Effect of maltitol-containing chewing gum use on the composition of dental plaque microbiota in subjects with active dental caries.

Background: Sugar alcohols such as xylitol are incorporated in a number of oral hygiene products for their anti-cariogenic properties while chewing gum is known to be beneficial to oral hygiene. Objective: The aim of this study was to determine the composition of the dental plaque microbiota in patients with active caries before and after using a chewing gum supplemented with maltitol. Design : Forty subjects with active caries were randomly allocated to chew maltitol gum or gum base for two weeks. A healthy control group used gum base for two weeks. Plaque samples were collected before and after treatment and the microbiota analysed by pyrosequencing of 16S rRNA genes. Results : A total of 773,547 sequences were obtained from 117 samples. There was no difference in structure of the bacterial communities between groups (AMOVA). There was a significant difference in community membership between groups, (AMOVA, p=0.009). There was a significant difference between the control group after treatment and the maltitol patient group after treatment (p<0.001). A. naeslundii HOT-176 and Actinomyces HOT-169 were significantly reduced following use of maltitol chewing gum in patients. Conclusions : This study has shown that chewing gum containing maltitol had minor effects on the composition of the plaque microbiome.

Spontaneous Preterm Birth Is Associated with Differential Expression of Vaginal Metabolites by Lactobacilli-Dominated Microflora.

A major challenge in preventing preterm birth (PTB) is identifying women at greatest risk. This pilot study prospectively examined the differences in vaginal microbiota and metabolite profiles of women who delivered prematurely compared to their term counterparts in a cohort of asymptomatic (studied at 20-22, n = 80; and 26-28 weeks, n = 41) and symptomatic women (studied at 24-36 weeks, n = 37). Using 16S rRNA sequencing, the vaginal microbiota from cervicovaginal fluid samples was characterized into five Community State Types (CST) dominated by Lactobacillus spp.: CSTI (Lactobacillus crispatus), CSTII (Lactobacillus gasseri), CSTIII (Lactobacillus iners), CSTV (Lactobacillus jensenii); and mixed anaerobes-CSTIV. This was then related to the vaginal metabolite profile and pH determined by 1H-Nuclear Magnetic Resonance spectroscopy and pH indicator paper, respectively. At 20-22 weeks, the term-delivered women (TDW) indicated a proportion of CSTI-dominated microbiota >2-fold higher compared to the preterm-delivered women (PTDW) (40.3 vs. 16.7%, P = 0.0002), and a slightly higher proportion at 26-28 weeks (20.7 vs. 16.7%, P = 0.03). CSTV was >2-fold higher in the PTDW compared to TDW at 20-22 (22.2 vs. 9.7%, P = 0.0002) and 26-28 weeks (25.0 vs. 10.3%, P = 0.03). Furthermore, at 26-28 weeks no PTDW had a CSTII-dominated microbiome, in contrast to 28% of TDW (P < 0.0001). CSTI-dominated samples showed higher lactate levels than CSTV at 20-22 weeks (P < 0.01), and 26-28 weeks (P < 0.05), while CSTII-dominated samples indicated raised succinate levels over CSTV at 26-28 weeks (P < 0.05). These were supported by Principal coordinates analysis, which revealed strong clustering of metabolites according to CST. In addition, the CSTI-dominated samples had an average pH of 3.8, which was lower than those of CSTII-4.4, and CSTV-4.2 (P < 0.05). Elevated vaginal lactate and succinate were associated with predominance of CSTI and II over CSTV in women who delivered at term compared with their preterm counterparts. This suggests that L. jensenii-dominance and decreased lactate and/or succinate could increase the risk of PTB, while L. crispatus/gasseri may confer some protection against inflammation-associated PTB and highlight the need for further study in this area.

The BBaRTS Healthy Teeth Behaviour Change Programme for preventing dental caries in primary school children: study protocol for a cluster randomised controlled trial.

