Ribotype Groups Research Articles

Genetic Diversity of the Species of the Genus Deschampsia P.Beauv. (Poaceae) Based on the Analysis of the ITS Region: Polymorphism Proves Distant Hybridization

The species of the genus Deschampsia are difficult for identification, and the genus is difficult for taxonomic treatment. The regions of 35S rRNA genes were studied for the species of the genus Deschampsia of different geographical origin with a method of sequencing by Sanger (ITS1–5.8S rRNA gene–ITS2, 14 species) and with a method of a locus-specific next-generation sequencing (NGS) on the Illumina platform (ITS1–5.8S rRNA, 7 species). All species of Deschampsia formed one clade; the species, referred by some authors on the basis of morphological characters to the species D. cespitosa s.l., entered one subclade. Subantarctic species formed a separate subclade and their ribotypes formed their own subnetwork. Avenella flexuosa, earlier referred to Deschampsia, entered the other clade, though this species contains some ribotypes common with some Deschampsia species. Deschampsia pamirica and related mountain species have their own specific ribotype groups. On the network of the ribotypes, one can see that D. cespitosa from Great Britain forms a network with some species, but D. cespitosa from the USA forms its own network. Ribotype analysis of each sample revealed traces of introgression with Deyeuxia/Calamagrostis in D. cespitosa and with A. flexuosa and probable introgression of Northern and subantarctic species.

Operculina and Neoassilina: A Revision of Recent Nummulitid Genera Based on Molecular and Morphological Data Reveals a New Genus

The genus Operculina, a large symbiont-bearing benthic foraminifer, is characterized by high morphological variability showing thick involute to intermediate semi-involute to flat evolute tests. Different morphotypes are either considered as ecophenotypes or distinct species. In order to test the hypothesis of ecophenotypes versus different species, a single cell high throughput sequencing approach was applied to assess the interspecific diversity of Operculina. This results in two groups of ribotypes, one corresponding to Operculina ammonoides/Operculina discoidalis, the other containing Operculina complanata/Operculina elegans. These groups can also be separated morphologically. Therefore, O. complanata and O. elegans represent a single species and the latter can be regarded as a junior synonym of O. complanata. Operculina ammonoides and O. discoidalis also form a single species, which makes the latter a junior synonym of O. ammonoides. Because generic differences in Operculina species are manifested in morphology and molecular genetics, the genus Neoassilina with the designated species Neoassilina ammonoides is installed. Additional analysis of ribosomal SSU rDNA data of eight recent nummulitid genera confirms the obtained high troughput sequencing results and further shows that Palaeonummulites venosus builds a clade with O. complanata that branches at the base of other Nummulitidae containing Planostegina, Planoperculina, Cycloclypeus, Heterostegina, Operculinella and Neoassilina.

