Characteristics of colistin-resistant Escherichia coli from pig farms in Central China

The emergence and dissemination of colistin resistance in Enterobacterioceae mediated by plasmid-borne mcr genes in recent years now pose a threat to public health.In this study,we isolated and characterized colistin-resistant and for mcr-positive£coli from pig farms in Central China.Between 2018 and 2019,594 samples were collected and recovered 445 E.coli isolates.Among them,33 with colistin resistance phenotypes and 37 that were positive for mcr genes were identified,including 34 positive for mcr-1,one positive for mcr-3,and two positive for both mcr-1 and mcr-3.An insertion of nine bases("CTGGATACG")into mcr-7 in four mcr-positive isolates led to gene dysfunction,and therefore did not confer the colistin resistance phenotype.Antimicrobial susceptibility testing revealed that 37 mcr-positive isolates showed severe drug resistance profiles,as 50% of them were resistant to 20 types of antibiotics.Multilocus sequence typing revealed a heterogeneous group of sequence types in mcr-positive isolates,among which ST10(5/37),ST156(5/37),and 5T617(4/37)were the predominant types.Plasmid conjugation assays showed that mcr-carrying plasmids of 25 mcr-positive isolates were conjugated with£coli recipient,with conjugation frequencies ranging from 1.7 × 10^(-6) to 4.1 × 10^(-3) per recipient.Conjugation of these mcr genes conferred a colistin resistance phenotype upon the recipient bacterium.PCR typing of plasmids harbored in the 25 transconjugants determined six types of plasmid replicons,including lncX4(14/25),FrepB(4/25),Incl2(3/25),lncHI2(2/25),FIB(1/25),and Inch(1/25).This study contributes to the current understanding of antibiotic resistance and molecular characteristics of colistin-resistant£coli in pig farms.

A New Evaluation Method for Antibiotic-Resistant Bacterial Groups in Environment

In the present manuscript it was presented whether spreading of antibiotic resistant bacterial groups in environment could be monitored by our newly developed method by enumerating antibiotic resistant bacterial groups in various biological wastes and composts. Although the numbers were not so high, diverse kinds of colistin resistant bacteria (25 mg·L-1) were included in row cattle feces (1.78 × 104 MPN g-1) and cattle feces manure (>3.84 × 104 MPN g-1). Compost originated from leftover food (>44.8 × 104 MPN g-1) and shochu lee (>320 × 104 MPN g-1) included higher numbers of chlortetracycline resistant Pseudomonas sp., (25 mg·L-1), and row cattle feces included higher numbers of chlortetracycline resistant Enterobacteriacea (15.7 × 104 MPN g-1), which mostly consisted from Pantoea sp. or Xenorhobdus doucetiae. Numbers of multi drug resistant bacteria, resistant to 25 mg·L-1 of ciprofloxacin, streptomycin, chloramphenicol, and ampicillin, were the highest in row cattle feces (>143.6 × 104 MPN g-1), followed by cattle feces manure (4.19 × 104 MPN g-1), and shochu lee (0.36 × 104 MPN g-1), which included diverse kinds of bacterial group. The present results indicated that higher numbers of multi drug resistant bacteria were typically found in row cattle feces, and the method was found suitable to enumerate and identify them. These results suggested that the method might become their environmental risk evaluation method.

A One-Health Sampling Strategy to Explore the Dissemination and Relationship Between Colistin Resistance in Human,Animal,and Environmental Sectors in Laos

This study was designed to investigate the molecular epidemiology of mobile colistin resistance(mcr)using a"One-Health"approach in Laos and to predict whether any dominant plasmid backbone and/or strain type influences the dissemination of mcr.We collected 673 samples from humans(rectal normal flora),poultry,and the environment(water,flies,birds,etc.)in Vientiane,Lao People’s Democratic Republic(Laos),from May to September 2018.A total of 238 Escherichia coli(E.coli)isolated from nonduplicative samples,consisting of 98 MCR-positive E.coli(MCRPEC)("mcr"denotes the gene encoding mobile colistin resistance,and"MCR"denotes the subsequent protein encoded by mcr)and 140 MCRnegative E.coli(MCRNEC),were characterized by phenotype and Illumina sequencing.A subset of MCRPEC was selected for Min ION sequencing,conjugation assay,plasmid stability,and growth kinetics in vitro.The prevalence of MCRPEC was found to be 14.6%(98/673),with the highest prevalence in human rectal swabs(45.9%(45/98),p<0.0001,odds ratio(OR):0.125,95% confidence interval(CI):0.077-0.202).The percentages of MCRPEC from other samples were 14.3%(2/14)in dog feces,12.0%(24/200)in flies,11.0%(11/100)in chicken meat,8.9%(8/90)in chicken cloacal,8.0%(4/50)in chicken caeca,and 7.5%(4/53)in wastewater.MCRPEC was significantly more resistant to co-amoxiclav,sulfamethoxazoletrimethoprim,levofloxacin,ciprofloxacin,and gentamicin than MCRNEC(p<0.05).Genomic analysis revealed the distribution of MCRPEC among diverse clonal types.The putative plasmid Inc types associated with mcr-1 were Inc X4,Inc HI2,Inc P1,Inc I2,and Inc FIA,and those associated with mcr-3 were Inc FII,Inc FIA,Inc FIB,Inc P1,and Inc R.Recovery of highly similar plasmids from both flies and other sampling sectors implied the role of flies in the dissemination of mcr-1.mcr-positive plasmids were shown to be conjugative,and a significantly high transfer rate into a hypervirulent clone ST1193 was observed.Plasmids containing mcr irrespective of Inc type were highly stable and invariably did not exert a fitness effect upon introduction into a new host.These findings signify the urgent need for a standard infection control program to radically decontaminate the source of resistance.

Discovery of multi-drug resistant,MCR-1 and ESBL-coproducing ST117 Escherichia coli from diseased chickens in northeast China

An endemic multi-drug resistant ST117 E. coil isolate coproducing MCR-I and 3 ESBL loci was, for the first time, detected from diseased chicken, Liaoning Province, in Northeast China, from 2011 to 2012. Whole- genome sequencing revealed 5 unique plasmids, namely pHXH-1, pHXH-2, pHXH-3, pHXH-4 and pHXH- 5）. Among them, pHXHI and pHXH4 encode ESBL, and pHXH-5 mediates MCR-1 colistin resistance. The results indicate that the potentially-national dissemination of MCR-l-positive pathogens with pan-drug resistance proceeds via food chains.

Genomic insights into the ESBL and MCR-l-producing ST648 Escherichia coli with multi-drug resistance

Polymyxin acts as an ultimate line of refuge against the severe infections by multidrug-resistant Gram- negative pathogens. This conventional idea is challenged dramatically by the recent discovery of mobile colistin resistance gene （mcr-1） is prevalent in food animals and human beings worldwide. More importantly, the mcr-1 gene was found to be co-localized with other antibiotic resistance genes, raising the possibility that super-bugs with pan-drug resistance are emerging. However, little is reported on the genomes of the mcr-l-positive bacterial host reservoirs. Here we report genome sequencing of three human isolates of the mcr-l-positive Escherichia coli （E15004, E15015 and E15017） and define general features through analyses of bacterial comparative genomics. Fur- ther genomic mining together with sequence typing allowed us to elucidate that the MCR-l-carrying E. coli E15017 belongs to the sequence type ST648 and copro- duces extended-spectrum β-1actamase （ESBL）. Given the fact that ST648 has been known to associate New Delhi metallo-β-1actamase 1 or ESBL, with either our results highlighted the possibility of ST648 as an epidemic clone with multidrug resistances.