Mcr-1 Gene Research Articles

Do Pathogenic Escherichia coli Isolated from Gallus gallus in South Africa Carry Co-Resistance Toward Colistin and Carbapenem Antimicrobials?

Colistin and carbapenems are critically important antimicrobials often used as a last resort to manage multidrug-resistant bacterial infections in humans. With limited alternatives, resistance to these antimicrobials is of concern as organisms could potentially spread horizontally rendering treatments ineffective. The aim of this study was to investigate co-resistance to colistin and carbapenems among Escherichia coli isolated from poultry in South Africa. Forty-six E. coli strains obtained from clinical cases of breeder and broiler chickens were used. In addition to other antibiotics, all the isolates were tested against colistin and carbapenems using broth microdilution. Multiplex polymerase chain reactions were used to investigate the presence of colistin (mcr-1 to 5) and carbapenem (blaOXA-48, blaNDM-1, and blaVIM) resistance genes. Isolates exhibiting colistin resistance (>2 μg/mL) underwent a whole-genome sequencing analysis. Resistance to colistin (10.9%) and cefepime (6.5%) was noted with all colistin-resistant strains harboring the mcr-1 gene. None of the E. coli isolates were resistant to carbapenems nor carried the other resistant genes (mcr-2 to 5, blaOXA-48, blaNDM-1, and blaVIM). The mcr-1-positive strains belonged to sequence types ST117 and ST156 and carried virulence genes ompA, aslA, fdeC, fimH, iroN, iutA, tsh, pic, ast A and set 1A/1B. In conclusion, clinical E. coli strains from chickens in this study possessed mobile resistance genes for colistin and several other clinically relevant antimicrobials but not carbapenems. Additionally, they belonged to sequence types in addition to carrying virulence factors often associated with human extraintestinal pathogenic E. coli infections. Thus, the potential risk of transmitting these strains to humans cannot be underestimated especially if sick birds are dispatched into the thriving poorly regulated Cornish hen industry. The need for routine veterinary surveillance and monitoring of antimicrobial resistance, antimicrobial use and the importance of strengthening regulations guiding the informal poultry sector remains important.

Chromosome-Mediated Colistin Resistance in Clinical Isolates of Klebsiella pneumoniae and Escherichia coli: Mutation Analysis in the Light of Genetic Background.

Colistin resistance mechanisms involving mutations in chromosomal genes associated with LPS modification are not completely understood. Mutations in genes coding for the MgrB regulator frequently account for colistin resistance in Klebsiella pneumoniae, whereas mutations in genes coding for PhoPQ and PmrAB are frequent in E. coli. Our aim was to perform a genetic analysis of chromosomal mutations in colistin-resistant (MIC ≥4 µg/mL) clinical isolates of K. pneumoniae (n = 8) and E. coli (n = 7) of different STs. Isolates were obtained in a 3-year period in a university hospital in Santiago, Chile. Susceptibility to colistin, aminoglycosides, cephalosporins, carbapenems and ciprofloxacin was determined through broth microdilution. Whole genome sequencing was performed for all isolates and chromosomal gene sequences were compared with sequences of colistin-susceptible isolates of the same sequence types. None of the isolates carried mcr genes. Most of the isolates were susceptible to all the antibiotics analyzed. E. coli isolates were ST69, ST127, ST59, ST131 and ST14, and K. pneumoniae isolates were ST454, ST45, ST6293, ST380 and ST25. All the isolates had mutations in chromosomal genes analyzed. K. pneumoniae had mutations mainly in mgrB gene, whereas E. coli had mutations in pmrA, pmrB and pmrE genes. Most of the amino acid changes in LPS-modifying enzymes of colistin-resistant isolates were found in colistin-susceptible isolates of the same and/or different ST. Eleven of them were found only in colistin-resistant isolates. Colistin resistance mechanisms depend on genetic background, and are due to chromosomal mutations, which implies a lower risk of transmission than plasmid-mediated genes. Colistin resistance is not associated with multidrug-resistance, nor to high-risk sequence types.

