Plasmid-mediated Colistin Resistance Research Articles

Vegetables and Fruit as a Reservoir of β-Lactam and Colistin-Resistant Gram-Negative Bacteria: A Review.

Antibacterial resistance is one of the 2019 World Health Organization’s top ten threats to public health worldwide. Hence, the emergence of β-lactam and colistin resistance among Gram-negative bacteria has become a serious concern. The reservoirs for such bacteria are increasing not only in hospital settings but in several other sources, including vegetables and fruit. In recent years, fresh produce gained important attention due to its consumption in healthy diets combined with a low energy density. However, since fresh produce is often consumed raw, it may also be a source of foodborne disease and a reservoir for antibiotic resistant Gram-negative bacteria including those producing extended-spectrum β-lactamase, cephalosporinase and carbapenemase enzymes, as well as those harboring the plasmid-mediated colistin resistance (mcr) gene. This review aims to provide an overview of the currently available scientific literature on the presence of extended-spectrum β-lactamases, cephalosporinase, carbapenemase and mcr genes in Gram-negative bacteria in vegetables and fruit with a focus on the possible contamination pathways in fresh produce.

Clonal Dissemination of Plasmid-Mediated Carbapenem and Colistin Resistance in Refugees Living in Overcrowded Camps in North Lebanon.

Carbapenem and colistin-resistant bacteria represent a global public health problem. Refugees carrying these bacteria and living in inadequate shelters can spread these microorganisms. The aim of this study was to investigate the intestinal carriage of these bacteria in Syrian refugees in Lebanon. Between June and July 2019, 250 rectal swabs were collected from two refugee camps in North Lebanon. Swabs were cultured on different selective media. Antibiotic susceptibility testing was performed using the disk diffusion method. Carbapenemase-encoding genes and mcr genes were investigated using real-time polymerase chain reaction (RT-PCR) and standard polymerase chain reaction (PCR). Epidemiological relatedness was studied using multilocus sequence typing (MLST). From 250 rectal swabs, 16 carbapenem-resistant, 5 colistin-resistant, and 4 colistin and carbapenem-resistant Enterobacteriaceae were isolated. The isolates exhibited multidrug-resistant phenotypes. Seven Klebsiella pneumoniae isolates harboured the blaOXA-48 gene, and in addition four K. pneumoniae had mutations in the two component systems pmrA/pmrB, phoP/phoQ and co-harboured the blaNDM-1 gene. Moreover, the blaNDM-1 gene was detected in six Escherichia coli and three Enterobacter cloacae isolates. The remaining five E. coli isolates harboured the mcr-1 gene. MLST results showed several sequence types, with a remarkable clonal dissemination. An urgent strategy needs to be adopted in order to avoid the spread of such resistance in highly crowded underserved communities.

PmrCAB Recombination Events among Colistin-Susceptible and -Resistant Acinetobacter baumannii Clinical Isolates Belonging to International Clone 7.

