Molecular characterization of Listeria monocytogenes of the serotype 4b complex (4b, 4d, 4e) from two turkey processing plants

Most foodborne outbreaks of listeriosis have been found to involve a small number of closely related strains of Listeria monocytogenes serotype 4b. The ecology of these organisms and their reservoirs in nature or in the processing plant environment, however, remain poorly understood. Surveys of environmental samples from two turkey processing plants in the United States indicated presence of L. monocytogenes of the serotype 4b complex (serotype 4b and the closely related serotypes 4d and 4e). In addition, environmental and raw product samples from one plant repeatedly yielded isolates with genetic markers typical of two major serotype 4b epidemic clonal groups, ECI and ECII. The pulsed field gel electrophoresis (PFGE) profiles of these isolates, however, were clearly distinct from those of confirmed epidemic-associated strains. Furthermore, we observed minor but consistent differences in PFGE profiles of isolates that harbored ECI- or ECII-specific genetic markers, and that were obtained at different sampling times from the same plant. The findings suggest processing plant persistence (or repeated introductions) and genomic diversification of L. monocytogenes serotype 4b isolates that harbor ECI- or ECII-specific genetic markers. Such diversification would need to be taken into consideration in further efforts to elucidate the evolution and epidemiology of these organisms.     

Association between asymptomatic carriage and sporadic (endemic) meningococcal disease in an open community.

We analysed a strain collection representative of the overall Neisseria meningitidis population circulating in an open community (46,000 inhabitants, Spain) during an endemic period (30 isolates from patients and 191 from throat cultures of healthy individuals) by both phenotypic and molecular techniques. Almost all patient isolates were assigned to three hyper-virulent lineages (ET-5 complex, ET-37 complex and cluster A4) by both multilocus enzyme electrophoresis (MEE) and pulsed-field gel electrophoresis (PFGE). In contrast, MEE and PFGE assigned 20% and 15% respectively of carrier isolates to the hyper-virulent clones (4% for both methods together). There was also a higher correlation between PFGE and phenotypes associated with virulent clones. These notable differences between the two molecular methods were further observed in more than half the carrier isolates, suggesting that the associations between these strains were distorted by recombination events. However, almost one-third of total endemic strains from symptom-free carriers and almost all patient strains belonged to clones defined by MEE and PFGE, with no known epidemiological connection. These data indicate low transmission and a weak clonal structure for N. meningitidis.     

深圳市1993-2002年霍乱弧菌的脉冲场凝胶电泳分析

目的 分析深圳市1993—2002年霍乱弧菌菌株的相关性。方法 60株霍乱弧菌基因组DNA经Not Ⅰ酶切，通过脉冲场凝胶电泳获得电泳图谱，利用BioNumerics软件对图谱进行聚类分析。结果 60株霍乱弧菌被分为39种脉冲场凝胶电泳图谱，聚类分析将大部分菌株分为A、B两群。结论 深圳地区霍乱弧菌存在紧密相关的流行克隆群。霍乱弧菌的脉冲场凝胶电泳分析，有助于霍乱的主动监测和传染来源追踪。     

MLEE and PFGE characterization of Neisseria meningitidis serogroup C and B isolated in the Slovak Republic in 1998

In the Slovak Republic the incidence and mortality of invasive meningococcal disease increased after 1995 when the new meningococcal clone of Neisseria meningitidis C:2a:P1.2,P1.5, ET-1.5/37 emerged. The new clone spread between 1995 and 1998 throughout the whole country. Morbidity of invasive meningococcal disease was 1.6/100,000 of the population and fatality reached the highest level of 23% in the Slovak Republic in 1998. The new clone caused a new emergent epidemiological and clinical situation. The occurrence of invasive meningococcal disease caused by this clone has continually risen since 1995. In 1998 72% of all diseases in Slovakia were caused by serogroup C. The emerging clone C:2a:Pl.2,P1.5 represented 74% of the serogroup C isolates. Clonality and genetic diversity of 15 selected meningococcal strains causing invasive meningococcal disease was compared by multilocus enzyme electrophoresis (MLEE) and DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE). The strains of serogroup C and B were isolated in all regions of Slovakia in 1998. The majority of isolates belong to hypervirulent clone ET-15 as determined by MLEE. By PFGE a higher degree of diversity was observed.     

