Novel xylan-degrading enzymes from polysaccharide utilizing loci of Prevotella copri DSM18205.

Prevotella copri is a bacterium that can be found in the human gastrointestinal tract (GIT). The role of P. copri in the GIT is unclear, and elevated numbers of the microbe have been reported both in dietary fiber-induced improvement in glucose metabolism but also in conjunction with certain inflammatory conditions. These findings raised our interest in investigating the possibility of P. copri to grow on xylan, and identify the enzyme systems playing a role in digestion of xylan-based dietary fibers. Two xylan degrading polysaccharide utilizing loci (PUL10 and 15) were found in the genome, with three and eight glycoside hydrolase (GH) -encoding genes, respectively. Three of them were successfully produced in Escherichia coli: One extracellular enzyme from GH43 (subfamily 12, in PUL10, 60 kDa) and two enzymes from PUL15, one extracellular GH10 (41 kDa), and one intracellular GH43 (subfamily 137 kDa). Based on our results, we propose that in PUL15, GH10 (1) is an extracellular endo-1,4-ß-xylanase, that hydrolazes mainly glucuronosylated xylan polymers to xylooligosaccharides (XOS); while, GH43_1 in the same PUL, is an intracellular ß-xylosidase, catalyzing complete hydrolysis of the XOS to xylose. In PUL10, the characterized GH43_12 is an arabinofuranosidase, with a role in degradation of arabinoxylan, catalyzing removal of arabinose-residues on xylan.

Comparing identification of clinically relevant Prevotella species by VITEK MS and MALDI biotyper.

In this multicenter study, we aimed to evaluate the performance of MALDI Biotyper and VITEK MS, for identification of Prevotella species. Three hundred and fourteen clinical isolates, collected in eight European countries between January 2014 and April 2016, were identified at the collecting sites by MALDI Biotyper (versions 3.0 and 3.1) and then reidentified by VITEK MS (version 3.0) in the central laboratory. 16S rRNA gene sequencing was used as a standard method. According to sequence analysis, the 314 Prevotella strains belonged to 19 species. MALDI Biotyper correctly identified 281 (89.5%) isolates to the species level and 33 (10.5%) only at the genus level. VITEK MS correctly identified 253 (80.6%) isolates at the species level and 276 (87.9%) isolates at the genus level. Thirty-three isolates belonging to P. bergensis, P. conceptionensis, P. corporis, P. histicola, and P. nanciensis, unavailable in the VITEK MS 3.0 database, were resulted in genus level or no identification. Six Prevotella strains, belonged to P. veroralis, P. timonensis, and P. conceptionensis not represented in the MALDI Biotyper system database, were misidentified at the genus level. In conclusion, both VITEK MS and MALDI Biotyper provided reliable and rapid identification. However, the permanent extension of the databases is needed.

Application of MALDI-TOF MS to rapid identification of anaerobic bacteria.

BACKGROUND: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been rapidly developed and widely used as an analytical technique in clinical laboratories with high accuracy in microorganism identification. OBJECTIVE: To validate the efficacy of MALDI-TOF MS in identification of clinical pathogenic anaerobes. METHODS: Twenty-eight studies covering 6685 strains of anaerobic bacteria were included in this meta-analysis. Fixed-effects models based on the P-value and the I-squared were used for meta-analysis to consider the possibility of heterogeneity between studies. Statistical analyses were performed by using STATA 12.0. RESULTS: The identification accuracy of MALDI-TOF MS was 84% for species (I2 = 98.0%, P < 0.1), and 92% for genus (I2 = 96.6%, P < 0.1). Thereinto, the identification accuracy of Bacteroides was the highest at 96% with a 95% CI of 95-97%, followed by Lactobacillus spp., Parabacteroides spp., Clostridium spp., Propionibacterium spp., Prevotella spp., Veillonella spp. and Peptostreptococcus spp., and their correct identification rates were all above 90%, while the accuracy of rare anaerobic bacteria was relatively low. Meanwhile, the overall capabilities of two MALDI-TOF MS systems were different. The identification accuracy rate was 90% for VITEK MS vs. 86% for MALDI biotyper system. CONCLUSIONS: Our research showed that MALDI-TOF-MS was satisfactory in genus identification of clinical pathogenic anaerobic bacteria. However, this method still suffers from different drawbacks in precise identification of rare anaerobe and species levels of common anaerobic bacteria.

Performance of mass spectrometric identification of clinical Prevotella species using the VITEK MS system: A prospective multi-center study.

