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The misuse of antibiotics in our daily lives has led to the emergence of antimicrobial resistance. As a result, many antibiotics are becoming ineffective. This phenomenon is linked with high rates of mortality and morbidity. Therefore, new approaches are required to address this major health issue. Leptotrichia buccalis is a Gram-negative, rod-shaped bacterium which normally resides in the oral and vaginal cavities. It is an emerging bacterial pathogen which is developing new antibiotic-resistance mechanisms. No approved vaccine is available against this pathogen, which is a cause for growing concern. In this study, an in silico-based, multi-epitopes vaccine against this pathogen was designed by applying reverse vaccinology and immunoinformatic approaches. Of a total of 2193 predicted proteins, 294 were found to be redundant while 1899 were non-redundant. Among the non-redundant proteins, 6 were predicted to be present in the extracellular region, 12 in the periplasmic region and 23 in the outer-membrane region. Three proteins (trypsin-like peptidase domain-containing protein, sell repeat family protein and TrbI/VirB10 family protein) were predicted to be virulent and potential subunit vaccine targets. In the epitopes prediction phase, the three proteins were subjected to B- and T-cell epitope mapping; 19 epitopes were used for vaccine design. The vaccine construct was docked with MHC-I, MHC-II and TLR-4 immune receptors and only the top-ranked complex (based on global energy value) was selected in each case. The selected docked complexes were examined in a molecular dynamic simulation and binding free energies analysis in order to assess their intermolecular stability. It was observed that the vaccine binding mode with receptors was stable and that the system presented stable dynamics. The net binding free energy of complexes was in the range of -300 to -500 kcal/mol, indicating the formation of stable complexes. In conclusion, the data reported herein might help vaccinologists to formulate a chimeric vaccine against the aforementioned target pathogen. King Saud University, Riyadh, Saudi Arabia [RSP2022R462] Published version This research was supporting by the Researchers Supporting Project number RSP2022R462, King Saud University, Riyadh, Saudi Arabia.

Made available in DSpace on 2021-06-25T11:05:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-05-01 Objectives: the objective of the present exploratory study was to determine bacterial diversity and endotoxin levels in deep carious lesions of teeth presenting symptoms of reversible pulpitis. Materials and methods: Twenty patients with deep carious lesions, reporting clinical symptomatology compatible with reversible pulpitis (n = 10) or not reporting clinical symptomatology (n = 10), were selected. Carious dentin samples were obtained with the aid of sterile and pyrogen-free spoon excavators and harvested in two steps: before and after infected dentin removal. Samples were collected for checkerboard and for kinetic chromogenic LAL assay for determination of microbial profile and quantitation of endotoxin, respectively. Data were analyzed by Mann Whitney for bacteria and two-way ANOVA for endotoxins (5%). Results: No difference on the studied bacteria was detected between the superficial and deep dentin layers. Symptomatic teeth showed greater presence of Lactobacillus species, Capnocytophaga sputigena, and Leptotrichia buccalis. For the endotoxins, symptomatic teeth resulted in greater quantity of endotoxins (p = 0.047), being 4.13 log10 EU/mL/μg dentin and 3.45 log10 EU/mL/μg dentin, for symptomatic and asymptomatic teeth, respectively. Dentin collected in different areas presented similar number of endotoxins (p = 0.139). Conclusion: The amount of the studied bacteria does not seem to be related to reported symptomatology of deep carious lesions, while endotoxins quantity is greater in symptomatic scenarios, regardless of the harvesting area. Clinical relevance: The understanding of bacterial amount in reversible pulpitis is important to establish a clinical protocol of treatment. Department of Restorative Dentistry Institute of Science and Technology São Paulo State University - UNESP Department of Biosciences and Oral Diagnosis Institute of Science and Technology São Paulo State University - UNESP Department of Restorative Dentistry Institute of Science and Technology São Paulo State University - UNESP Department of Biosciences and Oral Diagnosis Institute of Science and Technology São Paulo State University - UNESP

The in vitro antibacterial spectra and activities of five antimicrobial agents, including lascufloxacin (LSFX) and two quinolones, were investigated against 69 species of anaerobes in 31 genera and 188 strains in 9 genera, respectively. In this study, minimum inhibitory concentrations (MICs) of lascufloxacin against the reference strains associated with respiratory and head and neck infections. LSFX inhibited the growth of 33 gram-positive and gram-negative reference strains at ≤0.015–2 μg/mL, except for Leptotrichia buccalis. MICs ranges of LSFX against the clinical isolates of 44 Porphyromonas spp., 45 Prevotella spp., 25 Fusobacterium spp., 7 Leptotrichia spp., 25 Parvimonas micra, 25 other gram-positive anaerobic cocci, and 17 Veillonella spp., were ≤0.015–4, 0.125–4, 0.06–0.5, 2, 0.25–16, ≤0.015–2, ≤0.015–16 μg/mL, respectively. LSFX demonstrated potent antibacterial efficacy against a wide range of species isolated from specimens involved in respiratory as well as head and neck infections.

