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BACKGROUND: Little is known about the airway microbiome in intubated mechanically ventilated children. We sought to characterize the airway microbiome longitudinally and in association with clinical variables and possible ventilator-associated infection (VAI). METHODS: Serial tracheal aspirate samples were prospectively obtained from mechanically ventilated subjects under 3 years old from eight pediatric intensive care units in the United States from June 2017 to July 2018. Changes in the tracheal microbiome were analyzed by sequencing bacterial 16S ribosomal RNA gene relative to subject demographics, diagnoses, clinical parameters, outcomes, antibiotic treatment, and the Ventilator-Associated InfectioN (VAIN) score. RESULTS: A total of 221 samples from 58 patients were processed and 197 samples met the >1000 reads criteria (89%), with an average of 43,000 reads per sample. The median number of samples per subject was 3 (interquartile range [IQR]: 2–5), with a median VAIN score of 2 (IQR: 1–3). Proteobacteria was the highest observed phyla throughout the intubation period, followed by Firmicutes and Actinobacteria. Alpha diversity was negatively associated with days of intubation (p = .032) and VAIN score (p = .016). High VAIN scores were associated with a decrease of Mycobacterium obuense, and an increase of Streptococcus peroris, Porphyromonadaceae family (unclassified species), Veillonella atypica, and several other taxa. No specific pattern of microbiome composition related to clinically diagnosed VAIs was observed. CONCLUSIONS: Our data demonstrate decreasing alpha diversity with increasing VAIN score and days of intubation. No specific microbiome pattern was associated with clinically diagnosed VAI.

Bacterial vaginosis (BV) is a gynecologic disorder characterized by a shift in cervicovaginal microbiota from Lactobacillus spp. dominance to a polymicrobial biofilm composed of diverse anaerobes. We utilized a well-characterized human three-dimensional cervical epithelial cell model in conjunction with untargeted metabolomics and immunoproteomics analyses to determine the immunometabolic contribution of three members of the Veillonellaceae family: Veillonella atypica, Veillonella montpellierensis and Megasphaera micronuciformis at this site. We found that Veillonella spp. infections induced significant elevation of polyamines. M. micronuciformis infections significantly increased soluble inflammatory mediators, induced moderate levels of cell cytotoxicity, and accumulation of cell membrane lipids relative to Veillonella spp. Notably, both V. atypica and V. montpellierensis infections resulted in consumption of lactate, a key metabolite linked to gynecologic and reproductive health. Collectively our approach and data provide unique insights into the specific contributions of Veillonellaceae members to the pathogenesis of BV and women’s health.

Veillonella spp. have been reported to be the most prevalent nitrate-reducing bacterial species in the oral cavity. The purpose of this study was to examine the relationship between the abundance of Veillonella spp. and nitrite production after nitrate ingestion. Bacterial samples were obtained from the tongue surfaces of 50 university students. The predominant Veillonella spp., V. atypica, V. dispar, and V. rogosae were identified and enumerated using real-time polymerase chain reaction (qPCR). Salivary nitrate and nitrite were measured before and 30, 60, and 90 min after ingestion of 100 ml of beetroot juice. Increased nitrite concentrations were observed in all participants, with a mean increase of 0.61 (0.42-1.10) mM expressed as the median (interquartile range). Veillonella atypica was detected in 40 subjects (80%), V. dispar in 48 (96%), and V. rogosae in 48 (96%), at quantities ranging from 1.3 × 102 to 2.8 × 107 CFU/ml per subject. The strengths of the correlations of the log colony forming unit (CFU) values of V. atypica, V. dispar, V. rogosae, and the log CFU value of the three species together with the increase in nitrite levels were 0.091, 0.114, -0.228, and 0.060, respectively, none of which were significant (p > 0.05). Our results indicate that the abundance of Veillonella spp. is not related to salivary nitrite production after nitrate ingestion.

Background and AimsBiliary atresia is the most common cause of liver disease and liver transplantation in children. The accumulation of bile acids in hepatocytes and the stimulation of the intestinal microbiome can aggravate the disease progression. This study investigated changes in the composition of the gut microbiota and its metabolites in biliary atresia and the possible effects of these changes on disease progression.MethodsStool samples of biliary atresia at different disease stages and matched control individuals were collected (early stage: 16 patients, 16 controls; later stage: 16 patients, 10 controls). Metagenomic sequencing was performed to evaluate the gut microbiota structure. Untargeted metabolomics was performed to detect and analyze the metabolites and bile acid composition.ResultsA disturbed gut microbiota structure occurred in the early and later stages of biliary atresia. Klebsiella, Streptococcus, Veillonella, and Enterococcus have always been dominant. The abundance of V. atypica displayed significant changes between the early and later stages of biliary atresia. Combined with clinical indicators, Spearman’s analysis showed that Klebsiella and Veillonella atypica strongly correlated with liver enzymes. Enterococcus faecium had an enormously positive relationship with lithocholic acid derivatives. Metabolites involved in tryptophan metabolism were changed in the patients with biliary atresia, which had a significant association with stool V. atypica and blood total bilirubin (p < 0.05).ConclusionsThe liver damage of biliary atresia was directly or indirectly exacerbated by the interaction of enriched Klebsiella (K. pneumoniae), Veillonella (V. atypica), and Enterococcus (E. faecium) with dysmetabolism of tryptophan and bile acid.

The human gut microbiome is linked to many states of human health and disease1. The metabolic repertoire of the gut microbiome is vast, but the health implications of these bacterial pathways are poorly understood. In this study, we identify a link between members of the genus Veillonella and exercise performance. We observed an increase in Veillonella relative abundance in marathon runners postmarathon and isolated a strain of Veillonella atypica from stool samples. Inoculation of this strain into mice significantly increased exhaustive treadmill run time. Veillonella utilize lactate as their sole carbon source, which prompted us to perform a shotgun metagenomic analysis in a cohort of elite athletes, finding that every gene in a major pathway metabolizing lactate to propionate is at higher relative abundance postexercise. Using 13C3-labeled lactate in mice, we demonstrate that serum lactate crosses the epithelial barrier into the lumen of the gut. We also show that intrarectal instillation of propionate is sufficient to reproduce the increased treadmill run time performance observed with V. atypica gavage. Taken together, these studies reveal that V. atypica improves run time via its metabolic conversion of exercise-induced lactate into propionate, thereby identifying a natural, microbiome-encoded enzymatic process that enhances athletic performance.

