Clostridium sordellii genome analysis reveals plasmid localized toxin genes encoded within pathogenicity loci.

BACKGROUND: Clostridium sordellii can cause severe infections in animals and humans, the latter associated with trauma, toxic shock and often-fatal gynaecological infections. Strains can produce two large clostridial cytotoxins (LCCs), TcsL and TcsH, related to those produced by Clostridium difficile, Clostridium novyi and Clostridium perfringens, but the genetic basis of toxin production remains uncharacterised. RESULTS: Phylogenetic analysis of the genome sequences of 44 strains isolated from human and animal infections in the UK, US and Australia placed the species into four clades. Although all strains originated from animal or clinical disease, only 5 strains contained LCC genes: 4 strains contain tcsL alone and one strain contains tcsL and tcsH. Four toxin-positive strains were found within one clade. Where present, tcsL and tcsH were localised in a pathogenicity locus, similar to but distinct from that present in C. difficile. In contrast to C. difficile, where the LCCs are chromosomally localised, the C. sordellii tcsL and tcsH genes are localised on plasmids. Our data suggest gain and loss of entire toxigenic plasmids in addition to horizontal transfer of the pathogenicity locus. A high quality, annotated sequence of ATCC9714 reveals many putative virulence factors including neuraminidase, phospholipase C and the cholesterol-dependent cytolysin sordellilysin that are highly conserved between all strains studied. CONCLUSIONS: Genome analysis of C. sordellii reveals that the LCCs, the major virulence factors, are localised on plasmids. Many strains do not contain the LCC genes; it is probable that in several of these cases the plasmid has been lost upon laboratory subculture. Our data are consistent with LCCs being the primary virulence factors in the majority of infections, but LCC-negative strains may precipitate certain categories of infection. A high quality genome sequence reveals putative virulence factors whose role in virulence can be investigated.

Clostridium difficile infection among immunocompromised patients in Rio de Janeiro, Brazil and detection of moxifloxacin resistance in a ribotype 014 strain.

Clostridium difficile is a Gram-positive spore forming anaerobic bacterium, often associated with nosocomial diarrhea and pseudomembranous colitis. The acquisition of this organism occurs primarily in hospitals through accidental ingestion of spores, and its establishment and proliferation in the colon results from the removal of members of the normal intestinal flora during or after antibiotic therapy. In this study, stool samples from patients admitted to the University Hospital Clementino Fraga Filho (HUCCF/UFRJ) were screened for C. difficile toxins with an ELISA test and cultured with standard techniques for C. difficile isolation. A total of 74 stool samples were collected from patients undergoing antibiotic therapy between August 2009 and November 2010, only two (2.7%) were positive in the ELISA test and culture. A third isolate was obtained from a negative ELISA test sample. All cases of CDI were identified in patients with acute lymphoid or myeloid leukemia. Genotypic and phenotypic characterization showed that all strains carried toxins A and B genes, and belonged to PCR-ribotypes 014, 043 and 046. The isolated strains were sensitive to metronidazole and vancomycin, and resistant to ciprofloxacin and levofloxacin. Resistance to moxifloxacin, was present in the strain from PCR-ribotype 014, that showed an amino acid substitution in gyrB gene (Asp 426 â†’ Asn). This is the first time that this mutation in a PCR-ribotype 014 strain has been described in Brazil.

Varibaculum anthropi sp. nov. represented by three genetically different genomovars isolated from clinical material and emended description of the genus Varibaculum.

During the years 1994-2011 five strictly anaerobic, Gram-stain-positive, diphtheroid bacteria (strains CCUG 31793T, CCUG 44221, CCUG 61255, CCUG 45114, and CCUG 44993) were isolated from different clinical samples in Sweden and the United Kingdom. Comparative analysis of 16S rRNA gene sequences showed that the five strains shared 99-100% 16S rRNA gene sequence similarity among each other and 98.3-98.6% sequence similarity to Varibaculum cambriense DSM 15806T. Genomic fingerprint patterns generated with ERIC-, BOX-, and RAPD-PCR, and whole genome sequence (WGS) based comparison by in silico DNA-DNA hybridization (isDDH), average nucleotide identity (ANI) analysis and six housekeeping gene (atpA, rpoB, pgi, metG, gltA and gyrA) based multilocus sequence analysis (MLSA) showed that the strains could be differentiated from V. cambriense DSM 15806T and formed three genomic groups, which could only be differentiated at the species border level. Based on physiological characterizations the five strains could not be clearly distinguished among each other. Based on those data a new Varibaculum species, Varibaculum anthropi (type strain CCUG 31793T=JCM 19104T) is proposed including three genetically distinct genomovars (gv 1: CCUG 31793T, CCUG 44221, CCUG 61255; gv 2: CCUG 45114, and gv 3: CCUG 44993).

