Prevalence Of Mcr-1 Research Articles

Low prevalence of mcr-1 in Escherichia coli from food-producing animals and food products in China

Backgroundmcr-1-positive Escherichia coli has emerged as a significant threat to human health, veterinary health, and food safety in recent years. After the prohibition of colistin as a feed additive in animal husbandry in China, a noticeable reduction in both colistin resistance and the prevalence of mcr-1 was observed in E. coli from animals and humans.ObjectivesTo assess the prevalence of the colistin resistance gene mcr-1 and characterize its genetic context in E. coli strains derived from fecal and meat samples from food-producing animals in China.MethodsA total of 1,353 fecal samples and 836 food samples were collected between 2019 and 2020 in China. E. coli isolates were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and their susceptibility to colistin were determined using the broth microdilution method. The colistin-resistant E. coli isolates were screened for the presence of mcr by PCR analysis and sequencing. The minimal inhibitory concentrations (MICs) of 15 antimicrobial agents against the mcr-1-positive strains were further tested using the agar dilution method, conjugation assays were performed, and whole genome sequencing was performed using Illumina HiSeq.ResultsIn total, 1,403 E. coli strains were isolated. Thirteen isolates from chicken meat (n = 7), chickens (n = 3), and pigs (n = 3) were resistant to colistin with MIC values of 4 to 16 mg/L, and carried mcr-1. All mcr-1-positive strains, except for isolate AH20PE105, contained multiple resistance genes and exhibited multidrug-resistant phenotypes. They belonged to 10 sequence types (STs), including a novel ST (ST14521). mcr-1 was located on IncI2 (n = 9), IncX4 (n = 2), and IncHI2 (n = 2) plasmids, which were highly similar to other mcr-1-carrying plasmids sharing the same incompatibility type. Seven mcr-1-carrying plasmids could be successfully conjugally transferred to E. coli C600.ConclusionsWhile the low prevalence of mcr-1 (0.93%) identified in this study may not immediately seem alarming, the very emergence of this gene merits attention given its implications for colistin resistance and public health. Hence, ongoing surveillance of mcr-1 in E. coli remains crucial.

Characteristics, Whole-Genome Sequencing and Pathogenicity Analysis of Escherichia coli from a White Feather Broiler Farm.

Avian colibacillosis, caused by avian Escherichia coli (E. coli), has historically been one of the most prevalent infectious diseases in large-scale poultry production, causing growth delays and mortality in chickens, resulting in huge economic losses. In recent years, the widespread use of antibiotics has led to the emergence of multidrug resistance in E. coli as a significant global problem and long-term challenge. Resistant E. coli can be transmitted to humans through animal products or the environment, which presents significant public health concerns and food safety issues. In this study, we analyzed the features of 135 E. coli strains obtained from a white feather broiler farm in Shandong, China, including antimicrobial susceptibility tests, detection of class 1 integrons, drug resistance genes, virulence genes, and phylogenetic subgroups. It is particularly worrying that all 135 E. coli strains were resistant to at least five antibiotic agents, and 100% of them were multidrug-resistant (MDR). Notably, the resistance genes of blaTEM, blaCTX-M, qnrS, aaC4, tetA, and tetB exhibited a high prevalence of carriage among the tested resistance genes. However, mcr-2~mcr-9 were not detected, while the prevalence of mcr-1 was found to be 2.96%. The most common virulence genes detected were EAST1 (14.07%, encoding enterotoxins) and fyuA (14.81%, encoding biofilm formation). Phylogenetic subgroup analysis revealed that E. coli belonging to groups B2 and D, which are commonly associated with high virulence, constituted 2.22% and 11.11%, respectively. The positive rate of class 1 integrons was 31.1%. Whole-genome sequencing (WGS) and animal experiments were performed on a unique isolated strain called 21EC78 with an extremely strong membrane-forming capacity. The WGS results showed that 21EC78 carried 11 drug resistance genes and 16 virulence genes. Animal experiments showed that intraperitoneal injection with 2 × 105 CFU could cause the death of one-day-old SPF chickens in 3 days. However, the mortality of Luhua chickens was comparatively lower than that of SPF chickens. This study reports the isolation of multidrug-resistant E. coli strains in poultry, which may pose a potential threat to human health via the food chain. Furthermore, the findings of this study enhance our comprehension of the frequency and characteristics of multidrug-resistant E. coli in poultry farms, emphasizing the urgent need for improved and effective continuous surveillance to control its dissemination.

