1株藏猪源Moraxella pluranimalium的分离鉴定及生物学特性分析

doi:10.16431/j.cnki.1671-7236.2023.08.025

摘要/Abstract

摘要： 【目的】确定四川省甘孜藏族自治州发病藏猪的病原菌,探究各菌株之间的亲缘关系及耐药性,为对其进行有效防控提供科学依据。【方法】从病死猪肺脏中分离出1株可疑病原菌,通过细菌分离培养、16S rRNA PCR扩增测序、遗传演化分析、药敏试验、耐药基因与23S rRNA突变位点检测、生物被膜试验、小鼠致病试验对分离菌株进行鉴定和分析。【结果】经16S rRNA PCR扩增与遗传进化分析显示,该菌与西班牙菌株CR-18、LG6-7g、AR-5A、GM5-1的核苷酸序列相似性均为100%,并与猪源莫拉菌聚为一个遗传分支,表明该病原菌为Moraxella pluranimalium,命名为KD-4。形态学观察显示该菌为双肾型革兰阴性双球菌。生物被膜试验测定显示,该菌可形成生物被膜,使动物病变痊愈后仍有细菌定植在体内,长期成为该病原菌的宿主。药敏试验结果显示,该菌对抗菌药物复达欣、磺胺异噁唑高度敏感;对盐酸大观霉素、美罗培南中度敏感;对氨苄青霉素、庆大霉素、阿莫西林、头孢噻呋钠、氟苯尼考、四环素、恩诺沙星耐药。其中,复达欣、磺胺异噁唑、盐酸大观霉素和美罗培南对该菌的最小抑菌浓度(MIC)分别为2、128、64和2 μg/mL。耐药基因检测结果显示,ermF、ermB、tetB、tetD、bla_TEM、bla_CTX-M基因均未检出。23S rRNA序列分析显示,存在A2982T、C3132T等15个突变位点,推测与分离菌株出现多重耐药有关。小鼠致病性试验结果显示,该菌可引起小鼠肺脏出现少量出血点,脾脏明显肿大,但不致死,耐过后可恢复健康。【结论】本研究为国内首次报道藏猪源Moraxella pluranimalium,药敏试验结果可为该菌引起的疾病防治与流行病学调查提供参考依据。

关键词: 藏猪; Moraxella pluranimalium; 分离鉴定; 药敏试验; 致病性

Abstract: 【Objective】 This study was aimed to determine the pathogenic bacteria of Tibetan pigs in Ganzi Tibetan Autonomous Prefecture of Sichuan province,explore the genetic relationship and drug resistance among the strains,and provide scientific basis for effective prevention and control.【Method】 A suspicious pathogenic bacteria was isolated from lung of dead pig.The isolated strains were identified and analyzed by bacterial isolation and culture,16S rRNA PCR amplification and sequencing,genetic evolution analysis,drug sensitivity test,drug resistance gene and 23S rRNA mutation site detection,biofilm test and pathogenicity test of mice,respectively.【Result】 The results of 16S rRNA PCR amplification and genetic evolution analysis showed that the nucleotide sequence similarity between this strain and Spanish strain CR-18,LG6-7g,AR-5A and GM5-1 was 100%,respectively,and it was clustered into a genetic branch with Moraxella suis,indicating that this pathogen was Moraxella pluranimalium,named KD-4.Morphological observation result showed that this strain was a Gram negative diplococcus of kidney type.The biofilm test determined that the bacteria could form biofilm,so that the bacteria still colonized in the body after the animal disease healed,and became the host of the pathogen for a long time.The results of drug sensitivity test showed that this strain was highly sensitive to the antibiotics Fudaxin and sulfaisoxazole;Moderately sensitive to spectinomycin hydrochloride and meropenem;Resistant to ampicillin,gentamicin,amoxicillin,cefotaxifur sodium,florfenicol,tetracycline and enrofloxacin.Among them,the minimum inhibitory concentration (MIC) of Fudaxin,sulfaisoxazole,spectinomycin hydrochloride and meropenem against this bacterium were 2,128,64 and 2 μg/mL,respectively.The results of drug resistance gene detection showed that ermF,ermB,tetB,tetD,bla_TEM and bla_CTX-M genes were not detected.23S rRNA sequence analysis results showed that there were 15 mutation sites such as A2982T and C3132T,which might be related to the multiple drug resistance of the isolated strain.The pathogenicity test of mice showed that the bacteria could cause a small amount of bleeding spots in lung of mice,and the spleen was obviously swollen,but it was not fatal,and it could recover health after tolerance.【Conclusion】 This study was the first report of Moraxella pluranimalium from Tibetan pigs in China,and the results of drug sensitivity test could provide reference basis for disease prevention and epidemiological investigation caused by this bacterium.

Key words: Tibetan pigs; Moraxella pluranimalium; isolation and identification; drug susceptibility test; pathogenicity

中图分类号:

S852.61⁺5

马静, 杨丹娇, 韦龙拼, 甘代冰, 龙芷欣, 何宇琪, 卿霆, 范燕迪, 周泷. 1株藏猪源Moraxella pluranimalium的分离鉴定及生物学特性分析[J]. 中国畜牧兽医, 2023, 50(8): 3275-3285.

MA Jing, YANG Danjiao, WEI Longpin, GAN Daibing, LONG Zhixin, HE Yuqi, QING Ting, FAN Yandi, ZHOU Long. Isolation,Identification and Biological Characteristics Analysis of a Moraxella pluranimalium Strain from Tibetan Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(8): 3275-3285.

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