Plasmid-mediated Colistin Resistance Research Articles

Repeated Occurrence of Mobile Colistin Resistance Gene-Carrying Plasmids in Pathogenic Escherichia coli from German Pig Farms.

The global spread of plasmid-mediated mobile colistin resistance (mcr) genes threatens the vital role of colistin as a drug of last resort. We investigated whether the recurrent occurrence of specific E. coli pathotypes and plasmids in individual pig farms resulted from the continued presence or repeated reintroduction of distinct E. coli strains. E. coli isolates (n = 154) obtained from three pig farms with at least four consecutive years of mcr detection positive for virulence-associated genes (VAGs) predicting an intestinal pathogenic pathotype via polymerase chain reaction were analyzed. Detailed investigation of VAGs, antimicrobial resistance genes and plasmid Inc types was conducted using whole genome sequencing for 87 selected isolates. Sixty-one E. coli isolates harbored mcr-1, and one isolate carried mcr-4. On Farm 1, mcr-positive isolates were either edema disease E. coli (EDEC; 77.3%) or enterotoxigenic E. coli (ETEC; 22.7%). On Farm 2, all mcr-positive strains were ETEC, while mcr-positive isolates from Farm 3 showed a wider range of pathotypes. The mcr-1.1 gene was located on IncHI2 (Farm 1), IncX4 (Farm 2) or IncX4 and IncI2 plasmids (Farm 3). These findings suggest that various pathogenic E. coli strains play an important role in maintaining plasmid-encoded colistin resistance genes in the pig environment over time.

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.

An Intra-Hospital Spread of Colistin-Resistant K. pneumoniae Isolates-Epidemiological, Clinical, and Genetic Analysis.

Background and Objective: Klebsiella pneumoniae appears to be a significant problem due to its ability to accumulate antibiotic-resistance genes. After 2013, alarming colistin resistance rates among carbapenem-resistant K. pneumoniae have been reported in the Balkans. The study aims to perform an epidemiological, clinical, and genetic analysis of a local outbreak of COLr CR-Kp. Material and Methods: All carbapenem-resistant and colistin-resistant K. pneumoniae isolates observed among patients in the ICU unit of Military Medical Academy, Sofia, from 1 January to 31 October 2023, were included. The results were analyzed according to the EUCAST criteria. All isolates were screened for blaVIM, blaIMP, blaKPC, blaNDM, and blaOXA-48. Genetic similarity was determined using the Dice coefficient as a similarity measure and the unweighted pair group method with arithmetic mean (UPGMA). mgrB genes and plasmid-mediated colistin resistance determinants (mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5) were investigated. Results: There was a total of 379 multidrug-resistant K. pneumoniae isolates, 88% of which were carbapenem-resistant. Of these, there were nine (2.7%) colistin-resistant isolates in six patients. A time and space cluster for five patients was found. Epidemiology typing showed that two isolates belonged to clone A (pts. 1, 5) and the rest to clone B (pts. 2-4) with 69% similarity. Clone A isolates were coproducers of blaNDM-like and blaOXA-48-like and had mgrB-mediated colistin resistance (40%). Clone B isolates had only blaOXA-48-like and intact mgrB genes. All isolates were negative for mcr-1, -2, -3, -4, and -5 genes. Conclusions: The study describes a within-hospital spread of two clones of COLr CR-Kp with a 60% mortality rate. Clone A isolates were coproducers of NDM-like and OXA-48-like enzymes and had mgrB-mediated colistin resistance. Clone B isolates had only OXA-48-like enzymes and intact mgrB genes. No plasmid-mediated resistance was found. The extremely high mortality rate and limited treatment options warrant strict measures to prevent outbreaks.

A multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (mcr-1 to mcr-10) genes.