Changes in antibiotic susceptibility and ribotypes in Clostridium difficile isolates from southern Scotland, 1979-2004.

An increase in the incidence of clinical cases of Clostridium difficile infection has been reported in recent years, but few studies have examined changes in molecular epidemiology and antibiotic resistance over a long period of time. A collection of 179 isolates of C. difficile obtained from symptomatic adult patients in southern Scotland between 1979 and 2004 was used to determine changes in the prevalence of epidemiological types and antibiotic susceptibilities to common antibiotics. PCR ribotyping and MIC determination were performed on all isolates. A total of 56 different ribotypes were identified, among which ribotype 002 was the commonest type overall (14 .0%), followed by ribotypes 014 (7.3 %), 012 (5 .0%), 015 (5.0 %), 020 (5 .0%) and 001 (4.5 %). Ribotype 078 was also identified. The 10 commonest ribotypes comprised 55 % of the total isolates. Ribotype 001 increased in prevalence from 1.5 to 12.2 % over the study years, whereas the prevalence of ribotype 012 decreased from 8.7 to 2 .0%. Resistance to clindamycin, erythromycin and ceftriaxone was found in 95.5, 14.0 and 13.4 % of isolates, respectively. Resistance to vancomycin or metronidazole was not detected. Thirty-two (17.9 %) and 14 (7.8 %) isolates were resistant to two and three or more antibiotics, respectively. Ribotype 001 displayed maximum resistance, with 50 % of isolates resistant to erythromycin, moxifloxacin and ceftriaxone, and 100 % resistant to clindamycin. Over the 26 years of the study, antibiotic resistance and ribotype prevalence have changed, and antibiotic pressures may have been the major driver of this change.

Surface bacteriology of venous leg ulcers and healing outcome.

AIM: Bacteria can be cultured from all venous leg ulcers (VLUs) regardless of healing status, and the significance of a positive swab result in non-clinically infected ulcers is unknown. The aim of this study was to characterise the bacteriological flora of VLUs by routine culture to determine whether the data generated had prognostic value. METHODS: The ulcers of 178 patients were sampled weekly for 12 weeks and healing outcome monitored while the limb was treated with graduated compression. Wound bacteriology was assessed using culture methodology standardised to ensure data reproducibility. RESULTS: 153 individual bacterial species were identified. The species most frequently found were Staphylococcus aureus (64.3% of assessments), Corynebacterium striatum (60.6%), Pseudomonas aeruginosa (32.6%), Helcococcus kunzii (22.0%), Finegoldia magna (21.4%) and Proteus mirabilis (16.1%). No single species or the presence of anaerobes and increasing diversity of bacterial species, previously thought to be predictive of impaired healing, was shown to be associated with healing outcome. The presence of C striatum was associated with healing outcome but not after adjusting for the known prognostic factors of wound area and duration. CONCLUSION: Routine bacteriological culture analysis of the VLU wound surface may be used to identify diverse flora in all ulcers. However, the data generated are of no additional value as a prognostic indicator of healing outcome. The presence of C striatum may represent colonisation of non-healing VLU by normal skin flora.

Actinomyces--gathering evidence of human colonization and infection.

The roles of the 'classical'Actinomyces spp. as colonizers of oral cavities of man and animals, in development of intra-oral infections and as agents of actinomycosis have been well documented. This mini-review focuses on perceptions of human colonization and infection that have emerged in the past decade, largely as a result of advances in classification, identification and direct detection from clinical material. Arguably, of the greatest importance is the recognition of actinomycosis as a major factor and indicator of poor prognosis in both infected osteoradionecrosis and bisphosphonate-associated osteonecrosis of the jaws. Among recently described species, Actinomyces graevenitzii has been isolated almost exclusively from oral and respiratory sites and may be a causative agent of actinomycosis. Conversely, several other Actinomyces spp. are isolated commonly from superficial soft tissue infections. Members of the genus Actinobaculum, which is closely related to Actinomyces, are strongly associated with urosepsis. Isolation and identification of Actinomyces and related genera by conventional methods remain difficult. Diagnosis is commonly belated and based solely upon histological findings. Development of direct detection methods may aid patient management and further elucidate clinical associations.

