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.     

A Stenotrophomonas maltophilia Multilocus Sequence Typing Scheme for Inferring Population Structure

Stenotrophomonas maltophilia is an opportunistic, highly resistant, and ubiquitous pathogen. Strains have been assigned to genogroups using amplified fragment length polymorphism. Hence, isolates of environmental and clinical origin predominate in different groups. A multilocus sequence typing (MLST) scheme was developed using a highly diverse selection of 70 strains of various ecological origins from seven countries on all continents including strains of the 10 previously defined genogroups. Sequence data were assigned to 54 sequence types (ST) based on seven loci. Indices of association for all isolates and clinical isolates of 2.498 and 2.562 indicated a significant linkage disequilibrium, as well as high congruence of tree topologies from different loci. Potential recombination events were detected in one-sixth of all ST. Calculation of the mean divergence between and within predicted clusters confirmed previously defined groups and revealed five additional groups. Consideration of the different ecological origins showed that 18 out of 31 respiratory tract isolates, including 12 out of 19 isolates from cystic fibrosis (CF) patients, belonged to genogroup 6. In contrast, 16 invasive strains isolated from blood cultures were distributed among nine different genogroups. Three genogroups contained isolates of strictly environmental origin that also featured high sequence distances to other genogroups, including the S. maltophilia type strain. On the basis of this MLST scheme, isolates can be assigned to the genogroups of this species in order to further scrutinize the population structure of this species and to unravel the uneven distribution of environmental and clinical isolates obtained from infected, colonized, or CF patients.Stenotrophomonas maltophilia is ubiquitous in nature. It has, for instance, been isolated from the rhizosphere of various plants and animals (14, 27, 37). Due to its tolerance against cadmium and its ability to degrade xenobiotic compounds, it has been proposed for remediation of contaminated ground (9, 39). Increasingly, it is being isolated from immunosuppressed individuals and intensive care and cystic fibrosis (CF) patients and has been shown to be resistant to many antimicrobial agents (16, 17, 69). However, the role of this opportunistic pathogen as an innocent bystander or causative agent often remains unclear (30), and little is known about its virulence factors (20, 33).Recently, novel Stenotrophomonas species were described: Stenotrophomonas nitritireducens sp. nov. (24), Stenotrophomonas acidaminiphila (3), Stenotrophomonas rhizophila (73), and Stenotrophomonas africana sp. nov. (21). However, the latter is a synonym of S. maltophilia (10).Using amplified fragment length polymorphism (AFLP) fingerprinting and DNA-DNA hybridizations, remarkable diversity has been shown to exist among S. maltophilia isolates recovered from both the environment and human clinical samples. This species can be subdivided into 10 AFLP genomic groups (35) that comprise to various extents both clinical and environmental isolates. Similarly, different genomic groups of the genus Stenotrophomonas can be distinguished using restriction fragment length polymorphisms (RFLP) in the gyrB gene (11). Surprisingly, 36 out of 40 isolates from CF patients are grouped in just two clusters. However, no such differences were seen in other investigations using pulsed-field gel electrophoresis (PFGE) after DraI digestion, molecular typing by BOX-PCR, or temperature-gradient gel electrophoresis of 16S rRNA PCR fragments (7). Later DNA sequence analyses of the 16S rRNA revealed three clusters, with grouping of the strains according to their sources of isolation and signature sequences in the region V1, which distinguishes clinical from environmental isolates (44).The objective of this study was to develop a multilocus sequence typing (MLST) scheme on the basis of a diverse strain collection comprising isolates from different ecological origins, continents, and DNA hybridization groups (35). We then employed this scheme to start initial analyses of the population structure of this species.(This study was conducted by S. Kaiser in partial fulfillment of the requirements for a diploma thesis in biology from the Faculty of Biology, University of Freiburg, Freiburg, Germany, 2007.)     

