Effects of oral probiotic supplements on vaginal microbiota during pregnancy: a randomised, double-blind, placebo-controlled trial with microbiome analysis.

OBJECTIVE: To determine the effects on the vaginal microbiota of an oral probiotic preparation administered from early pregnancy. DESIGN: Randomised, double blind, placebo-controlled trial. SETTING: Four maternity units in the UK. POPULATION: Women aged 16 years or older recruited at 9-14 weeks' gestation. METHODS: Participants were randomly allocated to receive oral capsules of probiotic containing Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 each at 2.5 × 109 colony-forming units (CFUs) or placebo once daily from recruitment until the end of pregnancy. MAIN OUTCOME MEASURE: Rates of bacterial vaginosis (BV, defined as Nugent score ≥7) at 18-20 weeks' gestation compared by logistic regression adjusted for possible confounders. RESULTS: The primary analysis included 78% (238/304) of participants who initially consented (probiotic group 123, placebo group 115). Of these participants, 95% (227/238) reported an intake of 93% or more of the required number of capsules. The rates of BV did not differ between groups at 18-20 weeks' gestation (15% (19/123) in the probiotic group vs. 9% (10/115) in the placebo group, adjusted odds ratio 1.82, 95% confidence interval 0.64-5.19). There were also no differences between the groups in the proportion of women colonised with the probiotic strains, Escherichia coli, group B streptococci or other vaginal microbiota. There were no differences in the alpha diversity or composition of the bacterial communities between or within the probiotic and placebo groups at 9-14 and 18-20 weeks' gestation. CONCLUSIONS: Oral probiotics taken from early pregnancy did not modify the vaginal microbiota. TWEETABLE ABSTRACT: The oral probiotic preparation used in this study does not prevent BV in pregnant women.

Intravenous antibiotics in preterm infants have a negative effect upon microbiome development throughout preterm life.

BACKGROUND: Intestinal dysbiosis is implicated in the origins of necrotising enterocolitis and late-onset sepsis in preterm babies. However, the effect of modulators of bacterial growth (e.g. antibiotics) upon the developing microbiome is not well-characterised. In this prospectively-recruited, retrospectively-classified, case-control study, high-throughput 16S rRNA gene sequencing was combined with contemporaneous clinical data collection, to assess the within-subject relationship between antibiotic administration and microbiome development, in comparison to preterm infants with minimal antibiotic exposure. RESULTS: During courses of antibiotics, diversity progression fell in comparison to that seen outside periods of antibiotic use (-0.71units/week vs. + 0.63units/week, p < 0.01); Enterobacteriaceae relative abundance progression conversely rose (+ 10.6%/week vs. -8.9%/week, p < 0.01). After antibiotic cessation, diversity progression remained suppressed (+ 0.2units/week, p = 0.02). CONCLUSIONS: Antibiotic use has an acute and longer-lasting impact on the developing preterm intestinal microbiome. This has clinical implications with regard to the contribution of antibiotic use to evolving dysbiosis, and affects the interpretation of existing microbiome studies where this effect modulator is rarely accounted for.

Mechanisms of Atomization from Rotary Dental Instruments and Its Mitigation.