Longitudinal study of meningococcal carrier rates in teenagers

To gain actual information concerning the oropharyngeal carriage of Neisseria meningitidis among teenagers aged 15-18 years in Germany especially in a region with increased incidence of meningococal-related diseases prompted the study. Each teenager was swabbed three times with an interval of 2 months between the examinations. The 901 recovered N. meningitidis strains were characterized using serological (serogrouping, serotyping/serosubtyping) and molecular methods (PCR, PFGE) each. The results of the study demonstrate an overall average carrier rate of 18.8% for the three collection periods. There were, however, significant differences between the carrier rates within a given school and of different towns and counties. Of all isolates, 60.6% were not serogroupable. Serogroup B dominated (12.3%), followed by serogroup Y (9.0%) and serogroup C (3.6%). After PCR-based serogrouping of not serogroupable strains the percentages for serogroups enhanced to 18.8% for B, 10.8% for Y and 4.1% for C. Serotyping led to 305 different phenotypes with the most common being 29E:NT:P1.2,5 followed by Y:14:NST. In the 6 study towns the number of different N. meningitidis clones (PFGE types) isolated, varied between 30 and 87. In Wenden, where a prolonged outbreak had taken place, serogroup C (14.8%) was predominant. Only in this town C:2a isolates were found, all belonging to the ST-11/ET-37 complex and 12/13 matched identically to the ET-15 clone. Of the colonized teenagers, 26.7% were carriers over at least 23 weeks, 22.6% with the same strain, 36.0% were carrier for at least 15 weeks. Over all three collection periods 36.7% of the adolescents acquired a new strain. The highest acquisition rate was related to PFGE type 12.     

Genetic characterization of Mycobacterium avium isolates recovered from humans and animals in Australia.

Genetic relationships amongst 115 mainly Australian isolates of Mycobacterium avium were assessed using multilocus enzyme electrophoresis (MEE). The isolates were divided into 58 electrophoretic types (ETs), with a mean genetic diversity of 0.29. Isolates from humans were closely related to but distinct from those cultured from birds, whilst some porcine isolates belonged to the same ETs as certain human isolates. Pulsed field gel electrophoresis (PFGE) was used to differentiate related isolates, and those from birds and some from other animals, including pigs, were distinguished from the human isolates. The results of MEE and PFGE suggested that certain strains of M. avium may be transmitted between birds and pigs, but there was no clear evidence of transmission to humans. The serovar of the M. avium isolates was not obviously related to their ET assignment or their PFGE type.     

Genetic diversity of atypical Aeromonas salmonicida studied by pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) pattern analysis with XbaI restriction enzyme was used to study the genetic heterogeneity of 88 atypical Aeromonas salmonicida strains which were earlier or during this study characterized phenotypically, by ribotyping (ClaI/PstI) and by plasmid profile analysis. The strains of certain'ribotypes were also analysed by digestion with SpeI. The strains represented different geographic locations: Finland (72 strains), Iceland (5 strains), Norway (5 strains), Sweden (4 strains) and Denmark (2 strains), and they were from 17 fish species during 1981 97. Thirty-one PFGE genotypes found among these strains correlated well with the ribotypes, and in most cases PFGE pattern analysis subdivided ribotypes into several PFGE genotypes, and further within a PFGE genotype into subtypes. XbaI and SpeeI digests produced concordant results. In most cases, PFGE patterns of strains with the same ribotype shared many fragments, suggesting genetic relatedness. PFGE patterns of most Norwegian and Icelandic strains isolated during an approximately 10-year period had the same ribotype and their PFGE patterns shared most fragments, suggesting close genetic relatedness. Moreover, atypical strains of ribotypes B/B and H/H isolated from the same Finnish fish farms had closely related patterns suggesting genetic stability and persistence of these genotypes. Genotype 29 of Achromogenic strains was strongly associated with disease of Finnish arctic char and grayling. PFGE was shown to be a distinguishing method to study the genetic heterogeneity of atypical A. salmonicida. epidemiology of these infections.     

