, and colistin resistance was associated with mutations in the genes encoding the PmrA, PmrB, or PhoQ proteins. We demonstrate here the usefulness of whole-genome sequencing to exhaustively investigate the dissemination of MDR isolates at the sub-clone level. Thus, we suggest implementing such an approach to monitor the emergence and spread of new clones or sub-clones, which classical molecular analyses cannot detect. Moreover, we recommend increasing the surveillance of the endemic and problematic colistin resistance mcr-1 gene to avoid extensive dissemination.

Overcoming addition of phosphoethanolamine to lipid A mediated colistin resistance in Acinetobacter baumannii clinical isolates with colistin–sulbactam combination therapy

Overcoming colistin-resistant Acinetobacter baumannii (CoR-AB) has become a major concern due to the lack of effective antibiotics. This study aimed to explore the prevalence of CoR-AB clinical isolates in Thailand, their mechanisms of resistance, and test the efficacy of colistin plus sulbactam against CoR-AB isolates. The colistin resistance rate among carbapenem-resistant A. baumannii was 15.14%. The mcr gene or its variants were not detected in CoR-AB isolates by PCR screening. The lipid A mass spectra of CoR-AB isolates showed the additional [M–H]− ion peak at m/z = 2034 that correlated to the phosphoethanolamine (pEtN) addition to lipid A (N = 27/30). The important amino acid substitutions were found at position S14P, A138T, A227V in PmrB that are associated with overexpression of the pEtN transferase (PmrC) and contributed the pEtN addition. The lipopolysacccharide production genes (lpxACD) were not related to lipid A mass spectra. A colistin plus sulbactam combination exhibited the synergy rate at 86.7% against CoR-AB isolates compare to sulbactam (85.89% resistance) or colistin (15.14% resistance) alone. The excellent synergistic activity of colistin plus sulbactam combination has the potential for the treatment of CoR-AB infections.

Genomic Characterization of Two Escherichia fergusonii Isolates Harboring mcr-1 Gene From Farm Environment.

The prevalence and transmission of mobile colistin resistance (mcr) genes have led to a severe threat to humans and animals. Escherichia fergusonii is an emerging pathogen which is closely related to a variety of diseases. However, the report of mcr genes harboring E. fergusonii is still rare. One study in Brazil reported the E. fergusonii isolates with IncHI2-type plasmids harboring mcr-1. A Chinese study reported two strains carrying mcr-1 gene with the same plasmid type IncI2. Here, we identified two strains of E. fergusonii carrying mcr-1 gene from farm environments with IncX4-type and IncI2-type plasmids, respectively. To our best knowledge, this is the first report about mcr-1 gene located on IncX4-type plasmid in E. fergusonii. We investigate the resistance mechanism of colistin-resistant Escherichia fergusonii strains 6S41-1 and 5ZF15-2-1 and elucidate the genetic context of plasmids carrying mcr-1 genes. In addition, we also investigated chromosomal mutations mediated colistin resistance in these two strains. Species identification was performed using MALDI-TOF MS and 16S rRNA gene sequencing. The detection of mcr-1 gene was determined by PCR and Sanger sequencing. S1-pulsed-field gel electrophoresis (PFGE), Southern blotting, antimicrobial susceptibility testing, conjugation experiments, complete genome sequencing, and core genome analysis were conducted to investigate the characteristics of isolates harboring mcr-1. The mcr-1 genes on two strains were both plasmids encoded and the typical IS26-parA-mcr-1-pap2 cassette was identified in p6S41-1 while a nikA-nikB-mcr-1 locus sites on the conjugative plasmid p5ZF15-2-1. In addition, Core genome analysis reveals that E. fergusonii 6S41-1 and 5ZF15-2-1 have close genetic relationships. The mcr-1 gene is located on conjugative IncI2-type plasmid p5ZF15-2-1, which provides support for its further transmission. In addition, there’s the possibility of mcr-1 spreading to humans through farm environments and thereby threatening public health. Therefore, continuous monitoring and investigations of mcr-1 among Enterobacteriaceae in farm environments are necessary to control the spread.