Recently, 10 plasmid-mediated mobile colistin resistance genes, mcr-1 to mcr-10, and their variants have been identified, posing a new threat to the treatment of clinical infections caused by Gram-negative bacteria. Our objective was to develop a rapid, sensitive, and accurate molecular assay for detecting mcr genes in clinical isolates. The primers and corresponding TaqMan-MGB probes were designed based on the sequence characteristics of all reported MCR family genes, multiplex Taqman-MGB probe-based qPCR assays were developed and optimized, and the sensitivity, specificity and reproducibility of the method were evaluated. The assay contained 8 sets of primers and probes in 4 reaction tubes, each containing 2 sets of primers and probes. The standard curves for both the single and multiplex systems showed good linearity (R2 > 0.99) between the starting template amount and the Ct value, with a lower limit of detection of 102 copies/μL. The specificity test showed positive amplification results only for strains containing the mcr genes, whereas the other strains were negative. The results of intra-and inter-group repeatability experiments demonstrated the stability and reliability of the newly developed method. It was used to detect mcr genes in 467 clinically-obtained Gram-negative isolates, which were multidrug-resistant. Twelve strains containing the mcr genes were detected (seven isolates carrying mcr-1, four isolates carrying mcr-10, and one isolate carrying mcr-9). The products amplified by the full-length PCR primer were identified by sequencing, and the results were consistent with those of the multiplex qPCR method. The assay developed in this study has the advantages of high specificity, sensitivity, and reproducibility. It can be used to specifically detect drug-resistant clinical isolates carrying the mcr genes (mcr-1 to mcr-10), thus providing a better basis for clinical drug treatment and drug resistance research.

Molecular Detection of Plasmid-mediated colistin-resistance genes in Multi-drug resistant Escherichia coli isolated from human and poultry in Duhok city, Iraq

Molecular Detection of Plasmid-mediated colistin-resistance genes in Multi-drug resistant Escherichia coli isolated from human and poultry in Duhok city, Iraq

First Report of Colistin-Resistant Escherichia coli Carrying mcr-1 IncI2(delta) and IncX4 Plasmids from Camels (Camelus dromedarius) in the Gulf Region.

Addressing the emergence of antimicrobial resistance (AMR) poses a significant challenge in veterinary and public health. In this study, we focused on determining the presence, phenotypic background, and genetic epidemiology of plasmid-mediated colistin resistance (mcr) in Escherichia coli bacteria isolated from camels farmed in the United Arab Emirates (UAE). Fecal samples were collected from 50 camels at a Dubai-based farm in the UAE and colistin-resistant Gram-negative bacilli were isolated using selective culture. Subsequently, a multiplex PCR targeting a range of mcr-genes, plasmid profiling, and whole-genome sequencing (WGS) were conducted. Eleven of fifty camel fecal samples (22%) yielded colonies positive for E. coli isolates carrying the mcr-1 gene on mobile genetic elements. No other mcr-gene variants and no chromosomally located colistin resistance genes were detected. Following plasmid profiling and WGS, nine E. coli isolates from eight camels were selected for in-depth analysis. E. coli sequence types (STs) identified included ST7, ST21, ST24, ST399, ST649, ST999, and STdaa2. Seven IncI2(delta) and two IncX4 plasmids were found to be associated with mcr-1 carriage in these isolates. These findings represent the first identification of mcr-1-carrying plasmids associated with camels in the Gulf region. The presence of mcr-1 in camels from this region was previously unreported and serves as a novel finding in the field of AMR surveillance.

Hidden threats in the plastisphere: Carbapenemase-producing Enterobacterales colonizing microplastics in river water