BACKGROUND: Oral health behaviours such as establishing twice-daily toothbrushing and sugar control intake need parental self-efficacy (PSE) to prevent the development of childhood dental caries. A previous study has shown that behaviour change techniques (BCTs) delivered via a storybook can improve parental self-efficacy to undertake twice-daily toothbrushing. OBJECTIVE: to determine whether an intervention (BBaRTS, Bedtime Brush and Read Together to Sleep), designed to increase PSE; delivered through storybooks with embedded BCTs, parenting skills and oral health messages, can improve child oral health compared to (1) an exactly similar intervention containing no behaviour change techniques, and (2) the BBaRTS intervention supplemented with home supply of fluoride toothpaste and supervised toothbrushing on schooldays. METHODS/DESIGN: A 2-year, three-arm, multicentre, cluster randomised controlled trial. PARTICIPANTS: children (estimated 2000-2600) aged 5-7 years and their families from 60 UK primary schools. INTERVENTION: Test group 1: a series of eight children's storybooks developed by a psychologist, public health dentist, science educator, children's author and illustrators, with guidance from the Department for Education (England). The books feature animal characters and contain embedded dental health messages, parenting skills and BCTs to promote good oral health routines focused on controlling sugar intake and toothbrushing, as well as reading at bedtime. Books are given out over 2 years. Test group 2: as Test group 1 plus home supplies of fluoride toothpaste (1000 ppmF), and daily supervised toothbrushing in school on schooldays. Active Control group: series of eight books with exactly the same stories, characters and illustrations, but without BCTs, dental health messages or parenting skills. Annual child dental examinations and parental questionnaires will be undertaken. A sub-set of participants will be invited to join an embedded study of the child's diet and salivary microbiota composition. PRIMARY OUTCOME MEASURE: dental caries experience in permanent teeth at age 7-8 years. DISCUSSION: A multi-disciplinary team was established to develop the BBaRTS Children's Healthy Teeth Programme. The books were developed in partnership with the Department for Education (England), informed by a series of focus groups with children, teachers and parents. TRIAL REGISTRATION: ISRCTN21461006 (date of registration 23 September 2015).

The oral microbiome in human immunodeficiency virus (HIV)-positive individuals.

Human immunodeficiency virus (HIV) infection is associated with a range of oral conditions, and increased numbers of disease-associated microbial species have previously been found in HIV-positive subjects. The aim of this study was to use next-generation sequencing to compare the composition of the oral microbiome in HIV-positive and -negative individuals. Plaque and saliva were collected from 37 HIV-positive individuals and 37 HIV-negative individuals, and their bacterial composition determined by pyrosequencing of partial 16S rRNA genes. A total of 855,222 sequences were analysed. The number of species-level operational taxonomic units (OTUs) detected was significantly lower in the saliva of HIV-positive individuals (mean = 303.3) than in that of HIV-negative individuals (mean = 365.5) (P < 0.0003). Principal coordinates analysis (PCoA) based on community membership (Jaccard index) and structure (Yue and Clayton measure of dissimilarity) showed significant separation of plaque and saliva samples [analysis of molecular variance (AMOVA), P < 0.001]. PCoA plots did not show any clear separation based on HIV status. However, AMOVA indicated that there was a significant difference in the community membership of saliva between HIV-positive and -negative groups (P = 0.001). Linear discriminant analysis effect size revealed an OTU identified as Haemophilus parainfluenzae to be significantly associated with HIV-positive individuals, whilst Streptococcus mitis/HOT473 was most significantly associated with HIV-negative individuals. In conclusion, this study has confirmed that the microbial composition of saliva and plaque is different. The oral microbiomes of HIV-positive and -negative individuals were found to be similar overall, although there were minor but significant differences in the composition of the salivary microbiota of the two groups.

Development and pyrosequencing analysis of an in-vitro oral biofilm model.