In the present study, we characterized the microbiomes of acute leukemia (AL) patients who achieved complete remission following remission induction chemotherapy (RIC) as outpatients, but who did not receive antimicrobials to treat or prevent febrile neutropenia. Saliva and stool samples from 9 patients with acute myeloid leukemia, 11 patients with acute lymphoblastic leukemia, and 5 healthy controls were subjected to 16S ribosomal RNA sequencing at baseline and at 3 months following RIC. Only patients who achieved remission at 3 months post-treatment were included. We excluded anyone who used antimicrobials within 2 months of enrollment or at any time during the study period. At baseline, the relative abundances of species of Prevotella maculosa (P=0.001), Megasphaera micronuciformis (P=0.014), Roseburia inulinivorans (P=0.021), and Bacteroides uniformis (P=0.004) in saliva and Prevotella copri (P=0.002) in the stools of controls were significantly higher than in AL patients. Following RIC, the relative abundances of Eubacterium sp. oral clone DO008 (P=0.012), Leptotrichia sp. oral clone IK040 (P=0.002), Oribacterium sp. oral taxon 108 (P=0.029), Megasphaera micronuciformis (P=0.016), TM7 phylum sp. oral clone DR034 (P

BACKGROUND This study compared the composition of subgingival microbiota between obese and non-obese women with or without periodontal disease. METHODS Full-mouth periodontal clinical assessments were carried out in 76 obese women (17 periodontally healthy and 59 with periodontal disease), and 34 non-obese women (12 periodontally healthy, 22 with periodontal disease). Subgingival biofilm samples were individually obtained from seven sites of each individual, and the prevalence and counts of 40 bacterial taxa were determined by the checkerboard method. The frequency and counts of each species were computed for each individual and across the groups. Differences among and between groups were sought by the Kruskal-Wallis and Mann-Whitney tests, respectively. Possible correlations between obesity and clinical and microbiologic parameters were tested with Spearman correlation coefficient. RESULTS Streptococcus sanguinis, Streptococcus oralis, and Capnocytophaga ochracea were found in significantly higher levels in obese compared with non-obese women (P

Objective: To profile the salivary microbiomes of a Hong Kong Chinese cohort at a species-level resolution and determine species that discriminated clinically resolved periodontitis from periodontally healthy cases. Methods: Salivary microbiomes of 35 Hong Kong Chinese subjects' under routine supportive dental care were analyzed. All subjects had been treated for any dental caries or periodontal disease with all restorative treatment completed at least 1 year ago and had ≤3 residual pockets. They were categorized based on a past diagnosis of chronic periodontitis into “healthy” (H) or “periodontitis” (P) categories. Unstimulated whole saliva was collected, genomic DNA was isolated, and high throughput Illumina MiSeq sequencing of 16S rRNA (V3-V4) gene amplicons was performed. The sequences were assigned taxonomy at the species level by using a BLASTN based algorithm that used a combined reference database of HOMD RefSeqV14.51, HOMD RefSeqExtended V1.1 and GreenGeneGold. Species-level OTUs were subjected to downstream analysis in QIIME and R. For P and H group comparisons, community diversity measures were compared, differentially abundant species were determined using DESeq2, and disease indicator species were determined using multi-level pattern analysis within the R package “indicspecies.” Results: P subjects were significantly older than H subjects (p = 0.003) but not significantly different in their BOP scores (p = 0.82). No significant differences were noted in alpha diversity measures after adjusting for age, gender, and BOP or in the beta diversity estimates. Four species; Treponema sp. oral taxon 237, TM7 sp. Oral Taxon A56, Prevotella sp. oral taxon 314, Prevotella sp. oral taxon 304, and Capnocytophaga leadbetteri were significantly more abundant in P than in the H group. Indicator species analysis showed 7 significant indicators species of P group. Fusobacterium sp oral taxon 370 was the sole positive indicator of P group (positive predictive value = 0.9, p = 0.04). Significant indicators of the H category were Leptotrichia buccalis, Corynebacterium matruchotii, Leptotrichia hofstadii, and Streptococcus intermedius. Conclusion: This exploratory study showed salivary microbial species could discriminate treated, well-maintained chronic periodontitis from healthy controls with similar gingival inflammation levels. The findings suggest that certain salivary microbiome features may identify periodontitis-susceptible individuals despite clinical disease resolution.

Aim To study whether cytokine levels and bacterial counts in p atients with peri-implantitis reflect clinical treatment outcome following non-surgical management. Materials and Methods Luminex magnet bead technology and checkerboard DNA–DNA hybridization were used to assess treatment outcome after treatment at the implant with the most severe peri-implantitis in 41 participants. Results Study group mean age was 40.3 years (SD ± 9.9). Stable treatment outcome after 6 months (no further bone loss, probing pocket depth decrease ≥0.5 mm, no bleeding/suppuration) was identified in 9 of 41 (22%) participants. Peri-implant crevicular fluid (PICF) levels were also lower for Il-1β (P

Made available in DSpace on 2018-12-11T17:12:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-08-01 Introduction This clinical study was conducted to correlate the microbiological profile and levels of endotoxins found in primary endodontic infection with the presence of clinical features and to evaluate the removal of microorganisms and endotoxins using rotary, reciprocating, and hybrid systems for biomechanical preparation. Methods Thirty single root canals with primary endodontic infection were evaluated with signs and symptoms and were randomly divided into 3 groups according to the instrumentation system used (n = 10) as follows: rotary Mtwo instruments (VDW, Munich, Germany) with 8 files, the reciprocating Reciproc system (VDW) with a single file, and Genius hybrid instruments with 3 files (1 rotary and 2 reciprocating files) with irrigation using 24 mL 2.5% sodium hypochlorite. Samples were collected before (S1) and after instrumentation (S2) before being submitted to microbiological culture (colony-forming units/mL) and the checkerboard DNA-DNA hybridization test. Endotoxins were quantified using the limulus amebocyte lysate assay. Results Microbiological culture showed statistical differences in the reduction of colony-forming units/mL with all systems tested (P