Frequencies of mobilized colistin resistance (mcr-1, 2) genes in clinically isolated Escherichia coli; a cross sectional study

ObjectiveEscherichia coli (E. coli) is an opportunistic bacterium, which is globally recognized for its high prevalence and antimicrobial resistance (AMR). The presence of colistin-resistant representative mcr- 1, 2 genes in multi-drug resistant (MDR) clinically isolated E. coli is the main goal of this survey. After biochemically and molecular confirmation tests, susceptibility testing, biofilm formation, and minimum inhibitory concentration to colistin were performed on 100 E. coli isolates. Subsequently, taking advantage of uniplex-PCR, the presence of some responsible genes (mcr- 1, mcr- 2) to colistin-resistant phenotypes in mentioned bacterium was assessed.ResultsDisc diffusion methods indicated that the highest resistance rate was against ampicillin (80.0%), and trimethoprim/sulfamethoxazole (63%). Among the E. coli isolates, 72 (72.0%) was determined as MDR, respectively. Moreover, 47 (47%) strains were determined as extreme beta-lactamase (ESBL) phenotypes. Among 41 slime-producing E. coli strains, 7 (17.07%), 14 (34.14%), and 20 (48.78%) strains exhibited high, moderate, and weak levels of biofilm formation, respectively. Fifty-nine (81.94%), and 1(100%) of MDR isolates were assessed as colistin resistant (MIC > 2) and susceptible (MIC ≤ 2) as well. In 26(36.11%) of colistin-resistant isolates and 1(1.38%) of colistin, susceptible isolate mcr-1 gene was found. There is no mcr- 2 gene was detected in isolates.ConclusionThe diversity of high antibiotic-resistant rates could be avoided by developing appropriate healthcare policies and community awareness. Alarming resistance rates were observed in colistin and ampicillin, which should be taken into account in therapy guidelines.

Molecular Assessment of MCR-1 Gene among Pandrug-Resistant Acinetobacter baumannii

Background Antimicrobial resistance has become one of the most widespread threats to humans. Acinetobacter baumannii is one of the pathogens responsible for healthcare-associated infections (nosocomial). Colistin is considered the last resort antibiotic against infections with pandrug-resistant (PDR) pathogens. Results: Eleven isolates were detected phenotypically as PDR A. baumannii and were confirmed molecularly using 16S rDNA. The MCR-1 gene was not detected within the chromosomal DNA of the selected isolates. Plasmid bearing the MCR-1 gene was identified in 10 selected isolates of A. baumannii that had not been previously observed to carry the MCR-1 gene. Moreover, the use of colistin in combination with anionic antibiotics or natural compound pterostilbene poses a viable therapeutic alternative for PDR and revives colistin’s bactericidal effects on MCR-1-positive A. baumannii. Finally, the transmission electron microscopy studies proved the synergistic effect of these combinations and revealed the disruption of resistant A. baumannii’s outer membrane and alteration of the permeability properties that allowed overcoming the resistance of the isolates to colistin. Conclusions: Antimicrobial resistance of A. baumannii is related to the presence of the transferable plasmid-bearing MCR-1 gene. This study proved the ability of the combinations of colistin with anionic antibiotics and/or natural compound pterostilbene to restore the bactericidal effect of colistin. Overall, these combinations could be novel promising clinical alternatives against the increasing threat of the widespread multidrug-resistant A. baumannii.

The characteristics of mcr-bearing plasmids in clinical Salmonella enterica in Sichuan, China, 2014 to 2017.

Salmonella is one of the most important zoonotic pathogens and a major cause of foodborne illnesses, posing a serious global public health hazard. The emergence of plasmid-mediated mcr genes in Salmonella has greatly reduced the clinical choice of salmonellosis treatment. The aim of this study was to investigate the plasmid characteristics of mcr-positive Salmonella identified from patients in Sichuan, China during 2014 to 2017 by whole genomes sequencing. In this study, a total of 12 mcr-positive isolates (1.15%, ; mcr-1, n=10; mcr-3, n=2) were identified from 1046 Salmonella isolates using PCR. Further characterization of these isolates was performed through antimicrobial susceptibility testing, conjugation assays, whole genome sequencing, and bioinformatics analysis. The mcr-1 gene in these isolates were carried by three types of typical mcr-1-bearing plasmids widely distributed in Enterobacteriaceae (IncX4, IncI2 and IncHI2). Of note, two mcr-1-harboring IncHI2 plasmids were integrated into chromosomes by insertion sequences. Two mcr-3-bearing plasmids were IncC and IncFIB broad-host-range plasmids respectively. Genetic context analysis found that mcr-1 was mainly located in Tn6330 or truncated Tn6300, and mcr-3 shared a common genetic structure tnpA-mcr-3-dgkA-ISKpn40. Overall, we found that mcr gene in clinical Salmonella were commonly carried by broad-host plasmids and have potential to transfer into other bacteria by these plasmids. Continuous surveillance of MDR Salmonella in humans and investigation the underlying transmission mechanisms of ARGs are vital to curb the current severe AMR concern.