Background and Aims The human gastrointestinal tract harbours distinct microbial communities essential for health. Little is known about small intestinal communities, despite the small intestine playing a fundamental role in nutrient absorption and host-microbe immune homeostasis. We aimed to explore the small intestine microbial composition and metabolic potential, in the context of inflammatory bowel disease [IBD]. Methods Metagenomes derived from faecal samples and extensive phenotypes were collected from 57 individuals with an ileostomy or ileoanal pouch, and compared with 1178 general population and 478 IBD faecal metagenomes. Microbiome features were identified using MetaPhAn2 and HUMAnN2, and association analyses were performed using multivariate linear regression. Results Small intestinal samples had a significantly lower bacterial diversity, compared with the general population and, to a lesser extent, IBD samples. Comparing bacterial composition, small intestinal samples clustered furthest from general population samples and closest to IBD samples with intestinal resections. Veillonella atypica, Streptococcus salivarius, and Actinomyces graevenitzii were among the species significantly enriched in the small intestine. Predicted metabolic pathways in the small intestine are predominantly involved in simple carbohydrate and energy metabolism, but also suggest a higher pro-inflammatory potential. Conclusions We described the bacterial composition and metabolic potential of the small intestinal microbiota. The colonic microbiome of IBD patients, particularly with intestinal resections, showed resemblance to that of the small intestine. Moreover, several features characterising the small intestinal microbiome have been previously associated with IBD. These results highlight the importance of studying the small intestinal microbiota to gain new insight into disease pathogenesis.

Veillonella species are commensal bacteria of the human oral, gut, and vaginal microbiota that are rarely identified as clinically relevant pathogens. Here, we describe a novel case of Veillonella atypica bacteremia in a patient with biopsy-proven alcoholic hepatitis. Veillonella species have been correlated with disease severity and hepatic encephalopathy in liver diseases such as autoimmune hepatitis and cirrhosis. Their abundance has also been recently observed to be increased in alcoholic hepatitis, where postinflammatory infections are known to impact mortality. This case report highlights the possible clinical manifestations that result from significant gut dysbiosis in patients with severe alcoholic hepatitis. Early identification and treatment of Veillonella bacteremia in susceptible populations could be crucial to survival given this organism’s predilection for causing life-threatening infections, including meningitis, endocarditis, and osteomyelitis.

Veillonella species are one of the major anaerobes in the oral cavity and are frequently detected in both caries lesions and healthy oral microbiomes. They possess the ability to utilize lactate and convert nitrate (NO3-) into nitrite (NO2-). Recently, interest in NO2- has increased rapidly because of its beneficial effects on oral and general health; i.e., it inhibits the growth and metabolism of oral pathogenic bacteria, such as Streptococcus mutans, and lowers systemic blood pressure. However, there is only limited information about the biochemical characteristics of NO2- production by Veillonella species. We found that NO3- did not inhibit the growth of Veillonella atypica or Veillonella parvula, and it only inhibited the growth of Streptococcus mutans at a high concentration (100 mM). However, NO2- inhibited the growth of Streptococcus mutans at a low concentration (0.5 mM), while a higher concentration of NO2- (20 mM) was needed to inhibit the growth of Veillonella species. The NO2- production of Veillonella species was increased by environmental factors (lactate, acidic pH, and anaerobic conditions) and growth conditions (the presence of NO3-/NO2-), and was linked to anaerobic lactate metabolism. A stoichiometric evaluation revealed that NO3- is reduced to NO2- by accepting reducing power derived from the oxidization of lactate. These findings suggest that the biochemical characteristics of NO2- production from NO3- and its linkage with lactate metabolism in oral Veillonella species may play a key role in maintaining good oral and general health. IMPORTANCE The prevalence of dental caries is still high around the world. Dental caries is initiated when the teeth are exposed to acid, such as lactic acid, produced via carbohydrate metabolism by acidogenic microorganisms. Veillonella species, one of the major oral microorganisms, are considered to be beneficial bacteria due to their ability to convert lactic acid to weaker acids and to produce NO2- from NO3-, which is thought to be good for both oral and general health. Therefore, it is clear that there is a need to elucidate the biochemical characteristics of NO2- production in Veillonella species. The significance of our research is that we have found that lactate metabolism is linked to NO2- production in Veillonella species in the environment found in the oral cavity. This study suggests that Veillonella species are potential candidates for maintaining oral and general health.

Veillonella atypica is a bacterium that is present in the gut and the oral cavity of mammals and plays diverse roles in different niches. A recent study demonstrated that Veillonella is highly associated with marathon running and approved that V. atypica gavage improves treadmill run time in mice, revealing that V. atypica has a high biotechnological potential in improving athlete performance. However, a comprehensive analysis of the genetic diversity, function traits, and genome editing method of V. atypica remains elusive. In the present study, we conducted a systemically comparative analysis of the genetic datasets of nine V. atypica strains. The pan-genome of V. atypica consisted of 2,065 homologous clusters and exhibited an open pan-genome structure. A phylogenetic analysis of V. atypica with two different categories revealed that V. atypica OK5 was the most distant from the other eight V. atypica strains. A total of 43 orthologous genes were identified as CAZyme genes and grouped into 23 CAZyme families. The CAZyme components derived from accessory clusters contributed to the differences in the ability of the nine V. atypica strains to utilize carbohydrates. An integrated analysis of the metabolic pathways of V. atypica suggested that V. atypica strains harbored vancomycin resistance and were involved in several biosynthesis pathways of secondary metabolites. The V. atypica strains harbored four main Cas proteins, namely, CAS-Type IIIA, CAS-Type IIA, CAS-Type IIC, and CAS-Type IIID. This pilot study provides an in-depth understanding of and a fundamental knowledge about the biology of V. atypica that allow the possibility to increase the biotechnological potential of this bacterium.