Mcr Genes Conferring Colistin Resistance in Enterobacterales; a Five Year Overview.

The present review aims to study and detect the global emergence of mcr genes in E. coli, K. pneumoniae and Salmonella spp., isolates from human specimens over the last six years. Nowadays the rise of multidrug-resistant superbugs has made essential the return of drugs that were previously abandoned. A clear example is colistin, which acts against multidrug - resistant gram - negative pathogens, including Enterobacterales. Colistin resistance is an unfortunate fact, with the emergence of mcr genes conferring resistance to colistin in Enterobacterales posing the most recent threat. Literature about mcr genes and their spread in E. coli, K. pneumoniae and Salmonella spp. is cited, focusing on the emergence of mcr genes in human specimens since 2015. The data were taken from the PubMed and Scopus databases. It seems that the mcr-1 gene continues to be the protagonist among the three species. E. coli is the dominant species harbouring mcr genes. Moreover, plasmid - mediated colistin resistance is also conferred upon other species that carry different genes resistant to antibiotics. There are only scarse reports on human Salmonella spp isolates harbouring mcr genes. Finally, the emergence of the mcr-9 gene in all of them is quite remarkable. CONCLUSION: Plasmid - mediated colistin resistance in Enterobacterales is a global issue and has been worsening over the years. The continuous mutations of mcr gene subtypes underline the need for better surveillance, constant investigation and wise use of colistin, especially in countries with high levels of antibiotic resistance.

Colistin Resistance among Human Clinical Isolates from a Tertiary Care Hospital in Nepal

Background Colistin is increasingly being used for treatment of multidrug resistant (MDR) bacterial infections in human. The plasmid mediated mcr-1 gene was detected recently among the poultry in Nepal. Plasmid mediated colistin resistance among human isolates would severely compromise the treatment of MDR bacterial infections.
 Objective To find out the prevalence of colistin resistance among the gram-negative bacteria isolated from clinical specimen.
 Method A descriptive cross-sectional study was conducted in a tertiary care hospital in Kathmandu. Colistin resistance among all the gram negative bacilli (GNB), isolated in a year, from all clinical specimen subjected for aerobic bacterial isolation, was detected by disc diffusion and agar dilution methods. Those with a minimum inhibitory concentration (MIC) > 2 mcg/ml were subjected to polymerase chain reaction for detection of mcr-1 gene.
 Result A total of 1993 aerobic GNB were isolated from clinical samples received from February 2018 to January 2019. By the agar dilution screening method, 2.1% of the GNBs isolated had MIC of colistin > 2 mcg/ml. None of these possessed the mcr-1 gene.
 Conclusion Given the occurrence of increased MIC of colistin in the clinical isolates in our set up, nationwide active surveillance would generate data for Nepal. The study for the presence of other genes (chromosomal or plasmid mediated) responsible for the increased MIC of colistin would further guide the control measures.

The Attenuated Protective Effect of Outer Membrane Vesicles Produced by a mcr-1 Positive Strain on Colistin Sensitive Escherichia coli.

Resistance to colistin, especially mobilized colistin resistance (mcr), is a serious threat to public health since it may catalyze a return of the “pre-antibiotic era”. Outer membrane vesicles (OMVs) play a role in antibiotic resistance in various ways. Currently, how OMVs participate in mcr-1-mediated colistin resistance has not been established. In this study, we showed that both OMVs from the mcr-1 negative and positive Escherichia coli (E. coli) strains conferred dose-dependent protection from colistin. However, OMVs from the mcr-1 positive strain conferred attenuated protection when compared to the OMVs of a mcr-1 negative strain at the same concentration. The attenuated protective effect of OMVs was related to the reduced ability to absorb colistin from the environment, thus promoting the killing of colistin sensitive E. coli strains. Lipid A modified with phosphoethanolamine was presented in the OMVs of the mcr-1 positive E. coli strain and resulted in decreased affinity to colistin and less protection. Meanwhile, E. coli strain carrying the mcr-1 gene packed more unmodified lipid A in OMVs and kept more phosphoethanolamine modified lipid A in the bacterial cells. Our study provides a first glimpse of the role of OMVs in mcr-1 -mediated colistin resistance.