Multilocus Sequence Typing Scheme for Bacteria of the Bacillus cereus Group

In this study we developed a multilocus sequence typing (MLST) scheme for bacteria of the Bacillus cereus group. This group, which includes the species B. cereus, B. thuringiensis, B. weihenstephanensis, and B. anthracis, is known to be genetically very diverse. It is also very important because it comprises pathogenic organisms as well as bacteria with industrial applications. The MLST system was established by using 77 strains having various origins, including humans, animals, food, and soil. A total of 67 of these strains had been analyzed previously by multilocus enzyme electrophoresis, and they were selected to represent the genetic diversity of this group of bacteria. Primers were designed for conserved regions of housekeeping genes, and 330- to 504-bp internal fragments of seven such genes, adk, ccpA, ftsA, glpT, pyrE, recF, and sucC, were sequenced for all strains. The number of alleles at individual loci ranged from 25 to 40, and a total of 53 allelic profiles or sequence types (STs) were distinguished. Analysis of the sequence data showed that the population structure of the B. cereus group is weakly clonal. In particular, all five B. anthracis isolates analyzed had the same ST. The MLST scheme which we developed has a high level of resolution and should be an excellent tool for studying the population structure and epidemiology of the B. cereus group.     

Application of Multilocus Sequence Typing To Study the Genetic Structure of Megaplasmids in Medicago-Nodulating Rhizobia

