Sequence Analysis of 96 Genomic Regions Identifies Distinct Evolutionary Lineages within CC156, the Largest Streptococcus pneumoniae Clonal Complex in the MLST Database

Multi-Locus Sequence Typing (MLST) of Streptococcus pneumoniae is based on the sequence of seven housekeeping gene fragments. The analysis of MLST allelic profiles by eBURST allows the grouping of genetically related strains into Clonal Complexes (CCs) including those genotypes with a common descent from a predicted ancestor. However, the increasing use of MLST to characterize S. pneumoniae strains has led to the identification of a large number of new Sequence Types (STs) causing the merger of formerly distinct lineages into larger CCs. An example of this is the CC156, displaying a high level of complexity and including strains with allelic profiles differing in all seven of the MLST loci, capsular type and the presence of the Pilus Islet-1 (PI-1). Detailed analysis of the CC156 indicates that the identification of new STs, such as ST4945, induced the merging of formerly distinct clonal complexes. In order to discriminate the strain diversity within CC156, a recently developed typing schema, 96-MLST, was used to analyse 66 strains representative of 41 different STs. Analysis of allelic profiles by hierarchical clustering and a minimum spanning tree identified ten genetically distinct evolutionary lineages. Similar results were obtained by phylogenetic analysis on the concatenated sequences with different methods. The identified lineages are homogenous in capsular type and PI-1 presence. ST4945 strains were unequivocally assigned to one of the lineages. In conclusion, the identification of new STs through an exhaustive analysis of pneumococcal strains from various laboratories has highlighted that potentially unrelated subgroups can be grouped into a single CC by eBURST. The analysis of additional loci, such as those included in the 96-MLST schema, will be necessary to accurately discriminate the clonal evolution of the pneumococcal population.     

Multicolor Melting Curve Analysis-Based Multilocus Melt Typing of Vibrio parahaemolyticus

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis outbreaks. To track the source of these diseases in a timely manner, a high throughput typing method is critical. We hereby describe a novel genotyping method for V. parahaemolyticus, termed multilocus melt typing (MLMT), based on multilocus sequence typing (MLST). MLMT utilizes melting curve analysis to interrogate the allelic types of a set of informative single nucleotide polymorphisms (SNPs) derived from the housekeeping genes used in MLST. For each SNP, one allelic type generates distinct T_m values, which are converted into a binary code. Multiple SNPs thus generate a series of binary codes, forming a melt type (MT) corresponding with a sequence type (ST) of MLST. Using a set of 12 SNPs, the MLMT scheme could resolve 218 V.parahaemolyticus isolates into 50 MTs corresponding with 56 STs. The discriminatory power of MLMT and MLST was similar with Simpson’s index of diversity of 0.638 and 0.646, respectively. The global (adjusted Rand index = 0.982) and directional congruence (adjusted Wallace coefficient, MT→ST = 0.965; ST→MT = 1.000) between the two typing approaches was high. The entire procedure of MLMT could be finished within 3 h with negligible hands on time in a real-time PCR machine. We conclude that MLMT provides a reliable and efficient approach for V. parahaemolyticus genotyping and might also find use in other pathogens.     

Molecular Typing of Pathogenic Leptospira Serogroup Icterohaemorrhagiae Strains Circulating in China during the Past 50 Years

Background

Leptospirosis is one of the most important neglected tropical infectious diseases worldwide. Icterohaemorrhagiae has been throughout recent history, and still is, the predominant serogroup of this pathogen in China. However, very little in detail is known about the serovars or genotypes of this serogroup.

