Resistance Detection And Analysis Of Resistance Genes Spectrum Of Clinical Haemophilus Parasuis Isolates

Haemophilus parasuis is commonly found in the upper respiratory tract of swine,responsible for Gl?sser's disease,a disease with worldwide distribution characterized by polyarthritis,fibrinous polyserositis,and meningitis in swine,and it is one of the most serious diseases that can affect pig industry.Vaccination is an effective method of preventing Haemophilus parasuis diseases,however serovar diversity and the high number of non-typable isolates have hampered the development of effective crossprotective vaccines.Using antibacterials was a primary approach in disease control.Owing to blindly use of antimicrobial agents,Haemophilus parasuis had antibiotic resistance to varying degree,which creates huge challenges to cure this disease.To investigate the resistance to common antibiotics and to analyze the genetic correlations among Haemophius parasuis isolates isolated in Henan,Hubei,Hunan,Anhui,Jiangxi,Shanxi,Shanxi province.From 685 suspected pathological materials including lungs,weasand,spleen and lymph node in seven provinces,HPS isolates we obtained and identified with 16 s RNA.The resistance of HPS isolates to 18 antibiotics were determined by broth microdilution method.The study showed that a total of 206 isolates were obtained.The resistance rate of oxytetracycline,enrofloxacin,lincomycin,amoxicillin,amikacin,tylosin and trimethoprim-sulfamethoxazole were 48.5%,51.5%,26.2%,29.1%,73.8%,20.2% and 93.2% respectively.And the resistance rate of doxycycline,amoxicillin clavulanic acid,ceftiofur,cefquinome,gentamicin,florfenicol,and olaquindox were8.7%,10.7%,5.8%,1.0%,12.6%,4.9% and 4.9%,respectively.All of 206 isolates were sensitive to mequindox,tiamulin,tilmicosin and colistin.Among the 18 types of antibiotics selected,the clinical isolates that resistant to 2-5 antibiotics were 61.7%,however only 34 strains were resistant to one antibiotic and 8 strains were sensitive to all antibiotics.PCR amplification was carried out to specificly amplificate the 48 resistant genes of?-lactams,tetracyclines,aminoglycosides,macrolides,quinolones,lincosamide,oxazolidinone,amide alcohols,peptides,sulfonamides and others.Through sequences analysis in comparison with amoxillin-resistant isolates and amoxillin-sensitive isolates,nucleotide mutation sites were searched in oder to validate and describe the cause for amoxilin-resistance of HPS isolates.Among the 206 HPS isolates,the detection rate of bla TEM gene,bla Rob-1gene,bla CTX-M gene,tet A gene,tet B gene,tet C gene,tet M gene,tet H gene,oqx A gene,oqx B gene,mph E gene,msr A gene,flo R gene,qnr D gene,qnr S gene,sul1 gene and sul2 gene,were 17.5%,14.6%,2.4%,14.6%,20.4%,13.6%,37.9%,0.5%,0.5%,1.0%,2.4%,1%,4.9%,1.0%,24.3%,29.6% and 90.3%,respectively.While the bla OXA gene,bla SHV gene,tet D gene,erm A gene,erm B gene,erm C gene,erm D gene,erm42 gene,mef A gene,qnr A gene,qnr B gene,qnr C gene,qep A gene,aac(6')-Ib-cr gene,lnu A gene,lnu B gene,lnu C gene,lnu D gene,arm A gene,rmt A gene,rmt B gene,rmt C gene,rmt D gene,rmt E gene,rmt F gene,rmt G gene,rmt H gene,npm A gene,mbr A gene,mbr B gene,mbr C gene and mbr D gene didn't be detected in any isolates.And the variation of the amino acid mutation loci of 209 th,238th and 245 th in the transpeptidase region of PBP3 is the possible reason that HPS is resistant to amoxilin.The plasmid PEHP160723 carrying bla CTX-M was transformed into H.parasuis standard strain by electroporation.And the transformant showed 8-and 8-fold increases in the MICs of cefquinome and ceftiofur when compared with the recipient strain.The isolate EHP160904 carrying tet M gene was conjugant successfully indicating that tet M gene located in a plasmid which could be conjugated and transferred,and the transconjugants' s showed 8-increases in the MICs of Oxytetracycline when compared with the recipient strain.Overlapping PCR were used for tet M environment research,and the research showed that IS26,ORF13 and LP was located in the tet M upstream region of the YHP160513 which carrying tet M genes.While the downstream region was not found.206 H.parasuis strains were typed systematically with ERIC-PCR.The study showed that a total of 18 distinct ERIC patterns were observed among 199 of 206 isolates,and 49 strains belong to XIII-type which has the most strains,next were VI-type?VIII-type,IX-type,VII-type with 36,25,26,16 isolates,respectively.The strain anumber of the other types were between 2 to 8.Only 7of the 206 HPS could not be typed by ERIC-PCR.Except the Lincomycin-resistant isolates almost equally distributed 12 ERIC-PCR types,most of other antibiaotic-resistant isolates mainly were found to be distributed among 2 or 3 ERIC-PCR types(more than 12 strains each types).The ERIC-PCR results in this study indicated that most of Lincomycin-resistant isolates originated from various clones.The horizontal transmission of lincomycin-resistance may be the main spread mechanism in HPS isolates.And the genetic relationship among other antibiaotic-resistant isolates is pretty close,suggesting that cloned transmission of the resistance to other antibiotics may be the main mechanism.