Harmful algal blooms and their effects in coastal seas of Northern Europe

Harmful algal blooms (HAB) are recurrent phenomena in northern Europe along the coasts of the Baltic Sea, Kattegat-Skagerrak, eastern North Sea, Norwegian Sea and the Barents Sea. These HABs have caused occasional massive losses for the aquaculture industry and have chronically affected socioeconomic interests in several ways. This status review gives an overview of historical HAB events and summarises reports to the Harmful Algae Event Database from 1986 to the end of year 2019 and observations made in long term monitoring programmes of potentially harmful phytoplankton and of phycotoxins in bivalve shellfish. Major HAB taxa causing fish mortalities in the region include blooms of the prymnesiophyte Chrysochromulina leadbeateri in northern Norway in 1991 and 2019, resulting in huge economic losses for fish farmers. A bloom of the prymesiophyte Prymnesium polylepis (syn. Chrysochromulina polylepis) in the Kattegat-Skagerrak in 1988 was ecosystem disruptive. Blooms of the prymnesiophyte Phaeocystis spp. have caused accumulations of foam on beaches in the southwestern North Sea and Wadden Sea coasts and shellfish mortality has been linked to their occurrence. Mortality of shellfish linked to HAB events has been observed in estuarine waters associated with influx of water from the southern North Sea. The first bloom of the dictyochophyte genus Pseudochattonella was observed in 1998, and since then such blooms have been observed in high cell densities in spring causing fish mortalities some years. Dinoflagellates, primarily Dinophysis spp., intermittently yield concentrations of Diarrhetic Shellfish Toxins (DST) in blue mussels, Mytilus edulis, above regulatory limits along the coasts of Norway, Denmark and the Swedish west coast. On average, DST levels in shellfish have decreased along the Swedish and Norwegian Skagerrak coasts since approximately 2006, coinciding with a decrease in the cell abundance of D. acuta. Among dinoflagellates, Alexandrium species are the major source of Paralytic Shellfish Toxins (PST) in the region. PST concentrations above regulatory levels were rare in the Skagerrak-Kattegat during the three decadal review period, but frequent and often abundant findings of Alexandrium resting cysts in surface sediments indicate a high potential risk for blooms. PST levels often above regulatory limits along the west coast of Norway are associated with A. catenella (ribotype Group 1) as the main toxin producer. Other Alexandrium species, such as A. ostenfeldii and A. minutum, are capable of producing PST among some populations but are usually not associated with PSP events in the region. The cell abundance of A. pseudogonyaulax, a producer of the ichthyotoxin goniodomin (GD), has increased in the Skagerrak-Kattegat since 2010, and may constitute an emerging threat. The dinoflagellate Azadinium spp. have been unequivocally linked to the presence of azaspiracid toxins (AZT) responsible for Azaspiracid Shellfish Poisoning (AZP) in northern Europe. These toxins were detected in bivalve shellfish at concentrations above regulatory limits for the first time in Norway in blue mussels in 2005 and in Sweden in blue mussels and oysters (Ostrea edulis and Crassostrea gigas) in 2018. Certain members of the diatom genus Pseudo-nitzschia produce the neurotoxin domoic acid and analogs known as Amnesic Shellfish Toxins (AST). Blooms of Pseudo-nitzschia were common in the North Sea and the Skagerrak-Kattegat, but levels of AST in bivalve shellfish were rarely above regulatory limits during the review period. Summer cyanobacteria blooms in the Baltic Sea are a concern mainly for tourism by causing massive fouling of bathing water and beaches. Some of the cyanobacteria produce toxins, e.g. Nodularia spumigena, producer of nodularin, which may be a human health problem and cause occasional dog mortalities. Coastal and shelf sea regions in northern Europe provide a key supply of seafood, socioeconomic well-being and ecosystem services. Increasing anthropogenic influence and climate change create environmental stressors causing shifts in the biogeography and intensity of HABs. Continued monitoring of HAB and phycotoxins and the operation of historical databases such as HAEDAT provide not only an ongoing status report but also provide a way to interpret causes and mechanisms of HABs.

Intragenomic Polymorphism of the ITS 1 Region of 35S rRNA Gene in the Group of Grasses with Two-Chromosome Species: Different Genome Composition in Closely Related Zingeria Species

Zingeria (Poaceae) is a small genus that includes Z. biebersteiniana, a diploid species with the lowest chromosome number known in plants (2n = 4) as well as hexaploid Z. kochii and tetraploid Z. pisidica, and/or Z. trichopoda species. The relationship between these species and the other low-chromosomes species Colpodium versicolor are unclear. To explore the intragenomic polymorphism and genome composition of these species we examined the sequences of the internal transcribed spacer 1 of the 35S rRNA gene via NGS approach. Our study revealed six groups of ribotypes in Zingeria species. Their distribution confirmed the allopolyploid nature of Z. kochii, whose probable ancestors were Colpodium versicolor and Z. pisidica. Z. pisidica has 98% of rDNA characteristic only for this species, and about 0.3% of rDNA related to that of Z. biebersteiniana. We assume that hexaploid Z. kochii is either an old allopolyploid or a homodiploid that has lost most of the rRNA genes obtained from Z. biebersteiniana. In Z. trichopoda about 81% of rDNA is related to rDNA of Z. biebersteiniana and 19% of rDNA is derived from Poa diaphora sensu lato. The composition of the ribotypes of the two plants determined by a taxonomy specialist as Z. pisidica and Z. trichopoda is very different. Two singleton species are proposed on this base with ribotypes as discriminative characters. So, in all four studied Zingeria species, even if the morphological difference among the studied species was modest, the genomic constitution was significantly different, which suggests that these are allopolyploids that obtained genomes from different ancestors.

