肠道沙门菌O:4(B)菌群脉冲场凝胶电泳分子分型

为了明确2010年食源性疾病监测中分离的肠道沙门菌O:4(B)菌群的PFGE分子型别,探讨其多态性及其与分子流行病学关系,我们将分离获得的29株O:4(B)血清型沙门菌进一步鉴定培养,挑取单个菌落增菌,供试菌株基因组DNA用限制性内切酶Xba Ⅰ消化酶切后进行脉冲场凝胶电泳分型,所得结果用Bionumerics5.1软件进行聚类分析.实验结果表明,根据电泳指纹图谱,可将29株肠道沙门菌O:4(B)菌群分为24个PFGE型别,菌株间的相似值在56.31%~100%之间,同一血清型别的沙门菌有多个PFGE型别.脉冲场凝胶电泳对O:4(B)群沙门菌有较高的分型能力,可有效的应用于食物中沙门菌溯源分析及分子流行病学研究.     

辽宁食源性沙门菌血清型、 耐药谱及PFGE分型特征

目的分析辽宁食源性沙门菌血清型、耐药谱及脉冲场凝胶电泳(PFGE)型别,探讨辽宁沙门菌污染的同源性,为食源性疾病溯源和预警提供基础。方法对辽宁省2015年食品中、食源性疾病中分离的41株沙门菌进行血清学分型、耐药试验、PFGE分子分型,采用Bio Numerics version 6.6软件分析,比较同源性。结果 41株菌分为15个血清型,居前三位的是15株肠炎沙门菌、5株德尔卑沙门菌、5株姆班达卡沙门菌(辽宁省内少见血清型);对41株菌进行15种抗生素的耐药试验,对单一一种抗生素的耐药率为100.0%,其中红霉素97.6%,萘啶酸61.0%,氨苄西林53.7%;41株菌共分为18种PFGE带型,带型分布分散,只有两种优势,一种带型包含20株菌,有14株肠炎沙门菌,6株其他沙门菌,相似度为92.7%~100%;另一种包含5株菌,4株姆班达卡沙门菌,1株鼠伤寒沙门菌,相似度为96.6%~100.0%。结论辽宁省食源性沙门菌的血清型以肠炎沙门菌为主,生肉制品是其主要污染来源;血清型与PFGE图谱带型分布广泛,相同血清型沙门菌的PFGE带型聚集成簇、菌株具有高度同源性;相同PFGE型别的菌株耐药谱一致或相似;沙门菌的耐药情况较严重。     

2016‒2017年辽宁省沙门菌耐药菌谱与分子分型

摘要：目的 分析2016?2017年辽宁省沙门菌分离株的耐药特性与脉冲场凝胶电泳（PFGE）分子分型特征,为沙门菌引起的食源性疾病暴发、防控及抗生素使用提供参考数据。方法 对分离的54株沙门菌进行血清分型和药物敏感试验。根据PulseNet沙门菌标准PFGE分型技术,选取全部菌株进行PFGE分子分型分析,应用BioNumerics软件对菌株条带进行分析,确定菌株间的特征及相关性。结果 54株沙门菌血清型居首位的是肠炎沙门菌,占46.30％;其次是鼠伤寒沙门菌,占24.07％;共分为10个血清型。对13种抗生素的耐药分析显示多重耐药菌株为36株,占66.7％,其中耐3～5种的13株（24.1%）,耐6～8种的13株（24.1%）,耐9～11种的10株（18.5%）。54株沙门菌经聚类分析获得36种带型,相似度区间为49.7%～100.0％。结论 辽宁省沙门菌分离株多重耐药状况比较严重,相同血清型其PFGE带型相似度相对较高,同时具有较显著的优势带型特点;而且发现同一PFGE型菌株的耐药谱相对比较接近。     

辽宁省耐药沙门菌PFGE分子分型研究

目的对辽宁省2011-2012年间鸡肉中检测出的沙门菌进行耐药谱分析和PFGE分子分型分析,为沙门菌的监测、暴发预警提供依据。方法 Phoenix-100全自动微生物鉴定/药敏系统做耐药性分析;PFGE分型依据国际实验室分子分型监测网络PulseNet中沙门菌PFGE分型标准化方案进行。结果鸡肉中38株沙门菌均多重耐药,PFGE指纹图谱与血清具有一定的相关性,相同血清型的菌株聚类后,都能分到同一群。结论同一地区的沙门菌具有很高的相似带型,为以实验室为基础的食源性疾病暴发的发现及疫情控制提供依据。     

