Plasmid-mediated Colistin Resistance Gene Mcr-1 Research Articles

Investigation of Plasmid-Mediated Colistin Resistance Genes (mcr-1–8) in Enterobacterales Isolates

Investigation of Plasmid-Mediated Colistin Resistance Genes (mcr-1–8) in Enterobacterales Isolates

Development and evaluation of rapid and accurate one-tube RPA-CRISPR-Cas12b-based detection of mcr-1 and tet(X4).

The emergence and quick spread of the plasmid-mediated tigecycline resistance gene tet(X4) and colistin resistance gene mcr-1 have posed a great threat to public health and raised global concerns. It is imperative to develop rapid and accurate detection systems for the onsite surveillance of mcr-1 and tet(X4). In this study, we developed one-tube recombinase polymerase amplification (RPA) and CRISPR-Cas12b integrated mcr-1 and tet(X4) detection systems. We identified mcr-1- and tet(X4)-conserved and -specific protospacers through a comprehensive BLAST search based on the NCBI nt database and used them for assembling the detection systems. Our developed one-tube RPA-CRISPR-Cas12b-based detection systems enabled the specific detection of mcr-1 and tet(X4) with a sensitivity of 6.25 and 9 copies within a detection time of ~ 55 and ~ 40min, respectively. The detection results using pork and associated environmental samples collected from retail markets demonstrated that our developed mcr-1 and tet(X4) detection systems could successfully monitor mcr-1 and tet(X4), respectively. Notably, mcr-1- and tet(X4)-positive strains were isolated from the positive samples, as revealed using the developed detection systems. Whole-genome sequencing of representative strains identified an mcr-1-carrying IncI2 plasmid and a tet(X4)-carrying IncFII plasmid, which are known as important vectors for mcr-1 and tet(X4) transmission, respectively. Taken together, our developed one-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems show promising potential for the onsite detection of mcr-1 and tet(X4). KEY POINTS: • One-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems were developed based on identified novel protospacers. • Both detection systems exhibited high sensitivity and specification with a sample-to-answer time of less than 1 h. • The detection systems show promising potential for onsite detection of mcr-1 and tet(X4).

The prevalent dynamic and genetic characterization of mcr-1 encoding multi-drug resistant Escherichia coli strains recovered from poultry in Hebei, China

ObjectivesColistin is known as the last resort antibiotic to treat the infections caused by multi-drug resistant (MDR) foodborne pathogens. The emergence and widespread dissemination of plasmid-mediated colistin resistance gene mcr-1 in the E. coli incurs potential threat to public health. Here, we investigated the epidemiology, transmission dynamics, and genetic characterization of mcr-1 harboring E. coli isolates from poultry origin in Hebei province, China. MethodsA total of 297 fecal samples were collected from the two large poultry farms in Hebei province, China. The samples were processed for E. coli identification by MALDI-TOF-MS and 16S rD4A sequencing. Then, mcr-1 gene harboring E. coli strains were identified by PCR and subjected to antimicrobial susceptibility testing by broth microdilution assay. The genomic characterization of the isolates was done by whole genome sequencing using the various bioinformatics tools, and multi-locus sequence typing (MLST) was done by sequence analysis of the seven housekeeping genes. The conjugation experiment was done to check the transferability of mcr-1 along with the plasmid stability testing. ResultsA total of six mcr-1 E. coli isolates with MIC of 4 μg/mL were identified from 297 samples (2.02%). The mcr-1 harboring E. coli were identified as MDR and belonged to ST101 (n=4) and ST410 (n=2). The genetic environment of mcr-1 presented its position on IncHI2 plasmid in four isolates and p0111 in two isolates which is rarely reported plasmid type for mcr-1. Moreover, both type of plasmids was transferable to recipient J53, and mcr-1 was flanked by three mobile elements ISApl1, Tn3, and IS26 forming a novel backbone Tn3–IS26-mcr-1– pap2-ISApl1 on p0111 plasmid. The phylogenetic analysis shared a common lineage with mcr-1 harboring isolates from the environment, human and animals which indicate its horizontal spread among the diverse sources, species, and hosts. ConclusionThis study recommends the one health approach for future surveillance across multiple sources and bacterial species to adopt relevant measures and reduce global resistance crises.

