Research On The Rule Of Antibiotic Resistant Bacteria And Resistance Gene Pollution From Poultry Feeding Operations

In recent years,the large and even excessive use of antibiotics in livestock and poultry breeding has caused the pollution of antibiotic resistance bacteria(ARB)and antibiotic resistance genes(ARGs)in the environment to increase.As an important source of antibiotic resistant bacteria and resistance genes in the environment,there are relatively few researches on ARB and ARGs in livestock and poultry environments.Particularly,there are few systematic researches on the pollution rules of ARB and ARGs in the air environment of the breeding farms.In accordance with the above-mentioned deficiencies,this paper arranges to collect the feces of a typical layer farm in Beijing and the air samples inside and outside the house,and carries out an analysis of the concentration,the drug resistance and the influencing factors of ARB.Based on it,this paper surveys the types and concentration of ARGs.The results of this research will provide a basis for assessing the environmental hazards of ARBs and ARGs in the environment of the chicken farms and making the corresponding control measures.First,six-level impact sampler was used to collect total bacteria,staphylococcus aureus and MRSA in the air from inside and outside the layer,and feces samples were collected at the same time,and analyzed the trend of the concentration change and its possible influencing factors.By the drug sensitivity test with K-B method on the chicken farm air and feces of MRS A in the 7 categories of 14 antibiotics,Penicillin(PEN),Ampicillin(AMP),Cefoperazone(CFP),Streptomycin(STR),Gentamicin(GEN),Amikacin(AMK),Erythromycin(ERY),Ciprofloxacin(CIP),Levofloxacin(LEV),Norfloxacin(NOR),Tetracycline(TET),Doxycycline(DOX),Sulfonamide(SXT),and Vancomycin(VAN)were studied for drug resistance.By using of the micro-drop digital PCR(ddPCR)for the analysis of 6 classes of 22 antibiotic resistance genes,β-lactams(blaPSE,blaCTXM,blaTEM,blaOXA10,mecA),Aminoglycosides(aadA1,aadA2,strB,aac(6’)-Ⅱ,aphA3),Macrolides(mpha,mefA,ermA,ermB),Sulfonamides(sull,su12,dfrAl),Tetracyclines(tetG,tetW,tetQ),Vancomycin(VanA,VanB)and two kinds of gene integrons(intIl,intI2)were quantitatively analyzed.The results showed that the average concentration of MRSA in the air and feces was 4.31±14.56×102CFU·m-3 and 3.37±4.83×104CFU·m-3 with the ratio in the staphylococcus aureus of 41.99%±26.99%and 22.11%±20.06%,respectively.The total bacteria concentration increased linearly with the age increasing in both air and feces samples.The concentration of staphylococcus aureus and MRSA in the air increased with days,and was affected by temperature and particle concentration at the same time.The results of the drug sensitivity test with K-B method show that the MRSA and staphylococcus aureus isolated in this experiment reflect a significant drug resistance to β-lactams,Aminoglycosides,Macrolides,Quinolones and Tetracyclines,has a high sensitivity to Sulfamethoxazole and Amikacin,and has a full sensitivity to polypeptide Vancomycin.The drug resistance of staphylococcus aureus and MRSA to Cefoperazone in feces decreases with the age in days of laying hens,and shows a significantly negative correlation with the age in days of laying hens,shows a significant positive correlation between the drug resistance to Norfloxacin and the age in days of laying hens.When the age in days of laying hens is young,it shows a mild drug resistance,and shows a high degree of drug resistance and even full drug resistance when it is adult.The results of the resistance gene research show that most of the target genes of the six ARGs and the integron of two types of genes selected in this research can be detected in the air and feces of the chicken houses.Among them,blaTEM,aadAl,aadA2,ermB,tetW,sulI2,and sulll are genes with high detection rate and abundance in air and feces.From the point of the total amount of all kinds of resistance genes,sorted in order,Aminoglycosides＞Tetracyclines＞β-lactams＞Macrolides＞Sulfonamides＞Polypeptides.There are target genes that show significant positive correlations with the mobile genetic elements(intI1 intI2)in the six ARGs selected,and this positive correlation was detected in the fecal samples more frequently,followed by the indoor air samples.