Since the onset of coronavirus disease 2019, the potential risk of dental procedural generated spray emissions (including aerosols and splatters), for severe acute respiratory syndrome coronavirus 2 transmission, has challenged care providers and policy makers alike. New studies have described the production and dissemination of sprays during simulated dental procedures, but findings lack generalizability beyond their measurements setting. This study aims to describe the fundamental mechanisms associated with spray production from rotary dental instrumentation with particular focus on what are currently considered high-risk components-namely, the production of small droplets that may remain suspended in the room environment for extended periods and the dispersal of high-velocity droplets resulting in formites at distant surfaces. Procedural sprays were parametrically studied with variables including rotation speed, burr-to-tooth contact, and coolant premisting modified and visualized using high-speed imaging and broadband or monochromatic laser light-sheet illumination. Droplet velocities were estimated and probability density maps for all laser illuminated sprays generated. The impact of varying the coolant parameters on heating during instrumentation was considered. Complex structured sprays were produced by water-cooled rotary instruments, which, in the worst case of an air turbine, included droplet projection speeds in excess of 12 m/s and the formation of millions of small droplets that may remain suspended. Elimination of premisting (mixing of coolant water and air prior to burr contact) resulted in a significant reduction in small droplets, but radial atomization may still occur and is modified by burr-to-tooth contact. Spatial probability distribution mapping identified a threshold for rotation speeds for radial atomization between 80,000 and 100,000 rpm. In this operatory mode, cutting efficiency is reduced but sufficient coolant effectiveness appears to be maintained. Multiple mechanisms for atomization of fluids from rotatory instrumentation exist, but parameters can be controlled to modify key spray characteristics during the current crisis.

A systematic review of droplet and aerosol generation in dentistry.

OBJECTIVES: This review aimed to identify which dental procedures generate droplets and aerosols with subsequent contamination, and for these, characterise their pattern, spread and settle. DATA RESOURCES: Medline(OVID), Embase(OVID), Cochrane Central Register of Controlled Trials, Scopus, Web of Science and LILACS databases were searched for eligible studies from each database's inception to May 2020 (search updated 11/08/20). Studies investigating clinical dental activities that generate aerosol using duplicate independent screening. Data extraction by one reviewer and verified by another. Risk of bias assessed through contamination measurement tool sensitivity assessment. STUDY SELECTION: A total eighty-three studies met the inclusion criteria and covered: ultrasonic scaling (USS, n = 44), highspeed air-rotor (HSAR, n = 31); oral surgery (n = 11), slow-speed handpiece (n = 4); air-water (triple) syringe (n = 4), air-polishing (n = 4), prophylaxis (n = 2) and hand-scaling (n = 2). Although no studies investigated respiratory viruses, those on bacteria, blood-splatter and aerosol showed activities using powered devices produced greatest contamination. Contamination was found for all activities, and at the furthest points studied. The operator's torso, operator's arm and patient's body were especially affected. Heterogeneity precluded inter-study comparisons but intra-study comparisons allowed construction of a proposed hierarchy of procedure contamination risk: higher (USS, HSAR, air-water syringe, air polishing, extractions using motorised handpieces); moderate (slow-speed handpieces, prophylaxis, extractions) and lower (air-water syringe [water only] and hand scaling). CONCLUSION: Gaps in evidence, low sensitivity of measures and variable quality limit conclusions around contamination for procedures. A hierarchy of contamination from procedures is proposed for challenge/verification by future research which should consider standardised methodologies to facilitate research synthesis. CLINICAL SIGNIFICANCE: This manuscript addresses uncertainty around aerosol generating procedures (AGPs) in dentistry. Findings indicate a continuum of procedure-related aerosol generation rather than the common binary AGP or non-AGP perspective. The findings inform discussion around AGPs and direct future research to support knowledge and decision making around COVID-19 and dental procedures.

Cultivation of a Synergistetes strain representing a previously uncultivated lineage.

Subgingival plaque samples obtained from human subjects with periodontitis, shown to include previously uncultivable members of the phylum Synergistetes, were used to inoculate Cooked Meat Medium (CMM). The presence of Cluster A (uncultivable) Synergistetes was monitored by fluorescent in situ hybridization (FISH) and quantitative PCR (Q-PCR). Cluster A Synergistetes were found to grow in CMM in co-culture with other plaque bacteria and growth was stimulated by the addition of mucin and serum. Plaque samples were also used to inoculate Blood Agar (BA) plates and growth of Cluster A Synergistetes was revealed after anaerobic incubation, by colony hybridization with specific probes. Surface growth on the plates in regions identified by colony hybridization was harvested and used to inoculate fresh plates, thus enriching for Cluster A Synergistetes. Cross-streaks of other plaque bacteria were also used to stimulate Synergistetes growth. In the early passages, no discrete Synergistetes colonies were seen, but after eight passages and the use of cross-streaks of other bacteria present in the enriched community, colonies arose, which consisted solely of Cluster A Synergistetes cells, as determined by 16S rRNA gene PCR and cloning. This is the first report of the successful culture of a member of the uncultivable branch of this phylum.