Investigation of Escherichia coli isolates from pigs and humans for colistin resistance in Lao PDR- a cross-sectional study

BackgroundIn Laos, colistin is not currently registered for use in humans. This One Health study aimed to estimate the prevalence of meat-producing pigs carrying colistin-resistant Escherichia coli, and investigate if E. coli causing invasive human infections were colistin-resistant. MethodsBetween September 2022 and March 2023, rectal swabs were collected from 895 pigs from abattoirs in 9/17 Lao provinces. Pig rectal swabs and stored E. coli isolates from human blood cultures, submitted to Mahosot Hospital Microbiology laboratory between 2005 and 2022, were screened for colistin resistance on selective chromogenic agar with organism identification confirmed using MALDI-TOF MS. Suspected colistin-resistant isolates underwent colistin susceptibility testing by broth microdilution following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Isolates with MIC values of ≥2 μg/ml were tested for plasmid-mediated colistin resistance genes (mcr-1, mcr-2, and mcr-3) by multiplex SYBR Green PCR. ResultsA total of 15/620 (2.41%) invasive human E. coli isolates were phenotypically colistin-resistant by broth microdilution (MIC values 4 to 8 μg/ml). The earliest isolate was from 2015 in a patient from Phongsaly province in Northern Laos. A total of 582/895 (65.02%) pig rectal swab samples contained colistin-resistant E. coli. The detected colistin resistance genes were predominantly mcr-1 (57.8%, 346/598), followed by mcr-3 (20.23%,121/598), and 22.24% (133/598) were found to co-harbour mcr-1 and mcr-3. Among the 15 human isolates with colistin MIC values of ≥4 μg/ml, 12/15 were mcr-1. ConclusionsWe found that colistin resistant E. coli is causing invasive infection in humans in Laos despite the fact it is not available for human use. Use in animals seems to be widespread, confirmed by high carriage rates of colistin-resistant E. coli in pigs. It is probable that food-producing animals are the source of colistin-resistant E. coli bloodstream infection in Laos, although these have been infrequent to date. This is a serious public health concern in the region that needs to be addressed by appropriate enforceable legislation.

Gigantol restores the sensitivity of mcr carrying multidrug-resistant bacteria to colistin

BackgroundThe emergence and wide spread of plasmid-mediated colistin resistance gene (mcr-1) and its mutants have immensely limited the efficacy of colistin in treating multidrug-resistant (MDR) Gram-negative bacterial infections. The development of synergistic combinations of antibiotics with a natural product that coped with the resistance of MDR bacteria was an economic strategy to restore antibiotics activity. Herein, we investigated gigantol, a bibenzyl phytocompound, for restoring in vitro and in vivo, the sensitivity of mcr-positive bacteria to colistin. MethodsThe synergistic activity of gigantol and colistin against multidrug-resistant Enterobacterales was studied via checkerboard assay and time-killing curve. Subsequently, the transcription and protein expression levels of mcr-1 gene were determined by RT-PCR and Western blots. The interaction of gigantol and MCR-1 was simulated via molecular docking and verified via site-directed mutagenesis of MCR-1. Hemolytic activity and cytotoxicity assay were used to evaluate the safety of gigantol. Finally, the in vivo synergistic effect was evaluated via two animal infection models. ResultsGigantol restored the activity of colistin against mcr-positive bacteria E.coli B2 (MIC from 4 μg/ml to 0.25 μg/ml), Salmonella 15E343 (MIC from 8 μg/ml to 1 μg/ml), K. pneumoniae 19-2-1 (MIC from 32 μg/ml to 2 μg/ml) carrying mcr-1, mcr-3, mcr-8, respectively. Mechanistic studies revealed that gigantol down-regulated the expression of genes involved in LPS-modification, reduced the MCR-1 products and inhibited the activity of MCR-1 by binding to amino acid residues Tyr287 and Pro481 in its D-glucose-binding pocket. Safety evaluation showed that the addition of gigantol relieves the hemolysis caused by colistin. Compared with monotherapy, the combination of gigantol and colistin significantly improved the survival rate of Gallgallella mellonella larvae and mice infected by E.coli B2. Moreover, there was a considerable decrease in the bacterial load present in the viscera of mice. ConclusionOur results confirmed that gigantol was a potential colistin adjuvant, and could be used to tackle multi-drug resistant Gram-negative pathogen infections combined with colistin.