Actinomyces dentalis sp. nov., from a human dental abscess.

A previously undescribed filamentous, beaded, Gram-positive, rod-shaped bacterium was isolated from pus of a human dental abscess. Based on its cellular morphology and the results of biochemical testing the organism was tentatively identified as a member of the genus Actinomyces, but it did not correspond to any currently recognized species of this genus. Comparative 16S rRNA gene sequencing studies showed the bacterium represents a distinct subline within the genus Actinomyces, clustering within a group of species that includes Actinomyces bovis, the type species of the genus. Sequence divergence values of >8 % with other recognized species within this phylogenetic group clearly demonstrated that the organism represents a hitherto unknown species. Based on biochemical and molecular phylogenetic evidence, it is proposed that the unidentified organism recovered from a dental abscess be classified as a novel species, Actinomyces dentalis sp. nov. The type strain is R18165T (=CCUG 48064T=CIP 108337T).

Enzymatic/biochemical analysis of Actinomyces with commercial test kits with an emphasis on newly described species.

In clinical microbiology laboratories, the identification of Actinomyces-like bacteria can be very laborious and problematic. In the present study, we focused on reactivity patterns of 4 commercial test kits, RapID ANA II, RapID 32A, RapID CB Plus, and BBL Crystal ANR ID, that could be used for rapid preliminary identification of Actinomyces isolates belonging to newly described Actinomyces and closely related species. Out of the 54 strains tested, 25 strains (46%) were correctly identified to the genus/group level by BBL Crystal ANR ID system, 16 strains (30%) by RapID 32 A, 11 strains (20%) by RapID CB Plus, and 7 strains (13%) by RapID ANA II. The main problems with these kits were due to occasional weak enzymatic and sugar fermentation reactions. In conclusion, chromogenic substrate sensitivity and specificity need to be enhanced in order to improve the reliability of the test results of these kits, and the present database updated in order to more precisely identify newly described Actinomyces and closely related species.

Start your own support group.

I noted Katie Wood's comments on the need tor a support group for unemployed nurses, (Finished after finals? Campaign, February 17) and wondered whether she had considered starting her own.

Evaluation of four commercial test systems for identification of actinomyces and some closely related species.

We evaluated four commercially available kits for rapid identification of Actinomyces and related species. The kits identified correctly 26 to 65% of "classical" Actinomyces strains to the species level and 13 to 49% of newly described Actinomyces strains to the genus level, thus indicating relatively poor applicability and a need to update these kits.

Actinomyces cardiffensis sp. nov. from human clinical sources.

Eight strains of a previously undescribed catalase-negative Actinomyces-like bacterium were recovered from human clinical specimens. The morphological and biochemical characteristics of the isolates were consistent with their assignment to the genus Actinomyces, but they did not appear to correspond to any recognized species. 16S rRNA gene sequence analysis showed the organisms represent a hitherto unknown species within the genus Actinomyces related to, albeit distinct from, a group of species which includes Actinomyces turicensis and close relatives. Based on biochemical and molecular genetic evidence, it is proposed that the unknown isolates from human clinical sources be classified as a new species, Actinomyces cardiffensis sp. nov. The type strain of Actinomyces cardiffensis is CCUG 44997(T).

Actinomyces vaccimaxillae sp. nov., from the jaw of a cow.

A previously undescribed Actinomyces-like bacterium was isolated from a lesion in the jaw of a cow. Based on its cellular morphology and the results of biochemical testing, the organism was tentatively identified as a member of the genus Actinomyces. Comparative 16S rRNA gene sequencing studies showed that the bacterium represents a hitherto unknown species within the genus Actinomyces, and is related to a group of species that includes Actinomyces turicensis and its close relatives. It is proposed that the unknown organism be classified as Actinomyces vaccimaxillae sp. nov. (the type strain is CCUG 46091T =CIP 107423T).

Actinomyces nasicola sp. nov., isolated from a human nose.