A pleomorphic, Gram-negative, rod-shaped, indole-, oxidase- and catalase-negative, non-spore-forming, non-motile bacterium was originally isolated in 1992 from moribund, seawater farmed Atlantic salmon with multifocal tissue necrosis. Strain AVG 2115(T) displayed considerable similarities with Streptobacillus moniliformis, one of the two etiological agents of rat bite fever, and has been stored as Streptobacillus sp. NCIMB 703044(T). On the basis of 16S rRNA gene sequence analyses, this strain displayed >99 % sequence similarities with uncultured bacterial clones from the digestive tracts of marine mammals, followed by Sneathia sanguinegens CCUG 41628(T) (92.7 %), 'Sneathia amnii' Sn35 (92.5 %), Caviibacter abscessus CCUG 39713(T) (92.2 %), Streptobacillus rattiOGS16(T) (91.3 %), Streptobacillus notomytis AHL 370-1(T) (91.2 %), S. moniliformis DSM 12112(T) (91.0 %), Streptobacillus fells 131000547(T) (90.9 %) and Streptobacillus hongkongensis DSM 26322(T) (89.7 %). Sequence similarities to all other taxa were below 89 %. Phylogenetic analysis for strain NCIMB 703044(T) revealed highly similar results for gyrB, groEL and recA nucleotide and deduced amino acid sequence analyses independent of the employed treeing method. Average nucleotide identities (ANI) for complete genomes ranged from 66.00 % to 72.08 % between strain NCIMB 703044(T) and the type strains of Sebaldella termitidis, Leptotrichia buccalis, Streptobacillus moniliformis, Sneathia sanguinegens and Caviibacter abscessus. Chemotaxonomic and physiological data of strain NCIMB 703044(t) were in congruence with closely related members of the family Leptotrichiaceae, represented by highly similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis was capable to clearly discriminate strain NCIMB 703044(T) from all currently described taxa of the family Leptotrichiaceae. On the basis of these data we propose the novel taxon Oceanivirga salmonicida gen. nov. sp. nov. with the type strain AVG 2115(T) (= NCIMB 7030441(T)) (=DSM 101867(T)). The G+C content is 25.40 %, genome size is 1.77 Mbp.

Summary Leptotrichia buccalis ATCC 14201 is a gram-negative, anaerobic rod-shaped bacterium resident in oral biofilm at the tooth surface. The sequenced genome of this organism reveals three contiguous genes at loci: Lebu_1525, Lebu_1526 and Lebu_1527. The translation products of these genes exhibit significant homology with phospho-α-glucosidase (Pagl), a regulatory protein (GntR) and a phosphoenol pyruvate-dependent sugar transport protein (EIICB), respectively. In non-oral bacterial species, these genes comprise the sim operon that facilitates sucrose isomer metabolism. Growth studies showed that L. buccalis fermented a wide variety of carbohydrates, including four of the five isomers of sucrose. Growth on the isomeric disaccharides elicited expression of a 50-kDa polypeptide comparable in size to that encoded by Lebu_1525. The latter gene was cloned, and the expressed protein was purified to homogeneity from Escherichia coli TOP10 cells. In the presence of two cofactors, NAD+ and Mn2+ ions, the enzyme readily hydrolyzed p-nitrophenyl-α-glucopyranoside 6-phosphate (pNPαG6P), a chromogenic analogue of the phosphorylated isomers of sucrose. By comparative sequence alignment, immunoreactivity and signature motifs, the enzyme can be assigned to the phospho-α-glucosidase (Pagl) clade of Family 4 of the glycosyl hydrolase super family. We suggest that the products of Lebu_1527 and Lebu_1525, catalyze the phosphorylative translocation and hydrolysis of sucrose isomers in L. buccalis, respectively. Four genetically diverse, but 16S rDNA-related, species of Leptotrichia have recently been described: L. goodfellowii, L. hofstadii, L. shahii and L. wadei. The phenotypic traits of these new species, with respect to carbohydrate utilization, have also been determined.

Leptotrichia buccalis (Robin 1853) Trevisan 1879 is the type species of the genus, and is of phylogenetic interest because of its isolated location in the sparsely populated and neither taxonomically nor genomically adequately accessed family 'Leptotrichiaceae' within the phylum 'Fusobacteria'. Species of Leptotrichia are large, fusiform, non-motile, non-sporulating rods, which often populate the human oral flora. L. buccalis is anaerobic to aerotolerant, and saccharolytic. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of the order 'Fusobacteriales' and no more than the second sequence from the phylum 'Fusobacteria'. The 2,465,610 bp long single replicon genome with its 2306 protein-coding and 61 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

A lipopolysaccharide (LPS) from Leptotrichia buccalis LI 1 not sedimentable by ultracentrifugation at 100,000 × g was isolated, using gel filtration and ion-exchange chromatography. The final preparations were similar to the corresponding sedimented fraction, both serologically and with respect to the qualitative chemical composition, but had a far higher neutral sugar: fatty acid ratio (about 20:1) than the sedimented LPS (about 0.7:1).

Aim To examine the microbiological status of primary endodontic infections in teeth with and without a sinus tract. Methodology Samples were collected by means of a size 15 H-type file and two sterile paper points from 30 cases of primary endodontic infections with (n = 15) or without (n = 15) a sinus tract. The presence of 40 bacterial species was determined by the checkerboard DNA-DNA hybridization method. Results The species found at the highest levels and prevalence were Fusobacterium nucleatum sp. vincentii, Porphyromonas gingivalis, Veillonella parvula, Enterococcus faecalis, Campylobacter gracilis and Neisseria mucosa. Total bacterial counts were similar between teeth with (44 × 105) and without (50 × 105) a sinus tract (t-test: P > 0.05). E. faecalis, Streptococcus anginosus, Capnocytophaga sputigena and Capnocytophaga gingivalis had significantly higher counts in the absence of sinus tract (Mann–Whitney test, P

OBJECTIVES: To assess the microbiota at implants diagnosed with peri-implantitis, implant mucositis, or being clinically healthy. MATERIAL AND METHODS: Clinical and microbiological data were collected from 213 subjects (mean age: 65.7+/-14) with 976 implants in function (mean: 10.8 years, SD+/-1.5). Forty species were identified by the checkerboard DNA-DNA hybridization method. RESULTS: Implant mean % plaque score was 41.8+/-32.4%. Periodontitis defined by bone loss was found in 44.9% of subjects. Implant mucositis was diagnosed in 59% and peri-implantitis in 14.9% of all cases. Neisseria mucosa, Fusobacterium nucleatum sp. nucleatum, F. nucleatum sp. polymorphum, and Capnocytophaga sputigena dominated the implant sub-mucosal microbiota and the sub-gingival microbiota at tooth sites. Implant probing pocket depth at the implant site with the deepest probing depth was correlated with levels of Eikenella corrodens (r=0.16, P