Molecular Characterization of Escherichia coli Producing Extended-Spectrum ß-Lactamase and MCR-1 from Sick Pigs in a Greek Slaughterhouse.

The first prospective surveillance of ESBL and colistin-resistant Escherichia coli recovered from sick pigs from a slaughterhouse in Central Greece aimed to investigate the spread of relevant genetic elements. In February 2021, 25 E. coli isolates were subjected to antimicrobial susceptibility testing using disk diffusion and broth microdilution techniques. PCR screening was conducted to identify ESBLs and mcr genes. Additional assays, encompassing mating-out procedures, molecular typing utilizing Pulsed-Field Gel Electrophoresis, multilocus sequence typing analysis, and plasmid typing, were also conducted. A 40% prevalence of ESBLs and an 80% prevalence of MCR-1 were identified, with a co-occurrence rate of 32%. The predominant ESBL identified was CTX-M-3, followed by SHV-12. Resistance to colistin, chloramphenicol, cotrimoxazol, and ciprofloxacin was detected in twenty (80%), fifteen (60%), twelve (48%), and four (16%) isolates, respectively. All blaCTX-M-3 harboring plasmids were conjugative, belonging to the incompatibility group IncI1, and approximately 50 kb in size. Those carrying blaSHV-12 were also conjugative, classified into incompatibility group IncI2, and approximately 70 kb in size. The mcr-1 genes were predominantly located on conjugative plasmids associated with the IncX4 incompatibility group. Molecular typing of the ten concurrent ESBL and MCR-1 producers revealed seven multilocus sequence types. The heterogeneous population of E. coli isolates carrying resistant genes on constant plasmids implies that the dissemination of resistance genes is likely facilitated by horizontal plasmid transfer.

Characteristics of Two mcr-1-Harboring IncHI2 Plasmids from Clinical Salmonella Isolates in Jiaxing City.

Salmonella is a primary cause of foodborne diseases, and the increasing prevalence of mcr-1-carrying plasmids, which confer colistin resistance to Salmonella, poses significant global health concerns. As the frequency of occurrence of the mcr-1 gene is increasing globally, we studied the prevalence of mcr-1 in clinical Salmonella isolates by analyzing 195 clinical strains isolated in 2020. Of the 195 Salmonella isolates, 41 isolates were resistant to colistin. We found mcr-1 in two strains (Salmonella Typhimurium ZJJX20006 and Salmonella Kentucky ZJJX20014), which we analyzed in detail via whole-genome sequencing and antibiotic susceptibility testing. Two strains displayed resistance to ampicillin, ampicillin-sulbactam, tetracycline, chloramphenicol, and cotrimoxazole, while ZJJX20006 displayed resistance to colistin and ZJJX20014 was sensitive. Genomic analysis revealed that these strains had plasmid-encoded mcr-1 in IncHI2 plasmids, which were not similar to the mcr-1-IncX4 identified in 2016. These two strains also harbored other drug resistance genes, including blaOXA-1 and blaCTX-M-14. Our findings may help clarify the molecular mechanisms of mcr-1 dissemination among Salmonella strains in Jiaxing City and offer insights into the evolution of mcr-1 in Salmonella.

Prevalence of colistin-resistant mcr-1-positive Escherichia coli isolated from children patients with diarrhoea in Shanghai, 2016–2021

The emergence of the plasmid-mediated colistin resistance 1 (mcr-1) of Escherichia coli has become a global health concern. This study reports the prevalence of mcr-1 among E. coli isolates from diarrheal children under 5 years old in Shanghai and the genetic characterization of mcr-1-harbouring plasmid. A total of 1,723 E. coli strains were collected from the feces of diarrheal patients in all sentinel hospitals in Shanghai during 2016-2021. Antimicrobial susceptibility testing was performed by broth microdilution and plasmid conjunction transfer assay was carried out using E. coli C600 as the recipient. The mcr-1-positive E. coli strains (MCRPEC) were subjected to molecular characterization and bioinformatic analysis of the mcr-1-bearing plasmids that they harboured. Only 5 (0.28%) strains were found to harbour the mcr-1 gene using PCR screening. Plasmid conjugation assay and whole genome sequencing indicated EC16500, one MCRPEC strain, co-exhibited mcr-1, blaTEM-1, blaOXA-1, qnrS1, qnrS2, arr-3, and catB3, could be conjugated to EC C600 by horizontal transfer with an average efficiency of 3.2 × 10-5. The plasmid pEC16500 harboured the similar backbone as p70_2_15, pECGD-8-33, pNCYU-29-19-1_MCR1 and pIBMC_mcr1, and was shown to be encoded within a type IV secretion system (T4SS)-containing 32.6 kbp IncX4, next to the pap2-like membrane-associated gene to form a 2.4-kb cassette. Furthermore, sequencing and phylogenetic analyses revealed a similarity between other MCR-1-homolog proteins, indicating that the 5 E. coli isolates were colistin resistant. Our data represents a significant snapshot of colistin resistance mcr-1 genes and highlights the need to increase active surveillance especially among the children under 5 years old in Shanghai. Great effort needs to be taken to avoid further dissemination of plasmid-mediated colistin resistance among clinically relevant Gram-negative bacterial pathogens.

