| Bacterial diseases are an important group of epidemics that affect the goat industry.Bacterial diseases,either alone or in combination,cause serious economic losses to the goat industry in China.Currently,the prevention and control of bacterial diseases in goat is mainly through the use of antibiotics,but along with the increased use of antibiotics and irregular use of antibiotics,the problem of bacterial resistance is becoming more and more serious.More than half of antibiotics in China are used in animal husbandry industry such as pigs,cattle,goat and poultry.As the main source of human meat,there is also a very prominent drug resistance problem.With the increasing demand and the support of national policies,the number of goats shows an increasing trend year by year.As scale continues to be improved,epidemic diseases occur frequently,which are mostly mixed infections.Due to the change of production mode in goat industry,disease prevention and control technology can not meet the demand,and drug abuse and drug overuse are particularly prominent in goat industry.Compared with other animals,there are few studies on the drug resistance of goat-derived bacteria,so it is urgent to study the drug resistance of goat-derived bacteria in China.In order to investigate the drug resistance of goat-derived bacteria in China,analyze the mechanism of drug resistance and explore the technology of reducing resistance,this study collected respiratory tract,digestive tract and other samples of clinically diseased goat from large-scale goat farms of major large-scale goat breeding areas in China,isolated and identified bacteria.Drug sensitive slips method and broth method were used to determine bacterial drug resistance,PCR method was used to detect drug resistance genes,phages were isolated and its anti-resistance effect was tested,so as to provide theoretical basis and new ideas for the prevention and control of drug resistance of goat-derived bacteria in China.The research contents and conclusions are as follows:(1)From August 2018 to March 2022,54 batches of respiratory tract,digestive tract and other samples of clinically diseased goat were collected from large-scale goat farms in Jiangsu,Anhui,Zhejiang,Shandong,Jiangxi,Hebei,Shanxi and Qinghai,and 277 strains of goat-derived bacteria were isolated,including 191 Gram-negative bacteria and 86 Gram-positive bacteria.The drug susceptibility of 148 strains isolated from large-scale goat raising province was determined by K-B method.The results showed that the isolates showed the most serious resistance to macrolides and penicillins,the moderate resistance to β-lactam and cephalosporins,and the lowest resistance rate to aminoglycosides(less than 20%);84.25% of isolated bacteria were multi-drug resistant(more than 3 times),and 6 or more resistant strains accounted for 52.73%.30.8% of bacteria were resistant to more than 20 kinds of antibiotics,and most of them were Enterobacter.The susceptibility test of 98 Escherichia coli strains to 16 antibiotics was carried out by broth method.The results showed that the MIC values of 98 Escherichia coli strains to 16 antibiotics were mostly higher than the critical value of CLSI,and the MIC values of some Escherichia coli strains to ciprofloxacin were more than 200 times higher than the critical value of CLSI(1μg/m L).Comparative analysis of sampling provinces and seasons showed that the drug resistance rate in Jiangsu and Hebei was slightly higher than that in other provinces,and the drug resistance rate in spring and summer was higher than that in autumn and winter.In addition,a goat-derived Wohlfahrtiimonas chitiniclastica strain was isolated for the first time in this experiment,which could infect people and many animals.The drug resistance rate and drug resistance spectrum of the isolated strain were significantly higher than those reported in the past.(2)By designing primers and optimizing conditions,PCR detection methods for 8 drug resistance genes were established in this study,including sulfonamide resistance gene sul2,florfenicol resistance gene flor,β-lactam resistance genes TEM and mec A,macrolide resistance genes oqx A,erm A and erm C,tetracycline resistance gene tet A and others.Some antibioticresistant genes corresponding to severe antibiotic resistance in 277 isolated bacteria were detected together with the drug resistance genes reported in literature,and the results showed that quinolone resistance genes oqx A and qnr A,aminoglycoside and quinolone resistance gene aac(6’)-Ib,tetracycline resistance genes tet A and tet X,beta-lactam resistance genes TEM and CTXMU,sulfonamide resistance gene Sul-2 and florfenicol resistance gene flor were detected in 191 strains of gram-negative bacteria.The cfr resistance gene causing chloramphenicol and lincomide resistance was detected in a Proteus mirabilis strain.Among 86 strains of Grampositive bacteria,erm A gene was amplified from a Kosakonia cowanii strain,and erm C gene was amplified from a Bacillus subtilis strain of and a Enterococcus hirae strain.Optr A resistance gene was detected in 2 Enterococcus strains and 1 Bacillus strain.The cfr resistance gene was detected in 3 Staphylococcus martensii strains,and 1 Staphylococcus martensii strain carried optr A,cfr and mec A resistance genes at the same time.Optr A resistance gene was detected in1 Streptococcus strain.TEM,flor,aac(6’)-Ib-cr resistance genes were detected in a Wohlfahrtiimonas chitiniclastica strain for the first time.(3)Four strains of goat-derived bacteria,E.coli AY1,Klebsiella AY3,Pseudomonas aeruginosa K12-1 and Staphylococcus AI3,were only susceptible to amikacin.Secondgeneration whole-genome sequencing of these four strains revealed that they all carried more than eight resistance genes and were resistant to all the eight major classes of antibiotics tested.The three strains,AY1,AY3 and K12-1,carry more virulence genes and may possess virulent virulence.k12-1 is a new st-type strain and AI3 Staphylococcus lacks a st typing method.The results of this second-generation sequencing analysis provide research material and basis for subsequent research in the direction of drug resistance mechanism by third-generation sequencing.(4)With 98 E.coli strains as host bacteria,22 phages with different E.coli as host bacteria were isolated,among which 1 was virulent phage and the others were temperate phage;the drug sensitivity test of temperate bacteriophage combined with antibiotics by broth method showed that the MIC value of antibiotics could be reduced by adding corresponding bacteriophage to some bacteria and drugs,suggesting that adding bacteriophage may reduce the dosage of antibiotics;meanwhile,it was also found that the MIC value of some bacteria and drugs increased after adding corresponding phage,which provided a new basis and new material for exploring the mechanism of drug resistance.In conclusion,this study understood the background of bacterial drug resistance in largescale goat farms through isolation and identification of bacteria in goat farms and detection of bacterial drug resistance;through the detection of bacterial drug resistance genes,the drug resistance mechanism was analyzed;by isolating drug-resistant bacterial phages and using them in combination with drugs,the anti-drug technology was explored,which provides a new basis for the prevention and control of bacterial drug resistance in goat farms,new materials for the study of the formation mechanism of bacterial drug resistance,and new ideas for the research and development of anti-drug technology in goat farms. |
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