Carbapenem resistance poses a significant burden on healthcare systems worldwide. Microplastics (MPs) have emerged as potential contributors to antibiotic resistance spread in the environment. However, the link between MPs and carbapenem resistance remains unexplored. We investigated the prevalence of carbapenem-resistant bacteria colonizing MPs placed in a river. Three replicates of a mixture of polypropylene (PP), polyethylene (PE) and polyethylene terephthalate (PET) and of PET alone were placed both upstream and downstream a wastewater treatment plant (WWTP) discharge. Carbapenem-resistant Enterobacterales (CRE) were further characterized by phenotypic tests and whole-genome sequencing. The abundance of carbapenem-resistant bacteria on MPs increased significantly downstream the WWTP. Their prevalence was higher in the MPs mixture compared to PET alone. CRE strains colonizing MPs included Klebsiella pneumoniae (n = 3), Klebsiella quasipneumoniae (n = 3), Raoultella ornithinolytica (n = 2), Enterobacter kobei (n = 1) and Citrobacter freundii (n = 1), most (n = 8) recovered after the WWTP discharge. All strains exhibited at least one of the tested virulence traits (biofilm formation at 37 °C, haemolytic activity and siderophore production), were multi-drug resistant and carried carbapenemase-encoding genes [blaKPC-3 (n = 5), blaGES-5 (n = 2) or blaKPC-3 + blaGES-5 (n = 3)]. Uncommon phenotypes of resistance to imipenem/relebactam (n = 3) and ceftazidime/avibactam (n = 2) were observed. Two blaKPC−3-positive K. pneumoniae successfully transfer this gene trough conjugation. Genome analysis predicted all strains as human pathogens. The blaKPC−3 was associated with the Tn4401d transposon on a pBK30683-like plasmid in most of the isolates (n = 7). The blaGES-5 was mostly linked to class 3 integrons. A K. pneumoniae strain belonging to the outbreak-causing high-risk clone ST15 carried both blaKPC-3 and blaCTX-M-15. Two K. quasipneumoniae isolates carried the plasmid-mediated colistin resistance gene mcr-9. Our results underscore the role of MPs as vectors for CRE dissemination, particularly following WWTPs discharges. MPs may act as carriers, facilitating the dissemination of carbapenemase-encoding genes and potentially contributing to increased CRE incidence in the environment.

Detection of mcr-1 gene of plasmid-mediated colistin resistance in extended spectrum beta lactamase (ESBL) producing Escherichia coli in Khartoum State – Sudan

Detection of mcr-1 gene of plasmid-mediated colistin resistance in extended spectrum beta lactamase (ESBL) producing Escherichia coli in Khartoum State – Sudan

Bavachin Rejuvenates Sensitivity of Colistin against Colistin-Resistant Gram-Negative Bacteria.

The emergence of plasmid-mediated colistin resistance threatens the efficacy of colistin as a last-resort antibiotic used to treat infection caused by Gram-negative bacteria (GNB). Given the shortage of new antibiotics, the discovery of adjuvants to existing antibiotics is a promising strategy to combat infections caused by multidrug-resistant (MDR) GNB. This study was designed to investigate the potential synergistic antibacterial activity of bavachin, a bioactive compound extracted from the Psoralea Fructus, combined with colistin against MDR GNB. Herein, the synergistic efficacy in vitro and the therapeutic efficacy of colistin combined with bavachin in vivo were evaluated. The synergistic mechanism was detected by fluorescent probe and the transcript levels of mcr-1. Bavachin combined with colistin showed an excellent synergistic activity against GNB, as the FICI ≤ 0.5. In contrast to colistin alone, combination therapy dramatically increased the survival rate of Galleria mellonella and mice in vivo. Moreover, the combination of bavachin and colistin significantly reduced the amount of bacterial biofilm formation, improved the membrane disruption of colistin and inhibited mcr-1 transcription. These findings show that bavachin is a potential adjuvant of colistin, which may provide a new strategy to combat colistin-resistant bacteria infection with lower doses of colistin.

Genetic characterization of multidrug-resistant Escherichia coli harboring colistin-resistant gene isolated from food animals in food supply chain.

