Housekeeping Gene Fragments Research Articles

Association of Rahnella victoriana, Enterobacter hormaechei subsp. hoffmannii and Citrobacter braakii with walnut decline

Persian walnut (Juglans regia) has a considerable economic importance worldwide. However, the vigor and vitality of walnut trees were heavily affected by bark canker during the last few years. Irregular longitudinal cankers in the outer bark, stem tissue necrosis, and bleeding with black-colored exudates walnut trees were observed in Kermanshah, Hamedan, Markazi, Alborz, Isfahan, Qom, Semnan, and Razavi Khorasan provinces in western, central and eastern Iran during 2018 and 2019. A total of 150 symptomatic samples were collected from affected walnut trees in order to identify bacteria associated with walnut decline. Two-hundred sixty strains with a metallic green sheen were isolated on EMB-agar medium. The pathogenicity of all strains was proved by inoculating a suspension of the bacterial strains under the bark of immature walnut fruits cv. ‘Hartley’. Ninety-five strains caused necrosis and a dark-colored region in the mesocarp around the inoculation site 14 days post-inoculation. Moreover, 12 representative strains induced necrotic and black-colored tissues in the bark of young green twigs of two-year old walnut seedling cv. ‘Chandler’. The strains were classified into four categories based on conventional phenotypic characters confirmed with the 16S rRNA gene sequences. A phylogenetic tree based on the concatenated sequences of two housekeeping gene fragments, gyrB and infB, indicated that strains including I1, Q6, and S6 were grouped in a cluster with Gibbsiella quercinecans FBR97T as well as strains I2, I5, and KE6 were clustered with Rahnella victoriana FRB 225T. Moreover, strains MR1, MR3, and MR5 were grouped with the Enterobacter hormaechei subsp. hoffmannii DSM 14563T. The phylogenetic analyses based on the partial sequencing of housekeeping genes including fusA, pyrG, and leuS revealed that strains KH1, KH3, and KH7 belong to Citrobacter braakii species. To the best of our knowledge, this is the first report of C. braakii and E. hormaechei as plant pathogens and R. victoriana associated with walnut decline.

Identification of highly variable sequence fragments in unmapped reads for rapid bacterial genotyping

BackgroundBacterial genotyping is a crucial process in outbreak investigation and epidemiological studies. Several typing methods such as pulsed-field gel electrophoresis, multilocus sequence typing (MLST) and whole genome sequencing are currently used in routine clinical practice. However, these methods are costly, time-consuming and have high computational demands. An alternative to these methods is mini-MLST, a quick, cost-effective and robust method based on high-resolution melting analysis. Nevertheless, no standardized approach to identify markers suitable for mini-MLST exists. Here, we present a pipeline for variable fragment detection in unmapped reads based on a modified hybrid assembly approach using data from one sequencing platform.ResultsIn routine assembly against the reference sequence, high variable reads are not aligned and remain unmapped. If de novo assembly of them is performed, variable genomic regions can be located in created scaffolds. Based on the variability rates calculation, it is possible to find a highly variable region with the same discriminatory power as seven housekeeping gene fragments used in MLST. In the work presented here, we show the capability of identifying one variable fragment in de novo assembled scaffolds of 21 Escherichia coli genomes and three variable regions in scaffolds of 31 Klebsiella pneumoniae genomes. For each identified fragment, the melting temperatures are calculated based on the nearest neighbor method to verify the mini-MLST’s discriminatory power.ConclusionsA pipeline for a modified hybrid assembly approach consisting of reference-based mapping and de novo assembly of unmapped reads is presented. This approach can be employed for the identification of highly variable genomic fragments in unmapped reads. The identified variable regions can then be used in efficient laboratory methods for bacterial typing such as mini-MLST with high discriminatory power, fully replacing expensive methods such as MLST. The results can and will be delivered in a shorter time, which allows immediate and fast infection monitoring in clinical practice.

