Avirulent Bacillus anthracis Strain with Molecular Assay Targets as Surrogate for Irradiation-Inactivated Virulent Spores.

The revelation in May 2015 of the shipment of γ irradiation-inactivated wild-type Bacillus anthracis spore preparations containing a small number of live spores raised concern about the safety and security of these materials. The finding also raised doubts about the validity of the protocols and procedures used to prepare them. Such inactivated reference materials were used as positive controls in assays to detect suspected B. anthracis in samples because live agent cannot be shipped for use in field settings, in improvement of currently deployed detection methods or development of new methods, or for quality assurance and training activities. Hence, risk-mitigated B. anthracis strains are needed to fulfill these requirements. We constructed a genetically inactivated or attenuated strain containing relevant molecular assay targets and tested to compare assay performance using this strain to the historical data obtained using irradiation-inactivated virulent spores.

Demonstration of in vivo transfer of doxycycline resistance mediated by a novel transposon.

OBJECTIVES: The aim of this study was to investigate the transfer of bacterial doxycycline resistance between oral bacteria in subjects receiving systemic doxycycline for the treatment of periodontitis. PATIENTS AND METHODS: Streptococci were cultured before and after treatment from the subgingival plaque of two patients with periodontitis, genotyped and investigated for the presence of antimicrobial resistance determinants and conjugative transposons. RESULTS: In one subject, a strain of Streptococcus sanguinis resistant to doxycycline was a minor component of the pre-treatment streptococcal flora but dominated post-treatment. In a second subject, a strain of Streptococcus cristatus, which was sensitive to doxycycline before treatment, was found to have acquired a novel conjugative transposon during treatment, rendering it resistant to doxycycline and erythromycin. The novel transposon, named CTn6002, was sequenced and found to be a complex element derived in part from Tn916, and an unknown element which included the erythromycin resistance gene erm(B). A strain of Streptococcus oralis isolated from this subject pre-treatment was found to harbour CTn6002 and was therefore implicated as the donor. CONCLUSIONS: This is the first direct demonstration of transfer of antimicrobial resistance carried on a conjugative transposon between oral bacteria during systemic antimicrobial treatment of periodontitis in humans.

Genotypic and phenotypic characterization of fusobacteria from Chinese and European patients with inflammatory periodontal diseases.

Phylogenetic and antigenic studies were performed on 48 human oral Fusobacterium strains from Chinese patients with either necrotizing ulcerative gingivitis (NUG) or gingivitis and on 23 Fusobacterium nucleatum or Fusobacterium periodonticum strains from European periodontitis patients. Alignment of partial 16S rRNA gene sequences resulted in a phylogenetic tree that corresponded well with the current classification of oral fusobacteria into F. periodonticum and several subspecies of F. nucleatum, in spite of much minor genetic variability. F. periodonticum, F. nucleatum subsp. animalis and a previously undescribed phylogenetic cluster (C4), that may represent an additional F. nucleatum subspecies, constituted discrete clusters distinct from the remainder of F. nucleatum with high bootstrap values. Chinese and European strains differed markedly with regard to their respective classification patterns, suggesting a predominance of F. peridonticum and F. nucleatum susp. animalis over F. nucleatum subsp. nucleatum and F. nucleatum subsp. fusiforme/vincentii in samples from China. Antigenic typing enabled the association of many previously described serovars with distinct phylogenetic clusters and when applied directly to uncultured clinical samples confirmed the differential distribution of oral Fusobacterium taxa in Chinese and European samples. Bacteria from cluster C4 and F. nucleatum subsp. animalis were significantly more prevalent and accounted for higher cell numbers in NUG than in gingivitis samples, suggesting a possible association of these rarely observed taxa with NUG in Chinese patients.

Shuttleworthia satelles gen. nov., sp. nov., isolated from the human oral cavity.

Nine strains of anaerobic, non-spore-forming, gram-positive bacilli, isolated from the human oral cavity and provisionally identified as belonging to the genus Eubacterium, were subjected to a comprehensive range of phenotypic and genetic tests. Biochemically, they were found to comprise a homogeneous group, and phylogenetic analysis of their 16S rRNA sequences indicated that they constitute a unique branch within the Clostridium-Bacillus subphylum of the phylum Firmicutes. All of the isolates displayed an unusual colonial morphology after extended incubation. This resembled a contaminated culture in that small, secondary colonies were seen to arise around and from within the primary colony form, and a third, independent, colony type was also seen. However, inspection of the colonies by Gram-staining and scanning electron microscopy together with protein profile analysis and 16S rRNA gene sequence comparison of the two independent colony types revealed that only a single organism was present. A new genus, Shuttleworthia, and the species Shuttleworthia satelles gen. nov., sp. nov., are proposed. The cells are saccharolytic, and acetate, butyrate and lactate are produced as end products of glucose fermentation. Aesculin is hydrolysed and indole is produced. The G+C content of the DNA of the type strain is 51 mol%. The type strain is strain DSM 14600T (= CCUG 45864T = VPI D143K-13T).

Comparison of the molecular diversity of the methanogenic community at the brackish and marine ends of a UK estuary.

Abstract The 16S rRNA sequence diversity of the euryarchaeal community in a predominately freshwater sediment at East Hill Bridge (EHB) on the River Colne estuary, Essex, UK was investigated and compared to that from marine sediments at the mouth of the river (Colne Point). The East Hill Bridge sediments appear to support the full range of methanogen phenotypes with some genotypes similar to those previously detected at Colne Point. However, no Marine Benthic Group D or halophilic archaeal genotypes, both abundant in gene libraries at Colne Point, were detected at East Hill Bridge. Clones related to Methanosarcina and Methanocorpusculum were detected only at East Hill Bridge while clones closely related to Methanoculleus and Methanococcoides were detected only at Colne Point. The most common clones in the East Hill Bridge library were closely related to the obligate acetate-utilising Methanosaeta concilii, suggesting they may be important methanogens in these sediments. Clones that group closely with M. concilii appear to be ubiquitous in freshwater sediments and we suggest that they are prime candidates for a globally important acetoclastic methanogenic group. The distribution of clones in the East Hill Bridge and Colne Point libraries implies that certain methanogen groups are generalists, adapted to the range of conditions within an estuarine environment (e.g. Methanogenium) while others are more specialist (e.g. Methanosaeta).

Characterisation of Eubacterium-like strains isolated from oral infections.

The genus Eubacterium currently includes a heterogeneous group of gram-positive, non-spore-forming anaerobic bacilli, many of which are slow growing, fastidious and generally unreactive in biochemical tests. As a consequence, cultivation and identification of isolates are difficult and the taxonomy of the group remains indifferent. In this study, 105 isolates from odontogenic infections, infections associated with dental implants or saliva from healthy subjects and provisionally assigned to the genus Eubacterium were subjected to phenotypic and genotypic analysis. Ninety-one of the isolates were identified as belonging to one of 14 previously described species: Atopobium parvulum (5 isolates), A. rimae (29), Bulleidia extructa (2), Cryptobacterium curtum (1), Dialister pneumosintes (1), Eubacterium saburreum (2), E. sulci (8), E. yurii subsp. yurii (1), Filifactor alocis (3), Lactobacillus uli (1), Mogibacterium timidum (13), M. vescum (6), Pseudoramibacter alactolyticus (6) and Slackia exigua (13). The remaining 14 isolates did not correspond to existing species. This study confirms the diversity of organisms provisionally assigned to the genus Eubacterium by conventional identification methods. This group of organisms is frequently isolated from oral infections but their role in the aetiology of these conditions has yet to be determined.