Genomic epidemiology and transmission characteristics of mcr1-positive colistin-resistant Escherichia coli strains circulating at natural environment

MCR-positive Escherichia coli (MCRPEC) have been reported in humans worldwide. The high prevalence of mcr-1 poses clinical and environmental risks due to its diverse genetic mechanisms. Given the vital role of animals and the environment in the spread of antibiotic resistance, a “One Health” perspective should be taken when addressing antimicrobial resistance issues. This study conducted a prospective study in six farms (located in Jiaxing City, Zhejiang province, China) in 2019. MCRPEC strains were screened from samples of different sources. The molecular epidemiological surveys and transmission potential were investigated by whole-genome sequencing and phylogenetic analysis. MCRPEC were detected in different farms with various sources. Sequence type complex 10 was dominant and distributed widely in multiple sources. Core-genome multilocus sequence type (cgMLST) analysis indicated that clonal transmission could occur within and between farms. In addition, mcr-1 genes with different locations showed different transmission tendencies. The study indicated that interspecies and cross-regional transmission of MCRPEC could occur between different sectors in farms. Further surveillance and research of non-clinical MCRPEC strains are necessary to reduce the threat of MCRPEC.

Greater Invasion and Persistence of mcr-1-Bearing Plasmids in Escherichia coli than in Klebsiella pneumoniae.

The emergence of the plasmid-borne polymyxin resistance gene mcr-1 threatens the clinical utility of last-line polymyxins. Although mcr-1 has disseminated to various Enterobacterales species, the prevalence of mcr-1 is the highest among Escherichia coli isolates while remaining low in Klebsiella pneumoniae. The reason for such a difference in prevalence has not been investigated. In this study, we examined and compared the biological characteristics of various mcr-1 plasmids in these two bacterial species. Although mcr-1-bearing plasmids were stably maintained in both E. coli and K. pneumoniae, the former presented itself to be superior by demonstrating a fitness advantage while carrying the plasmid. The inter- and intraspecies transferability efficiencies were evaluated for common mcr-1-harboring plasmids (IncX4, IncI2, IncHI2, IncP, and IncF types) with native E. coli and K. pneumoniae strains as donors. Here, we found that the conjugation frequencies of mcr-1 plasmids were significantly higher in E. coli than in K. pneumoniae, regardless of the donor species and Inc types of the mcr-1 plasmids. Plasmid invasion experiments revealed that mcr-1 plasmids displayed greater invasiveness and stability in E. coli than in K. pneumoniae. Moreover, K. pneumoniae carrying mcr-1 plasmids showed a competitive disadvantage when cocultured with E. coli. These findings indicate that mcr-1 plasmids could spread more easily among E. coli than among K. pneumoniae isolates and that mcr-1 plasmid-carrying E. coli has a competitive advantage over K. pneumoniae, leading to E. coli being the main mcr-1 reservoir. IMPORTANCE As infections caused by multidrug-resistant "superbugs" are increasing globally, polymyxins are often the only viable therapeutic option. Alarmingly, the wide spread of the plasmid-mediated polymyxin resistance gene mcr-1 is restricting the clinical utility of this last-line treatment option. With this, there is an urgent need to investigate the factors contributing to the spread and persistence of mcr-1-bearing plasmids in the bacterial community. Our research highlights that the higher prevalence of mcr-1 in E. coli than in K. pneumoniae is attributed to the greater transferability and persistence of mcr-1-bearing plasmid in the former species. By gaining these important insights into the persistence of mcr-1 in different bacterial species, we will be able to formulate effective strategies to curb the spread of mcr-1 and prolong the clinical life span of polymyxins.

Conjugative IncHI2/HI2A plasmids harbouring mcr-9 in colistin-susceptible Escherichia coli isolated from diseased pigs in Japan.