Prevotella species, members of the human microbiota, can cause opportunistic infections. Rapid and accurate identification of Prevotella isolates plays a critical role in successful treatment, especially since the antibiotic susceptibility profile differs between species. Studies, mostly carried out using the Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) Biotyper system, showed that MALDI-TOF MS is an accurate, rapid and satisfactory method for the identification of clinically important anaerobes. In this multi-center study, we assessed the performance of the MALDI-TOF MS VITEK MS system for the identification of clinical Prevotella isolates. A total of 508 Prevotella isolates, representing 19 different species, collected from 11 European countries, Kuwait and Turkey between January 2014 and April 2016, were identified using VITEK MS (v3.0). The reliability of the identification was assessed by 16S rRNA gene sequencing. Using VITEK MS, 422 (83.1%) of the 508 isolates were identified on the species level, 459 (90.4%) on the genus level. A total of 49 (9.6%) isolates were not identified correctly. 16S rRNA gene sequencing results showed that this was partly due to the fact that several species were not represented in the database. However, some species that were represented in the database were also not identified. Five Prevotella strains were misidentified at the genus level, 2 of these strains belonged to a species not represented in the database. In general, the VITEK MS offers a reliable and rapid identification of Prevotella species, however the databases needs to be expanded.

Performance of MALDI-TOF MS for identification of oral Prevotella species.

During the past decade, the clinically relevant genus Prevotella has expanded considerably. Prevotella species can be isolated from nearly all types of oral infections but also from various non-oral infections. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introduced in clinical microbiology laboratories as a convenient method for identifying bacterial isolates from clinical specimens. Here we tested the diagnostic accuracy of a total of 123 oral Prevotella isolates, selected based on their biochemical profile, by Bruker MALDI-TOF MS. Partial 16S rRNA sequencing was used as a reference method. The performance of MALDI-TOF MS to identify the isolates to the genus level was excellent with 100.0% accuracy, while a good identification rate of 88.6% was achieved to the species level with a log score of ≥2.0. The isolates representing P. aurantiaca and P. jejuni, which are currently missing from the MALDI BioTyper database, were identified correctly to the genus level. Of the 123 isolates, one P. pallens isolate (0.8%) was identified with a score variation of 1.7-1.999. Overall, biochemical testing produced a high proportion (70.7%) of incorrect identifications within different species. MALDI-TOF MS offers a reliable and rapid method for the identification of Prevotella species included in the database.

Prevotella falsenii sp. nov., a Prevotella intermedia-like organism isolated from monkey dental plaque.

Eight anaerobic, pigmented, non-spore-forming, Gram-negative, rod-shaped strains isolated from monkey oral cavities were characterized phenotypically and chemotaxonomically and their phylogenetic positions were determined using 16S rRNA gene sequence analysis. The 16S rRNA gene sequence analysis showed that these isolates represent a single species of the genus Prevotella. These strains were most closely related to Prevotella intermedia ATCC 25611(T), with 95.0 % 16S rRNA gene sequence similarity. The next most closely related species were Prevotella pallens and Prevotella nigrescens (92.7 and 92.1 % similarity to the respective type strains). The phenotypic and biochemical characteristics of the isolates were the same as those of P. intermedia JCM 12248(T) and P. nigrescens JCM 12250(T). The isolates could be differentiated from P. pallens JCM 11140(T) on the basis of mannose fermentation and alpha-fucosidase activity. The isolates could not be distinguished from P. intermedia or P. nigrescens using conventional biochemical tests. DNA-DNA hybridization experiments revealed the genomic distinctiveness of these eight strains with respect to P. pallens JCM 11140(T), P. intermedia JCM 12248(T) and P. nigrescens JCM 12250(T). On the basis of these data, strains 04013, 04021, 04043, 04052(T), 0406, 04113, 04111 and 04161 represent a novel Prevotella species, for which the name Prevotella falsenii sp. nov. is proposed. The type strain is 04052(T) (=JCM 15124(T) =CCUG 56137(T)).

Prevotella amnii sp. nov., isolated from human amniotic fluid.

