| With the continuous development of intensive aquaculture, the drug resistance of pathogenic bacteria has become increasingly prominent. Due to irrational use of antibiotics, the drug resistant strains have emerged, deteriorating phenomenon of multidrug resistance, resistance mechanism is more and more complicated, which to the clinical treatment of bacterial diseases of livestock brought great difficulties, a serious threat to public health and safety, but also brought great pressure to the development of new drugs. There are few reports about the bacterial drug resistance system of livestock and poultry in Guizhou, this research is based on the analysis of the research data and the investigation report, The aim of this study is on the foundation of analyzing research data and investigation reports, to carry out rapid detection technology, gene phenotype and genotype of resistance, resistance gene sequence evolution characteristics and other related research of livestock and poultry pathogenic bacteria in Guizhou, and it will bring great meaning in theory for exploring the molecular mechanism of bacterial drug resistance.1. Epidemiologic study on main pathogenic bacteria in Guizhou:Collected related medical information from 2008 to 2014 in Guizhou Province, analysis of the epidemic characteristics of bacterial blight in livestock and poultry farms from the aspects of time distribution, the distribution of animal species, geographical distribution and so on, and the prevention and control of bacterial diseases of livestock and poultry disease cases were investigated and analyzed.The results shown that Escherichia coli, salmonella, pasteurella, staphylococcus and streptococcus is the leading pathogenic bacteria of Guizhou here.Its have an obvious seasonal, May to September happened in frequency and October to December in low incidence.Bacterial blights occurred mainly in sheep, the rest are pigs, cattle, poultry and other animals in turn. There are some differences in the number of bacterial diseases in allparts of Guizhou province. Among them, the number of major bacterial diseases in Tongren is higher than other areas, the rate of immune control of bacterial blight is low, the rate of immune in 2.3%~20.6%, the main way of prevention and control is still the prevention and control of antimicrobial agents. Beta-lactam, aminoglycosides,sulfonamides, macrolide and quinolone antibiotics are the most commonly been used in farm, and the beta-lactam use most frequently.2. Establishment and application of multiplex PCR detection method of main pathogenic bacteria from Livestock and Poultry:Design and synthesis of primers for the main epidemic pathogens(Laboratory of early separation, and pathogenicity of Escherichia coli, Salmonella, Pasteurella, Streptococcus,Staphylococcus) in Guizhou province, establish PCR reaction system and reaction conditions, and evaluate the performance of multiplex PCR method, required for the study of isolated strains were reinspection analysis. The results showed that all kinds of bacterial PCR primers were designed to amplify the target gene effectively, the amplified gene fragment of 23 S gene of Escherichia coli, KMT of Pasteurella, invA of Salmonella, nuc of Staphylococcus and EF-TU of Streptococcus is 663 bp, 456 bp, 284 bp, 500 bp and 197 bp respectively. The optimal reaction conditions of duplex PCR that can detect staphylococcus and streptococcus simultaneously is the best primer concentrations were 0.5 ?mol/L and 0.5 ?mol/L, the best annealing temperature is54?. Under these conditions, the sensitivity of the detection of Staphylococcus and Streptococcus were 1.50 ng/ L and 1.44 ng/ L, respectively. The optimal reaction conditions of Triplex PCR that can detect Escherichia coli, Pasteurella and Salmonella simultaneously is the best primer concentrations were 1.0?mol/L, 1.5?mol/L and1.0?mol/L, respectively, the best annealing temperature is 56?. Under these conditions, the sensitivity of the detection of Escherichia coli, Pasteurella and Salmonella were 114.00 pg/?L, 1.50 ng/?L and 1.44 ng/?L, respectively. Detection of147 strains of clinical isolated strains showed the duplex PCR and triplex PCR detection results with the biochemical identification results coincidence rate was98.72% and 97.26% respectively. Results show that the established PCR method to detect the results with traditional biochemical identification results are highlyconsistent.3. Study on the drug resistance phenotype and drug resistance of main pathogenic bacteria in livestock and poultry:A total of 147 strains were randomly selected from livestock and poultry 5 kinds of pathogenic bacteria, with K-B of 5kinds of 21 kinds of antibacterial drugs on the for drug sensitivity test, and design synthesized 23 pairs of specific primers for beta-lactam, quinolones, macrolide,streptomycin and sulfanilamide resistance genes, detection of a variety of bacterial drug resistance related genes. Analysis associated with bacterial resistance phenotypic and genotypic resistance. The results showed that all the tested strains showed obvious resistance, the total resistance rate was 54.42%~93.20%, and the multiple drug resistance was mainly between 6-11 drugs. In selected 21 kinds of antibiotics, to the poor the bacteriostatic effect of penicillin, amoxicillin, streptomycin and sulfamethoxazole isoxazole; 23 resistance genes were detected 19, The highest detection rate was Escherichia coli quinolones aac(6')-Ib-cr in 77.59%. The resistance gene mediated by Escherichia coli resistant to sulfonamides is sul1, to quinolones is mainly aac(6')-Ib-cr, to beta-actam drugs is mainly TEM in Guizhou; the resistance gene mediated by Pasteurella resistant to sulfonamides is sul2, to streptomycin is mainly Str A in Guizhou; the resistance gene mediated by Salmonella resistant to sulfonamides is sul2, to streptomycin is mainly StrA; the resistance gene mediated by Staphylococcus resistant to macrolide is mainly ermB, to beta-lactam is mainly bla Z;the resistance gene mediated by Streptococcus resistant to macrolide is mainly ermB.through the analysis of 151 strains of clinically isolated strain resistance phenotype and drug resistance genes associated. Results show: streptomycin resistant gene and resistance phenotypes of the highest coincidence rate was 100%, Pasteurella multocida of sulfa drug resistance phenotype and drug resistance base due to type with the lowest rate, was 71.43%.4. Analysis of drug resistance related gene sequences of main pathogenic bacteria in livestock and poultry:Using molecular cloning technology to clone and sequence for the main resistance genes of all kinds of bacteria, and using MegAlign software to analyze the homology, genetic identity and genetic evolution of thesequences. The results showed that except aac(6')-Ib-cr gene of Escherichia coli, gyrA and parC gene of Pasteurella, gyr A and par C gene of Streptococcus has many variations and other resistance genes showed a high conservation, among them, the most conservative is the Escherichia coli CTX-M gene, the sul2 gene and Streptococcus mefA gene, and the sequence homology are 100% comparing with the reference strains at home and abroad.Conclusion:1. The main pathogenic bacteria of animal bacterial diseases in Guizhou were Escherichia coli, Salmonella, Pasteurella, Staphylococcus and streptococcus; has time and regional differences occur in bacterial disease.2. Established a triple PCR method for rapid identification of Escherichia coli,Salmonella, Pasteurella and a double PCR method for rapid identification Staphylococcus and Streptococcus, the results of the two methods were consistent with the results of clinical isolates and biochemical tests.3. There are extensive drug resistance in five main pathogenic bacteria of Guizhou, and the resistance phenotype and genotype are highly unified.4. The five main pathogenic bacteria of livestock and poultry in Guizhou are relatively conservative, and some drug resistance genes have some mutations. |
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