ABSTRACTAcinetobacter baumannii is a successful nosocomial pathogen due to its genomic plasticity. Homologous recombination allows genetic exchange and allelic variation among different clonal lineages and is one of the mechanisms associated with horizontal gene transfer (HGT) of resistance determinants. The main mechanism of colistin resistance in A. baumannii is mediated through mutations in the pmrCAB operon. Here, we describe two A. baumannii clinical isolates belonging to International Clone 7 (IC7) that have undergone recombination in the pmrCAB operon and evaluate the contribution of mobile genetic elements (MGE) to this phenomenon. Isolates 67569 and 72554 were colistin susceptible and resistant, respectively, and were submitted for short- and long-read genome sequencing using Illumina MiSeq and MinION platforms. Hybrid assemblies were built with Unicycler, and the assembled genomes were compared to reference genomes using NUCmer, Cortex, and SplitsTree. Genomes were annotated using Prokka, and MGEs were identified with ISfinder and repeat match. Both isolates presented a 21.5-kb recombining region encompassing pmrCAB. In isolate 67659, this region originated from IC5, while in isolate 72554 multiple recombination events might have happened, with the 5-kb recombining region encompassing pmrCAB associated with an isolate representing IC4. We could not identify MGEs involved in the mobilization of pmrCAB in these isolates. In summary, A. baumannii belonging to IC7 can present additional sequence divergence due to homologous recombination across clonal lineages. Such variation does not seem to be driven by antibiotic pressure but could contribute to HGT mediating colistin resistance.IMPORTANCE Colistin resistance rates among Acinetobacter baumannii clinical isolates have increased over the last 20 years. Despite reports of the spread of plasmid-mediated colistin resistance among Enterobacterales, the presence of mcr-type genes in Acinetobacter spp. remains rare, and reduced colistin susceptibility is mainly associated with the acquisition of nonsynonymous mutations in pmrCAB. We have recently demonstrated that distinct pmrCAB sequences are associated with different A. baumannii International Clones (IC). In this study, we identified the presence of homologous recombination as an additional cause of genetic variation in this operon, which, to the best of our knowledge, was not mediated by mobile genetic elements. Even though this phenomenon was observed in both colistin-susceptible and -resistant isolates, it has the potential to contribute to the spread of resistance-conferring alleles, leading to reduced susceptibility to this last-resort antimicrobial agent.

Development and validation of novel PCR primers for identification of plasmid-mediated colistin resistance (mcr) genes in various environmental settings

Antibiotic resistance is considered one of the biggest threats to public health and has become a major concern for governments and international organizations. Combating this problem starts with improving global surveillance of antibiotic resistance genes (ARGs) and applying standardized protocols, both in a clinical and environmental context, in agreement with the One Health approach. Exceptional efforts should be directed to controlling ARGs conferring resistance to Critically Important Antimicrobials (CIA). In this study, a systematic literature review to synthesize data on the identification of mcr genes using a PCR technique was performed. Additionally, a novel set of PCR primers for mcr-1 – mcr-9 genes detection was proposed. The developed primers were in silico and experimentally validated by comparison with mcr-specific PCR primers reported in the literature. This validation, besides being a proof-of-concept for primers’ usefulness, provided insight into the distribution of mcr genes in municipal wastewater, clay and river sediments, glacier moraine, manure, seagulls and auks feces and daphnids from four countries. This analysis proved that commonly used primers may deliver false results, and some mcr genes may be overlooked in tested samples. Newly-developed PCR primers turned out to be relevant for the screening of mcr genes in various environments.

Virulence Determinants and Plasmid-Mediated Colistin Resistance mcr Genes in Gram-Negative Bacteria Isolated From Bovine Milk.