Colistin is a last resort antimicrobial used for the treatment of gram-negative bacterial infections. Plasmid-mediated colistin resistance (mcr) genes are a cause of global concern, and, thus far, mcr-1-10 have been identified. In a previous study, we screened mcr-1-5 in Escherichia coli derived from diseased pigs in Japan and reported a high prevalence of mcr-1, -3 and -5. However, the previous report on the prevalence of mcr genes was inaccurate. In the present study, we aimed to clarify the prevalence of all reported variants of mcr in E. coli derived from the diseased pigs, which were previously screened for mcr-1-5. Additionally, we also characterized the mcr-9-positive E. coli , which was detected in this study. We screened mcr in 120 E. coli strains from diseased pigs and mcr-positive E. coli and an mcr-carrying plasmid were also characterized. One mcr-9-positive colistin-susceptible E. coli strain was detected (0.8 %). Plasmid-mediated mcr-9 was transferred to E. coli ML4909 as the recipient strain, and it was located on IncHI2/HI2A plasmid p387_L with other antimicrobial resistance genes (ARGs). The region harbouring ARGs including mcr-9, was similar to that on the Klebsiella pneumoniae chromosome harbouring mcr-9 isolated in Japan. mcr-3, -5 and -9 were detected (4.2 %) in colistin-susceptible strains. mcr-9 was found to be disseminated via the plasmid IncHI2/HI2A p387_L and transferred and inserted into chromosomes via a transposon. Our results suggest that mcr genes should be monitored regularly, regardless of their susceptibility to colistin.

Genetic and drug susceptibility profiles of mcr-1-bearing foodborne Salmonella strains collected in Shenzhen, China during the period 2014–2017

Colistin resistance mediated by mcr-1-bearing plasmids poses a new challenge to treatment of Salmonella infections. To probe the scale of the problem that colistin resistance mediated by mcr-1 plasmids among Salmonella, the prevalence of mcr-1 in foodborne Salmonella recovered from 2014 to 2017 in Shenzhen, China and genetic profile of mcr-1 positive isolates were investigated. All mcr-1 positives Salmonella strains were collected from food products, characterized by PCR and MALDI-TOF, and subjected to antimicrobial susceptibility testing, whole-genome sequencing, bioinformatics analysis, and conjugation. Twenty-eight mcr-1-positive Salmonella strains were recovered from pork. The rate of recovery displayed an increasing trend and was often accompanied by multidrug resistance. Salmonella Typhimurium was the most prevalent serotypes. Comparative genomic analysis indicated that the mcr-1 gene was located on the transferable IncX4 plasmids, as well as the IncHI2 plasmids, in which the gene was associated with ISApl1. All two types of plasmids were often detected in zoonotic pathogen. Transferable 251K mcr-1-bearing IncHI2 type plasmids were frequently reported in human and food-producing animals, but this is first time to detect a certain number in food. These findings show that dissemination of these two types of plasmids is responsible for the increase in the prevalence of colistin resistance in Salmonella strains in recent years, leading to rapid emergence of MDR Salmonella upon acquisition of these two mcr-1-bearing plasmids. Transmission of IncX4 and IncHI2 plasmids in Salmonella would cause huge public health concerns in controlling foodborne infections caused by Salmonella.

Antimicrobial resistance surveillance of Escherichia coli from chickens in the Qinghai Plateau of China.

Antimicrobial resistance (AMR) may lead to worldwide epidemics through human activities and natural transmission, posing a global public safety threat. Colistin resistance mediated by the mcr-1 gene is the most prevalent among animal-derived Escherichia coli, and mcr-1-carrying E. coli have been frequently detected in central-eastern China. However, animal-derived E. coli with AMR and the prevalence of mcr-1 in the Qinghai Plateau have been rarely investigated. Herein, 375 stool samples were collected from 13 poultry farms in Qinghai Province and 346 E. coli strains were isolated, of which eight carried mcr-1. The AMR rates of the E. coli strains to ampicillin, amoxicillin/clavulanic acid, and tetracycline were all above 90%, and the resistance rates to ciprofloxacin, cefotaxime, ceftiofur, and florfenicol were above 70%. Multidrug-resistant strains accounted for 95.66% of the total isolates. Twelve E. coli strains showed colistin resistance, from which a total of 46 AMR genes and 36 virulence factors were identified through whole-genome sequencing. The mcr-1 gene resided on the IncHI2, IncI2-type and IncY-type plasmids, and mcr-1 was located in the nikA-nikB-mcr-1-pap2 gene cassette (three strains) or the pap2-mcr-1-ISApl1 structure (one strain). Completed IncI2-type plasmid pMCR4D31–3 sequence (62,259 bp) revealed that it may cause the horizontal transmission of mcr-1 and may increase the risk of its spread through the food chain. Taken together, the AMR of chicken-derived E. coli in the plateau is of concern, suggesting that it is very necessary for us to strengthen the surveillance in various regions under the background of one health.