ITS1–5.8S rDNA–ITS2 and trnL-trnF Sequences as Markers for the Study of Species Diversity of Altai Feather Grasses

The ITS1–5.8S rDNA–ITS2 sequence of the 35S rRNA genes of 16 species of feather grasses and 2 species of false needlegrasses of the flora of the Altai Republic and Altai krai (Stipabaicalensis, S. borysthenica, S. capillata, S. consanguinea, S. dasyphylla, S. desertorum, S. glareosa, S. grandis, S. korshinskyi, S. krylovii, S. lessingiana, S. orientalis,S. pennata, S. praecapillata, S. pulcherrima, S. zalesskii, Ptilagrostisjunatovii, and P. mongholica), as well as four feather grass species from other regions of Russia (S. pontica, S. rubens, S. tirsa, and S. ucrainica), was sequenced. The trnL-trnF chloroplast sequences of S. capillata, S. borysthenica, S. glareosa, S. krylovii, S. lessingiana, S. orientalis, S. pulcherrima, and S. zalesskii were also determined. The trnL-trnF region, as well as the 5.8S rDNA, is highly conserved in feather grasses and cannot be used to differentiate sections and species within the genus. The ITS1 and ITS2 sequences, on the contrary, have some parsimony-informative substitutions and indels, which makes it possible to identify groups of ribotypes corresponding to sections and individual species within some sections. The clades corresponding to the Stipa and Leiostipa sections were separated; the species of the Barbatae and Subbarbatae sections composed the same clade, while the section Hemibarbatae turned out to be polyphyletic. Only in the section Stipa were no species-specific nucleotide substitutions and indels revealed. The isolation of the species S. desertorum, the taxonomic rank of which was previously unclear, was shown. The origin of polymorphic sites in the ITS sequences of feather grasses is discussed as possible evidence of distant hybridization.

Plankton Multiproxy Analyses in the Northern Patagonian Shelf, Argentina: Community Structure, Phycotoxins, and Characterization of Toxic Alexandrium Strains

The extensive Argentine continental shelf supports high plankton productivity and fish catches. In particular, El Rincon coastal area and the adjacent shelf fronts (38.5–42o S, 58.5–62o W) comprise diverse habitats and hold species of economic and ecological value. So far, studies of the microbial community at the base of the food web remain scarce. Here we describe the early spring plankton (>5-200 µm) structure in terms of abundance, biomass, species composition, functional groups and phycotoxin profiles in surface waters of El Rincon in September 2015. Diatoms were the most abundant and the largest contributors to carbon biomass at most stations. They dominated in coastal and inner-shelf (depths 20 µm) heterotrophic protists, e.g. various ciliates and dinoflagellates species were more abundant offshore. Scanning of phycotoxins in the field disclosed that paralytic shellfish poisoning (PSP) toxins were dominated by gonyautoxins-1/4 (GTX1/4). Lipophilic toxins were detected in low abundance, e.g. domoic acid (DA), although a bloom of Pseudo-nitzschia spp. (up to 3.6 x 105 cells L-1) was detected at inner-shelf stations. Pectenotoxin-2 (PTX-2) and 13-desmethyl spirolide C (SPX-1) were the most abundant in the field. PTX-2 co-occurred with Dinophysis spp., mainly D. tripos, while SPX-1 dominated at middle-shelf stations, where cells of Alexandrium catenella (1 strain) and A. ostenfeldii (3 strains) were isolated. The quantitative PSP profiles of the Alexandrium strains differed significantly from the field profiles. Moreover, the three A. ostenfeldii strains proved to be PSP producers and additionally produced 5 novel spirolides. Phylogenetic analyses of these first strains from the South Atlantic disclosed a new ribotype group suggesting a biogeographical distinction of the population. The plankton survey presented here contributes with baseline knowledge to evaluate potential ecosystem changes and to track the global distribution of toxigenic species.