Genomic characterization of oxacillin-resistant Staphylococcus epidermidis and Staphylococcus haemolyticus isolated from Brazilian medical centres

Studies on the genetic diversity of oxacillin-resistant coagulase-negative staphylococcal (CNS) isolates are important for the control and prevention of infections. The present study evaluated the clonal diversity of oxacillin-resistant Staphylococcus epidermidis (ORSE) and Staphylococcus haemolyticus (ORSH) strains, isolated from patients in nine Brazilian medical centres by using pulsed-field gel electrophoresis (PFGE) after digestion of bacterial DNA using SmaI. PFGE analysis of ORSE (N=44) and ORSH (N=25) strains showed the presence of 29 restriction profiles clustered in 16 PFGE types, and 21 distinct profiles in 15 PFGE types, respectively, indicating a large genetic diversity among isolates of both of these species. Among the ORSE isolates, 23 (52%) strains belonged to two predominant PFGE types (named A and B), which were observed in most of the hospitals assessed, indicating the spread of these PFGE types in hospitals located in Rio de Janeiro. The spread of PFGE types of ORSH was also detected in some of the hospitals investigated. The results show that PFGE is a suitable tool for epidemiological studies of oxacillin-resistant CNS, and can be used as a basis for infection control procedures for these multiresistant organisms.     

Interpretation of band differences to distinguish strains of Serratia marcescens by pulsed-field gel electrophoresis of XbaI DNA digests

The number of band differences in DNA macrorestriction profiles required to distinguish unrelated strains from an index strain varies in an outbreak with the species and restriction enzyme used. In order to define this difference for epidemiological studies of Serratia marcescens, we produced DNA fingerprints from 57 isolates of the organism using the restriction enzyme XbaI and pulsed-field gel electrophoresis (PFGE). The isolates were selected on the basis of their epidemiology, serotype and phage-typing patterns to include 28 unrelated strains and 29 representatives from 2 distinct outbreaks. One of the outbreaks was prolonged. lasting for several years. Electrophoretic profiles consisting of 20 or more clearly resolved bands were obtained for all isolates. Twenty-six of the unrelated strains had unique profiles with over 10 band differences from all other strains, while 27 of the outbreak representatives could be assigned to the appropriate outbreak with confidence. The majority of the outbreak isolates had none or 2 band differences from the index profile, although 3 isolates differed by 5-7 bands. The 2 exceptions among the unrelated strains differed by 4 bands, and 3 phage typing reactions, and were isolated from London and Berlin 3 years apart, while the 2 exceptions among the outbreak collection had clearly unique profiles with over 20 band differences from each other and the outbreak profiles. Cluster analysis using Dice coefficient and UPGMA gave cut-off values of 75-78% similarity overall for related isolates, while the closest similarity for unrelated strains was 70%. The results of this study together with those of the 6 previous reports of PFGE for S. marcescens (which used either enzymes XbaI or SpeI) confirm that this technique is of value for this species and that with XbaI at least, most epidemiologically related strains will only differ by 3-4 bands. However, on occasion up to 7 band differences can be found within an apparent outbreak, which may be suggestive of genetic drift.     

Meningococci throughout the world : Phenotypic and molecular markers.

The first markers of meningococci were serogroup, defined by different polyosidic capsular immunospecifities (12 are described at present), and are still of great importance. Several other antigenic structures such as outer membrane proteins (OMPs) are used as markers : OMP serotypes of classes 2 and 3, OMP subtype of class 1. Serogroups, serotypes, subtypes and sometimes immunotypes (based on LPS) are associated in an antigenic formula (AF). At a world-wide level, the Electrophoretic Type (ET) defined by Multilocus Enzyme Electrophoresis (MLEE) is the most useful marker. For instance, the ET-5 and ET-37 have been described. The ET-5 was constituted primarily, but not exclusively, by strains of AF:B:15:P1.7,16 and B:4:P1.15. The ET-37 was constituted mostly by strains C:2a:P1.2,5. Two pandemics were due to Neisseria meningitidis serogroup A. They were mainly defined by MLEE. The first began in China in 1966, crossed Europe, and ended in Brazil in 1974 where it was responsible for a particularly widespread outbreak. The second pandemic, due to the same epidemic invasive strain A:4:P1.9/clone III-1 also began in China in 1983, spreading through Nepal, northern India. It was responsible for a severe oubreak in Mecca in August of 1987. It spread all around the world when the pilgrims returned to their countries. In countries with adequate health care facilities, the pandemic was stropped within two or three weeks. Unfortunately, in countries without these health care facilities, the spreading continues. For instance in Africa, specifically Niger, strains of this type continued to be isolated through the beginning of 1996. Molecular epidemiology markers like pulsotype and ribotype for instance, are able to demonstrate genetic variability between strains.     