The anaerobic bacterium, Leptotrichia buccalis (Lb), belongs to the normal oral flora of humans and is seldom found in clinically significant specimens. However, on rare occasions, Lb has been isolated from blood cultures from patients with lesions of the oral mucosa, in particular from patients with neutropenia. Over a period of 6 months, Lb was isolated from blood cultures in our laboratory from 2 immunocompetent patients. The identification characteristics of Lb are described including the typical morphology at microscopy. Furthermore, we review the bacteraemic cases reported so far together with present knowledge of the pathogenicity and epidemiology of Lb.

The incidence of anaerobic bloodstream infections (BSI) in patients who underwent bone marrow transplantation (BMT) recently increased at our institution. A retrospective case-control study of patients undergoing BMT from January 1995 through December 1998 was performed to determine the microbiological characteristics, epidemiology, and outcome of anaerobic BSI and to identify independent risk factors for infection. Anaerobic BSI occurred in 23 patients, for a rate of 4 BSIs per 100 BMT procedures, and it accounted for 17% of all BSIs that occurred during the study period. Infection occurred at a mean (+/- standard deviation) of 7+/-4 days after BMT and 7+/-5 days after the onset of neutropenia. Fusobacterium nucleatum was the most frequently isolated pathogen (in 17 patients), followed by Leptotrichia buccalis (in 4), Clostridium septicum (in 1), and Clostridium tertium (in 1). Two case patients (9%) died. Severity of mucositis was an independent predictor of anaerobic BSI (odds ratio, 4.4; P=.01). Controlling mucositis is critical for the prevention of anaerobic BSI in this patient population.

Introduction The aim of the present study was to investigate the composition of the root canal microbiota in endodontic failures in order to identify and quantify these microorganisms. Methods Microbiological samples were taken from 36 root canals with persistent endodontic infection. The presence, levels, and proportions of 79 bacterial species were determined by checkerboard DNA-DNA hybridization. The Pearson correlation coefficient was used to investigate the relations between bacterial counts and clinical conditions ( P ≤ .05). Results Enterococcus faecium (36%), Streptococcus epidermidis (36%), Eubacterium saburreum (28%), Parvimonas micra (28%), Streptococcus sanguis (28%), Capnocytophaga sputigena (28%), Leptotrichia buccalis (28%), Enterococcus faecalis (28%), and Staphylococcus warneri (28%) were the most prevalent species; and there was a low prevalence of Treponema socranskii (3%), Fusobacterium periodonticum (3%), Capnocytophaga gingivalis (3%), and Spiroplasma ixodetis (3%). The highest mean levels were found for the following species: E. faecium , Dialister pneumosintes , Staphylococcus epidermidis and Helicobacter pylori . There was a statistically significant difference between the levels of gram-negative species and gram-positive species (13.5 × 10 5 vs 6.5 × 10 5 , respectively). A positive correlation was found between the area of the periapical lesion and the levels of gram-negative and rod species ( P Conclusions The microbiota from teeth with persistent apical periodontitis presents a mixed and complex profile, hosting E. faecium and S. epidermidis as the most highly prevalent species. No correlation was found between any of the species tested and clinical findings; however, periapical lesions with the largest areas presented higher counts of gram-negative and rod species.

We report here on a new case of L. buccalis bacteraemia. To our knowledge 16 other cases of L. buccalis bacteraemia have been reported in the literature, most of them in neutropenic patients. However L buccalis endocarditis does occasionally occur in non-neutropenic patients. The identification of L. buccalis is based on well known phenotypic features and confirmed by the detection of a large peak of lactic acid by gas chromatography for non volatile organic acids. L. buccalis is sensitive to a wide range of antibiotics including beta-lactam antibiotics, but it is resistant to aminoglycosides and macrolides.

Summary A previously unknown bacterial disease of seawater farmed Atlantic salmon ( Salmo salar ) has recently been described. Standard biochemical tests were carried out on the isolated bacterium but it was not possible to assign the organism to a genus on the basis of these tests. We determined the nucleotide sequence of the 16S ribosomal RNA (rRNA) gene of this organism. The phylogenetic relationship of this organism was then inferred by comparison with other published sequences and was found to be most similar to Streptobacillus moniliformis, Sebaldella termitidis (= Bacteroides termitidis ), Leptotrichia buccalis, Propionigenium modestum and to Fusobacterium spp.

Cell free extracts from a wide variety of Gram-negative, anaerobic, non-sporeforming rods were screened for dehydrogenase enzymes of carbohydrate and nitrogen metabolism. Four enzymes, malate dehydrogenase, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were found to be of diagnostic importance. The ‘Bacteroides fragilis’ group possessed all four enzymes whereas the ‘B. melaninogenicus-B. oralis’ group and the asaccharolytic, pigmented species were characterised by the presence of only malate and glutamate dehydrogenases. The saccharolytic and asaccharolytic pigmented species could be differentiated by the wide difference in pH optimum for malate dehydrogenase. Fusobacterium species and Leptotrichia buccalis both possessed only glutamate dehydrogenase, but there were differences in enzyme activity between both taxa. Other genera such as Anaerorhabdus, Megamonas, Mitsuokella. Rikenella, Sebaldella and Tissierella had characteristic enzyme profiles. These enzymatic data are of diagnostic value within the Bacteroidaceae and may serve as useful markers for studying the ecological interrelationships of these bacteria.Keywords: Bacteroidaceae; Dehydrogenases; Enzyme patterns.