As Clear as Mud? Determining the Diversity and Prevalence of Prophages in the Draft Genomes of Estuarine Isolates of Clostridium difficile.

The bacterium Clostridium difficile is a significant cause of nosocomial infections worldwide. The pathogenic success of this organism can be attributed to its flexible genome which is characterized by the exchange of mobile genetic elements, and by ongoing genome evolution. Despite its pathogenic status, C. difficile can also be carried asymptomatically, and has been isolated from natural environments such as water and sediments where multiple strain types (ribotypes) are found in close proximity. These include ribotypes which are associated with disease, as well as those that are less commonly isolated from patients. Little is known about the genomic content of strains in such reservoirs in the natural environment. In this study, draft genomes have been generated for 13 C. difficile isolates from estuarine sediments including clinically relevant and environmental associated types. To identify the genetic diversity within this strain collection, whole-genome comparisons were performed using the assemblies. The strains are highly genetically diverse with regards to the C. difficile “mobilome,” which includes transposons and prophage elements. We identified a novel transposon-like element in two R078 isolates. Multiple, related and unrelated, prophages were detected in isolates across ribotype groups, including two novel prophage elements and those related to the transducing phage φC2. The susceptibility of these isolates to lytic phage infection was tested using a panel of characterized phages found from the same locality. In conclusion, estuarine sediments are a source of genetically diverse C. difficile strains with a complex network of prophages, which could contribute to the emergence of new strains in clinics.

Disentangling relationships among the diploid members of the intricate genus Knautia (Caprifoliaceae, Dipsacoideae)

The genus Knautia (Caprifoliaceae, Dipsacoideae) encompasses 40–60 species mainly distributed in western Eurasia, with highest species diversity in the Alps and the Balkan Peninsula. It is traditionally regarded as one of the taxonomically most challenging European genera due to the widespread occurrence of polyploidy, the high incidence of hybridisation and the maintenance of morphologically intermediate forms. A prerequisite for assessing the complex spatiotemporal diversification of a polyploid group is a comprehensive hypothesis of the phylogenetic relationships among its diploid members. To this end, DNA sequence data (nrDNA ITS and plastid petN(ycf6)-psbM) combined with AFLP fingerprinting were performed on 148 diploid populations belonging to 35 taxa. Phylogenies obtained by maximum parsimony and Bayesian analyses were used to test the monophyly of the genus and its three sections Trichera, Tricheroides and Knautia, to provide insights into its evolutionary history and to test previous hypotheses of inter- and intrasectional classification. Both nuclear and chloroplast datasets support the monophyly of Knautia and its three sections, with ambiguous placement of K. cf. degenii. The majority of species belong to the nearly exclusively perennial section Trichera (x=10). Within section Trichera all markers revealed largely unresolved phylogenetic relationships suggesting rapid radiation and recent range expansion. In addition, extensive sharing of plastid haplotypes across taxa and wide geographic ranges of plastid haplotypes and ribotype groups were observed. The molecular data are partly at odds with the traditional informal grouping of taxa within section Trichera. Whereas the traditional groups of K. dinarica, K. drymeia and K. montana can be maintained, the new, smaller and well supported Midzorensis and Pancicii Groups as well as the SW European Group are separated from the heterogeneous traditional K. longifolia group. The former groups of K. arvensis, K. dalmatica, K. fleischmannii and K. velutina are clearly polyphyletic. Their diploid members have to be rearranged into the Xerophytic Group, the Carinthiaca Group, and the Northern and Southern Arvensis Groups. The annual sections Tricheroides (x=10) and Knautia (x=8) with only a few taxa are resolved in the ITS and plastid trees on long branches as early diverging lineages within the genus.