A multilocus sequence typing (MLST) analysis was used to examine the genetic structure and diversity within the two large extrachromosomal replicons in Medicago-nodulating rhizobia (Sinorhizobium meliloti and Sinorhizobium medicae). The allelic diversity within these replicons was high compared to the reported diversity within the corresponding chromosomes of the same strains (P. van Berkum et al., J. Bacteriol. 188:5570-5577, 2006). Also, there was strong localized linkage disequilibrium (LD) between certain pSymA loci: e.g., nodC and nifD. Although both of these observations could be explained by positive (or diversifying) selection by plant hosts, results of tests for positive selection did not provide consistent support for this hypothesis. The strong LD observed between the nodC and nifD genes could also be explained by their close proximity on the pSymA replicon. Evidence was obtained that some nodC alleles had a history of intragenic recombination, while other alleles of this locus had a history of intergenic recombination. Both types of recombination were associated with a decline in symbiotic competence with Medicago sativa as the host plant. The combined observations of LD between the nodC and nifD genes and intragenic recombination within one of these loci indicate that the symbiotic gene region on the pSymA plasmid has evolved as a clonal segment, which has been laterally transferred within the natural populations.Plants of the genus Medicago are legumes that often benefit from a mutualistic symbiosis with rhizobia. The most agriculturally significant species of rhizobia that nodulate these plants are Sinorhizobium meliloti (9) and Sinorhizobium medicae (22). Previously reported population genetic analyses of these bacteria have focused on the study of how allelic variants at multiple loci are distributed within and among natural populations (2, 3, 10, 26, 31, 32). This was also the focus of the present study, but it was extended by examining more loci in many more strains of both species of Sinorhizobium coupled with an analysis having a range of symbiotic genotypes. One goal was to determine if there were any obvious correlations between the megaplasmid genotypes observed and their symbiotic competence. A second goal was to determine if selection by their host plants may have influenced the evolution of their symbiotic relationships.The genes for symbiosis reside on the extrachromosomal replicons pSymA (1,354,226 nucleotides [nt]) and pSMED02 (1,245,408 nt) in the genomes of S. meliloti Rm1021 and S. medicae WSM419, respectively (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE006469","term_id":"25168258","term_text":"AE006469"}}AE006469 and {"type":"entrez-nucleotide","attrs":{"text":"CP000740","term_id":"150031715","term_text":"CP000740"}}CP000740, respectively). Besides these two plasmids, these two strains each harbor one other large extrachromosomal replicon, pSymB (1,683,333 nt) and pSMED01 (1,570,951 nt), respectively (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AL591985","term_id":"30407151","term_text":"AL591985"}}AL591985 and {"type":"entrez-nucleotide","attrs":{"text":"CP000739","term_id":"150030273","term_text":"CP000739"}}CP000739, respectively).Multilocus sequence typing (MLST) (16) is a form of genomic indexing that is commonly used to study the population genetic structure and phylogenetic relatedness within diverse groups of bacteria. In this method, nucleotide sequences of a fixed set of common loci are obtained from a collection of strains, and polymorphic sites among these sequences are used to derive an allelic profile or sequence type (ST) for each genome. Comparisons of the resulting data can be used to infer phylogenetic relationships among the organisms in the sample population, and they also can be used to infer how evolutionary processes, such as recombination and selection, have shaped the genetic structure of the population. For example, levels of intergenic recombination among chromosomal genes in natural populations of Neisseria meningitidis reportedly are relatively high, while corresponding levels within populations of Staphylococcus aureus were low (28). Depending on the specific pairs of loci examined, the levels of linkage disequilibrium (LD) (a lack of intergenic recombination) among several chromosomally carried core genes of S. meliloti were reported to be generally moderate to high (26).The MLST approach has been used to confirm that the chromosomes of S. meliloti and S. medicae are sexually isolated (2, 3, 31) and to provide evidence that horizontal gene transfer (HGT) does occur between the symbiotic megaplasmids of these species (3, 32). It has also been used to demonstrate that levels of intergenic recombination, as indicated by linkage disequilibrium, differ between the three replicons of S. meliloti (26). Levels of intergenic recombination within the pSymB replicons of these strains are generally high, unlike the chromosomes and pSymA replicons within the same strains (26). Bailly et al. (3) hypothesized that the region of the pSymA plasmid that contains the nodulation (nod) genes is frequently transferred in natural populations. They also suggested that selective pressures from the host plant may have influenced both nod gene diversity and patterns of polymorphism across the entire nod gene region.In the present study, multilocus allelic variation of the two megaplasmids was examined among 231 Medicago-nodulating rhizobia that originated primarily from southwest Asia (10). Previously, 91 different chromosomal sequence types (STs) were identified among the same strains from sequence variation in 10 loci (31). This collection of strains had earlier been divided into two closely related groups based on results of multilocus enzyme electrophoresis (10), and this result was subsequently cited in support of separating the Medicago-nodulating rhizobia into the two species S. meliloti and S. medicae (22).The objectives of this study were (i) to use MLST to examine the genetic relationships within and among the large extrachromosomal replicons in S. meliloti and S. medicae, (ii) to estimate levels of intergenic and intragenic recombination in these replicons, (iii) to evaluate the nitrogen-fixing competence of representative symbiotic genotypes with Medicago sativa, and (iv) to determine whether positive (or diversifying) selection may have influenced the genetic structure of the megaplasmids.     

Genome Sequence and Comparative Genomics Analysis of a Vibrio cholerae O1 Strain Isolated from a Cholera Patient in Malaysia

The genome sequence analysis of a clinical Vibrio cholerae VC35 strain from an outbreak case in Malaysia indicates multiple genes involved in host adaptation and a novel Na⁺-driven multidrug efflux pump-coding gene in the genome of Vibrio cholerae with the highest similarity to VMA_001754 of Vibrio mimicus VMA223.     

Optimized Multilocus Sequence Typing (MLST) Scheme for Trypanosoma cruzi

Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared.     