A major increase of bacterial resistance to colistin, a last-resort treatment for severe infections, was observed globally. Using colistin in livestock rearing is believed to be the ground of mobilized colistin resistance (mcr) gene circulation and is of crucial concern to public health. This study aimed to determine the frequency and virulence characteristics of colistin-resistant Gram-negative bacteria from the milk of mastitic cows and raw unpasteurized milk in Egypt. One hundred and seventeen strains belonging to Enterobacteriaceae (n = 90), Pseudomonas aeruginosa (n = 10), and Aeromonas hydrophila (n = 17) were screened for colistin resistance by antimicrobial susceptibility testing. The genetic characteristics of colistin-resistant strains were investigated for mcr-1–9 genes, phylogenetic groups, and virulence genes. Moreover, we evaluated four commonly used biocides in dairy farms for teat disinfection toward colistin-resistant strains. Multidrug-resistant (MDR) and extensive drug-resistant (XDR) phenotypes were detected in 82.91% (97/117) and 3.42% (4/117) of the isolates, respectively. Of the 117 tested isolates, 61 (52.14%) were colistin resistant (MIC >2 mg/L), distributed as 24/70 (34.29%) from clinical mastitis, 10/11 (90.91%) from subclinical mastitis, and 27/36 (75%) from raw milk. Of these 61 colistin-resistant isolates, 47 (19 from clinical mastitis, 8 from subclinical mastitis, and 20 from raw milk) harbored plasmid-borne mcr genes. The mcr-1 gene was identified in 31.91%, mcr-2 in 29.79%, mcr-3 in 34.04%, and each of mcr-4 and mcr-7 in 2.13% of the colistin-resistant isolates. Among these isolates, 42.55% (20/47) were E. coli, 21.28% (10/47) A. hydrophila, 19.12% (9/47) K. pneumoniae, and 17.02% (8/47) P. aeruginosa. This is the first report of mcr-3 and mcr-7 in P. aeruginosa. Conjugation experiments using the broth-mating technique showed successful transfer of colistin resistance to E. coli J53-recipient strain. Different combinations of virulence genes were observed among colistin-resistant isolates with almost all isolates harboring genes. Hydrogen peroxide has the best efficiency against all bacterial isolates even at a low concentration (10%). In conclusion, the dissemination of mobile colistin resistance mcr gene and its variants between MDR- and XDR-virulent Gram-negative isolates from dairy cattle confirms the spread of mcr genes at all levels; animals, humans, and environmental, and heralds the penetration of the last-resort antimicrobial against MDR bacteria. Consequently, a decision to ban colistin in food animals is urgently required to fight XDR and MDR bacteria.

Risk Factors and Prevalence of mcr-1-Positive Escherichia coli in Fecal Carriages Among Community Children in Southern Taiwan

Colistin is the last resort antimicrobial for treating multidrug-resistant gram-negative bacterial infections. The plasmid-mediated colistin resistance gene, mcr-1, crucially influences colistin’s resistance transmission. Human fecal carriages of mcr-1-positive Escherichia coli (E. coli) were detected in many regions worldwide; however, only a few studies have focused on children. Therefore, we identified the prevalence and risk factors of mcr-1-positive E. coli in fecal carriages among community children in Southern Taiwan. In this study, 510 stool samples were collected from April 2016 to August 2019 from the pediatric department at a medical center in Southern Taiwan. These samples were collected within 3 days after admission and were all screened for the presence of the mcr-1 gene. Diet habits, travel history, pet contact, and medical history were also obtained from participants to analyze the risk factors of their fecal carriages to mcr-1-positive E. coli. Antimicrobial susceptibility testing was determined using the VITEK 2 system and the broth microdilution test. Twelve mcr-1-positive E. coli. were isolated from 2.4% of the fecal samples. Through multivariate analysis, frequent chicken consumption (at least 3 times per week) had a significantly positive association with the presence of mcr-1-positive E. coli in fecal carriages (adjust odds ratio 6.60, 95% confidence interval1.58– 27.62, p = 0.033). Additionally, multidrug resistance was more common in mcr-1-positive E. coli. (75.0% vs. 39.5%, p = 0.031) than in non-mcr-1-positive Escherichia coli. Furthermore, the percentage of extraintestinal pathogenic E. coli in mcr-1-positive isolates was 83.3%. Some multi-locus sequence types in our mcr-1-positive E. coli were also similar to those isolated from food animals in the literature. The prevalence of fecal carriages of mcr-1-positive E. coli was low among community children in Southern Taiwan. Our data shows that chicken consumption with a higher frequency increases the risk of mcr-1-positive E. coli. in fecal carriages.

Prevalence and Characterization of ESBL/AmpC Producing Escherichia coli from Fresh Meat in Portugal.