Sequence Similarity of Clostridium difficile Strains by Analysis of Conserved Genes and Genome Content Is Reflected by Their Ribotype Affiliation

PCR-ribotyping is a broadly used method for the classification of isolates of Clostridium difficile, an emerging intestinal pathogen, causing infections with increased disease severity and incidence in several European and North American countries. We have now carried out clustering analysis with selected genes of numerous C. difficile strains as well as gene content comparisons of their genomes in order to broaden our view of the relatedness of strains assigned to different ribotypes. We analyzed the genomic content of 48 C. difficile strains representing 21 different ribotypes. The calculation of distance matrix-based dendrograms using the neighbor joining method for 14 conserved genes (standard phylogenetic marker genes) from the genomes of the C. difficile strains demonstrated that the genes from strains with the same ribotype generally clustered together. Further, certain ribotypes always clustered together and formed ribotype groups, i.e. ribotypes 078, 033 and 126, as well as ribotypes 002 and 017, indicating their relatedness. Comparisons of the gene contents of the genomes of ribotypes that clustered according to the conserved gene analysis revealed that the number of common genes of the ribotypes belonging to each of these three ribotype groups were very similar for the 078/033/126 group (at most 69 specific genes between the different strains with the same ribotype) but less similar for the 002/017 group (86 genes difference). It appears that the ribotype is indicative not only of a specific pattern of the amplified 16S–23S rRNA intergenic spacer but also reflects specific differences in the nucleotide sequences of the conserved genes studied here. It can be anticipated that the sequence deviations of more genes of C. difficile strains are correlated with their PCR-ribotype. In conclusion, the results of this study corroborate and extend the concept of clonal C. difficile lineages, which correlate with ribotypes affiliation.

Molecular evidence for reticulate speciation in Astragalus (Fabaceae) as revealed by a case study from sect. Dissitiflori

Although hybridization has long been recognized as a major force driving speciation in land plants, it has not yet been evidenced in Astragalus, the largest angiosperm genus. Here, we reveal the possible contribution of hybridization to speciation in Astragalus by employing cloning of the nrDNA ITS region and sampling three plastid regions (ycf1, ndhF–rpl32, and rpl32–trnL) in taxa belonging to sect. Dissitiflori. Phylogenetic network and tree analyses uncovered various levels of intra-individual and intraspecific polymorphism of ITS in most of the taxa investigated. Two distantly related ribotype groups were found to be shared by the closely related polyploids Astragalus pallescens M.Bieb., Astragalus peterfii Jáv., and Astragalus pseudoglaucus Klokov suggesting ancient hybridization followed by incomplete lineage sorting (i.e., shared ancestral polymorphism) in nrDNA ITS. Reticulation is also invoked as an underlying evolutionary process behind the statistically highly supported incongruent placement of A. pseudoglaucus and Astragalus vesicarius subsp. pastellianus (Pollini) Arcang. in nuclear versus plastid phylogenies. The phylogenetic results also shed light on taxonomic controversies in the section, such as the false synonimization of A. peterfii under A. vesicarius s.l. Our results provide evidence for the (at least past) existence of speciation processes driven by hybridization in Astragalus.

Germination efficiency of clinical Clostridium difficile spores and correlation with ribotype, disease severity and therapy failure

Spore germination is an important part of the pathogenesis of Clostridium difficile infection (CDI). Spores are resistant to antibiotics, including those therapeutically administered for CDI and strains with a high germination rate are significantly more likely to be implicated in recurrent CDI. The role of germination efficiency in cases of refractory CDI where first-line therapy fails remains unclear. We investigated spore germination efficiencies of clinical C. difficile isolates by measuring drop in OD600 and colony forming efficiency. Ribotype 027 isolates exhibited significantly higher germination efficiencies in the presence of 0.1 % (w/v) sodium taurocholate (51.66 ± 8.75 %; 95 % confidence interval (CI) 47.37-55.95 %) than ribotype 106 (41.91 ± 8.35 %; 95 % CI 37.82-46 %) (P<0.05) and ribotype 078 (42.07 ± 8.57 %, 95 % CI 37.22-46.92 %) (P<0.05). Spore outgrowth rates were comparable between the ribotype groups but the exponential phase occurred approximately 4 h later in the absence of sodium taurocholate. Spore germination efficiencies for isolates implicated in severe CDI were significantly higher (49.68 ± 10.00 %, 95 % CI 47.06-52.30 %) than non-severe CDI (40.92 ± 9.29 %, 95 % CI 37.48-44.36 %); P<0.01. Germination efficiencies were also significantly higher in recurrent CDI or when metronidazole therapy failed than when therapy was successful [(49.00 ± 10.49 %, 95 % CI 46.25-51.75 %) versus (41.42 ± 9.43 %, 95 % CI 37.93-44.91 %); P<0.01]. This study suggests an important link between C. difficile spore germination, CDI pathogenesis and response to treatment; however, further work is warranted before the complex interplay between germination dynamics and CDI outcome can be fully understood.