副溶血弧菌脉冲场凝胶电泳技术分子分型分析

目的分析副溶血弧菌菌株之间的相关性,建立深圳市副溶血弧菌的DNA指纹图谱数据库。方法56株副溶血弧菌基因组DNA经NotI酶切,通过脉冲场凝胶电泳获得电泳图谱,利用BioNumerics软件对图谱进行聚类分析。结果56株副溶血弧菌被分为34种脉冲场凝胶电泳技术（PFGE）图谱,聚类分析显示,在90%的相似性水平上,28株细菌谱型属于同一个群,其中27株均为食物中毒患者分离株,并各年度均有分离。结论深圳地区存在遗传谱系紧密相关的副溶血弧菌流行克隆。建立DNA指纹图谱数据库为建立分子分型监测网络打下了良好的基础,有助于副溶血弧菌所致食源性疾病的及时主动监测和传染来源追踪。     

Genome fingerprinting of Salmonella typhi by pulsed-field gel electrophoresis for subtyping common phage types.

The genomic DNA of 39 strains of Salmonella typhi isolated from local residents and patients who had visited countries in the Asian region was analysed for restriction fragment length polymorphisms (RFLP). Pulsed-field gel electrophoretic (PFGE) analysis of Xba I- and Spe I-generated genomic restriction fragments established 22 PFGE types whereas phage typing differentiated the 39 isolates into 9 distinct phage types. This study showed that PFGE is more discriminatory than phage typing as it is capable of subtyping S. typhi strains of the same phage types. Genetic relatedness among the isolates was determined. Seven major clusters were identified at SABs of > 0.80 and the remaining 13 isolates were distributed into minor clusters which were related at SABs of less than 0.80. In conclusion, PFGE analysis in conjunction with distance matrix analysis served as a useful tool for delineating common S. typhi phage types of diverse origins from different geographical locales and separated in time.     

Human antibody responses to A and C capsular polysaccharides, IgA1 protease and transferrin-binding protein complex stimulated by infection with Neisseria meningitidis of subgroup IV-1 or ET-37 complex

The protein sequences of the IgA1 protease, TbpA and TbpB proteins differ between meningococci representative of serogroup A, subgroup IV-1 from epidemic disease in The Gambia and serogroup C, ET-37 complex from endemic disease in Mali. The uniformity of restriction endonuclease sites was determined for the iga, tbpA and thpB genes among strains of both clonal lineages. Rare isolates had acquired a variant thpAB operon by horizontal genetic exchange but all other strains were uniform within each clonal lineage. The quantitative levels of IgG to capsular polysaccharide, IgA1 protease and TBP complex were measured in paired acute phase and convalescent phase sera from The Gambia and from Mali using antigens from the homologous clonal lineages. IgG levels to these antigens were also measured in paired sera from healthy Gambians who permanently carried meningococci in the nasopharynx or did not. The results showed that disease stimulated IgG to each antigen in Mali and to all but TBP complex in The Gambia. Similarly, higher levels of IgG were found in sera from permanent carriers than in sera from permanent non-carriers. Acute phase sera from Mali contained low levels of IgG to C capsular polysaccharide (geometric mean value of 0.3 microg ml(-1)) while such sera from The Gambia contained higher and potentially protective levels of IgG to A polysaccharide (geometric mean of 5.5 microg ml(-1)). The concentrations of IgG to TBP complex in acute phase sera were higher and IgG to IgA1 protease was even higher, suggesting that intermediate levels of IgG to these proteins do not protect against disease.     

