| Antibiotic Resistance Genes naturally exist in the natural environment before human or animal drugs are used,and a variety of ARGs have been detected in environmental media such as water,soil,and air.It has been defined as a new type of environmental pollutant at home and abroad in recent years.Intestinal microbes of livestock and poultry are also an important repository of ARGs.Not only enteropathogenic bacteria contain high abundance and high diversity of ARGs,but also intestinal probiotic symbiotic bacteria contain diverse ARGs.After they are excreted with feces,they become an important source of ARGs accumulation pollution in the natural environment,threatening ecological security and human and animal health.Intestinal Lactobacillus is a major type of symbiotic probiotics in the intestinal tract of livestock and poultry,and it is also an important source for screening feed Lactobacillus.At present,many studies have reported the prevalence of ARGs in enteric pathogenic bacteria in livestock and poultry,but few studies on ARGs contamination abundance,diversity and antibiotic resistance phenotype monitoring of intestinal commensal Lactobacillus commensal in livestock and poultry nationwide.To monitor the overall contamination characteristics and background data of intestinal symbiotic Lactobacillus ARGs in pigs and chickens in farms in different regions of China,Lactobacillus was isolated from 5 laying hen farms and 5 pig farms in East China（Shandong Province,Jiangsu Province）,South China（Guangxi）,Southwest China（Sichuan Province）,North China（Tianjin City,Hebei Province）and Central China（Hubei Province）.The abundance and distribution characteristics of 21 ARGs corresponding to 7 commonly used antibiotics in Lactobacillus were determined by real-time quantitative PCR.The resistance phenotype of Lactobacillus to 7 kinds of commonly used antibiotics was determined by the micro-broth dilution method.The main findings are as follows:（1）The relative abundances ofβ-lactam resistance gene bla Z,tetracycline resistance gene tet W,drug and fungicide resistance genes acr R,acr D and oqx B in Lactobacillus from pigs and chickens were relatively high,and their relative abundances in Lactobacillus from pigs were 0.60-1.94（copies/16S r RNA）,0.14-0.51（copies/16S r RNA）,1.98×10-5-2.33×10-4（copies/16S r RNA）,1.81×10-4-4.83×10-4（copies/16S r RNA）,2.05×10-5-2.31×10-4（copies/16S r RNA）,the relative abundances in Lactobacillus from chickens were 0.42-0.82（copies/16S r RNA）,3.90×10-3-0.03（copies/16S r RNA）,6.16×10-5-8.05×10-5（copies/16S r RNA）,5.03×10-5-1.19×10-4（copies/16S r RNA）,9.22×10-6-6.02×10-4（copies/16S r RNA）.The relative abundances of aminoglycoside resistance genes arm A,rmt B,macrolide-lincosamide-streptavidin（MLS）resistance genes mef A,ere A and fluoroquinolone resistance gene mex A in Lactobacillus from pigs and chickens were low,ranging from 10-8 to 10-6（copies/16S r RNA）.（2）The total relative abundance of 21 ARGs in Lactobacillus from pigs is higher than that of Lactobacillus from chickens in different regions.The total relative abundance of 21 ARGs ranged from 0.24 to 3.50（copies/16S r RNA）in Lactobacillus from pigs and 0.11 to 1.29（copies/16S r RNA）in Lactobacillus from chickens.（3）Except for Hubei Province,the relative abundance ofβ-lactam resistance gene bla Z in Lactobacillus from pigs from the other four regions was significantly higher than that of Lactobacillus from chickens（P<0.05）;the relative abundance of tetracycline-resistant gene tet W in Lactobacillus from pigs in East China,South China,Southwest China and Central China was significantly higher than that of chicken-derived Lactobacillus（P<0.05）;the relative abundance of MLS resistance gene mph A in intestinal Lactobacillus from pigs in other 4 regions was significantly higher than that in intestinal Lactobacillus from chickens（P<0.05）,except in Sichuan Province.In addition,the relative abundance of drug and fungicide resistance gene acr R in intestinal Lactobacillus from pigs was significantly higher than that in intestinal Lactobacillus from chickens in five different regions of China（P<0.05）.（4）In this study,11 strains with low abundance of bla Z,tet W,acr R,acr D and oqx B resistance genes were screened from 100 Lactobacillus strains.Among them,3strains of intestinal Lactobacillus in pigs and 8 strains of intestinal Lactobacillus in laying hens were used for subsequent in-depth experiments to develop candidate Lactobacillus strains for feed.（5）Most lactobacilli have high resistance to kanamycin,tetracycline and chloramphenicol,the MIC90 of kanamycin was 512μg/m L,and the MIC50 of tetracycline was 128μg/m L,the MIC50 of chloramphenicol was 16μg/m L,and the MIC90 of ciprofloxacin was 128μg/m L.At the same time,it was also found that some strains showed resistance to ampicillin and amoxicillin.The MIC90 of ampicillin was 32μg/m L,and the MIC90 of amoxicillin was 64μg/m L.In conclusion,the relative abundances ofβ-lactams,tetracyclines and drug-fungicide resistance genes in intestinal Lactobacillus in pigs and chickens were relatively high,and the total relative abundance of 21 representative ARGs corresponding to 7 antibiotics in intestinal Lactobacillus in pigs was higher than that in chickens.Among them,bla Z,tet W,mph A and acr R were more prevalent in swine intestinal Lactobacillus than chicken intestinal Lactobacillus.Pig and chicken intestinal lactobacilli were highly resistant to kanamycin,tetracycline and chloramphenicol,but less resistant to ampicillin and amoxicillin.This study provides background data for large-scale monitoring of ARGs contamination characteristics and antibiotic resistance phenotypes of Lactobacillus in the intestinal tract of livestock and poultry and its risk assessment,and also reserves strain resources for the development of feed Lactobacillus. |
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