2016-2019年辽宁省肠炎沙门菌分离株分子分型及其耐药性

目的分析辽宁省肠炎沙门菌分离株的分子分型特征及耐药情况,为辽宁省肠炎沙门菌的分子流行病学及防控措施提供参考依据。方法采用PFGE分子分型方法对辽宁省2016-2019年肠炎沙门菌分离株进行分子分型,应用BioNumerics 7.6软件对酶切片段进行聚类分析,明确菌株的特征及同源性;采用最低抑菌浓度(MIC)法测定菌株对14种药物敏感性。结果共获得49株肠炎沙门菌,分子分型结果证明其呈17种PFGE带型,相似度区间为77.4%~100.0%,有2种优势带型;对萘啶酸的耐药率最高,达89.80%,其次氨苄西林的耐药率为69.39%,对3种以上抗生素的耐药率为55.10%。结论辽宁省肠炎沙门菌PFGE分子分型具有独特的优势带型,存在带型较多的特点;肠炎沙门菌分离株多重耐药状况比较严重,对萘啶酸的耐药率最高。     

辽宁省食源性副溶血性弧菌PFGE分子分型及血清型、耐药谱

目的分析辽宁省副溶血性弧菌脉冲场凝胶电泳(PFGE)型别及血清型、耐药谱,探讨辽宁省副溶血性弧菌污染的同源性,为食源性疾病溯源和预警提供基础。方法对50株从辽宁省2015年食源性疾病和食品中分离的副溶血性弧菌进行PFGE分子分型、血清学分型、药物敏感试验,采用BioNumerics version 6.6软件进行分析,比较同源性。结果 50株副溶血性弧菌共分为19个血清型,食源性疾病分离株分为5个血清群,主要的血清型为O3群,其次为O1群;食品分离株分为9个血清群,主要的血清型为O3群,其次为O1和O4群。50株副溶血性弧菌对15种抗生素的耐药试验,对头孢唑啉耐药率最高达72%。50株副溶血性弧菌共分为4种PFGE优势带型,相似度为90.8%~100.0%。结论辽宁省食源性副溶血性弧的血清型以O3群和O1群为主,致病菌流行株血清型为O3:K6;食品中分离菌株血清型和PFGE带型非常分散,提示这些多为遗传不相关菌株。PFGE图谱可看出,绝大部分相同血清型菌株的PFGE带型相似度很高,O3与O1血清型菌株有高度同源性密切相关,耐药菌株间PFGE带型相似度很高,均具有高度同源性。相比2014年的耐药情况,对头孢唑啉耐药率显著增高。另外,少数菌株出现多重耐药,耐药情况不容乐观。     

2014年大连市伤寒沙门菌耐药及 分子分型研究

摘要：目的 了解大连市伤寒沙门菌耐药性及分子分型特点,建立沙门菌分子特征本底信息,为今后防治工作提供科学依据。方法 采用微量肉汤稀释法对46株伤寒沙门菌进行8种抗生素敏感试验;运用脉冲场凝胶电泳（PFGE）方法对46株伤寒沙门菌进行分子分型及聚类分析。结果 46株伤寒沙门菌对萘啶酸（NAL）100%敏感,对氯霉素（CHL）和甲氧苄啶/磺胺甲噁唑（TMP/SMZ）耐药率为4.35%,对庆大霉素（GEN）耐药率为47.83%,发现多重耐药株1株;BioNumerics分析结果显示,46株伤寒沙门菌共产生30种PFGE带型,有7株表现为同一PFGE型别。结论 大连地区存在耐庆大霉素的伤寒沙门菌;PFGE结果表明这些菌株存在遗传多态性,并有优势菌株的存在。     

重度肥胖患者肠道内机会性致病菌Escherichia coli微多样性分析

本研究利用志贺氏菌显色培养基和ERIC-PCR指纹图谱技术,从一名重度肥胖患者肠道内分离出19种不同ERIC类型的分离物。选取不同ERIC类型的代表菌株进行16S r RNA基因分子鉴定并构建系统发育树,发现19种代表菌株的16S r RNA基因和埃希氏菌属(Escherichia)、志贺氏菌属(Shigella)的相似性达到99%。经生理生化反应鉴定,19种代表菌株均为肠杆菌科的肠埃希氏菌(Escherichia coli)。体外生物学分析发现19种E.coli代表菌株在生化特性、耐药性、毒性方面有明显差异。本研究表明来自一名重度肥胖患者肠道内同一种群的E.coli在菌株水平上具有丰富的微多样性,不同ERIC类型的E.coli在生理生化特性上存在较大的差异,且部分菌株具有潜在致病性。     