Methodology/Principal Findings

In this study, 120 epidemic strains from five geographically diverse regions in China collected over a 50 year period (1958~2008), and 8 international reference strains characterized by 16S rRNA sequencing and MLST analysis. 115, 11 and 2 strains were identified as L. interrogans, L. borgpetersenii, and L. kirschneri, respectively. 17 different STs were identified including 69 ST1 strains, 18 ST17, 18 ST128, 9 ST143 and 2 ST209. The remaining 12 strains belonged to 12 different STs. eBURST analysis demonstrated that, among the clonal complexes isolated (CCs), CC1 accounted for 73.3% (88/120) strains representing three STs: ST1, ST128 and ST98. ST1 was the most likely ancestral strain of this CC, followed by singleton CC17 (17/120) and CC143 (11/120). Further analysis of adding 116 serogroup Icterohaemorrhagiae strains in the MLST database and studies previously described using global eBURST analysis and MST dendrogram revealed relatively similar ST clustering patterns with five main CCs and 8 singletons among these 244 strains. CC17 was found to be the most prevalent clone of pathogenic Leptospira circulating worldwide. This is the first time, to our knowledge, that ST1 and ST17 strains were distributed among 4 distinct serovars, indicating a highly complicated relationship between serovars and STs.

Conclusions/Significance

Our studies demonstrated a high level of genetic diversity in the serogroup Icterohaemorrhagiae strains. Distinct from ST17 or ST37 circulating elsewhere, ST1 included in CC1, has over the past 50 years or so, proven to be the most prevalent ST of pathogenic leptospires isolated in China. Moreover, the complicated relationship between STs and serovars indicates an urgent need to develop an improved scheme for Leptospira serotyping.     

Population Structure of Clinical and Environmental Vibrio parahaemolyticus from the Pacific Northwest Coast of the United States

Vibrio parahaemolyticus is a common marine bacterium and a leading cause of seafood-borne bacterial gastroenteritis worldwide. Although this bacterium has been the subject of much research, the population structure of cold-water populations remains largely undescribed. We present a broad phylogenetic analysis of clinical and environmental V. parahaemolyticus originating largely from the Pacific Northwest coast of the United States. Repetitive extragenic palindromic PCR (REP-PCR) separated 167 isolates into 39 groups and subsequent multilocus sequence typing (MLST) separated a subset of 77 isolates into 24 sequence types. The Pacific Northwest population exhibited a semi-clonal structure attributed to an environmental clade (ST3, N = 17 isolates) clonally related to the pandemic O3:K6 complex and a clinical clade (ST36, N = 20 isolates) genetically related to a regionally endemic O4:K12 complex. Further, the identification of at least five additional clinical sequence types (i.e., ST43, 50, 65, 135 and 417) demonstrates that V. parahaemolyticus gastroenteritis in the Pacific Northwest is polyphyletic in nature. Recombination was evident as a significant source of genetic diversity and in particular, the recA and dtdS alleles showed strong support for frequent recombination. Although pandemic-related illnesses were not documented during the study, the environmental occurrence of the pandemic clone may present a significant threat to human health and warrants continued monitoring. It is evident that V. parahaemolyticus population structure in the Pacific Northwest is semi-clonal and it would appear that multiple sequence types are contributing to the burden of disease in this region.     

Genetic diversity of Staphylococcus equorum isolates from Saeu-jeotgal evaluated by multilocus sequence typing

Staphylococcus equorum, the predominant bacterial species detected in Saeu-jeotgal, a Korean high-salt fermented seafood, is a candidate starter bacterium for Saeu-jeotgal fermentation. A multilocus sequence typing (MLST) scheme was developed to evaluate the genetic diversity and background of S. equorum strains isolated from Saeu-jeotgal. A total of 135 strains, including 117 isolates from Saeu-jeotgal, and others from Myeolchi-jeotgal, sausage, cheese and horse skin, were subjected to MLST, and the internal fragments of seven housekeeping genes, aroE, dnaJ, glpF, gmk, hsp60, mutS, and pta, were compared. This MLST scheme produced 45 sequence types (STs) and the eBURST algorithm clustered the STs into nine clonal groups and seven singletons. Clonal group 1, the major group, consisted of 30 isolates from cheese, Saeu-jeotgal and sausages, which were classified into 12 STs. The predominant ST, ST26, comprised 25 isolates and presented as a singleton. Most of the isolates from Myeolchi-jeotgal and sausages clustered on two different branches of a phylogenetic tree generated with a cluster analysis using the maximum likelihood algorithm. This MLST scheme established the genetic backgrounds of S. equorum strains isolated from different types of food. Among the housekeeping genes used for MLST, gmk had the fewest allele types and fairly low sequence identities (74.0–90.0 %) within the Staphylococcus species. Therefore, sequence analyses of the gmk gene and 16S rRNA gene can be used for the accurate and rapid identification of S. equorum.     