Antibiotic resistance and virulence genes profiling of Vibrio cholerae and Vibrio mimicus isolates from some seafood collected at the aquatic environment and wet markets in Eastern Cape Province, South Africa.

The current study determines the density of Vibrio spp. and isolates V. cholerae and Vibrio mimicus from fish-anatomical-sites, prawn, crab and mussel samples recovered from fish markets, freshwater and brackish water. Virulence and antibiotic resistance profiling of isolates were carried out using standard molecular and microbiology techniques. Vibrio spp. was detected in more than 90% of samples [134/144] and its density was significantly more in fish than in other samples. Vibrio. cholerae and V. mimicus were isolated in at least one sample of each sample type with higher isolation frequency in fish samples. All the V. cholerae isolates belong to non-O1/non-O139 serogroup. One or more V. cholerae isolates exhibited intermediate or resistance against each of the eighteen panels of antibiotics used but 100% of the V. mimicus were susceptible to amikacin, gentamycin and chloramphenicol. Vibrio cholerae exhibited relatively high resistance against polymyxin, ampicillin and amoxicillin/clavulanate while V. mimicus isolates exhibited relatively high resistance against nitrofurantoin, ampicillin and polymixin. The multiple-antibiotic-resistance-index [MARI] for isolates ranges between 0 and 0.67 and 48% of the isolates have MARI that is >0.2 while 55% of the isolates exhibit MultiDrug Resistance Phenotypes. The percentage detection of acc, ant, drf18, sul1, mcr-1, blasvh, blaoxa, blatem, blaoxa48, gyrA, gyrB and parC resistance-associated genes were 2%, 9%, 14%, 7%, 2%, 25%, 7%, 2%, 2%, 32%, 25% and 27% respectively while that for virulence-associated genes in increasing other was ace [2%], tcp [11%], vpi [16%], ompU [34%], toxR [43%], rtxC [70%], rtxA [73%] and hyla [77%]. The study confirmed the potential of environmental non-O1/non-O139 V. cholerae and V. mimicus to cause cholera-like infection and other vibriosis which could be difficult to manage with commonly recommended antibiotics. Thus, regular monitoring of the environment to create necessary awareness for this kind of pathogens is important in the interest of public health.

Antimicrobial resistance- and pathogen patterns in the fecal microbiota of sows and their offspring in German commercial pig farms.

Reducing antibiotic use is one of the biggest challenges in pig farming, as antibiotics have been used for years to control typical problems such as newborn or post-weaning diarrhea. The pressure a one health approach has created on animal production regarding antimicrobial resistance is an opportunity to find other strategies against enterobacterial pathogens in suckling and weaned piglets. A farm-specific approach could have a good success due to the individual farm structures in Germany and other countries. In this study, non-metric multidimensional scaling, hierarchical clustering, and latent class analysis were used to determine the impact of antibiotic use on antibiotic resistance patterns and pathogen prevalence in 20 German pig farms. This may help to develop individualized health strategies. 802 fresh fecal samples were collected from sows and piglets from 20 piglet production and rearing farms at different production times (sows antepartum and postpartum, suckling piglets, weaned piglets). In addition, the use of antibiotics was recorded. DNA extracts were subjected to quantitative real-time qPCR with primers specific for antibiotic resistance genes (int1, sul1-3, dfrA1, mcr-1, blaCTX-M), and virulence factors of relevant bacteria (C. difficile, C. perfringens, Salmonella, Escherichia/Shigella/Hafnia, E. coli). Linear and logistic regression models were used to analyze the relationship between different antibiotics and the major genes contributing to the clustering of observations for the different animal groups. Clustering revealed different farm clusters for sows, suckling piglets, and weaned piglets, with the most remarkable diversity in antibiotic use among weaned piglets. Amoxicillin, lincomycin, and enrofloxacin were identified as the most probable cause of increased odds of the presence of relevant antibiotic resistance genes (mcr1, dfrA1, blaCTX-M). Still, direct effects of a specific antibiotic on its associated resistance gene were rare. Enrofloxacin and florfenicol favored the occurrence of C. difficile in sows. The E. coli fimbriae genes were less affected by antibiotic use in sows and piglets, but the F4 fimbriae gene could be associated with the integrase 1 gene in piglets. The results confirm that multidrug-resistant enterobacteria are widespread in German pig farms and give awareness of the impact of current antibiotic use while searching for alternative health strategies.