Colistin is widely used for the prophylaxis and treatment of infectious disease in humans and livestock. However, the global food chain may actively promote the dissemination of colistin-resistant bacteria in the world. Mobile colistin-resistant (mcr) genes have spread globally, in both communities and hospitals. This study sought to genomically characterize mcr-mediated colistin resistance in 16 Escherichia coli strains isolated from retail meat samples using whole genome sequencing with short-read and long-read platforms. To assess colistin resistance and the transferability of mcr genes, antimicrobial susceptibility testing and conjugation experiments were conducted. Among the 16 isolates, 11 contained mcr-1, whereas three carried mcr-3 and two contained mcr-1 and mcr-3. All isolates had minimum inhibitory concentration (MIC) for colistin in the range 1-64 μg/mL. Notably, 15 out of the 16 isolates demonstrated successful transfer of mcr genes via conjugation, indicative of their presence on plasmids. In contrast, the KK3 strain did not exhibit such transferability. Replicon types of mcr-1-containing plasmids included IncI2 and IncX4, while IncFIB, IncFII, and IncP1 contained mcr-3. Another single strain carried mcr-1.1 on IncX4 and mcr-3.5 on IncP1. Notably, one isolate contained mcr-1.1 located on a chromosome and carrying mcr-3.1 on the IncFIB plasmid. The chromosomal location of the mcr gene may ensure a steady spread of resistance in the absence of selective pressure. Retail meat products may act as critical reservoirs of plasmid-mediated colistin resistance that has been transmitted to humans.

Convergence of plasmid-mediated Colistin and Tigecycline resistance in Klebsiella pneumoniae.

The co-occurrence of colistin and tigecycline resistance genes in Klebsiella pneumoniae poses a serious public health problem. This study aimed to characterize a K. pneumoniae strain, K82, co-harboring a colistin resistance gene (CoRG) and tigecycline resistance gene (TRG), and, importantly, investigate the genetic characteristics of the plasmid with CoRG or TRG in GenBank. K. pneumoniae strain K82 was subjected to antimicrobial susceptibility testing, conjugation assay, and whole-genome sequencing (WGS). In addition, comparative genomic analysis of CoRG or TRG-harboring plasmids from K82 and GenBank was conducted. K. pneumoniae strain K82 was resistant to all the tested antimicrobials including colistin and tigecycline, except for carbapenems. WGS and bioinformatic analysis showed that K82 belonged to the ST656 sequence type and carried multiple drug resistance genes, including mcr-1 and tmexCD1-toprJ1, which located on IncFIA/IncHI2/IncHI2A/IncN/IncR-type plasmid pK82-mcr-1 and IncFIB/IncFII-type plasmid pK82-tmexCD-toprJ, respectively. The pK82-mcr-1 plasmid was capable of conjugation. Analysis of the CoRG/TRG-harboring plasmid showed that mcr-8 and tmexCD1-toprJ1 were the most common CoRG and TRG of Klebsiella spp., respectively. These TRG/CoRG-harboring plasmids could be divided into two categories based on mash distance. Moreover, we found an IncFIB/IncHI1B-type plasmid, pSYCC1_tmex_287k, co-harboring mcr-1 and tmexCD1-toprJ1. To the best of our knowledge, this is the first report on the co-occurrence of mcr-1 and tmexCD1-toprJ1 on a single plasmid. Our research expands the known diversity of CoRG and TRG-harboring plasmids in K. pneumoniae. Effective surveillance should be implemented to assess the prevalence of co-harboring CoRG and TRG in a single K. pneumoniae isolate or even a single plasmid.

Reference gene catalog and metagenome-assembled genomes from the gut microbiome reveal the microbial composition, antibiotic resistome, and adaptability of a lignocellulose diet in the giant panda

