甘肃省及周边地区畜禽屠宰及市售环节沙门菌的分离鉴定及生物学特性分析

Isolation,Identification and Biological Characterization Analysis of Salmonella from Livestock and Poultry Slaughtering and Marketing in Gansu Province and Neighboring Areas

[目的]为合理防控沙门菌感染,本试验针对甘肃省及周边地区畜禽屠宰及市售环节沙门菌的感染情况开展研究。[方法]通过细菌分离培养、生化试验及分子生物学方法进行细菌分离鉴定,采用玻片凝集法和多位点序列分型(MLST)确定沙门菌血清型。采用结晶紫染色定量法评价分离株生物被膜形成能力,通过药敏试验检测分离菌对常用抗菌药的敏感性,评估4种市售消毒剂对沙门菌的灭菌效果,并确定其最小抑菌浓度(MIC)。[结果]细菌分离培养结果显示,疑似沙门菌分离株在BS平板上菌落呈黑色有金属光泽、棕褐色或灰色;在XLD平板上菌落有光泽且中心呈黑色,或呈现全部黑色的菌落;镜检显示为革兰阴性短杆菌。生化鉴定结果显示,分离菌硫化氢、葡萄糖、鸟氨酸、赖氨酸脱羧酶反应结果呈阳性;尿素酶、氰化钾、乳糖、蛋白胨水反应结果呈阴性。PCR扩增沙门菌特异性基因invA获得大小约为284 bp的目的条带,共分离到95株沙门菌,均属于肠道沙门菌的3种血清型——鼠伤寒沙门菌、德尔卑沙门菌、里森沙门菌。多位点序列分型共鉴定出3种,分别为ST34、ST469、ST40型。所有分离株均可形成生物被膜。药敏试验结果显示,所有菌株对米诺环素、万古霉素完全耐药,对阿米卡星、头孢曲松、卡那霉素、头孢唑啉、头孢氨苄、庆大霉素等敏感性较强,75.78%的菌株具有多重耐药性。4种消毒剂对分离株MIC存在差异,0.2%甲醛溶液的MIC为0.125~0.25 mg/mL,0.2%苯扎溴铵的MIC为0.0625~0.125 mg/mL,0.5%碘溶液的MIC为0.3125~0.625 mg/mL,2%氢氧化钠的MIC为1.25~2.5 mg/mL。[结论]德尔卑沙门菌、鼠伤寒沙门菌和里森沙门菌是当前屠宰场中沙门菌的主要血清型,具有生物被膜形成能力和多重耐药性,给公共卫生带来很大威胁。本研究为加强甘肃省及周边地区屠宰场及市售环节卫生管理提供科学依据。

[Objective]In order to rationally prevent and control Salmonella infection,an experiment was conducted to study the prevalence of Salmonella in livestock and poultry slaughtering and marketing in Gansu province and neighboring areas.[Method]Bacterial isolation and identification were carried out through bacterial culture,biochemical experiments,and molecular biology methods.The serotype of Salmonella was determined using slide agglutination and multilocus sequence typing(MLST).The quantitative method of crystal violet staining was used to evaluate the biofilm formation ability of the isolated strains.The sensitivity of isolated bacteria to commonly used antibiotics was tested through drug sensitivity tests.The sterilization effect of four commercially available disinfectants on Salmonella was evaluated,and their minimum inhibitory concentration(MIC)was determined.[Result]The results of bacterial isolation and culture showed that the suspected Salmonella isolates showed black colonies with metallic luster,tan or gray on BS plates.In XLD plate,the colonies showed glossy and black center,or all black colonies,and the microscopic examination showed that they were Gram-negative short bacilli.The biochemical identification results showed that the isolates reacted positively with hydrogen sulfide,glucose,ornithine,and lysine decarboxylase,and reacted negatively with urease,potassium cyanide,lactose and peptone water.PCR amplification of the Salmonella-specific gene invA yielded a target band of approximately 284 bp in size,confirming that a total of 95 Salmonella strains were isolated.All the isolates belonged to three serotypes of Salmonella enterica:Salmonella Typhimurium,Salmonella Derby and Salmonella Rissen.Multilocus sequence typing identified 3 species,ST34,ST469 and ST40.All the isolates could form biofilm,and drug sensitivity test showed that all strains were completely resistant to minocycline and vancomycin,and antibiotics such as amikacin,ceftriaxone,kanamycin,cefazolin,cefadroxil,and gentamicin were more susceptible to Salmonella isolates,and 75.78%of the strains were multidrug-resistant.The MIC of the four disinfectants against the isolates varied from 0.125 to 0.25 mg/mL for 0.2%formaldehyde solution,0.0625 to 0.125 mg/mL for 0.2%benzalkonium bromide,0.3125 to 0.625 mg/mL for 0.5%iodine solution,1.25 to 2.5 mg/mL for 2%sodium hydroxide.[Conclusion]Salmonella Typhimurium,Salmonella Derby and Salmonella Rissen were the main serotypes of Salmonella in current slaughterhouses,and they possessd the ability to form biofilms and exhibited multidrug resistance,posing a significant threat to public health.This study provided a scientific basis for strengthening hygiene management in slaughterhouses and agricultural markets in Gansu province and surrounding areas.