BACKGROUND: Dental caries and periodontal disease are the commonest bacterial diseases of man and can result in tooth loss. The principal method of prevention is the mechanical removal of dental plaque augmented by active agents incorporated into toothpastes and mouthrinses. In-vitro assays that include complex oral bacterial biofilms are required to accurately predict the efficacy of novel active agents in vivo. The aim of this study was to develop an oral biofilm model using the Calgary biofilm device (CBD) seeded with a natural saliva inoculum and analysed by next generation sequencing. The specific objectives were to determine the reproducibility and stability of the model by comparing the composition of the biofilms over time derived from (i) the same volunteers at different time points, and (ii) different panels of volunteers. RESULTS: Pyrosequencing yielded 280,093 sequences with a mean length of 432 bases after filtering. A mean of 320 and 250 OTUs were detected in pooled saliva and biofilm samples, respectively. Principal coordinates analysis (PCoA) plots based on community membership and structure showed that replicate biofilm samples were highly similar and clustered together. In addition, there were no significant differences between biofilms derived from the same panel at different times using analysis of molecular variance (AMOVA). There were significant differences between biofilms from different panels (AMOVA, P < 0.002). PCoA revealed that there was a shift in biofilm composition between seven and 14 days (AMOVA, P < 0.001). Veillonella parvula, Veillonella atypica/dispar/parvula and Peptostreptococcus stomatis were the predominant OTUs detected in seven-day biofilms, whilst Prevotella oralis, V. parvula and Streptococcus constellatus were predominant in 14-day biofilms. CONCLUSIONS: Diverse oral biofilms were successfully grown and maintained using the CBD. Biofilms derived from the same panel of volunteers were highly reproducible. This model could be used to screen both antimicrobial-containing oral care products and also novel approaches aiming to modify plaque composition, such as pre- or probiotics.

Bacterial community development in experimental gingivitis.

Current knowledge of the microbial composition of dental plaque in early gingivitis is based largely on microscopy and cultural methods, which do not provide a comprehensive description of oral microbial communities. This study used 454-pyrosequencing of the V1-V3 region of 16S rRNA genes (approximately 500 bp), and bacterial culture, to characterize the composition of plaque during the transition from periodontal health to gingivitis. A total of 20 healthy volunteers abstained from oral hygiene for two weeks, allowing plaque to accumulate and gingivitis to develop. Plaque samples were analyzed at baseline, and after one and two weeks. In addition, plaque samples from 20 chronic periodontitis patients were analyzed for cross-sectional comparison to the experimental gingivitis cohort. All of the healthy volunteers developed gingivitis after two weeks. Pyrosequencing yielded a final total of 344,267 sequences after filtering, with a mean length of 354 bases, that were clustered into an average of 299 species-level Operational Taxonomic Units (OTUs) per sample. Principal coordinates analysis (PCoA) plots revealed significant shifts in the bacterial community structure of plaque as gingivitis was induced, and community diversity increased significantly after two weeks. Changes in the relative abundance of OTUs during the transition from health to gingivitis were correlated to bleeding on probing (BoP) scores and resulted in the identification of new health- and gingivitis-associated taxa. Comparison of the healthy volunteers to the periodontitis patients also confirmed the association of a number of putative periodontal pathogens with chronic periodontitis. Taxa associated with gingivitis included Fusobacterium nucleatum subsp. polymorphum, Lachnospiraceae [G-2] sp. HOT100, Lautropia sp. HOTA94, and Prevotella oulorum, whilst Rothia dentocariosa was associated with periodontal health. Further study of these taxa is warranted and may lead to new therapeutic approaches to prevent periodontal disease.

Effects of the UK Biobank collection protocol on potential biomarkers in saliva.

BACKGROUND: The UK Biobank (UKB) is a national epidemiological study of the health of 500 000 people, aged 40-69 years, who completed health-related tests and a questionnaire and gave samples of blood and urine. Salivas collected from 120 000 of these subjects were transported at 4°C and were placed in ultra-low temperature archives at up to 24 h after collection. The present study assessed how changes in saliva composition under UKB conditions influence a range of potential biomarkers resulting from holding saliva at 4°C for 24 h. METHODS: Unstimulated whole-mouth saliva samples were collected from 23 volunteers aged 45-69 years. Salivas were split into aliquots some of which were immediately frozen at -80°C, whereas others were stored at 4°C for 24 h and then frozen at -80°C, mimicking the UKB protocol. RESULTS: Assessment of mRNA by real-time polymerase chain reaction revealed no difference between samples that were analysed after the UKB protocol and those that were immediately preserved. Immunochemical analysis showed some loss of ß-Actin under UKB conditions, whereas other salivary proteins including cytokines and C-reactive protein appeared to be unaffected. Cortisol and showed no reduction by UKB conditions, but salivary nitrite was reduced by 30%. The oral microbiome, as revealed by sequencing 16S rRNA genes, showed variations between subjects, but paired samples within subjects were very similar. CONCLUSIONS: Our results suggest that many salivary components remain little affected under UKB collection and handling protocols, suggesting that the resource of 120 000 samples held in storage will be useful for phenotyping subjects and revealing potential prognostic disease biomarkers.