The giant panda, a strict herbivore that feeds on bamboo, still retains a typical carnivorous digestive system. Reference catalogs of microbial genes and genomes are lacking, largely limiting the antibiotic resistome and functional exploration of the giant panda gut microbiome. Here, we integrated 177 fecal metagenomes of captive and wild giant pandas to construct a giant panda integrated gene catalog (GPIGC) comprised of approximately 4.5 million non-redundant genes and reconstruct 393 metagenome-assembled genomes (MAGs). Taxonomic and functional characterization of genes revealed that the captivity of the giant panda significantly changed the core microbial composition and the distribution of microbial genes. Higher abundance and prevalence of antibiotic resistance genes (ARGs) were detected in the guts of captive giant pandas, and ARG distribution was influenced by geography, for both captive and wild individuals. Escherichia, as the prevalent genus in the guts of captive giant pandas, was the main carrier of ARGs, meaning there is a high risk of ARG transmission by Escherichia. We also found that multiple mcr gene variants, conferring plasmid-mediated mobile colistin resistance, were widespread in the guts of captive and wild giant pandas. There were low proportions of carbohydrate-active enzyme (CAZyme) genes in GPIGC and MAGs compared with several omnivorous and herbivorous mammals. Many members of Clostridium MAGs were significantly enriched in the guts of adult, old and wild giant pandas. The genomes of isolates and MAGs of Clostridiaceae harbored key genes or enzymes in complete pathways for degrading lignocellulose and producing short-chain fatty acids (SCFAs), indicating the potential of these bacteria to utilize the low-nutrient bamboo diet. Overall, our data presented an exhaustive reference gene catalog and MAGs in giant panda gut and provided a comprehensive understanding of the antibiotic resistome and microbial adaptability for a high-lignocellulose diet.

A new variant of the colistin resistance gene MCR-1 with co-resistance to β-lactam antibiotics reveals a potential novel antimicrobial peptide.

The emerging and global spread of a novel plasmid-mediated colistin resistance gene, mcr-1, threatens human health. Expression of the MCR-1 protein affects bacterial fitness and this cost correlates with lipid A perturbation. However, the exact molecular mechanism remains unclear. Here, we identified the MCR-1 M6 variant carrying two-point mutations that conferred co-resistance to β-lactam antibiotics. Compared to wild-type (WT) MCR-1, this variant caused severe disturbance in lipid A, resulting in up-regulation of L, D-transpeptidases (LDTs) pathway, which explains co-resistance to β-lactams. Moreover, we show that a lipid A loading pocket is localized at the linker domain of MCR-1 where these 2 mutations are located. This pocket governs colistin resistance and bacterial membrane permeability, and the mutated pocket in M6 enhances the binding affinity towards lipid A. Based on this new information, we also designed synthetic peptides derived from M6 that exhibit broad-spectrum antimicrobial activity, exposing a potential vulnerability that could be exploited for future antimicrobial drug design.

A new function of thymol nanoemulsion for reversing colistin resistance in Salmonella enterica serovar Typhimurium infection.

Adjuvant addition of approved drugs or foodborne additives to colistin might be a cost-effective strategy to overcome the challenge of plasmid-mediated mobile colistin resistance gene emergence, which poses a threat in the clinic and in livestock caused by infections with Gram-negative bacteria, especially carbapenem-resistant Enterobacteriaceae. Chequerboard assay was applied to screen the colistin adjuvants from natural compounds. The killing-time curve, combined disc test and membrane permeation assay were conducted to identify the synergy efficacy of thymol and colistin in vitro. Thin-layer chromatography (TLC), LC-MS and fluorescence spectra were used to indicate the interaction of thymol and MCR-1. The potential binding sites were then investigated by molecular simulation dynamics. Finally, a thymol nanoemulsion was prepared with high-pressure homogenization as the clinical dosage form. Thymol presented an excellent synergistic effect in vitro with colistin against Salmonella enterica serovar Typhimurium and Escherichia coli bacteria. Thymol addition, forming a complex with MCR-1, might interfere with the efficacy of MCR-1. Moreover, thymol strengthened colistin activity associated with potentiating membrane damage, destroying the biofilm and enhancing reactive oxygen species-mediated oxidative damage. Thymol nanoemulsion combined with colistin remarkably prevented the intestinal damage caused by S. Typhimurium infection, resulting in a survival rate higher than 60%. This study achieved a promising thymol oral formulation as colistin adjuvant to combat S. Typhimurium infection, which could be used to extend the lifespan of colistin in clinical veterinary medicine.

Evaluation of different methods for in vitro susceptibility testing of colistin in carbapenem resistant Gram-negative bacilli.