Carbapenem-Resistant Gram-Negative Bacilli Characterization in a Tertiary Care Center from El Bajio, Mexico.

Carbapenem-resistant Gram-negative bacilli (CR-GNB) are a major public health concern. We aimed to evaluate the prevalence of CR-GNB and the frequency of carbapenemase-encoding genes in a tertiary referral center from El Bajio, Mexico. A cross-sectional study was conducted between January and October 2022; Gram-negative bacilli (GNB) were screened for in vitro resistance to at least one carbapenem. CR-GNB were further analyzed for carbapenemase-production through phenotypical methods and by real-time PCR for the following genes: blaKPC, blaGES, blaNDM, blaVIM, blaIMP, and blaOXA-48. In total, 37 out of 508 GNB were carbapenem-resistant (7.3%, 95% CI 5.2-9.9). Non-fermenters had higher rates of carbapenem resistance than Enterobacterales (32.5% vs. 2.6%; OR 18.3, 95% CI 8.5-39, p < 0.0001), and Enterobacter cloacae showed higher carbapenem resistance than other Enterobacterales (27% vs. 1.4%; OR 25.9, 95% CI 6.9-95, p < 0.0001). Only 15 (40.5%) CR-GNB had a carbapenemase-encoding gene; Enterobacterales were more likely to have a carbapenemase-encoding gene than non-fermenters (63.6% vs. 30.8%, p = 0.08); blaNDM-1 and blaNDM-5 were the main genes found in Enterobacterales; and blaIMP-75 was the most common for Pseudomonas aeruginosa. The mcr-2 gene was harbored in one polymyxin-resistant E. cloacae. In our setting, NDM was the most common carbapenemase; however, less than half of the CR-GNB showed a carbapenemase-encoding gene.

Structural-Activity Relationship-Inspired the Discovery of Saturated Fatty Acids as Novel Colistin Enhancers.

The emergence and prevalence of mobile colistin resistance gene mcr have dramatically compromised the clinical efficacy of colistin, a cyclopeptide antibiotic considered to be the last option for treating different-to-treat infections. The combination strategy provides a productive and cost-effective strategy to expand the lifespan of existing antibiotics. Structural-activity relationship analysis of polymyxins indicates that the fatty acyl chain plays an indispensable role in their antibacterial activity. Herein, it is revealed that three saturated fatty acids (SFAs), especially sodium caprate (SC), substantially potentiate the antibacterial activity of colistin against mcr-positive bacteria. The combination of SFAs and colistin effectively inhibits biofilm formation and eliminates matured biofilms, and is capable of preventing the emergence and spread of mobile colistin resistance. Mechanistically, the addition of SFAs reduces lipopolysaccharide (LPS) modification by simultaneously promoting LPS biosynthesis and inhibiting the activity of MCR enzyme, enhance bacterial membrane damage, and impair the proton motive force-dependent efflux pump, thereby boosting the action of colistin. In three animal models of infection by mcr-positive pathogens, SC combined with colistin exhibit an excellent therapeutic effect. These findings indicate the therapeutic potential of SFAs as novel antibiotic adjuvants for the treatment of infections caused by multidrug-resistant bacteria in combination with colistin.

The occurrence and molecular detection of mcr-1 and mcr-5 genes in Enterobacteriaceae isolated from poultry and poultry meats in Malaysia.