Molecular Diversity and Evolutionary Relatedness of Paulownia Witches’-Broom Phytoplasma in Different Geographical Distributions in China

To reveal the distribution and transmission pathway of Paulownia witches'-broom (PaWB) disease, which is caused by phytoplasmas related to genetic variation, and the adaptability to the hosts and environments of the pathogenic population in different geographical regions in China, in this study, we used ten housekeeping gene fragments, including rp, fusA, secY, tuf, secA, dnaK, rpoB, pyrG, gyrB, and ipt, for multilocus sequence typing (MLST). A total of 142 PaWB phytoplasma strains were collected from 18 provinces or municipalities. The results showed that the genetic diversity was comparatively higher among the PaWB phytoplasma strains, and substantially different from that of the other 16SrI subgroup strains. The number of gene variation sites for different housekeeping genes in the PaWB phytoplasma strains ranged from 1 to 14 SNPs. Among them, rpoB (1.47%) and dnaK (1.12%) had higher genetic variation, and rp (0.20%) had the least genetic variation. The tuf and rpoB genes showed the fixation of positively selected beneficial mutations in the PaWB phytoplasma populations, and all housekeeping genes except tuf followed the neutral evolutionary model. We found an absence of recombination among PaWB phytoplasma sequence types (STs) for each housekeeping gene except dnaK, and no evidence for such recombination events for concatenated sequences of PaWB phytoplasma strains. The 22 sequence types were identified among the concatenated sequences of seven housekeeping genes (rp, fusA, secY, secA, tuf, dnaK, and rpoB) from 105 representative strains. We analyzed all 22 STs by goeBURST algorithm, forming two clonal complexes (CCs) and three singletons. Among them, ST1, as the primary founder of CC1, had the widest geographical distribution, accounting for 72.38% of all strains, with a high frequency of shared sequence type. The results of phylogenetic analysis of the concatenated sequences further revealed that the 105 strains were clustered into two representative lineages of PaWB phytoplasma, with obvious geographical differentiation. The ST1 strains of highly homogeneous lineage-1 were a widespread and predominant population in diseased areas. Lineage-2 contained strains from Jiangxi, Fujian, and Shaanxi provinces, highlighting the close genetic relatedness of the strains in these regions, which was also consistent with the results of most single-gene phylogenetic analysis of each gene. We also found that the variability in the northwest China population was higher than in other geographical populations; the range of genetic differentiation between the south of the Yangtze River population and the Huang-huai-hai Plain (or southwest China) population was relatively large. The achieved diversity and evolution data, as well as the MLST technique, are helpful for epidemiological studies and guiding PaWB disease control decisions.

Multilocus sequence typing for Candida albicans strains from the intestinal tract of patients with cirrhosis.

Multilocus sequence typing (MLST) was applied to investigate the genetic diversity of Candida albicans in the intestinal tract of cirrhosis patients. We used CHROM agar Candida medium to obtain 105 Candida sp. isolates from fecal samples (276 subjects), including 63 isolates from the cirrhosis group (141 subjects) and 42 isolates from the healthy control group (135 subjects). Among the 105 Candida strains isolated, 60 strains were identified as Candida albicans. Patients with cirrhosis had significantly higher rates of colonization by Candida sp. (44.68% vs. 31.11%, p < 0.05) and C. albicans (27.66% vs. 15.56%, p < 0.05) relative to healthy controls. In the cirrhosis group, the rate of colonization further increased with disease progression and antibiotic treatment (p < 0.01). Sixty C. albicans isolates were analyzed by MLST. Fifty diploid sequence types (DST) were observed, and 26 new DSTs and 3 novel alleles were found. The majority of isolates were distributed among three clades, clade 8 (31.67%), clade 14 (15.00%) and clade 18 (21.67%). Among 39 strains from the cirrhosis group, 16 strains (41.02%) belonged to clade 8, while only 3 strains (14.29%) from healthy group belonged to clade 8 (p < 0.05). In addition, concatenated sequences of the 7 housekeeping gene fragments were analyzed for all the different DSTs in clade 8 to evaluate the loss of heterozygosity (LOH), which indicates C. albicans microvariation in the gut of cirrhosis patients. This study suggests that cirrhosis disease progression and antibiotic treatment is associated with increased colonization by Candida sp. and C. albicans. We are the first to provide MLST-based genotype profiles for C. albicans Guizhou China, and to identify clade 8 as the potential main clade of C. albicans colonization in the gut of cirrhosis patients.