The increasing antibiotic resistance like the advent of carbapenem resistant Enterobactarales (CRE), Carbapenem Resistant Acinetobacter baumanii (CRAB), and Carbapenem Resistant Pseudomonas aeruginosa (CRPA) has led to to the use of toxic and older drugs like colistin for these organisms. But worldwide there is an increase in resistance even to colistin mediated both by chromosomes and plasmids. This necessitates accurate detection of resistance. This is impeded by the unavailability of a user-friendly phenotypic methods for use in routine clinical microbiology practice. The present study attempts to evaluate two different methods - colistin broth disc elution and MIC detection by Vitek two in comparison to CLSI approved broth microdilution (BMD) for colistin for Enterobactarales, Pseudomonas aeruginosa , and Acinetobacter baumanii clinical isolates. Colistin susceptibility of 6013 carbapenem resistant isolates was determined by BMD, Colistin Broth Disc Elution (CBDE), and Vitek two methods and was interpreted as per CLSI guidelines. The MIC results of CBDE, Vitek two were compared with that of BMD and essential agreement (EA), categorical agreement (CA), sensitivity, specificity, very major error (VME), major error (ME) and Cohen's kappa (CK) was calculated. The presence of any plasmid-mediated colistin resistance (mcr-1, 2, 3, 4 and 5) was evaluated in all colistin-resistant isolates by conventional polymerase chain reaction. Colistin resistance was found in 778 (12.9 %) strains among the carbapenem resistant isolates. Klebsiella pneumoniae had the highest (18.9 %) colistin resistance by the BMD method. MIC of Vitek two had sensitivity ranging from 78.2-84.8% and specificity of >92 %. There were 171 VMEs and 323 MEs by Vitek two method, much more than CLSI acceptable range. The highest percentage of errors was committed for Acinetobacter baumanii (27.8 % of VME and 7.9 % ME). On the other hand, the CBDE method performed well with EA, CA, VME and ME within acceptable range for all the organisms. The sensitivity of the CBDE method compared to gold standard BMD varied from 97.5-98.8 % for different strains with a specificity of more than 97.6 %. None of the isolated colistin resistant organisms harboured mcr plasmids. As BMD has many technical complexities, CBDE is the best viable alternative available for countries like India. A sensitive MIC reported by Vitek two needs to be carefully considered due high propensity for VMEs particularly for Klebsiella spp.

Plasmid-mediated colistin resistance from fresh meat and slaughtered animals in the Czech Republic: nation-wide surveillance 2020-2021.

The aim of this study was to determine the occurrence of plasmid-mediated colistin resistance in domestic and imported meat and slaughter animals in the Czech Republic during 2020-2021 by using selective cultivation and direct PCR testing. A total of 111 colistin-resistant Escherichia coli isolates with mcr-1 gene were obtained from 65 (9.9%, n = 659) samples and subjected to whole-genome sequencing. Isolates with mcr were frequently found in fresh meat from domestic production (14.2%) as well as from import (28.8%). The mcr-1-positive E. coli isolates predominantly originated from meat samples (16.6%), mainly poultry (27.1%), and only minor part of the isolates came from the cecum (1.7%). In contrast to selective cultivation, 205 (31.1%) samples of whole-community DNA were positive for at least one mcr variant, and other genes besides mcr-1 were detected. Analysis of whole-genome data of sequenced E. coli isolates revealed diverse sequence types (STs) including pathogenic lineages and dominance of ST1011 (15.6%) and ST162 (12.8%). Most isolates showed multidrug-resistant profile, and 9% of isolates produced clinically important beta-lactamases. The mcr-1 gene was predominantly located on one of three conjugative plasmids of IncX4 (83.5%), IncI2 (7.3%), and IncHI2 (7.3%) groups. Seventy-two percent isolates of several STs carried ColV plasmids. The study revealed high prevalence of mcr genes in fresh meat of slaughter animals. Our results confirmed previous assumptions that the livestock, especially poultry production, is an important source of colistin-resistant E. coli with the potential of transfer to humans via the food chain. IMPORTANCE We present the first data on nation-wide surveillance of plasmid-mediated colistin resistance in the Czech Republic. High occurrence of plasmid-mediated colistin resistance was found in meat samples, especially in poultry from both domestic production and import, while the presence of mcr genes was lower in the gut of slaughter animals. In contrast to culture-based approach, testing of whole-community DNA showed higher prevalence of mcr and presence of various mcr variants. Our results support the importance of combining cultivation methods with direct culture-independent techniques and highlight the need for harmonized surveillance of plasmid-mediated colistin resistance. Our study confirmed the importance of livestock as a major reservoir of plasmid-mediated colistin resistance and pointed out the risks of poultry meat for the transmission of mcr genes toward humans. We identified several mcr-associated prevalent STs, especially ST1011, which should be monitored further as they represent zoonotic bacteria circulating between different environments.

