Dectection And Dissemination Mechanism Of 16S RRNA Methylase Genes Among Proteus Miralis And Salmonella Isolates In Chickens

Plasmid-encoded 16S rRNA methylase, which confer high-level resistance to 4, 6-disubstituted deoxystreptamine aminoglycosides (such as amikacin and gentamicin) in many Gram-negative pathogens, is a novel discovered resistant determinant. Therefore, it has the significance in the antimicrobial fields in veterinary clinic to investigate epidemiology and dissemination mechanism of these 16S rRNA methyltransferase genes among Enterobacteriaceae strains isolated from chickens in chicken farm391 samples including feces and liver between May 2009 and Nov 2010 were collected for the bacterial isolation. After the initial bacterial isolation and further identification by API system and PCR, 20 proteus mirabilis strains and 21 salmonella strains were obtained. Firstly, the drug susceptibility tests were performed for them. Secondly, these isolates were subjected to PCR for the presence of 16S rRNA methylase genes (armA, rmtA, rmtB, rmtC, rmtD and npmA). In addition, ESBL genes (TEM, SHV and CTX-M) and plasmid-mediated quinolone resistant deteriminants (qnr, qepA and aac(6)-ib-cr) were also detected among these strains producing 16S rRNA methylase. Finially, conjugation (Escherichia coli J53AzR or EC600 as the recipient), transformation (E. coli DH10B as the recipient) and PFGE were performed to clarify the dissemination mechanism of 16S rRNA methyltransferase genes among these isolates.The results showed that, 20 Proteus mirabilis isolates were all resistant to seven commonly used antimicrobial agents, the 16S rRNA methylase gene rmtB was detected in all isolates; most of 21 sallonella isolates were susceptible to seven commonly used antimicrobial agents, the 16S rRNA methylase gene armA was detected in one isolate. Of 20 rmtB-producing Proteus mirabilis isolates, there also coexistence with CTX-M-2 and/or CTX-M-9 group ESBLs, the positive rates for CTX-M CTX-M-2 and CTX-M-9 group ESBLs were 90.0% and 35%, respectively. While the armA-producing salmonella isolates, there also coexistence with TEM-1, CTX-M-2 type ESBLs, qnrB and aac(6')-Ib-cr.Many attempts for conjugation and transformation experiment in proteus mirabilis were unsuccessful. This may suggest the vertical transfer but not the horizontal transfer has played an important role in the dissemination of the 16S rRNA methylase gene rmtB in proteus mirabilis. The PFGE results further confirm this situation, which indicated the close relationship exists among these rmtB-producing proteus mirabilis and they may evolve from a common ancestor. The attempts for conjugation experiment in Salmonella were also unsuccessful. The reasons remains further elucidated. However, the transformants were succussfully obtained in Salmonella by electroporation using the E coli DH10B as the recipient. This suggested the 16S rRNA methylase gene armA located in plasmid and the horizontal transfer could have an important role in the dissemination of the 16S rRNA methylase gene armA in Salmonella.In conclusion, the prevalence and molecular epidemiology of 16S rRNA methylase genes among proteus mirabilis and salmonella strains of animal origin were investigated. To our knowledge, the rmtB in Proteus mirabilis and armA in salmonella were first found in isolates of animal origin. The data primarily indicated that the vertical transfer could have played an important role in the dissemination of 16S rRNA methylase gene rmtB among these Proteus mirabilis isolates, while the 16S rRNA methylase gene armA was located on the plasmid in salmonella isolates and horizontal transfer could have played an important role in the dissemination of armA.