Involvement of closely related strains of a new clonal group of Listeria monocytogenes in the 1998-99 and 2002 multistate outbreaks of foodborne listeriosis in the United States

In 1998-99, a multistate outbreak of listeriosis in the United States was associated with contaminated hot dogs and was caused by a strain of Listeria monocytogenes serotype 4b that had been only rarely encountered before in the national PulseNet database. Upon further characterization, the strains from this outbreak were designated as Epidemic Clone II (ECII). ECII isolates exhibited diversification in a genomic region ("region 18") that was otherwise conserved among L. monocytogenes of serotype 4b. Additional unique genetic markers were identified through genome sequencing of one of the isolates from the 1998-99 outbreak. In 2002, another multistate outbreak of listeriosis also involved bacteria of serotype 4b and was attributed to contaminated turkey deli meats. Molecular subtyping data revealed that the macrorestriction patterns of the isolates from the 1998-99 and 2002 outbreaks were closely related. In addition, the 2002 outbreak isolates harbored chromosomal genetic markers found to be unique to, and typical of, the 1998-99 outbreak isolates, including diversification in genomic region 18. Macroarray- based subtyping using chromosomal sequences confirmed the close genetic relatedness between the isolates from the two outbreaks. Genomic content was highly conserved among isolates from each outbreak, with differences detected only in prophage and internalin-like gene sequences. However, since these differences were observed among isolates from each of the outbreaks, they did not differentiate the 1998-99 isolates as a group from those of the 2002 outbreak. Two of 15 randomly chosen serotype 4b clinical isolates from a non-outbreak period (calendar year 2003) appeared to be closely related to the 1998-99 and 2002 outbreak isolates. These findings suggest that both multistate outbreaks of listeriosis in the United States involved closely related members of a single clonal group (ECII) that had not been identified in outbreaks prior to 1998. Since the outbreaks involved different food vehicles and processing plants, the findings suggest establishment of ECII in a still unidentified reservoir in the United States, from which the organisms were introduced to different processing plants.     

A comparison of Listeria monocytogenes serovar 4b isolates of clinical and food origin in Austria by automated ribotyping and pulsed-field gel electrophoresis

In this study, two typing methods, automated ribotyping and pulsed-field gel electrophoresis (PFGE), were evaluated for the subtyping of Listeria monocytogenes serotype 4b. The strains originated from patients and food samples collected in Austria during 2001-2005 and from Europe and North America in the World Health Organization collaborative study on the subtyping of this species. The largest group of Austrian clinical isolates was of the same PFGE subtype as those isolated from foodborne outbreaks in Switzerland and in the United States. Another subtype of clinical isolates from Austria was indistinguishable to that obtained from isolates responsible for a foodborne outbreak in the United States in 1985. Although the discriminatory power of PFGE was higher than that of automated ribotyping, some PFGE types were differentiated by ribotyping. Thus, combining data obtained by both automated ribotyping and PFGE increases the strain discrimination. Still, many of the Austrian strains remain indistinguishable from strains of foodborne outbreaks in other countries although there is no known epidemiological relation. This complies to previous studies which show the highly clonal nature of L. monocytogenes 4b strains which are responsible for both large outbreaks and sporadic cases.     

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.     

Listeria monocytogenes serotypes 1/2c and 3c possess inlH

To examine the serotype specificity of inlH, which encodes the virulence-associated surface protein InlH related to the intracellular survival of Listeria monocytogenes in mice, the presence of inlH in 337 L. monocytogenes strains, representing 11 different serotypes, was studied. A total of 106 strains representing 3 serotypes and 14 pulsed-field gel electrophoresis (PFGE) types were positive for inlH by polymerase chain reaction. inlH was present in all 99 serotype 1/2c and 3 serotype 3c strains. Moreover, 4 out of 129 (3%) serotype 1/2a strains carried inlH. All 106 strains representing serotypes 1/2b, 3a, 3b, 4a, 4b, 4c, 4d, and 7 and 125 out of 129 (97%) serotype 1/2a strains were inlH-negative. The coding sequences of the inlH genes of eight L. monocytogenes strains representing three serotypes and five PFGE types were identical. These results suggest that inlH is specifically present in serotype 1/2c, 3c, and a small fraction of 1/2a L. monocytogenes strains and exists as a single allele.     