A previously undescribed facultatively anaerobic, catalase-negative, Actinomyces-like bacterium was isolated from the nose of a human. On the basis of its cellular morphology and the results of biochemical testing, the micro-organism was tentatively identified as a member of the genus Actinomyces, but it did not correspond to any currently recognized species. Comparative 16S rRNA gene sequencing studies showed the bacterium to be a hitherto unknown subline within the genus Actinomyces, displaying sequence divergence values of more than 6 % with respect to recognized species of the genus. On the basis of biochemical, molecular chemical and molecular phylogenetic evidence, it is proposed that the unknown organism, strain R2014(T) (=CCUG 46092(T)=CIP 107668(T)), be classified as the type strain of a novel species, Actinomyces nasicola sp. nov.

Actinobaculum urinale sp. nov., from human urine.

A hitherto undescribed Actinomyces-like bacterium was isolated from human urine. Based on its biochemical characteristics, the unidentified bacterium did not correspond to any currently described Actinomyces species or related taxa. Comparative 16S rRNA gene sequencing showed that the unknown bacterium exhibits a specific phylogenetic association with the genus Actinobaculum, but a sequence divergence of > 5% from the two currently recognized members of this genus, Actinobaculum schaalii and Actinobaculum suis, demonstrates that it represents a distinct species. Based on both phenotypic and 16S rRNA gene sequence considerations, it is proposed that the unknown bacterium from urine should be classified as a novel species, Actinobaculum urinale sp. nov. The type strain of Actinobaculum urinale is CCUG 46093(T) (= CIP 107424(T)).

Characterization of some actinomyces-like isolates from human clinical sources: description of Varibaculum cambriensis gen nov, sp nov.

Fifteen strains of an anaerobic, catalase-negative, gram-positive diphtheroid-shaped bacterium recovered from human sources were characterized by phenotypic and molecular chemical and molecular genetic methods. The unidentified bacterium showed some resemblance to Actinomyces species and related taxa, but biochemical testing, polyacrylamide gel electrophoresis analysis of whole-cell proteins, and amplified 16S ribosomal DNA restriction analysis indicated the strains were distinct from all currently named Actinomyces species and related taxa. Comparative 16S rRNA gene sequencing studies showed that the bacterium represents a hitherto-unknown phylogenetic line that is related to but distinct from Actinomyces, Actinobaculum, Arcanobacterium, and Mobiluncus: We propose, on the basis of phenotypic and phylogenetic evidence, that the unknown bacterium from human clinical specimens should be classified as a new genus and species, Varibaculum cambriensis gen. nov., sp. nov. The type strain of Varibaculum cambriensis sp. nov. is CCUG 44998(T) = CIP 107344(T).

Actinomyces oricola sp. nov., from a human dental abscess.

A previously undescribed Actinomyces-like bacterium was isolated from a human dental abscess. Based on its cellular morphology and the results of biochemical testing the organism was tentatively identified as a member of the genus Actinomyces, but it did not correspond to any currently recognized species of this genus. Comparative 16S rRNA gene sequencing studies showed the bacterium represents a hitherto unknown subline within the genus Actinomyces, clustering within a group of species, which includes Actinomyces bovis, the type species of the genus. Based on biochemical and molecular phylogenetic evidence, it is proposed that the unknown organism recovered from a dental abscess be classified as a new species, Actinomyces oricola sp. nov. The type strain of Actinomyces oricola is R5292(T) (=CCUG 46090(T)=CIP 107639(T)).

Corynebacterium atypicum sp. nov., from a human clinical source, does not contain corynomycolic acids.

An unusual gram-positive, facultatively anaerobic, catalase-positive, diphtheroid-shaped organism originating from an unknown human clinical source was characterized by biochemical, molecular chemical and molecular phylogenetic methods. Based on its morphological and biochemical characteristics and the presence of a murein based on meso-diaminopimelic acid, the unidentified organism was tentatively assigned to the genus Corynebacterium. However, the unknown organism was found to lack the distinctive, short-chain corynomycolic acids that are considered to be characteristic of this genus. Despite the absence of these characteristic lipids, comparative 16S rRNA gene sequencing showed that the unknown bacterium was phylogenetically a member of the genus Corynebacterium and was distinct from all currently known species. Based on both phenotypic and 16S rRNA sequence considerations, it is proposed that the unknown organism be classified as a novel species, Corynebacterium atypicum sp. nov. The type strain of C. atypicum is strain R2070T (=CCUG 45804T=CIP 107431T).