Multiplex ligation-dependent probe amplification (MLPA): a reliable alternative for fetal chromosome analysis?

OBJECTIVE: To determine whether molecular karyotyping using multiple ligation probe amplification (MLPA) is a reliable alternative for quick and accurate diagnosis of fetal chromosomal abnormalities. METHODS: MLPA, using specialised probe sets designed to detect aneuploidy, major chromosomal rearrangements and recognised microdeletion syndromes, was used to analyse chorionic villi or amniocytes left after traditional karyotyping of 476 fetuses for clinical indications. RESULTS: An abnormal result was obtained in 190 cases, including 124 trisomies, 21 sex chromosome anomalies, 14 triploidies, and 31 rearrangements or mosaics. All trisomies were detected by all three techniques, but triploidies were only detected by karyotyping and QF-PCR. In 19 of the 31 cases of rearrangements or mosaicism there was an uncertain or high risk of adverse outcome. Traditional karyotyping detected 13 of the 19 pathogenic rearrangements, MLPA detected 18, and QF-PCR did not detect any. CONCLUSION: MLPA, using specialized probe sets, detects more chromosomal rearrangements, conferring significant risk of adverse outcome than karyotyping. A combination of qfPCR and MLPA could be a good, rapid alternative to current practice. In the future, used in conjunction with non-invasive prenatal diagnosis based on cell free fetal DNA it might provide a rapid and efficient approach to fetal karyotyping.

Wide geographic spread of diverse acquired AmpC beta-lactamases among Escherichia coli and Klebsiella spp. in the UK and Ireland.

OBJECTIVES: To determine the distribution of acquired AmpC beta-lactamases in 173 isolates of Escherichia coli and Klebsiella spp. submitted to the UK's national reference laboratory for antibiotic resistance. METHODS: MICs were determined and interpreted according to BSAC guidelines. Candidate isolates were those resistant to cefotaxime and/or ceftazidime, irrespective of addition of clavulanic acid. Genes encoding six phylogenetic groups of acquired AmpC enzymes were sought by PCR. Selected isolates were compared by pulsed-field gel electrophoresis (PFGE), and one bla(AmpC) amplicon was sequenced. RESULTS: Genes encoding acquired AmpC enzymes were detected in 67 (49%) candidate E. coli and 21 (55%) Klebsiella spp. Sixty isolates produced CIT-type enzymes, 14 had ACC types, 11 had FOX types and 3 had DHA enzymes. The low-level cephalosporin resistance of the remaining isolates (n = 85; 49%) was inferred to result from reduced permeability or, in E. coli, from hyperexpression of chromosomal ampC. Twenty-four E. coli isolates from one hospital produced a CIT-type enzyme, with 20 of these additionally producing a group 1 CTX-M extended-spectrum beta-lactamase. PFGE indicated that these isolates belonged to UK epidemic strain A, which normally produces CTX-M-15, but no acquired AmpC. Sequencing a representative bla(AmpC) amplicon indicated that in one centre this strain had acquired a novel CMY-2 variant, designated CMY-23. CONCLUSIONS: Diverse acquired AmpC enzymes occur in E. coli and Klebsiella spp. isolates in the UK and Ireland, with CIT types the most common. Producers are geographically scattered, but with some local outbreaks. Acquisition of a CMY-2-like enzyme by E. coli epidemic strain A suggests that these enzymes may be poised to become an important public health issue.