Characterization and Antimicrobial Resistance of Listeria monocytogenes Isolated from Retail Beef Meat in Poland

Abstract Four hundred seventeen retail beef meat samples purchased in the eastern part of Poland during October 2009 to January 2011 were tested for the presence of Listeria monocytogenes. It was found that 81 (19.4%) of them were positive for this microorganism as identified by the culture and polymerase chain reaction (PCR) methods. Molecular serotyping performed by PCR revealed that the majority of the isolates (50 strains; 61.7%) were of 1/2a serotype. Furthermore, 26 (32.1%) L. monocytogenes strains were classified as 1/2c serotype, and only five strains belonged to serotypes 1/2b or 4b (four and one isolates, respectively). All the isolates were positive for the inlA, inlC, inlJ, and lmo2672 sequences, whereas two L. monocytogenes (both of 4b serotype) had another virulence marker gene-llsX. The results of the antimicrobial resistance revealed that the strains were sensitive to most of the antimicrobials used in the study except oxacillin (62.7% resistant strains). Several isolates (17.3%) were also resistant to ceftriaxone. Our results indicate that L. monocytogenes identified in raw beef meat possessed virulence markers that make them potentially pathogenic for humans. Therefore, this kind of food may create a public health concern.     

Genetic typing of human and food isolates of Listeria monocytogenes from episodes of listeriosis

Ten clinical and food Listeria monocytogenes strains isolated during the epidemiological investigations of episodes of listeriosis (one outbreak and two sporadic cases) that occurred in northern Italy during 1993–1995 have been examined by DNA macrorestriction pattern analysis obtained by PFGE and RAPD typing, in order to confirm the food vehicle of infections. The same DNA profiles within the isolates from the three episodes were obtained by both techniques. The ApaI and SmaI PFGE profiles and RAPD patterns with primer OPM-01 confirmed the close relationship between strains from two distinct episodes. However, RAPD analysis with primer UBC-127 distinguished between these L. monocytogenes isolates.     

Antimicrobial susceptibility, serotype and genotype distribution of meningococci in Portugal, 2001-2002

One hundred and eighteen Neisseria meningitidis isolates were recovered from patients with invasive meningococcal disease in Portugal, over one year. Our study was undertaken to evaluate antimicrobial susceptibility, serogroup, serotype and genotype of isolates. One quarter (24.6%) of the isolates showed moderate resistance to penicillin and 47.4% were resistant to sulphadiazine. The two most common serosubtypes were C:2b:P1.5,2 (31.3%) and B:4:P1.15 (3.4%). Half (53.6%) of the isolates with moderate resistance to penicillin were phenotype C:2b:P1.5,2 (n=14), C:2b:P1.2 (n=1) or C:2b:NST (n=1); Pulsed-field gel electrophoresis (PFGE) showed that all these isolates were genetically related. Multilocus sequence typing (MLST) analysis of representative clones from each PFGE pattern showed the predominance of the ST-8 complex/cluster A4 among N. meningitidis with moderate resistance to penicillin. This clonal complex has been principally found in Southern Europe. The apparent emergence and dissemination of the hypervirulent ST-8 complex/cluster A4 among serogroup C strains increases the need for a continued surveillance of antimicrobial susceptibility of meningococci and of genotypic markers in Portugal.     

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.     

单核细胞增生李斯特菌脉冲场凝胶电泳分型技术的内切酶质量评估

目的对单核细胞增生李斯特菌脉冲场凝胶电泳(PFGE)分子分型的相关影响因素进行分析和探讨,并建立该菌的PFGE双酶切技术。方法分别利用限制性内切酶AscⅠ和ApaⅠ对1/2a、1/2b、1/2c、3a和4b型的单核细胞增生李斯特菌进行PFGE分析(包括1种品牌的AscⅠ和5种品牌的ApaⅠ),通过指纹图谱的比较,优化各种内切酶的实验参数。结果AscⅠ酶切产生11条左右清晰的DNA指纹图谱条带,ApaⅠ酶切产生的条带数量与之相似,但其中约10%的菌株(1/2a、1/2b血清型)均产生消化不完全的DNA片段。同时用5种品牌的ApaⅠ酶消化,并改变消化温度、提高酶浓度、延长酶消化时间均无法克服此现象。结论AscⅠ酶适于1/2a、1/2b、1/2c、3a和4b血清型的单核细胞增生李斯特菌的分型,但ApaI酶适于多数1/2a和1/2b血清型的分型。     

Genetic characterization of a new variant within the ET-37 complex of Neisseria meningitidis associated with outbreaks in various parts of the world

A new variant within the electrophoretic type (ET)-37 complex of Neisseria meningitidis, ET-15, first detected in Canada in 1986, has been associated with severe clinical infections and high mortality rates in several European countries, Israel and Australia. To ascertain the genetic and epidemiological relationships of ET-15 strains from different geographical areas, 72 ET-15 isolates from 10 countries were compared to 13 isolates representing other clones of the ET-37 complex. The 85 strains were analysed by pulsed-field gel electrophoresis (PFGE) using 2 restriction endonucleases and Southern hybridization with 10 genetic markers. Four ET-15 strains and 4 other strains of the ET-37 complex were further examined using an additional restriction enzyme and a total of 18 genetic markers. PFGE fingerprints of the ET-15 strains were closely related. Strains within each country were even more closely related, suggesting single introductions of the clone. Physical mapping of genes in ET-15 and other strains of the ET-37 complex demonstrated that large genetic rearrangements of the genome have occurred in association with the appearance of the ET-15 variant.     

