| Background:With the rapid development of the livestock farming industry,the amount of fecal matter discharged from forms in our Region is as high as 4 million tons per year.The pathogenic microorganisms,drug-resistant bacteria,and antibiotics resistance genes carried in the cow's excrement will cause health problems among dairy cows.And the use of faeces returning to land will be a potential source of food-borne and environmental pollution.At present,the excrement discharged from dairy farming has become one of the serious threats to healthy farming and public health security.Objective:The objective of this research is to identify the diversity of environmental microbial communities and bacterial drug resistance in large-scale dairy cattle forms in Yinchuan City so as to provide effective data for risk assessment with respect to environmental protection and public health safety of dairy cattle breeding industry.And it is also to provide theoretical basis for quick detection of pathogenic microorganisms and for the application of manure-free treatments.Method:Through the collection of fresh faeces,manure,waste water and soil samples in a scaled dairy cow breeding farm in Yinchuan(referred to as "dairy cow farm"),16S rDNA V4 region sequencing and metagenome sequencing methods were used.From a macro point of view,dairy cattle farms The diversity of environmental micro-organisms was studied,and the diversity of the environment-cultivable micro-organisms in dairy farms was studied from the perspective of culturable microorganisms using classical purification and separation,bacterial biochemical detection and full-length sequencing of 16S rDNA;Sensitive disk detection and PCR detection of antibiotics resistance genes Seven clinically used antimicrobial agents(Penicillin,Amikacin,Lincomycin,Ciprofloxacin,Doxycycline,Cotrimoxazole and Erythromycin)were tested on dairy farm environmental cultivatable strains from the perspective of drug resistant phenotypes and genotypes,and 15 antibiotics resistance genes(Sulfonamides:sull,sul2,sul3;Tetracyclines:tetA,tetB,tetD;Aminoglycosides:aacC2,aacC4,aadD;?-lactams:CTX-M,OXA,TEM;Quinolones:qnrA,aac(6')-Ib-cr,gyrA)resistance studies.Results:(1)Research results based on 16S rDNA sequencing and metagenomic sequencing indicated that the composition of microbial communities in fresh faeces,manure,waste water,and soil samples in dairy farms in Yinchuan area is rich and diverse.Among which the Actinobacteria,Firmicutes and Bacteroidetes are dominant phyla,accounting for more than 70%of all bacterial communities.Further research on their microbial composition found that:Bacillus,Corynebacterium,Staphylococcus and other bacteria in the manure samples and waste water samples were significantly higher than those in the fresh faeces and soil samples.(2)Through isolation and purification to obtain a total of 629 culturable strains under the dairy cattle environment,from which a total of 560 strains were identified,336 strains were Gram-positive bacteria and 224 strains were Gram-negative bacteria.All of them could be categorised in 52 genera and 144 species and accounted for 83.04%of pathogenic bacteria(465/560).In this study,three methods were conducted from macroscopic to specific perspectives to reveal the diversity and abundance of environmental micro-organisms in dairy cattle farms from culturable and non-culturable perspectives.(3)The results of the study on the resistance of 560 strains of daily cows to the identified strains showed that the resistance rate of the 7 types of commonly used antibiotics in dairy farms was higher than 50%.Among them,the accumulated strains of the stool samples were penicillin-and The resistance rate of the compound sulfamethoxazole was 100%,and the isolates of the waste water samples were resistant to penicillin,lincomycin,and cotrimoxazole 100%;and all the isolates were multidrug resistant strains,of which 6 were resistant The most strains were found,accounting for 38.21%.Among the waste water samples,7 resistant strains were the most,accounting for 44.12%.(4)The results of drug resistance gene detection showed that 15 antibiotics resistance genes were all positive,and 249 positive strains were detected.The highest positive rate was ?-lactam TEM gene,which was 10.71%.The lowest was sulfamethoxazole.The drug sul3 gene was 0.89%,the total of 115 drug-resistant genotypes,and up to 9 drug-resistance genes(sull/sul3/tetA/tetD/aacC2/aadD/TEM/qnrA/gyrA)were detected in a single strain.The results of genome sequencing indicated that 1138 antibiotics resistance genes were annotated,and the amount of drug-resistant gene annotation in the manure samples was significantly higher(P<0.05)than those in fresh faeces and soil samples.Conclusions:(1)The samples in the scaled dairy cow farm in Yinchuan area are diverse in composition of the micro-organisms and there are differences in abundance within each sample.Among them the dominant strains in the fresh feces samples were Bacteroides,Prevotella and Clostridium;in the manure samples were Pseudomonas,Streptomyces and Halomonas;in the waste water samples were Rhodoferax,Bacteroides,Psychrobacter and Flavobacterium;in soil samples were Mycobacterium,Nocardioides,Streptomyces and Koculia.The composition of microbial communities in fresh feces samples and waste water samples is similar.The microbial community structure of the manure and soil samples is similar,but as the classification hierarchy decreases,the complexity of the microbial community composition within the sample increases and passes through the 16S rDNA V4 region.The results of sequencing and metagenomic sequencing showed that the abundance of conditional pathogens in manure and waste water samples was higher than that of in fresh feces and soil samples.(2)Based on the isolation and identification of culturable bacteria sample from dairy farms,a total of 560 isolates were identified,of which 83.04%were conditional pathogens,revealing that the conditional pathogens can be cultivated in the feces of dairy cattle farms in Yinchuan.The variety and quantity are abundant,suggesting that farm waste is a potential source of pathogenic microorganisms,which poses a great potential threat to the healthy breeding of cows and the safety of human food.(3)The microbial resistance in the fresh faeces,manure,waste water,and soil samples in the scaled daily cow farms in Yinchuan area was severe.All 560 strains isolated were multidrug-resistant strains,and the resistance rate to penicillin and compound sulfamethoxazole was as high as 100%;Within all four types of samples,the accumulation of feces and waste water samples was the most severe with respect to drug resistance.(4)The composition of drug resistance genes in the environment of large-scale dairy cows in the Yinchuan area is abundant,and the abundance of drug resistance genes in the samples of feces(fresh feces,manure,and waste water)is high,which is the source and repository of potential drug resistance genes;and it would therefore pose a potential threat not only to the health of cows,but also to the health of humans and the safety of the environment. |
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