Antibiotic Resistance And Epidemiological Characteristics Of Klebsiella Pneumoniae And Staphylococcus Aureus Isolated From Dairy Breeding Environment In Shanghai

Klebsiella pneumoniae and Staphylococcus aureus are the main pathogenic bacteria in milk cow mastitis,to treat cow mastitis,most cow breeding in the process of long-term use of antibiotics,can make the S.aureus and K.pneumoniae resistant to antibiotics,and serious damage to the dairy farming and food safety.In order to understand the antibiotic resistance and epidemiological characteristics of K.pneumoniae and S.aureus in intensive dairy breeding environment and raw milk,this study identified K.pneumoniae and S.aureus in dairy breeding environmental samples and raw milk samples.The resistance of K.pneumoniae and S.aureus isolates to antibiotics was detected by broth dilution method,and the strain producing extended-spectrum β-lactamase K.pneumoniae(ESBL-KP)was identified.Antibiotic resistance genes(ARGs)and virulence genes carried by K.pneumoniae and S.aureus,serotype of K.pneumoniae and methicillin-resistant S.aureus(MRSA)were detected by PCR.The genetic diversity of K.pneumoniae and S.aureus were analyzed by enterobacterial repetitive intergenic consensus sequence PCR(ERIC-PCR)and multi-locus sequence typing(MLST),and the key links leading to the infection of pathogenic bacteria in dairy cows and the contamination of raw milk by pathogenic bacteria in intensive dairy breeding were deduced.1.Antibiotic resistance of K.pneumoniae and S.aureus isolated from dairy breeding environment and raw milkA total of 98 strains of K.pneumoniae and 23 strains of S.aureus were identified from 157 isolates in intensive dairy farming environment in Shanghai.K.pneumoniae was distributed throughout the whole production process from breeding materials to milk storage.S.aureus is mainly found in raw milk.Antibiotics sensitivity test showed that K.pneumoniae and S.aureus were 100% of resistance to ampicillin,resistance to ciprofloxacin and maintain is extremely low(< 10%).Until effective alternatives to antibiotics are developed,quinolones can be an effective means of controlling K.pneumoniae and S.aureus.The MDR strains of K.pneumoniae and S.aureus accounted for 76.5% and 73.9%,respectively.The drug-resistance phenotypes were highly diverse,indicating that the drug-resistance of pathogenic bacteria in dairy cow breeding environment was complex,and the use of antibiotics should be strictly monitored.With the development of raw milk production,the antibiotic resistance index gradually increased from 0.07 to 0.64,and the resistance level of nipple milk and milk can milk isolates was significantly higher than that of the environmental strains.The detection rate of ESBL-KP was 53.1% and all the 23 strains of S.aureus were identified as MRSA.Both pathogens had the potential to spread multiple drug resistance.In the process of intensive dairy farming,it is necessary to monitor the development of bacterial resistance and put more efforts into cleaning dairy sheds,rational use of antibiotics,and management of milk storage equipment.2.Determinants of antibiotic resistance and virulence potential of K.pneumoniae and S.aureusExcept for chloramphenicol,the average detection rate of ARGs in other categories was higher than 30%,indicating that there was serious ARGs pollution in dairy farming environment.The resistance mechanism of the two pathogens was complex,but there were some differences.The detection rate of K.pneumoniae efflox pump resistance gene was the highest(62.1%),while the detection rate of S.aureus antibiotic inactivation gene was the highest(57.8%).The distribution of ARGs in different strains showed great differences,and the detection rate of ARGs in raw milk isolates was generally higher than that in environmental isolates.Except for chloramphenicol,the other five types of drug resistance phenotypes showed a coincidence rate of more than75% with ARGs detection results in the two strains.There are strains that carry ARGs without the corresponding resistance phenotype and are at risk of becoming resistant strains.K.pneumoniae were identified as K2 and K3 serotypes common in clinical infections,and virulence gene test results showed that all K.pneumoniae carried at least three virulence genes.All the five enterotoxin genes of S.aureus tested negative,leading to a low risk of foodborne outbreaks.3.Genetic diversity of K.pneumoniae and S.aureusIn the molecular typing of K.pneumoniae and S.aureus,the resolution of ERIC-PCR was slightly higher than that of MLST,and both methods showed a high degree of genetic diversity in the bacterial populations.ERIC-PCR classified K.pneumoniae into 7 populations and 49 genotypes.MLST classified K.pneumoniae into67 ST types,of which 41 ST types have appeared in clinical infections.Part of K.pneumoniae comes from environmental samples such as feces,soil,bedding,etc.,in dairy breeding and raw milk production activities through direct or indirect contact with the surface of the breast,and then infects the mammary glands of cows.Another part of K.pneumoniae has adapted to the environment in dairy cows,causing contamination of raw milk during colonization and increasing the risk of outbreaks of cow mastitis.S.aureus was divided into 4 populations and 18 genotypes by ERIC-PCR,and was divided into 5 ST types by MLST,among which there was an important sequence type ST398 leading to dairy cow mastitis and foodborne disease.The main way to contaminate raw milk is from the bovine mammary gland colonized strain.