广东省食品中单核细胞增多性李斯特菌脉冲场凝胶电泳技术分子分型的研究

目的应用脉冲场凝胶电泳技术（PFGE）对广东省食品中分离的单核细胞增多性李斯特菌（Lm）进行分子分型,并建立PFGE数据库。方法参照美国PulseNet的Lm PFGE分子分型标准方法进行检测。应用BioNumerics软件对不同食物种类、时间和地点的分离株进行比对,分析菌株之间的相关性。结果107株Lm分成41个PFGE型,型别较为分散;从鸡肉中分离的菌株数最多（37株）,PFGE分型也最多（19个）;冷藏和冷冻食品的分离率较高,其中有26株菌仅存在1-2个电泳条带的差异,相似度极高;韶关和惠州地区分离的Lm存在着地区特异性;随时间变迁,部分菌株仍存在不同程度的相关性。结论广东省食品中分离的Lm在整体上表现出较大的遗传多样性,但部分菌株有不同程度的相关性。     

广州地区小川型霍乱弧菌的脉冲场凝胶电泳分析

目的分析小川型霍乱弧菌的致病相关基因型,探讨广州地区小川型霍乱流行趋势和规律。方法采用多重PCR方法检测小川型菌株的4种致病相关基因,应用脉冲场凝胶电泳技术（PFGE）对菌株进行分子分型,对PFGE图谱采用分子分型软件BioNumerics Version 4.0进行聚类分析。结果广州地区小川型菌株中存在3种致病相关基因型,即A型、B型和C型,20%感染者分离株为致病相关基因A型,80%环境分离株为致病相关基因C型。25株霍乱弧菌分为14个不同的PFGE型,归为3个聚类群（Ⅰ、Ⅱ、Ⅲ群）。每一次小川型暴发中分离的菌株PFGE克隆型相同或相近,而散发病例分离株与暴发株的PFGE型有些相同,有的差异较大。环境小川型菌株中存在PFGE克隆型的多样性,部分环境株与感染者分离株具有相近的PFGE型。结论应建立广州地区霍乱菌株的PFGE分子分型数据库,加强霍乱的预防、控制和预警。     

Pulsed-Field Gel Electrophoresis, Conventional, and Molecular Serotyping of Listeria monocytogenes from Food Proficiency Testing Trials Toward an Harmonization of Subtyping at European Level

Abstract The European Union Reference Laboratory for Listeria monocytogenes (EURL for L. monocytogenes) coordinates a European network of 29 National Reference Laboratories (NRLs). Depending on a national decision, NRLs undertake food, environmental, and veterinary L. monocytogenes strain surveillance in their respective countries. In the framework of the PulseNet Europe network, two pulsed-field gel electrophoresis (PFGE) subtyping proficiency testing (PT) trials were carried out in 2003 and 2006. The obtained data showed that PFGE profiles can be compared and exchanged between laboratories. However, no further PT trial had been performed since 2006. In this context, two PT trials were organized by the EURL to evaluate the ability of NRLs to perform conventional serotyping, molecular serotyping and PFGE subtyping. Eleven well-characterized isolates of L. monocytogenes were used: six and nine isolates were tested in 2009 and 2010, respectively. Three isolates were repeated between the two studies. In the 2010 panel, a strain was tested in duplicate, and two strains were related to the same epidemiological group. The strains were analyzed blind in different laboratories (17 in 2009 and 25 in 2010) using (1) their own in-house method for serotyping methods and (2) standardized protocols based on the PulseNet protocol for PFGE. For conventional serotyping, 86.0% in 2009 and 91.0% in 2010 of the serotypes obtained were in agreement with the EURL data. For molecular serotyping, 93.5% of the results in 2009 and 95.2% in 2010 matched the EURL data. For PFGE, 68.9% in 2009 and 81.7% of the combined AscI/ApaI profiles were indistinguishable from the EURL reference profiles. The variations observed could be attributed to slight standardization defaults or, in a few cases, to a failure in DNA extraction. These PT trials provided a valuable opportunity to improve the subtyping ability of NRLs and facilitate exchanges of subtyping data in the future.     