We report the first case of bacteremia caused by Veillonella atypica in a morbid elderly female patient who developed obstructive pyelonephritis. She was treated with ceftriaxone and ureteral stenting; this is the first report of V. atypica infection in humans. Species identification was performed by multiplex PCR and sequencing of rpoB. The strain was susceptible to metronidazole and clindamycin but resistant to benzylpenicillin, ampicillin, ampicillin/sulbactam, and moxifloxacin.

In a recent Nature Medicine article Scheiman and colleagues studied the influence of the gut microbiome on exercise performance in elite runners and identified a performance-enhancing microbe belonging to the genus Veillonella .1 The bacterium Veillonella atypica was enriched in marathon runners after a race and inoculation of the same strain into mice increased exhaustive treadmill run time. The authors suggest that V. atypica improves run time via its metabolic conversion of exercise-induced lactate into propionate, thereby identifying a natural, microbiome-encoded enzymatic process that enhances athletic performance. Here we highlight an additional means by which Veillonella may help to achieve the same result (figure 1). Figure 1 Inorganic nitrate from endogenous and dietary sources is metabolised in humans to nitrite and then nitric oxide and other bioactive nitrogen oxides with effects on mitochondrial function and skeletal muscle contraction. Oral nitrate reducing bacteria …

Highlights • In this commentary, we discuss some controversial issues about the performance-enhancing role of the lactate-consuming bacteria Veillonella. • The relative abundance of Veillonella in human stool samples was not associated with training status or endurance performance. • In studying the effect on performance of Veillonella supplementation in mice, we found that Lactobacillus bulgaricus creates a biased control due to its interference in lactate metabolism and its influence on endurance performance. • In the absence of a vehicle-treated mouse group and ignoring the response to exercise of the native microbiota, the role of Veillonella atypica administration in maximal endurance performance in nontrained mice cannot be elucidated.

Actinomycosis is a rare, chronic granulomatous infection, frequently associated with immunocompromised states, but it can also affect healthy people. Here, we report a case of a pulmonary infection by Actinomyces odontolitycus and Veillonella atypica due to a dental caries in an immunocompetent 65‐year‐old man patient.

Veillonella atypica within the human gut converts lactate to propionate to improve marathoners’ endurance.

Purpose. This study aimed to investigate the effect of smoking on the buccal microbiome and to analyse the descriptive ability of each of the seven hypervariable regions in their 16S rRNA genes. Methodology. Microbiome compositions of 40 buccal swab samples collected from smokers (n =20) and non-smokers (n =20) were determined using 16S rRNA sequencing. Seven different 16S rRNA hypervariable regions (V2, V3, V4, V6-7, V8 and V9) in each sample were amplified using the Ion Torrent 16S Metagenomics kit and were sequenced on the Ion S5 instrument. Results. Seven hypervariable regions in the 16S rRNA gene were successfully sequenced for all samples tested. The data obtained with the V2 region was found to be informative but the consensus data generated according to a number of operational taxonomic unit reads gathered from all seven hypervariable regions gave the most accurate result. At the phylum level, no statistically significant difference was found between smokers and non-smokers whereas relative abundances of Veillonella atypica , Streptococcus australis , Prevotella melaninogenica , Prevotella salivae and Rothia mucilaginosa showed significant increases in the smoker group (P-adj=0.05). Alpha diversity results did not show a significant difference between the two groups; however, beta diversity analysis indicated that samples of smoker and non-smoker groups had a tendency to be clustered within themselves. Conclusion. The results of the current study indicate that smoking is a factor influencing buccal microbiome composition. In addition, sequencing of all seven hypervariable regions yielded more accurate results than those with any of the single variable regions.

The human gut microbiome encodes a vast metabolic repertoire with direct impacts on many aspects of host physiology, yet it is unknown whether it has any bearing on physical performance or exercise. To begin to address this question, we performed a longitudinal metagenomic analysis on runners in the 2015 Boston Marathon to identify microbiome features associated with intense physical activity. The strongest microbiome feature enriched post-marathon was an increase in the abundance of the bacterial genus Veillonella. We isolated Veillonella atypica directly from the marathon runners, and found that upon inoculation in laboratory mice in an AB/BA crossover study testing treadmill runtime to exhaustion, runtime was increased 13% in a V. atypica-dependent manner. V. atypica has a preference for lactate as its primary carbon source, and we find using shotgun metagenomic analysis in a cohort of elite athletes that every differentially expressed gene family in the pathway metabolizing lactate to the short-chain fatty acid propionate is at higher relative abundance post-exercise. Using 13C3-labeled lactate in mice we demonstrate that serum lactate crosses the epithelial barrier into the lumen of the gut. We also show that intrarectal instillation of propionate is sufficient to reproduce the increased treadmill runtime performance observed with V. atypica gavage. Taken together, these studies reveal that V. atypica improves runtime via its metabolic conversion of exercise-induced lactate into propionate, thereby identifying a natural, microbiome-encoded enzymatic process that enhances athletic performance. Disclosure A. Kostic: Stock/Shareholder; Self; DeepBiome Therapeutics, Inc., FitBiomics, Inc. Funding American Diabetes Association/Pathway to Stop Diabetes (1-17-INI-13)