Profiling of Oral Bacterial Communities.

The profiling of bacterial communities by the sequencing of housekeeping genes such as that encoding the small subunit ribosomal RNA has revealed the extensive diversity of bacterial life on earth. Standard protocols have been developed and are widely used for this application, but individual habitats may require modification of methods. This review discusses the sequencing and analysis methods most appropriate for the study of the bacterial component of the human oral microbiota. If possible, DNA should be extracted from samples soon after collection. If samples have to be stored for practical reasons, precautions to avoid DNA degradation on freezing should be taken. A critical aspect of profiling oral bacterial communities is the choice of region of the 16S rRNA gene for sequencing. The V1-V2 region provides the best discrimination between species of the genus Streptococcus, the most common genus in the mouth and important in health and disease. The MiSeq platform is most commonly used for sequencing, but long-read technologies are now becoming available that should improve the resolution of analyses. There are a variety of well-established data analysis pipelines available, including mothur and QIIME, which identify sequence reads as phylotypes by comparing them to reference data sets or grouping them into operational taxonomic units. DADA2 has improved sequence error correction capabilities and resolves reads to unique variants. Two curated oral 16S rRNA databases are available: HOMD and CORE. Expert interpretation of community profiles is required, both to detect the presence of contaminating DNA, which is commonly present in the reagents used in analysis, and to differentiate oral and nonoral bacteria and determine the significance of findings. Despite advances in shotgun whole-genome metagenomic methods, oral bacterial community profiling via 16S rRNA sequence analysis remains a valuable technique for the characterization of oral bacterial populations.

Perinatal inflammation influences but does not arrest rapid immune development in preterm babies.

Infection and infection-related complications are important causes of death and morbidity following preterm birth. Despite this risk, there is limited understanding of the development of the immune system in those born prematurely, and of how this development is influenced by perinatal factors. Here we prospectively and longitudinally follow a cohort of babies born before 32 weeks of gestation. We demonstrate that preterm babies, including those born extremely prematurely (<28 weeks), are capable of rapidly acquiring some adult levels of immune functionality, in which immune maturation occurs independently of the developing heterogeneous microbiome. By contrast, we observe a reduced percentage of CXCL8-producing T cells, but comparable levels of TNF-producing T cells, from babies exposed to in utero or postnatal infection, which precedes an unstable post-natal clinical course. These data show that rapid immune development is possible in preterm babies, but distinct identifiable differences in functionality may predict subsequent infection mediated outcomes.

Diversity and morphology of members of the phylum "synergistetes" in periodontal health and disease.

Members of the phylum "Synergistetes" have frequently been detected in the human oral cavity at sites of dental disease, but they have rarely been detected in studies of oral health. Only two oral "Synergistetes" taxa are cultivable. The aims of this study were to investigate the diversity of "Synergistetes" in the oral cavity, to establish whether "Synergistetes" taxa are more strongly associated with periodontitis than with oral health, and to visualize unculturable "Synergistetes" in situ. Sixty samples (saliva, dental plaque, and mucosal swabs) were collected from five subjects with periodontitis and five periodontally healthy controls. Using phylum-specific 16S rRNA gene primers, "Synergistetes" were identified by PCR, cloning, and sequencing of 48 clones per PCR-positive sample. Subgingival plaque samples were labeled with probes targeting rRNA of unculturable oral "Synergistetes" using fluorescent in situ hybridization (FISH). Analysis of 1,664 clones revealed 12 "Synergistetes" operational taxonomic units (OTUs) at the 99% sequence identity level, 5 of which were novel. "Synergistetes" OTU 4.2 was found in significantly more subjects with periodontitis than controls (P = 0.048) and was more abundant in subgingival plaque at diseased sites than at healthy sites in subjects with periodontitis (P = 0.019) or controls (P = 0.019). FISH analysis revealed that unculturable oral "Synergistetes" cells were large curved bacilli. The human oral cavity harbors a diverse population of "Synergistetes." "Synergistetes" OTU 4.2 is associated with periodontitis and may have a pathogenic role.