Determination of molecular phylogenetics of Vibrio parahaemolyticus strains by multilocus sequence typing

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. There is a growing public health concern due to the emergence of a pandemic strain causing severe outbreaks worldwide. Many questions remain unanswered regarding the evolution and population structure of V. parahaemolyticus. In this work, we describe a multilocus sequence typing (MLST) scheme for V. parahaemolyticus based on the internal fragment sequences of seven housekeeping genes. This MLST scheme was applied to 100 V. parahaemolyticus strains isolated from geographically diverse clinical (n = 37) and environmental (n = 63) sources. The sequences obtained from this work were deposited and are available in a public database (http://pubmlst.org/vparahaemolyticus). Sixty-two unique sequence types were identified, and most (50) were represented by a single isolate, suggesting a high level of genetic diversity. Three major clonal complexes were identified by eBURST analysis. Separate clonal complexes were observed for V. parahaemolyticus isolates originating from the Pacific and Gulf coasts of the United States, while a third clonal complex consisted of strains belonging to the pandemic clonal complex with worldwide distribution. The data reported in this study indicate that V. parahaemolyticus is genetically diverse with a semiclonal population structure and an epidemic structure similar to that of Vibrio cholerae. Genetic diversity in V. parahaemolyticus appears to be driven primarily by frequent recombination rather than mutation, with recombination ratios estimated at 2.5:1 and 8.8:1 by allele and site, respectively. Application of this MLST scheme to more V. parahaemolyticus strains and by different laboratories will facilitate production of a global picture of the epidemiology and evolution of this pathogen.     

Global optimal eBURST analysis of multilocus typing data using a graphic matroid approach

Background  

Multilocus Sequence Typing (MLST) is a frequently used typing method for the analysis of the clonal relationships among strains of several clinically relevant microbial species. MLST is based on the sequence of housekeeping genes that result in each strain having a distinct numerical allelic profile, which is abbreviated to a unique identifier: the sequence type (ST). The relatedness between two strains can then be inferred by the differences between allelic profiles. For a more comprehensive analysis of the possible patterns of evolutionary descent, a set of rules were proposed and implemented in the eBURST algorithm. These rules allow the division of a data set into several clusters of related strains, dubbed clonal complexes, by implementing a simple model of clonal expansion and diversification. Within each clonal complex, the rules identify which links between STs correspond to the most probable pattern of descent. However, the eBURST algorithm is not globally optimized, which can result in links, within the clonal complexes, that violate the rules proposed.     

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations.     

Multilocus Sequence Typing of Leuconostoc gelidum subsp. gasicomitatum,a Psychrotrophic Lactic Acid Bacterium Causing Spoilage of Packaged Perishable Foods

Leuconostoc gelidum subsp. gasicomitatum is a psychrotrophic lactic acid bacterium (LAB) that causes spoilage of a variety of modified-atmosphere-packaged (MAP) cold-stored food products. During the past 10 years, this spoilage organism has been increasingly reported in MAP meat and vegetable products in northern Europe. In the present study, the population structure within 252 L. gelidum subsp. gasicomitatum strains was determined based on a novel multilocus sequence-typing (MLST) scheme employing seven housekeeping genes. These strains had been isolated from meat and vegetable sources over a time span of 15 years, and all 68 previously detected pulsed-field gel electrophoresis (PFGE) genotypes were represented. A total of 46 sequence types (STs) were identified, with a majority of the strains (>60%) belonging to three major STs, which were grouped into three clonal complexes (CCs) and 17 singletons by Global Optimal eBURST (goeBURST). The results by Bayesian analysis of population structure (BAPS) mostly correlated with the grouping by goeBURST. Admixture analysis by BAPS indicated a very low level of exchange of genetic material between the subpopulations. Niche specificity was observed within the subpopulations: CC1 and BAPS cluster 1 consisted mostly of strains from a variety of MAP meats, whereas vegetable strains grouped together with strains from MAP poultry within CC2 and BAPS cluster 2. The MLST scheme presented in this study provides a shareable and continuously growing sequence database enabling global comparison of strains associated with spoilage cases. This will further advance our understanding of the microbial ecology of this industrially important LAB.     