多位点序列分型在食源性金黄色葡萄球菌分型中的应用研究

对食源性金黄色葡萄球菌进行多位点序列分型(MLST)分析,了解其基因型特征,并与流行病学资料进行对比分析。应用MLST方法对2012年石家庄市分离出的18株食源性金葡菌进行基因分型,并对该地区食源性金葡菌分子特性和流行病学特性进行分析。18株食源性金葡菌通过MLST分析得到10个ST序列型,其中ST5序列型最多,共5株;其次为ST464序列型,共3株;ST7型和ST15各2株;ST6型、ST9型、ST59型和ST2138型各1株,有2个菌株是2个新的ST型其ST码分别为287-1-1-8-1-1-1和10-14-8-6-278-3-2。本地区食源性金葡菌的ST型别丰富,主要流行克隆系为ST5和ST464,ST6、ST7、ST9、ST15、ST59和ST2138等克隆系也有分布。     

Development of a Tiered Multilocus Sequence Typing Scheme for Members of the Lactobacillus acidophilus Complex

Members of the Lactobacillus acidophilus complex are associated with functional foods and dietary supplements because of purported health benefits they impart to the consumer. Many characteristics of these microorganisms are reported to be strain specific. Therefore, proper strain typing is essential for safety assessment and product labeling, and also for monitoring strain integrity for industrial production purposes. Fifty-two strains of the L. acidophilus complex (L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum, L. gasseri, and L. johnsonii) were genotyped using two established methods and compared to a novel multilocus sequence typing (MLST) scheme. PCR restriction fragment length polymorphism (PCR-RFLP) analysis of the hsp60 gene with AluI and TaqI successfully clustered 51 of the 52 strains into the six species examined, but it lacked strain-level discrimination. Random amplified polymorphic DNA PCR (RAPD-PCR) targeting the M13 sequence resulted in highly discriminatory profiles but lacked reproducibility. In this study, an MLST scheme was developed using the conserved housekeeping genes fusA, gpmA, gyrA, gyrB, lepA, pyrG, and recA, which identified 40 sequence types that successfully clustered all of the strains into the six species. Analysis of the observed alleles suggests that nucleotide substitutions within five of the seven MLST loci have reached saturation, a finding that emphasizes the highly diverse nature of the L. acidophilus complex and our unconventional application of a typically intraspecies molecular typing tool. Our MLST results indicate that this method could be useful for characterization and strain discrimination of a multispecies complex, with the potential for taxonomic expansion to a broader collection of Lactobacillus species.     

多位点测序分型技术在病原微生物分型鉴定中的应用

多位点测序分型(Multilocus sequence typing,MLST)技术是一种以核苷酸序列为基础的病原菌分型方法,它是高通量测序技术与成熟的群体遗传学相结合的产物。该方法简单易行,重复性强,可以通过国际互联网对某一致病菌株在全球范围内的传播分布情况进行追踪监控。目前,MLST技术已被广泛应用于原核病原菌及一些真核病原菌(如真菌)的分型鉴定中。主要对MLST技术的原理及其在一些常见病原菌分型鉴定中的应用进行了简要的阐述。     

Meddling Vibrio cholerae Murmurs: A Neoteric Advancement in Cholera Research

Cholera, a known diarrheal disease is associated with various risk factors like hypovolemic shock, rice watery stools, and death in developing countries. The overuse of antibiotics to treat cholera imposed a selective pressure for the emergence and spread of multi-drug resistant Vibrio cholerae strains. The failure of conventional antimicrobial therapy urged the researchers to find an alternative therapy that could meddle the cholera murmurs (Quorum Sensing). It seems to effectively overcome the conventional cholera therapies in parallel to decrease the morbidity and mortality rate in the developing countries. The paramount objective of this review essentially focuses on the different Quorum Sensing (QS) regulatory switches governing virulence and pathogenicity of Vibrio cholerae. This review also provides an insight into the plausible QS targets that could be exploited to bring about a breakthrough to the prevailing cholera therapy.     

Multilocus Sequence Typing of Clinical Isolates of Cryptococcus from India

Mycopathologia - Cryptococcosis is a life-threatening infection caused by Cryptococcus neoformans and C. gattii species complex. In the present study, to understand the molecular epidemiology of...     