Two Gram-negative, anaerobic, non-spore-forming, rod-shaped organisms were isolated from human amniotic fluid. Based on morphological and biochemical criteria, the strains were tentatively identified as Bacteroidaceae but they did not appear to correspond to any recognized species of this family. Comparative 16S rRNA gene sequencing studies showed the strains were highly related to each other and confirmed their placement in the genus Prevotella, but sequence divergence values of >4% with reference Prevotella species demonstrated that the organisms from human clinical sources represent a novel species. Phylogenetic analysis revealed the novel organism to be most closely related to Prevotella bivia, an organism frequently associated with pelvic inflammatory diseases. The major long-chain cellular fatty acids of the novel species consist of iso-C(14:0), anteiso-C(15:0), iso-C(15:0), C(16:0), iso-C(16:0) and iso-3-OH-C(17:0). Based on biochemical criteria and phylogenetic considerations, it is proposed that the unknown isolates from human amniotic fluid be assigned to a new species of the genus Prevotella, as Prevotella amnii sp. nov. The type strain of Prevotella amnii is CCUG 53648(T) (=JCM 14753(T)).

Prevotella pleuritidis sp. nov., isolated from pleural fluid.

A strain isolated from pleural fluid of a patient with suppurative pleuritis (strain GTC 3021(T)) was characterized in terms of its phenotypic and biochemical features, cellular fatty acid profile and phylogenetic position based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that the isolate was a member of the genus Prevotella. The isolate was related to Prevotella enoeca ATCC 51261(T) with about 92 % 16S rRNA gene sequence similarity. The strain was an obligately anaerobic, non-pigmenting, non-spore-forming, non-motile, Gram-negative rod. Although the phenotypic and biochemical characteristics of the strain were similar to those of P. enoeca JCM 12259(T), the cellular fatty acid composition of the isolate was significantly different from that of P. enoeca JCM 12259(T) (C(18 : 1) omega 9c and anteiso-C(15 : 0) fatty acid content). Based on these data, we propose a novel Prevotella species, Prevotella pleuritidis sp. nov., with the type strain GTC 3021(T) (=JCM 14110(T) =CCUG 54350(T)). The G+C content of the type strain is 45.4 mol%.

Prevotella copri sp. nov. and Prevotella stercorea sp. nov., isolated from human faeces.

Six strains (CB7(T), CB18, CB23, CB26, CB28 and CB35(T)) were isolated from human faeces. Based on phylogenetic analysis, phenotypic characteristics, cellular fatty acid profiles and menaquinone profiles, these strains could be included within the genus Prevotella and made up two clusters. 16S rRNA gene sequence analysis indicated that five strains were most closely related to Prevotella veroralis, sharing about 92 % sequence similarity; the remaining strain was most closely related to Prevotella shahii, sharing about 90 % sequence similarity. All six strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods. The cellular fatty acid compositions of the six strains differed significantly from those of other Prevotella species. Five strains (CB7(T), CB18, CB23, CB26 and CB28) contained dimethyl acetals and the major menaquinones of these strains were MK-11, MK-12 and MK-13. The major menaquinones of CB35(T) were MK-12 and MK-13. Based on phenotypic and phylogenetic findings, two novel species, Prevotella copri sp. nov. and Prevotella stercorea sp. nov., are proposed, representing the two different strain clusters. The DNA G+C contents of strains CB7(T) and CB35(T) were 45.3 and 48.2 mol%, respectively. The type strains of P. copri and P. stercorea are CB7(T) (=JCM 13464(T)=DSM 18205(T)) and CB35(T) (=JCM 13469(T)=DSM 18206(T)), respectively.

Evaluation of the effects of short-chain fatty acids and extracellular pH on bovine neutrophil function in vitro.

OBJECTIVE: To investigate the effects of short-chain fatty acids (SCFAs) and pH on neutrophil oxidative burst, phagocytosis, and morphology after exposure to acetate, propionate, butyrate, or succinate at pH 5.5 and 6.7. SAMPLE POPULATION: Neutrophils isolated from bovine blood samples and Porphyromonas levii, Prevotella spp, and Bacteroides fragilis isolated from lesions of cattle with acute interdigital phlegmon (foot rot). PROCEDURES: Bacteria were cultured in strictly anaerobic conditions. Bacterial SCFA production was measured with high-performance liquid chromatography. Neutrophils were isolated, stimulated with phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OZ), and incubated with dihydroethidium or dichlorofluorescein diacetate to measure production of O(2)and H(2)O(2), respectively. Phagocytosis was assessed after exposure to serum-opsonized bacteria. Cellular morphology was assessed with differential staining. RESULTS: All bacteria produced at least 3 of the 4 SCFAs. Production of both O(2) and H(2)O(2) was markedly curtailed in PMA-stimulated neutrophils exposed to SCFA at pH 5.5, compared with production at pH 6.7. Succinate caused a significant dose-dependent decrease in O(2) production at pH 6.7 in OZ-stimulated neutrophils. Monoprotic SCFAs elicited a significant increase in H(2)O(2) production in OZ-stimulated neutrophils at pH 6.7 but a significant decrease at pH 5.5. Monoprotic SCFAs significantly increased phagocytosis at pH 6.7 but decreased phagocytic activity at pH 5.5. Cellular necrosis was observed in cells exposed to SCFAs at pH 5.5. CONCLUSIONS AND CLINICAL RELEVANCE: Establishment and persistence of anaerobic bacteria in cattle with foot rot infection may result in part from neutrophil dysfunction secondary to the effects of bacterially secreted SCFA in acidotic microenvironments.