Cajanin stilbene acid: A direct inhibitor of colistin resistance protein MCR-1 that restores the efficacy of polymyxin B against resistant Gram-negative bacteria

BackgroundThe resistance of Gram-negative bacteria to polymyxin B, caused by the plasmid-mediated colistin resistance gene mcr-1, which encodes a phosphoethanolamine transferase (MCR-1), is a serious threat to global public health. Therefore, it is urgent to find new drugs that can effectively alleviate polymyxin B resistance. Through the screening of 78 natural compounds, we found that cajanin stilbene acid (CSA) can significantly restore the susceptibility of polymyxin B to mcr-1 positive Escherichia coli (E. coli). PurposeIn this study, we tried to evaluate the ability of CSA to restore the susceptibility of polymyxin B towards the E. coli, and explore the mechanism of sensitivity recovery. Study design and methodsCheckerboard MICs, time-killing curves, scanning electron microscope, lethal and semi-lethal models of infection in mice were used to assess the ability of CSA to restore the susceptibility of polymyxyn to E. coli. The interaction between CSA and MCR-1 was evaluated using surface plasmon resonance (SPR), and molecular docking experiments. ResultsHere, we find that CSA, a potential direct inhibitor of MCR-1, effectively restores the sensitivity of E. coli to polymyxin B. CSA can restore the sensitivity of polymyxin B to drug-resistant E. coli, and the MIC value can be reduced to 1 μg/ml. The time killing curve and scanning electron microscopy results also showed that CSA can effectively restore polymyxin B sensitivity. In vivo experiments showed that the simultaneous use of CSA and polymyxin B can effectively reduce the infection of drug-resistant E. coli in mice. SPR and molecular docking experiments confirmed that CSA strongly bound to MCR-1. The 17-carbonyl oxygen and 12- and 18‑hydroxyl oxygens of CSA were the key sites binding to MCR-1. ConclusionCSA is able to significantly restore the sensitivity of polymyxin B to E. coli in vivo and in vitro. CSA inhibits the enzymatic activity of the MCR-1 protein by binding to key amino acids at the active center of the MCR-1 protein.

Mcr-1 colistin resistance gene sharing between Escherichia coli from cohabiting dogs and humans, Lisbon, Portugal, 2018 to 2020.

BackgroundThe emergence of colistin resistance is a One Health antimicrobial resistance challenge worldwide. The close contact between companion animals and humans creates opportunities for transmission and dissemination of colistin-resistant bacteria.AimTo detect potential animal reservoirs of colistin-resistant Escherichia coli and investigate the possible sharing of these bacteria between dogs, cats and their cohabiting humans in the community in Lisbon, Portugal.MethodsA prospective longitudinal study was performed from 2018 to 2020. Faecal samples from dogs and cats either healthy or diagnosed with a skin and soft tissue or urinary tract infection, and their cohabiting humans were screened for the presence of colistin-resistant E. coli. All isolates were tested by broth microdilution against colistin and 12 other antimicrobials. Colistin-resistant isolates were screened for 30 resistance genes, including plasmid-mediated colistin resistance genes (mcr-1 to mcr-9), and typed by multilocus sequence typing. Genetic relatedness between animal and human isolates was analysed by whole genome sequencing.ResultsColistin-resistant E. coli strains harbouring the mcr-1 gene were recovered from faecal samples of companion animals (8/102; 7.8%) and humans (4/125; 3.2%). No difference between control and infection group was detected. Indistinguishable multidrug-resistant E. coli ST744 strains harbouring the mcr-1 gene were found in humans and their dogs in two households.ConclusionsThe identification of identical E. coli strains containing the plasmid-mediated mcr-1 gene in companion animals and humans in daily close contact is of concern. These results demonstrate the importance of the animal-human unit as possible disseminators of clinically important resistance genes in the community setting.

