Microbial population shift and metabolic characterization of silver diamine fluoride treatment failure on dental caries.

The objective of this pilot study was to describe the microbial profiles present in the plaque and saliva of children who continued to develop new carious lesions following treatment with silver diamine fluoride ("nonresponders") compared to caries active, caries-free, and children immediately receiving SDF treatment for untreated caries in order to identify potential microbial differences that may relate to a re-incidence of caries. Saliva and plaque samples from infected and contralateral sites were obtained from twenty children who were either caries free, had active carious lesions, were caries active and received SDF treatment immediately before sampling, or had previously received SDF treatment and developed new caries. In total, 8,057,899 Illumina-generated sequence reads from 60 samples were obtained. Reads were processed using the Quantitative Insights Into Microbial Ecology pipeline. Group differences were assessed using Analysis of Variance Models and Tukey Honest Significant Differences. To identify significant taxa between treatment groups, Linear discriminant analysis Effect Size (LefSe) and Analysis of Differential Abundance Taking Sample Variation Into Account were used. Differential abundant analysis indicated that members of the Lachnospiraceae family were significantly enriched in non-responders and the genus Tannerella and species Granulicatella adiances were also highly abundant in this group. LefSe analysis between non-responders and SDF-treated groups revealed that genera Leptotrichia and Granulicatella were enriched in non-responders. We observed the highest abundance of phosphotransferase system and lowest abundance of lipopolysaccharide synthesis in non-responders. The microbiome in dental biofilms is responsible for initiation and progression of dental caries. SDF has been shown to be effective in arresting the progression carious lesions, in part due to its antimicrobial properties. Findings suggest that the differential abundance of select microbiota and specific pathway functioning in individuals that present with recurrent decay after SDF treatment may contribute to a potential failure of silver diamine fluoride to arrest dental caries. However, the short duration of sample collection following SDF application and the small sample size emphasize the need for further data and additional analysis.

Identification of Leptotrichia goodfellowii infective endocarditis by next-generation sequencing of 16S rDNA amplicons.

The oral aerotolerant anaerobe Leptotrichia goodfellowii is an unusual cause of endocarditis and is amenable to treatment with ß-lactam antibiotics. Because this organism is difficult to identify by conventional methods, molecular detection is a key diagnostic modality. Broad-range 16S rDNA PCR followed by Sanger sequencing constitute the first-line molecular approach, yet poor DNA quality, contaminating DNA, or low template quantity make identification challenging. Here we report a case of culture-negative, aortic and mitral valve endocarditis in a 66-yr-old woman with a history of cardiomyopathy, atrial fibrillation with intracardiac pacer, poor dentition, and recent tooth infection. In this case, 16S rDNA amplicon Sanger sequencing was not sufficient for pathogen identification because of interfering DNA, but deconvolution of the clinical sample using reflexive next-generation amplicon sequencing enabled confident identification of a single pathogenic organism, L. goodfellowii The patient developed a sigmoid colon perforation and died despite additional surgical treatment. Most Leptotrichia endocarditis cases have been subacute and have been successfully treated with antibiotics, with or without valve replacement. This case highlights both an unusual etiologic agent of endocarditis, as well as the rational utilization of advanced molecular diagnostics tools for characterizing serious infections.

Shock séptico por Leptotrichia buccalis en paciente neutropénico por quimioterapia / Septic shock caused by Leptotrichia buccalis in a neutropenic patient secondary to chemotherapy

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Airway microbiome composition correlates with lung function and arterial stiffness in an age-dependent manner.