Prevotella bergensis sp. nov., isolated from human infections.

Eight strains of anaerobic Gram-negative bacilli isolated from infections of the skin and soft tissues were subjected to a comprehensive range of phenotypic and genotypic tests. 16S rRNA gene sequence analysis revealed the strains to constitute a homogeneous group, distinct from species with validly published names but related to a cluster including Prevotella buccae, Prevotella dentalis and Prevotella baroniae. A novel species, Prevotella bergensis sp. nov., is proposed to accommodate these strains. Prevotella bergensis is saccharolytic and produces acetic and succinic acids as end products of fermentation. The G + C content of the DNA of the type strain is 48 mol%. The type strain of Prevotella bergensis is 94067913T (= DSM 17361T = CCUG 51224T).

Molecular analysis of the bacterial microbiota in the human stomach.

The microbiota of the human stomach and the influence of Helicobacter pylori colonization on its composition remain largely unknown. We characterized bacterial diversity within the human gastric mucosa by using a small subunit 16S rDNA clone library approach and analyzed 1,833 sequences generated by broad-range bacterial PCR from 23 gastric endoscopic biopsy samples. A diverse community of 128 phylotypes was identified, featuring diversity at this site greater than previously described. The majority of sequences were assigned to the Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Fusobacteria phyla. Ten percent of the phylotypes were previously uncharacterized, including a Deinococcus-related organism, relatives of which have been found in extreme environments but not reported before in humans. The gastric clone libraries from 19 subjects contained H. pylori rDNA; however, only 12 of these subjects tested positive for H. pylori by conventional laboratory methods. Statistical analysis revealed a large degree of intersubject variability of the gastric ecosystem. The presence of H. pylori did not affect the composition of the gastric community. This gastric bacterial rDNA data set was significantly different from sequence collections of the human mouth and esophagus described in other studies, indicating that the human stomach may be home to a distinct microbial eco-system. The gastric microbiota may play important, as-yet-undiscovered roles in human health and disease.

Prevotella multiformis sp. nov., isolated from human subgingival plaque.

Four bacterial strains isolated from the human oral cavity, PPPA19, PPPA21(T), PPPA28 and PPPA30, were characterized by determining phenotypic and biochemical features, cellular fatty acid profiles and the phylogenetic position based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that each of the isolates was a member of the genus Prevotella. These strains were related to Prevotella denticola with about 95 % similarity. The strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods. However, the cells of these strains were often cocci (coccobacilli), depending on the cultivation time. Colonies of different sizes were detected on Eggerth Gagnon agar plates for these strains. The cells forming large colonies were cocci, whereas those forming small colonies were cocci and rods. However, 16S rRNA gene sequence comparison of colonies of different sizes revealed that only a single organism was present. Although these strains had phenotypic characteristics that were similar to those of P. denticola JCM 8528, they could be differentiated from P. denticola JCM 8528 by aesculin hydrolysis and d-cellobiose fermentation in API 20A tests. DNA-DNA hybridization experiments revealed the genomic distinction of these four strains with respect to P. denticola JCM 8528. On the basis of these data, a novel Prevotella species, Prevotella multiformis sp. nov., is proposed, with PPPA21(T) (=JCM 12541(T)=DSM 16608(T)) as the type strain.

The haem pigment of the oral anaerobes Prevotella nigrescens and Prevotella intermedia is composed of iron(III) protoporphyrin IX in the monomeric form.