Analyzing degraded evidence is an important challenge in forensic casework. Saliva remaining at a crime scene may deteriorate, due to various factors, making it difficult to identify. This study aims to clarify the efficacy of oral gram-positive and -negative bacterial DNA-based identification of saliva for analyzing highly degraded samples. Saliva samples were subjected to three different degradation treatments (heat denaturation: 40–80 °C in wet conditions; microbial decomposition: 1–5 days in humid soil; and ultraviolet (UV) irradiation: 0.01–1 J/cm2). We compared saliva markers’ detectability from the degraded samples—oral gram-positive bacterial DNA (Streptococcus salivarius and Streptococcus oralis), oral gram-negative bacterial DNA (Veillonella atypica and Prevotella maculosa) and salivary α-amylase. Oral bacterial DNA was detected using a melting curve analysis following real-time PCR. The efficacy of short tandem repeats (STR) and mitochondrial DNA (mtDNA) analyses were also compared. All oral bacterial DNA were detected with specific melting peaks from the heat-denatured samples, while neither catalytic nor immunochromatographic tests detected salivary α-amylase from the heat (80 °C) samples. The gram-positive bacterial DNA (S. salivarius and S. oralis) was detected from the microbial degradation (1–5 days) samples. In contrast, the gram-negative bacterial DNA (V. atypica and P. maculosa) and salivary α-amylase were not detected from samples treated for more than two days. UV exposure made bacterial DNA-based saliva identification difficult in a dose–dependent manner; however, UV irradiation did not influence protein-based saliva tests using salivary α-amylase as an indicator. As a result of STR and mtDNA typing, partial or null STR profiles were generated from the severely degraded (microbial (2–5 days) and UV (0.1–1 J/cm2) degradation) samples, but full mtDNA profiles were obtained from all degraded samples. The forensic applicability of bacterial DNA test evaluated, using mock case samples, indicates that the oral gram-positive bacterial DNA was more resistant to degradation than the other markers. We conclude that the oral gram-positive bacterial DNA-based examination could be useful for identifying saliva from severely environmentally-exposed forensic samples as well as mtDNA typing.

Aspiration of oral contents can lead to pneumonia, which is a major cause of death among elderly adults susceptible to swallowing impairments. Tongue microbiota are a dominant source of oral microbial populations that are ingested with saliva. This large-scale population-based study revealed variations in the tongue microbiota among community-dwelling elderly adults. The total bacterial density was independent of the conditions of teeth surrounding the tongue, whereas the microbiota composition, especially the relative abundances of predominant commensals, showed an association with tooth conditions. Our results demonstrate that the elderly with fewer teeth, poorer dental hygiene, and more dental caries experience constantly ingest more dysbiotic microbiota, which could be harmful for their respiratory health., Tongue microbiota are a dominant source of oral microbial populations that are ingested with saliva, and therefore careful attention is required for the maintenance of health of elderly adults, who are susceptible to aspiration of oral contents. This study aimed to investigate the variation in tongue microbiota among community-dwelling elderly adults. Following a dental examination, tongue coating was collected from a 15-mm-diameter circular area at the center of the tongue dorsum of 506 elderly adults aged 70 to 80 years inhabiting the town of Hisayama, Japan. The microbial composition and density were determined by a 16S rRNA gene sequencing approach using a next-generation sequencer and quantitative PCR analysis, respectively. Co-occurrence network analysis identified two cohabiting groups of predominant commensals, one of which was primarily composed of Prevotella histicola, Veillonella atypica, Streptococcus salivarius, and Streptococcus parasanguinis; these organisms have been previously associated with an increased risk of mortality due to pneumonia in the frail elderly. This bacterial group was more predominant in the elderly with fewer teeth, a higher plaque index, and more dental caries experience, whereas the total bacterial density was independent of these traits. A higher density of fungi was also observed in the elderly with these traits, as well as in individuals who wore dentures. These results suggest that elderly adults with poorer oral health swallow a more dysbiotic microbiota formed on the tongue. IMPORTANCE Aspiration of oral contents can lead to pneumonia, which is a major cause of death among elderly adults susceptible to swallowing impairments. Tongue microbiota are a dominant source of oral microbial populations that are ingested with saliva. This large-scale population-based study revealed variations in the tongue microbiota among community-dwelling elderly adults. The total bacterial density was independent of the conditions of teeth surrounding the tongue, whereas the microbiota composition, especially the relative abundances of predominant commensals, showed an association with tooth conditions. Our results demonstrate that the elderly with fewer teeth, poorer dental hygiene, and more dental caries experience constantly ingest more dysbiotic microbiota, which could be harmful for their respiratory health.

Objective: Since dentures can serve as a reservoir for halitosis-causing oral bacteria, halitosis development is a concern for denture wearers. In this study, we surveyed the prevalence of four selected halitosis-related species (Fusobacterium nucleatum, Tannerella forsythia,Veillonella atypicaandKlebsiella pneumoniae) in clinical denture plaque samples, and developed denture biofilm models for these speciesin vitroto facilitate assessment of antimicrobial treatment efficacy.Design:Denture plaque from ten healthy and ten denture stomatitis patients was screened for the presence of aforementioned four species by PCR. Biofilm formation by these halitosis-associated species on the surfaces of denture base resin (DBR) discs was evaluated by crystal violet staining and confocal laser scanning microscopy. The efficacy of denture cleanser treatment on these mono-species biofilms was evaluated by colony counting.Results:80% of the subjects in the denture stomatitis group and 60% in the healthy group contained at least one of the targeted halitosis-related species in their denture plaque. All halitosis species tested were able to form biofilms on DBR disc surfaces to varying degrees. Thesein vitromono-species resin biofilm models were used to evaluate the efficacy of denture cleansers, which exhibited differential efficacies. When forming biofilms on resin surfaces, the halitosis-related species displayed enhanced resistance to denture cleansers compared with their planktonic counterparts.Conclusion:The four selected halitosis-related bacterial species examined in this study are present on the majority of dentures. The mono-species biofilm models established on DBR discs for these species are an efficient screening tool for dental product evaluation.

