新疆地区某奶牛场都柏林与姆班达卡沙门氏菌的致病力与耐药性分析

Pathogenicity and antibiotic resistance of Salmonella dublin and Salmonella mbandaka from a dairy farm in Xinjiang

摘要:

【背景】沙门氏菌(Salmonella)是重要的人兽共患病病原菌，也是重要的食源性致病菌，其在新疆地区缺少奶牛源的流行菌株信息，对其耐药情况和危害的研究相对缺乏。【目的】了解石河子地区某犊牛腹泻牛场沙门氏菌感染情况，揭示感染病原分型、致病力和耐药性。【方法】采集了腹泻牛肛拭子、环境拭子55份。按照国标方法GB 4789.4—2016从样本中分离可疑菌株并进行纯化培养，使用革兰氏染色镜检、全自动生化鉴定系统、stn基因、16S rRNA基因等方法进行鉴定；分析其血清型及多位点序列分型，使用药敏试验(微量肉汤稀释法)、耐药基因、小鼠致病力试验和毒力基因检测进行耐药性和毒力分析。【结果】分离获得4株疑似沙门氏菌的病原菌，在沙门氏菌显色培养基上显紫色，镜检可见革兰氏阴性短杆菌；生化鉴定结果与沙门氏菌的相似性为98%，stn基因扩增片段大小与预期一致。16S rRNA基因系统进化分析显示分离株与沙门氏菌参考株的相似性为99%以上。病死牛组织源分离株命名为Sal135，肛拭子源分离株命名为Sal141、Sal142、Sal143。Sal135、Sal143属于都柏林沙门氏菌(Salmonella dublin)，ST10型；Sal141、Sal142属于姆班达卡沙门氏菌(Salmonella mbandaka)，ST413型；4株分离株对头孢呋辛、庆大霉素、链霉素均耐药，对氨苄西林、替加环素、氟苯尼考、多黏菌素E和阿奇霉素耐药率为25%-50%，其中菌株Sal143为七重耐药菌株。所有菌株均携带aac(6')-Iaa基因，此外，菌株Sal143还携带有aph(3'')-Ib、bla_CTX-M、floR、sul2和tetA。以约2×10⁷ CFU/mL剂量对小鼠腹腔注射后，菌株Sal135组死亡率为60%，菌株Sal143组死亡率为40%。16种毒力基因检测结果显示，invA、sipA、avrA、mgtCB、sipC、bcfA、fimA、sopB基因的检出率为100%，除pefA基因外其他毒力基因均被检出。【结论】发现该牛场4头犊牛感染了沙门氏菌，分离株Sal143显示多重耐药，都柏林沙门氏菌菌株的毒力较强且携带spv族毒力基因，推测其为致犊牛腹泻的主要病原。

Abstract:

[Background] Salmonella is a genus of major zoonotic agents and foodborne pathogens. However, there is limited data on cow-derived strains of Salmonella in Xinjiang, and studies remain to be carried out for their antibiotic resistance and pathogenicity. [Objective] To investigate Salmonella infections in calves from a dairy farm with the outbreak of diarrhea in Shihezi and reveal the types, virulence factors, and antibiotic resistance of the pathogens. [Methods] A total of 55 anal and environmental swabs were subsequently collected. The isolation of suspected strains were obtained from the samples according to the national standard method GB 4789.4—2016, and purified and cultured. The isolates were identified by Gram staining, microscopic examination, an automated biochemical identification system, and molecular techniques including amplification of stn and the 16S rRNA gene. Following serotyping and multi-locus sequence typing, the antibiotic susceptibility testing via minimal inhibitory concentration determination, detection of resistance genes, testing of pathogenicity in mice, and virulence gene detection were carried out for the isolates. [Results] Four suspected Salmonella strains were isolated, exhibiting purple colonies on Salmonella chromogenic media and appearing as Gram-negative short rods in microscopy. Biochemical identification results indicated 98% similarity to Salmonella, and the amplified fragment of stn matched the expected size. The phylogenetic analysis of the isolates based on the 16S rRNA gene revealed over 99% sequence homology with the reference strains of Salmonella. The isolate from the tissue of the deceased calf was designated Sal135, while those from anal swabs were named Sal141, Sal142, and Sal143. Sal135 and Sal143 were identified as Salmonella dublin, ST10, while Sal141 and Sal142 were identified as Salmonella mbandaka, ST413. The four isolates exhibited resistance to cefuroxime, gentamicin, and streptomycin, with the resistance rates to ampicillin, tigecycline, florfenicol, colistin, and azithromycin ranging from 25% to 50%. Among the isolates, Sal143 demonstrated the resistance to seven categories of antibiotics. All the strains carried aac(6')-Iaa, and Sal143 additionally carried aph(3'')-Ib, bla_CTX-M, floR, sul2, and tetA. Following intraperitoneal injection into mice at a dose of approximately 2×10⁷ CFU/mL, Sal135 and Sal143 induced the mortality rates of 60% and 40%, respectively. Detection of 16 virulence genes revealed that invA, sipA, avrA, mgtCB, sipC, bcfA, fimA, and sopB were present in all strains, and other virulence gens but pefA were detected. [Conclusion] Four calves in this farm were found to be infected with Salmonella. The isolate Sal143 exhibited multidrug resistance, and the strains of Salmonella dublin demonstrated strong virulence and carried the virulence gene cluster spv, being the primary pathogens responsible for calf diarrhea.