Multi-drug Resistance And PFGE Analysis Of Actinobacillus Pleuropneumoniae Isolates

The purpose of this study was to investigate the antimicrobial susceptibility,resistance gene distribution and genetic similarities of Actinobacillus pleuropneumoniae(APP)isolates,so as to provide theoretical guidance for the treatment and prevention and control of APP infection,and to provide partial basis for the establishment of livestock and poultry drug resistance database.In this study,the APP isolates from Henan,Hubei,Jiangxi,Shaanxi,Hunan,Shanxi and Anhui were isolated and identified by PCR.Microbroth dilution method was used to test the susceptibility of the APP to 19 antibacterial drugs including ?-lactams,tetracyclines,quinolones,aminoglycosides,macrolides,aminoalcohols,sulfonamides,etc.,and PCR and sequencing were used to further detect resistance genes related to various antimicrobials and genetic environment.Southern blot hybridization was used to confirm genetic location of the novel gene.Genotyping of APP isolates was performed by PFGE methods,and then the genetic relatedness of APP isolates was analyzed.One hundred and four APP isolates were successfully obtained from 1156 samples,with an isolation rate of 9.00%.The isolation rate of APP from Henan was highest and reached 24.04%(25/1156),and Jiangxi was the least,with an isolation rate of 5.77%(6/1156).The isolation rate of other provinces ranged from 8.65%to 21.15%.The results showed that the resistance of APP to oxytetracycline,sulfamonomethoxine,florfcnicol,cnrofloxacin,doxycycline,tylosin and amoxicillin was 75.96%,51.92%,18.27%,18.27%,7.69%,4.81%and 3.85%,respectively.But it is susceptible to gentamicin,amikacin,tilmicosin,olaquindox,lincomycin,colistin,compound amoxicillin and ceftiofur sodium.Analysis of antimicrobial resistance patterns found the number of isolates resistant to 2-3 drugs was the largest,of which OTC+SMM was the largest at 20.19%(21/104),while the number of isolates resistant to 4-5 drugs was the lowest,namely OTC+FFC+ENR+SMM and OTC+DOX+FFC+ENR+SMM 0.96%(1/104).Detection of resistance genes showed that the detection rate of tet(B),Sul2,floR,Sul3,Sul1,tet(L),tet(O),blaROB-1 and tet(M)was 64.42%,51.92%,46.15%,43.27%,30.77%,9.61%,6.73%,3.85%and 1.92%,respectively.In addition,plasmids carrying blaROB-1 and floR genes,namely p1144 and pA1144,were obtained by reverse PCR,and the genetic environment of blaROB-1 was determined to be blaROB-1-ISApl1,and it was proved that p1144 could be transferred to the susceptible APP strains and the amoxicillin resistance was increased by 8 folds.However,through Southern blot hybridization,tet(M)gene was found to be located on the genome in isolate 1144.PFGE typing showed that 99 out of 104 APP isolates could be divided into 83 PFGE types,suggesting that the isolates were mainly spread horizontally.In conclusion,the isolation rate of APP is low,while the resistance of APP isolates to oxytetracycline,sulfamonomethoxine,florfenicol and enrofloxacin was serious.And these antimicrobial agents should be prudent to use.Meanwhile,doxycycline,tylosin and amoxicillin should be used reasonably to prevent further enhancement of APP resistance.The detection rate of tet(B)was the highest,and blaROB-1 gene is an important cause of APP resistance to ?-lactam antibiotics.In this study,it is the first time that the tet(M)gene was detected and located on the chromosomal genome in APP,which indicated that tet(M)has been present in APP isolates and plays an important role in resistance to doxycycline.