Summary Dental biofilm development is a sequential process, and adherence between microbes and the salivary pellicle (adhesion) as well as among different microbes (co-adhesion or coaggregation) plays a critical role in building a biofilm community. The Veillonella species are among the most predominant species in the oral cavity and coaggregate with many initial, early, middle, and late colonizers. Similar to oral fusobacteria, they are also considered bridging species in biofilm development. However, the mechanism of this ability has yet to be reported, due to the previous lack of a genetic transformation system in the entire genus. In this study, we used our recently discovered transformable Veillonella strain, Veillonella atypica OK5, to probe the mechanism of coaggregation between Veillonella species and other oral bacteria. By insertional inactivation of all eight putative hemagglutinin genes, we identified one gene, hag1, which is involved in V. atypica coaggregation with the initial colonizers Streptococcus gordonii, Streptococcus oralis and Streptococcus cristatus, and the periodontal pathogen Porphyromonas gingivalis. The hag1 mutant also abolished adherence to human buccal cells. Inhibition assays using various chemical or physiological treatments suggest different mechanisms being involved in coaggregation with different partners. The entire hag1 gene was sequenced and shown to be the largest known bacterial hemagglutinin gene.

The oral biofilm is a multispecies community in which antagonism and mutualism coexist among friends and foes to keep an ecological balance of community members. The pioneer colonizers, such asStreptococcus gordonii, produce H2O2to inhibit the growth of competitors, like the mutans streptococci, as well as strict anaerobic middle and later colonizers of the dental biofilm. Interestingly,Veillonellaspecies, as early colonizers, physically interact (coaggregate) withS. gordonii. A putative catalase gene (catA) is found in most sequencedVeillonellaspecies; however, the function of this gene is unknown. In this study, we characterized the ecological function ofcatAfromVeillonella parvulaPK1910 by integrating it into the only transformable strain,Veillonella atypicaOK5, which iscatAnegative. The strain (OK5-catA) became more resistant to H2O2. Further studies demonstrated that thecatAgene expression is induced by the addition of H2O2or coculture withS. gordonii. Mixed-culture experiments further revealed that the transgenic OK5-catAstrain not only enhanced the growth ofFusobacterium nucleatum, a strict anaerobic periodontopathogen, under microaerophilic conditions, but it also rescuedF. nucleatumfrom killing byS. gordonii. A potential role of catalase in veillonellae in biofilm ecology and pathogenesis is discussed here.IMPORTANCEVeillonellaspecies, as early colonizers, can coaggregate with many bacteria, including the initial colonizerStreptococcus gordoniiand periodontal pathogenFusobacterium nucleatum, during various stages of oral biofilm formation. In addition to providing binding sites for many microbes, our previous study also showed thatVeillonellaproduces nutrients for the survival and growth of periodontal pathogens. These findings indicate thatVeillonellaplays an important “bridging” role in the development of oral biofilms and the ecology of the human oral cavity. In this study, we demonstrated that the reducing activity ofVeillonellacan rescue the growth ofFusobacterium nucleatumnot only under microaerophilic conditions, but also in an environment in whichStreptococcus gordoniiis present. Thus, this study will provide a new insight for future studies on the mechanisms of human oral biofilm formation and the control of periodontal diseases.

The Veillonella atypica strain OK5 was isolated from a human saliva sample and was the first strain shown to be genetically transformable in the Veillonella genus. Genetic studies using this strain have helped us gain much insight into the ecology of human oral biofilms. Here, we report the complete genome sequence of V. atypica OK5.

Oral Veillonella, Veillonella atypica, Veillonella denticariosi, Veillonella dispar, Veillonella parvula, Veillonella rogosae, and Veillonella tobetsuensis are known as early colonizers in oral biofilm formation. To investigate the role of oral Veillonella, biofilms formed by the co-culture of Streptococcus gordonii, Streptococcus mutans, Streptococcus salivarius, or Streptococcus sanguinis, with oral Veillonella were examined at the species level. The amount of biofilm formed by S. mutans, S. gordonii, and S. salivarius in the presence of the six Veillonella species was greater than that formed in the control experiments, with the exception of S. mutans with V. dispar. In contrast, in the case of biofilm formation by S. sanguinis, the presence of Veillonella species reduced the amount of the biofilm, with the exception of V. parvula and V. dispar. The time-dependent changes in the amount of biofilm and the number of planktonic cells were grouped into four patterns over the 24 combinations. Only that of S. gordonii with V. tobetsuensis showed a unique pattern. These results indicate that the mode of action of this combination differed from that of the other combinations with respect to biofilm formation. It is possible that there may be several factors involved in the interaction between Streptococcus and Veillonella species.

Indigenous oral bacteria in the tongue coating such as Veillonella have been identified as the main producers of hydrogen sulfide (H 2 S), one of the major components of oral malodor. However, there is little information on the physiological properties of H 2 S production by oral Veillonella such as metabolic activity and oral environmental factors which may affect H 2 S production. Thus, in the present study, the H 2 S-producing activity of growing cells, resting cells, and cell extracts of oral Veillonella species and the effects of oral environmental factors, including pH and lactate, were investigated. Type strains of Veillonella atypica , Veillonella dispar , and Veillonella parvula were used. These Veillonella species produced H 2 S during growth in the presence of l -cysteine. Resting cells of these bacteria produced H 2 S from l -cysteine, and the cell extracts showed enzymatic activity to convert l -cysteine to H 2 S. H 2 S production by resting cells was higher at pH 6 to 7 and lower at pH 5. The presence of lactate markedly increased H 2 S production by resting cells (4.5- to 23.7-fold), while lactate had no effect on enzymatic activity in cell extracts. In addition to H 2 S, ammonia was produced in cell extracts of all the strains, indicating that H 2 S was produced by the catalysis of cystathionine γ-lyase (EC 4.4.1.1). Serine was also produced in cell extracts of V. atypica and V. parvula , suggesting the involvement of cystathionine β-synthase lyase (EC 4.2.1.22) in these strains. This study indicates that Veillonella produce H 2 S from l -cysteine and that their H 2 S production can be regulated by oral environmental factors, namely, pH and lactate.