The prevalence, antibiotic resistance and multilocus sequence typing of colistin-resistant bacteria isolated from Penaeus vannamei farms in earthen ponds and HDPE film-lined ponds in China.

The aquaculture environment, especially the culture ponds and aquaculture products, is considered to be an important reservoir of colistin resistance genes. However, systematic investigations of colistin resistance in Penaeus vannamei farming in different culture modes are scarce. In this study, a total of 93 non-duplicated samples were collected from P. vannamei farms in five cities in China from 2019 to 2021. The prevalence, antibiotic resistance and multilocus sequence typing (MLST) of colistin-resistant bacteria were measured and analysed. The results showed that among the 1601 isolates in P. vannamei and its environmental samples, the pollution of colistin-resistant bacteria was serious (the overall prevalence was 37.3% and 28.8%, respectively), regardless of the earthen pond or high-density polyethylene (HDPE) film-lined pond. Among 533 isolates, the prevalence of mobile colistin resistance (mcr) genes, mcr-1, was the highest (60%, 320/533), followed by mcr-4 (1.5%, 8/533), mcr-8 (0.9%, 5/533), mcr-10 (0.6%, 3/533) and mcr-7 (0.4%, 2/533). The prevalence of mcr-1 in earthen ponds was significantly higher than that in HDPE film-lined ponds (67.5% vs. 49.1%, p < .001). The dominant strain carrying mcr-1 was Bacillus spp. (54.1%, 173/320), followed by Enterobacter spp. (8.1%, 26/320), Staphylococcus spp. (6.3%, 20/320) and Aeromonas spp. (5.3%, 17/320). The antibiotic resistance profiles of 173 Bacillus spp. varied among different sampling locations and culture types. These isolates were highly resistant to cefepime, ceftriaxone, trimethoprim-sulfamethoxazole and ceftiofur (>45%), and multidrug-resistant isolates were common (62.4%, 108/173). Sequence type (ST) 26 (37/66, 56%) was found to be the most prevalent ST in mcr-1-positive Bacillus cereus isolated from the aquaculture environment. In summary, our study pointed out that it is necessary to continuously monitor antibiotic usage and its residues regardless of the pond types, especially with regard to critical drugs such as colistin.

Carriage of Plasmid-Mediated Colistin Resistance-1-Positive Escherichia coli in Humans, Animals, and Environment on Farms in Vietnam.

Plasmid-Mediated Colistin Resistance 1 (mcr-1) was first reported in 2015 and is a great concern to human health. In this study, we investigated the prevalence of mcr-1 and mcr-1-positive Escherichia coli (MCRPEC) and the association in infection status among various reservoirs connected to livestock. The study was conducted in 70 poultry and swine farms in a commune in Ha Nam province, northern Vietnam. Samples were collected from farmers, food animals, domestic animals, and farm environments (flies and wastewater) for polymerase chain reaction (PCR) screening for mcr-1 gene and species identification of PCR positive isolates. Among 379 obtained mcr-1 positives isolates, Escherichia coli was the major identified, varying from 50% (2/4) in dog feces to 100% (31/31) in humans feces isolates. The prevalence of MCRPEC was 14.4% (20/139), 49.7% (96/193), 31.3% (25/80), 36.7% (40/109), 26.9% (18/67), and 3.9% (2/51) in humans, chickens, pigs, flies, wastewater, and dogs, respectively. The study identified association between MCRPEC infection status in humans and flies (OR = 3.4), between flies and chickens (OR = 5.3), and between flies and pigs (OR = 9.0). Farmers' age and farm livestock unit were also associated factors of MCRPEC infection status in humans (OR = 5.1 and 1.05, respectively). These findings bring new knowledge on antibiotic resistance in livestock setting and important suggestions on potential role of flies in the transmission of mcr-1 resistance gene.

