Study On Bacterial Polymyxin-resistance Genes And Drug-resistance Phenotype In Guangzhou And Zhongshan

Polymyxin is known as the last line of defense against multi-drug resistant Gram-negative bacteria.Research on polymyxin-resistance genes has been hot in recent years because the discovery of plasmid-mediated gene(mcr-1).In order to further improve the polymyxin-resistance genes spectrum in Guangzhou and Zhongshan,this study collected the clinical isolates of Gram-negative strains from the top three hospital in Guangzhou,and the Proteus mirabilis in fresh meat from some meat markets in Zhongshan,and the P.mirabilis in pigs and environment of some livestock farms in Zhongshan.The drug-resistant phenotype,detection of polymyxin-resistance genes and related researches were then carried out.The clinical isolates were identified and drug-resistant profiles were defined by using a fully automated microbial identification and drug susceptibility system and some auxiliary experiments.Clinical isolates of Gram-negative bacterias were collected and the results were statistically analyzed.The research strains were subjected to genomic DNA extraction by boiling.The polymyxin-resistance genes were amplified by polymerase chain reaction(PCR),and the PCR products with positive results were sent to sequencing to ensure the accuracy.At the same time,drug-resistant profiles were defined according to the standards established by the Clinical and Laboratory Standards Institute(CLSI).The Kirby-Bauer disk diffusion method and broth dilution method were carried out.Subsequently,conjugation experiments and the others were performed to study the transferability of the drug-resistance genes.The ERIC-PCR was carried out to to analyze clonal relationship of mcr-1-positive isolates.Results are as follows:(1)A total of 1190 strains of clinically isolated Gram-negative bacteria were collected,including: 363 strains of Pseudomonas aeruginosa,221 strains of Escherichia coli,206 strains of Acinetobacter baumannii,204 strains of Klebsiella pneumoniae,75 strains of P.mirabilis,and the other 121 strains.According to the strains,the corresponding antimicrobial susceptibility tests were carried out,and the results showed that the drugresistant phenomenon was not serious.(2)There was a mcr-1-carrying isolate E.coli 5798 in 1190 clinical isolates,and no strain carrying mcr-2,mcr-3,mcr-4,or mcr-5 was found;and there were nine mcr-1-carrying strains detected in 1114 isolates of P.mirabilis: 1DM18 b,1acx3a,1acx46 a,1bcm6b,1ccx14 b,1Y33a,1acD2 a,19Pb and 10 Cb.(3)All of the 10 mcr-1-carrying isolates were resistant to 3 or more antibiotics,the drug-resistance phenomenon was serious.5798 was sensi-tive to polymyxin B(PLB)and polymyxin E(colistin,COL).1acx3 a,1bcm6b,1acD2 a and 19 Pb were not resistant to PLB and COL,and the other 5 strains were resistant to PLB and COL.(4)The mcr-1-carrying isolate 5798 was successfully transferred by conjugation and a plasmid was got,and the plasmid was larger than 15 kb.The other 9 P.mirabilis were failed by conjugation.The size of plasmids extracted from 1acx46 a,1bcm6b and 1ccx14 b were more than 15 kb,the plasmids of 1acD2 a,1Y33a,1DM18 b and 19 Pb were about 15 kb,and the fragment of 10 Cb was less than 2.5 kb,and 1acx3 a did not extract the plasmid.It was verified that the resistance genes of 5798,1acD2 a,1DM18b,19 Pb and 1ccx14 b were located in plasmid,and 1acx46 a,1acx3a,1bcm6 b,1Y33a and 10 Cb were located in chromosome.(5)10 mcr-1-carrying isolates were successfully typed by ERIC-PCR,with similar values of 0.5-0.85.The similarity of 75% or more can be divided into 7 clusters of A-G,and the similarity of more than 90% can be judged as the same genotype.No strain of the same genotype was found in this classification.In summary,the drug-resistance phenomenon of 1190 strains was not serious.The detection rate of polymyxin-resistance genes in this study was very low,but the drug-resistance phenomenon of 10 mcr-1-carrying isolates were severely.The 5 strains of mcr-1 were located in the plasmid and the other 5 strains were located in the chromosome.The genotypes of 10 mcr-1-carrying isolates were different.Through the research in this paper,the polymyxin-resistance genes spectrum in Guangdong was further completed.The results of antimicrobial susceptibility tests provided a scientific reference for the use of antibiotics and drug-resistance monitoring.