The present study aimed to characterize the extended-spectrum β-lactamases and plasmid-mediated AmpC β-lactamases (ESBL/PMAβ) among Escherichia coli producers isolated from beef, pork, and poultry meat collected at retail, in Portugal. A total of 638 meat samples were collected and inoculated on selective medium for the search of E. coli resistant to 3rd generation cephalosporins. Isolates were characterized by antimicrobial susceptibility testing, molecular assays targeting ESBL/AmpC, plasmid-mediated quinolone resistance (PMQR), and plasmid-mediated colistin resistance (PMCR) encoding genes. The highest frequency of E. coli non-wild type to 3rd generation cephalosporins and fluoroquinolones was observed in broiler meat (30.3% and 93.3%, respectively). Overall, a diversity of acquired resistance mechanisms, were detected: blaESBL [blaCTX-M-1 (n = 19), blaCTX-M-15 (n = 4), blaCTX-M-32 (n = 12), blaCTX-M-55 (n = 8), blaCTX-M-65 (n = 4), blaCTX-M-27 (n = 2), blaCTX-M-9 (n = 1), blaCTX-M-14 (n = 11), blaSHV-12 (n = 27), blaTEM-52 (n = 1)], blaPMAβ [blaCMY-2 (n = 8)], PMQR [qnrB (n = 27), qnrS (n = 21) and aac(6’)-Ib-type (n = 4)] and PMCR [mcr-1 (n = 8)]. Our study highlights that consumers may be exposed through the food chain to multidrug-resistant E. coli carrying diverse plasmid-mediated antimicrobial resistance genes, posing a great hazard to food safety and a public health risk.

Comparative genome analysis of colistin-resistant OXA-48-producing Klebsiellapneumoniae clinical strains isolated from two Iranian hospitals

BackgroundCarbapenemase-producing Klebsiella pneumoniae (CP-KP) is becoming extensively disseminated in Iranian medical centers. Colistin is among the few agents that retains its activity against CP-KP. However, the administration of colistin for treatment of carbapenem-resistant infections has increased resistance against this antibiotic. Therefore, the identification of genetic background of co-carbapenem, colistin-resistance K.pneumoniae (Co-CCRKp) is urgent for implementation of serious infection control strategies.MethodsFourteen Co-CCRKp strains obtained from routine microbiological examinations were subjected to molecular analysis of antimicrobial resistance (AMR) using whole genome sequencing (WGS).ResultsNine of 14 K.pneumoniae strains belonged to sequence type (ST)-11 and 50% of the isolates had K-locus type 15. All strains carried blaOXA-48 except for P26. blaNDM-1 was detected in only two plasmids associated with P6 and P26 strains belonging to incompatibility (Inc) groups; IncFIB, IncHI1B and IncFII. No blaKPC, blaVIM and blaIMP were identified. Multi-drug resistant (MDR) conjugative plasmids were identified in strains P6, P31, P35, P38 and P40. MICcolistin of K. pneumoniae strains ranged from 4 to 32 µg/ml. Modification of PmrA, PmrB, PhoQ, RamA and CrrB regulators as well as MgrB was identified as the mechanism of colistin resistance in our isolates. Single amino acid polymorphysims (SAPs) in PhoQ (D150G) and PmrB (R256G) were identified in all strains except for P35 and P38. CrrB was absent in P37 and modified in P7 (A200E). Insertion of ISKpn72 (P32), establishment of stop codon (Q30*) (P35 and P38), nucleotides deletion (P37), and amino acid substitution at position 28 were identified in MgrB (P33 and P42). None of the isolates were positive for plasmid-mediated colistin resistance (mcr) genes. P35 and P38 strains carried iutA, iucD, iucC, iucB and iucA genes and are considered as MDR-hypervirulent strains. P6, P7 and P43 had ICEKp4 variant and ICEKp3 was identified in 78% of the strains with specific carriage in ST11.ConclusionIn our study, different genetic modifications in chromosomal coding regions of some regulator genes resulted in phenotypic resistance to colistin. However, the extra-chromosomal colistin resistance through mcr genes was not detected. Continuous genomic investigations need to be conducted to accurately depict the status of colistin resistance in clinical settings.

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.