Multi-locus variable number tandem repeat analysis for Clostridium botulinum type B isolates in Japan: Comparison with other isolates and genotyping methods

Clostridium botulinum produces botulinum neurotoxin (BoNT) and causes botulism in humans and animals. Recently, 15-loci multi-locus variable number tandem repeat analysis (MLVA) for C. botulinum was developed for high-resolution and inter-lab comparative genotyping. This study examines the relation between MLVA and other genotyping methods such as pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), BoNT/B subtyping and bont/b gene location to evaluate MLVA as a method applicable to the genetic markers for C. botulinum type B. Japanese isolates were genotyped using MLVA and were compared with strains from other sources reported previously. Results show that the discriminatory power of MLVA was comparable to that of PFGE and higher than that of MLST. The topology of the minimum spanning tree (MST) constructed using MLVA data was very consistent with the phylogenetic classifications of PFGE and MLST. The MST topology also represented genetic diversity between the strains possessing bont/b gene on chromosomes and plasmids. Some Japanese isolates including those associated with infant botulism were inferred to be related to isolates of Europe origin from MLVA genotyping results. The MLVA scheme used for this study is apparently useful not only for high-resolution molecular typing, but also for phylogenetic characterization of C. botulinum type B.     

Molecular subtyping of Salmonella enterica serovar Typhi isolates from Colombia and Argentina

Salmonella Typhi is the etiological agent of typhoid fever with 16 million annual cases estimated worldwide. In Colombia and Argentina it is a notifiable disease but many cases have only a clinical diagnosis. Molecular subtyping of S. Typhi is necessary to complement epidemiologic analysis of typhoid fever. The aims of this study were to determine the genetic relationships between the strains circulating in both countries and to evaluate possible variations in the distribution of 12 virulence genes. A total of 136 isolates were analyzed by pulsed-field gel electrophoresis (PFGE) with XbaI following PulseNet protocols and analysis guidelines. Eighty-three different PFGE patterns were identified, showing high diversity among the strains from both countries. Three outbreaks, two in Colombia and one in Argentina, were caused by strains of different PFGE types. In Colombia, two PFGE patterns were found predominantly, which included 36.6% of the isolates from that country. No association was found between the PFGE patterns and the year or place of isolation of the strains, the age of the patients or type of sample. However, several clusters were detected, which included isolates recovered predominantly either from Colombia or Argentina. Most of the strains (97%) exhibited a single virulence profile, suggesting that the pathogenicity markers analyzed are of limited value for strain discrimination and do not correlate with the origin of the isolates (intestinal vs. extra-intestinal). Since the creation of PulseNet Latin America, this was the first international study conducted in South America. The PFGE types identified were incorporated into the Regional S. Typhi PulseNet Database and are now available for comparison with those of strains isolated in other regions. This information will be used for active surveillance, future studies, and outbreak investigations.     

钩端螺旋体脉冲场凝胶电泳标准化技术的建立及谱型特征初步分析

目的建立中国钩端螺旋体（钩体）脉冲场凝胶电泳（PFGE）的标准化操作程序及中国15群15型代表菌株的图谱数据库。方法参照目前美国疾病控制中心及亚太地区PulseNet提供的其他病原体PFGE标准化操作程序，结合钩体菌株的特性，对菌体基因组染色体DNA纯化技术、限制性内切酶消解方案及脉冲场电泳参数进行优化。结果利用标准化PFGE操作程序，建立了中国15群15型钩体代表菌株基因组DNA的NotI酶切图谱，并对历年从四川、安徽省钩体病监测工作现场分离的部分黄疸出血群菌株进行PFGE分析，结果发现中国15群15型钩体代表菌株各自具有独特的谱型特征，24株黄疸出血群分离株存在3种谱型特征，91．67％（22／24）的分离株与黄疸出血群赖型（56601）的谱型匹配。结论建立的PFGE标准化操作程序制作钩体菌株的酶切图谱，具有图像清晰、分辨率高、各种大小的片段分布均匀等特点，能较好地反映出中国钩体菌株的分子遗传学特征，与传统的血清学分类存在较好的相关性。     

