Case report of bacteremia due to Neisseria mucosa.

Neisseria mucosa, a Gram-negative diplococcus, is part of normal nasopharyngeal flora. We report a case of bacteremia caused by N. mucosa in a 50-year-old neutropenic patient suffering from non-secretory multiple myeloma stage IIIA. This case underscores that mostly nonpathogenic N. mucosa can cause bacteremia in neutropenic patients who developed mucositis after hematopoietic stem cell transplantation.

Genome sequence analyses show that Neisseria oralis is the same species as 'Neisseria mucosa var. heidelbergensis'.

Phylogenies generated from whole genome sequence (WGS) data provide definitive means of bacterial isolate characterization for typing and taxonomy. The species status of strains recently defined with conventional taxonomic approaches as representing Neisseria oralis was examined by the analysis of sequences derived from WGS data, specifically: (i) 53 Neisseria ribosomal protein subunit (rps) genes (ribosomal multi-locus sequence typing, rMLST); and (ii) 246 Neisseria core genes (core genome MLST, cgMLST). These data were compared with phylogenies derived from 16S and 23S rRNA gene sequences, demonstrating that the N. oralis strains were monophyletic with strains described previously as representing 'Neisseria mucosa var. heidelbergensis' and that this group was of equivalent taxonomic status to other well-described species of the genus Neisseria. Phylogenetic analyses also indicated that Neisseria sicca and Neisseria macacae should be considered the same species as Neisseria mucosa and that Neisseria flavescens should be considered the same species as Neisseria subflava. Analyses using rMLST showed that some strains currently defined as belonging to the genus Neisseria were more closely related to species belonging to other genera within the family; however, whole genome analysis of a more comprehensive selection of strains from within the family Neisseriaceae would be necessary to confirm this. We suggest that strains previously identified as representing 'N. mucosa var. heidelbergensis' and deposited in culture collections should be renamed N. oralis. Finally, one of the strains of N. oralis was able to ferment lactose, due to the presence of ß-galactosidase and lactose permease genes, a characteristic previously thought to be unique to Neisseria lactamica, which therefore cannot be thought of as diagnostic for this species; however, the rMLST and cgMLST analyses confirm that N. oralis is most closely related to N. mucosa.

Amplification of minute amounts of oral bacterial DNA for real-time quantitative PCR analysis.

BACKGROUND: High-throughput technologies for typing caries or health-associated bacterial populations including PCR, DNA microarrays and next-generation sequencing techniques require significant amounts of bacterial DNA. In clinical settings, the amount of sampled DNA is often limited and amplification is therefore essential. Protocols should be able to reproducibly amplify sequences in order to maintain initial sequence ratios and should not bias the representation of particular DNA sequence types. METHODS: A linear amplification protocol using DNA polymerase I was modified to permit the amplification and subsequent analysis of small amounts of bacterial DNA. The protocol was tested on human oral bacterial biofilms from different sources, including carious dentine and plaque, and compared to amplification by degenerate PCR of 16S rDNA sequences. Real-time quantitative PCR of 24 bacterial species was used as a readout system to test amplified DNA against unamplified DNA. RESULTS: The amplification protocol reliably yielded 5-10 µg DNA from as little as 12.5 ng of template DNA. Correlation coefficients between real-time quantitative PCR results from amplified and unamplified DNA were between 0.78 and 0.98. CONCLUSION: The optimized protocol consistently produced amplification products from minute amounts of bacterial DNA from caries and plaque; the amplification products are suitable for downstream genetic analyses.

Evaluation of the microbiota of primary endodontic infections using checkerboard DNA-DNA hybridization.

BACKGROUND/AIMS: The aim of this study was to evaluate the composition of the microbiota of primary endodontic infections in 111 selected cases of single-rooted teeth with necrotic pulp. METHODS: Samples were collected from the root canals using #15 Hedströen-type files and two sterile paper points, which were introduced 1 mm short of the apical foramen. The presence, levels, and proportions of 40 different bacterial species in each sample were determined using DNA probes and checkerboard DNA-DNA hybridization techniques. RESULTS: The mean number of species per sample was 22. Enterococcus faecalis (89.3%), Campylobacter gracilis (89.3%), Leptotrichia buccalis (89.3%), Neisseria mucosa (87.5%), Prevotella melaninogenica (86.6%), Fusobacterium nucleatum ssp. vincentii (85.7%), Eubacterium saburreum (75.9%), Streptococcus anginosus (75%), and Veillonella parvula (74.1%) were the most prevalent species. The species found in highest mean counts (over 10(5)) were F. nucleatum ssp. vincentii (13.14 x 10(5)), E. saburreum (5.67 x 10(5)), E. faecalis (5.38 x 10(5)), N. mucosa (4.19 x 10(5)), V. parvula (3.63 x 10(5)), C. gracilis (3.46 x 10(5)), Treponema socranskii (3.34 x 10(5)), Porphyromonas endodontalis (2.96 x 10(5)), Porphyromonas gingivalis (2.85 x 10(5)), Micromonas micros (2.81 x 10(5)), Prevotella nigrescens (2.68 x 10(5)) and Fusobacterium nucleatum ssp. nucleatum (2.64 x 10(5)). Most of these species were also found in high proportions. CONCLUSIONS: Our results suggest that several bacterial species considered to be oral pathogens seem to be implicated in the etiology of primary endodontic infections.

Improved accuracy in terminal restriction fragment length polymorphism phylogenetic analysis using a novel internal size standard definition.

BACKGROUND: Terminal restriction fragment length polymorphism (T-RFLP) analysis is commonly used to analyze microbial communities, including oral microflora. However, accurate identification of terminal restriction fragment (T-RF) origins is prevented by unpredictable errors in sizing, thus necessitating the clone library analysis. To minimize sizing errors, we proposed optimizing the size definition of internal standards. METHODS: GeneScan-1000 ROX was regenerated as an internal standard by redefining the fragment sizes in terms of molecular weight (MW) based on their mobility relative to 6-carboxyfluorescein (FAM) -labeled restriction fragments derived from the 16S recombinant RNA gene of Porphyromonas gingivalis. Using the new size definition, the average sizing error among eight oral bacteria from six phyla was estimated and compared with that of the conventional method. Microbial communities isolated from saliva were analyzed using the new MW size definition. Bacterial species were assigned to peaks using TRFMA, a Web-based tool for T-RFLP analysis, and compared with those identified in a clone library analysis. RESULTS: Using the new size definition, the average sizing error for 40 T-RFs was drastically reduced from 2.42 to 0.62 bases, and large sizing errors (more than two bases) were eliminated. More than 90% of the total bacterial clones detected by the clone library analysis were assigned by T-RFLP. CONCLUSION: The size definition of the newly constructed internal standards reduced fragment sizing errors and allowed for accurate assignment of bacteria to peaks by the T-RFLP analysis. This provided a more effective means for studying microbial communities, including the oral microflora.