Horizontal and Vertical Transfer of Oral Microbial Dysbiosis and Periodontal Disease.

One of the hallmark features of destructive periodontal disease, well documented over the last 50 y, is a change to the quantitative and qualitative composition of the associated microbiology. These alterations are now generally viewed as transformational shifts of the microbial populations associated with health leading to the emergence of bacterial species, which are only present in low abundance in health and a proportionate decrease in the abundance of others. The role of this dysbiosis of the health associated microbiota in the development of disease remains controversial: is this altered microbiology the driving agent of disease or merely a consequence of the altered environmental conditions that invariably accompany destructive disease? In this work, we aimed to address this controversy through controlled transmission experiments in the mouse in which a dysbiotic oral microbiome was transferred either horizontally or vertically into healthy recipient mice. The results of these murine studies demonstrate conclusively that natural transfer of the dysbiotic oral microbiome from a periodontally diseased individual into a healthy individual will lead to establishment of the dysbiotic community in the recipient and concomitant transmission of the disease phenotype. The inherent resilience of the dysbiotic microbial community structure in diseased animals was further demonstrated by analysis of the effects of antibiotic therapy on periodontally diseased mice. Although antibiotic treatment led to a reversal of dysbiosis of the oral microbiome, in terms of both microbial load and community structure, dysbiosis of the microbiome was reestablished following cessation of therapy. Collectively, these data suggest that an oral dysbiotic microbial community structure is stable to transfer and can act in a similar manner to a conventional transmissible infectious disease agent with concomitant effects on pathology. These findings have implications to our understanding of the role of microbial dysbiosis in the development and progression of human periodontal disease.

First Cultivation of Health-Associated Tannerella sp. HOT-286 (BU063).

Despite significant advances in recent years in culture-independent molecular microbiology methods, the detailed study of individual bacterial species still relies on having pure cultures in the laboratory. Yet, more than a third of the approximately 700 bacterial taxa found in the human oral cavity are as yet uncultivated in vitro. One such taxon, Tannerella sp. HOT-286 (phylotype BU063), is the focus of much interest since it is associated with periodontal health, while Tannerella forsythia, its closest phylogenetic neighbor, is strongly associated with periodontal disease. HOT-286, however, has remained uncultivated despite the efforts of several research groups, spanning over a decade. The aim of this study was to cultivate Tannerella sp. HOT-286. A heavily diluted sample of subgingival plaque was inoculated onto culture plates supplemented with siderophores (pyoverdines-Fe complex or desferricoprogen) or a neat plaque suspension. After 8 d of anaerobic incubation, microcolonies and colonies showing satellitism were passaged onto fresh culture plates cross-streaked with potential helper strains or onto cellulose-acetate membranes placed over lawn cultures of helper strains. Subcultured colonies were identified by 16S rRNA gene sequencing, and purity was confirmed by sequencing 20 clones per library prepared from a single colony. Three colonies of interest (derived from pyoverdines- and plaque-supplemented plates) were identified as Tannerella sp. HOT-286. The isolates were found to be incapable of independent growth, requiring helpers such as Propionibacterium acnes and Prevotella intermedia for stimulation, with best growth on membranes over "helper" lawns. A representative isolate was subjected to phenotypic characterization and found to produce a range of glycosidic and proteolytic enzymes. Further comparison of this novel "periodontal health-associated" taxon with T. forsythia will be valuable in investigating virulence factors of the latter and possible health benefits of the former.

The oral microbiome - an update for oral healthcare professionals.

