| Mastitis is a kind of common disease that restricts the development of dairy cattle breeding industry.It is easy to cause problems such as the decline of fecundity of cows,shortening of service life,reduction of milk production,and reduction of milk quality.The adverse effects caused by this disease have long hindered the economic benefits of pastures around the country.The etiology of dairy cow mastitis is much and complicated,pathogen infection is one of the main reasons.On the basis of strengthening the prevention and control of contagious mastitis,caused by environment pathogenic bacteria of mastitis than up,gradually to the attention of the people.Among them,Klebsiella pneumoniae(KP)is the second largest environmental conditional pathogen after Escherichia coli,which has been isolated from milk samples of mastitis cases in many literature reports.Nowadays,in order to effectively control the incidence rate of cow mastitis,the use of antibiotics has become the preferred treatment.However,with its extensive and frequent use and abuse,the phenomenon of antibiotic resistance of pathogenic bacteria has become increasingly significant,and experts and scholars at home and abroad have conducted multiple active studies on this issue.This experiment isolated and identified pathogenic bacteria from mastitis milk samples collected from four pastures in northern Jiangsu,explored the multi site sequence typing characteristics(MLST)of KP isolates,the carrying status of virulence and resistance genes,and their relationship with antibiotic resistance.The aim was to understand the prevalence of pathogenic bacteria from mastitis in dairy cows in some pastures in northern Jiangsu,the distribution of KP virulence and resistance genes,and the relationship between virulence and resistance genes.In order to explore the key molecular targets in bacteria that regulate the virulence and drug resistance characteristics of KP,and provide scientific theoretical basis for precise prevention and control of cow mastitis disease and drug guidance.208 clinical/occult mastitis milk samples were collected from 4 pastures in northern Jiangsu from March to August 2021.Through a combination of traditional bacterial culture and 16S rDNA isolation and identification,16 pathogenic bacteria were detected from 186 positive milk samples,totaling 438 strains.Among them,the detection rate of KP(15.53%)is the highest.The experiment used MLST method to obtain 23 ST types of 68 KP isolates,with the highest KP(19.1%)belonging to ST 2661 type;The specific PCR method was used to detect the carrying status of 11 common virulence genes in KP.The results showed that the virulence gene allS was not detected,and the detection rates of fimH,ureA,uge,and wabG were 100.0%.The detection rates of rmpA,wcaG,kfuBC,ybtA,iucB,and iroNB were 4.4%,11.8%,50.0%,23.5%,52.9%,and 4.4%,respectively.All isolates carried multiple virulence genes.K-B disk agar diffusion method was used to analyze the drug resistance of KP.It was found that KP was highly sensitive to ciprofloxacin,nitrofurantoin and meropenem;The highest resistance rate to penicillin(85.3%);The KP with simultaneous resistance to penicillin and amoxicillin was the highest(23.5%);Among the 13 multi drug resistant isolates,5 different drug resistance profiles were observed;Eight strains producing ESBLs KP were detected.Detection of 9 species in isolated strains using specific PCR method β-The results showed that five kinds of resistance genes were detected.The positive rate of ESBLs resistance gene blaSHV was 100.0%,followed by ESBLs resistance gene blaTEM,which was 64.7%;The detection rates of carbapenem resistant genes blaNDM-1,blaVIM,and blaOXA-10 were all below 50%;All isolates carry multiple drug resistance genes.Chi-square test and Spearman correlation coefficient method were used to analyze the correlation between resistance genes,virulence genes,and drug resistance in KP.The results showed that the positive rates of resistance genes blaNDM-1,blaVIM,blaTEM,and virulence genes kfuBC,rmpA,iroNB,and iucB were correlated to varying degrees with the resistance of multiple antibiotics.It is worth noting that the virulence gene kfuBC showed a highly significant negative correlation with the number of KP resistance.To further explore the relationship between the virulence and drug resistance of KP,the dominant virulence gene kfuBC was selected based on the analysis results of virulence genes and drug resistance.The relative expression level of kfuBC in KP was detected using real-time fluorescence quantitative PCR(qRT PCR).Three KP strains with no expression,low expression,and high expression of kfuBC were randomly selected to infect primary mammary epithelial cells(pbMEC)of cows and construct a mouse pathogenic model.The CCK-8 method was used to detect the cell survival rate of each group,and the results showed that the cell survival rate of the kfuBC high expression group was significantly lower than that of the low expression group and the non expression group(P<0.05).Using enzyme-linked immunosorbent assay(ELISA)to detect interleukin-6(IL-6)and interleukin-1 in cell culture media of each group β(IL-1β)And tumor necrosis factor-α(TNF-α)Concentration,the results showed that the high expression group of kfuBC had inflammatory factors IL-6 and TNF-α.The concentration was significantly higher than that of the low expression group and the non expression group(P<0.05).The content of SOD,MDA,ALP,ALT,and AST in the serum of mice was measured using a correlation detection kit.The results showed that the MDA content in the serum of mice in the kfuBC high expression group and low expression group was significantly higher than that in the non expression group(P<0.05);Compared with the kfuBC non expression group and the low expression group,the SOD activity in the serum of the high expression group mice was significantly reduced,while the levels of ALP,ALT,and AST were significantly increased(P<0.05).Aseptically collected liver and spleen tissues from mice for pathological sectioning,the results showed that the high expression group of kfuBC had more severe damage to the liver and spleen tissues.The experimental results suggest that KP with high expression of kfuBC has higher toxicity and causes stronger inflammatory reactions.This experiment evaluated the prevalence of pathogenic bacteria causing mastitis in dairy cows from four pastures in northern Jiangsu Province,revealing the distribution of virulence and resistance genes of KP and their relationship with drug resistance.Key virulence genes were discovered to regulate KP virulence and resistance,providing theoretical support for guiding rational drug use and prevention and treatment of mastitis in pastures. |
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