Listeriosis associated with gorgonzola (Italian blue-veined cheese)

We describe a case of listeriosis in Italy associated with the consumption of cheese. Opened samples of two brands of gorgonzola (Italian blue-veined cheese; referred to as brands "B" and "C") were collected from the patient's refrigerator. Unopened samples of the brand suspected to be the source of infection (brand B) were taken from the store where the cheese had been purchased, other local stores, and the production plant. Listeria monocytogenes serotype 1/2b was isolated from the patient and from the opened and unopened cheese samples. The contamination level varied from <100 to 1,200 cfu g(-1). Molecular typing of the isolates, using both randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE), demonstrated that the isolates from the patient's refrigerator, food stores, and production-plant samples were indistinguishable from the clinical isolate. Molecular typing verified the peristence of closely related L. monocytogenes isolates in the production plant B for 5 months. The results stress the importance of developing a code of hygienic practice for preventing, limiting, and where possible, eliminating this pathogen in processed foods and of educating at-risk persons on foods likely to be contaminated.     

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.     

1998至2001年四川省霍乱流行中6b型菌株的分子特征

目的 分析1998-2001年四川省霍乱流行中噬菌体-生物分型为6b的特殊菌型霍乱弧菌的分子特征.方法 选择1998-2001年四川省01群霍乱弧菌流行菌株以及同时期其他省份的流行菌株共45株,进行噬菌体-生物分型、脉冲场凝胶电泳(PFGE)分析以及突变基因的序列测定比较.结果 四川在1998-2001年期间流行的菌株存在1b型和6b型菌株,这些菌株均为产毒株;PFGE分析结果显示菌株呈现24种带型,其中13株菌表现为相同带型;来自四川的部分1b和6b型菌株以及部分其他省份的1b型菌株具有相同的PFGE带型;与1b型菌株相比,6b型菌株(共22株)的ompW基因ORF内存在11 bp缺失,而其余23株菌非6b型菌株的ompW基因ORF是完整的.结论 1998-2001年四川流行的O1群霍乱弧菌6b菌株是一类具有特殊遗传背景的菌型,可能与同期流行的1b型菌株有进化上的关联.     

Genetic relatedness of group A streptococci of the newly designated serotype M90 causing a food-borne outbreak and sporadic infections

Twenty-six isolates of the newly designated M90 serotype group A Streptococcus (GAS) from a large food-borne outbreak of pharyngitis in Greece and six M90 sporadic isolates from UK, were typed by pulsed-field gel electrophoresis (PFGE). Twenty-four outbreak isolates were identical and two closely related. The Greek isolates were possibly related with one UK isolate, while other sporadic isolates exhibited distinct PFGE profiles from the former isolates.     

脉冲场凝胶电泳在一起慕尼黑沙门菌食源性暴发中的应用研究

目的 脉冲场凝胶电泳(PFGE)在食源性疾病病原菌诊断中的应用.方法 采集19例患者和18名厨师的肛拭子、9份自制冷饮、2份井水以及5例患者的急性期和恢复期血清.同时采集未发病学生大便10份,血清5份,作为正常人对照.用传统的方法进行致病菌的分离和表型鉴定,对所分离的菌株参照美国疾病预防控制中心(CDC)的实验方法用PFGE进行DNA分子分型,使用BioNumerics软件进行聚类分析.方法 从19份食物中毒患者肛拭子中分离出14株慕尼黑沙门菌、从9份可疑食物中分离出3株慕尼黑沙门菌、从18名厨师肛拭子中分离出7株慕尼黑沙门菌,所分离菌株生化结果一致、耐药性相同,患者恢复期血清比急性期血清对所分离的菌株抗体有4倍以上增长.23株分离株的PFGE型完全一致.结论PFGE能直观判断肠道致病菌的亲缘关系,及时确定传染源、传播途径和流行范围,是有效控制食源性疾病大面积暴发的早期预警手段.     

沈阳地区副溶血弧菌脉冲场凝胶电泳分析

目的 运用脉冲场凝胶电泳技术(pulsed-fieldgelelectro phoresis,PFGE)和PCR技术对辽宁沈阳市2010年7-8月份临床粪便标本分离的副溶血性弧菌进行毒力基因检测和分子分型分析.方法 运用PCR技术检测24株副溶血弧菌的耐热溶血素基因(tdh)、耐热溶血素相关溶血素基因(trh)和不耐热溶血素基因(tl);用PFGE技术对其进行分子分型和聚类分析.结果 24株副溶血弧菌均含有tdh和tl基因,表明均为有毒株;PFGE结果显示,24株菌株可分为10种图谱型,其中17株O3:K6被分为4种型别(仅相差1～3条带),表明17株O3:K6在流行病学上密切相关,提示沈阳地区可能存在副溶血弧菌腹泻病的局部暴发或流行.结论 沈阳地区流行的食源性副溶血弧菌存在基因型的多样性,但以遗传关系密切的O3:K6型为优势型别.     

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
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