NMPA-approved traditional Chinese medicine-Pingwei Pill: new indication for colistin recovery against MCR-positive bacteria infection

BackgroundThe wide spread of plasmid-mediated colistin resistance by mobile colistin resistance (MCR) in Enterobacteriaceae severely limits the clinical application of colistin as a last-line drug against bacterial infection. The identification of colistin potentiator from natural plants or their compound preparation as antibiotic adjuncts is a new promising strategy to meet this challenge.MethodsHerein, the synergistic activity, as well as the potential mechanism, of Pingwei pill plus antibiotics against MCR-positive Gram-negative pathogens was examined using checkerboard assay, time-killing curves, combined disk test, western blot assay, and microscope analysis. Additionally, the Salmonella sp. HYM2 infection models of mouse and chick were employed to examine the in vivo efficacy of Pingwei pill in combination with colistin against bacteria infection. Finally, network pharmacology and molecular docking assay were used to predicate other actions of Pingwei pill for Salmonella infection.ResultsOur results revealed that Pingwei Pill synergistically potentiated the antibacterial activity of colistin against MCR-1-positive bacteria by accelerating the damage and permeability of the bacterial outer membrane with an FIC (Fractional Inhibitory Concentration) index less than 0.5. The treatment of Pingwei Pill neither inhibited bacterial growth nor affected MCR production. Notably, Pingwei Pill in combination with colistin significantly prolonged the median survival in mouse and chick models of infection using the Salmonella sp. strain HYM2, decreased bacteria burden and organ index of infected animal, alleviated pathological damage of cecum, which suggest that Pingwei Pill recovered the therapeutic performance of colistin for MCR-1- positive Salmonella infection in mice and the naturally infected host chick. Pharmacological network topological analysis, molecular docking, bacterial adhesion, and invasion pathway verification assays were performed to identify the other molecular mechanisms of Pingwei Pill as a colistin potentiator against Gram-negative bacteria infection.ConclusionTaken together, NMPA (National Medical Products Administration)-approved Pingwei Pill is a promising adjuvant with colistin for MCR-positive bacterial infection with a shortened R&D (research and development) cycle and affordable R&D cost and risk.

Genome analysis of an OXA-48-producing carbapenem- and colistin-resistant Klebsiella pneumoniae sequence type 11 clone isolated from an inpatient

BackgroundColistin is one of the last-resort therapeutic antibiotics used to control carbapenem-resistant Klebsiella pneumoniae. However, the emergence of colistin-resistant strains with high mortality and morbidity has become a new challenge. Therefore, it is crucial to characterize the genotypic features of carbapenem- and colistin-resistant K. pneumoniae (CCKP) isolates to implement public health intervention strategies. MethodsIn this study, we analyzed the whole genome of an OXA-48-producing CCKP strain for molecular characterization of antimicrobial resistance (AMR) as well as comparative genome and plasmid analysis. ResultsThe whole-genome analysis showed that IRAN-43 genome possessed 140 contigs with 5,585,691 bp length, GC content of 56.9% and N50 score of 200,728. Multi-locus sequence typing (MLST) analysis showed that this K. pneumoniae strain belonged to sequence type (ST) 11. Single amino acid substitutions associated with colistin resistance were found in PhoP (D150G), PmrB (R256G) and RamA (I25T). In addition, two plasmids harboring qnrS1, tetR, tetB (plasmid-1) and blaOXA-48 (plasmid-2) were discovered. No plasmid-mediated colistin resistance (mcr) genes were detected. Integrative conjugative element of K. pneumoniae (ICEKp)4 variant was detected as well as cps locus length of 22,156 bp encoding 17 open reading frames (ORFs) which belonged to K. pneumoniae capsular locus (KL)15-1. ConclusionCCKP has been increasingly reporting from Iran. High frequency of antibiotic resistance genes among ST11 suggests that stricter control measures are needed to prevent the spread of this clone in hospital settings.