The haem pigment of Porphyromonas gingivalis is composed of micro -oxo bishaem, [Fe(III)PPIX](2)O, but the nature of that generated by Prevotella species has not been established. Mössbauer, Raman and UV-visible spectrophotometry were used to characterize the haem pigment of Prevotella intermedia and Prevotella nigrescens. Mössbauer and Raman spectroscopy revealed the major haem species to be monomeric iron protoporphyrin IX, Fe(III)PPIX.OH (haematin). The terminal growth pH of both species on blood agar was between 5.8 and 6.0, which favours the formation and maintenance of monomeric Fe(III)PPIX.OH. Incubation of Pr. nigrescens and Pr. intermedia with oxyhaemoglobin at pH 6.5 resulted in formation of aquomethaemoglobin which was degraded to generate Fe(III)PPIX.OH which in turn became cell-associated, whilst incubation at pH 7.5 resulted in formation of [Fe(III)PPIX](2)O. It is concluded that both Prevotella species degrade oxyhaemoglobin to form [Fe(III)PPIX](2)O as an intermediate, which is converted to Fe(III)PPIX.OH through a depression in pH. The low pH encourages cell-surface deposition of insoluble Fe(III)PPIX.OH which would act as a barrier against oxygen and reactive oxygen species, and also protect against H(2)O(2) through its inherent catalase activity.

Reliable identification of Prevotella and Butyrivibrio spp. from rumen by fatty acid methyl ester profiles.

Data for bacterial identification were provided by culturing anaerobic bacteria under standardized conditions followed by extraction and methylation of cellular long-chain fatty acids and gas chromatographic analysis. The databases of fatty acid methyl ester (FAMEs) profiles for two predominant ruminal genera, Prevotella and Butyrivibrio, were created. Major long-chain cellular fatty acids found in the 23 analyzed Prevotella strains were 15:0 (anteiso), 15:0, 15:0 (iso) and 16:0. The strains of Prevotella could be well identified on species level by the characteristic ratios among major fatty acids and by acids unique fatty for each species. The 45 Butyrivibrio strains were grouped into 4 major and 2 minor groups according to FAMEs profiles. The major fatty acids for the bulk of the Butyrivibrio strains were 14:0, 15:1, 16:0 and 16:0 (iso). This groups corresponded to those based on 16S rDNA sequences.

Possible potentiation of toxins from Prevotella intermedia, Prevotella nigrescens, and Porphyromonas gingivalis by cotinine.

BACKGROUND: Smoking is a recognized risk factor for the initiation and progression of periodontitis. However, the mechanism by which smoking induces its negative effects on the periodontium is not clear. This study aimed to test the hypothesis that synergy may occur between cotinine and bacterial products isolated from 3 putative periodontopathogens. METHODS: A chick embryo toxin assay was used to investigate bacterial toxins (cell-free extracellular toxins and cell-free cell lysates) from 5 species with and without cotinine. A total of 9 putative periodontopathogens (3 species) and 2 non-oral controls (2 species) were studied. The periodontal species were: Prevotella intermedia (n = 4), Prevotella nigrescens (n = 4), and Porphyromonas gingivalis (n = 1). The control species tested were: Staphylococcus aureus (n = 1) and Escherichia coli (n = 1). RESULTS: The toxicity kill was significantly greater than expected by simple addition alone (P <0.05, Fisher's exact test) between cotinine (800 ng/ml) and 1) the cell-free extracellular toxins of P. nigrescens MH1 and 2) the cell-free cell lysates of P. intermedia MH2. Synergy occurred with cotinine plus the cell-free extracellular toxins in all but 3 periodontal isolates, and the cell-free cell lysates in all but 2 periodontal isolates. Cotinine significantly (P <0.05, Fisher's exact test) enhanced the effects of cell-free extracellular toxins and cell lysates from one control species (E. coli), but not the other (S. aureus). CONCLUSIONS: These findings indicate that synergy in an in vitro assay can occur between cotinine and toxins from putative periodontopathogens. This may be one important mechanism by which smoking increases the severity of periodontitis.

Increase of CD26/dipeptidyl peptidase IV expression on human gingival fibroblasts upon stimulation with cytokines and bacterial components.