The aim of this study was to determine the effects of short-term xylitol gum chewing on the salivary microbiota of children. The study was a randomised, controlled, double-blind trial. Healthy children used xylitol chewing gum (xylitol group, n = 35) or sorbitol chewing gum (control group, n = 38) for 5 weeks. The daily dose of xylitol/sorbitol was approximately 6 g/day. At baseline and at the end of the test period, unstimulated and paraffin-stimulated saliva were collected. The microbial composition of the saliva was assessed using human oral microbe identification microarray (HOMIM). Mutans streptococci (MS) were plate cultured. As judged by HOMIM results, no xylitol-induced changes in the salivary microbiota took place in the xylitol group. In the control group, Veillonella atypica showed a significant decrease (p = 0.0001). The xylitol gum chewing decreased viable counts of MS in both stimulated (p = 0.006) and unstimulated (p = 0.002) saliva, but similar effects were also seen in the control group. The use of xylitol gum decreased MS, in general, but did not change the salivary microbial composition. Short-term consumption of xylitol had no impact on the composition of the salivary microbiota, but resulted in a decrease in the levels of MS.

The aim of this study was to learn whether presence of caries in an adult population was associated with a salivary bacterial profile different from that of individuals without untreated caries. Stimulated saliva samples from 621 participants of the Danish Health Examination Survey were analyzed using the Human Oral Microbe Identification Microarray technology. Samples from 174 individuals with dental caries and 447 from a control cohort were compared using frequency and levels of identified bacterial taxa/clusters as endpoints. Differences at taxon/cluster level were analyzed using Mann-Whitney's test with Benjamini-Hochberg correction for multiple comparisons. Principal component analysis was used to visualize bacterial community profiles. A reduced bacterial diversity was observed in samples from subjects with dental caries. Five bacterial taxa (Veillonella parvula, Veillonella atypica, Megasphaera micronuciformis, Fusobacterium periodontium and Achromobacter xylosoxidans) and one bacterial cluster (Leptotrichia sp. clones C3MKM102 and GT018_ot417/462) were less frequently found in the caries group (adjusted p value Solobacterium moorei and Streptococcus salivarius) and three bacterial clusters (Streptococcus parasanguinis I and II and sp. clone BE024_ot057/411/721, Streptococcus parasanguinis I and II and sinensis_ot411/721/767, Streptococcus salivarius and sp. clone FO042_ot067/755) were present at significantly higher levels (adjusted p value

Veillonella atypica, Veillonella denticariosi, Veillonella dispar, Veillonella parvula, and Veillonella rogosae have been reported to be isolated from human oral cavities. The recently detected Veillonellatobetsuensis in human tongue biofilms was proposed as a novel Veillonella sp. In this study, to determine the distribution and frequency of V. tobetsuensis, we established a method for the detection and identification of V. tobetsuensis by using polymerase chain reaction (PCR) using a species-specific primer pair. The primer pair for V. tobetsuensis was designed on the basis of the nucleotide sequence of the 70-kDa heat shock protein (dnaK) gene of V. tobetsuensis JCM 17976T (=ATCC BAA-2400T). The primer pair generated a specific PCR product for V. tobetsuensis but not for other oral Veillonella spp. With the PCR procedure using the primer pair, we could detect less than 10 ng of genomic DNA extracted from V. tobetsuensis. Thus, the PCR method using this primer pair is suitable for the specific detection and identification of V. tobetsuensis. The distribution and frequency of V. tobetsuensis were investigated by PCR using this species-specific primer pair. V. tobetsuensis was detected in 5 of 27 subjects. V. tobetsuensis was recovered from 19% (5/27) of subjects with other Veillonella species. And, prevalence of V. tobetsuensis ranged from 7.6% to 20.0% in these subjects. V. tobetsuensis is likely to coexist with other Veillonella spp. in tongue biofilm. In this study, the species-specific PCR primer pair for V. tobetsuensis was designed using partial sequences of the dnaK gene. This is the first report using a species-specific primer pair for PCR to determine the distribution and frequency of V. tobetsuensis in tongue biofilm.

Haemin/haem is one of the essential nutrients required by periodontopathogens such as Porphyromonas gingivalis to grow in vitro. In the oral cavity, this nutrient is believed to be provided by the crevicular fluid, a serum-like exudate produced during gum inflammation. However, P. gingivalis is also present in the healthy dental biofilm where inflammation is absent. This study was designed to answer the question: what organism(s) in the healthy dental biofilm provides haemin/haem to those periodontal pathogens? We report here that veillonellae, a group of bridging species in dental biofilm development, harbour a complete gene cluster for haem biosynthesis. Haemin production was detected from cell lysate, suggesting that the haem biosynthesis pathway is functional in veillonellae. Using the only transformable strain Veillonella atypica OK5, we inactivated specific key genes in the haem biosynthesis pathway. Inactivation of hemE, encoding the enzyme uroporphyrinogen decarboxylase, not only abolished haemin production but also significantly decreased OK5-supported growth of P. gingivalis. A luciferase gene reporter to the hemEHG operon demonstrated up-regulation of operon expression by P. gingivalis. Analysis of all sequenced genomes of oral bacteria in the HOMD database identified three genera (Veillonella, Propionibacterium and Aggregatibacter) that have a complete haem biosynthesis gene cluster, suggesting that they all could be potential haemin/haem providers in the dental biofilm.

Antibacterial activity of sodium citrate against tongue bacteria, especially hydrogen-sulfide (H2S) producers, was examined. Based on 16s rDNA partial sequences analysis, 691 isolates from 6 subjects were identified. In the present study, Prevotella histicola, P. melaninogenica, Veillonella atypica and V. dispar/V. parvula were detected as predominant H2S producing bacteria. Antibacterial assay showed that sodium citrate inhibited the growth of the representative strains of H2S producers at low concentrations although it similarly inhibited that of commensal bacteria, viz., Streptococcus parasanguinis and S. salivarius. When the effects of several pH conditions on antibacterial activity of sodium citrate against P. melaninogenica JCM6325T were examined, the activity was effective under neutral pH as well as under acidic conditions.

