Molecular Characterization Of Multidrug-resistant Salmonella Isolated From Poultry

Salmonella was an important and intestinal pathogen of zoonosis,It threatened the health and safety of human beings and animals seriously.It has always been the focus of attention in medicine,veterinary medicine and public health.In recent years,with the widespread use and abuse of antimicrobial agents for prevention and therapy in infected animals,the rapid dissemination of antimicrobial resistance,especially Multidrug-resistant(MDR),is increasing among poultry-original Salmonella in the worldwide,which is largely inseparable from the bacterial Multidrug-resistance through conjugation and transposition.The purpose of this experiment was to study the antimicrobial susceptibility testing,resistant genes screening and epidemic characteristics of Salmonella isolated from poultry,and the upstream and downstream environments of a tet M gene resistant to tetracycline antibiotics.The Multidrug-resistant isolates were examined by isolation and identification,antimicrobial susceptibility testing,resistant genes screening.ERIC-PCR was used to study the genetic relatedness of a collection of isolates of Salmonella.Conjugation experiments,plasmid extraction,analysis of plasmid incompatibility and PCR mapping were performed to investigate the genetic environment of tet M.1.860 samples from different chicken farms in Henan,Hubei,Shanxi,Jiangxi,Hunan,Anhui,and Shanxi,were isolated from liver of dead chicken suspected to be infected with Salmonella.Reference to food hygiene Hygienic examination standard of Salmonella(GB/T4789.4-2010),156 isolates of Salmonella were identified with bacterial screening by SS agar and CHROMagar TM Salmonella,and amplification and sequencing of 16 S r RNA and specific invasion gene(inv A).2.According to the standards recommended by the CLSI,156 strains of Salmonella isolated from poultry were tested for susceptibility testing of 13 antimicrobial drug.The results showed that the isolates had the highest rates of resistance to amoxicillin,oxytetracycline,sulfonamides and doxycycline,which were 100%,99%,97%,and 87%,respectively,followed by florfenicol?enrofloxacin,gentamicin,the resistance rates were 57%,49%,and 49%,respectively.The resistance rates for ceftiofur,cefquinome,olaquindox and mequindox were 27%,26%,22%,15%.And the isolates were most sensitive to Amikacin and Colistin,the sensitivity rates were 97%,99%,respectively.The isolates were all Multi-drug resistant strains,which had the lowest resistance to three antimicrobial agents and the highest resistance to 11 antimicrobial agents,87% of which were resistance to five or more antibiotics,indicating that there were more Multidrug resistant strains.3.PCR was used to amplify the corresponding drug resistance genes,and some of the isolates were subjected to a conjugation experiments.As for isolates producing the ESBL,bla CTX-M genes were detected in 10 of the 156 Salmonella isolates(6.4%).The most common CTX-M types were bla CTX-M-65,followed by bla CTX-M-14,bla CTX-M-55.Bla TEM was detected in 41 isolates,bla OXA-1 was detected in 29 isolates.bla OXA-2 was detected in 18 isolates.As for isolates producing the Amp Cs,blacmy was detected in 4 isolates,which belonged to blacmy-2 by sequencing.Bla SHV was not detected in all isolates.As for resistant genes of tetracycline,the most common genes were tet A(n=79),tet B(n=60),followed by tet C(n=8),tet M(n=1).tet D gene was not detected.Only rmt B gene was detected among the strains resistant to aminoglycoside.flo R gene was detected in 55 of the 156 Salmonella isolates(35%).As for plasmid-mediated Multidrug efflux pump resistance gene,both oqx A and oqx B genes had a detection rate of 13%.Sul 1 gene had a highest detection rate of 96%.A series of plasmid-mediated quinolone resistance gene was not detected.1 transconjugant containing tet M gene was successfully obtained by conjugation experiments,and the plasmid types belonged to FIA,FIB,and FII.4.CTX-M-producing Salmonella have increased in prevalence and diversity in poulty.The downstream of the bla CTX-M gene were shown specific for the subtype of the gene.Nucleotide sequence analysis revealed that a mobile genetic element ISEcp1 were detected upstream of bla CTX-M,insertion sequence IS903 was located downstream of CTX-M-1 group gene,and orf477 was detected downstream of CTX-M-9 group gene.5.PCR mapping revealed that tet M gene was detected upstream of tnp A1 gene,incomplete fragment of orf13.And tnp R gene was detected downstream.