安徽部分地区动物源弯曲菌的分离鉴定及多位点序列分型

【背景】弯曲菌是一种重要的食源性人兽共患病原菌,革兰氏阴性、微需氧、弯曲螺旋状。【目的】为了解安徽地区弯曲菌流行状况和分子遗传特征,对安徽6个不同地区动物源的弯曲菌进行分离鉴定,并研究分离株分子分型。【方法】通过形态学及培养特性观察、生化试验、PCR方法对菌株进行鉴定。以弯曲菌7个管家基因asp A、gln A、glt A、gly A、pgm、tkt和unc A为目的基因对分离株进行多位点序列分型,并制成遗传进化树。【结果】共分离到42株弯曲菌菌株,源自6个地区的分离株具有较为一致的形态特性和相似的生化特性。多位点序列分型结果显示,本研究中共获得32种ST型,共发现9种新的ST型(8190、8222、8223、8831、8833、8841、8832、8834和8843)和6个新的等位基因(gln A606、gln A607、glt A518、gly A680、pgm863和unc A541)。进化树结果显示,空肠弯曲菌与结肠弯曲菌遗传关系相差甚远,聚集归为两个大群,分别有5个分支和3个分支。【结论】安徽6个地区不同来源的空肠弯曲菌与结肠弯曲菌均有丰富的基因型,且没有明显优势的基因型。从遗传变异的角度来看,空肠弯曲菌复杂多样,结肠弯曲菌相对保守。     

47株空肠弯曲菌湖北禽源株的多位点序列分型

【目的】为了解湖北地区家禽空肠弯曲菌的流行状况及其分子特征,应用多位点序列分型方法对2013–2014年的47株禽源空肠弯曲菌湖北分离株进行分子分型研究。【方法】以空肠弯曲菌的7个管家基因aspA、glnA、gltA、glyA、pgm、tkt和uncA为目的基因,提取样本基因组后PCR扩增,测序和分析。将测序结果上传数据库进行比对,制作成多位点序列分型(Multilocus sequence typing,MLST)遗传进化树。【结果】分离株共有38个ST型,10个克隆群,其中最多的克隆群为ST-353CC和ST-464CC,发现2个新的等位基因编号和25个新的ST型。遗传进化树显示,不同家禽宿主中空肠弯曲菌序列型存在一定的差异,不同地区和来源的空肠弯曲菌呈现出遗传多样性。【结论】本研究对湖北分离的47株禽源空肠弯曲菌进行了MLST分析,其结果显示菌株多样性较为丰富,将为我国家禽空肠弯曲菌的流行病学调查提供科学的数据。     

多位点序列分型分析空肠弯曲菌华东动物源分离株

【目的】研究空肠弯曲菌菌株间的分子特征,对不同宿主来源的空肠弯曲菌进行分子分型研究。【方法】选择空肠弯曲菌的7个看家基因gltA、aspA、glnA、glyA、pgm、tkt和uncA作为目的基因,对2006-2008年间华东地区分离的42株空肠弯曲菌样本进行PCR扩增后测序。将测序结果软件分析并上传到数据库进行比对,将结果制作多位点序列分型(multilocus sequence typing,MLST)遗传进化树并进行分析。【结果】与数据库已有类型比对,发现了24个新的ST型,通过进化树得到其遗传关系。【结论】MLST方法对于研究空肠弯曲菌的菌株群体基因差异与进化趋势具有重要意义。     

基于全基因组序列的弯曲菌特征分析

空肠弯曲菌(Campylobacter jejuni)和结肠弯曲菌(Campylobacter coli)是引起人类腹泻的主要致病菌。传统生化方法在鉴定弯曲菌时存在步骤多、耗时长、通量低等问题。本研究通过利用生物信息学方法对弯曲菌全基因组进行序列、基因注释、耐药基因、多位点序列分型以及CRISPR-Cas系统等分析,挖掘能够有效区分空肠弯曲菌和结肠弯曲菌的高分辨力特征。实验结果表明,空肠弯曲菌和结肠弯曲菌在基因组序列长度、GC含量、基因数量、多位点序列分型以及CRISPR-Cas系统等方面存在显著差异。同时,研究还发现了一段在空肠弯曲菌基因组中广泛存在的高分辨力CRISPR重复序列。这些特征可用于构建能够准确鉴别空肠弯曲菌和结肠弯曲菌的生物信息学方法。     