Characteristics and genomic epidemiology of colistin-resistant Enterobacterales from farmers, swine, and hospitalized patients in Thailand, 2014–2017

BackgroundColistin is one of the last resort therapeutic options for treating carbapenemase-producing Enterobacterales, which are resistant to a broad range of beta-lactam antibiotics. However, the increased use of colistin in clinical and livestock farming settings in Thailand and China, has led to the inevitable emergence of colistin resistance. To better understand the rise of colistin-resistant strains in each of these settings, we characterized colistin-resistant Enterobacterales isolated from farmers, swine, and hospitalized patients in Thailand.MethodsEnterobacterales were isolated from 149 stool samples or rectal swabs collected from farmers, pigs, and hospitalized patients in Thailand between November 2014–December 2017. Confirmed colistin-resistant isolates were sequenced. Genomic analyses included species identification, multilocus sequence typing, and detection of antimicrobial resistance determinants and plasmids.ResultsThe overall colistin-resistant Enterobacterales colonization rate was 26.2% (n = 39/149). The plasmid-mediated colistin-resistance gene (mcr) was detected in all 25 Escherichia coli isolates and 9 of 14 (64.3%) Klebsiella spp. isolates. Five novel mcr allelic variants were also identified: mcr-2.3, mcr-3.21, mcr-3.22, mcr-3.23, and mcr-3.24, that were only detected in E. coli and Klebsiella spp. isolates from farmed pigs.ConclusionOur data confirmed the presence of colistin-resistance genes in combination with extended spectrum beta-lactamase genes in bacterial isolates from farmers, swine, and patients in Thailand. Differences between the colistin-resistance mechanisms of Escherichia coli and Klebsiella pneumoniae in hospitalized patients were observed, as expected. Additionally, we identified mobile colistin-resistance mcr-1.1 genes from swine and patient isolates belonging to plasmids of the same incompatibility group. This supported the possibility that horizontal transmission of bacterial strains or plasmid-mediated colistin-resistance genes occurs between humans and swine.

Detection of Plasmid-Mediated Resistance against Colistin in Multi-Drug-Resistant Gram-Negative Bacilli Isolated from a Tertiary Hospital.

The aim of this study was to determine the prevalence of plasmid-mediated colistin resistance mcr-1 to mcr-5 genes among colistin and multi-drug-resistant Gram-negative bacilli strains isolated from patients in a tertiary hospital in Toluca, Mexico. The presence of mcr genes among the 241 strains collected was assessed by PCR. In the case of mcr-carrying E. coli, further PCR tests were performed to determine the presence of blaCTX-M and whether the strains belonged to the O25b-ST131 clone. Conjugation experiments were also carried out to assess the horizontal transmission of colistin resistance. A total of twelve strains (5.0%), of which four were E. coli; four were P. aeruginosa; three were K. pneumoniae, and one E. cloacae, were found to be resistant to colistin. Of these strains, two E. coli isolates were found to carry mcr-1, and Southern blot hybridization demonstrated its presence on an approximately 60 kb plasmid. Both mcr-1-carrying E. coli strains were found to co-express blaCTX-M, belong to the O25b-ST131 clone, and horizontally transmit their colistin resistance. The results of this study confirm the presence of plasmid-mediated colistin resistance in hospitalized patients in Mexico and demonstrated that the multi-drug-resistant O25b-ST131 E. coli clone can acquire mcr genes and transmit such resistance traits to other bacteria.