CD26/dipeptidyl peptidase IV (DPPIV) is a cell surface ectoenzyme which participates in immune and inflammatory reactions. We found that CD26 was only partially expressed on human fibroblasts from periodontal tissues, whereas fibroblasts from lung and skin expressed CD26 constitutively as revealed by flow cytometry. We examined the possible upregulation of CD26 expression on human gingival fibroblasts in response to various stimulants. Interleukin-1alpha (IL-1alpha); tumor necrosis factor alpha; gamma interferon; lipopolysaccharide from Porphyromonas gingivalis, Prevotella intermedia, and Escherichia coli; and Prevotella glycoprotein augmented CD26 expression on gingival fibroblasts. Among the stimulants, IL-1alpha exhibited the most potent activity. Enzymatic activity of CD26 induced by IL-1alpha on fibroblasts was determined colorimetrically in terms of Gly-Pro hydrolysis of a synthetic chromogenic substrate, Gly-Pro p-nitroanilide. Among various inhibitors tested, diprotin A and phenylmethylsulfonyl fluoride inhibited the enzymatic activity, suggesting that the enzyme induced by IL-1alpha was DPPIV. The upregulation of CD26 mRNA expression upon stimulation with IL-1alpha was also revealed by a quantitative reverse transcription-PCR assay. In the kinetic experiment, 48 h and several days were required for maximum CD26 mRNA accumulation and CD26 molecule expression on the cell surface, respectively. The addition of cycloheximide at 2 h before IL-1alpha stimulation almost completely inhibited the accumulation of CD26 mRNA. These results suggested that induction of CD26 on human gingival fibroblasts is regulated at the transcriptional level and is also dependent on a de novo-synthesized protein factor(s).

The translational hop junction and the 5' transcriptional start site for the Prevotella loescheii adhesin encoded by plaA.

The Prevotella loescheii adhesin gene, plaA, contains a coding gap between a small open reading frame (ORF-1) and a large open reading frame (ORF-2). Translation of the plaA mRNA requires bypassing this 29-nt coding gap on the plaA transcript. We have determined the N-terminal peptide sequence of the SO34 adhesin beyond the gap sequence. This sequence shows that the peptide junction between ORF-1 and ORF-2 is continuous in the adhesin and supports the conclusion that synthesis of the SO34 adhesin occurs by a ribosomal hop mechanism. To elucidate upstream signals, we used the 5' RACE (rapid amplification of cDNA ends) technique to map the start point of the plaA mRNA. DNA sequencing of plasmids with the 5' RACE products placed the 5' end of plaA mRNA 270 nt upstream from the plaA start codon. A region corresponding to a Bacteroides fragilis promoter consensus sequence precedes this start site.

Anaerobic bacteremia and fungemia in patients undergoing endodontic therapy: an overview.

Oral focal infection, a concept neglected for several decades, is a subject of controversy. Recent progress in classification and identification of oral microorganisms has renewed interest in focal infection. The aim of this study was to use phenotypic and genetic methods to trace microorganisms released into the bloodstream during and after endodontic treatment back to their presumed source--the root canal. Microbiological samples were taken from the root canals of 26 patients with asymptomatic apical periodontitis of single-rooted teeth. The blood of the patients was drawn during and 10 minutes after endodontic therapy. Microorganisms in blood were collected after anaerobic lysis filtration and cultured anaerobically on blood agar plates. The phenotypic methods used for characterization and tracing of microorganisms in blood and root canals were: biochemical and antimicrobial susceptibility test, SDS-PAGE of whole-cell soluble proteins, and gas chromatography of cellular fatty acids. Phenotypic data were verified by DNA restriction patterns and corresponding ribotypes of the root canal and blood isolates by using a computer-assisted system fro gel analysis. All root canals contained anaerobic bacteria. The frequency of bacteremia varied from 31% to 54%. The microorganisms from the root canal and blood presented identical phenotype and genetic characteristics within the patients examined. These characteristics differed between patients. The present study demonstrated that endodontic treatment can be the cause of anaerobic bacteremia and fungemia. The phenotypic and genetic methods used appeared valuable for tracing microorganisms in the blood back to their origin.

Phospholipid molecular species distribution of oral Prevotella corporis clinical isolates.

The aim of this study was to determine the distribution of phospholipid molecular species within Prevotella corporis of oral origin. Phospholipids of fresh clinical isolates were extracted and analysed by fast atom bombardment mass spectrometry (FAB-MS) in negative-ion mode. The major monocarboxylate anion peaks, with putative identification, observed for Prevotella corporis were m/z 241, C(15:0); 255, C(16:0); 269, C(17:1); 277, C(18:3); 279, C(18:2); 281, C(18:1). In the high mass region, major anion peaks putatively identified as individual phospholipid (PL) molecular species of Prevotella corporis were of m/z 677, PG(29:1); 691, PG(30:1); 705, PG(31:1); 706, first isotope peak of PG(31:1); and 707, PG(31:0). Related species have a different distribution of PL analogues. Separation of extracted lipid families by TLC confirmed that phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) are the major polar lipids (PLs) in Prevotella corporis. Thus Prevotella corporis has a unique combination of phospholipid analogues of chemosystematic significance.