139株空肠弯曲菌耐药性分析

空肠弯曲菌(Campylobacter jejuni)是最常见的食源性病原菌之一。本研究采用微量肉汤稀释法对分离得到的139株空肠弯曲菌(117株为禽源样本分离株,22株为人源样本分离株)进行耐药性检测。通过对最小抑菌浓度(MIC)的判定结果得出:120株(86. 33%)空肠弯曲菌分离株对6类9组临床常用的抗生素表现出不同程度的耐药,其中禽源空肠弯曲菌耐药率为83. 76%,22株人源空肠弯曲菌均表现出耐药性。对喹诺酮类抗生素表现出高度耐药(环丙沙星80. 58%,萘啶酸77. 70%);对四环素类表现为中等耐药(四环素53. 24%);对部分大环内酯类、氨基糖苷类、林可酰胺类表现为低耐药(庆大霉素7. 19%,阿奇霉素5. 76%,克林霉素6. 47%);对酰胺醇类、部分大环内酯类表现为敏感(氟苯尼考0%,红霉素0%、泰利霉素0%)。139株空肠弯曲菌共产生14种耐药谱型,以TET-CIP-NAL谱型最多,占比38. 13%,耐三重及以上抗生素的多重耐药菌株占比53. 24%。禽源菌株中多重耐药占比46. 15%,人源菌株中多重耐药占比90. 91%。研究结果显示空肠弯曲菌耐药现状不容乐观,尤其对喹诺酮类与四环素类抗生素耐药性较为突出,且过半数菌株为多重耐药。本研究为食源性空肠弯曲菌的防控及临床用药提供参考。     

儿童感染青霉素耐药肺炎链球菌的多位点序列分型

目的 对儿童感染的青霉素耐药肺炎链球菌进行多位点序列分型,了解厦门地区肺炎链球菌青霉素耐药菌株遗传背景。方法 采用多位点序列分型法对2012年1月至2014年12月期间分离的60株青霉素耐药肺炎链球菌进行分子分型。结果 60株青霉素耐药肺炎链球菌MLST法共检出24个ST型,其中发现6个新的ST型,分别被命名为ST10004、ST10005、ST10006、ST10007、ST10008和ST10009。存在一个优势型别ST271,占31.7%（19/60）,发现了4个克隆群和20种单一克隆,其中主要克隆群为国际流行耐药克隆群Taiwan19F-14,占41.7%（25/60）。结论 本地区分离的儿童青霉素耐药肺炎链球菌主要以ST271型为主,属国际流行耐药克隆群Taiwan19F-14,是引起儿童呼吸道感染肺炎链球菌多重耐药的主要原因。     

天津地区气单胞菌分离株的鉴定与多位点序列分型

[目的]研究气单胞菌菌株分类情况,并分析其致病性.[方法]采集环境样品和鱼类标本,分离并鉴定气单胞菌菌株,并运用多位点序列分型(Multilocus sequence typing,MLST)方法进行分类研究,利用PCR和测序方法分析毒力基因Aera、Hly、Aha1、GCAT和Nuc的分布.[结果]通过对分离菌株的16S rRNA基因进行分析,确认属于4种不同气单胞菌的7个分离株.发现所有菌株至少有1种毒力基因阳性,其中3株具有4种毒力基因.药物敏感实验显示,6株分离株对3种或3种以上抗菌素具有多重耐药性.最后,对看家基因gyrB、groL、gltA、metG、ppsA和recA进行分析,与MLST数据库中的等位基因序列比对,发现7株分离株均为新的不同的序列型(Sequence type,ST).[结论]气单胞菌具有较高的遗传多样性.     

