畜牧兽医学报  2023, Vol. 54 Issue (11): 4880-4888. DOI: 10.11843/j.issn.0366-6964.2023.11.042    PDF    
鸽源鼠伤寒沙门菌的分离鉴定及致病性分析
杨梦林1, 郑世奇1, 彭凯2, 王玮1, 黄燕华1,3, 彭杰1     
1. 仲恺农业工程学院健康养殖创新研究院, 广州 510225;
2. 广东省农业科学院动物科学研究所, 广东省畜禽育种与营养研究重点实验室, 农业农村部华南动物营养与饲料重点实验室, 广州 510640;
3. 岭南现代农业科学与技术广东省实验室, 广州 510642
摘要:本研究旨在探究广东某地区鸽场沙门菌的系统进化分群、耐药性、耐药基因和毒力基因的携带情况及其致病性, 为该地区沙门菌病的防控提供理论依据。试验将72份鸽病变组织样品进行分离纯化以及生化鉴定, 通过16S rRNA测序、血清型鉴定、鼠伤寒沙门菌特异性引物PCR鉴定结果判断菌株类型, 检测分离菌耐药性、耐药基因、毒力基因, 并利用小鼠开展动物致病性试验。结果显示, 12株分离菌镜检形态、生化指标、16S rRNA序列比对、血清型鉴定及特异性引物PCR结果均符合鼠伤寒沙门菌特性; 耐药结果显示, 12株菌对萘啶酸和利福平耐药, 对甲氧苄啶、复方新诺明、恩诺沙星、氟苯尼考、氨苄西林、美罗培南高度敏感; 15种耐药基因检测结果显示7株菌含有tetA、5株菌含有sul-11, 其他耐药基因均未检测出; 17种毒力基因检测结果显示, 5株菌毒力基因携带率为100%, 其余菌株毒力基因携带率也较高; 在动物致病性试验中, 试验组小鼠体重、采食量显著下降, HE染色观察肝脏枯否细胞数目增加, 空肠杯状细胞数目减少, 绒隐比下降。综上, 本研究分离的12株菌均属于鼠伤寒沙门菌, 均出现多重耐药, 并且毒力基因携带率较高, 动物试验观察到致病菌对动物体器官能够造成较强的炎性损伤, 致病性强。建议当地使用甲氧苄啶等高敏感药物治疗鸽沙门菌病, 或使用抗生素替代物治疗, 定期检测细菌耐药性, 及时调整治疗方案。
关键词鼠伤寒沙门菌        耐药基因    毒力基因    致病性    
Isolation and Identification of Pigeon-derived Salmonella Typhimurium and Pathogenic Analysis
YANG Menglin1, ZHENG Shiqi1, PENG Kai2, WANG Wei1, HUANG Yanhua1,3, PENG Jie1     
1. Innovative Institute of Animal Healthy Breeding, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China;
2. Key Laboratory of Animal Nutrition and Feed Science in South China of Ministry of Agriculture in Rural Affairs/Guangdong Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
3. Guangdong Laboratory for Lingnan Modern Agricultural, Guangzhou 510642, China
Abstract: The aim of this study was to investigate the phylogenetic grouping, drug resistance, virulence and resistance gene carriage and pathogenicity of Salmonella spp. carried by diseased pigeons in a region of Guangdong, and to propose a basis for the prevention and control of Salmonella spp. diseases in the region. In this study, 72 samples of diseased pigeon tissues were isolated, purified and identified. We determined the strain type by 16S rRNA sequencing, serotype identification, and PCR identification of Salmonella typhimurium-specific primers, and detected the drug resistance, resistance gene, and virulence genes of isolated bacteria, and carried out animal pathogenicity tests using mice. We found that the morphology, biochemical indexes, 16S rRNA sequence ratio, serotype identification and specific primer PCR results of the 12 isolates were consistent with the characteristics of Salmonella typhimurium; Resistance results showed that 12 strains were resistant to nalidixic acid and rifampicin, and highly sensitive to trimethoprim, co-trimoxazole, enrofloxacin, florfenicol, ampicillin, and meropenem; We found that 15 drug resistance gene test results showed that 7 strains contained tetA, 5 strains contained sul-11, and other resistance genes were not detected. The results of 17 virulence gene tests showed that the virulence gene carrier rate of 5 strains was 100%, and the virulence gene carrier rate of the remaining strains was also high; In addition, we found that in the animal pathogenicity test, the body weight and feed intake of mice in the experimental group decreased significantly, HE staining observed that the number of liver withered cells increased, the number of jejunal goblet cells decreased, and VH/CD decreased. The 12 strains isolated in this study all belonged to Salmonella typhimurium, all of which showed multi-drug resistance and high virulence gene carrier rate. Animal tests have observed that pathogenic bacteria can cause strong inflammatory damage to animal body organs and are highly pathogenic. We recommend the use of highly sensitive drugs such as trimethoprim for the treatment of salmonellosis in pigeons, or treatment with antibiotic substitutes, regular detection of bacterial resistance, and timely adjustment of treatment regimens.
Key words: Salmonella typhimurium    pigeon    resistance genes    virulence genes    pathogenicity    

