Study On The Resistance Genes Of A Multidrug-Resistant Listeria Monocytogenes

The use of antimicrobials in both animals and humans can select for resistant bacterialpopulations. In food animals, antimicrobials are used for the control and treatment of bacterialassociated infectious diseases as well as for growth promotion purposes, An undesiredconsequence of antimicrobial use in animals is the potential development ofantimicrobial-resistant foodborne bacterial pathogens and subsequent transmission to humansas food contaminants. In addition, spontaneous mutation in foodborne bacteria or the spreadof resistant bacteria in the absence of selective pressure may also contribute to theantimicrobial resistance burden in food. In fact, emerging antimicrobial resistance phenotypeshave been recognized among multiple foodborne pathogens including Listeriamonocytogenes.90foodborne L. monocytogenes strains isolated from Hebei province were tested for theirresistance to18antimicrobial agents (β-lactam, quinolone, tetracycline, macrolide,aminoglycoside, glycopeptide, sulfonamides, rifampin) currently used in veterinary andhuman therapy by the broth microdilution method. Among90isolates33(36.7%) displayedresistance to one or more antimicrobials. Antimicrobial resistance was most frequentlyobserved for ciprofloxacin (17.8%), tetracycline (15.6%) and streptomycin (12.2%). Among6multidrug resistant isolates detected,2were resistant to5or more antimicrobials.One isolate, named L78, which showed resistance to five antimicrobials(chloramphenicol, erythromycin, streptomycin, tetracycline, andtrimethoprim-sulfamethoxazole), was then subjected to PCR screening to identify genesresponsible for the multidrug resistance phenotype. Three antibiotic resistancedeterminants(cat, ermB, tetS)were detected. The cat gene encoding an acetyltransferase whichcatalyzes acetyl-S-coenzyme A(CoA)-dependent acetylation of chloramphenicol at the3-hydroxyl group, ermB gene encoding a23S rRNA methyltransferase that modifies themacrolide-lincosamide-streptogramin B antibiotic binding site, while tetS gene coding fortetracycline and minocycline resistance by ribosome protection.PCR primers were designed based on the conserved regions of each determinant toobtain the whole sequence of three resistance genes. Analysis of the sequencing data showed that there were several base differences between cat/tetS sequence of L78and the sequencereported in LM previously by other researchers. However, the cat/tetS sequence showed100%homology with relevant sequences detected in Lactococcus lactis(X92946.1)/Enterococcusfaecalis(HQ426665.1), Lactococcus garvieae(AB290882.1). To date, there were no ermBsequences of LM has been reported in GenBank yet. The ermB sequence of L78we obtainedshowed100%homology with relevant sequences detected in Lactococcusgarvieae(AB290882.1).To assess whether the resistance is plasmid associated, we studied the plasmid content ofthe L78strain and cured the plasmid by novobiocin.The results showed that cat, ermB, tetSwere all located on plasmid and likely to be located on a same multidrug-resistant plasmid.The MICs of chloramphenicol, erythromycin, clindamycin, streptomycin, tetracycline,minocycline, trimethoprim-sulfamethoxazole in L78were all significantly decreased afterplasmid curing.The plasmid with3antibiotic resistance determinants of L78strain showed high stabilitywhen consecutively transferred for more than400generations in Brain Heart Infusion Brothat the absence of the selective pressure.In a word, the antimicrobial resistance of L. monocytogenes has been emerging. Study onthe antibiotic resistance genes of multidrug-resistant strain L78showed that the resistance islikely to be associated with gene transmission, suggesting that widespread surveillance ofantibiotic resistance should be conducted at the gene level to provide proper strategies forlimiting the emergence of antimicrobial-resistant L. monocytogenes.