Isolation,Identification And Drug Resistance Analyis Of Haemophilus Parasuis And Streptococcus Suis

Haemophilus parasuis（HPS）,a gram-negative bacillus,is the causative agent of Gl?sser’s disease characterized by fibrinous polyserositis,polyarthritis and meningitis.Streptococcus suis（SS）belongs to the gram-positive bacteria,mainly causing meningitis,arthritis,septicemia and other diseases.These two kinds of pathogens are popular in the world,which have brought huge impacts and economic losses to the pig industry.At present,there are 15 serotypes in HPS and 35 serotypes in SS.Although HPS and SS can be prevented by vaccination,but the practicle situation is that there are a large number of serotypes and a considerable portion of the disease can not be carried out in serotyping,as well as the presence of cross protections between different serotypes,which make the commercial vaccines diffficult to,not play a role for all bacteiral strains.The best choice for prevention and treatment is still antibiotics.The long period of nonstandard and abuse of antibiotics lead to the evolution of bacterial resistance,which not only cause clinical failure,but also threat the human health through the food chain.In this study,the serotyping,drug-resistant phenotype and resistance genes of HPS and SS isolated from 10provinces in China were detected.The purpose of this study was to demonstrate the prevalence of serotypes and the distribution and prevalence of drug resistance genes throughout the country,and to provide scientific basis for the two pathogens in drug control and risk assessment.The results were as follows.（1）Isolation and identification of HPS and SS and serotype classificationA total of 19 HPS strains,36 SS strains,were determined form the suspected pigs through the biological characteristics of isolation and culture,Gram’s staining,biochemical test and so on,and the identification by PCR and sequencing.Combined with preserved strians 71strains of HPS and 110 strains of SS were carried out in serotyping by multiplex PCR.The results showed that the main epidemic serotypes of HPS were type 4（30.99%）,type 5 or 12（14.08%）and type 13（7.04%）,and he main epidemic serotypes of SS were 2 or type 1/2（40.91%）,1 or 14（11.82%）and 29（6.36%）.There were differences in the main epidemic serotypes between the two pathogens in different regions.（2）Drug sensitivity test of HPS and SSThe minimum inhibitory concentrations of 71 strains of HPS and 98 strains of SS were determined by agar dilution method for 13 kinds of antibiotics.The results showed that all isolates of HPS were highly sensitive to cefotaxime（100%）,tigocycline（100%）,meropenem（98.57%）and ampicillin（97.14%）,and also sensitive to some drugs with a relatively low percentage,such as 84.29%of myxomycin E and chlortetracycline,81.43%of Amikacin and 82.86%of florfenicol In addition,the phenomenon of drug resistance in different degrees also appeared.The resistance to sulfamethoxazole and trimethoprim was most obvious（92.86%）,followed by fosfomycin（71.43%）,ciprofloxacin（42.86%）and gentamicin（21.43%）.Moreover,multiple drug resistance was evident,and the number of double drug-resistant and multiple drug-resistant isolates was as high as 51（72.86%）.All SS isolates were highly sensitive to tigocycline（100%）,Mei Lopez Nan（98.98%）,cefotaxime（97.96%）and ampicillin（96.94%）,and were reletively low sensitive to florfenicol（87.76%）.At the same time,different degrees of drug resistance were detected,The resistance to sulfamethoxazole,trimethoprim and fosfomycin was most significant（97.96%）,followed by chlortetracycline（90.82%）,Amikacin（78.57%）,ciprofloxacin（62.24%）and gentamycin（48.06%）.Multi-drug resistance was also obvious,and 97 strains（98.98%）were found double drug-resistant or multiple drug-resistant.（3）Detection resistance gene of HPS and SSThe resistance genes of tetracycline,quinolones,beta lactam,aminoglycoside,florfenicol,sulfonamides,and macrolides were detected by regular PCR.Of the 71 HPS,the detection rate of tetracycline resistance genes was 94.37%94.37%for tet B gene,40.85%for tet M gene and 1.41%for tet L.The detection rate ofquinolones qeq A gene detection andβ-lactam bla_tem gene was 100%and 4.23%respectively.Aminoglycosides detected 3 kinds of resistance genes str A,str B,aph（3’）-I a,the detection rates were 92.96%,97.18%and95.77%respectively.The detection rates of florfenicol fex（A）gene and cml A gene were7.04%and 25.35%respectively.The detection rate of the sul 2 resistant gene of sulfonamides was 98.59%.All the HPS could detect 3 or more than 3 resistant genes（100%）.Among 110 strains of SS,the detection rate of tetracycline resistance gene tet M was 96.36%,and the tet L gene detection rate was 7.27%.The detection rate of florfenicol resistant gene fex（A）was 29.09%,and the detection rate of cml A gene was 56.36%.The detection rate of the sulfonamide resistant gene sul 2 was 53.64%.The detection rate of macrolide resistance gene erm A,erm B gene and mef A/E gene was 3.64%,93.64%and11.82%,respectively.A total of 103 SS were schlepping 2 or more resistance genes（93.64%）.