病原微生物基因多位点序列分型的研究进展

多位点序列分型(Multilocus sequence typing,MLST)是近年来发展很快的分子生物学分析方法,具有很高的分辨能力,既适于分子流行病学研究,也可用于分子进化学的研究。通过分析多个管家基因(Housekeeping gene)450bp左右的核心片段的核酸序列,从而对菌株的等位基因进行多样性的比较,不同的菌株对应不同的序列型(Se-quence type)。所以MLST越来越多的被作为能进行国际间菌株比较的常用工具,建立一种更为准确的分型系统方法。并且应用于研究出现的不同的抗生素抵抗株,毒力或抗原的相关特殊基因型,及新的变异株引起的疾病流行等流行病学分析,进行生物进化和种群结构的研究。     

四川地区猪源艰难梭菌分子分型调查

[背景] 艰难梭菌是一种重要的人畜共患肠道病原菌,可引起人和多种动物抗生素相关性腹泻或假膜性肠炎。四川作为我国主要的生猪产区,还未有猪源艰难梭菌流行病学调查的相关报道,对猪源艰难梭菌的防控及保障猪肉安全带来挑战。[目的] 调查四川地区猪源艰难梭菌的感染、流行情况,并对分离出的艰难梭菌进行分子分型研究。[方法] 收集来自四川生猪主要产区6个养殖场中猪的粪便标本（n=110）,采用厌氧培养技术在艰难梭菌鉴别培养基上进行分离培养;采用PCR方法扩增艰难梭菌4个毒素基因（tcdA、tcdB、cdtA、cdtB）和7个管家基因（adk、atpA、dxr、glyA、recA、sodA、tpi）,对分离株进行毒素基因分型和多位点序列分型。[结果] 从110份样品中,经革兰氏染色镜检及PCR鉴定,共分离出20株艰难梭菌,分离率高达18.18%;毒素基因分型结果显示共获得3种毒素基因型,包括tcdA⁺tcdB⁺cdtA/cdtB⁺（n=3）、tcdA⁺tcdB⁺cdtA/cdtB^—（n=6）、tcdA^—tcdB^—cdtA/cdtB^—（n=11）;多位点序列分型结果显示获得5个ST型,包括ST11（n=3）、ST3（n=1）、ST35（n=2）、ST36（n=4）、ST109（n=10）;进化树结果显示,所有分离株聚集为2个大群,分别为3个分支和17个分支。[结论] 四川主要生猪产区猪群存在艰难梭菌感染,分离株的分子分型呈多样性,主要流行型为ST11、ST3、ST35、ST36、ST109型,并且存在ST11型高毒力菌株流行的风险。     

海南岛光滑假丝酵母菌多位点序列分型研究

目的:对海南省光滑假丝酵母菌临床分离株进行基因分型研究,了解菌株的遗传特征及遗传进化关系.方法:采用多位点序列分型(Multilocus sequence typing,MLST)技术,对临床分离的25株光滑假丝酵母菌6个管家基因序列进行测定;并将各个基因的序列与MLST数据库中储存的序列比对,确定其等位基因谱型及菌株序列型(STs).结果:25株临床分离的光滑假丝酵母菌通过MLST产生ST7、ST19、ST15、ST26、ST45共5个不同的序列型,其中ST7为主要序列型.结论:海南省光滑假丝酵母菌感染型别丰富,具有多样性;MLST分型具有较高的分辨力,可用于流行病学和菌群多态性的研究.     

Genotyping of Campylobacter coli isolated from humans and retail meats using multilocus sequence typing and pulsed-field gel electrophoresis

Aims:  To determine the antimicrobial resistant profiles and clonality of Campylobacter coli isolated from clinically ill humans and retail meats.
Methods and Results:  A total of 98 C. coli isolates (20 from humans and 78 from retail meats) were phenotypically characterized. Antimicrobial susceptibility testing was done using agar dilution method for ciprofloxacin, gentamicin, erythromycin and doxycycline. Seventy C. coli isolates including humans ( n  = 20) and retail meats ( n  = 50) were genotyped by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Resistance to ciprofloxacin was found in 29% and 15% of isolates from retail meats and humans. We observed 61 PFGE profiles using two enzymes ( Sma I, Kpn I) with an Index of discrimination of 0·99, whereas MLST generated 37 sequence types. Two clonal complexes were identified with 58 (82%) C. coli isolates clustered in the ST-828 complex.
Conclusions:  Resistance to ciprofloxacin and erythromycin was identified in C. coli obtained from retail meats and ill humans. PFGE typing of C. coli isolates was more discriminatory than MLST. Grouping of C. coli isolates (82%) by MLST in ST-828 clonal complex indicates a common ancestry.
Significance and Impact of the Study:  A high frequency of resistance found to ciprofloxacin and erythromycin is concerning from food safety perspective. PFGE using single or double restriction enzymes was found to be more discriminatory than MLST for genotyping C. coli . Overall, the C. coli populations recovered from humans and retail meats were genotypically diverse.     