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 Hedstroen-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.

Intra-amniotic Leptotrichia buccalis has not been previously associated with adverse pregnancy outcome. We report a case of chorioamnionitis and pregnancy loss associated with this primarily oral commensal. We review Leptotrichia buccalis and other oral commensals that have been identified in cases of intraamniotic infection.

BackgroundAortic aneurysms are common vascular conditions that cause considerable morbidity and mortality. Understanding of the mechanisms involved in the pathogenesis of the condition remains limited. Recently, infection has been suggested as possible contributor in the development of the disease. The aim of the present study was to examine aortic aneurysms for the presence of bacterial DNA using polymerase chain reaction (PCR) targeting the 16S ribosomal RNA (rRNA) gene, followed by cloning and sequencing.MethodsUniversal eubacterial primers were used to amplify 16S rRNA bacterial genes in 10 specimens from arterial walls of aortic aneurysms. Subsequently, PCR amplicons were cloned into Escherichia coli and sequencing of the cloned inserts was used to determine species identity or closest relatives by comparison with known sequences in GenBank.ResultsSequences of Stenotrophomonas spp., including S. maltophilia (formerly Pseudomonas homology group V) were detected in six aneurysm samples. Propionibacterium acnes was identified in five samples, and Brevundimonas diminuta (formerly P. diminuta) in four samples. Other species previously assigned to the Pseudomonas genus such as Comamonas testosteroni, Delftia acidovorans, Burkholderia cepacia, Herbaspirillum sp., and Acidovorax sp. were also detected. Some clones fell into other environmental species, including Methylobacterium sp. and Bradyrhizobium elkanii, and others represented bacteria that have not yet been cultivated. DNA sequences from oral bacteria, including Streptococcus sanguinis, Tannerella forsythia, and Leptotrichia buccalis were detected. Sequences from Prevotella melaninogenica and Lactobacillus delbrueckii, which are commonly found in both mouth and gastrointestinal tract, were also detected. Additional species included Dermacoccus spp. and Corynebacterium vitaeruminis.ConclusionsA wide variety of bacteria, including oral bacteria, was found to colonize aortic aneurysms and may play a role in their development. Several of these microorganisms have not yet been cultivated.Clinical RelevanceAlthough Chlamydophila pneumoniae has been detected in aneurysmal walls, its exact role in the condition remains inconclusive. Overall, there is scarce information about the role of microorganisms in aneurysmal disease. In the present study, we used molecular genetics to detect a diversity of bacteria in arterial walls of aortic aneurysms. The presence of multiple microorganisms in aneurysmal disease may have implications for chemoprophylaxis and antibiotic treatment if directed only at C. pneumoniae.

Background: Previous studies have shown differences in the mean proportions of subgingival species in samples from periodontitis subjects in different countries, which may relate to differences in diet, genetics, disease susceptibility and manifestation. The purpose of the present investigation was to determine whether there were differences in the subgingival microbiotas of Swedish and American subjects who exhibited periodontal health or minimal periodontal disease. Method: One hundred and fifty eight periodontally healthy or minimally diseased subjects (N Sweden=79; USA=79) were recruited. Subjects were measured at baseline for plaque, gingivitis, BOP, suppuration, pocket depth and attachment level at 6 sites per tooth. Subgingival plaque samples taken from the mesial aspect of each tooth at baseline were individually analyzed, in one laboratory, for their content of 40 bacterial species using checkerboard DNA–DNA hybridization (total samples=4345). % DNA probe counts comprised by each species was determined for each site and averaged across sites in each subject. Significance of differences in proportions of each species between countries was determined using ancova adjusting for age, mean pocket depth, gender and smoking status. p values were adjusted for multiple comparisons. Cluster analysis was performed to group subjects based on their subgingival microbial profiles using a chord coefficient and an average unweighted linkage sort. Results: On average, all species were detected in samples from subjects in both countries. After adjusting for multiple comparisons, 5 species were in significantly higher adjusted mean percentages in Swedish than American subjects: Actinomyces naeslundii genospecies 1 (9.7, 3.3); Streptococcus sanguis (2.5, 1.2); Eikenella corrodens (1.7, 1.0); Tannerella forsythensis (3.5, 2.3) and Prevotella melaninogenica (6.3, 1.8). Leptotrichia buccalis was in significantly higher adjusted mean percentages in American (5.5) than Swedish subjects (3.0). Cluster analysis grouped 121 subjects into 8 microbial profiles. Twenty four of the 40 test species examined differed significantly among cluster groups. Five clusters were dominated by American subjects and 2 clusters by Swedish subjects. Fifty eight of 79 (73%) of the Swedish subjects fell into 1 cluster group dominated by high proportions of A. naeslundii genospecies 1, Prevotella nigrescens, T. forsythensis and P. melaninogenica. Other clusters were characterized by high proportions of Actinomyces gerencseriae, Veillonella parvula, Capnocytophaga gingivalis, Prevotella intermedia, Eubacterium saburreum, L. buccalis and Neisseria mucosa. Conclusions: The microbial profiles of subgingival plaque samples from Swedish and American subjects who exhibited periodontal health or minimal disease differed. The heterogeneity in subgingival microbial profiles was more pronounced in the American subjects, possibly because of greater genetic and microbiologic diversity in the American subjects sampled.