OBJECTIVE: To investigate age-associated changes in airway microbiome composition and their relationships with lung function and arterial stiffness among genetically matched young and elderly pairs. METHODS: Twenty-four genetically linked family pairs comprised of younger (≤40 years) and older (≥60 years) healthy participants were recruited (Total n = 48). Lung function and arterial stiffness (carotid-femoral pulse wave velocity (PWV) and augmentation index (AIx)) were assessed. Sputum samples were collected for targeted 16S rRNA gene amplicon sequencing and correlations between microbiome composition, lung function and arterial stiffness were investigated. RESULTS: Elderly participants exhibited reductions in lung function (FEV1 (p<0.001), FVC (p<0.001) and percentage FEV1/FVC (p = 0.003)) and a 1.3-3.9-fold increase in arterial stiffness (p<0.001) relative to genetically related younger adults. Elderly adults had a higher relative abundance of Firmicutes (p = 0.035) and lower relative abundance of Proteobacteria (p = 0.014), including specific genera Haemophilus (p = 0.024) and Lautropia (p = 0.020) which were enriched in the younger adults. Alpha diversity was comparable between young and elderly pairs (p>0.05) but was inversely associated with lung function (FEV1%Predicted and FVC %Predicted) in the young (p = 0.006 and p = 0.003) though not the elderly (p = 0.481 and p = 0.696). Conversely, alpha diversity was negatively associated with PWV in the elderly (p = 0.01) but not the young (p = 0.569). Specifically, phylum Firmicutes including the genus Gemella were correlated with lung function (FVC %Predicted) in the young group (p = 0.047 and p = 0.040), while Fusobacteria and Leptotrichia were associated with arterial stiffness (PWV) in the elderly (both p = 0.004). CONCLUSION: Ageing is associated with increased Firmicutes and decreased Proteobacteria representation in the airway microbiome among a healthy Asian cohort. The diversity and composition of the airway microbiome is independently associated with lung function and arterial stiffness in the young and elderly groups respectively. This suggests differential microbial associations with these phenotypes at specific stages of life with potential prognostic implications.

Emergent genital infection by Leptotrichia trevisanii.

We report the first case of an association between Leptotrichia trevisanii and an episode of pelvic inflammatory disease (PID) and the second case of the isolation of this infection in the cervical canal. A 45-yr-old woman was admitted to our emergency department with clinical and radiological signs and symptoms compatible with an episode of PID. She was hospitalized for intravenous antibiotic control and treatment and the subsequent surgical drainage of abscesses. Cultures were taken throughout the process, but only cultures from cervical canal exudate were positive, with the growth of L. trevisanii species. It appears important to carry out a complete microbiological screening, not limited to conventional agents, on adequate clinical samples to detect possible infectious agents that may be missed in these cases.

Bacteriemia por Leptotrichia trevesanii. Descripción de un caso / Bacteremia by Leptotrichia trevesanii. Case report

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Leptrotrichia Amnionii, an emerging pathogen of postpartum endometritis.

Leptotrichia amnionii, a recently described fastidious gram-negative anaerobic bacterium, is an opportunistic pathogen of the female urogenital tract. We report a rare case of L. amnionii bacteremia in a patient with postpartum endometritis which was successfully treated by amoxicilline-clavunalate. There is more and more evidence that L. amnonii has its role in Pelvic Inflammatory Disease and postpartum endometritis.

Chorioamnionitis due to Leptotrichia trevisanii.

Very long fusiform gram-negative bacilli were observed after Gram staining of amniotic fluid from a 36-year-old multigravida woman. At 24 hours, pure, abundant growth of smooth, gray, only slightly convex catalase-positive and oxidase-negative colonies measuring about 2 mm were observed. Growth was greater in anaerobic than in aerobic conditions. The bacterium was identified as Leptotrichia trevisanii by matrix-assisted laser desorption ionization time of flight mass spectrometry. Ampicillin and gentamicin were prescribed for chorioamnionitis, and vaginal prostaglandins were administered to terminate the pregnancy. The patient remained afebrile throughout 48 hours and was discharged. Microscopic examination of the placenta revealed severe acute chorioamnionitis with a maternal inflammatory response and abundant bacillary-shaped microorganisms. To our knowledge, this isolate constitutes the first reported case of chorioamnionitis caused by L. trevisanii.

Cytological and cytomorphometric characteristics of buccal mucosa cells from smokeless tobacco users.

