| Both colistin and polymyxin B belong to the polymyxin group,which is one of the main antibiotics against most gram-negative bacteria.Polymyxin is considered as a treatment of last resort for multidrug-resistant Enterobacter infections due to its superior anti-infective action against Klebsiella pneumoniae,Pseudomonas aeruginosa and Acinetobacter baumannii.In addition,polymyxin E(Colistin)acts as a growth promoter and was commonly used as an antibiotic in livestock production until 2017.A variety of reasons have led to the overuse and even abuse of polymyxins,which has led to the increasingly serious crisis of polymyxinresistant bacteria from human and animal sources around the world.Since polymyxin was widely used in animal husbandry,strains of Enterobacteriaceae from animal origin were considered to be the "source" and "reservoir" of many important polymyxin resistance genes.In recent years,the original mcr-1 gene and several subtypes of the mcr family were also found in animal bacteria isolates.The presence of these resistant strains and components not only harms livestock production,but also poses a potential threat to people and other wildlife if they spread into the surrounding environment.In 2017,China stipulated that polymyxins are not allowed to be used in animal husbandry.Multiple studies have shown increasing reports of animal-derived mcr-positive strains,possibly due to the illegal use of antibiotics and the presence of horizontal transfer elements that allow polymyxin resistance genes to spread between strains,species and even regions.The rapid and extensive spread of polymyxin-resistant strains indicates that a large number of studies are still needed to elucidate the transmission status,causes,characteristics and intricate resistance mechanisms of polymyxin-resistant strains of animal origin.Most of the reports of polymyxin-resistant strains,especially mcr-positive strains,are concentrated in the southeast coastal area.Regional investigation and research in other areas are relatively scarce.In this study,anal swab samples were collected from farmers(pigs,sheep,chickens)and crested ibis breeding and protection centers in central and southern Shaanxi Province,gram-negative bacteria were isolated,antibiotic-resistant strains were detected,and the isolation rate and transmission of antibiotic-resistant strains were analyzed.Focus on mcr gene positive strains,combined with whole genome sequencing technology,to preliminarily explore the strain characteristics and gene environment of epidemic antibiotic-resistant strains.In order to provide more theoretical basis for the epidemic characteristics of mcr-positive strains in China,the mechanism of mcr gene mediated antibiotic resistance and its transmission mechanism.The following results are obtained:(1)A total of 124 Gram-negative strains,80 Escherichia coli,33 Klebsiella pneumoniae,3 Acinetobacter,2 Klebsiella ornitholyticus,1 Praeversia sp.,4 Pseudomonas aeruginosa and1 Enterobacter Hawkella were isolated from 4 different sources.Among them,23 strains were isolated from pig samples,with a separation rate of 33.33%.25 strains were isolated from chicken samples,and the isolation rate was 33.33%.Sixteen isolates were isolated from sheep samples,the separation rate was 34.04%.Sixty strains were isolated from crested ibis samples,and the isolation rate was 87.14%.One strain of K.pneumoniae resistant to polymyxin B was detected,and there were also 28 strains(22.58%)that were critical to polymyxin B and intermediate to multiple antibiotics.The resistance rate of the crested ibis strain to nearly half of the antibiotics was close to or even partially exceeded that of the other three strains.(2)18 strains of E.coli and 17 strains of K.pneumoniae were selected for whole genome sequencing.Based on the WGS data,multi-site sequence typing(MLST),antibiotic resistance genes,virulence genes and plasmid types were analyzed.8 genotypes were identified in E.coli,and many of them were unknown ST type.6 genotypes were identified in K.pneumoniae,among which ST542 was a relatively large genotype.One mcr-9 and phoP/phoQ gene-positive strain was found in E.coli;one mcr-1.1-positive strain was found in K.pneumoniae,and one strain was found in all K.pneumoniae carrying acrA/acrB gene Carry the phoP/phoQ gene.(3)A preliminary study on the resistance mechanism of R42 showed that R42 could be induced to resist polymyxin B.The adaptation cost of host bacteria to mcr-9 gene is small.There was no significant difference in the copy number of mcr-9 gene in the strains with different MIC results for polymyxin B,indicating that the copy number was not the reason for the different resistance of the strains to polymyxin B.Further analysis of R42(mcr-9 positive strain)by third generation whole genome sequencing technology revealed that the R42 strain contained four plasmids,two of which contained the tet(X4)or mcr-9 gene,respectively.pR42＿mcr-9-26 R 793 and pR42＿tet(X4)-26 R 793 were successfully conjured and transferred.The isolated strain R42 belonged to ST86 and serotype O23: H16.Containing a large number of virulence genes and a variety of antibiotic resistance genes;The gene environment of pR42＿mcr-9 gene in pR42＿mcr-9 was relatively simple after comparing with the reference sequence.Two multi-resistant regions(MRRS)were identified in different regions of pR42＿mcr-9,both of which were flanked by IS26 sequences.To sum up,this study analyzed antibiotic-resistant isolations isolated from samples in Shaanxi Province through MIC phenotype analysis,WGS analysis,conjugation transfer and mechanism analysis.We found that although only one strain was resistant to polymyxin-B,there were 28 strains that were in the critical value of polymyxin-B and in the intermediate range of multiple antibiotics.The strains involved carried multiple virulence genes and other important antibiotic resistance genes at the same time.The risk of transmission,disease and resistance should be vigilant.As a new mcr family gene,mcr-9 has been reported in animal and human isolates recently.Multiple studies have shown that its mediated antibiotic resistance mechanism is different from other mcr family genes,which brings new challenges to its monitoring and control.Due to its large stock and complex "communication" with the environment,polymyxin-resistant bacteria of animal origin need more powerful supervision and regulatory measures and more in-depth mechanism research. |
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