Genetic structure and phylogeny of Italian and Czech populations of the cucurbit powdery mildew fungus Golovinomyces orontii inferred by multilocus sequence typing

Cucurbit powdery mildew caused by Golovinomyces orontii is a serious disease that affects cucurbit crops in temperate areas. In northern Italy, the species is responsible for the early infections at the beginning of the growing season. However, chasmothecia have never been recorded in Italy and the impact of either asexual or sexual reproduction of G. orontii remains to be determined. To investigate and compare the genetic structure of Italian populations with those from the Czech Republic, where chasmothecia are sporadically recorded, seven housekeeping gene fragments (tef‐1α, csI, ITS, H3, tub2, IGS and mtLSU) were amplified and used in a multilocus sequence typing (MLST) approach. The four‐gamete test was performed to detect recombination between and within populations. Phylogeny was inferred using both Bayesian and minimum evolution analyses. Results from MLST revealed the presence of 141 single nucleotide polymorphisms and the existence of 13 different haplotypes. Phylogenetic analysis revealed the existence of two main distinct phylogenetic groups that were also highly genetically different, confirming that G. orontii is a species complex. Recombination was detected within both the phylogenetic groups and the geographical populations, indicating that sexual reproduction could have occurred. However, considering the lower haplotype diversity and the high frequency of ST3 (17 isolates) and ST7 (eight isolates) haplotypes, the Italian population could be predominantly clonal, while sexual reproduction may occasionally occur due to the introduction of genotypes similar to those from the Czech Republic.

The evolution and population structure of Lactobacillus fermentum from different naturally fermented products as determined by multilocus sequence typing (MLST).

BackgroundLactobacillus fermentum is economically important in the production and preservation of fermented foods. A repeatable and discriminative typing method was devised to characterize L. fermentum at the molecular level. The multilocus sequence typing (MLST) scheme developed was based on analysis of the internal sequence of 11 housekeeping gene fragments (clpX, dnaA, dnaK, groEL, murC, murE, pepX, pyrG, recA, rpoB, and uvrC).ResultsMLST analysis of 203 isolates of L. fermentum from Mongolia and seven provinces/ autonomous regions in China identified 57 sequence types (ST), 27 of which were represented by only a single isolate, indicating high genetic diversity. Phylogenetic analyses based on the sequence of the 11 housekeeping gene fragments indicated that the L. fermentum isolates analyzed belonged to two major groups. A standardized index of association (IAS) indicated a weak clonal population structure in L. fermentum. Split decomposition analysis indicated that recombination played an important role in generating the genetic diversity observed in L. fermentum. The results from the minimum spanning tree strongly suggested that evolution of L. fermentum STs was not correlated with geography or food-type.ConclusionsThe MLST scheme developed will be valuable for further studies on the evolution and population structure of L. fermentum isolates used in food products.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0447-z) contains supplementary material, which is available to authorized users.

Genetic diversity analysis of the cucurbit powdery mildew fungus Podosphaera xanthii suggests a clonal population structure

The sexual stage of Podosphaera xanthii is rarely found worldwide. However, chasmothecia are frequently recorded in northern Italy, suggesting the presence of an actively mating population. With the aim of investigating the genetic structure of the Italian population with respect to populations from other countries, genetic diversity analysis was performed both on 92 isolates from European and American countries by multilocus sequence typing (MLST) and on 59 isolates by amplified fragment length polymorphism (AFLP) methods. Mating type frequencies were tested for random mating and two-locus linkage disequilibrium (LD) analysis was performed. Results showed very low levels of genetic diversity: MLST showed no variations in eight housekeeping gene fragments and, accordingly, UPGMA dendrogram from AFLP data showed a high similarity (0.91–1.00 simple matching similarity coefficient) between isolates. Moreover, the random mating test showed no deviations from mating-type 1:1 ratio in the Italian population but deviations were observed in populations from Europe and American countries while two-locus LD analysis showed the presence of significant LD. The results suggest that the populations of P. xanthii are likely to be predominantly clonal, and asexual reproduction, producing a huge amount of conidia, appears to be the predominant type of reproduction of the species.

A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3min.