Genetic diversity and antimicrobial resistance? in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.     

江苏水产养殖区拟态弧菌种群结构及遗传多样性

【背景】拟态弧菌(Vibrio mimicus)是一种常见的革兰氏阴性病原菌,广泛分布于水环境和水生动物体内,可导致多种水产动物和人类感染。多位点序列分型(multilocus sequence typing, MLST)已被应用于多种病原菌的分子分型,其通过分析不同菌株之间的遗传关系,监测细菌传播的时间和地理分布,确定感染和传播途径,但目前未见有关拟态弧菌MLST的报道。【目的】开发一种基于MLST的拟态弧菌分型方法,并用于江苏水产养殖区拟态弧菌的种群结构和遗传进化分析,为拟态弧菌感染所引起的疾病防治提供理论基础。【方法】选择拟态弧菌的7个管家基因dnaE、gyrB、mdh、recA、rpoD、pntA和pyrH作为靶点,对江苏水产养殖区分离的155株拟态弧菌进行PCR扩增和测序。将测序结果分配等位基因,制作等位基因谱,分配不同的序列类型(sequence type, ST),利用软件goeBURST-1.2.1和MEGA-X对分配的ST型进行克隆复合体和遗传进化树聚类分析;此外,利用Kirby-Bauer圆盘扩散法测试155株拟态弧菌的药敏特性。【结果】155株拟态弧菌被分为56个STs,其中ST11占比最高;在双位点变异(double locus variants, DLV)水平分析发现56个STs分为3个克隆复合体和3个单体;系统发育树显示,56个STs被分为3个集群(cluster I、cluster II、cluster III)。药敏结果显示,155株拟态弧菌对红霉素类抗生素的耐药性最高(88.39%, 137/155),对氯霉素类抗生素敏感性最高(91.61%, 142/155)。【结论】本研究建立的MLST方法具有良好的分辨率,可作为拟态弧菌系统发育和未来流行病学调查有用的分子分型工具。根据抗生素耐药谱结果,提示在养殖过程中可选用氟苯尼考等国家批准使用的专用抗菌药对拟态弧菌进行防治。     

Multilocus sequence typing reveals high genetic diversity and epidemic population structure for the fish pathogen Yersinia ruckeri

Yersinia ruckeri is the causative agent of enteric redmouth in fish and one of the major bacterial pathogens causing losses in salmonid aquaculture. Previously typing methods, including restriction enzyme analysis, pulsed-field gel electrophoresis and multilocus enzyme electrophoresis (MLEE) have indicated a clonal population structure. In this work, we describe a multilocus sequence typing (MLST) scheme for Y.ruckeri based on the internal fragment sequence of six housekeeping genes. This MLST scheme was applied to 103 Y.ruckeri strains from diverse geographic areas and hosts as well as environmental sources. Sequences obtained from this work were deposited and are available in a public database (http://publmst.org/yruckeri/). Thirty different sequence types (ST) were identified, 21 of which were represented by a single isolate, evidencing high genetic diversity. ST2 comprised more than one-third of the isolates and was most frequently observed among isolates from trout. Two major clonal complexes (CC) were identified by eBURST analysis showing a common evolutionary origin for 94 isolates forming 21 STs into CC1 and for 6 isolates of 6 STs in the CC2. It was also possible to associate some unique ST with isolates from recent outbreaks in vaccinated salmonid fish.     

