[Contamination of Conjugative Antibiotic-resistant Plasmids in Large-scale Livestock and Poultry Manure and Their Occurrence Characteristics of Antibiotic Resistance Gene].

Abstract

The emergence and spread of antibiotic resistance in bacterial pathogens are now becoming a serious threat to public health globally. To study the occurrence and transfer characteristics of plasmid-mediated mobile antibiotic resistance genes （ARGs） in manure, antibiotic-resistant plasmids were captured from layers, broilers, and pig farms in Beijing, Hebei, and Ningxia provinces. The conjugative antibiotic-resistant plasmids （CARP） were captured by a filter membrane conjugation experiment, and the transfer frequency of plasmids from feces to recipient bacteria was tested. The ARGs-carried plasmid was extracted and sequenced with the Illumina sequencer, and the replicon types of CARP were identified with the PlasimidFinder database. The antibiotic-resistant phenotype of the captured conjugative plasmid was determined using the disk diffusion method （Kirby-Bauer）. In general, the 125 CARPs that were captured from 35 farms carried 13 types and 65 subtypes of ARGs. The most common ARGs were floR, aac （6'）-lb7, and TEM-150. Certain differences exist in the prevalence of CARP in the manure of different animals. The CARP in layer manure had a higher transfer frequency and prevalence than those in other animal farms, but the level of multidrug resistance conferred by these plasmids was lower than that of broilers and pigs. The 49 shared ARGs were observed from plasmids of broilers, layers, and pigs, of which 64% belonged to aminoglycosides, β-lactamase, fluoroquinolones, and trimethoprim-resistant genes. The antibiotic-resistant phenotypic characteristics of conjugants were similar with genotypes, and the dominant resistant conjugants belonged to β-lactamase （95.31%）, tetracyclines （89.06%）, aminoglycosides （87.5%）, and fluoroquinolones （68.75%）. IncH, IncN, and IncR plasmids were detected in pig manure, and 12.5% of the conjugants carried five or more high-risk ARGs in pig farms. The proportion of high-risk ARGs in pig farms was higher than that in other animal farms. In conclusion, the CARP in livestock manure commonly carries multiple types of ARGs, which can provide multiple antibiotic resistance capabilities for host bacteria. In addition, high-risk ARGs are carried by the broad host range of conjugative plasmids, which can promote the spread of high-risk ARGs in livestock farms to the surrounding environment.