Carina Öhman

Oral microbial profile discriminates breast-fed from formula-fed infants

Objectives: Little is known about the effect of diet on the oral microbiota of infants, although diet is known to affect the gut microbiota. The aims of the present study were to compare the oral microbiota in breast-fed and formula-fed infants, and investigate growth inhibition of streptococci by infant-isolated lactobacilli. Methods: A total of 207 mothers consented to participation of their 3-month-old infants. A total of 146 (70.5%) infants were exclusively and 38 (18.4%) partially breast-fed, and 23 (11.1%) were exclusively formula-fed. Saliva from all of their infants was cultured for Lactobacillus species, with isolate identifications from 21 infants. Lactobacillus isolates were tested for their ability to suppress Streptococcus mutans and S sanguinis. Oral swabs from 73 infants were analysed by the Human Oral Microbe Identification Microarray (HOMIM) and by quantitative polymerase chain reaction for Lactobacillus gasseri. Results: Lactobacilli were cultured from 27.8% of exclusively and partially breast-fed infants, but not from formula-fed infants. The prevalence of 14 HOMIM-detected taxa, and total salivary lactobacilli counts differed by feeding method. Multivariate modelling of HOMIM-detected bacteria and possible confounders clustered samples from breast-fed infants separately from formula-fed infants. The microbiota of breast-fed infants differed based on vaginal or C-section delivery. Isolates of L plantarum, L gasseri, and L vaginalis inhibited growth of the cariogenic S mutans and the commensal S sanguinis: L plantarum >L gasseri >L vaginalis. Conclusions: The microbiota of the mouth differs between 3-month-old breast-fed and formula-fed infants. Possible mechanisms for microbial differences observed include species suppression by lactobacilli indigenous to breast milk.

Characterization and in vitro properties of oral lactobacilli in breastfed infants

BackgroundLactobacillus species can contribute positively to general and oral health and are frequently acquired by breastfeeding in infancy. The present study aimed to identify oral lactobacilli in breast and formula-fed 4 month-old infants and to evaluate potential probiotic properties of the dominant Lactobacillus species detected. Saliva and oral swab samples were collected from 133 infants who were enrolled in a longitudinal study (n=240) examining the effect of a new infant formula on child growth and development. Saliva was cultured and Lactobacillus isolates were identified from 16S rRNA gene sequences. Five L. gasseri isolates that differed in 16S rRNA sequence were tested for their ability to inhibit growth of selected oral bacteria and for adhesion to oral tissues. Oral swab samples were analyzed by qPCR for Lactobacillus gasseri.Results43 (32.3%) infants were breastfed and 90 (67.7%) were formula-fed with either a standard formula (43 out of 90) or formula supplemented with a milk fat globule membrane (MFGM) fraction (47 out of 90). Lactobacilli were cultured from saliva of 34.1% breastfed infants, but only in 4.7% of the standard and 9.3% of the MFGM supplemented formula-fed infants. L. gasseri was the most prevalent (88% of Lactobacillus positive infants) of six Lactobacillus species detected. L. gasseri isolates inhibited Streptococcus mutans binding to saliva-coated hydroxyapatite, and inhibited growth of S. mutans, Streptococcus sobrinus, Actinomyces naeslundii, Actinomyces oris, Candida albicans and Fusobacterium nucleatum in a concentration dependent fashion. L. gasseri isolates bound to parotid and submandibular saliva, salivary gp340 and MUC7, and purified MFGM, and adhered to epithelial cells. L. gasseri was detected by qPCR in 29.7% of the oral swabs. Breastfed infants had significantly higher mean DNA levels of L. gasseri (2.14 pg/uL) than infants fed the standard (0.363 pg/uL) or MFGM (0.697 pg/uL) formula.ConclusionsLactobacilli colonized the oral cavity of breastfed infants significantly more frequently than formula-fed infants. The dominant Lactobacillus was L. gasseri, which was detected at higher levels in breastfed than formula-fed infants and displayed probiotic traits in vitro.

Maturation of Oral Microbiota in Children with or without Dental Caries

Background The aim of this longitudinal study was to evaluate the oral microbiota in children from age 3 months to 3 years, and to determine the association of the presence of caries at 3 years of age. Methods and findings Oral biofilms and saliva were sampled from children at 3 months (n = 207) and 3 years (n = 155) of age, and dental caries was scored at 3 years of age. Oral microbiota was assessed by culturing of total lactobacilli and mutans streptococci, PCR detection of Streptococcus mutans and Streptococcus sobrinus, 454 pyrosequencing and HOMIM (Human Oral Microbe Identification Microarray) microarray detection of more then 300 species/ phylotypes. Species richness and taxa diversity significantly increased from 3 months to 3 years. Three bacterial genera, present in all the 3-month-old infants, persisted at 3 years of age, whereas three other genera had disappeared by this age. A large number of new taxa were also observed in the 3-year-olds. The microbiota at 3 months of age, except for lactobacilli, was unrelated to caries development at a later age. In contrast, several taxa in the oral biofilms of the 3-year-olds were linked with the presence or absence of caries. The main species/phylotypes associated with caries in 3-year-olds belonged to the Actinobaculum, Atopobium, Aggregatibacter, and Streptococcus genera, whereas those influencing the absence of caries belonged to the Actinomyces, Bergeyella, Campylobacter, Granulicatella, Kingella, Leptotrichia, and Streptococcus genera. Conclusions Thus, during the first years of life, species richness and taxa diversity in the mouth increase significantly. Besides the more prevalent colonization of lactobacilli, the composition of the overall microbiota at 3 months of age was unrelated to caries development at a later age. Several taxa within the oral biofilms of the 3-year-olds could be linked to the presence or absence of caries.