沙门菌在全世界广泛分布,自1885年沙门菌首次被分离后,截至目前为止,已发现的血清型已经超过2 600种[1]。鸽沙门菌病是肉鸽养殖业中常见的细菌病之一,主要病原菌为鼠伤寒沙门菌,通过消化道、呼吸道等途径传播,症状表现为精神状态萎靡、羽毛凌乱、下痢、行为失常等。被感染的种鸽能够通过垂直传播将细菌传递给种蛋,引起种蛋坏死,降低孵化率,还能通过哺乳等方式感染乳鸽,引起乳鸽生长速度减慢甚至死亡[2]。此外,沙门菌作为人畜共患病原菌,能够通过污染鸽的加工产品传播给人类和动物,对公共卫生安全造成威胁。目前抗生素滥用情况较为严重,造成了细菌的耐药性形成速度加快[3],因此了解沙门菌的耐药性对于科学用药有重要意义。

广东某地区规模化肉鸽养殖场出现肉鸽下痢、消瘦、精神异常、关节肿大等症状,临床观察发现疑似感染鸽沙门菌。试验采集病死鸽病变组织,进行细菌分离、纯化、鉴定、耐药性分析、耐药基因和毒力基因检测并建立感染啮齿动物模型,分析沙门菌的毒力和致病性,以期为沙门菌病防治提供一定的科学依据。

1 材料与方法 1.1 病料收集

从广东某地区三个大规模肉鸽养殖场收集72只病死鸽,根据病鸽临床症状在无菌条件下分别采集36份肝、23份脾、13份膝关节样品,共计72份待检测样本。

1.2 试剂和培养基

麦康凯培养基、XLD琼脂、SS琼脂、LB培养基、生化鉴定管购自广州环凯微生物科技有限公司产品;药敏纸片购自杭州微生物试剂有限公司;2×F8 Master Mix购自艾德莱有限公司;DNA标准DL2000,核酸染料E×Red(10 000×)购自北京庄盟国际生物基因科技有限公司产品;沙门菌属诊断血清试剂盒购自宁波天润生物药业有限公司。

1.3 病原菌分离与鉴定

在无菌条件下将72份病变组织进行研磨,先后接种于XLD琼脂、麦康凯琼脂和SS琼脂培养基分离纯化,记录菌落形态和颜色并进行革兰染色和镜检。对分离菌进行生化鉴定以及生长曲线观察;采用沙门菌属诊断血清,观察分离菌与O价血清凝集情况;鼠伤寒沙门菌特异性STM4497基因PCR鉴定[4]及16S rRNA鉴定[5],扩增片段送往生工生物工程(上海)股份有限公司测序,测序结果在NCBI数据库中进行Blast序列相似性对比分析,并构建系统进化树。

1.4 药敏试验

参照美国临床实验室标准委员会(NCCLS-2018) 描述的K-B法的标准,根据抑菌圈直径进行判定分离菌株的耐药情况。药敏试验用药包括氨苄西林、阿莫西林等20种药物。

1.5 耐药基因和毒力基因检测

试验挑取单菌落溶解于装有50 μL无菌水的EP管中,振荡混匀后作DNA模板,检测耐药基因(tetAtetBsul-11、aphA3、aacC4、aadAFaacC2、qnrAqnrBAac(6′)-IbcatIfloRcmlAblaTEMblaSHV)[6-14]和毒力基因(mogAsseLmgtCbcfAaraBspvAspvBspvCspvDspvRstnfliCfimAavrAinvHsopAvirK)[15-18]