A combined approach of VNTR and MLST analysis: improving molecular typing of Argentinean isolates of Leptospira interrogans

Leptospirosis is an emerging infectious disease that has been identified as both a human and animal health problem worldwide. Regular outbreaks associated with specific risk factors have been reported in Argentina. However, there are no available data concerning the genetic population level for this pathogen. Therefore, the aim of this work was to describe the genetic diversity of Leptospira interrogans through the application of two molecular typing strategies: variable number of tandem repeats (VNTR) and multilocus sequence typing (MLST). For this purpose, seven reference strains and 18 non-epidemiologically related isolates from diverse hosts and Argentinean regions were analysed. Among them, nine genotypes and seven sequence types (STs), including three unreported STs, were described using VNTR and MLST, respectively. eBURST analysis demonstrated that ST37 was the most frequent and founder genotype of a clonal complex (CCs) containing STN1 and STN3, suggesting the importance of studying the serovars belonging to this CC in Argentina. The data from maximum parsimony analysis, which combined both techniques, achieved intra-serovar discrimination, surmounted microscopic agglutination test discrepancies and increased the discriminatory power of each technique applied separately. This study is the first to combine both strategies for L. interrogans typing to generate a more comprehensive molecular genotyping of isolates from Argentina in a global context.     

Genetic diversity and structure of Rhizobium leguminosarum populations associated with clover plants are influenced by local environmental variables

The identification and conservation of indigenous rhizobia associated with legume plants and their application as biofertilizers is becoming an agricultural worldwide priority. However, little is known about the genetic diversity and phylogeny of rhizobia in Romania. In the present study, the genetic diversity and population composition of Rhizobium leguminosarum symbiovar trifolii isolates from 12 clover plants populations located across two regions in Romania were analyzed. Red clover isolates were phenotypically evaluated and genotyped by sequencing 16S rRNA gene, 16S-23S intergenic spacer, three chromosomal genes (atpD, glnII and recA) and two plasmid genes (nifH and nodA). Multilocus sequence typing (MLST) analysis revealed that red clover plants are nodulated by a wide genetic diversity of R. leguminosarum symbiovar trifolii sequence types (STs), highly similar to the ones previously found in white clover. Rhizobial genetic variation was found mainly within the two clover populations for both chromosomal and plasmid types. Many STs appear to be unique for this region and the genetic composition of rhizobia differs significantly among the clover populations. Furthermore, our results showed that both soil pH and altitude contributed to plasmid sequence type composition while differences in chromosomal composition were affected by the altitude and were strongly correlated with distance.     

Population Structure and Evolution of Non-O1/Non-O139 Vibrio cholerae by Multilocus Sequence Typing

Pathogenic non-O1/non-O139 Vibrio cholerae strains can cause sporadic outbreaks of cholera worldwide. In this study, multilocus sequence typing (MLST) of seven housekeeping genes was applied to 55 non-O1/non-O139 isolates from clinical and environmental sources. Data from five published O1 isolates and 17 genomes were also included, giving a total of 77 isolates available for analysis. There were 66 sequence types (STs), with the majority being unique, and only three clonal complexes. The V. cholerae strains can be divided into four subpopulations with evidence of recombination among the subpopulations. Subpopulations I and III contained predominantly clinical strains. PCR screening for virulence factors including Vibrio pathogenicity island (VPI), cholera toxin prophage (CTXΦ), type III secretion system (T3SS), and enterotoxin genes (rtxA and sto/stn) showed that combinations of these factors were present in the clinical isolates with 85.7% having rtxA, 51.4% T3SS, 31.4% VPI, 31.4% sto/stn (NAG-ST) and 11.4% CTXΦ. These factors were also present in environmental isolates but at a lower frequency. Five strains previously mis-identified as V. cholerae serogroups O114 to O117 were also analysed and formed a separate population with V. mimicus. The MLST scheme developed in this study provides a framework to identify sporadic cholera isolates by genetic identity.     