Potential sources and characteristic occurrence of mobile colistin resistance (mcr) gene-harbouring bacteria recovered from the poultry sector: a literature synthesis specific to high-income countries.

Understanding the sources, prevalence, phenotypic and genotypic characteristics of mcr gene-harbouring bacteria (MGHB) in the poultry sector is crucial to supplement existing information. Through this, the plasmid-mediated colistin resistance (PMCR) could be tackled to improve food safety and reduce public health risks. Therefore, we conducted a literature synthesis of potential sources and characteristic occurrence of MGHB recovered from the poultry sector specific to the high-income countries (HICs). Colistin (COL) is a last-resort antibiotic used for treating deadly infections. For more than 60 years, COL has been used in the poultry sector globally, including the HICs. The emergence and rapid spread of mobile COL resistance (mcr) genes threaten the clinical use of COL. Currently, ten mcr genes (mcr-1 to mcr-10) have been described. By horizontal and vertical transfer, the mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, and mcr-9 genes have disseminated in the poultry sector in HICs, thus posing a grave danger to animal and human health, as harboured by Escherichia coli, Klebsiella pneumoniae, Salmonella species, and Aeromonas isolates. Conjugative and non-conjugative plasmids are the major backbones for mcr in poultry isolates from HICs. The mcr-1, mcr-3 and mcr-9 have been integrated into the chromosome, making them persist among the clones. Transposons, insertion sequences (IS), especially ISApl1 located downstream and upstream of mcr, and integrons also drive the COL resistance in isolates recovered from the poultry sector in HICs. Genes coding multi-and extensive-drug resistance and virulence factors are often co-carried with mcr on chromosome and plasmids in poultry isolates. Transmission of mcr to/among poultry strains in HICs is clonally unrestricted. Additionally, the contact with poultry birds, manure, meat/egg, farmer’s wears/farm equipment, consumption of contaminated poultry meat/egg and associated products, and trade of poultry-related products continue to serve as transmission routes of MGHB in HICs. Indeed, the policymakers, especially those involved in antimicrobial resistance and agricultural and poultry sector stakeholders-clinical microbiologists, farmers, veterinarians, occupational health clinicians and related specialists, consumers, and the general public will find this current literature synthesis very useful.

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.

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.

Propagation of mcr-1 through Inc FIA in Escherichia coli ST5162 in a tertiary referral hospital of North-East India

Colistin is regarded as the ultimate choice of antibiotic to use against gram-negative bacteria which are resistant to multiple antibiotics. The plasmid-mediated colistin resistance gene, mcr-1, which can transfer horizontally poses a notable risk to public health. Therefore, this study aimed at investigating the occurrence of mcr-1 harboring colistin-resistant Escherichia coli isolates in a tertiary referral hospital in the North-Eastern part of India. A total of 126 E. coli isolates were obtained from different wards of Silchar Medical College and Hospital during August 2018 to February 2019. Phenotypic resistance to colistin was determined by the broth microdilution method and polymyxin NP test. Genotypic characterization was done by PCR assay followed by sequencing. Co-occurrence of other resistance determinants was determined. Transferability was determined by transformation and conjugation assay. PCR- based plasmid incompatibility typing and Multilocus sequence typing (MLST) were performed. 29 E. coli isolates were screened positive for colistin resistance. The mcr-1 gene was found in two E. coli isolates belonging to sequence type ST5162. Co-occurrence of other antibiotic resistance genes namely aph(4)Ia, ant(2″)-Ia, aac(3)-I, aac(3)-IIc, sul1, and qepA were observed. The resistance determinants were found to be horizontally transferable and located within the IncFIA plasmid. This study revealed the propagation and adaptation of colistin resistance through IncFIA in a single clone of E. coli within a clinical setting which is of great concern for infection control and therapeutic choices. Therefore, urgent need for investigation is required to track down their source and continuance within hospital settings to minimize more lateral transfer.

Genetic Comparison of ESBL-Producing Escherichia coli from Workers and Pigs at Vietnamese Pig Farms.