Molecular Detection of &lt;i&gt;Clostridium&lt;/i&gt; Species in Beef Obtained from Retail Shops in North West Province, South Africa

<i>Clostridium</i> species cause diseases in humans that result from consumption of undercooked beef. The objectives of this study were to isolate <i>Clostridium</i> species from beef then the detection of the presence of <i>tpi</i> housekeeping gene as well as determination of the antibiotic resistant profile of the isolates. Twenty six (26) beef samples were bought from butcheries, supermarkets and street vendors. The samples were analyzed for the characteristics of <i>Clostridium</i> species and a total of 78 presumptive isolates were subjected to Gram-staining, catalase test, API 20A sugar fermentation profiles 16S rRNA and <i>tpi</i> species specific PCR analysis. Susceptibility profiles to 8 antibiotics were determined and antibiotic resistance patterns were compiled. Large proportions (93.3%-100%) of the isolates were penicillin, vancomycin and erythromycin resistant. PCR were performed to amplify species-specific 16S rRNA gene to confirm the identity of the isolates and 44.7% of the isolates were positively identified as <i>Clostridium</i> species. PCR were performed to amplify <i>tpi</i> housekeeping gene fragments. The <i>tpi</i> housekeeping gene produced amplicons of 501bp after PCR amplification and 19% of the isolates possess <i>tpi</i> housekeeping gene which confirmed the presence of <i>Clostridium</i> species in beef.

Using multilocus sequence typing to study bacterial variation: prospects in the genomic era.

Multilocus sequence typing (MLST) indexes the sequence variation present in a small number (usually seven) of housekeeping gene fragments located around the bacterial genome. Unique alleles at these loci are assigned arbitrary integer identifiers, which effectively summarizes the variation present in several thousand base pairs of genome sequence information as a series of numbers. Comparing bacterial isolates using allele-based methods efficiently corrects for the effects of lateral gene transfer present in many bacterial populations and is computationally efficient. This 'gene-by-gene' approach can be applied to larger collections of loci, such as the ribosomal protein genes used in ribosomal MLST (rMLST), up to and including the complete set of coding sequences present in a genome, whole-genome MLST (wgMLST), providing scalable, efficient and readily interpreted genome analysis.

Classification, identification, and clinical significance of Haemophilus and Aggregatibacter species with host specificity for humans.

The aim of this review is to provide a comprehensive update on the current classification and identification of Haemophilus and Aggregatibacter species with exclusive or predominant host specificity for humans. Haemophilus influenzae and some of the other Haemophilus species are commonly encountered in the clinical microbiology laboratory and demonstrate a wide range of pathogenicity, from life-threatening invasive disease to respiratory infections to a nonpathogenic, commensal lifestyle. New species of Haemophilus have been described (Haemophilus pittmaniae and Haemophilus sputorum), and the new genus Aggregatibacter was created to accommodate some former Haemophilus and Actinobacillus species (Aggregatibacter aphrophilus, Aggregatibacter segnis, and Aggregatibacter actinomycetemcomitans). Aggregatibacter species are now a dominant etiology of infective endocarditis caused by fastidious organisms (HACEK endocarditis), and A. aphrophilus has emerged as an important cause of brain abscesses. Correct identification of Haemophilus and Aggregatibacter species based on phenotypic characterization can be challenging. It has become clear that 15 to 20% of presumptive H. influenzae isolates from the respiratory tracts of healthy individuals do not belong to this species but represent nonhemolytic variants of Haemophilus haemolyticus. Due to the limited pathogenicity of H. haemolyticus, the proportion of misidentified strains may be lower in clinical samples, but even among invasive strains, a misidentification rate of 0.5 to 2% can be found. Several methods have been investigated for differentiation of H. influenzae from its less pathogenic relatives, but a simple method for reliable discrimination is not available. With the implementation of identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry, the more rarely encountered species of Haemophilus and Aggregatibacter will increasingly be identified in clinical microbiology practice. However, identification of some strains will still be problematic, necessitating DNA sequencing of multiple housekeeping gene fragments or full-length 16S rRNA genes.