1.6 动物致病性试验

将6周龄C57BL/6小鼠随机分为5组,每组6只,选取携带毒力基因数量最多的致病菌分别以103、105、107、109 CFU ·mL-1浓度的攻毒剂量处理试验组,每只灌服0.2 mL,设置阴性对照组,以PBS代替菌液,每只0.2 mL,仅第1日灌服,饲养期为6 d,试验期结束后观察小鼠器官病变。

2 结果 2.1 分离菌分离鉴定

本研究在72份样本共分离鉴定出12株病原菌,在XLD培养基均长出边缘整齐、圆形粉红色菌落,中心呈黑色(图 1A);在麦康凯培养基均长出表面光滑、圆形半透明灰白色菌落(图 1B);在SS琼脂均长出无色半透明状菌落,中心呈黑色(图 1C)。镜检观察到两端钝圆、没有芽胞和荚膜的革兰阴性杆状菌(图 1D)。生化鉴定结果均符合沙门菌特性,血清型鉴定结果显示沙门菌A~F多价O血清和沙门菌O4单价血清因子与12株分离菌发生凝集反应,鼠伤寒沙门菌特异性序列STM4497经PCR扩增得到一条接近523 bp DNA目的条带,并将16S rRNA测序结果在NCBI数据库中进行Blast分析对比并建立系统进化树(图 1E),综上,说明12株分离菌均属于鼠伤寒沙门菌。

A. XLD培养基上的菌落形态;B. 麦康凯培养基上的菌落形态;C. SS琼脂上的菌落形态;D. 镜检分离菌形态;E. 基于16S rRNA的系统进化树 A. Colony morphology on Xylose lysine Deoxycholate agar medium; B. Colony morphology on MacConey agar medium; C. Colony morphology on SS agar; D. Microscopic picture of isolated bacteria; E. Phylogenetic tree based on 16S rRNA 图 1 分离菌镜检及系统进化树构建 Fig. 1 Microscopic examination of isolated bacteria and construction of phylogenetic tree
2.2 分离菌耐药性检测

药敏结果显示,12株菌分离菌对恩诺沙星、甲氧苄啶、复方新诺明、氟苯尼考、氯霉素、氨苄西林、阿莫西林、头孢噻吩、美罗培南、头孢曲松耐药率为0.0%,对卡那霉素、呋喃妥因、头孢噻肟、丁胺卡那、环丙沙星、庆大霉素的耐药率为8.3%~33.3%,对多西环素、米诺环素的耐药率为58.3%~83.3%,对萘啶酸和利福平耐药率为100.0%。

2.3 分离菌耐药基因和毒力基因检测

12株分离菌耐药基因检测结果显示,tetA携带率为58.3%,sul-11携带率为41.7%,其他耐药基因未检出,结合“2.2”药敏试验结果发现,四环素类耐药基因与其耐药表型符合率100%,但磺胺类耐药基因与耐药表型符合率为0%。毒力基因检测结果显示,mgtCbcfAspvAspvDspvRstnavrAinvHsopA携带率为100%,sseLspvCfliC携带率为91.7%,mogAvirK基因携带率为83.3%,araB为75.0%,spvB为66.7%,fimA为58.3%,说明该地区流行沙门菌毒力基因携带较多,详见表 1

表 1 12株鼠伤寒沙门菌耐药基因与毒力基因结果 Table 1 Results of 12 strains of Salmonella typhimurium resistance genes and virulence genetic
2.4 分离菌致病性研究

试验随机选取携带检测毒力基因最多的1株分离菌进行攻毒小鼠试验,结果显示,随着灌菌浓度增大,小鼠体重和采食量显著下降(图 2AB),脾、肝肿大,结肠长度缩减(图 2CDE)。观察空肠HE切片发现鼠伤寒沙门菌引起绒毛长度与隐窝深度比值下降(图 2F),杯状细胞逐渐减少(图 2GI);观察肝HE切片发现大量免疫细胞汇集肝,并且肝枯否细胞(Kupffer cell)数目逐渐增加(图 2H图 3)。

