The Transmittion Mechanism And Gene Environment Analysis Of Mefa/Msrd In Streptococcus Suis

A mechanism of resistance to macrolides based on an efflux system has recently emerged among clinical isolates of Streptococcus suis. It is due to the presence of macrolide efflux (mef) genes which is characterized by low resistance to macrolides. The transmittion mechanism and genetic environment of mef/msr in other Streptococcus speciese have been reported, while the study in S. suis was lack. Here we report the first characterization of a mef/msr-carrying genetic element in S. suis as well as the molecular typing by PFGE and MLST.In this study, the macrolide resistance genes mefA/msrD in strains of Streptococcus suis was detected by PCR method. And the MICs of8antimicrobial agents were determined by microdilution method recommended by CLSI. Then prophage related genes in the strains carrying mefA/msrD gene were detected and prophage was induced with mitomycin C, purified and observed by electron microscopy. Long range PCR was used to amplify upstream and downstream the sequence of mefA/msrD and the products were sequenced. The software Vector NTI was used to analysis ORF of the obtained sequence, and to locate mefA through BLAST way. At last, the molecular types of resistant strains were determined by pulsed-field gel electrophoresis (PFGE) with Smal digestion and MLST (multiple sites sequence typing) with sequence7housekeeping genes.PCR resuslts showed that4strains were detected with mefA and msrD in Streptococcus suis strains isolated from2005-2007. Of four strains, one strain is type2(YY060816) and the others are type9(NJ-2, NJ-3and NJ-5). Three strains presented low resisitance to both14-and15-membered macrolide antibiotics, but one strain YY060816showed highly resistant to all macrolide antibiotics. The four strains with mefA/msrD carried lysogenic prophage, but the difference among four strains was that YY060816does not have phage capsid and phage terminase small subunit. Induction assays by exposure of strains to mitomycin C demonstrated that the mefA/msrD carrying element of NJ-2and NJ-3strains were present. But it could not be induced in the strain YY060816and NJ-5. The induced prophage showed a regular hexahedron body and a tail observed by transmission electron microscope. Analysis of sequence with size of about23kb obtained by long range PCR presented24ORFs, where mefA and msrD genes were fifth and sixth ORF respectively. The mefA/msrD chimeric element showed more related with phage construction like phage polymerase, phage related DNA helicase, phage terminase small subunit, phage DNA polymerase, which has a high homology with ORF15-34of ΦSsUD.1. The four strains belong to three different PFGE subtypes, as the strains digested by SmaI with5-13bands at size of10-913kb and dividatured cloning subtype according to90%similarity. Based on allele serial number and UPGMA system phylogenetic tree map, the four strains belong to three ST types, which consistent with the PFGE typing results. The homology of strain NJ-2and NJ-3is higher than90%, and both belong to ST240, most likely derived from the strain clone for same popular pathogen. The strainYY060816belongs to ST7type, which have high homology with published outbreak strains in Sichuan, only30%homology with the NJ-2and NJ-3. The homology of strain NJ-5(ST222type) and NJ-2as well as NJ-3.was close to50%.The above results indicate that the mefA/msrD gene were probably carried by phage-like chimeric element and inserted into chromosome genome of S. suis at an insertional hot spot site of the3’end of a conserved tRNA uracil methyltransferase (rum) gene. And the prevalence and dissemination of mefA/msrD in strains of S. suis was by horizontal and clonal transmittion.