Characteristics of the plasmid-mediated colistin-resistance gene mcr-1 in Escherichia coli isolated from a veterinary hospital in Shanghai.

The mobile colistin-resistance (mcr)-1 gene is primarily detected in Enterobacteriaceae species, such as Escherichia coli and Salmonella enterica, and represents a significant public health threat. Herein, we investigated the prevalence and characteristics of mcr-1-positive E. coli (MCRPEC) in hospitalized companion animals in a pet hospital in Shanghai, China, from May 2021 to July 2021. Seventy-nine non-duplicate samples were collected from the feces (n = 52) and wounds (n = 20) of cats and dogs and the surrounding hospital environment (n = 7). Seven MCRPEC strains, identified using screening assays and polymerase chain reaction, exhibited multidrug-resistant phenotypes in broth-microdilution and agar-dilution assays. Based in whole-genome sequencing and bioinformatics analyses, all seven isolates were determined to belong to sequence type (ST) 117. Moreover, the Incl2 plasmid was prevalent in these MCRPEC isolates, and the genetic environment of the seven E. coli strains was highly similar to that of E. coli SZ02 isolated from human blood. The isolates also harbored the β-lactamase gene bla CTX-M-65, and florfenicol resistance gene floR, among other resistance genes. Given that horizontal transfer occurred in all seven strains, E. coli plasmid transferability may accelerate the emergence of multidrug-resistant bacteria and may be transmitted from companion animals to humans. Therefore, the surveillance of MCRPEC isolates among companion animals should be strengthened.

Pogostone Enhances the Antibacterial Activity of Colistin against MCR-1-Positive Bacteria by Inhibiting the Biological Function of MCR-1.

The emergence of the plasmid-mediated colistin resistance gene mcr-1 has resulted in the loss of available treatments for certain severe infections. Here we identified a potential inhibitor of MCR-1 for the treatment of infections caused by MCR-1-positive drug-resistant bacteria, especially MCR-1-positive carbapenem-resistant Enterobacteriaceae (CRE). A checkerboard minimum inhibitory concentration (MIC) test, a killing curve test, a growth curve test, bacterial live/dead assays, scanning electron microscope (SEM) analysis, cytotoxicity tests, molecular dynamics simulation analysis, and animal studies were used to confirm the in vivo/in vitro synergistic effects of pogostone and colistin. The results showed that pogostone could restore the bactericidal activity of colistin against all tested MCR-1-positive bacterial strains or MCR-1 mutant–positive bacterial strains (FIC < 0.5). Pogostone does not inhibit the expression of MCR-1. Rather, it inhibits the binding of MCR-1 to substrates by binding to amino acids in the active region of MCR-1, thus inhibiting the biological activity of MCR-1 and its mutants (such as MCR-3). An in vivo mouse systemic infection model, pogostone in combination with colistin resulted in 80.0% (the survival rates after monotherapy with colistin or pogostone alone were 33.3% and 40.0%) survival at 72 h after infection of MCR-1-positve Escherichia coli (E. coli) ZJ487 (blaNDM-1-carrying), and pogostone in combination with colistin led to one or more order of magnitude decreases in the bacterial burdens in the liver, spleen and kidney compared with pogostone or colistin alone. Our results confirm that pogostone is a potential inhibitor of MCR-1 for use in combination with polymyxin for the treatment of severe infections caused by MCR-1-positive Enterobacteriaceae.

Genetic Characterization of mcr-1-Positive Multidrug-Resistant Salmonella enterica Serotype Typhimurium Isolated From Intestinal Infection in Children and Pork Offal in China.