林麝肠道乳酸菌及肠杆菌多态性分析及耐药性研究

为研究林麝肠道中乳酸菌和肠杆菌基因型及表型多样性并对其耐药性进行分析,本研究从10头健康成年林麝粪便样品中分离到22株乳酸菌和16株肠杆菌,通过16S rDNA进化分析和生理生化测定对分离菌株进行了鉴定,采用脉冲场凝胶电泳(PFGE)技术分析了分离菌株的基因型并利用药敏纸片法进行了分离菌株的耐药性研究。本研究共分离到了11种细菌,分别为大肠杆菌Escherichia coli、阴沟肠杆菌Enterobacter cloaca、河生肠杆菌E.amnigenus、肺炎克雷伯氏菌Klebsiella pneumoniae、蒙氏肠球菌Enterococcus mundtii、耐久肠球菌E.durans、明串球菌Leuconostoc fallax、植物乳杆菌Lactobacillus plantarum、嗜酸乳杆菌L.acidipiscis、屎肠球菌E.faecium和食窦魏斯氏菌Weissella cibaria。其中大肠杆菌(n=11,28.9%)是林麝肠道中的优势菌群,肠球菌(n=10,26.3%)是林麝肠道中的优势乳酸菌群。PFGE分型结果表明肠杆菌分为7个基因型,乳酸菌分为9个基因型。本研究首次分析了林麝肠道中可培养细菌的基因型及表型并且对其耐药性进行了测定,结果表明林麝肠道中的原生菌群多态性明显,并且对目前常用的抗生素敏感,未检测到耐药菌株。     

秦岭地区野生细鳞鲑肠道微生物多样性及产酶活性分析

为探究秦岭地区野生细鳞鲑(Brachymystax lenok)肠道细菌组成多样性,筛选出产胞外酶菌株,利用传统分离培养并分子鉴定的方法和基于16S r RNA基因克隆的现代分子生物技术相结合测定秦岭野生细鳞鲑肠道细菌菌群多样性并构建系统发育树,利用淀粉酶、蛋白酶、纤维素酶及脂肪酶4种胞外酶筛选培养基筛选出产上述酶的细菌。细菌传统分离培养并分子鉴定法从细鳞鲑肠道获得18个属的细菌类群,分别归属于变形菌门、拟杆菌门和厚壁菌门,其中,气单胞菌属(Aeromonas)为优势菌群。基于16S r RNA基因克隆的现代分子方法获得22个属的细菌类群,分别归属于变形菌门、拟杆菌门、厚壁菌门和放线菌门,其中,鞘氨醇杆菌属(Sphingomonas)为优势菌群。4种胞外酶筛选获得53株细菌产胞外酶,其中21株可在低温(10℃)环境下产胞外酶。结果表明,传统分离培养法与基于16S r RNA基因克隆的现代分子生物技术相结合能够更有效全面地分析细鳞鲑鱼肠道微生物的多样性,并且细鳞鲑肠道微生物具有一定的产酶活性。     

Limited genetic diversity in <Emphasis Type="Italic">Salmonella enterica</Emphasis> Serovar Enteritidis PT13

Background  

Salmonella enterica serovar Enteritidis has emerged as a significant foodborne pathogen throughout the world and is commonly characterized by phage typing. In Canada phage types (PT) 4, 8 and 13 predominate and in 2005 a large foodborne PT13 outbreak occurred in the province of Ontario. The ability to link strains during this outbreak was difficult due to the apparent clonality of PT13 isolates in Canada, as there was a single dominant pulsed-field gel electrophoresis (PFGE) profile amongst epidemiologically linked human and food isolates as well as concurrent sporadic strains. The aim of this study was to perform comparative genomic hybridization (CGH), DNA sequence-based typing (SBT) genomic analyses, plasmid analyses, and automated repetitive sequence-based PCR (rep-PCR) to identify epidemiologically significant traits capable of subtyping S. Enteritidis PT13.     