The advent of antimicrobials-resistant (AMR), including colistin-resistant bacteria, poses a significant challenge to animal and human health, food safety, socio-economic growth, and the global environment. This study aimed to ascertain the colistin resistance prevalence and molecular mechanisms of colistin resistance in Enterobacteriaceae. The colistin resistance was determined using broth microdilution assay, PCR; and Sanger sequencing of mcr genes responsible for colistin resistance in Enterobacteriaceae (n = 627), including Escherichia coli (436), Salmonella spp. (n = 140), and Klebsiella pneumoniae (n = 51), obtained from chicken and chicken meats. Out of 627 Enterobacteriaceae, 8.6% of isolates exhibited colistin resistance phenotypically. Among these colistin resistant isolates, 9.3% (n = 37) were isolated from chicken meat, 7.2% (n = 11) from the cloacal swab of chicken and 7.9% (n = 6) from the litter samples. Overall, 12.96% of colistin-resistant isolates were positive with mcr genes, in which mcr-1 and mcr-5 genes were determined in 11.11% and 1.85% of colistin-resistant isolates, respectively. The E. coli isolates obtained from chicken meats, cloacal swabs and litter samples were found positive for mcr-1, and Salmonella spp. originated from the chicken meat sample was observed with mcr-5, whereas no mcr genes were observed in K. pneumoniae strains isolated from any of the collected samples. The other colistin resistance genes, including mcr-2, mcr-3, mcr-4, mcr-6, mcr-7, mcr-8, mcr-9, and mcr-10 were not detected in the studied samples. The mcr-1 and mcr-5 genes were sequenced and found to be 100% identical to the mcr-1 and mcr-5 gene sequences available in the NCBI database. This is the first report of colistin resistance mcr-5 gene in Malaysia which could portend the emergence of mcr-5 harboring bacterial strains for infection. Further studies are needed to characterize the mr-5 harbouring bacteria for the determination of plasmid associated with mcr-5 gene.

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.

Prevalence of AmpC, ESBL, and colistin resistance genes in Enterobacterales isolated from ready-to-eat food in Algeria.

Antimicrobial resistance among bacteria present in ready-to-eat foods is an emerging concern. Hence, this study investigated the presence of extended-spectrum and AmpC β-lactamases (ESBL/AmpC)-producing Enterobacterales (ESBL-E) and the dissemination of mcr-1 in ESBL-E from ready-to-eat food samples (RTE) in Algeria. RTE food samples (n = 204) were aseptically collected and selectively cultured using MacConkey agar. The isolates were screened for ESBL production using the DDST test, confirmed ESBL-E isolates were identified using different conventional methods and MALDI-TOF MS, antibiotic susceptibility was determined using the disc diffusion and broth microdilution assay, ESBL-E isolates were analyzed for colistin and ESBL/AmpC encoding genes by PCR, and food samples were analyzed by univariate and multiple logistic regression. Overall, 48 (17.4%) of the 276 Enterobacterales were confirmed as ESBL producers, with a high prevalence in soups (40%), salads (25%), and cream-filled pastries (23.8%). Antibiotic susceptibility testing revealed that all the ESBL-E isolates were found multi-drug resistant. PCR revealed that blaTEM, blaCTX-M, blaCMY-2, blaOXA-1, and blaSHV were the most frequently detected. blaCTX-M-9 and blaCTX-M-1 were the predominant CTX-M types. Furthermore, four isolates were positive for mcr-1; three of them harbored the colistin resistance gene and ESBL/AmpC genes (2 E. cloacae and 1 S. enterica). To the best of our knowledge, this is the first report that detects the presence of the mcr-1 gene in ESBL-E strains isolated from RTE foods in Algeria. These findings suggest an urgent need for strict policies that prevent the spread and transmission of ESBL-E in food.

Colistin Resistance in Aquaculture Environment: An Emerging Challenge

AbstractThe emergence of colistin resistance is one of the most serious future issues since it is a last‐resort antibiotic used to treat infections caused by multidrug‐resistant (MDR) gram‐negative bacteria, particularly those resistant to carbapenems. The aquatic environment acts as a reservoir for the emergence and spread of colistin resistant pathogens. The emergence and transmission of a plasmid mediated mcr gene is one of the most important mechanism of colistin resistance in gram negative bacteria. Several mcr variants have been identified in zoonotic pathogens of aquatic origin that may easily transmit this gene to human pathogens. Currently, very few studies have been conducted to test colistin susceptibility in aquaculture associated bacteria. This may be attributed to a limited technical resource for screening colistin‐resistant microorganisms. As a result, to reduce the emergence and spread of colistin resistance in human and animal health, as well as in the environment, the One Health strategy and laboratory methodologies must be expanded.