Molecular Evidence for the Thriving of Campylobacter jejuni ST-4526 in Japan

Campylobacter jejuni is a leading cause of human gastroenteritis worldwide. This study aimed at a better understanding of the genetic diversity of this pathogen disseminated in Japan. We performed multilocus sequence typing (MLST) of Campylobacter jejuni isolated from different sources (100 human, 61 poultry, and 51 cattle isolates) in Japan between 2005 and 2006. This approach identified 62 sequence types (STs) and 19 clonal complexes (CCs), including 11 novel STs. These 62 STs were phylogenetically divided into 6 clusters, partially exhibiting host association. We identified a novel ST (ST-4526) that has never been reported in other countries; a phylogenetic analysis showed that ST-4526 and related STs showed distant lineage from the founder ST, ST-21 within CC-21. Comparative genome analysis was performed to investigate which properties could be responsible for the successful dissemination of ST-4526 in Japan. Results revealed that three representative ST-4526 isolates contained a putative island comprising the region from Cj0737 to Cj0744, which differed between the ST-4526 isolates and the reference strain NCTC11168 (ST-43/CC-21). Amino acid sequence alignment analyses showed that two of three ST-4526 isolates expressed 693aa- filamentous hemagglutination domain protein (FHA), while most of other C. jejuni strains whose genome were sequenced exhibited its truncation. Correspondingly, host cell binding of FHA-positive C. jejuni was greater than that of FHA-truncated strains, and exogenous administration of rFHA protein reduced cell adhesion of FHA-positive bacteria. Biochemical assays showed that this putative protein exhibited a dose-dependent binding affinity to heparan sulfate, indicating its adhesin activity. Moreover, ST-4526 showed increased antibiotic-resistance (nalidixic acid and fluoroquinolones) and a reduced ability for DNA uptake. Taken together, our data suggested that these combined features contributed to the clonal thriving of ST-4526 in Japan.     

Transoceanic Spreading of Pathogenic Strains of Vibrio parahaemolyticus with Distinctive Genetic Signatures in the recA Gene

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. Consistent multilocus sequence typing for V. parahaemolyticus has shown difficulties in the amplification of the recA gene by PCR associated with a lack of amplification or a larger PCR product than expected. In one strain (090–96, Peru, 1996), the produced PCR product was determined to be composed of two recA fragments derived from different Vibrio species. To better understand this phenomenon, we sequenced the whole genome of this strain. The hybrid recA gene was found to be the result of a fragmentation of the original lineage-specific recA gene resulting from a DNA insertion of approximately 30 kb in length. This insert had a G+C content of 38.8%, lower than that of the average G+C content of V. parahaemolyticus (45.2%), and contained 19 ORFs, including a complete recA gene. This new acquired recA gene deviated 24% in sequence from the original recA and was distantly related to recA genes from bacteria of the Vibrionaceae family. The reconstruction of the original recA gene (recA3) identified the precursor as belonging to ST189, a sequence type reported previously only in Asian countries. The identification of this singular genetic feature in strains from Asia reveals new evidence for genetic connectivity between V. parahaemolyticus populations at both sides of the Pacific Ocean that, in addition to the previously described pandemic clone, supports the existence of a recurrent transoceanic spreading of pathogenic V. parahaemolyticus with the corresponding potential risk of pandemic expansion.     

Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys

Commercial turkey flocks in North Carolina have been found to be colonized frequently with Campylobacter coli strains that are resistant to several antimicrobials (tetracycline, streptomycin, erythromycin, kanamycin, and ciprofloxacin/nalidixic acid). Such strains have been designated multidrug resistant (MDR). However, the population structure of MDR C. coli from turkeys remains poorly characterized. In this study, an analysis of multilocus sequence typing (MLST)-based sequence types (STs) of 59 MDR strains from turkeys revealed that the majority of these strains corresponded to one of 14 different STs, with three STs accounting for 41 (69%) of the strains. The major STs were turkey specific, and most (87%) of the strains with these STs were resistant to the entire panel of antibiotics mentioned above. Some (13%) of the strains with these STs were susceptible to just one or two of the antibiotics in this panel. Further subtyping using fla typing and pulsed-field gel electrophoresis with SmaI and KpnI revealed that the major MDR STs corresponded to strains of related but distinct subtypes, providing evidence for genomic diversification within these STs. These findings suggest that MDR strains of C. coli from turkeys have a clonal population structure characterized by the presence of a relatively small number of clonal groups that appear to be disseminated in the turkey production system. In addition, the observed correlation between STs and the MDR profiles of the microbes indicates that MLST-based typing holds potential for source-tracking applications specific to the animal source (turkeys) and the antimicrobial resistance profile (MDR status) of C. coli.     