MCR-1-dependent lipid remodelling compromises the viability of Gram-negative bacteria

ABSTRACT The global dissemination of the mobilized colistin resistance gene, mcr-1, threatens human health. Recent studies by our group and others have shown that the withdrawal of colistin as a feed additive dramatically reduced the prevalence of mcr-1. Although it is accepted that the rapid reduction in mcr-1 prevalence may have resulted, to some extent, from the toxic effects of MCR-1, the detailed mechanism remains unclear. Here, we found that MCR-1 damaged the outer membrane (OM) permeability in Escherichia coli and Klebsiella pneumonia and that this event was associated with MCR-1-mediated cell shrinkage and death during the stationary phase. Notably, the capacity of MCR-1-expressing cells for recovery from the stationary phase under improved conditions was reduced in a time-dependent manner. We also showed that mutations in the potential lipid-A-binding pocket of MCR-1, but not in the catalytic domain, restored OM permeability and cell viability. During the stationary phase, PbgA, a sensor of periplasmic lipid-A and LpxC production that performed the first step in lipid-A synthesis, was reduced after MCR-1 expression, suggesting that MCR-1 disrupted lipid homeostasis. Consistent with this, the overexpression of LpxC completely reversed the MCR-1-induced OM permeability defect. We propose that MCR-1 causes lipid remodelling that results in an OM permeability defect, thus compromising the viability of Gram-negative bacteria. These findings extended our understanding of the effect of MCR-1 on bacterial physiology and provided a potential strategy for eliminating drug-resistant bacteria.

Prevalence of colistin resistance gene mcr-1 in Escherichia coli isolated from chickens in central China, 2014 to 2019

This study investigated the prevalence and characteristics of mcr-1-harbouring Escherichia coli isolated from chickens in central China from 2014 to 2019. A total of 1132 E. coli isolated from 1647 chicken swabs were analysed for colistin susceptibility by broth microdilution method and prevalence of mcr-1 gene by PCR. The colistin-resistant E. coli isolates were typed by multi-locus sequence typing (MLST) and tested with 12 antimicrobial agents. The transconjugation assay was conducted for the mcr-1-positive isolates using the transconjugant E. coli C600. Of the 1132 E. coli isolated from chickens, 131 isolates (11.6%) exhibited colistin resistance, and 51 isolates (4.5%) were mcr-1 positive. The mcr-1-positive rate was quite low in 2014 (2.3%) and 2015 (1.7%), increased to peak in 2016 (12.6%) and 2017 (11.4%), and then decreased significantly in 2018 (1.7%) and 2019 (0.9%). The 131 colistin resistant isolates were assigned to 66 unique sequence types (STs), 27 of which contained mcr-1-positive isolates. Compared with mcr-1-negative E. coli, mcr-1-positive E. coli showed higher resistance rates to nalidixic acid, ciprofloxacin, ceftriaxone, cefotaxime, and tetracycline. Furthermore, 30 of the 51 mcr-1 positive isolates transduced their mcr-1 gene into E. coli C600, and 13 of the 30 transconjugants carried more than one replicon types. The mcr-1 positive rate varied enormously during 2014-2019 in central China. The ban on colistin likely decreased the dissemination of mcr-1 in E. coli isolates from chickens. Multidrug-resistant trait is observed in mcr-1 positive E. coli isolates and can be transferred into other transconjugants.

Molecular Epidemiology and Colistin-Resistant Mechanism of mcr-Positive and mcr-Negative Escherichia coli Isolated From Animal in Sichuan Province, China.

Colistin is the last line of defense for the treatment of multidrug-resistant gram-negative bacterial infections. However, colistin resistance is gradually increasing worldwide, with resistance commonly regulated by two-component system and mcr gene. Thus, this study aimed to investigate molecular epidemiology and colistin-resistant mechanism of mcr-positive and mcr-negative Escherichia coli isolates from animal in Sichuan Province, China. In this study, a total of 101 colistin-resistant E. coli strains were isolated from 300 fecal samples in six farms in Sichuan Province. PCR was used to detect mcr gene (mcr-1 to mcr-9). The prevalence of mcr-1 in colistin-resistant E. coli was 53.47% (54/101), and the prevalence of mcr-3 in colistin-resistant E. coli was 10.89% (11/101). The colistin-resistant E. coli and mcr-1–positive E. coli showed extensive antimicrobial resistance profiles. For follow-up experiments, we used 30 mcr-negative and 30 mcr-1–positive colistin-resistant E. coli isolates and E. coli K-12 MG1655 model strain. Multi-locus sequence typing (MLST) of 30 strains carrying mcr-1 as detected by PCR identified revealed six strains (20%) of ST10 and three strains (10%) of each ST206, ST48, and ST155 and either two (for ST542 and 2539) or just one for all other types. The conjugation experiment and plasmid replicon type analysis suggest that mcr-1 was more likely to be horizontally transferred and primarily localized on IncX4-type and IncI2-type plasmid. The ST diversity of the mcr-1 indicated a scattered and non-clonal spreading in mcr-1–positive E. coli. Twenty-eight mcr-negative colistin-resistant E. coli isolates carried diverse amino acid alterations in PmrA, PmrB, PhoP, PhoQ, and MgrB, whereas no mutation was found in the remaining isolates. The finding showed the high prevalence of colistin resistance in livestock farm environments in Sichuan Province, China. Our study demonstrates that colistin resistance is related to chromosomal point mutations including the two-component systems PhoP/PhoQ, PmrA/PmrB, and their regulators MgrB. These point mutations may confer colistin resistance in mcr-negative E. coli. These findings help in gaining insight of chromosomal-encoded colistin resistance in E. coli.