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

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

Genomic characterisation of multi-drug resistant Escherichia coli and Klebsiella pneumoniae co-harbouring mcr-1 and mcr-3 genes on a single plasmid from paediatric clinical cases

Emergence of plasmid-born mobile colistin resistance (mcr) gene, a growing concern in healthcare. Therefore, this study aims to genomically characterize multidrug-resistant Escherichia coli and Klebsiella pneumoniae co-harboring the mcr-1 and mcr-3 genes in young children. E. coli (n=3) and K. pneumoniae (n=2) were collected from abdominal secretion and blood, respectively. The isolates were screened using tryptone soya broth (TSB) with 4µl/ml polymyxin-B. Growing bacteria were identified using the VITEK-2 system, MALDI-TOF and 16s RNA sequencing, followed by antibiotic susceptibility testing, as well as metallo-β-lactamase (MBL) and extended spectrum β-lactamase (ESBL) production was detected. Afterwards, strains were subjected to molecular screening targeting mcr variants, and ESBLs/MBL encoding genes. Conjugation, PFGE, southern hybridisation, MLST, and Phylogenic group detection were performed. Plasmid-genome sequencing and bioinformatic analysis were performed. E. coli named EC-19-322, 323, and 331 and K. pneumoniae isolates named KP-19-225 and 226 harboured both mcr-1 and mcr-3 genes. These strains were also found to be resistant to more than three classes of antibiotics. The conjugation experiment revealed the presence of mcr-1 and mcr-3 on the single plasmid and the transmission frequency was 10-2 to 10-3. Both strains were found to be able to produce ESBLs and MBL. E. coli EC-19-322 and 323 were identified as an ST131(O25a:H41), SP-19-331as an ST1577 (O16:H30) and K. pneumoniae as an ST231 (K2). All E. coli strains belonged to phylogenetic group B2, and the results of PFGE supported the MLST findings. This study, reported the co-occurrence of mcr-1 and mcr-3 genes on single plasmid in pathogenic ESBLs/MBL-producing E. coli and K. pneumoniae isolated from young children.

Plasmid-Borne Mobile Colistin Resistant Gene (Mcr-1) Detection and Multidrug Resistant Bacteria Isolated from Some Abattoir Environments in Benin City, Edo State, Nigeria

Antibiotics resistance is an increasing public health challenge globally, and very recently, global attention has focused on colistin, which is termed “last resort antibiotics”. This study was aimed at investigating the prevalence of plasmid-borne mobile colistin resistant and multidrug resistant bacteria from 6 major abattoirs located in Benin City, Edo State, Nigeria. Two hundred and eighty-eight samples from fresh water, wastewater, utensils, and handlers were obtained over a 6-months period. Mean mesophilic aerobic bacteria (MAB) and thermotolerant coliform bacteria (TCB) were determined by pour plate method, while the indoor air of the abattoirs was sampled using passive sedimentation technique. Bacterial isolates were identified by morphological, biochemical and 16S rRNA analysis. Phenotypic detection of colistin-resistance as well as multi-drug resistant profile of all isolates was done by the modified Kirky Bauer method. The presence and/or absence of colistin-resistance gene (mcr-1 to mcr-8) were investigated by polymerase chain reaction. The MAB ranged from 0.3 ± 0.0 cfu/m3 in indoor air from both Holy Ghost B and Bob Izua abattoir to 2.6 ± 0.3 cfu/ml in wash water from Holy Ghost A abattoir, while the TCB ranged from 0.0 ± 0.0 cfu/ml in wastewater from Lawal and Sons abattoir to 0.6 ± 0.1 cfu/ml in wash water from Osazee abattoir. A total of 149 bacterial isolates, belonging to 6 different species (Pseudomonas aeruginosa PA01, Enterobacter ludwigii EN-119, Providencia stuartii PRV00010, Klebsiella quasipneumoniae strain KqPF26, Enterococcus saccharolyticus ATCC 43076 and Provincia rettgeri strain AR_0082) were obtained with the majority (&gt;90%) being multidrug resistant. Seven (4.7%) of the isolates were phenotypically resistant to colistin, while only 3 harbored the mcr-1 gene. This result shows that plasmid-borne colistinresistant and multidrug resistant bacteria are prevalent in abattoir environment located in Benin City, Edo State, Nigeria. This is an indication that abattoir facilities could be a source of human exposure to colistin resistant bacteria, and efforts must be made at reducing the high dependence of antibiotics in farm animals.