With the rapid emergence of plasmid-mediated colistin resistance gene mcr-1, the increased resistance of Salmonella has attracted extensive attention. This study reports on 11 multidrug-resistant Salmonella enterica serovar Typhimurium strains harboring mcr-1 in China. They all presented resistance to colistin, and additionally, one that was isolated from a child’s stool sample was also resistant to ceftriaxone and azithromycin. We screened 1454 strains of Salmonella for mcr-1 gene through PCR, and these strains are all preserved in our laboratory. Antimicrobial sensitivity analysis was carried out for the screened mcr-1 positive strains. Genetic polymorphism analysis of S. Typhimurium was performed by using the Pulsed-Field Gel Electrophoresis (PFGE). The plasmids harboring mcr-1 were identified by S1-PFGE and southern blotting. Plasmid conjugation assays were used to analyze the transferability of colistin resistance. The plasmids harboring mcr-1 were characterized by sequencing and bioinformatic analysis. Eleven S. Typhimurium strains harboring mcr-1 with colistin resistance (MICs 4μg/ml) were detected, which were isolated from children and pig offal in China. All of them were multidrug-resistant strains. PFGE results revealed that the strains isolated from different samples or locations have identical genotypes. S1-PFGE and southern blotting experiments showed that three plasmids of different sizes (33, 60, and 250 kb) all carried the mcr-1 gene. The plasmid conjugation assays revealed that Salmonella acquired mcr-1 harboring plasmids by horizontal transfer. Sequencing and plasmid type analysis revealed that these plasmids were types IncX4, IncI2, and IncHI2. Among them, IncX4 and IncI2 plasmids had extremely similar backbones and contained one resistant gene mcr-1. IncHI2 plasmid contained multiple resistant genes including blaCTX–M, oqxB, sul, aph, aadA, and blaTEM. We identified 11 mcr-1 harboring S. Typhimurium strains in China and described their characteristics. Our findings indicate that the mcr-1 gene can effectively spread among intestinal bacteria by horizontal transfer of three types of plasmids. Moreover, the IncHI2 plasmid can also mediate the transfer of other drug resistance genes. These results reveal that constant surveillance of mcr-1 harboring S Typhimurium is imperative to prevent the spread of colistin resistance.

Identification of the Plasmid-Mediated Colistin Resistance Gene mcr-1 in Escherichia coli Isolates From Migratory Birds in Guangdong, China.

We determined the prevalence and transmission characteristics of mcr-1-positive Escherichia coli (MCRPEC) isolates from migratory birds Anser indicus in Guangdong, China. We identified 22 MCRPEC from 303 A. indicus fecal samples (7.3%) in Guangzhou, Zhaoqing, and Futian. The mcr-1 gene coexisted with 24 other types of antibiotic resistance genes (ARG), and 11 ARGs were highly prevalent at levels >50%. The MCRPEC displayed a diversity of sequence types (ST), and 19 distinct STs were identified with ST10, ST1146, and ST1147 as the most prevalent. In addition, these MCRPEC from birds were closely related phylogenetically to those from other sources in China. Whole-genome sequencing analysis demonstrated that mcr-1 was located on IncX4 (n=9, 40.9%), IncI2 (n=5, 22.7%) and IncP (n=1, 4.5%) plasmids and the latter shared an identical plasmid backbone with other sources. These results highlight the significance of migratory birds in the transmission of antibiotic resistance and provide powerful evidence that migratory birds are potential transmitters of antibiotic resistance.

Widespread prevalence and molecular epidemiology of tet(X4) and mcr-1 harboring Escherichia coli isolated from chickens in Pakistan

