牛源嗜麦芽寡养单胞菌的分离鉴定、毒力、耐药基因及生物学特性分析

Isolation,identification,virulence,drug resistance gene and biological characteristics analysis of Stenotrophomonas maltophilia from cows

旨在明确荷斯坦奶牛瘤胃内嗜麦芽寡养单胞菌的生物学特性、耐药性以及携带的毒力和耐药基因等信息。采集荷斯坦奶牛瘤胃内容物,经富集、分离和纯化等步骤获得菌株,通过16S rDNA测序以及同源性分析确定菌株种属,纸片扩散法检测菌株耐药性,PCR扩增和琼脂糖凝胶电泳检测菌株携带的毒力和耐药基因,通过小鼠灌喂试验检测菌株的致病性。最后从荷斯坦奶牛瘤胃内共分离到17株嗜麦芽寡养单胞菌PMIK-2(Stenotrophomonas maltophilia),其中高产奶牛组4株、低产奶牛组13株。生物学特性结果表明,分离菌株的最佳生长温度为38℃,最适生长pH为7.0,对胆盐有较强的耐受性。药敏结果显示,分离菌株对卡那霉素、头孢氨苄、头孢唑啉、克拉霉素、红霉素、环丙沙星、诺氟沙星、氧氟沙星高度敏感,对链霉素、氨苄西林、四环素、亚胺培南、庆大霉素具有耐药性。此外,在分离株的基因中检测到了fimH、mrkD和wabG 3种毒力基因和AadA1、BlaSHV1、TetD 3种耐药基因。菌株致病性结果显示,灌喂PMIK-2菌液1.0×10^(7),1.0×10^(9),1.0×10^(11) CFU/mL 3个浓度的致死率分别为28.6%~42.9%,42.9%~57.1%和71.4%~85.7%。嗜麦芽寡养单胞菌PMIK-2的生化特性基本符合伯杰氏系统细菌学手册,最适生长温度和pH值以及对胆盐的耐受能力也说明其可以在奶牛肠道中生存。在治疗感染PMIK-2引起的疾病时,应当减少或者避免使用链霉素、氨苄西林、四环素、亚胺培南、庆大霉素等药物。PMIK-2具有较强的致病性,高产组与低产组奶牛瘤胃内分离PMIK-2菌株数量的不同可能是其生产状况产生差异的原因之一。

The objective of this experiment was to study the biochemical characteristics of Stenotrophomonas maltophilia in the rumen of Holstein cows and the virulence genes and drug resistance genes carried by the bacteria.Rumen fluid of Holstein cows with low production status was collected,and the strains were obtained by enrichment,separation and purification.The strains were identified by 16 S rDNA sequencing and homology analysis.The drug resistance of the strains was detected by paper diffusion method.The virulence and drug resistance genes carried by the strains were detected by PCR amplification and agarose gel electrophoresis.The pathogenicity of the strain was detected by mouse feeding test.Finally,seventeen strains of Stenotrophomonas maltophilia(PMIK-2)were isolated from rumen of Holstein dairy cows,including 4 strains from high-yielding dairy cows and 13 strains from low-yielding dairy cows.The results of biological characteristics showed that the optimum growth temperature was 38℃,the optimum pH was 7.0,and the strain had strong tolerance to bile salts,and the isolates had strong tolerance to bile salt.The results of drug sensitivity showed that the isolates were highly sensitive to kanamycin,cephalexin,cefazolin,clarithromycin,erythromycin,ciprofloxacin,norfloxacin,ofloxacin,and resistant to streptomycin,ampicillin,tetracycline,imipenem,and gentamicin.In addition,three virulence genes of fimH、mrkD and wabGand three drug resistance genes of AadA1,BlaSHV1,TetD were detected in the isolates.The pathogenicity of the strain showed that the 7-day fatality rates of PMIK-2were 71.4%and 85.7%,respectively.The biochemical characteristics of Stenotrophomonas maltophilia PMIK-2are basically in line with Berger’s manual of systematic bacteriology.The optimal growth temperature and pH as well as the tolerance to bile salts also indicated that PMIK-2could survive in the intestinal tract of dairy cows.The use of streptomycin,ampicillin,tetracycline,imipenem,gentamicin and other drugs should be reduced or avoided in the treatment of diseases caused by PMIK-2infection.In conclusion,PMIK-2has strong pathogenicity,and the difference in the number of isolated PMIK-2in rumen between high-yield and low-yield groups may be one of the reasons for the difference in production status of dairy cows.