Comparative Genotyping of Campylobacter jejuni Strains from Patients with Guillain-Barré Syndrome in Bangladesh

Background

Campylobacter jejuni is a common cause of acute gastroenteritis and is associated with post-infectious neuropathies such as the Guillain-Barré syndrome (GBS) and the Miller Fisher syndrome (MFS). We here present comparative genotyping of 49 C. jejuni strains from Bangladesh that were recovered from patients with enteritis or GBS. All strains were serotyped and analyzed by lipo-oligosaccharide (LOS) genotyping, amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE).

Methodology/Principal Findings

C. jejuni HS:23 was a predominant serotype among GBS patients (50%), and no specific serotype was significantly associated with GBS compared to enteritis. PCR screening showed that 38/49 (78%) of strains could be assigned to LOS classes A, B, C, or E. The class A locus (4/7 vs 3/39; p<0.01) was significantly associated in the GBS-related strains as compared to enteritis strains. All GBS/oculomotor related strains contained the class B locus; which was also detected in 46% of control strains. Overlapping clonal groups were defined by MLST, AFLP and PFGE for strains from patients with gastroenteritis and GBS. MLST defined 22 sequence types (STs) and 7 clonal complexes including 7 STs not previously identified (ST-3742, ST-3741, ST-3743, ST-3748, ST-3968, ST-3969 and ST-3970). C. jejuni HS:23 strains from patients with GBS or enteritis were clonal and all strains belonged to ST-403 complex. Concordance between LOS class B and ST-403 complex was revealed. AFLP defined 25 different types at 90% similarity. The predominant AFLP type AF-20 coincided with the C. jejuni HS:23 and ST-403 complex.

Conclusion/Significance

LOS genotyping, MLST, AFLP and PFGE helped to identify the HS:23 strains from GBS or enteritis patients as clonal. Overall, genotypes exclusive for enteritis or for GBS-related strains were not obtained although LOS class A was significantly associated with GBS strains. Particularly, the presence of a clonal and putative neuropathogenic C. jejuni HS:23 serotype may contribute to the high prevalence of C. jejuni related GBS in Bangladesh.     

Understanding the Molecular Epidemiology and Global Relationships of Brachyspira hyodysenteriae from Swine Herds in the United States: A Multi-Locus Sequence Typing Approach

Outbreaks of mucohemorrhagic diarrhea in pigs caused by Brachyspira hyodysenteriae in the late 2000s indicated the re-emergence of Swine Dysentery (SD) in the U.S. Although the clinical disease was absent in the U.S. since the early 1990s, it continued to cause significant economic losses to other swine rearing countries worldwide. This study aims to fill the gap in knowledge pertaining to the re-emergence and epidemiology of B. hyodysenteriae in the U.S. and its global relationships using a multi-locus sequence typing (MLST) approach. Fifty-nine post re-emergent isolates originating from a variety of sources in the U.S. were characterized by MLST, analyzed for epidemiological relationships (within and between multiple sites of swine systems), and were compared with pre re-emergent isolates from the U.S. Information for an additional 272 global isolates from the MLST database was utilized for international comparisons. Thirteen nucleotide sequence types (STs) including a predominant genotype (ST93) were identified in the post re-emergent U.S. isolates; some of which showed genetic similarity to the pre re-emergent STs thereby suggesting its likely role in the re-emergence of SD. In the U.S., in general, no more than one ST was found on a site; multiple sites of a common system shared a ST; and STs found in the U.S. were distinct from those identified globally. Of the 110 STs characterized from ten countries, only two were found in more than one country. The U.S. and global populations, identified as clonal and heterogeneous based on STs, showed close relatedness based on amino acid types (AATs). One predicted founder type (AAT9) and multiple predicted subgroup founder types identified for both the U.S. and the global population indicate the potential microevolution of this pathogen. This study elucidates the strain diversity and microevolution of B. hyodysenteriae, and highlights the utility of MLST for epidemiological and surveillance studies.