Multilocus sequence typing scheme for the Mycobacterium abscessus complex

We developed a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus sensu lato, based on the partial sequencing of seven housekeeping genes: argH, cya, glpK, gnd, murC, pta and purH. This scheme was used to characterize a collection of 227 isolates recovered between 1994 and 2010 in France, Germany, Switzerland and Brazil. We identified 100 different sequence types (STs), which were distributed into three groups on the tree obtained by concatenating the sequences of the seven housekeeping gene fragments (3576bp): the M. abscessus sensu stricto group (44 STs), the “M. massiliense” group (31 STs) and the “M. bolletii” group (25 STs). SplitTree analysis showed a degree of intergroup lateral transfers. There was also evidence of lateral transfer events involving rpoB. The most prevalent STs in our collection were ST1 (CC5; 20 isolates) and ST23 (CC3; 31 isolates). Both STs were found in Europe and Brazil, and the latter was implicated in a large post-surgical procedure outbreak in Brazil. Respiratory isolates from patients with cystic fibrosis belonged to a large variety of STs; however, ST2 was predominant in this group of patients.Our MLST scheme, publicly available at www.pasteur.fr/mlst, offers investigators a valuable typing tool for M. abscessus sensu lato in future epidemiological studies throughout the world.

Development of a multilocus sequence typing scheme for Streptococcus gallolyticus

Streptococcus gallolyticus is often found as a member of the normal gut microflora in various animals. However, it has been reported to cause mastitis in cattle, septicaemia in pigeons, and meningitis, septicaemia and endocarditis in humans. However, little is known about the epidemiology and crucial virulence factors of S. gallolyticus. To help address these issues, we developed a multilocus sequence typing (MLST) scheme for S. gallolyticus. Seven housekeeping gene fragments were sequenced from each of 58 S. gallolyticus isolates collected from diverse origins and sources. The MLST scheme had good discriminatory ability. The 63 strains, including the 5 whole genome sequenced strains examined, resolved into 57 sequence types (STs), with 52 STs represented by only a single strain. With respect to the identification of S. gallolyticus subspecies (i.e. S. gallolyticus subsp. gallolyticus, S. gallolyticus subsp. pasteurianus and S. gallolyticus subsp. macedonicus), the results of biochemical tests and DNA-DNA hybridization were in high concordance with those of the MLST scheme. The MLST scheme developed in this study may be a useful tool capable of replacing the conventional methods used for S. gallolyticus subspecies identification. The results of this study suggest that the biology and virulence of two pathogenic S. gallolyticus subspecies (i.e. S. gallolyticus subsp. gallolyticus and S. gallolyticus subsp. pasteurianus) are very different. The MLST scheme offers researchers a valuable typing tool that will promote further investigation of the epidemiology of S. gallolyticus.

Multiple gene genealogical analyses of a nematophagous fungus Paecilomyces lilacinus from China

Paecilomyces lilacinus is a geographically widespread nematophagous fungus and a promising biological control agent against plant parasitic nematodes. However, relatively little is known about its patterns of genetic variation through its broad geographic and ecological contexts. In this study, we analyzed the genetic variation of 2 virulence-associated genes (PLS and PLC) and 4 housekeeping gene fragments (ITS, RPB1, RPB2, and β-tubulin) among 80 P. lilacinus specimens collected from 7 locations in China. Various degrees of polymorphism and haplotype diversity were observed among the six gene fragments. However, no genetic differentiation was observed among the geographic populations, consistent with extensive gene flow among these geographic populations of P. lilacinus in China. Our analysis also suggested that clonal reproduction was the predominant mode of reproduction in natural populations of P. lilacinus.

Multilocus sequence typing and further genetic characterization of the enigmatic pathogen, Staphylococcus hominis.