福建省非典型肠致病性大肠埃希菌的分子特征研究

摘要：目的　研究福建省非典型肠致病性大肠埃希菌（ａＥＰＥＣ）菌株的毒力基因特征和菌株间的遗传相似
度;结合菌株的背景资料分析不同的分子特征对ａＥＰＥＣ 流行的影响。方法　运用ＰＣＲ 技术进行系列的相
关毒力基因扩增;同时运用脉冲场凝胶电泳分子分型技术,对福建省２０１０－２０１２年分离的ａＥＰＥＣ 菌株进
行脉冲场凝胶电泳（ＰＦＧＥ）分子分型。结果　分离３０株实验菌株毒力基因的检测呈阳性的分别为：犫１１２１
５３．３％ （１６）,狔犻犪犃３６．７％ （１１）,狊犲狋／犲狀狋、狀犾犲犅、狀犾犲犈均为３０％ （９）,犾狆犳犃犚１４１２３．３％ （７）,犲犳犪／犾犻犳犃
２０％ （６）,犲犺狓犃３．３３％ （１）;其余毒力基因均未检出;９３．３％ （２８）菌株不同程度地携带有相关的毒力因
子。２９株菌按照１００％的相似度分为１５个ＰＦＧＥ 型别（Ｐ１ Ｐ１５）;其中存在４组不同的ＰＦＧＥ 簇（Ｉ Ⅳ）,
相同ＰＦＧＥ 簇的病例在发病的时间和地区上有聚集性,同时相同簇内菌株的毒力基因谱也表现相同。结论
　犫１１２１、狔犻犪犃和ＥＨＥＣ 毒力岛ＯＩ １２２ 相关基因（犲犳犪１／犾犻犳犃,狀犾犲犅,狀犾犲犈,狊犲狋／犲狀狋） 在福建省ａＥＰＥＣ 菌
株中携带率较高。非典型肠致病性大肠埃希菌的毒力谱表现为多样性, 基因组也呈现遗传多态性;同时
ＰＦＧＥ 分析发现福建省存在由ａＥＰＥＣ 新发病原菌引起的聚集性病例。
关键词：非典型肠致病性大肠埃希菌;毒力基因;ＰＦＧＥ
中图分类号：Ｒ３７８　　文献标识码：Ａ　　文章编号：１００９ ６６３９ （２０１４）０３ ０２１６ ０５
犕狅犾犲犮狌犾犪狉犮犺犪狉犪犮狋犲狉犻狊狋犻犮狊狅犳犪狋狔狆犻犮犪犾犲狀狋犲狉狅狆犪狋犺狅犵犲狀犻犮     

Clonal identification of Aeromonas hydrophila strains using randomly amplified polymorphic DNA analysis

The suitability of arbitrary primer polymerase chain reaction (RAPD) as a typing technique was evaluated by comparing it with pulsed-field gel electrophoresis (PFGE) to characterize Aeromonas hydrophila strains isolated from a cluster of hospital-acquired infections. Five isolates from patients and 10 isolates from the water supply were compared to 10 epidemiologically unrelated strains isolated from patients and rivers. Two methods were used to prepare DNA and two primers (AP3 and AP5) were selected. The discriminatory power was better with the extractive DNA preparation than the boiling method. The discrimination of closely related from less related strains by PCR using AP3 was consistent with that by PFGE: water supply of Cholet hospital contaminated with Aeromonas species was not the source of the cluster of hospital infections and only two patients were infected with clonally-related strains. RAPD using primer AP3 was simpler, cheaper, and quicker to perform than pulsed-field gel electrophoresis and is well suited for the epidemiological study of A. hydrophila isolates.     

Molecular epidemiological investigation of an outbreak of Campylobacter jejuni identifies a dominant clonal line within Scottish serotype HS55 populations.

Three molecular typing methods, pulsed-field gel electrophoresis (PFGE), ribotyping, and flagellin (flaA) gene typing, were used to discriminate within a group of 28 Campylobacter jejuni, heat-stable serotype 55 (HS55) isolates derived from cases of campylobacter enteritis occurring throughout Scotland, including 9 isolates associated with an outbreak. PFGE was found to be most discriminatory, identifying 6 distinct profiles, followed by ribotyping (5 profiles), and then flagellin gene typing (4 profiles). The coincidence of all three genotypic markers identified a dominant clonal line within the HS55 group, accounting for each of the outbreak strains, and for 9 of the 19 sporadic strains. A second, closely related, clonal line accounted for a further 5 of the sporadic strains, and also included the HS55 reference strain. Identification and monitoring of such clonal lines should facilitate more effective future epidemiological surveillance of C. jejuni.