禽源肠炎沙门菌的分离鉴定及耐药性与致病性分析

doi:10.16431/j.cnki.1671-7236.2023.04.037

摘要/Abstract

摘要： 【目的】为合理防控肠炎沙门菌感染,本试验针对四川4个地区禽源肠炎沙门菌的感染情况开展研究。【方法】通过细菌分离培养、生化试验及分子学方法进行细菌分离鉴定;进一步通过K-B法检测分离菌对14种抗菌药物的敏感性,并对分离菌进行耐药基因、毒力基因及致病性研究。【结果】细菌分离培养结果显示,分离菌符合沙门菌的培养特性,镜检为革兰氏阴性短杆菌,生化鉴定结果均符合沙门菌生化特性。沙门菌特异性毒力靶标基因invA和肠炎沙门菌特异性毒力靶标基因sdfI PCR扩增结果显示,获得大小分别为571和304 bp的目的条带,与预期大小相符,确认分离到10株肠炎沙门菌,总分离率为2.8%(10/361)。药敏试验结果显示,分离菌对氨基糖苷类、磺胺类、多肽类抗菌药耐药率较高;氨基糖苷类药物中,对庆大霉素和链霉素的耐药率分别为50%(5/10)和70%(7/10);磺胺类药物中,对复方新诺明和磺胺异噁唑的耐药率分别为40%(4/10)和100%(10/10);多肽类药物中,对多黏菌素B耐药率达90%(9/10);分离菌对喹诺酮类药物均表现敏感,对头孢类、β-内酰胺类和四环素类较敏感。分离菌普遍存在多重耐药现象,14种耐药基因中检出四环素类耐药基因tetB(40%)和tetM(90%),氨基糖苷类耐药基因aadA1(80%)和aph(3’)-Ⅱa(90%),β-内酰胺类耐药基因bla_TEM-1(100%),磺胺类耐药基因sul3(100.0%);6种毒力相关基因中,ssaQ(SPI-2)、mgtC(SPI-3)、spi4D(SPI-4)、pipA(SPI-5)、invA(SPI-1)和spvB的检出率分别为60%、80%、70%、60%、100%和80%。致病性试验结果显示,小鼠灌胃分离菌菌液24 h后出现死亡,剖检及病理切片观察到脏器淤血、出血,细胞变性等,肠段出现绒毛断裂脱落。【结论】本研究成功分离得到10株禽源肠炎沙门菌,分离菌具有较强耐药性和多重耐药现象,毒力基因检出率较高且有较强的致病性,本研究结果可为肠炎沙门菌病的防控及临床治疗提供参考。

关键词: 肠炎沙门菌; 耐药性; 耐药基因; 毒力基因; 致病性

Abstract: 【Objective】 In order to reasonably prevent and control Salmonella Enteritidis (S.Enteritidis) infection,this experiment was conducted on the infection of S.Enteritidis from avian in four regions of Sichuan.【Method】 Bacterial isolation and identification was carried out by bacterial isolation and culture,biochemical test and molecular method.The sensitivity of isolated bacteria to 14 antibacterial drugs was further detected by K-B method,and drug resistance genes,virulence genes and pathogenicity of isolated bacteria were studied.【Result】 The results of bacterial isolation and culture showed that the isolated bacteria conformed to the culture characteristics of Salmonella,the microscopic examination was Gram-negative short bacilli,and the biochemical identification results were consistent with the biochemical characteristics of Salmonella.The PCR amplification results of Salmonella specific virulence target gene invA and S. Enteritidis specific virulence target gene sdfI showed that the target bands of 571 and 304 bp were obtained,which were consistent with the expected size.It was confirmed that 10 strains of S.Enteritidis were isolated,with a total isolation rate of 2.8% (10/361).The results of drug sensitivity test showed that the isolated bacteria had a high resistance rate to aminoglycosides,sulfonamides and peptides.Among aminoglycosides,the drug resistance rates to gentamicin and streptomycin were 50% (5/10) and 70% (7/10), respectively.Among sulfonamides,the drug resistance rates to compound sulfamethoxazole and sulfamethoxazole were 40% (4/10) and 100% (10/10) respectively.Among polypeptide drugs,the resistance rate to polymyxin B was 90% (9/10).The isolated bacteria were sensitive to quinolones,and moderately sensitive to cephalosporins β-lactamides and tetracyclines.Multiple drug resistance was common in isolated bacteria.Tetracycline resistance genes tetB (40.0%) and tetM (90%),aminoglycoside resistance genes aadA1 (80%) and aph(3')-Ⅱa (90%),β-lactam resistance gene bla_TEM-1 (100%),sulfonamide resistance gene sul3 (100.0%) were detected in 14 drug resistance genes.Among the six virulence related genes,the detection rates of ssaQ (SPI-2),mgtC (SPI-3),spi4D (SPI-4),pipA (SPI-5),invA (SPI-1) and spvB were 60%,80%,70%,60%,100% and 80%,respectively.The results of the pathogenicity test showed that the mice died after 24 h of intragastric administration of isolated bacterial solution.Blood stasis,hemorrhage,cell degeneration,etc.were observed in the organs and pathological sections,and the villi in the intestinal segment were broken and shed.【Conclusion】 In this study,10 strains of S.Enteritidis from avian were successfully isolated,and the isolated bacteria had strong drug resistance and multiple drug resistance,and the detection rate of virulence genes of isolated bacteria was high and the pathogenicity was strong,which could provide reference for the prevention and control and clinical treatment of S.Enteritidis.

Key words: Salmonella Enteritidis; drug resistance; drug resistance gene; virulence gene; pathogenicity

中图分类号:

S852.61

王路才, 撒朗文朱, 张焕容. 禽源肠炎沙门菌的分离鉴定及耐药性与致病性分析[J]. 中国畜牧兽医, 2023, 50(4): 1663-1674.

WANG Lucai, SALANG Wenzhu, ZHANG Huanrong. Isolation,Identification and Drug Resistance and Pathogenicity Analysis of Salmonella Enteritidis from Avian[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1663-1674.

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