BACKGROUND: Use of smokeless tobacco (ST) is increasing in many communities. We investigated whether ST alters the cytological and cytomorphometric features of buccal mucosa cells. METHODS: Twenty male participants who had used Nicotiana rustica Linn.-containing ST (Maras powder) for at least 10 years, and 20 healthy male controls who did not use ST, were included in this study. After rinsing the mouth with water, samples were taken using a toothbrush from the buccal mucosa of subjects in both groups. Samples were gently spread over a glass slide. After applying a cytofixative spray, the Papanicolaou method was used to stain the slides. The presence of dysplasia, dyskeratosis, parakeratosis, hyperkeratosis, hypergranulosis, karyorrhexis, and pyknosis was evaluated by light microscopy, as were the increment amount of candida, cocco-bacillus, and Leptotrichia buccalis. Cytomorphometric analysis was performed and at least 20 cells with well-defined borders were evaluated from each slide, and the cellular diameter (CD), nuclear diameter (ND), and nucleus/cytoplasm (N/C) ratio of the cells were analyzed using a 60× objective. RESULTS: Other than the presence of dysplasia and candida, all measured cytological parameters were significantly higher in the ST users than in the non-ST users. Furthermore, CD was lower while nuclear/cytoplasmic ratio was higher in the ST users than in those non-ST users. CONCLUSION: Cytological changes associated with the use of ST, include dyskeratosis, parakeratosis, hyperkeratosis, hypergranulosis, karyorrhexis, pyknosis together with increase in the bacterial population of cocco-bacillus and L. buccalis. There were no significant differences in patients with dysplasia in spite of reduction of CD, increased nuclear size and N/C ratio.

First report of Sneathia sanguinegens together with Mycoplasma hominis in postpartum prosthetic valve infective endocarditis: a case report.

BACKGROUND: The presence of more than one bacterial agent is relatively rare in infective endocarditis, although more common in prosthetic cases. Molecular diagnosis from a removed heart tissue is considered a quick and effective way to diagnose fastidious or intracellular agents. CASE PRESENTATION: Here we describe the case of postpartum polymicrobial prosthetic valve endocarditis in a young woman. Sneathia sanguinegens and Mycoplasma hominis were simultaneously detected from the heart valve sample using broad range 16S rRNA polymerase chain reaction (PCR) followed by sequencing while culture remained negative. Results were confirmed by independent PCR combined with denaturing gradient gel electrophoresis. Before the final agent identification, the highly non-compliant patient left from the hospital against medical advice on empirical intravenous treatment with aminopenicillins, clavulanate and gentamicin switched to oral amoxycillin and clavulanate. Four months after surgery, no signs of inflammation were present despite new regurgitation and valve leaflet flail was detected. However, after another 5 months the patient died from sepsis and recurrent infective endocarditis of unclarified etiology. CONCLUSIONS: Mycoplasma hominis is a rare causative agent of infective endocarditis. To the best of our knowledge, presented case is the first report of Sneathia sanguinegens detected in this condition. Molecular techniques were shown to be useful even in polymicrobial infective endocarditis samples.

Bacteriemia por Leptotrichia trevisanii en una paciente sometida a trasplante alogénico de médula ósea / Bacteremia due to Leptotrichia trevisanii after an allogeneic bone marrow transplant

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Laboratory Identification of Leptotrichia Species Isolated From Bacteremia Patients at a Single Institution.

We describe the laboratory identification of Leptotrichia species from clinical isolates collected over a six-year period. Five isolates from blood cultures were identified as Leptotrichia species. Gram stain showed large, fusiform, gram-negative or -variable bacilli. Identification based on biochemical testing was unsuccessful; however, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry proved to be a useful tool for identifying Leptotrichia species to the genus level. Species level identification was successfully achieved by using 16S ribosomal RNA gene sequencing.

Impact of Periodic Presumptive Treatment for Bacterial Vaginosis on the Vaginal Microbiome among Women Participating in the Preventing Vaginal Infections Trial.