The emergence and spread of plasmid-mediated tigecycline resistance gene tet(X4) and colistin resistance gene mcr-1 in Escherichia coli (E. coli) pose a potential threat to public health, due to the importance of colistin and tigecycline for treating serious clinical infections. However, the characterization of bacteria coharboring both genes was few reported. Here, we described the molecular epidemiology of tet(X4) and mcr-1 harboring E. coli strains of chicken origin in Pakistan, with methods including PCR, antimicrobial susceptibility testing, DNA transfer assays, plasmid replicon typing, whole-genome sequencing and bioinformatics analysis. The tet(X4) gene was identified in 36 isolates exhibiting high levels of tigecycline resistance (MICs, 16–128 mg/L). Worryingly, 24 of the 36 tet(X4)-bearing isolates were confirmed as colistin resistance, positive for plasmid-borne mcr-1. We observed the prevalence of tet(X4)-bearing IncFII plasmid with mcr-1-bearing IncI2 plasmid in 12 E. coli isolates, with a high co-transfer frequency except for one strain PK8233, in which tet(X4)- and mcr-1-bearing plasmids were non-transferable. Coexistence of tet(X4)-bearing IncFII plasmid with mcr-1-carrying multidrug-resistant (MDR) IncHI2 plasmid was also identified in 10 E. coli isolates, and a relatively low co-transfer frequency was obtained except PK8575, in which mcr-1 was non-transferable. The transferability of pPK8275-tetX in PK8275 and pPK8233-tetX in PK8233, that could transfer from E. coli J53 to C600 by conjugation, was interfered by certain factors in PK8275 and PK8233. This may provide new insights to prevent and control the spread of antibiotic resistance genes. Two strains were reported to co-carry tet(X4)-positive IncQ1 plasmid and mcr-1-positive IncI2 plasmid. Convergence of tet(X4) and mcr-1 genes in E. coli by conjugative or mobilizable plasmids may lead to potentially widespread transmission of such resistance genes, which may incur antibiotic-resistance crisis globally.

Acquisition of the mcr-1 Gene Lowers the Target Mutation to Impede the Evolution of a High-Level Colistin-Resistant Mutant in Escherichia coli.

ObjectiveThe spread of the plasmid-mediated colistin resistance gene mcr-1 poses a significant public health threat. Little information is available on the development of high-level colistin-resistant mutants (HLCRMs) in MCR-1-producing Escherichia coli (MCRPEC). The present study was designed to evaluate the impact of chromosomal modifications in pmrAB, phoPQ, and mgrB combined with mcr-1 on colistin resistance in E. coli.MethodsFive MCRPEC and three non-MCRPEC (E. coli ATCC25922 and two plasmid-curing) strains were used. The HLCRMs were selected through multi-stepwise colistin exposure. Moreover, two E. coli C600-pMCRs were constructed and used for selection of HLCRMs. Further analysis included mutation rates and DNA sequencing. Transcripts of pmrABC, phoP, mgrB, and mcr-1 were quantified by real-time quantitative PCR.ResultsAll tested HLCRMs were successfully isolated from their parental strains. Non-MCRPEC strains had higher minimum inhibitory concentrations (MICs) and mutation rates than MCRPEC strains. Nineteen amino acid substitutions were identified: seven in PmrA, six in PmrB, one in PhoP, three in PhoQ, and two in MgrB. Most were detected in non-MCRPEC strains. Sorting Intolerant From Tolerant predicted that four substitutions, PmrA Gly15Arg, Gly53Arg, PmrB Pro94Gln, and PhoP Asp86Gly, affected protein function. Two HLCRM isolates did not show amino acid substitutions in contrast to their parental MCRPEC isolates. No further mutations were detected in the second- and third-step mutants. Further transcriptional analysis showed that the up-regulation of pmrCAB expression was greater in the mutant of E. coli C600 than in E. coli C600-pMCR.ConclusionAcquisition of the mcr-1 gene had a negative impact on the development of HLCRMs in E. coli, but was associated with low-level colistin resistance. Thus, colistin-based combination regimens may be effective against infections with MCR-1-producing isolates.

Detection of Plasmid-Mediated Mobile Colistin Resistance Gene (mcr-1) in Enterobacterales Isolates from a University Hospital.