Genome sequence of the human pathogen Vibrio cholerae Amazonia

Vibrio cholerae O1 Amazonia is a pathogen that was isolated from cholera-like diarrhea cases in at least two countries, Brazil and Ghana. Based on multilocus sequence analysis, this lineage belongs to a distinct profile compared to strains from El Tor and classical biotypes. The genomic analysis revealed that it contains Vibrio pathogenicity island 2 and a set of genes related to pathogenesis and fitness, such as the type VI secretion system, present in choleragenic V. cholerae strains.     

海南岛光滑假丝酵母菌多位点序列分型研究

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

Analysis of the Population Structure of Anaplasma phagocytophilum Using Multilocus Sequence Typing

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophils. It is transmitted via tick-bite and causes febrile disease in humans and animals. Human granulocytic anaplasmosis is regarded as an emerging infectious disease in North America, Europe and Asia. However, although increasingly detected, it is still rare in Europe. Clinically apparent A. phagocytophilum infections in animals are mainly found in horses, dogs, cats, sheep and cattle. Evidence from cross-infection experiments that A. phagocytophilum isolates of distinct host origin are not uniformly infectious for heterologous hosts has led to several approaches of molecular strain characterization. Unfortunately, the results of these studies are not always easily comparable, because different gene regions and fragment lengths were investigated. Multilocus sequence typing is a widely accepted method for molecular characterization of bacteria. We here provide for the first time a universal typing method that is easily transferable between different laboratories. We validated our approach on an unprecedented large data set of almost 400 A. phagocytophilum strains from humans and animals mostly from Europe. The typability was 74% (284/383). One major clonal complex containing 177 strains was detected. However, 54% (49/90) of the sequence types were not part of a clonal complex indicating that the population structure of A. phagocytophilum is probably semiclonal. All strains from humans, dogs and horses from Europe belonged to the same clonal complex. As canine and equine granulocytic anaplasmosis occurs frequently in Europe, human granulocytic anaplasmosis is likely to be underdiagnosed in Europe. Further, wild boars and hedgehogs may serve as reservoir hosts of the disease in humans and domestic animals in Europe, because their strains belonged to the same clonal complex. In contrast, as they were only distantly related, roe deer, voles and shrews are unlikely to harbor A. phagocytophilum strains infectious for humans, domestic or farm animals.     

Fine-Scale Phylogeographic Structure of Borrelia lusitaniae Revealed by Multilocus Sequence Typing

Borrelia lusitaniae is an Old World species of the Lyme borreliosis (LB) group of tick-borne spirochetes and prevails mainly in countries around the Mediterranean Basin. Lizards of the family Lacertidae have been identified as reservoir hosts of B. lusitaniae. These reptiles are highly structured geographically, indicating limited migration. In order to examine whether host geographic structure shapes the evolution and epidemiology of B. lusitaniae, we analyzed the phylogeographic population structure of this tick-borne bacterium using a recently developed multilocus sequence typing (MLST) scheme based on chromosomal housekeeping genes. A total of 2,099 questing nymphal and adult Ixodes ricinus ticks were collected in two climatically different regions of Portugal, being ∼130 km apart. All ticks were screened for spirochetes by direct PCR. Attempts to isolate strains yielded 16 cultures of B. lusitaniae in total. Uncontaminated cultures as well as infected ticks were included in this study. The results using MLST show that the regional B. lusitaniae populations constitute genetically distinct populations. In contrast, no clear phylogeographic signals were detected in sequences of the commonly used molecular markers ospA and ospC. The pronounced population structure of B. lusitaniae over a short geographic distance as captured by MLST of the housekeeping genes suggests that the migration rates of B. lusitaniae are rather low, most likely because the distribution of mediterranean lizard populations is highly parapatric. The study underlines the importance of vertebrate hosts in the geographic spread of tick-borne microparasites.     

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.     