Carriage of Clostridium difficile in outpatients with irritable bowel syndrome

Irritable bowel syndrome (IBS) is a common, typically chronic and sometimes disabling gastrointestinal condition of uncertain aetiology. Recently, a variety of links to gastrointestinal infections have been described including the onset of IBS following exposure to enteric pathogens and an apparent predisposition to gastrointestinal infection. The prevalence of Clostridium difficile in a population of IBS outpatients (n = 87) in the absence of established risk factors for the acquisition of C. difficile infection was examined. Overall, 5.7 % of patients (n = 5) carried culturable C. difficile and 4.6 % (n = 4) of isolates were toxigenic, belonging to toxinotype group 0, compared with 1.1 % (n = 1) for the healthy control group (n = 88). These isolates were members of toxigenic PCR ribotype groups 005 and 050 (IBS group) and 062 (control group) and were identified further as three individual strains by PFGE. Although no significant difference was observed between IBS patients and healthy volunteers, these findings support the concept that a subpopulation of IBS patients may be susceptible to gastrointestinal infection.

Emergence of fluoroquinolone‐resistant clonal group A: clonal analysis of Norwegian and Russian E. coli isolates

We describe a study of urinary tract and intestinal isolates of Escherichia coli from Norway and Russia using automated ribotyping, single nucleotide polymorphism analysis for clonal group A (CgA) supplemented with phylogrouping, virulence gene profiling and resistance profiling. CgA comprised 19% of the Norwegian UTI isolates from 2001. Two highly multiresistant fluoroquinolone-resistant CgA isolates were found. Ribotypes clustered into four major and six minor groups (ribogroups). Fluoroquinolone-resistant isolates and phylogroups A and B1 were associated with ribogroup (R)A. Ribogroup (R)B predominated among Russian UTI isolates and was predominantly phylogroup A and depleted in P-fimbriae. Ribogroup (R)C predominated among Norwegian UTI isolates and was rich in virulence factors (S-fimbriae, haemagglutinin and haemolysin) and predominantly phylogroup B2 and D. Ribogroup (R)G was associated with CgA and predominantly phylogroup D. Ribogroups (R)D, (R)E and (R)F had too few members for statistical analysis. The correlation between ribotype and phylogenetic group was not as strong as reported in other studies.

Genetic Diversity of Antifungi-Producing Rhizobacteria of Pseudomonas sp. Isolated from Rhizosphere of Soybean Plant

Antifungi-producing rhizobacteria have been recognized playing an important role in plant disease suppression. In our laboratory, 13 indigenous soybeans' rhizobacteria Pseudomonas sp. that showed strong growth inhibition of root pathogenic fungi, Rhizoctonia solani , Fusarium oxysporum and Sclerotium rolfsii, have been isolated from rhizosphere of soybean plant. For further understanding, the genetic diversity of the antifungi-producing Pseudomonas sp. was investigated using Amplified 16S rDNA Restriction Analysis (ARDRA) and 16S rRNA gene sequences analysis. 16S rDNA were amplified by PCR technique and digested with restriction endonuclease HaeIII, RsaI and AluI. Sequences of 16S rRNA gene were analyzed using the BLAST program for similarity searches on sequence databases. ARDRA based dendrogram analysis was carried out by neighbor-joining of TREECON 1.3b software package. ARDRA indicated the variability of Pseudomonas sp. based on the digestion sites. Dendrogram clustering analysis based on the restriction enzymes profile of the amplified rDNA distinguished Pseudomonas sp. into 7 ribotype groups. The sequences of 16S rRNA gene confirmed that the isolates belonging to Pseudomonas sp. and the phylogenetic tree formed 4 clusters. There was a quite overlap among ARDRA groups and 16S rRNA sequence clusters. This finding suggested that antifungal producing Pseudomonas sp. were present in the rhizosphere of soybean plant and the level of genetic diversity exist within these species. Sequence analysis of the 16S rRNA gene of the Pseudomonas sp. with an identical ARDRA pattern confirmed that members of an ARDRA group were closely related to each other.