We analyzed and compared genomes of Extended Spectrum Beta-Lactamase (ESBL)-producing Escherichia coli from pigs and pig farm workers at 116 farms in Vietnam. Analyses revealed the presence of blaCTX-M-55, blaCTX-M-27, blaCTX-M-15, blaCTX-M-14, blaCTX-M-3, blaCTX-M-65, blaCTX-M-24, blaDHA-1, and blaCMY2 in both hosts. Most strains from pigs contained quinolones (qnr) and colistin resistance genes (mcr-1 and mcr-3). Isolates predominantly harbored more than one plasmid replicon and some harbored plasmid replicons on the same contigs as the ESBL genes. Five strains from farm workers of ST38 (2), ST69 (1), and ST1722 (2) were classified as either uropathogenic E. coli (UPECHM)/extraintestinal pathogenic E. coli (ExPECJJ) or UPECHM, and the remaining were genetically distinct commensals. A high heterogeneity was found among the ESBL-producing E. coli from pigs and workers, with most isolates belonging to unrelated phylogroups, serogroups, and sequence types with >4046 Single-Nucleotide Polymorphisms-(SNPs). In comparing the genomes of pig isolates to those from humans, it appeared that ESBL-producing E. coli in workers did not predominantly originate from pigs but were rather host-specific. Nevertheless, the occurrence of ESBL-producing E. coli carrying plasmid-mediated colistin and quinolone resistance genes in pigs could represent a potential source for horizontal transmission to humans through food rather than direct contact.

Global distribution of plasmid-mediated colistin resistance mcr gene in Salmonella: A systematic review.

This systematic review focuses on obtaining the most relevant information from multiple studies that detected a mobilized colistin resistance mcr gene in Salmonella for a better comprehension of its global distribution. A group of strategic and systematic keywords were combined to retrieve research data on the detection frequency of the mcr gene globally from four database platforms (Google Scholar, Science Direct, PubMed and Scielo). Forty-eight studies attended all the eligibility criteria and were selected. China was the country with the highest frequency of Salmonella strains with the mcr gene, and Europe exhibited a wide diversity of countries with positive mcr strains. In addition, animals and humans carried the highest frequency of positive strains for the mcr gene. Salmonella Typhimurium was the most frequent serovar carrying the mcr gene. Apparently, colistin overuse in animal husbandry has increased the selective pressure of antimicrobial resistance, resulting in the emergence of a plasmid-mediated colistin resistance mcr gene in China. The mcr-positive Salmonella strains are recently predominant worldwide, which is probably due to the capacity of this gene to be swiftly horizontally transmissible. The transmission ability of mcr-positive Salmonella strains to humans through the consumption of contaminated animal-based food is a public health concern.

Subinhibitory concentration stress of colistin enhanced PhoPQ expression in Escherichia coli harboring mcr‐1

The increased and inappropriate use of colistin led to the emergence of its resistance among Gram-negative bacterial isolates and the most common mechanism of colistin resistance in Gram-negative bacteria is the modification of the lipopolysaccharide mediated by two-component regulatory systems, PhoPQ and PmrAB. The aim of the present study was to investigate the transcriptional expression of the PhoPQ system against colistin stress in clinical isolates of Escherichia coli with colistin-resistant phenotype. Six colistin-resistant E. coli isolates were obtained from Silchar Medical College and Hospital, Silchar that were of clinical origin and received for routine culture and sensitivity testing. Screening for colistin resistance was done by broth microdilution method and further screened for the presence of the different types of plasmid-mediated colistin resistance mcr genes namely, mcr-1 to mcr-10 by polymerase chain reaction (PCR). The screened positive isolates were subjected to PCR assay targeting phoP and phoQ genes and their expression was measured by quantitative real-time PCR. The results of this study revealed that two E. coli isolates (TS2 and TS4) were found to carry the mcr-1 gene. PhoP and PhoQ gene amplification was observed in all the isolates. Transcriptional analysis showed that the isolates harboring the mcr-1 gene showed an enhanced level of expression in the PhoP, PhoQ genes in the presence of a subinhibitory concentration of colistin whereas no significant expression was observed for the isolates which were devoid of the mcr gene. This study demonstrates the involvement of mcr-1 in the PhoPQ system in clinical isolates of colistin-resistant E. coli which will help in designing a molecular marker for detecting colistin-resistant E. coli and contribute to the assessment of resistance burden and infection control strategy.