Introduction: Members of the genus Veillonella cannot be reliably distinguished by their biochemical characteristics and phenotypic features. Moreover, DNA–DNA hybridization and sequence analyses of the 16S ribosomal RNA gene including random fragment length polymorphism analysis, are complex and time-consuming procedures that are not well-suited to identifying oral species of Veillonella: Veillonella atypica, Veillonella denticariosi, Veillonella dispar, Veillonella parvula, and Veillonella rogosae. Methods: In this study, five forward primers and a reverse primer were designed for polymerase chain reaction (PCR) according to the partial sequences of the rpoB genes of these oral Veillonella species. Results: The forward primers were species-specific for these five Veillonella species, and could produce specific amplicons when used together with reverse primer and individual DNA templates of these species in PCR. These primer pairs were also found to discriminate between the respective species, and the Veillonella strains isolated from human oral cavities were successfully assigned to one of the five oral species of the genus Veillonella based on their specific products by PCR. Conclusion: A simple two-step PCR procedure using the five sets of primer pairs developed in the present study is a rapid and reliable method for the identification of the recognized oral Veillonella species.

The predominant Veillonella spp. were isolated from the dorsum surface of the tongues of 11 healthy adults and identified to species level using rpoB sequencing because 16S ribosomal RNA sequence analysis does not reliably differentiate between all members of this genus. In all, 253 isolates were identified and the mean proportion (+/- SE) of Veillonella spp. per sample was 16.2 (+/- 3.6) with a range of 3.0% to 36.3% of the total anaerobic colony count. The predominant species were Veillonella atypica (10/11), Veillonella dispar (9/11) and Veillonella rogosae (8/11) because they were isolated from the majority of subjects. Veillonella parvula was isolated from only one subject while Veillonella dentocariosi and Veillonella montpelleriensis were not isolated from any subject.

Background : Reduced salivation is considered a major clinical feature of most but not all cases of primary Sjogren’s syndrome (pSS). Reduced saliva flow may lead to changes in the salivary microbiota. These changes have mainly been studied with culture that typically recovers only 65% of the bacteria present. Objective : This study was to use high throughput sequencing, covering both cultivated and not-yet-cultivated bacteria, to assess the bacterial microbiota of whole saliva in pSS patients with normal salivation. Methods : Bacteria of whole unstimulated saliva from nine pSS patients with normal salivation flow and from nine healthy controls were examined by high throughput sequencing of the hypervariable region V1V2 of 16S rRNA using the 454 GS Junior system. Raw sequence reads were subjected to a species-level, reference-based taxonomy assignment pipeline specially designed for studying the human oral microbial community. Each of the sequence reads was BLASTN-searched against a database consisting of reference sequences representing 1,156 oral and 12,013 non-oral species. Unassigned reads were then screened for high-quality non-chimeras and subjected to de novo species-level operational taxonomy unit (OTU) calling for potential novel species. Downstream analyses, including alpha and beta diversities, were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline. To reveal significant differences between the microbiota of control saliva and Sjogren’s saliva, a statistical method introduced in Metastats www.metastats.cbcb.umd.edu was used. Results : Saliva of pSS patients with normal salivation had a significantly higher frequency of Firmicutes compared with controls ( p =0.004). Two other major phyla, Synergistetes and Spirochaetes, were significantly depleted in pSS ( p =0.001 for both). In addition, we saw a nearly 17% decrease in the number of genera in pSS (25 vs. 30). While Prevotella was almost equally abundant in both groups (25% in pSS and 22% in controls), about a twofold increase in pSS of Streptococcus (28% vs. 17%) and Veillonella (26% vs. 12%) was detected. Prevotella melaninogenica was the major species in controls (13%) while Veillonella atypica and the Veillonella parvula groups dominated in patient samples (14 and 14%). The scarcity in bacterial species in pSS compared with controls was also demonstrated by alpha and beta diversity analyses, as well as read abundance depicted in a phylogenetic tree. Conclusions : While Firmicutes was significantly higher in pSS patients than in controls, Synergistetes and Spirochaetes were significantly lower. The number of bacterial genera and species was also lower. These data showed that microbial dysbiosis is another key characteristic of pSS whole saliva which can occur independent of hyposalivation. Keywords: primary Sjogren’s syndrome; whole saliva; normal salivary flow rate; bacteria; high throughput sequencing; pyrosequencing (Published: 20 October 2016) Citation: Journal of Oral Microbiology 2016, 8: 31119 - http://dx.doi.org/10.3402/jom.v8.31119

Using an alternative sigma factor ecf3 as target, we successfully established the first markerless mutagenesis system in the Veillonella genus. This system will be a valuable tool for mutagenesis of multiple genes for gene function analysis as well as for gene regulation studies in Veillonella.

Anaerobic gram-negative cocci belonging to the genus Veillonella are currently subdivided into eight species. Among them, four have been isolated either from human flora or from clinical samples during infectious processes: Veillonella atypica, V. dispar, V. parvula and V. montpellierensis. To date, the four other species have only been found in animals. In particular, V. ratti and V. criceti, since their characterization, have been exclusively reported from mouth and intestine of rodents. We report here for the first time a human isolate belonging to the V. ratti-V. criceti group recovered in mixed aerobic-anaerobic flora from a semen sample in a 24-year-old man attending the urology unit of our hospital for infertility. Identification of the isolate was based on 16S ribosomal DNA and dnaK gene sequencing. Since then, two strains of Veillonella sp. recovered from semen samples remained unidentified to the species level, and 16S rDNA-based phylogenetic analysis revealed that they might represent novel taxa within the genus Veillonella. Taken together, these observations suggest that host restriction may not exist in the genus Veillonella and that bacterial diversity remains underestimated both within this genus and in human semen.