Characterization of a mcr-1 and CRISPR-Cas System Co-harboring Plasmid in a Carbapenemase-Producing High-Risk ST11 Klebsiella pneumoniae Strain.

We set out to study the prevalence of the mcr-1 gene in carbapenemase-producing Klebsiella pneumoniae (CPKP) strains, and to determine whether its presence is associated with a fitness cost. A total of 234 clinical CPKP isolates were collected from a tertiary medical center in Taiwan from January 2018 to January 2019. The mcr-1 and carbapenemase genes were detected by polymerase chain reaction (PCR) followed by Sanger sequencing. The mcr-1-positive carbapenemase-producing strain was characterized by whole genome sequencing, a plasmid stability test and a conjugation assay. In vitro growth rate and an in vivo virulence test were compared between the parental mcr-1-positive strain and its mcr-1 plasmid-cured strain. We identified only one mcr-1 positive strain (KP2509), co-harboring blaKPC–2 and blaOXA–48, among 234 (1/234, 0.43%) CPKP strains. KP2509 and its Escherichia coli mcr-1 transconjugant showed moderate colistin resistance (MIC = 8 mg/L). The mcr-1 is located on a large conjugative plasmid (317 kb), pKP2509-MCR, with three replicons, IncHI, IncFIB, and IncN. Interestingly, a complete Type IV-A3 CRISPR-Cas system was identified in pKP2509-MCR. Plasmid pKP2509-MCR was highly stable in KP2509 after 270 generation of passage, and the pKP2509-MCR cured strain PC-KP2509 showed similar growth rate and in vivo virulence in comparison to KP2509. The prevalence of mcr-1 in CPKP strains remains low in our center. Notably, we identified a large plasmid with multiple replicons containing both the mcr-1 and the Type IV-3A CRISPR-Cas genes. The further spread of this highly stable plasmid raises concern that it may promote the increase of mcr-1 prevalence in CPKP.

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

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

Prevalence and risk factors of mcr-1-positive volunteers after colistin banning as animal growth promoter in China: a community-based case–control study

ObjectivesChina banned the use of colistin as animal growth promoter in April 2017. Herein, we report the prevalence of mcr-1 in the intestine of healthy humans and risk factors associated with mcr-1 carriage after the implementation of the ban. MethodsWe recruited 719 healthy volunteers from Shenzhen City from 1 March 2018 to 31 December 2019 to investigate the prevalence of mcr-1 in human intestine, and undertook a case–control study to ascertain the risk factors associated with the mcr-1-positive population. A further comparative study was conducted to identify differences between genetic characteristics of mcr-1-positive and mcr-1-negative Escherichia coli. ResultsOverall, 56 (7.8%, 95% CI 5.9%–10.0%, n = 719) individual faecal samples were positive for mcr-1, and prevalence of mcr-1 among individuals in 2019 (2.4%, 95% CI 8.7%–15.0%, 7/294) was significantly lower than that in 2018 (11.5%, 95% CI 1.0%–4.8%, 49/425) (p < 0.0001). After the colistin ban, animal-derived food (pork and chicken meat) was no longer a risk factor for mcr-1 carriage in human intestine, whereas a higher intake of fish and seafood (>75 g/day) and whole grains (>150 g/day) was associated with higher and lower risk of mcr-1 carriage, respectively (OR 2.175, 95% CI 1.047–4.517; OR 0.045, 95% CI 0.004–0.567). Compared with mcr-1-negative E. coli, the mcr-1-positive E. coli had different patterns of resistance genes and genetic heterogeneity. ConclusionsOur study implicates aquatic food as beeing associated with mcr-1 carriage in the healthy population, even after the ban on colistin. Dietary modification (e.g. whole grains) may help to combat mcr-1-positive bacterial colonization of the gut.

