| BackgroundWith the widespread use of antibiotics,bacterial resistance has become a serious public health problem worldwide.In recent years,the resistance rate of Gram-negative bacilli to common clinical antibiotics has been increasing.Escherichia coli is the most common bacteria.The main reason for its resistance to beta-lactams is the production of extended-spectrum beta-Lactamases(ESBLs).Surveillance of bacterial resistance from 2015 to 2016 in China showed that the detection rate of ESBLs in clinical isolates of Escherichia coli was 59.4%.Increased bacterial resistance will lead to prolonged hospitalization and increased medical costs,which will cause a great burden on patients.It has been pointed out that the large-scale application of antibiotics in animal husbandry has led to the increase of bacterial resistance and accelerated the spread of drug-resistant strains in animals and people.In Shandong Province,the detection rate of the bacteria was 42%in rural residents of non-farming areas,and 82.5%in rural residents of a pig farming county.Objectives1.To understand the drug susceptibility of ESBLs-producing Escherichia coli and the characteristics of beta-lactamase resistance genes among rural residents in Shandong province.2.To describe the molecular typing characteristics of ESBLs-producing Escherichia coli in rural areas of Shandong Province,and to analyze the possible transmission routes of drug-resistant bacteria,so as to provide theoretical basis for the use of antibiotics and the prevention and control of drug-resistant bacteria in this area.Methods1.Source of strain:The research is based on Sino-Swedish Integrated Multisectorial Partnership for Antibiotic Resistance Containment(IMPACT).611 strains of ESBLs-producing Escherichia coli were isolated from the feces of residents in 12 villages of a pig farming county in Shandong Province.8 villages were randomly selected and 30 plants were randomly selected from each village.A total of 240 strains.Sex and age of residents,Whether to raise pigs or not were collected.2.Drug sensitivity test:According to CLSI M100-S27 standard,agar dilution method was used to detect antibiotic sensitivity.The breakpoint values of antibiotics refer to the CLSI standard M100-S27.Since CLSI does not give the standard of tegacycline,the breakpoint values refer to the European Union drug sensitivity test standard EUCAST 2017.3.Detection of beta-lactamase genotype:BlaCTX-M,blaSHV and blaTEM genotypes were detected by PCR.BlaCTX-M strains were further tested for CTX-M-1 group and CTX-M-9 group.4.Molecular typing:DNA extraction kit was used to extract nucleic acid.Molecular typing experiments were carried out on target bacteria using MLST website.Seven housekeeping genes of Escherichia coli were amplified and sequenced by PCR.The target length sequence was obtained by splicing and splicing with Contig software and CLC sequence software.The sequence was uploaded to PubMLST international unified database to obtain the ST sequence of Escherichia coli.The cluster analysis was carried out by eBURSTv3.0 software.5.Genome sequencing:Among the relatively dominant ST10 strains,the most resistant strains were selected for genome sequencing analysis,i.e.all 12 strains resistant to 5 or 6 drugs.At the same time,three strains of non-multidrug resistant strains were randomly selected for comparison.A total of 15 strains of ESBL-producing Escherichia coli ST10 strains were included.DNA was extracted and amplified by PCR.The whole genome of the products was sequenced.The strain sequence was spliced on PATRIC website.The spliced sequence was uploaded to Center for Genomic Epidemiology website for analysis of drug resistance and virulence genes.CLC Genomics Workbench software was used for clustering analysis by the shortest distance method.Main results1.General condition240 strains of ESBLs-producing Escherichia coli were isolated from 240 residents in 8 villages,with 30 people in each village.Among them,men accounted for 55.0%(132/240),women accounted for 45.0%(108/240);the age range was between 18 and 94 years old;pig farmers accounted for 37,5%(90/240),non-pig farmers accounted for 62.5%(150/240).2.Drug sensitivity of ESBL-producing Escherichia coli(1)ESBLs-producing Escherichia coli was resistant to a variety of antimicrobial agents.The resistance rates to cefotaxime,florfenicol,tetracycline and compound neotamine were 100.0%(240/240),90.8%(21]8/240),80.4%(193/240),79.6%(191/240),imipenem,meropenem,tegacycline,cefoxitin,ceftazidime and amikacin were higher,respectively.Lower than 5%.(2)The multidrug resistance rate of ESBLs-producing Escherichia coli was 84.2%(202/240).76.7%(184/240)were resistant to 3-5 drugs and 7.5%(18/240)were resistant to 6 drugs.3.Detection of beta-lactamase genotypeThe positive rate of CTX-M gene was 98.8%(237/240),TEM gene was 40.4%(97/240),and SHV gene was 1.7%(4/240).The main subtypes of CTX-M gene were CTX-M-9 group and CTX-M-1 group,accounting for 22.9%(55/240)and 18.8%(45/240),respectively.4.Molecular typingAmong 240 ESBLs-producing Escherichia coli strains,108 ST-forms were detected.Among them,ST10 was the most,accounting for 13.3%,followed by ST38(9/240),ST648(8/240),ST206(7/240),ST48(7/240),ST46(6/240),ST6338(6/240),ST744(6/240),ST162(5/240),ST349(5/240).The other ST types correspond to 5 or less strains respectively.The results of cluster analysis by eBURSTv3.0 software showed that 14 clones were formed in some of the tested strains.Among them,CC10 was the most important clone group in this study,including 32 ST types,including ST10,ST48,ST167,accounting for 36.7%(88/240).5.Genome-wide SequencingAmong 15 strains of ESBL-producing Escherichia coli ST10,the detection rates of resistance genes to beta-lactamases and tetracyclines were 100.0%(15/15),aminoglycosides and trimethoprim were 80.0%(12/15),sulfonamide resistance genes were 73.3%(11/15),macrolide-lincomide-streptomycin resistance genes were 60.0%(11/15),chloramphenicol resistance genes were 80.0%(12/15).The detection rate of antibiotic resistance genes was 53.3%(8/15),and the detection rate of other types of antibiotic resistance genes was below 50.0%.Virulence genes include gad,iss,cma,iroN,capU,etc.The detection rate of GAD gene is 100.0%(15/15),ISS gene is 60.0%(9/15),and other virulence genes are under 20.0%.Cluster analysis showed that 15 strains of ESBL-producing Escherichia coli ST10 were clustered into two groups,of which E004 and A012,G055 and A022,E042 and A025 had relatively high homology with other strains,and each group of strains came from different villages.Conclusion1.The drug resistance of cefotaxime,florfenicol,tetracycline and trimethoprim to ESBLs-producing Escherichia coli in rural areas of the farming area is serious,and the application and management of these four kinds of antibiotics should be strengthened.2.The resistance genes of beta-lactamase in ESBLs-producing Escherichia coli in the farming area were mainly CTX-M,and the main subtypes were CTX-M-9 and CTX-M-1.3.The molecular typing of ESBLs-producing Escherichia coli carried by rural residents in this farming area has a small aggregation of CC10 and may have a route transmission between animals and humans. |
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