Multiphasic approach reveals genetic diversity of environmental and patient isolates of Mycobacterium mucogenicum and Mycobacterium phocaicum associated with an outbreak of bacteremias at a Texas hospital

Between March and May 2006, a Texas hospital identified five Mycobacterium mucogenicum bloodstream infections among hospitalized oncology patients using fluorescence high-performance liquid chromatography analysis of mycolic acids. Isolates from blood cultures were compared to 16 isolates from environmental sites or water associated with this ward. These isolates were further characterized by hsp65, 16S rRNA, and rpoB gene sequencing, hsp65 PCR restriction analysis, and molecular typing methods, including repetitive element PCR, random amplified polymorphic DNA PCR, and pulsed-field gel electrophoresis (PFGE) of large restriction fragments. Three of five patient isolates were confirmed as M. mucogenicum and were in a single cluster as determined by all identification and typing methods. The remaining two patient isolates were identified as different strains of Mycobacterium phocaicum by rpoB sequence analysis. One of these matched an environmental isolate from a swab of a hand shower in the patient's room, while none of the clinical isolates of M. mucogenicum matched environmental strains. Among the other 15 environmental isolates, 11 were identified as M. mucogenicum and 4 as M. phocaicum strains, all of which were unrelated by typing methods. Although the 16S rRNA gene sequences matched for all 14 M. mucogenicum isolates, there were two each of the hsp65 and rpoB sequevars, seven PCR typing patterns, and 12 PFGE patterns. Among the seven M. phocaicum isolates were three 16S rRNA sequevars, two hsp65 sequevars, two rpoB sequevars, six PCR typing patterns, and six PFGE patterns. This outbreak represents the first case of catheter-associated bacteremia caused by M. phocaicum and the first report of clinical isolates from a U.S. hospital. The investigation highlights important differences in the available typing methods for mycobacteria and demonstrates the genetic diversity of these organisms even within narrow confines of time and space.     

Application of pulsed-field gel electrophoresis to laboratory surveillance of small community outbreaks of Salmonella enterica serovar Typhimurium

Infectious diarrhea syndrome is an important cause of human morbidity around the world, and Salmonella genus remains one of the most prevalent etiology. Salmonella enterica serovar Typhimurium outbreak-associated isolates received by the Laboratory for Enteric Pathogens from N.I.R.D.M.I. "Cantacuzino" for confirmation and typing were analyzed by genomic pulsed-field gel electrophoresis (PFGE) and phage susceptibility testing to establish their relatedness. Both typing methods proved to have similar discriminatory power. The isolates originating from the same outbreak belonged to the same phage type and showed indistinguishable PFGE profiles. The molecular characterization of autochthonal Salmonella enterica Typhimurium outbreak human isolates provided laboratory evidence that epidemiologically related isolates collected from community outbreaks of disease were also genetically related. In order to improve the national and international surveillance of major foodborne pathogens the reference laboratory centers are required to establish and maintain the capacity to perform a wide range of both phenotypic and genotypic methods to support outbreak investigations.     

Genotypes and toxin gene profiles of Staphylococcus aureus clinical isolates from China

A total of 108 S. aureus isolates from 16 major hospitals located in 14 different provinces in China were characterized for the profiles of 18 staphylococcal enterotoxin (SE) genes, 3 exfoliatin genes (eta, etb and etd), and the toxic shock syndrome toxin gene (tsst) by PCR. The genomic diversity of each isolate was also evaluated by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and accessory gene regulator (agr) typing. Of these strains, 90.7% (98/108) harbored toxin genes, in which tsst was the most prevalent toxin gene (48.1%), followed by sea (44.4%), sek (42.6%) and seq (40.7%). The see and etb genes were not found in any of the isolates tested. Because of high-frequency transfer of toxin gene-containing mobile genetic elements between S. aureus strains, a total of 47 different toxin gene combinations were detected, including a complete egc cluster in 19 isolates, co-occurrence of sea, sek and seq in 38 strains, and sec and sel together in 11 strains. Genetic typing by PFGE grouped all the strains into 25 clusters based on 80% similarity. MLST revealed 25 sequence types (ST) which were assigned into 16 clonal complexes (CCs) including 2 new singletons. Among these, 11 new and 6 known STs were first reported in the S. aureus strains from China. Overall, the genotyping results showed high genetic diversity of the strains regardless of their geographical distributions, and no strong correlation between genetic background and toxin genotypes of the strains. For genotyping S. aureus, PFGE appears to be more discriminatory than MLST. However, toxin gene typing combined with PFGE or MLST could increase the discriminatory power of genotyping S. aureus strains.     