PurposeColistin represents one of the last treatment options for infections caused by multi-drug resistant (MDR) Enterobacterales. The emergence of a plasmid-mediated mobile colistin resistance-1 (mcr-1) gene has raised serious concerns about its potential dissemination among bacteria.MethodsIn this study, we evaluated the chromogenic medium, CHROMID® Colistin Resistance (COLR) agar, for the rapid detection of colistin-resistant Enterobacterales using broth microdilution (BMD) as a reference method. We also attempted to detect mcr-1, −2, −3, −4, and −5 genes, as well as the insertion sequence ISApl1 via polymerase chain reaction (PCR), followed by sequencing of mcr gene(s).ResultsAmong the 100 studied Enterobacterales isolates, 53% of them were colistin-resistant, with higher rate among Klebsiella pneumoniae (75%) as compared to Escherichia coli (44.4%). The COLR agar showed 83.2% sensitivity and 97.9% specificity for the detection of colistin resistance. Among colistin-resistant isolates, mcr-1 gene was only detected in four (7.5%) E. coli isolates. The ISApl1 was not found among mcr-1 positive isolates. Sequencing of mcr-1 gene revealed nucleotide sequence homogeneity with the wild-type mcr-1 gene in BLAST.ConclusionThe COLR agar is a promising phenotypic method for the detection of colistin-resistant Enterobacterales. Multiplex PCR followed by sequencing can be used for mcr genes’ detection and characterization.

Prevalence and Characteristic of Swine-Origin mcr-1-Positive Escherichia coli in Northeastern China.

The emergence of the plasmid-mediated colistin resistance gene mcr-1 is threatening the last-line role of colistin in human medicine. With mcr-1-positive Escherichia coli (E. coli) isolated from food animal being frequently reported in China, the prevalence of mcr-1 in food animal has attracted public attention. In the present study, a total of 105 colistin-resistant E. coli strains were isolated from 200 fecal samples collected from six swine farms in northeastern China. mcr-PCR revealed that the prevalence of mcr-1 in colistin-resistant E. coli was 53.33% (56/105). mcr-1-positive E. coli showed extensive antimicrobial resistance profiles with the presence of additional resistance genes, increased expression of multidrug efflux pump-associated genes, and increased biofilm formation ability. MLST differentiated all the mcr-1-positive E. coli into 25 sequence types (STs) and five unknown ST, and the most common ST was ST10 (n = 11). By phylogenetic group classification, the distribution of all mcr-1-positive E. coli belonging to groups A, B1, B2, and D was 46.43, 35.71, 5.36, and 5.36%, respectively. Conjugation experiment demonstrated that most of the mcr-1 were transferable at frequencies of 2.68 × 10–6–3.73 × 10–3 among 30 representative mcr-1-positive E. coli. The plasmid replicon types IncI2 (n = 9), IncX4 (n = 5), IncHI2 (n = 3), IncN (n = 3), and IncP (n = 1) were detected in the transconjugants. The results of growth assay, competition experiment, and plasmid stability testing showed that acquisition of mcr-1-harboring plasmids could reduce the fitness of bacterial hosts, but mcr-1 remained stable in the recipient strain. Due to the potential possibility of these mcr-1-positive E. coli being transmitted to humans through the food chain or through horizontal transmission, therefore, it is necessary to continuously monitor the prevalence and dissemination of mcr-1 in food animal, particularly in swine.

Development of a loop-mediated isothermal amplification assay combined with a nanoparticle-based lateral flow biosensor for rapid detection of plasmid-mediated colistin resistance gene mcr-1.

A loop-mediated isothermal amplification assay combined with a nanoparticle-based lateral flow biosensor (LAMP-LFB) was established for the rapid and accurate detection of the mobilized colistin resistance gene (mcr-1), which causes the loss of colistin antibacterial efficacy in clinical treatments. The amplification stage of the assay was completed in 60 min at 63°C, and the reaction products could be visually detected by employing the LFB, which provided a fast (within 2 min) and objective method to evaluate the amplification results. The LAMP assay amplified the target sequences of mcr-1 with high specificity. In pure strains, the detection limit of the LAMP-LFB assay was 360 fg plasmid DNA/reaction, and in spiked feces samples the value was approximately 6.3×103 CFU/mL (~6.3 CFU/reaction), which was tenfold more sensitive than the PCR assay. The results show that the developed LAMP-LFB assay will be a worthy tool for the simple, rapid, specific, and sensitive detection of mcr-1 gene in clinical settings and resource-limited areas.