Phenotypic and genotypic characterization of mcr-1-positive multidrug-resistant Escherichia coli ST93, ST117, ST156, ST10, and ST744 isolated from poultry in Poland

BackgroundA plasmid-mediated mechanism of bacterial resistance to polymyxin is a serious threat to public health worldwide. The present study aimed to determine the occurrence of plasmid-mediated colistin resistance genes and to conduct the molecular characterization of mcr-positive Escherichia coli strains isolated from Polish poultry.MethodsIn this study, 318 E. coli strains were characterized by the prevalence of mcr1–mcr5 genes, antimicrobial susceptibility testing by minimal inhibitory concentration method, the presence of antimicrobial resistance genes was screened by PCR, and the biofilm formation ability was tested using the crystal violet staining method. Genetic relatedness of mcr-1-positive E. coli strains was evaluated by multilocus sequence typing method.ResultsAmong the 318 E. coli isolates, 17 (5.35%) harbored the mcr-1 gene. High antimicrobial resistance rates were observed for ampicillin (100%), tetracycline (88.24%), and chloramphenicol (82.35%). All mcr-1-positive E. coli strains were multidrug-resistant, and as many as 88.24% of the isolates contained the blaTEM gene, tetracycline (tetA and tetB), and sulfonamide (sul1, sul2, and sul3) resistance genes. Additionally, 41.18% of multidrug-resistant, mcr-1-positive E. coli isolates were moderate biofilm producers, while the rest of the strains showed weak biofilm production. Nine different sequence types were identified, and the dominant ST was ST93 (29.41%), followed by ST117 (17.65%), ST156 (11.76%), ST 8979 (11.76%), ST744 (5.88%), and ST10 (5.88%). Moreover, the new ST was identified in this study.ConclusionsOur results showed a low occurrence of mcr-1-positive E. coli strains isolated from Polish poultry; however, all the isolated strains were resistant to multiple antimicrobial agents and were able to form biofilms at low or medium level.

Mcr-1 Identified in Fecal Escherichia coli and Avian Pathogenic E. coli (APEC) From Brazil.

Colisitin-associated resistance in bacteria of food producing animals has gained significant attention with the mcr gene being linked with resistance. Recently, newer variants of mcr have emerged with more than nine variants currently recognized. Reports of mcr associated resistance in Escherichia coli of poultry appear to be relatively limited, but its prevalence requires assessment since poultry is one of the most important and cheapest sources of the world’s protein and the emergence of resistance could limit our ability to treat disease outbreaks. Here, 107 E. coli isolates from production poultry were screened for the presence of mcr 1–9. The isolates were collected between April 2015 and June 2016 from broiler chickens and free-range layer hens in Rio de Janeiro, Brazil. All isolates were recovered from the trachea and cloaca of healthy birds and an additional two isolates were recovered from sick birds diagnosed with colibacillosis. All isolates were screened for the presence of mcr-1 to 9 using PCR and Sanger sequencing for confirmation of positive genes. Additionally, pulse field gel electrophoresis (PFGE) analysis, avian fecal E. coli (APEC) virulence associated gene screening, plasmid replicon typing and antimicrobial resistance phenotype and resistance gene screening, were also carried out to further characterize these isolates. The mcr-1 gene was detected in 62 (57.9%) isolates (61 healthy and 1 APEC) and the mcr-5 gene was detected in 3 (2.8%) isolates; mcr-2, mcr-3, mcr-4, mcr-6, mcr-7, mcr-8, and mcr-9 were not detected in any isolate. In addition, mcr 1 and 5 positive isolates were phenotypically resistant to colistin using the agar dilution assay (> 8ug/ml). PFGE analysis found that most of the isolates screened had unique fingerprints suggesting that the emergence of colistin resistance was not the result of clonal dissemination. Plasmid replicon types IncI2, FIB, and B/O were found in 38, 36, and 34% of the mcr positive isolates and were the most prevalent replicon types detected; tetA and tetB (32 and 26%, respectively) were the most prevalent antimicrobial resistance genes detected and iutA, was the most prevalent APEC virulence associated gene, detected in 50% of the isolates. Approximately 32% of the isolates examined could be classified as APEC-like, based on the presence of 3 or more genes of APEC virulence associated path panel (iroN, ompT, hlyF, iss, iutA). This study has identified a high prevalence of mcr-1 in poultry isolates in Brazil, suggesting that animal husbandry practices could result in a potential source of resistance to the human food chain in countries where application of colistin in animal health is practiced. Emergence of the mcr gene and associated colisitin resistance in production poultry warrants continued monitoring from the animal health and human health perspective.