High prevalence of colistin heteroresistance in specific species and lineages of Enterobacter cloacae complex derived from human clinical specimens

BackgroundColistin (CST) is a last-line drug for multidrug-resistant Gram-negative bacterial infections. CST-heteroresistant Enterobacter cloacae complex (ECC) has been isolated. However, integrated analysis of epidemiology and resistance mechanisms based on the complete ECC species identification has not been performed.MethodsClinical isolates identified as “E. cloacae complex” by MALDI-TOF MS Biotyper Compass in a university hospital in Japan were analyzed. Minimum inhibitory concentrations of CST were determined by the broth microdilution method. The population analysis profiling (PAP) was performed for detecting the heteroresistant phenotype. The heat shock protein 60 (hsp60) cluster was determined from its partial nucleotide sequence. From the data of whole-genome sequencing, average nucleotide identity (ANI) for determining ECC species, multilocus sequence type, core genome single-nucleotide-polymorphism-based phylogenetic analysis were performed. phoPQ-, eptA-, and arnT-deleted mutants were established to evaluate the mechanism underlying colistin heteroresistance. The arnT mRNA expression levels were determined by reverse transcription quantitative PCR.ResultsThirty-eight CST-resistant isolates, all of which exhibited the heteroresistant phenotype by PAP, were found from 138 ECC clinical isolates (27.5%). The prevalence of CST-resistant isolates did not significantly differ among the origin of specimens (29.0%, 27.8%, and 20.2% for respiratory, urine, and blood specimens, respectively). hsp60 clusters, core genome phylogeny, and ANI revealed that the CST-heteroresistant isolates were found in all or most of Enterobacter roggenkampii (hsp60 cluster IV), Enterobacter kobei (cluster II), Enterobacter chuandaensis (clusters III and IX), and Enterobacter cloacae subspecies (clusters XI and XII). No heteroresistant isolates were found in Enterobacter hormaechei subspecies (clusters VIII, VI, and III) and Enterobacter ludwigii (cluster V). CST-induced mRNA upregulation of arnT, which encodes 4-amino-4-deoxy-l-arabinose transferase, was observed in the CST-heteroresistant isolates, and it is mediated by phoPQ pathway. Isolates possessing mcr-9 and mcr-10 (3.6% and 5.6% of total ECC isolates, respectively) exhibited similar CST susceptibility and PAP compared with mcr-negative isolates.ConclusionsSignificant prevalence (approximately 28%) of CST heteroresistance is observed in ECC clinical isolates, and they are accumulated in specific species and lineages. Heteroresistance is occurred by upregulation of arnT mRNA induced by CST. Acquisition of mcr genes contributes less to CST resistance in ECC.

Occurrence of mcr genes and alterations in mgrB gene in intrinsic colistin- resistant Enterobacterales isolated from chicken meat samples