The emergence of Klebsiella pneumoniae sequence type 395 non-susceptible to carbapenems and colistin from Turkey

PurposeOXA-48 producing Klebsiella pneumoniae is an emerging threat and outbreaks due to specific sequence types have been commonly reported. Here, we report an outbreak due to multidrug-resistant ST395 K. pneumoniae ST395. To the best of our knowledge, this is the first outbreak of K. pneumoniae ST395 harbouring blaOXA-48 genes in our country. MethodsThe strains were characterized by antimicrobial susceptibility, extended-spectrum β-lactamase (ESBL) and carbapenemase production, plasmid-mediated colistin, high-level aminoglycoside, and quinolone resistance. Also multidrug efflux pumps and porin coding genes were investigated. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), wzi typing and plasmid analysis were used for the epidemiological relationships. ResultsAll strains were positive for blaOXA-48 with at least one of the β-lactamase genes (blaCTX-M, blaTEM, blaSHV) and harboured IncL plasmids. 16 of 20 (80%) isolates carried qnrA. All isolates were positive for aac(6’)-1b, acrAB-tolC, ompK35, and ompK36 genes but none of them harboured 16s rRNA methyltransferase, mcr-1-5, qepA, oqxAB, and mdtK genes. All strains had the same PFGE pattern, that is, wzi type K2 and found to be ST395 with MLST. ConclusionThe association of ST395 with OXA-48-producers could be an emerging threat for Turkey and continuous monitoring is crucial to prevent the spread of these powerful strains.

Genomic Evidence of mcr-1.26 IncX4 Plasmid Transmission between Poultry and Humans.

Colistin is still commonly used and misused in animal husbandry driving the evolution and dissemination of transmissible plasmid-mediated colistin resistance (mcr). mcr-1.26 is a rare variant and, so far, has only been detected in Escherichia coli obtained from a hospitalized patient in Germany in 2018. Recently, it was also notified in fecal samples from a pigeon in Lebanon. We report on the presence of 16 colistin-resistant, mcr-1.26-carrying extended-spectrum beta-lactamase (ESBL)-producing and commensal E. coli isolated from poultry samples in Germany, of which retail meat was the most common source. Short- and long-read genome sequencing and bioinformatic analyses revealed the location of mcr-1.26 exclusively on IncX4 plasmids. mcr-1.26 was identified on two different IncX4 plasmid types of 33 and 38 kb and was associated with an IS6-like element. Based on the genetic diversity of E. coli isolates, transmission of the mcr-1.26 resistance determinant is mediated by horizontal transfer of IncX4 plasmids, as confirmed by conjugation experiments. Notably, the 33-kb plasmid is highly similar to the plasmid reported for the human sample. Furthermore, we identified the acquisition of an additional beta-lactam resistance linked to a Tn2 transposon on the mcr-1.26 IncX4 plasmids of three isolates, indicating progressive plasmid evolution. Overall, all described mcr-1.26-carrying plasmids contain a highly conserved core genome necessary for colistin resistance development, transmission, replication, and maintenance. Variations in the plasmid sequences are mainly caused by the acquisition of insertion sequences and alteration in intergenic sequences or genes of unknown function. IMPORTANCE Evolutionary events causing the emergence of new resistances/variants are usually rare and challenging to predict. Conversely, common transmission events of widespread resistance determinants are quantifiable and predictable. One such example is the transmissible plasmid-mediated colistin resistance. The main determinant, mcr-1, has been notified in 2016 but has successfully established itself in multiple plasmid backbones in diverse bacterial species across all One Health sectors. So far, 34 variants of mcr-1 are described, of which some can be used for epidemiological tracing-back analysis to identify the origin and transmission dynamics of these genes. Here, we report the presence of the rare mcr-1.26 gene in E. coli isolated from poultry since 2014. Based on the temporal occurrence and high similarity of the plasmids between poultry and human isolates, our study provides first indications for poultry husbandry as the primary source of mcr-1.26 and its transmission between different niches.