Antibiotic resistance and detection of plasmid mediated colistin resistance mcr-1 gene among Escherichia coli and Klebsiella pneumoniae isolated from clinical samples

BackgroundThe prevalence of antimicrobial resistance (AMR) among Gram-negative bacteria is alarmingly high. Reintroduction of colistin as last resort treatment in the infections caused by drug-resistant Gram-negative bacteria has led to the emergence and spread of colistin resistance. This study was designed to determine the prevalence of drug-resistance among beta-lactamase-producing strains of Escherichia coli and Klebsiella pneumoniae, isolated from the clinical specimens received at a tertiary care centre of Kathmandu, Nepal during the period of March to August, 2019.MethodsA total of 3216 different clinical samples were processed in the Microbiology laboratory of Kathmandu Model Hospital. Gram-negative isolates (E. coli and K. pneumoniae) were processed for antimicrobial susceptibility test (AST) by using modified Kirby-Bauer disc diffusion method. Drug-resistant isolates were further screened for extended-spectrum beta-lactamase (ESBL), metallo-beta-lactamase (MBL), carbapenemase and K. pneumoniae carbapenemase (KPC) production tests. All the suspected enzyme producers were processed for phenotypic confirmatory tests. Colistin resistance was determined by minimum inhibitory concentration (MIC) using agar dilution method. Colistin resistant strains were further screened for plasmid-mediated mcr-1 gene using conventional polymerase chain reaction (PCR).ResultsAmong the total samples processed, 16.4% (529/3216) samples had bacterial growth. A total of 583 bacterial isolates were recovered from 529 clinical samples. Among the total isolates, 78.0% (455/583) isolates were Gram-negative bacteria. The most predominant isolate among Gram-negatives was E. coli (66.4%; 302/455) and K. pneumoniae isolates were 9% (41/455). In AST, colistin, polymyxin B and tigecycline were the most effective antibiotics. The overall prevalence of multidrug-resistance (MDR) among both of the isolates was 58.0% (199/343). In the ESBL testing, 41.1% (n = 141) isolates were confirmed as ESBL-producers. The prevalence of ESBL-producing E. coli was 43% (130/302) whereas that of K. pneumoniae was 26.8% (11/41). Similarly, 12.5% (43/343) of the total isolates, 10.9% (33/302) of E. coli and 24.3% of (10/41) K. pneumoniae were resistant to carbapenem. Among 43 carbapenem resistant isolates, 30.2% (13/43) and 60.5% (26/43) were KPC and MBL-producers respectively. KPC-producers isolates of E. coli and K. pneumoniae were 33.3% (11/33) and 20% (2/10) respectively. Similarly, 63.6% (21/33) of the E. coli and 50% (5/10) of the K. pneumoniae were MBL-producers. In MIC assay, 2.2% (4/179) of E. coli and 10% (2/20) of K. pneumoniae isolates were confirmed as colistin resistant (MIC ≥ 4 µg/ml). Overall, the prevalence of colistin resistance was 3.1% (6/199) and acquisition of mcr-1 was 16.6% (3/18) among the E. coli isolates.ConclusionHigh prevalence of drug-resistance in our study is indicative of a deteriorating situation of AMR. Moreover, significant prevalence of resistant enzymes in our study reinforces their roles in the emergence of drug resistance. Resistance to last resort drug (colistin) and the isolation of mcr-1 indicate further urgency in infection management. Therefore, extensive surveillance, formulation and implementation of effective policies, augmentation of diagnostic facilities and incorporation of antibiotic stewardship programs can be some remedies to cope with this global crisis.