Genomic characterisation of a multidrug-resistant Escherichia coli strain carrying the mcr-1 gene recovered from a paediatric patient in China

ObjectivesThe global spread of carbapenem-resistant Enterobacterales is a leading public-health threat. Lack of effective treatment has resulted in use of colistin as a last-resort therapeutic option for multidrug-resistant (MDR) bacterial infections. Here we report the complete genome sequence of a MDR Escherichia coli strain carrying a plasmid-mediated colistin resistance gene mcr-1 recovered from a Chinese paediatric patient. MethodsWhole-genome sequencing of E. coli strain 1506 was performed using both Oxford Nanopore MinION and Illumina NovaSeq 6000 platforms. De novo hybrid assembly of short Illumina reads and long MinION reads was performed using Unicycler. In silico multilocus sequence typing (MLST), antimicrobial resistance genes (ARGs) and plasmid replicons were identified from the genome sequence. Core genome multilocus sequence typing (cgMLST) analysis between E. coli 1506 and all of the ST48 E. coli strains retrieved from the NCBI GenBank database was performed using BacWGSTdb 2.0 server. ResultsThe complete genome sequence of E. coli 1506 consists of six contigs comprising 4 849 058 bp, including one chromosome and five plasmids, and was assigned to ST48. Fourteen ARGs were identified, including mcr-1 located on a 33 309-bp IncX4 plasmid. The closest relative of E. coli 1506 was another isolate originating from livestock in Australia, which differed by 614 cgMLST alleles. ConclusionOur study reports the genome sequence of a MDR E. coli carrying mcr-1 isolated from a Chinese paediatric patient. These data may help to understand the antimicrobial resistance mechanisms and genomic features and highlight the growing threat of antimicrobial resistance in children.

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.

Widespread Prevalence of Plasmid-Mediated Colistin Resistance Gene mcr-1 in Escherichia coli from Père David's Deer in China.

Père David's deer (Elaphurus davidianus or milu) is an endangered species, and the prevalence of antimicrobial resistance (AMR) such as mcr-1-positive strains among them has been unknown. In this study, we aimed to investigate the genomic characterizations of mcr-1-positive strains and provide insight into the dissemination of AMR in nature reserve settings. Sixty-seven mcr-1-positive Escherichia coli isolates from 97 fecal samples were identified by PCR and found resistant to colistin. The prevalence of β-lactam resistance was very high, and there were 64 mcr-1-positive isolates containing β-lactamase genes. Transconjugants of 66 mcr-1-positive isolates were acquired through conjugation experiments. PCR-based replicon typing (PBRT) showed that 44 strains harbored IncI2 mcr-1-bearing plasmids, eight strains harbored IncX4 mcr-1-carrying plasmids, and 14 strains harbored IncHI2 mcr-1-positive plasmids. Notably, mcr-1 was located in the chromosome of LD27-1. Clonal dissemination and horizontal dissemination of mcr-1 by plasmids coexist. We first report the prevalence of plasmid-mediated mcr-1 in E. coli from Père David's deer in China. mcr-1-bearing IncI2 plasmid was the most frequent plasmid type, and the first IncI2 plasmid harboring both blaCTX-M-132 and mcr-1 is characterized here. Our results support the implication of Père David's deer as a potential reservoir for MCR-1-producing E. coliIMPORTANCE The mcr-1 gene is widely reported around the world and has been identified on various plasmids with different replicon types. Resistance to the last-line antibiotic colistin mediated by mcr-1 still represents a threat to global public health. Père David's deer is a highly endangered species originating in China, and many deer are currently being raised in captivity for gradual reintroduction to the wild. If this species carries AMR bacteria, it will pose a potential threat to the environment. Therefore, research on the dissemination of mcr-1-positive E. coli from Père David's deer is of great significance. This is the first study to investigate the microbiological and genomic surveillance of MCR-1-producing bacteria colonized among Père David's deer in China, and we uncovered a high prevalence of MCR-1-producing E. coli The importance of constant surveillance for AMR bacteria in nature reserve settings is emphasized.

First detection of plasmid-mediated colistin-resistance gene (mcr-1, mcr-2 and mcr-3) in Escherichia coli isolated from breeder poultry of Bangladesh

First detection of plasmid-mediated colistin-resistance gene (mcr-1, mcr-2 and mcr-3) in Escherichia coli isolated from breeder poultry of Bangladesh