A Repetitive DNA Element Regulates Expression of the Helicobacter pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism

During persistent infection, optimal expression of bacterial factors is required to match the ever-changing host environment. The gastric pathogen Helicobacter pylori has a large set of simple sequence repeats (SSR), which constitute contingency loci. Through a slipped strand mispairing mechanism, the SSRs generate heterogeneous populations that facilitate adaptation. Here, we present a model that explains, in molecular terms, how an intergenically located T-tract, via slipped strand mispairing, operates with a rheostat-like function, to fine-tune activity of the promoter that drives expression of the sialic acid binding adhesin, SabA. Using T-tract variants, in an isogenic strain background, we show that the length of the T-tract generates multiphasic output from the sabA promoter. Consequently, this alters the H. pylori binding to sialyl-Lewis x receptors on gastric mucosa. Fragment length analysis of post-infection isolated clones shows that the T-tract length is a highly variable feature in H. pylori. This mirrors the host-pathogen interplay, where the bacterium generates a set of clones from which the best-fit phenotypes are selected in the host. In silico and functional in vitro analyzes revealed that the length of the T-tract affects the local DNA structure and thereby binding of the RNA polymerase, through shifting of the axial alignment between the core promoter and UP-like elements. We identified additional genes in H. pylori, with T- or A-tracts positioned similar to that of sabA, and show that variations in the tract length likewise acted as rheostats to modulate cognate promoter output. Thus, we propose that this generally applicable mechanism, mediated by promoter-proximal SSRs, provides an alternative mechanism for transcriptional regulation in bacteria, such as H. pylori, which possesses a limited repertoire of classical trans-acting regulatory factors.

Oral Microbiota Shift after 12-Week Supplementation with Lactobacillus reuteri DSM 17938 and PTA 5289; A Randomized Control Trial

Background Lactobacillus spp. potentially contribute to health by modulating bacterial biofilm formation, but their effects on the overall oral microbiota remain unclear. Methods and Findings Oral microbiota was characterized via 454-pyrosequencing of the 16S rDNA hypervariable region V3-V4 after 12 weeks of daily Lactobacillus reuteri DSM 17938 and PTA 5289 consumption. Forty-four adults were assigned to a test group (n = 22) that received lactobacilli lozenges (108 CFU of each strain/lozenge) or a control group that received placebo (n = 22). Presence of L. reuteri was confirmed by cultivation and species specific PCR. Tooth biofilm samples from 16 adults before, during, and after exposure were analyzed by pyrosequencing. A total of 1,310,292 sequences were quality filtered. After removing single reads, 257 species or phylotypes were identified at 98.5% identity in the Human Oral Microbiome Database. Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria, and Actinobacteria were the most abundant phyla. Streptococcus was the most common genus and the S. oralis/S. mitis/S. mitis bv2/S. infantis group comprised the dominant species. The number of observed species was unaffected by L. reuteri exposure. However, subjects who had consumed L. reuteri were clustered in a principal coordinates analysis relative to scattering at baseline, and multivariate modeling of pyrosequencing microbiota, and culture and PCR detected L. reuteri separated baseline from 12-week samples in test subjects. L. reuteri intake correlated with increased S. oralis/S. mitis/S. mitis bv2/S. infantis group and Campylobacter concisus, Granulicatella adiacens, Bergeyella sp. HOT322, Neisseria subflava, and SR1 [G-1] sp. HOT874 detection and reduced S. mutans, S. anginosus, N. mucosa, Fusobacterium periodicum, F. nucleatum ss vincentii, and Prevotella maculosa detection. This effect had disappeared 1 month after exposure was terminated. Conclusions L. reuteri consumption did not affect species richness but induced a shift in the oral microbiota composition. The biological relevance of this remains to be elucidated. Trial Registration ClinicalTrials.gov NCT02311218

Metabolite quantification by NMR and LC-MS/MS reveals differences between unstimulated, stimulated, and pure parotid saliva

&NA; Saliva is a readily available biofluid that is sensitive to metabolic changes and can be collected through rapid and non‐invasive collection procedures, and it shows great promise for clinical metabolomic studies. This work studied the metabolite composition of, and the differences between, saliva samples collected by unstimulated spitting/drooling, paraffin chewing‐stimulated spitting, and parotid gland suction using targeted nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS) for metabolite quantification. As applied here, these two analytical techniques provide complementary metabolite information and together extend the metabolome coverage with robust NMR quantification of soluble metabolites and sensitive targeted LC‐MS/MS analysis of bioactive lipids in specific metabolic pathways. The NMR analysis was performed on ultrafiltrated (3 kDa cutoff) saliva samples and resulted in a total of 45 quantified metabolites. The LC‐MS/MS analysis was performed on both filtered and unfiltered samples and resulted in the quantification of two endocannabinoids (AEA and PEA) and 22 oxylipins, which at present is the most comprehensive targeted analysis of bioactive lipids in human saliva. Important differences in the metabolite composition were observed between the three saliva sample collection methods, which should be taken into consideration when designing metabolomic studies of saliva. Furthermore, the combined use of the two metabolomics platforms (NMR and LC‐MS/MS) proved to be viable for research and clinical studies of the salivary metabolome. Graphical abstract Figure. No caption available. HighlightsUnstimulated, stimulated and parotid saliva were analyzed by NMR and LC‐MS/MS.NMR quantified up to 45 metabolites.LC‐MS/MS quantified 24 bioactive lipids, at present the largest panel in saliva.Clear differences in the metabolite composition between saliva types were observed.The outcome will affect future study design of metabolomics in clinical research.