Sixty strains of Gram-negative, anaerobic, rod-shaped bacteria from human sources initially assigned to Leptotrichia buccalis (n=58) and ‘Leptotrichia pseudobuccalis’ (n=2) have been subjected to polyphasic taxonomy. Full-length 16S rDNA sequencing, DNA–DNA hybridization, RAPD, SDS-PAGE of whole-cell proteins, cellular fatty acid analysis and enzymic/biochemical tests supported the establishment of four novel Leptotrichia species from this collection, Leptotrichia goodfellowii sp. nov. (type strain LB 57T=CCUG 32286T=CIP 107915T), Leptotrichia hofstadii sp. nov. (type strain LB 23T=CCUG 47504T=CIP 107917T), Leptotrichia shahii sp. nov. (type strain LB 37T=CCUG 47503T=CIP 107916T) and Leptotrichia wadei sp. nov. (type strain LB 16T=CCUG 47505T=CIP 107918T). Light and electron microscopy showed that the four novel species were Gram-negative, non-spore-forming and non-motile rods. L. goodfellowii produced arginine dihydrolase, β-galactosidase, N-acetyl-β-glucosaminidase, arginine arylamidase, leucine arylamidase and histidine arylamidase. L. shahii produced α-arabinosidase. L. buccalis and L. goodfellowii fermented mannose and were β-galactosidase-6-phosphate positive. L. goodfellowii, L. hofstadii and L. wadei were β-haemolytic. L. buccalis fermented raffinose. With L. buccalis, L. goodfellowii showed 3·8–5·5 % DNA–DNA relatedness, L. shahii showed 24·5–34·1 % relatedness, L. hofstadii showed 27·3–36·3 % relatedness and L. wadei showed 24·1–35·9 % relatedness. 16S rDNA sequencing demonstrated that L. hofstadii, L. shahii, L. wadei and L. goodfellowii each formed individual clusters with 97, 96, 94 and 92 % similarity, respectively, to L. buccalis.

In a polyphasic approach to improve the taxonomy of Leptotrichia we have recently demonstrated heterogeneity in the enzymatic/biochemical reactions and cellular fatty acid content of Leptotrichia isolates. In the present study, SDS-PAGE of whole-cell proteins and random amplified polymorphic DNA (RAPD) analyses were used to further characterize and distinguish these isolates of which 57 had been assigned as Leptotrichia buccalis and two as ‘Leptotrichia pseudobuccalis’. The phylogenetically closely related Sebaldella termitidisT and Fusobacterium perfoetensT were included for comparison. Two major protein bands at approximately 30–40 and 70–80 kDa, respectively, were characteristic for almost all Leptotrichia strains examined as well as for S. termitidisT while F. perfoetensT exhibited only the 70–80 kDa major band. Numerical data analysis of the gel patterns obtained with SDS-PAGE assigned most of the strains into 12 clusters at 70% similarity. On RAPD gels with the primer OPA 3 most of the strains shared only one band at approximately 0.40–0.50 kb. With OPA 5 almost all the strains showed two major bands at approximately 0.40 and 0.85 kb, respectively. Numerical analysis of RAPD gels at 70% similarity assigned most strains into ten clusters when the OPA 3 had been used and into 14 clusters with OPA 5. Accordingly, both SDS-PAGE and RAPD demonstrated considerable heterogeneity among Leptotrichia isolates. OPA 5 provided better strain distinction than did OPA 3.Keywords: Leptotrichia, cellular proteins, SDS-PAGE, RAPD.

The bacteria on the dorsum of the tongue are the most frequent cause of oral malodor; however, the bacterial flora of the tongue has not been well defined. Although recent studies have used DNA probes to detect the presence of certain periodontal pathogens, cultural studies have been limited because of the complexity of the flora of the tongue dorsum. The purpose of this study was to grow and to identify maximum numbers of capnophylic Gram-negative bacilli and anaerobic micro-organisms by culturing tongue samples on to several selective and non-selective media. The most frequently isolated species included Peptostreptococcus anaerobius, Collinsella aerofaciens, Eubacterium group, Actinomyces spp., Eikenella corrodens, Veillonella spp., Fusobacterium nucleatum, pigmented Prevotella spp. and Selenomonas spp. Reported for the first time are Actinomyces turicensis, Collinsella aerofaciens, Eubacterium saburreum, E. timidum, Prevotella tannerae, Campylobacter concisus, Campylobacter mucosalis, Leptotrichia buccalis, Selenomonas flueggei, and Centipeda periodontii. Species not previously reported in studies that used only molecular techniques were identified in the present study.

Phospho-beta-galactosidase (P-beta-gal; EC 3.2.1.85) is induced during growth of Leptotrichia buccalis ATCC 14201 on lactose and lactulose. The enzyme has been purified to electrophoretic homogeneity (M(r) approximately 53 kDa, pI approximately 4.8), and kinetic parameters have been determined using the chromogenic analog o-nitrophenyl-beta-D-galactopyranoside-6-phosphate as substrate. Both ATP and galactose-6-phosphate are inhibitors of P-beta-gal activity. Microsequence analysis has identified the first 32 residues from the N-terminus of the protein, and by comparative sequence alignment the enzyme can be assigned to Family 1 of the glycosylhydrolase superfamily. Polyclonal antibody against the enzyme permits the highly specific immuno-detection of P-beta-gal in cell-free extracts of L. buccalis. Although described previously in several Gram-positive species, this is the first reported purification of P-beta-gal from a Gram-negative organism.