Background: Evidence suggests that specific vaginal bacteria associated with bacterial vaginosis (BV) may increase the risk of adverse health outcomes in women. Among women participating in a randomized, double-blinded trial, we assessed the effect of periodic presumptive treatment (PPT) on detection of select vaginal bacteria. Methods: High-risk women from the United States and Kenya with a recent vaginal infection received intravaginal metronidazole 750 mg plus miconazole 200 mg or placebo for 5 consecutive nights each month for 12 months. Vaginal fluid specimens were collected via polyester/polyethylene terephthalate swabs every other month and tested for bacteria, using quantitative polymerase chain reaction (PCR) assays targeting the 16S ribosomal RNA gene. The effect of PPT on bacterium detection was assessed among all participants and stratified by country. Results: Of 234 women enrolled, 221 had specimens available for analysis. The proportion of follow-up visits with detectable quantities was lower in the PPT arm versus the placebo arm for the following bacteria: BVAB1, BVAB2, Atopobium vaginae, Leptotrichia/Sneathia, and Megasphaera. The magnitude of reductions was greater among Kenyan participants as compared to US participants. Conclusions: Use of monthly PPT for 1 year reduced colonization with several bacteria strongly associated with BV. The role of PPT to improve vaginal health should be considered, and efforts to improve the impact of PPT regimens are warranted.

High Prevalence of Leptotrichia amnionii, Atopobium vaginae, Sneathia sanguinegens, and Factor 1 Microbes and Association of Spontaneous Abortion among Korean Women.

Objective. The purpose of this study was to (i) determine the cervical microbial composition in different abortion samples and to (ii) investigate the correlation between spontaneous abortion and cervical microbes in Korean women. Methods. We collected cervical swabs from women who had never undergone abortion (N = 36), had spontaneous abortion (N = 23), and had undergone induced abortion (N = 88) and subjected those samples to 16S rRNA pyrosequencing. Further, factor analysis and correlation between cervical microbiota and spontaneous abortion were evaluated by logistic regression analysis. Results. In spontaneous abortion women, 16 S rRNA gene sequences showed significant increases in Atopobium vaginae, Megasphaera spp., Gardnerella vaginalis, Leptotrichia amnionii, and Sneathia sanguinegens compared to women in nonabortion group. In multivariate logistic regression analysis, A. vaginae (OD = 11.27; 95% = 1.57-81), L. amnionii (OD = 11.47; 95% = 1.22-107.94), S. sanguinegens (OD = 6.89; 95% = 1.07-44.33), and factor 1 microbes (OD = 16.4; 95% = 1.88-42.5) were strongly associated with spontaneous abortion. Conclusions. This study showed a high prevalence of L. amnionii, A. vaginae, S. sanguinegens, and factor 1 microbes in spontaneous abortion and association with spontaneous abortion in Korean women.

[Analysis of causes and whole microbial structure in a case of rampant caries].

OBJECTIVE: To analyze the whole microbial structure in a case of rampant caries to provide evidence for its prevention and treatment. METHODS: Clinical samples including blood, supragingival plaque, plaque in the caries cavity, saliva, and mucosal swabs were collected with the patient's consent. The blood sample was sent for routine immune test, and the others samples were stained using Gram method and cultured for identifying colonies and 16S rRNA sequencing. DNA was extracted from the samples and tested for the main cariogenic bacterium (Streptococcus mutans) with qPCR, and the whole microbial structure was analyzed using DGGE. RESULTS: The patient had a high levels of IgE and segmented neutrophils in his blood. Streptococci with extremely long chains were found in the saliva samples under microscope. Culture of the samples revealed the highest bacterial concentration in the saliva. The relative content of hemolytic bacterium was detected in the samples, the highest in the caries cavity; C. albicans was the highest in the dental plaque. In addition, 33 bacterial colonies were identified by VITEK system and 16S rDNA sequence phylogenetic analysis, and among them streptococci and Leptotrichia wade were enriched in the dental plaque sample, Streptococcus mutans, Fusobacterium nucleatum, and Streptococcus tigurinus in the caries cavity, and Lactobacillus in the saliva. S. mutans was significantly abundant in the mucosal swabs, saliva and plaque samples of the caries cavity as shown by qPCR. Compared to samples collected from a healthy individual and another two patients with rampant caries, the samples from this case showed a decreased bacterial diversity and increased bacterial abundance shown by PCR-DGGE profiling, and multiple Leptotrichia sp. were detected by gel sequencing. CONCLUSION: The outgrowth of such pathogenic microorganisms as S. mutans and Leptotrichia sp., and dysbiosis of oral microbial community might contribute to the pathogenesis of rampant caries in this case.