Molecular fingerprinting of some clinically significant Nocardia and related strains by restriction polymorphism ribosomal RNA analyses

Fifty-three Nocardia strains were the subject of a restriction polymorphic ribosomal RNA analysis (ribotyping) designed to distinguish between representatives of clinically significant species and related strains. The organisms were assigned to 19 groups using a combination of EcoRV gene restriction endonuclease patterns and a digoxigenin-labelled Streptomyces violaceoruber TK21 rDNA probe. Each ribotype group contained 4 to 13 restriction fragments that ranged in size from 20.7 to 0.9 kb. The N. brasiliensis, N. crassostreae, N. farcinica, N. otitidiscaviarum, and N. seriolea strains showed distinct ribotype patterns. Unique banding patterns were also seen for the type strains of N. brevicatena, N. carnea, N. salmonicida, N. uniformis, and N. vaccinii, and for the single representatives of "N. fusca", "N. pseudosporangifera", and "N. violaceofusca". More than one banding pattern was detected for the N. asteroides, N. flavorosea, N. nova, N. pseudobrasiliensis, and N. transvalensis strains. The results are in line with current trends in nocardial systematics thereby indicating that restriction polymorphism ribosomal RNA analyses provide valuable data for the classification and identification of novel and pathogenic nocardiae at the species level.

The Vexed Relationship Between Clostridium Difficile and Inflammatory Bowel Disease: An Assessment of Carriage in an Outpatient Setting Among Patients in Remission

Comorbidity with Clostridium difficile may cause diagnostic delay in newly presenting inflammatory bowel disease (IBD) patients, trigger relapse in established disease, confound therapies, and serve as an indicator of an underlying defect in innate immunity. Retrospective analyses have suggested community acquisition; to address this we conducted a prospective analysis of C. difficile carriage in IBD patients using molecular methods specifically in an outpatient setting. Recruited participants had long-standing diagnoses of ulcerative colitis (n = 64) and Crohn's disease (n = 58), were in clinical remission, and had no recent exposure to antibiotics, corticosteroids, immunomodulatory drugs or recent hospitalization. Isolates were cultured from stools and confirmed by 16S sequencing. The antibiotic susceptibilities of the isolates were tested followed by further strain characterization by toxinotyping, ribotyping, and pulsed-field gel electrophoresis (PFGE). The frequency of toxigenic C. difficile was higher in IBD patients than in healthy volunteers at 8.2 and 1.0%, respectively (P = 0.02 Fisher's exact test). All strains belonged to toxinotype 0 with rare subtypes of this group noted in five isolates and represented by an altered repressor genotype. Patients harbored a diverse range of toxigenic ribotype groups, including those previously associated with C. difficile-associated disease (CDAD) (R015, R005, and R020) and the rarer types R062, R050, and R003. Interestingly, common nosocomial groups were not identified. The considerable nonclonal distribution of distinct strains was further demonstrated by PFGE genomic fingerprinting. None of the study subjects experienced a clinical episode of CDAD during a 6-month period of follow-up. Detection of C. difficile is increased in IBD outpatients in remission, and strain diversity is consistent with community acquisition from a multitude of sources.

