| Salmonella is an important zoonotic and foodborne pathogen that poses a serious threat to animal health and human health.Broad-spectrum cephalosporins,especially the third-generation cephalosporins,are among the drugs of preferred choice for the treatment of salmonella infections,and polymyxin is considered to be the“last resort of defense”for the treatment of serious infections caused by Gram-negative bacteria.Recent years,studies at home and abroad have shown that the condition of salmonella resistant to these two types of antimicrobial drugs is becoming increasing serious.Plasmid is an independent,reproducible,mobile genetic factor that carries genes that mediate resistance to cephalosporins and colistin,and mediates the horizontal transfer of antimicrobial resistance genes among different bacteria.Therefore,it is of great theoretical and practical significance to strengthen the research on the the mechanism of salmonella resistant to these two types of drugs,especially the plasmid-mediated resistance mechanism.This study intends to screen cefotaxime-resistant strains from multi-drug resistant salmonella isolated and identified by our laboratory,and to work out plasmid-mediated broad-spectrum cephalosporin and colistin resistance mechanisms.The main results are as follows:1.Screening of cefotaxime-resistant salmonella and its antimicrobial susceptibility evaluation110 multi-drug resistant salmonella strains were screened using MHA plates containing4?g/mL cefotaxime,and 16 strains were found to be resistant to cefotaxime.The minimum inhibitory concentration?MIC?of the 16 salmonella strains against cefotaxime,cefepime,aztreonam,ceftazidime,meropenem and colistin was detected by micro-broth dilution method.The results showed that MIC of cefotaxime were all greater than 128?g/mL.16 strains were sensitive to meropenem,11 strains were resistant to cefepime,10strains were resistant to aztreonam,10 strains were resistant to colistin,and only 2 strains were resistant to ceftazidime.When cefotaxime,cefepime and ceftazidime were used in combination with clavulanic acid,16 strains were all sensitive,suggesting that these salmonella are strains that producing extended-spectrum?-lactamase?ESBLs?.2.Extraction and typing of plasmids of salmonellaPlasmid extraction of 16 salmonella was carried out,plasmid bands were detected in12 salmonella,and 10 of them were colistin-resistant strains.The number of plasmid bands was 2-6,the band sizes were between 1.39-19.56 kb,and a plasmid bands of 19.56kb was present in all 10 colistin-resistant strains.The detection of the plasmid replicon types revealed that the type Inc N replicon was detected in 15 strains,the type Inc HI2replicon was detected in 13 strains,and the type Inc I1 replicon was detected in 1 strain.3.Mechanism of broad-spectrum cephalosporin resistance mediated by plasmid6 types of ESBLs resistance genes were detected by PCR for 16 salmonella strains.The results showed that the gene CTX-M could be detected by all strains,but other genes such as SHV,CMY-2,PER,IMP and KPC were not detected.Furthermore,the CTX-M gene was sequenced,14 strains were subtype CTX-M-14 of CTX-M-9 group,and 2 strains were subtype CTX-M-15 of CTX-M-1 group.16 salmonella strains were used as the donor strain and Escherichia coli NK5449 was used as the recipient strain,the conjugative transfer experiment was carried out by broth method.The conjugants was screened by cefotaxime-rifampin double-drug plates.The results showed that only 10 colistin-resistant strains were capable of transferring the gene CTX-M to the recipient strain by conjugative transfer.4.Mechanism of colistin resistance mediated by plasmidThe results of PCR showed that all the 10 colistin-resistant salmonella strains carried the gene mcr-1,while none of the 6 colistin-sensitive strains carried the gene mcr-1.16porcine salmonella were used as the donor strains,and rifampicin-resistant Escherichia coli NK5449 was used as the recipient strain,the conjugative transfer experiment was carried out by broth method,the conjugants was screened by colistin-rifampin double-drug plates.The results showed that,the conjugative transfer only occurred in 10colistin-resistant strains,and the transfer frequency was between 2.00×10-6 and 7.21×10-5.The full-length of gene mcr-1 was amplified from the colistin-resistant strain FA56,and ligated into the vector pHSG396 by double enzyme digestion,and the recombinant vector was transformed into DH5?competent cells.As a result,the recombinant strain possessed colistin resistance,and the study indicated that the gene mcr-1 was the main factor of colistin resistance of salmonella stains. |
PDF Full Text Request