For millions of years, our resident microbes have coevolved and coexisted with us in a mostly harmonious symbiotic relationship. We are not distinct entities from our microbiome, but together we form a 'superorganism' or holobiont, with the microbiome playing a significant role in our physiology and health. The mouth houses the second most diverse microbial community in the body, harbouring over 700 species of bacteria that colonise the hard surfaces of teeth and the soft tissues of the oral mucosa. Through recent advances in technology, we have started to unravel the complexities of the oral microbiome and gained new insights into its role during both health and disease. Perturbations of the oral microbiome through modern-day lifestyles can have detrimental consequences for our general and oral health. In dysbiosis, the finely-tuned equilibrium of the oral ecosystem is disrupted, allowing disease-promoting bacteria to manifest and cause conditions such as caries, gingivitis and periodontitis. For practitioners and patients alike, promoting a balanced microbiome is therefore important to effectively maintain or restore oral health. This article aims to give an update on our current knowledge of the oral microbiome in health and disease and to discuss implications for modern-day oral healthcare.

Class-specific antibodies to Streptococcus mutans in human serum, saliva and breast milk.

Previous techniques used for the detection and quantitation of antibodies in body fluids may be inappropriate where only small volumes are available, or may not be sensitive enough to detect low levels of specific antibodies. An indirect ELISA technique has successfully been employed to estimate class-specific antibody levels to Streptococcus mutans in serum and secretions in a group of mothers and their neonates, and an attempt has been made to relate such levels to the presence or absence of active caries in the mothers. A high maternal serum IgG antibody level appears to exert a protective effect against dental caries. Antibody levels in maternal saliva and colostrum/breast milk showed no differences between the 2 groups. The presence of active caries in mothers was associated with an elevated IgA antibody level in neonatal saliva. Although ELISA permitted the detection of low levels of antibody in the small volumes of neonatal saliva collected, a further increase in sensitivity and specificity of the assay would be advantageous.

Comparison of solid media for cultivation of anaerobes.

Two commercial agar media for the cultivation of anaerobes were compared with four other media for their ability to support the growth of a wide range of anaerobes from clinical specimens of subgingival plaque. Fastidious anaerobe agar (FAA, Lab M) and anaerobe agar (GAA, Gibco) allowed better growth of the pure cultures than the other media. FAA recovered the highest numbers of bacteria from subgingival plaque specimens which were composed predominantly of anaerobes. GAA performed poorly with these samples. It is concluded that FAA seemed to be superior to the other media tested for the cultivation and recovery of anaerobes.

The deconvolution of pyrolysis mass spectra using genetic programming: application to the identification of some Eubacterium species.

Pyrolysis mass spectrometry was used to produce complex biochemical fingerprints of Eubacterium exiguum, E. infirmum, E. tardum and E. timidum. To examine the relationship between these organisms the spectra were clustered by canonical variates analysis, and four clusters, one for each species, were observed. In an earlier study we trained artificial neural networks to identify these clinical isolates successfully; however, the information used by the neural network was not accessible from this so-called 'black box' technique. To allow the deconvolution of such complex spectra (in terms of which masses were important for discrimination) it was necessary to develop a system that itself produces 'rules' that are readily comprehensible. We here exploit the evolutionary computational technique of genetic programming; this rapidly and automatically produced simple mathematical functions that were also able to classify organisms to each of the four bacterial groups correctly and unambiguously. Since the rules used only a very limited set of masses, from a search space some 50 orders of magnitude greater than the dimensionality actually necessary, visual discrimination of the organisms on the basis of these spectral masses alone was also then possible.

Bacteroides ureolyticus (NTU) medium for the selective recovery of Bacteroides gracilis.