Sequence and phylogenetic analysis of the gene for surface layer protein, slpA, from 14 PCR ribotypes of Clostridium difficile

Clostridium difficile is the commonest cause of antibiotic-associated diarrhoea, with the hospitalized elderly being at particular risk. The organism makes a crystalline surface protein layer (S-layer), encoded by the slpA gene, the product of which is cleaved to give two mature peptides which associate to form the layer. The larger peptide (high molecular weight; HMW), derived from the C-terminal portion of the precursor, is relatively conserved, whereas the smaller peptide (low molecular weight; LMW), derived from the N-terminal portion of the precursor, is a dominant antigen which substantially forms the basis for serotyping of isolates. PCR ribotyping is a more discriminatory typing method, based on the intergenic rRNA. We obtained the sequence for slpA and some flanking DNA from a collection of C. difficile strains of 14 ribotypes isolated from elderly patients. Sequences from different ribotypes were compared with one another and with published sequences. Sequences from C. difficile ribotypes 046 and 092 were identical. Sequences from ribotype pairs 005 and 054, 012 and 046/092, 014 and 066 and 031 and 094 differed by 1-3 nt in the slpA gene. There were ultimately nine ribotypes or groups of ribotypes with very different slpA sequences, particularly in the region encoding the LMW peptide. The sequence from ribotype 002 was very different from previously published sequences. The DNA segment sequenced included the 5' 315 bp of a secA homologue, encoding a putative transport protein required for peptide secretion across the plasma membrane. The amino acid sequences of the predicted HMW peptides were aligned and a neighbour-joining tree was produced using 10,000 bootstrap replicates. The predicted SecA N-terminal region was similarly analysed. For both SlpA and SecA, a strong association was found between ribotypes 012, 046/092, 017, 031 and 094. Ribotypes 001 and 078 formed part of this clade for SlpA but not SecA, indicating independent evolution for slpA and secA, presumably because they come under different selection pressures.

The use of ribotyping and antibiotic resistance patterns for identification of host sources of Escherichia coli strains

To compare antibiotic resistance and ribotyping patterns ability to identify triplicate isolates sent from a group of 40 Escherichia coli taken from seven host sources. Of the 120 isolates, 22 isolates were resistant to ampicillin, streptomycin, tetracycline and trimethoprim and 98 isolates were susceptible. Antibiotic patterns identified 33 of the triplicates and three of the six groups had isolates from multiple hosts. Ribotyping divided the isolates into 27 ribotype groups with all triplicates grouped into the same ribotype group with one host per group. Antibiotic susceptibility pattern placed 98 of the isolates in a single group with 50% of the antibiotic susceptibility pattern groups containing multiple host species. Ribotyping groups were host specific with each host having one to seven ribotype groups. Antibiotic susceptibility pattern groups have been used for environmental source identification and faecal pollution tracking, however these groups do not always distinguish between host species. Stability of the markers is a potential concern and this system can only be used if antibiotic resistance levels are high in the isolates studied. All isolates have a ribotype group which was stable and like other molecular methods has advantages over antibiotic susceptibility pattern groups which uses a phenotypic method.

Phenotypic and genetic data supporting reclassification of Butyrivibrio fibrisolvens isolates.

Among 55 Butyrivibrio fibrisolvens strains five ribotypes of B. fibrisolvens were described on the basis of RFLP profiles of 16S rDNA regions obtained with restriction endonuclease HaeIII. In the phylogenetic tree, these ribotypes were located in the XIVa cluster of Gram-positive bacteria. Phenotypic differences of selected ribotype groups became the basis for further reclassification of B. fibrisolvens.

Prevalence of Lyme disease spirochetes in Ixodes persulcatus and wild rodents in far eastern Russia.

Borrelia spirochetes were isolated from the adult ixodid tick (Ixodes persulcatus) in three areas of far eastern Russia, namely, Khabarovsk, Vladivostok, and Yuzhno-Sakhalinsk. Borrelia infective rates of ticks in those areas were 24.5, 41.4, and 25.1%, respectively (total rate was 26.6%). Spirochetes were also isolated from the tissues of small mammals captured at Khabarovsk (infective rate was 20.8%). Samples were classified by rRNA gene restriction fragment length polymorphism (RFLP) analysis. The isolated spirochetes from ticks were classified mainly RFLP ribotype group IV (B. garinii), followed by groups II (B. garinii), III (B. afzelii), and V (B. garinii), showing that B. garinii is a dominant species among them. Both B. garinii and B. afzelii were also found in rodents, and multiple infections with those two species were observed in some rodents. B. burgdorferi sensu stricto (group I) was not isolated from either ticks or rodents.