Detection of plasmid-mediated colistin resistance in carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates in Suez Canal University Hospitals

Background: The polymyxins including colistin are the “last resort” antibiotics for treatment of infections with carbapenem resistant Enterobacteriaceae. Recently, transferable colistin resistance mediated by the mcr-genes has been described in Enterobacteriaceae. The Testing of colistin susceptibility is challenging. The aim of the work is to detect the occurence of mcr-1 and mcr-2 genes in phenotypically colistin-resistant carbapenem resistant Escherichia coli and Klebsiella pneumoniae isolates using conventional PCR. Methods: One hundred and sixteen carbapenem-resistant E.coli and K.pneumoniae isolates were collected from patients admitted to different wards of Suez Canal University Hospitals in Ismailia. Urine, endotracheal aspirates, blood, pus and sputum specimens were collected from different patients. Minimal inhibitory concentration by broth microdiluton method was done to assess phenotypic colistin resistance. The colistin resistant E.coli and K.pneumoniae isolates were tested by conventional PCR to detect plasmid mediated mcr-1 and mcr-2 genes. Chi-square test was applied and p -value < 0.05 was considered statistically significant. Results: Forty three isolates out of 116 carbapenem resistant isolates (37.1%) were colistin resistant as shown by MIC by broth microdiluton method. The 43 colistin resistant E.coli and K.pneumoniae isolates were tested by conventional PCR to detect plasmid mediated mcr-1 and mcr-2 genes. Two mcr-2 genes and one mcr-1 gene were detected. Conclusion: Results revealed that the prevalence of colistin resistance among carbapenem-resistant E.coli and K.pneumoniae in SCUHs is concerning; further limiting potential therapeutic options. Plasmid mediated colistin resistance genes mcr-1 and mcr-2 is emerging in SCUHs which refer to a problem in the hospital as by horizontal transfer of this plasmid, the resistance can spread to many isolates in the hospital.

Investigation of Phenotypic and mcr-1–Mediated Colistin Resistance in Escherichia coli

Investigation of Phenotypic and mcr-1–Mediated Colistin Resistance in Escherichia coli

Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane.

Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane (OM) by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than OM. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane (CM). We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the CM of Pseudomonas aeruginosa, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.

Epidemiological Characterization of Colistin and Carbapenem Resistant Enterobacteriaceae in a Tertiary: A Hospital from Anhui Province.

PurposeAntimicrobial resistance, especially carbapenem resistance Enterobacteriaceae and plasmid mediated mobile colistin resistance, is a serious issue worldwide. This study was designed to determine the epidemiological characteristics of plasmid mediated colistin resistance and carbapenem resistant Enterobacteriaceae from tertiary A hospital located in Hefei, China.MethodsTotally, 158 carbapenems resistant Enterobacteriaceae (CRE) were screened for antibiotic susceptibility, mcr-1, extended spectrum β-lactamases (ESBLs), metallo-β-lactamases (MBLs), and fosfomycin resistance genes using PCR and sequencing. The sequence types were identified by multilocus sequence typing (MLST). Plasmid profiles were determined by PCR based replicon typing (PBRT), and the plasmid sizes were confirmed by southern blotting.ResultsThe isolates showed high MIC50 and MIC90 for all antimicrobials, except tigecycline, meropenem, and colistin. The main Carbapenemase genes were blaKPC-2 (90.5%), blaNDM-1(3.7%), blaOXA-48(5.6%) and fosA3 (14.5%). The blaCTXM-15 found 36.7%, mcr-1 (3.7%) recorded in six isolates. PBRT revealed blaKPC-2 in K. pneumoniae on IncR, IncFII, and IncA/C. blaNDM-1 in E. coli on IncFII, whereas in E. cloacae noticed on IncHI2 plasmid. mcr-1 was recorded among IncFIIK, IncFII, and IncF in E. coli, K. pneumoniae, and E. cloacae. Resistance genes (mcr-1, blaNDM-1, blaKPC-2) harboring plasmids are successfully trans-conjugant to EC-600. A high incidence of ST11 was observed in K. pneumoniae carbapenem resistant isolates. While in E. coli, multiple STs were identified. However, mcr-1 in ST23 was identified for the first time in Anhui Province. Among Enterobacter cloacae, ST270 detected carrying blaNDM-1. Southern-hybridization confirmed the plasmid sizes 35–150kb.ConclusionThis study indicates the co-carrying of mcr-1, blaKPC-2, and blaNDM-1 among clinical isolates, the prevalence of different Enterobacteriaceae STs is alarming, especially in E. coli. Holding such a resistance profile is a threat for humans and animals, which may be transferred between the strains through plasmid transfusion. Persistent control actions are immediately necessary to combat this hazard.