A.体重;B.采食量;C.脾脏重;D.肝脏重;E.结肠长度;F.空肠绒毛长度/隐窝深度;G.空肠杯状细胞数;H.肝脏枯否细胞数;I.空肠与肝脏HE染色(n=4~6),红色箭头所指分别为杯状细胞和枯否细胞。以上试验组与NC组均进行t检验(P<0.05表示差异显著,P>0.05表示差异不显著,其中*、**、***、****分别表示P<0.05、0.01、0.001、0.000 1) A. Weight; B. Intake; C. Spleen weight; D. Liver weigh; E. Colon length; F. VH/CD; G. Number of goblet cells in jejunum; H. Number of hepatic Kupffer cell; I. HE staining of jejunum and liver (n=4-6), The red arrow to goblet cells and kupffer cells. The above experimental group and NC group were subjected to t-test (P<0.05 means the difference is significant, P>0.05 means the difference is not significant, among*, **, ***, **** respective P<0.05, 0.01, 0.001, 0.000 1) 图 2 相关指标数据统计 Fig. 2 The related index data were counted
图 3 空肠和肝脏病理切片(HE染色) Fig. 3 The pathological section of jejunum and liver (HE staining)
3 讨论

本研究发现从样本中分离出的12株致病菌均为鼠伤寒沙门菌,除本研究外,在多地报告中也指出鸽沙门菌病是由鼠伤寒沙门菌感染引起,这提示了鸽子对鼠伤寒沙门菌易感。细菌耐药性形成与抗生素产生的环境压力有着紧密联系,细菌能够通过整合子、质粒等从外界获取抗性基因,同时还能够通过质粒、转座子等多种可移动原件发生水平传播[3, 19-20]。试验检测出5株菌具有磺胺类耐药基因sul-11,但对磺胺类药物却保持高度敏感,与相关报道中磺胺类耐药基因sul-11与磺胺异噁唑耐药表型符合程度较高[21]具有差异性,这可能与本研究使用的药物为甲氧苄啶和复方新诺明有关,不同的药物作用机制存在差异,此外,sul-11基因能够表达出具有磺胺类药物抗性的二氢蝶酸合成酶,使细菌的生长不受磺胺类药物影响。但在细菌内部由于sul-11基因存在多种类型的质粒中[22-24],其表达水平以及作用机制并不清晰,需要进一步探究。除此之外,部分菌种对氨基糖苷类和喹诺酮类具有耐药性,但并未检出氨基糖苷类和喹诺酮类耐药基因,可能与本研究检测耐药基因种类不全有关,亦或者产生了新的耐药基因。

细菌是通过多个毒力因子之间的协同作用对机体器官造成炎性损伤的,因此检测毒力基因携带量有利于评估沙门菌致病能力。本研究发现,毒力岛基因sselmogAmgtCbcfAaraB检出率高于75.0%,毒力质粒基因、肠毒素毒力基因、鞭毛基因以及其他毒力基因检出率均高于66.0%,其中11株菌含有sseL毒力基因,在诸多研究中发现,携带毒力岛基因sseL的沙门菌毒性更强,沙门菌分泌的sseL蛋白能够抑制机体炎症反应,并且还能损伤吞噬细胞,有利于沙门菌逃逸[25-26]。除此之外,还有magtCavrAspvBspvCsopA等主要毒力因子[27],在本试验分离菌中携带率也较高,说明该地区沙门菌的致病性较强。

在动物致病性试验结果中发现灌服致病菌浓度高于105 CFU ·mL-1时,小鼠空肠绒隐比及杯状细胞数目随着灌菌浓度升高逐渐下降。杯状细胞分泌的黏蛋白是肠道机械屏障的重要组成部分,当沙门菌定植于肠道后,毒力岛SPI-1和SPI-2向肠上皮细胞输送效应蛋白,引起肠上皮细胞损伤,破坏肠道屏障的完整性[28-31],研究结果说明沙门菌对肠道吸收功能和屏障功能具有负面影响。除此之外,研究发现随着灌服致病菌浓度升高,肝脏中枯否细胞数量逐渐增多,有研究指出革兰阴性菌细胞壁组分脂多糖能够活化枯否细胞,使其炎症因子分泌增加,引起其他免疫细胞向肝脏聚集,共同清除血液中的细菌[32],然而这个过程是否有枯否细胞前体细胞募集进入肝脏发生分化增殖这一机制并不清晰,本研究枯否细胞数量增加可能与上述过程有关,此机制还有待进一步探究。

4 结论

成功分离出12株鼠伤寒沙门菌,通过药敏测试、耐药和毒力基因检测以及6周龄小鼠致病性研究发现分离菌均出现多重耐药,并且毒力基因携带量较高,致病性强,建议在该地区治疗鸽沙门菌病中使用甲氧苄啶、复方新诺明等高敏感药物,同时要提高公共卫生安全意识,注重食品安全,避免向人群传播。

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