Microbial communities within the human oral cavity are dynamic associations of more than 500 bacterial species that form biofilms on the soft and hard tissues of the mouth. Understanding the development and spatial organization of oral biofilms has been facilitated by the use of in vitro models. We used a saliva-conditioned flow cell, with saliva as the sole nutritional source, as a model to examine the development of multispecies biofilm communities from an inoculum containing the coaggregation partners Streptococcus gordonii , Actinomyces naeslundii , Veillonella atypica , and Fusobacterium nucleatum . Biofilms inoculated with individual species in a sequential order were compared with biofilms inoculated with coaggregates of the four species. Our results indicated that flow cells inoculated sequentially produced biofilms with larger biovolumes compared to those biofilms inoculated with coaggregates. Individual-species biovolumes within the four-species communities also differed between the two modes of inoculation. Fluorescence in situ hybridization with genus- and species-specific probes revealed that the majority of cells in both sequentially and coaggregate-inoculated biofilms were S. gordonii , regardless of the inoculation order. However, the representation of A. naeslundii and V. atypica was significantly higher in biofilms inoculated with coaggregates compared to sequentially inoculated biofilms. Thus, these results indicate that the development of multispecies biofilm communities is influenced by coaggregations preformed in planktonic phase. Coaggregating bacteria such as certain streptococci are especially adapted to primary colonization of saliva-conditioned surfaces independent of the mode of inoculation and order of addition in the multispecies inoculum. Preformed coaggregations favor other bacterial strains and may facilitate symbiotic relationships.

Clinical microorganisms isolated during a 5-year study in our hospital that could not be identified by conventional criteria were studied by 16S rRNA gene sequence analysis. Each isolate yielded a ≥1,400-bp sequence containing Actinobaculum massiliae ,” “ Candidatus Actinobaculum timonae,” Paenibacillus sanguinis , “ Candidatus Bacteroides massiliae,” Chryseobacterium massiliae , “ Candidatus Chryseobacterium timonae,” Paenibacillus massiliensis , “ Candidatus Peptostreptococcus massiliae,” “ Candidatus Prevotella massiliensis,” Rhodobacter massiliensis , and “ Candidatus Veillonella atypica” were identified. Sixteen species were obtained from humans for the first time. Our results show the important role that 16S rRNA gene sequence-based bacterial identification currently plays in recognizing unusual and emerging bacterial diseases.

Among the seven species characterized within the genus Veillonella, three (Veillonella dispar, Veillonella parvula and Veillonella atypica) have so far been isolated from human flora and during infectious processes. Sequencing and analysis of 16S rDNA (rrs) has been described as the best method for identification of Veillonella strains at the species level since phenotypic characteristics are unable to differentiate between species. rrs sequencing for the three species isolated from humans showed more than 98 % identity between them. Four rrs copies were found in the reference strains and in all the clinical isolates studied. The sequences of each rrs were determined for the clinical strain ADV 360.1, and they showed a relatively high level of heterogeneity (1·43 %). In the majority of cases, polymorphic positions corresponded to nucleotides allowing differentiation between the three species isolated from humans. Moreover, variability observed between rrs copies was higher than that between 16S rDNA sequences of V. parvula and V. dispar. Phylogenetic analysis showed that polymorphism between rrs copies affected the position of strain ADV 360.1 in the tree. Variable positions occurred in stems and loops belonging to variable and hypervariable regions of the 16S rRNA secondary structure but did not change the overall structure of the 16S rRNA. PCR-RFLP experiments performed on 27 clinical isolates of Veillonella sp. suggested that inter-rrs heterogeneity occurs widely among the members of the genus Veillonella. These results, together with the lack of phenotypic criteria for species differentiation, give preliminary arguments for unification of V. dispar and V. parvula.

The molybdenum-containing iron-sulfur flavoprotein xanthine dehydrogenase from the anaerobic bacterium Veillonella atypica has been purified approximately 800-fold with a yield of approximately 40% and a specific activity of approximately 70 micromol ferricyanide reduced x min(-1) x mg protein(-1) with xanthine as electron donor, which corresponds to approximately 30 micromol xanthine oxidized x min(-1) x mg protein(-1) with methylene blue as electron acceptor. The 129-kDa enzyme was a non-covalent heterotrimer with large (82.4 kDa), medium (28.5 kDa) and small (18.4 kDa) subunits. The N-termini of the small and medium polypeptides of V. atypica xanthine dehydrogenase and the corresponding domains of eukaryotic xanthine dehydrogenases were similar, whereas the N-terminus of the large polypeptide was unrelated to eukaryotic xanthine dehydrogenases. The enzyme contained 0.86 atoms Mo, 1.75 atoms Fe, 1.61 atoms acid-labile sulfur and 0.68 molecules FAD/molecule, which corresponds to a 1:2.0:1.9:0.8 molar ratio. Acid hydrolysis revealed 0.95 mol CMP and 0.80 mol AMP/mol xanthine dehydrogenase. After treatment of the enzyme with iodoacetamide, di(carboxamidomethyl)molybdopterin cytosine dinucleotide was identified, which indicates that molybdopterin cytosine dinucleotide is the organic portion of the V. atypica xanthine dehydrogenase molybdenum cofactor. The enzyme and its molybdenum cofactor occurred in a 1:1 molar ratio. Xanthine dehydrogenases from eukaryotic sources are characterized by a domain structure and the presence of duplicate copies of two types of [2Fe-2S) clusters. In contrast, the xanthine dehydrogenase from V. atypica had a heterotrimeric subunit structure and a single [2Fe-2S] cluster. In addition, the enzyme indicates the presence of a molybdopterin dinucleotide as a constituent of a xanthine dehydrogenase molybdenum cofactor.