Identification of Functional Interactome of Colistin Resistance Protein MCR-1 in Escherichia coli.

The emergence and worldwide dissemination of plasmid-mediated colistin resistance gene mcr-1 has attracted global attention. The MCR-1 enzyme mediated colistin resistance by catalyzing phosphoethanolamine (PEA) transfer onto bacterial lipid A. However, the interaction partners of MCR-1 located in membrane protein in E. coli are unknown. Co-immunoprecipitation (Co-IP) and Mass Spectrometry were performed to define the interacting proteins of MCR-1. A total of three different anti-MCR-1 monoclonal antibody (mAbs) were prepared and 3G4 mAb was selected as the bait protein by compared their suitability for Co-IP. We identified 53, 13, and 14 interacting proteins in E. coli BL21 (DE3) (pET28a-mcr-1), E. coli BL21 (DE3) (pET28a-mcr-1-200), and E. coli DH5α (pUC19-mcr-1), respectively. Six proteins, including the stress response proteins DnaK (chaperone protein) and SspB (stringent starvation protein B), the transcriptional regulation protein H-NS, and ribosomal proteins (RpsE, RpsJ, and RpsP) were identified in all these three strains. These MCR-1-interacting proteins were mainly involved in ribosome and RNA degradation, suggesting that MCR-1 influences the protein biosynthesis through the interaction with ribosomal protein. Multidrug efflux pump AcrA and TolC were important interacting membrane proteins of MCR-1 referred to drug efflux during the PEA modification of the bacterial cell membrane. Overall, we firstly identified the functional interactome profile of MCR-1 in E. coli and discovered that two-component AcrA-TolC multidrug efflux pump was involved in mcr-1-mediated colistin resistance.

Detection of plasmid mediated colistin resistance mcr-1 gene in ESBL producing Escherichia coli isolated from raw milk hard cheese in Egypt

The mcr-1 gene in 125 ESBL-producing Gram-negative bacteria obtained from animal products was screened and the location and transferability of the mcr-1 gene were examined. The mcr-1 gene was detected by PCR in one Escherichia coli isolate (RC263) obtained from hard cheese made from raw milk. Antimicrobial susceptibility test showed that the strain RC263 was phenotypically multidrug-resistant with colistin and nine other antimicrobials. Further genetic analysis indicated that this isolate also harboured the blaTEM and blaCTX-M-9 genes. S1-PFGE followed by Southern blotting verified that the mcr-1 gene was located on an approximately 153 kb plasmid. The plasmid was transferred to a recipient E. coli strain by conjugation, suggesting that the mcr-1 gene may spread to other Gram-negative bacteria. In conclusion, ESBL-producing E. coli carrying a transferable plasmid with mcr-1 gene was present in hard cheese in Egypt.

Whole genome sequence of colistin-resistant Escherichia coli from western India

BackgroundWith virtually dried out new antibiotic discovery pipeline, emergence and spread of antimicrobial resistance is a cause for global concern. Colistin, a cyclic polypeptide antibiotic, often regarded as last resort for multi drug resistance gram-negative bacteria, is also rendered ineffective by horizontal transfer of resistance genes. Surveillance of colistin resistance in GNB is essential to ascertain molecular epidemiology. MethodsWhole genome sequencing (WGS) of an unusual colistin resistant urinary isolate of Escherichia coli was performed using Illumina MiSeq platform using 2x250bp V2 chemistry by following the manufactures protocol (Illumina Inc. USA). Multiple web-based bio-informatic tools were utilized to ascertain antibiotic resistant genes. ResultsAn approximate 5.4 Mb of genome of the urinary isolate AFMC_UC19 was sequenced successfully. Mobile colistin resistance gene (mcr) on the plasmid responsible for horizontal spread was absent in the isolate. ConclusionColistin resistance has been reported previously in Klebsiella pneumoniae and it is a rare occurrence in Escherichia coli in Indian setting. Although the isolate lack mcr mediated colistin resistance, emergence and spread of colistin resistant in gram-negative bacteria pose a threat.