Multilocus sequence typing performed on Campylobacter coli isolates from humans, broilers, pigs and cattle originating in Denmark

AIMS: To assess whether Campylobacter coli isolated from different sources in Denmark constitute separate populations. METHODS AND RESULTS: Multilocus sequence typing (MLST) was applied to 160 C. coli isolates from animal origin, food products and human cases of gastroenteritis. The isolates were collected in Denmark over a 2-year period. In total, 84 sequence types (STs) were obtained and 57 of these STs were novel to this study. Ten per cent of the isolates possessed STs that were found in both human, poultry and pig isolates. Only 10% of the isolates from pigs shared ST with isolates from humans, and these shared STs were found in poultry isolates as well. CONCLUSIONS: Great genetic diversity was seen within the Danish C. coli population. Furthermore, we found that the C. coli types isolated from Danish pigs constitute a small fraction of the C. coli causing human disease. SIGNIFICANCE AND IMPACT OF THE STUDY: C. coli isolates from pigs is presumably not a significant source of human campylobacteriosis in Denmark. The Danish C. coli isolates include 68% STs novel to this study, showing a great diversity compared with studies from other countries.     

Multilocus sequence typing of Campylobacter jejuni isolates from New South Wales, Australia

AIMS: Multilocus sequence typing (MLST) was used to examine the diversity and population structure of Campylobacter jejuni isolates associated with sporadic cases of gastroenteritis in Australia, and to compare these isolates with those from elsewhere. METHODS AND RESULTS: A total of 153 Camp. jejuni isolates were genotyped. Forty sequence types (STs) were found, 19 of which were previously undescribed and 21 identified in other countries. The 19 newly described STs accounted for 43% of isolates, 16 of which were assigned to known clonal complexes. Eighty-eight percent of isolates were assigned to a total of 15 clonal complexes. Of these, four clonal complexes accounted for 60% of isolates. Three STs accounted for nearly 40% of all isolates and appeared to be endemic, while 21 STs were represented by more than one isolate. Seven infections were acquired during international travel, and the associated isolates all had different STs, three of which were exclusive to the travel-acquired cases. Comparison of serotypes among isolates from clonal complexes revealed further diversity. Eight serotypes were identified among isolates from more than one clonal complex, while isolates from six clonal complexes displayed serotypes not previously associated with those clonal complexes. CONCLUSIONS: Multilocus sequence typing is a useful tool for the discrimination of subtypes and examination of the population structure of Camp. jejuni associated with sporadic infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the genotypic diversity of Camp. jejuni in Australia, demonstrating that STs causing disease have both a global and a local distribution evident from the typing of domestically and internationally acquired Camp. jejuni isolates.     

Ribosomal operon intergenic sequence region (ISR) heterogeneity in Campylobacter coli and Campylobacter jejuni

Aims: The intergenic sequence regions (ISR) between the 16S and 23S genes of Campylobacter jejuni and Campylobacter coli are markedly different for each species. However, in the genomic sequence for Camp. coli RM2228 , two rRNA operons have an ISR that is characteristic of Camp. coli, and the third operon is characteristic of Camp. jejuni. The aim of this study was to determine the prevalence of ISR heterogeneity in these organisms. Methods and Results: PCR primers were designed to yield a 327‐base pair (bp) product for Camp. coli and 166‐bp product for Camp. jejuni. A strain like Camp. coli RM2228 should yield products of both sizes. DNA from a panel of Camp. coli (n = 133) and Camp. jejuni (n = 134) isolates were tested. All of the isolates yielded products of the predicted size for the species. To verify the data for Camp. coli RM2228 , each ribosomal operon from the isolate was individually amplified by PCR and tested with the ISR primer pair. Products of both sizes were produced as predicted. Conclusions: The cross‐species heterogeneity of the ISR seen in Camp. coli RM2228 is uncommon. Significance and Impact of the Study: The heterogeneity must have been caused by horizontal gene transfer at a frequency lower than predicted from housekeeping gene data. Thus, it can be expected that species identification based on the ISR can be confused in rare isolates.