Leptotrichia goodfellowii Infection: Case Report and Literature Review.

Leptotrichia is a pencil-shaped Gram-negative rod and is known as an uncommon pathogen of bacteremia in immunocompromised patients. However, because culture and identification of Leptotrichia species is difficult in clinical laboratories, Leptotrichia goodfellowii is grossly underestimated as a human pathogen. In this study, we report a case of L. goodfellowii bacteremia in an immunocompetent patient and review 5 previously reported cases describing infection with L. goodfellowii.

Pyrosequencing Analysis of Subgingival Microbiota in Distinct Periodontal Conditions.

Subgingival microorganisms are potentially associated with periodontal diseases. However, changes in the subgingival microbiota during the progress of periodontal diseases are poorly understood. In this study, we analyzed bacterial communities in the subgingival paper point samples from 32 Korean individuals with no sign of disease, gingivitis, or periodontitis using 454 FLX Titanium pyrosequencing. A total of 256,113 reads representing 26 phyla, 433 genera, and 1,016 species were detected. Bacteroidetes, Fusobacteria, Synergistetes, and Spirochaetes were the abundant phyla in periodontitis subjects, whereas Firmicutes and Proteobacteria were identified as the dominant phyla in the gingivitis and healthy subjects, respectively. Although high levels of Porphyromonas, Fusobacterium, Fretibacterium, Rothia, Filifactor, and Treponema genera were observed in the periodontitis subjects, Streptococcus, Capnocytophaga, Leptotrichia, and Haemophilus genera were found at high frequency in the gingivitis subjects. Species including Porphyromonas gingivalis, Fusobacterium nucleatum, and Fretibacterium fastidiosum were significantly increased in periodontitis subjects. On the other hand, Streptococcus pseudopneumoniae, Haemophilus parainfluenzae, and Leptotrichia hongkongensis were preferentially observed in the gingivitis subjects. Intriguingly, the halophile Halomonas hamiltonii was revealed as a predominant species in the healthy subjects. Based on Fast UniFrac analysis, distinctive bacterial clusters were classified for the healthy, gingivitis, and periodontitis state. The current findings might be useful for understanding the pathogenesis, diagnosis, and treatment of periodontal diseases.

Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women.

OBJECTIVE: The objective of the study was to evaluate the upper genital tract (UGT) presence of vaginal bacterial species using sensitive molecular methods capable of detecting fastidious bacterial vaginosis (BV)-associated bacteria. STUDY DESIGN: Vaginal swabs were collected prior to hysterectomy. The excised uterus was sterilely opened and swabs collected from the endometrium and upper endocervix. DNA was tested in 11 quantitative polymerase chain reaction (PCR) assays for 12 bacterial species: Lactobacillus iners, L crispatus, L jensenii, Gardnerella vaginalis, Atopobium vaginae, Megasphaera spp, Prevotella spp, Leptotrichia/Sneathia, BVAB1, BVAB2, BVAB3, and a broad-range16S ribosomal ribonucleic acid gene assay. Endometrial fluid was tested with Luminex and an enzyme-linked immunosorbent assay for cytokines and defensins and tissue for gene expression of defensins and cathelicidin. RESULTS: We enrolled 58 women: mean aged 43±7 years, mostly white (n=46; 79%) and BV negative (n=43; 74%). By species-specific quantitative PCR, 55 (95%) had UGT colonization with at least 1 species (n=52) or were positive by 16S PCR (n=3). The most common species were L iners (45% UGT, 61% vagina), Prevotella spp (33% UGT, 76% vagina) and L crispatus (33% UGT, 56% vagina). Median quantities of bacteria in the UGT were lower than vaginal levels by 2-4 log10 ribosomal ribonucleic acid gene copies per swab. There were no differences in the endometrial inflammatory markers between women with no bacteria, Lactobacillus only, or any BV-associated species in the UGT. CONCLUSION: Our data suggest that the endometrial cavity is not sterile in most women undergoing hysterectomy and that the presence of low levels of bacteria in the uterus is not associated with significant inflammation.

Microbial diversity in persistent root canal infections investigated by checkerboard DNA-DNA hybridization.

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 < .05). 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.