Emergence of colistin resistance genes (mcr-1) in Escherichia coli among widely distributed wild ungulates

The environment is considered a major reservoir of antimicrobial resistant microorganisms (AMR) and antimicrobial resistance genes (ARG). Colistin, a “last resort” antibiotic, is used for the treatment of severe infections caused by multidrug-resistant Gram-negative bacteria. The global dissemination of mobile colistin resistance genes (mcr) in natural and non-natural environments is a major setback in the fight against antimicrobial resistance. Hitherto, there is a limited number of studies screening this resistance determinant in bacteria from wildlife. In this study, we describe for the first time the detection of plasmid-mediated colistin resistance in Escherichia coli from wild ungulates in Portugal, which are also widely distributed across Europe. This information is critical to identify the importance of ungulates in the dissemination of resistant bacteria, and their corresponding genes, across the environment. Here, 151 resistant-Enterobacteriaceae isolated from 181 samples collected from different wild ungulate species throughout Portugal were screened for mcr genes. Four mcr-1-positive Escherichia coli were detected from four fallow deer individuals that were sampled in the same hunting ground. These four isolates harboured mcr-1-related IncP plasmids belonging to sequencing types ST155, ST533 and ST345 (n = 2), suggesting bacterial and/or plasmid circulation. All mcr-1-positive E. coli also showed other resistance phenotypes, including MDR, including the B1 commensal phylogenetic profile. All mcr-1-positive E. coli show additional resistance phenotypes, including MDR, including the B1 commensal phylogenetic profile. Our findings are upsetting, highlighting the global dissemination of colistin resistance genes in the whole ecosystem, which, under the One Health framework, emphasizes the urgent need for effective implementation of AMR surveillance and control in the human-animal-environment interfaces.

Withdrawal of Colistin Reduces Incidence of mcr-1-Harboring IncX4-Type Plasmids but Has Limited Effects on Unrelated Antibiotic Resistance.

The global dissemination of plasmid-mediated colistin resistance gene mcr and its variants have posed a great threat to public health. Therefore, the Chinese government banned the use of colistin as a feed additive in livestock in April 2017. To explore the dynamic changes of overall antibiotic resistance genes (ARGs) and phylogenetic relationship of bacteria from a single pig farm before and after the withdrawal of colistin, fecal swab samples were collected from a large-scale pig farm before (n = 32; 2 months pre-withdrawal of colistin) and after withdrawal of colistin (n = 30; 13 months post-withdrawal of colistin). Escherichia coli and Klebsiella pneumoniae were isolated. Whole-genome sequencing (Illumina, MiSeq) was performed to examine ARGs, plasmids and the genetic relationship of the isolates. The overall SNP results indicated all isolates had high genetic diversity, and the evolutionary relationship across isolates was not influenced by the ban of colistin. However, the prevalence of mcr-1.1 (5.6%, p < 0.01) was significantly lower than before the ban (86.4%). Plasmid profiling analysis showed that 17 of 20 (85.0%) observed mcr-1.1 genes reside on IncX4-type plasmids, 16 of which (94.1%) were from isolates before the ban. On the contrary, the presence of blaCTX-M gene was significantly increased (p = 0.0215) post-withdrawal of colistin. Our results showed that withdrawal of colistin reduced the incidence of mcr-1-harboring IncX4-type plasmids, but had limited influences on unrelated ARGs.