BackgroundTolerance of gram negative pathogens toward last resort colistin is mediated by mcr genes and alterations in chromosomal mgrB via modification of lipopolysaccharide through the PmrAB and PhoPQ component system. Proteus sp., Morganella sp., Neisseria sp., Burkholderia sp. and Providencia sp. are intrinsic resistant to colistin drug. Recent reports have shown that colistin intrinsic resistant organisms harbor and act as reservoirs for mcr genes. AimTo evaluate the presence of mcr-1 to mcr-5 genes and alterations in mgrB gene in intrinsic colistin-resistant gram negative bacteria isolated from chicken meat samples in Coimbatore district, Tamil Nadu, India. MethodsOne hundred chicken meat samples were collected during 2019–20. Samples were enriched and plated on MacConkey agar supplemented with colistin (2 μg/ml). The bacterial isolates were then identified using biochemical tests. DNA were extracted from isolates using the thermal lysis method. mcr-1 to mcr-5 and mgrB genes was detected using conventional PCR and agarose gel electrophoresis methods. Result and conclusionThe presence of mcr-1 to mcr-5 genes was found to be 23 % (23/100). mcr-1 and mcr-5 genes were not detected in sample isolates. 17/23 samples positive for mcr genes were also found to be carrying alterations in mgrB gene. Phenotypic characterization of these isolates revealed that these bacteria were belonging to colistin intrinsic resistant gram negative bacteria such as Proteus sp., Providencia sp., and Morganella sp. Intrinsic resistant bacteria could act as a potential reservoir and disseminate mcr genes to the colistin sensitive non-intrinsic pathogens of clinical importance in the environment.

Multiplex Hybrid Capture Improves the Deep Detection of Antimicrobial Resistance Genes from Wastewater Treatment Plant Effluents to Assess Environmental Issues.

Metagenomic sequencing (mDNA-seq) is one of the best approaches to address antimicrobial resistance (AMR) issues and characterize AMR genes (ARGs) and their host bacteria (ARB); however, the sensitivity provided is insufficient for the overall detection in wastewater treatment plant (WWTP) effluents because the effluent is well treated. This study investigated the multiplex hybrid capture (xHYB) method (QIAseq × HYB AMR Panel) and its potential to increase AMR assessment sensitivity. The mDNA-Seq analysis suggested that the WWTP effluents had an average of 104 reads per kilobase of gene per million (RPKM) for the detection of all targeted ARGs, whereas xHYB significantly improved detection at 601,576 RPKM, indicating an average 5,805-fold increase in sensitivity. For instance, sul1 was detected at 15 and 114,229 RPKM using mDNA-seq and xHYB, respectively. The blaCTX-M, blaKPC, and mcr gene variants were not detected by mDNA-Seq but were detected by xHYB at 67, 20, and 1,010 RPKM, respectively. This study demonstrates that the multiplex xHYB method could be a suitable evaluation standard with high sensitivity and specificity for deep-dive detection, highlighting a broader illustration of ongoing dissemination in the entire community.

Genomic analysis of multidrug-resistant Escherichia coli strains carrying the mcr-1 gene recovered from pigs in Lima-Peru

Antibiotic resistance is a current problem that significantly impacts overall health. The dissemination of antibiotic resistance genes (ARGs) to urban areas primarily occurs through ARG-carrying bacteria present in the gut microbiota of animals raised in intensive farming settings, such as pig production. Hence, this study aimed to isolate and analyzed 87 Escherichia coli strains from pig fecal samples obtained from intensive farms in Lima Department. The isolates were subjected to Kirby-Bauer-Disk Diffusion Test and PCR for mcr-1 gene identification. Disk-diffusion assay revealed a high level of resistance among these isolates to oxytetracycline, ampicillin, cephalothin, chloramphenicol, ciprofloxacin, and doxycycline. PCR analysis identified the mcr-1 gene in 8% (7/87) E. coli isolates. Further, whole genome sequencing was conducted on 17 isolates, including multidrug resistance (MDR) E. coli and/or mcr-1 gene carriers. This analysis unveiled a diverse array of ARGs. Alongside the mcr-1 gene, the blaCTX-M55 gene was particularly noteworthy as it confers resistance to third generation cephalosporins, including ceftriaxone. MDR E. coli genomes exhibited other ARGs encoding resistance to fosfomycin (fosA3), quinolones (qnrB19, qnrS1, qnrE1), tetracyclines (tetA, tetB, tetD, tetM), sulfonamides (sul1, sul2, sul3), amphenicols (cmlA1, floR), lincosamides (inuE), as well as various aminoglycoside resistance genes. Additionally, Multi Locus Sequence Typing (MLST) revealed a high diversity of E. coli strains, including ST10, a pandemic clone. This information provides evidence of the dissemination of highly significant ARGs in public health. Therefore, it is imperative to implement measures aimed at mitigating and preventing the transmission of MDR bacteria carrying ARGs to urban environments.