Leptotrichia buccalis varies with respect to cellular and colonial morphology, fermentation of sugars and presence of glucosidases. It can be confused with Fusobacterium spp. and gram-positive bacilli related to Lactobacillus. The aim of the present study was to examine the heterogeneity of 60 Leptotrichia isolates, of which 58 had been assigned as L. buccalis and two as ‘Leptotrichia pseudobuccalis ’ by using enzymatic/biochemical and cellular fatty acid analyses. The rapid ID 32 A kit (API, bioMérieux, Marcy-l’Etoile, France) revealed considerable strain heterogeneity. Most frequently detected were strains producing α-glucosidase, alkaline phosphatase and β-glucosidase. Three other prominent strain clusters generated β-galactosidase-6-phosphate, β-galactosidase and α-galactosidase, respectively. β-Nacetyl- glucosaminidase was also produced by many strains. Mannose and raffinose fermentation was common and all strains were indole negative. Four strains reduced nitrate, and one strain produced urease. The diagnosis L. buccalis was achieved for 36 of the 60 (60%) Leptotrichia isolates. The MIDI (Microbial ID, Inc., Newark, DE, USA) system demonstrated that the most abundant cellular fatty acids were C16:0 FA (32–59%) and C18:1-cis 11/t9/t6 FA or an unidenti? ed compound with an equivalent chain length (ECL) of 17.8 (21–42%). C12:0 FA (3–16%), C15:0 DMA or 3-OH C14:0 FA (4–14%), C14:0 FA (3–20%), and C16:1-cis 9 FA (1–5%) were also detected. Forty-two of the strains (70%) contained C18:1-cis 9 FA (1–9%) and 37 (62%) C18:2-cis 9,12 FA (1–6%). The MIDI system came up with the diagnosis L. buccalis in 57 of the 60 isolates (95%). There was complete agreement in diagnosis between the two systems in 34 of the 60 isolates (57%). The heterogeneity in enzymatic/biochemical reactions and cellular fatty acid composition may indicate that the genus Leptotrichia contains hitherto unrecognized species.Keywords: enzymatic reactions, biochemical tests, fatty acid analysis, gas chromatography, Leptotrichia.

The aim of this study was to assess levels of oral bacteria and their correlations in paired samples of saliva and subgingival plaque in a population of adult Sudanese. Whole saliva and pooled subgingival plaque samples from six probing sites of one tooth in each jaw were obtained from 56 Sudanese adults (mean age 35.2+/-8.9 years). Levels of 24 oral bacteria in the autologous saliva and pooled plaque sample of each subject were assessed by DNA probes and checkerboard DNA-DNA hybridization. There were significantly ( Por =0.01) higher percentages of subjects withor =10(5) bacterial cells of Prevotella intermedia, Campylobacter rectus, Veillonella parvula, Streptococcus mutans, Lactobacillus acidophilus, Streptococcus anginosus, Streptococcus salivarius, and Leptotrichia buccalis and significantly ( Por =0.01) lower percentages with Treponema denticola in saliva than in subgingival plaque. The detection frequencies ator =10(6) bacterial cells were significantly higher for Selenomonas sputigena, S. anginosus, Streptococcus sanguis, and S. salivarius and significantly lower for Porphyromonas gingivalis in saliva than in subgingival plaque ( Por =0.01). Porphyromonas gingivalis, Fusobacterium nucleatum, S. sputigena, S. sanguis, and Streptococcus mitis demonstrated significant ( Por =0.05) positive correlations between their levels in plaque and saliva. This study indicates that the levels of P. gingivalis, F. nucleatum, S. sputigena, S. sanguis, and S. mitis correlate significantly in saliva and subgingival plaque and that higher accuracy of detection and assessment of the levels of these bacteria in the oral cavity may be achieved by concurrent sampling of saliva and subgingival plaque.

Four strains of fastidious gram-negative rods, thought to be Capnocytophaga species (formerly CDC group DF-1 or Bacteroides ochraceus) or CDC group DF-3 on the basis of conventional phenotypic criteria, were also analyzed for cellular fatty acid (CFA) composition. It was found that the CFA compositions of these strains were qualitatively incorrect for those taxa. Subsequently, it was determined that all four bacteria were in fact aerotolerant strains of Leptotrichia buccalis, based on biochemical reactions, CFA composition, and lactic acid as the major end product of glucose fermentation. It is recommended that, in addition to conventional cultural and biochemical criteria, all strains of Capnocytophaga or CDC group DF-3 should also be tested for metabolic end products of fermentation and CFA composition as essential adjuncts for identification.

We present the case of a 64-year-old previously healthy man admitted for long evoluating fever. Buccal examination showed severe parodontitis. Blood cultures were positive for Leptotrichia buccalis and computed abdominal tomography discovered a hepatic abscess. So far, only exceptional cases of systemic diseases associated with Leptotrichia buccalis infections have been described in immunocompetent patients. Thus, in cases of severe parodontitis and long evoluating fever, attention must be paid to secondary anaerobic infections even in immunocompetent subjects.

We describe four cases of bacteremia due to Leptotrichia buccalis (an organism that is part of the normal human oral flora) that occurred in a bone marrow transplant unit over a 3-month period. All of the patients were neutropenic, all had mucositis or esophagitis, and all were receiving antimicrobial prophylaxis with ciprofloxacin and vancomycin (drugs to which Leptotrichia is resistant). One patient died of adult respiratory distress syndrome; the others had minimal symptoms. Pulsed field gel electrophoresis of bacterial DNA digested with Sma 1 demonstrated a unique banding pattern for each isolate, indicating that the isolates belonged to distinct strains. Quantitative gas-liquid chromatography of whole-cell free fatty acids confirmed the uniqueness of the strains, obviating the need to search for a common source of infection. We postulate that this outbreak resulted from antibiotic selection pressure on the oral flora in patients who had been compromised by severe neutropenia and mucosal disruption.

Oral cancers are amongst the ten most prevalent forms of cancer worldwide and one of the most common in India. Various etiological factors like smoking, alcohol, tobacco consumption have been attributed as thmain causative factors for cancer. However, studies being carried out have revealed an emerging role of various bacteria as an etiological factor in oral cancer. Different bacteria have been proposed to induce carcinogenesis either through induction of chronic inflammation or by interference, either directly or indirectly, with eukaryotic cell cycle and signaling pathways, or by metabolism of potentially carcinogenic substances like acetaldehyde causing mutagenesis. The most common bacteria implicated include P. melaninogenica, C. gingivalis, Capnocytophaga ochracea, Eubacterium saburreum, Leptotrichia buccalis and S. mitis. This paper gives a review on the various the various bacteria involved and the possible pathway for bacterial carcinogenesis.