Bacteroides gracilis is a gram-negative anaerobic bacillus which requires formate and fumarate for growth; it has been implicated in periodontal disease and serious infections of the head and neck. In this study, Bacteroides ureolyticus (NTU) medium was tested for its ability to allow the growth of B. gracilis and other formate-fumarate requiring gram-negative anaerobes and to enable the recovery of these organisms from clinical specimens. All reference strains grew on NTU medium with the exception of Wolinella recta and formate-fumarate requiring organisms were isolated from 18 of 20 samples of subgingival dental plaque from patients with chronic periodontitis. B. gracilis was the commonest species isolated (14 of the 29 isolates); B. ureolyticus was not found.

An improved medium for isolation of Streptococcus mutans.

A medium based upon tryptone, yeast extract, cystine (TYC) agar and incorporating bacitracin and sucrose has been evaluated for selective isolation of Streptococcus mutans. The effect of varying the concentrations of sucrose and bacitracin on the recovery of two standard strains was investigated. Growth of S. mutans NCTC 10449 was significantly inhibited by increasing concentrations of sucrose but was not affected by bacitracin; the reverse was seen with S. sobrinus strain 6715. The best compromise between recovery of the streptococci and growth of other organisms was obtained with a final sucrose concentration of 20% and bacitracin 0.2 units/ml. In comparison with three other selective media, this medium gave the highest recovery rate of standard strains, indicating that it is superior to mitis-salivarius bacitracin (MSB) agar for the recovery of S. mutans from saliva.

Comparison of identification methods for oral asaccharolytic Eubacterium species.

Thirty one strains of oral, asaccharolytic Eubacterium spp. and the type strains of E. brachy, E. nodatum and E. timidum were subjected to three identification techniques--protein-profile analysis, determination of metabolic end-products, and the API ATB32A identification kit. Five clusters were obtained from numerical analysis of protein profiles and excellent correlations were seen with the other two methods. Protein profiles alone allowed unequivocal identification.

Rapid differentiation of Prevotella intermedia and P. nigrescens by 16S rDNA PCR-RFLP.

Prevotella intermedia and the newly described P. nigrescens cannot be reliably distinguished by phenotypic tests. In this study, restriction endonuclease digestion of amplified 16S rDNA (16S rDNA PCR-RFLP) was used to generate restriction profiles of the type strains of P. intermedia and P. nigrescens and 43 fresh isolates identified as belonging to one of the two species. Whole-cell protein profiles were obtained by SDS-PAGE for comparative purposes. The type strains of P. intermedia and P. nigrescens were easily distinguished by 16S rDNA PCR-RFLP and the fresh isolates were assigned to either species on the basis of their restriction profiles. The identifications obtained were identical to those obtained by protein profiles. 16S rDNA PCR-RFLP is a rapid and reliable method for the differentiation of P. intermedia and P. nigrescens.

An unclassified Eubacterium taxon in acute dento-alveolar abscess.

The microflora of pus samples aspirated from 50 acute dento-alveolar abscesses was examined. A total of 143 bacterial strains was isolated, consisting predominantly of Prevotella spp., alpha-haemolytic Streptococcus spp., Peptostreptococcus spp. and Eubacterium spp. An unclassified asaccharolytic Eubacterium taxon was encountered in 17 (34%) of the abscesses. This taxon was found to have a positive association with Fusobacterium spp. and a negative association with alpha-haemolytic Streptococcus spp.

Molecular and cultural analysis of the microflora associated with endodontic infections.

Cultural studies have indicated that a subset of the oral microflora is responsible for endodontic infections. Approximately 50% of oral bacteria are unculturable, so it is likely that currently unknown bacteria are present in such infections. In this study, cultural and molecular analyses were performed on the microflora in aspirate samples collected from 5 infected root canals. 16S rDNA sequences from 261 isolates and 624 clones were identified by comparison with database sequences. Sixty-five taxa were identified, of which 26 were found by the molecular method alone. A mean of 20.2 taxa was found in each sample. A new species of Dialister was the only organism present in all 5 samples. Twenty-seven novel taxa were detected, 18 of which belonged to the phylum Firmicutes and 8 to Bacteroidetes. Culture-independent, molecular analysis has revealed a more diverse microflora associated with endodontic infections than that revealed by cultural methods alone.