Differentiation of Environmental and Clinical Isolates of Vibrio mimicus from Vibrio cholerae by Multilocus Enzyme Electrophoresis

In this study, we demonstrated that analyzed strains of Vibrio mimicus and Vibrio cholerae could be separated in two groups by using multilocus enzyme electrophoresis (MEE) data from 14 loci. We also showed that the combination of four enzymatic loci enables us to differentiate these two species. Our results showed that the ribosomal intergenic spacer regions PCR-mediated identification system failed, in some cases, to differentiate between V. mimicus and V. cholerae. On the other hand, MEE proved to be a powerful molecular tool for the discrimination of these two species even when atypical strains were analyzed.     

Multilocus Sequence Typing and Phylogenetic Analyses of Pseudomonas aeruginosa Isolates from the Ocean

Recent isolation of Pseudomonas aeruginosa strains from the open ocean and subsequent pulsed-field gel electrophoresis analyses indicate that these strains have a unique genotype (N. H. Khan, Y. Ishii, N. Kimata-Kino, H. Esaki, T. Nishino, M. Nishimura, and K. Kogure, Microb. Ecol. 53:173-186, 2007). We hypothesized that ocean P. aeruginosa strains have a unique phylogenetic position relative to other strains. The objective of this study was to clarify the intraspecies phylogenetic relationship between marine strains and other strains from various geographical locations. Considering the advantages of using databases, multilocus sequence typing (MLST) was chosen for the typing and discrimination of ocean P. aeruginosa strains. Seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) were analyzed, and the results were compared with data on the MLST website. These genes were also used for phylogenetic analysis of P. aeruginosa. Rooted and unrooted phylogenetic trees were generated for each gene locus and the concatenated gene fragments. MLST data showed that all the ocean strains were new. Trees constructed for individual and concatenated genes revealed that ocean P. aeruginosa strains have clusters distinct from those of other P. aeruginosa strains. These clusters roughly reflected the geographical locations of the isolates. These data support our previous findings that P. aeruginosa strains are present in the ocean. It can be concluded that the ocean P. aeruginosa strains have diverged from other isolates and form a distinct cluster based on MLST and phylogenetic analyses of seven housekeeping genes.     

An Expanded Multilocus Sequence Typing Scheme for Propionibacterium acnes: Investigation of 'Pathogenic', 'Commensal' and Antibiotic Resistant Strains

The Gram-positive bacterium Propionibacterium acnes is a member of the normal human skin microbiota and is associated with various infections and clinical conditions. There is tentative evidence to suggest that certain lineages may be associated with disease and others with health. We recently described a multilocus sequence typing scheme (MLST) for P. acnes based on seven housekeeping genes (http://pubmlst.org/pacnes). We now describe an expanded eight gene version based on six housekeeping genes and two 'putative virulence' genes (eMLST) that provides improved high resolution typing (91eSTs from 285 isolates), and generates phylogenies congruent with those based on whole genome analysis. When compared with the nine gene MLST scheme developed at the University of Bath, UK, and utilised by researchers at Aarhus University, Denmark, the eMLST method offers greater resolution. Using the scheme, we examined 208 isolates from disparate clinical sources, and 77 isolates from healthy skin. Acne was predominately associated with type IA(1) clonal complexes CC1, CC3 and CC4; with eST1 and eST3 lineages being highly represented. In contrast, type IA(2) strains were recovered at a rate similar to type IB and II organisms. Ophthalmic infections were predominately associated with type IA(1) and IA(2) strains, while type IB and II were more frequently recovered from soft tissue and retrieved medical devices. Strains with rRNA mutations conferring resistance to antibiotics used in acne treatment were dominated by eST3, with some evidence for intercontinental spread. In contrast, despite its high association with acne, only a small number of resistant CC1 eSTs were identified. A number of eSTs were only recovered from healthy skin, particularly eSTs representing CC72 (type II) and CC77 (type III). Collectively our data lends support to the view that pathogenic versus truly commensal lineages of P. acnes may exist. This is likely to have important therapeutic and diagnostic implications.