Leptotrichia buccalis shows a mosaic of surface structure on its outer membrane consisting of curved ridges 35 nm high and 22 nm apart, and erect on that surface. Fimbriae (common pili) are not present and nor is an S layer. The flap-like ridges consist of strings of macromolecules radiating from the cell surface. This ridge structure is not soluble in any of the usual chaotropes and can only be released when the outer membrane has been damaged or dispersed by extracting envelope preparations with 0.5% SDS at room temperature. The ridge is then found to be attached firmly to the peptidoglycan sacculus, which may be the point of origin of the structure. When so prepared the macromolecules forming the ridge can be removed from the sacculus by treatment with 6 M guanidine HCl, and SDS-PAGE analysis of the extract reveals a 210-kDa polypeptide as a major component and a 15-kDa minor component. The latter is probably a peptidoglycan-associated protein and much of it remains with the sacculus. Each string forming the ridge is of a volume consistent with being made of three elongated 210-kDa molecules, which are united in series by strong hydrophobic association and laterally with neighboring strings by slightly weaker forces. We confirm that L. buccalis causes haemagglutination and the bacteria are known to attach to various tissue cells. Human group A red blood corpuscles remove both of the proteins from solution, which supports the hypothesis that the ridges are adhesin structures. It is likely but not proven that the 210-kDa molecule is the adhesin.Key words: Leptotrichia buccalis, cell wall, S layer, protein array, adhesin.

The isolation of a Leptotrichia buccalis-like bacterium from the blood of a patient with endocarditis associated with a prosthetic aortic valve is reported. The organism was isolated from different blood cultures. It was similar but not completely identical to Leptotrichia buccalis. The microbiological identification was confirmed independently by three laboratories, including two reference institutions. The case and the characteristics of the microorganism are reported.

The guanine plus cytosine content of the DNA of Leptotrichia buccalis varied from 28.4 to 29.5 mol% (three strains). Eleven strains examined grew well under anaerobic and microaerobic conditions, but slowly in air in the presence of CO2. When examined for preformed enzymes in the APIZYM Complete Research Kit, positive reactions were obtained for several glucosidases and carboxylic ester hydrolases, and for a few peptidases.

The oral cavity of humans is the natural habitat of several different kinds of filamentous organisms, including the Gram-negative anaerobic bacterium Leptotrichia buccalis. This organism, which is the only species of the genus Leptotrichia, was among the first microorganisms to be recognizably described and drawn in the letters of Antoni van Leeuwenhoek.

The ability of three different oligonucleotide probes to identify different oral strains of Fusobacterium nucleatum was tested. Probes 119 and 214 were designed on the basis of known amino acid sequences from the N-terminal end of an outer membrane protein of F. nucleatum strain Fev1. Probe H 2.1, was a randomly cloned chromosomal DNA fragment of 2.1 kbp. The specificities of the probes were examined by testing each of them against a panel of five other species of Fusobacterium (six strains), oral and non-oral, and also against seven different oral species of six other genera. The chromosomal DNA of the bacteria to be examined was digested to completion using the restriction enzymes HincII, HindIII, EcoRI, EcoRV and Sau3AI. Digests were run on agarose gels and subjected to southern blotting before being hybridized with the probe in question. The results were confirmed by running a slot blot of genomic DNA from all of the 19 strains and hybridizing with a probe H 2.1. Probe H 2.1 turned out to be the most specific and useful of the three probes investigated. By use of this probe, interstrain identification of the six different strains of F. nucleatum could be performed and the strains could be distinguished from other oral species found in the oral cavity, such as Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Capnocytophaga sputigena, Porphyromonas (Bacteroides) gingivalis, Eikenella corrodens and Leptotrichia buccalis. The probe reacted weakly with other non-oral species of the genus Fusobacterium, and there was no problem in distinguishing between the different strains.

Leptotrichia buccalis, an anaerobic gram-negative rod, is part of the normal oral flora and has rarely been isolated from clinical material. We describe four patients with neutropenia and progressive malignancy who had symptomatic L. buccalis bacteremia, and we review an additional four cases from the literature. The mean age of the patients was 31 years (range, 7-73 years), with an equal number of males and females. The number of positive blood cultures in each case ranged from one to four (mean, two); these cultures became positive after 48-120 hours (median, 54 hours). All tested isolates were sensitive to the beta-lactam agents, clindamycin, tetracycline, and metronidazole; five of seven were sensitive to chloramphenicol; and not one was sensitive to the aminoglycosides, vancomycin, ciprofloxacin, or erythromycin. Seven patients had one or more possible portals of entry for bacteremia, including mucositis (four patients), mucositis plus esophageal lesions (two), and possible mucositis plus diverticulitis (one). L. buccalis should be considered a potential pathogen in neutropenic patients, especially when breaks in the mucosal breaks in the mucosal barriers are present.

Lesions showing clinical signs of foot-rot from 120 goats were cultured on six selective media during October 1987 to November 1988. A total of 582 strictly anaerobic microorganisms belonging to 50 different species was isolated and identified. The anaerobes most frequently isolated belonged to the following genera: Bacteroides (80%), Peptostreptococcus (63.6%), Megasphaera (40%), Fusobacterium (29.2%), Clostridium (22.5%), Propionibacterium (12.5%), Eubacterium (11.7%) and Leptotrichia (10.8%). Percentages for Acidaminococcus, Peptococcus, Tissierella, Wolinella and Veillonella were below 10%. The following species were identified in 10% or more of cases: Peptostreptococcus anaerobius (61.7%), Bacteroides buccae (51.7%), Bacteroides nodosus (42.5%), Megasphaera elsdenii (40%), Bacteroides ruminicola subsp. brevis (22.5%), Fusobacterium necrophorum (19.2%), Leptotrichia buccalis (11.7%) and Clostridium perfringens (10%). Lower percentages were obtained for the remaining species. The efficiency and selectivity of the six culture media used for isolation are discussed.