Clonal relations among Salmonella enteritidis phage type 3 outbreak isolates traced by DNA fingerprinting.

Isolates of Salmonella enteritidis PT3, a rare phage type, were recovered from patients and strains were isolated from an outbreak of gastroenteritis that occurred during the summer of 1997 in North-East Sardinia, Italy. To investigate possible clonal involvement in the outbreak and to evaluate the capacity to discriminate among S. enteritidis PT3 strains, a number of molecular typing methods including ribotyping with a mixture of PstI and SphI (PS-ribotyping), PFGE with endonuclease XbaI and RAPD typing with four arbitrary primers was used. The typical XbaI endonuclease generated PFGE pattern also explained the prevalence of highly clonal S. enteritidis PT3 strains in the outbreak and adjacent areas. RAPD fingerprinting with primers OPA 4, OPB 15, OPB17 and P1254 exhibited a single but unique RAPD profile among the outbreak strains from various sources that differed significantly from control strains. The results of this study showed that when an appropriately chosen set of primers is employed, RAPD fingerprinting can be used as an alternative, rapid, highly reproducible technique for tracing the clonal relations of S. enteritidis PT3, and can be more discriminatory than PFGE. Furthermore, this study revealed the possibility of PT3 causing outbreak.     

Prolonged incubation period of salmonellosis in an outbreak of Salmonella enteritidis infection

An outbreak of Salmonella enteritidis infections occurred in Otaru, Japan, in September 1997. A total of 143 cases of salmonellosis were reported to the local Public Health Center. In this outbreak, one case had a 214-hr incubation period. We investigated 5 isolates including this case by phage typing and pulsed-field gel electrophoresis (PFGE) to determine the genetic heterogeneity of S. enteritidis. Five isolates were phage typed as reacted, but did not conform (RDNC) with identical reaction patterns and had quite similar PFGE patterns. Thus, the prolonged incubation period may not be attributed to genetic heterogeneity of the organism but rather to other factors.     

Comparison of four methods, including semi-automated rep-PCR, for the typing of vancomycin-resistant Enterococcus faecium

We have assessed the performance of semi-automated rep-PCR (Diversilab®) and multilocus sequence typing (MLST) in comparison to pulsed-field gel electrophoresis (PFGE) for typing a collection of 29 epidemiologically characterized vancomycin-resistant Enterococcus faecium (VRE). Sixteen strains that harbored the Tn1546 element were typed by PCR mapping. The discriminative power of the typing methods was calculated by the Simpson's index of diversity, and the concordance between methods was evaluated by the Kendall's coefficient of concordance. Semi-automated rep-PCR appeared as discriminative as PFGE and was further compared with PFGE for typing 67 VRE isolated during a hospital outbreak. Rep-PCR appeared to be more discriminative than PFGE for this second set of strains. Reproducibility of DiversiLab® was also tested against 35 selected isolates. Only three showed less than 97% similarity, indicating high reproducibility at this level of discrimination. In conclusion, semi-automated rep-PCR is a useful tool for rapid screening of VRE isolates during an outbreak, although cost of the system may be limiting for routine implementation. PFGE, which remains the reference method, should be used for confirmation and evaluation of the genetic relatedness of epidemic isolates.     

Clonal multidrug-resistant Corynebacterium striatum within a nosocomial environment,Rio de Janeiro,Brazil

Corynebacterium striatum is a potentially pathogenic microorganism with the ability to produce outbreaks of nosocomial infections. Here, we document a nosocomial outbreak caused by multidrug-resistant (MDR) C. striatum in Rio de Janeiro, Brazil. C. striatum identification was confirmed by 16S rRNA and rpoB gene sequencing. Fifteen C. striatum strains were isolated from adults (half of whom were 50 years of age and older). C. striatum was mostly isolated in pure culture from tracheal aspirates of patients undergoing endotracheal intubation procedures. The analysis by pulsed-field gel electrophoresis (PFGE) indicated the presence of four PFGE profiles, including two related clones of MDR strains (PFGE I and II). The data demonstrated the predominance of PFGE type I, comprising 11 MDR isolates that were mostly isolated from intensive care units and surgical wards. A potential causal link between death and MDR C. striatum (PFGE types I and II) infection was observed in five cases.