Staphylococcus hominis is a commensal resident of human skin and an opportunistic pathogen. The species is subdivided into two subspecies, S. hominis subsp. hominis and S. hominis subsp. novobiosepticus, which are difficult to distinguish. To investigate the evolution and epidemiology of S. hominis, a total of 108 isolates collected from 10 countries over 40 years were characterized by classical phenotypic methods and genetic methods. One nonsynonymous mutation in gyrB, scored with a novel SNP typing assay, had a perfect association with the novobiocin-resistant phenotype. A multilocus sequence typing (MLST) scheme was developed from six housekeeping gene fragments, and revealed relatively high levels of genetic diversity and a significant impact of recombination on S. hominis population structure. Among the 40 sequence types (STs) identified by MLST, three STs (ST2, ST16 and ST23) were S. hominis subsp. novobiosepticus, and they distinguished between isolates from different outbreaks, whereas 37 other STs were S. hominis subsp. hominis, one of which was widely disseminated (ST1). A modified PCR assay was developed to detect the presence of ccrAB4 from the SCCmec genetic element. S. hominis subsp. novobiosepticus isolates were oxacillin-resistant and carriers of specific components of SCCmec (mecA class A, ccrAB3, ccrAB4, ccrC), whereas S. hominis subsp. hominis included both oxacillin-sensitive and -resistant isolates and a more diverse array of SCCmec components. Surprisingly, phylogenetic analyses indicated that S. hominis subsp. novobiosepticus may be a polyphyletic and, hence, artificial taxon. In summary, these results revealed the genetic diversity of S. hominis, the identities of outbreak-causing clones, and the evolutionary relationships between subspecies and clones. The pathogenic lifestyle attributed to S. hominis subsp. novobiosepticus may have originated on more than one occasion.

Multilocus Sequence Analysis Provides Insights into Molecular Epidemiology of Chlamydia pecorum Infections in Australian Sheep, Cattle, and Koalas

Chlamydia pecorum is a significant pathogen of domestic livestock and wildlife. We have developed a C. pecorum-specific multilocus sequence analysis (MLSA) scheme to examine the genetic diversity of and relationships between Australian sheep, cattle, and koala isolates. An MLSA of seven concatenated housekeeping gene fragments was performed using 35 isolates, including 18 livestock isolates (11 Australian sheep, one Australian cow, and six U.S. livestock isolates) and 17 Australian koala isolates. Phylogenetic analyses showed that the koala isolates formed a distinct clade, with limited clustering with C. pecorum isolates from Australian sheep. We identified 11 MLSA sequence types (STs) among Australian C. pecorum isolates, 10 of them novel, with koala and sheep sharing at least one identical ST (designated ST2013Aa). ST23, previously identified in global C. pecorum livestock isolates, was observed here in a subset of Australian bovine and sheep isolates. Most notably, ST23 was found in association with multiple disease states and hosts, providing insights into the transmission of this pathogen between livestock hosts. The complexity of the epidemiology of this disease was further highlighted by the observation that at least two examples of sheep were infected with different C. pecorum STs in the eyes and gastrointestinal tract. We have demonstrated the feasibility of our MLSA scheme for understanding the host relationship that exists between Australian C. pecorum strains and provide the first molecular epidemiological data on infections in Australian livestock hosts.

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.

Identification of haemolytic Haemophilus species isolated from human clinical specimens and description of Haemophilus sputorum sp. nov

Haemolytic Haemophilus strains with no requirement for X factor are regularly isolated from sputum and throat swabs and occasionally from invasive infections, but the classification of such strains is not clear. We characterized 56 strains with a phenotype concordant with Haemophilus parahaemolyticus (V, but not X factor-dependent; urease-positive; tryptophanase-negative; ornithine decarboxylase-negative) by extended phenotypic testing and 16S rRNA gene sequencing. In addition, 31 of the strains and representative type strains were investigated by multilocus sequence analysis based on 3 housekeeping gene fragments. Most strains could be assigned to H. parahaemolyticus and were characterized by expression of IgA1 protease and a negative test for β-galactosidase. Isolation of H. parahaemolyticus from various infections and its absence among more than 300 commensal Haemophilus isolates suggests a pathogenic potential of this organism. The majority of haemolytic strains with β-galactosidase activity did not cluster with the type strain of H. paraphrohaemolyticus, but constituted a distinct and coherent novel taxon. Ten strains of this new taxon proved to be genetically and phenotypically homogeneous. Few biochemical characters discriminate the new taxon from related Haemophilus species, but identification is easily accomplished by routine matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Genetic, biochemical, and spectrometry data show that the taxon merits recognition as a novel species of Haemophilus. The name Haemophilus sputorum is proposed, with CCUG 13788T (=DSM 24472T=NCTC 13537T) as the type strain.

Multilocus Sequence Typing of Pseudomonas syringae Sensu Lato Confirms Previously Described Genomospecies and Permits Rapid Identification of P. syringae pv. coriandricola and P. syringae pv. apii Causing Bacterial Leaf Spot on Parsley

Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.

A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacteriumPasteuria penetrans

To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans. An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.