Salivary microbiota - how to measure it and its associations with body size and antimicrobial use

The human microbiota, i.e. the microbes living in or on humans, plays an important role in health and disease. Lifestyle factors, such as diet and physical activity as well as environmental factors, and exposure to antimicrobials (AMs) are likely among the important factors shaping the microbiota. Several studies have reported that the gut microbiota may be associated with overweight and obesity. Fewer studies have investigated associations between body mass index (BMI) and the salivary microbiota. There is a worldwide epidemic of obesity and the number of overweight and obese people reached 1.9 billion in 2016. Overweight and obese children are likely to stay obese into adulthood and tend to develop diseases more frequently and at a younger age. Thus, identification of the associations between microbiota and body size in young individuals are of great importance. The increased use of AMs rises concern of increasing antibiotic resistance resulting in lack of treatment options for many diseases. Exposure to AMs affects the microbial diversity and composition in the gut microbiota, but less is known about salivary microbiota. Since AM use is frequent in children, it is vital to study its associations with saliva microbiota at this age. The objectives of this doctoral thesis were to develop cost effective protocols to assess the salivary microbiota profiles for large-scale epidemiological studies and, with the new protocol, to determine its association with body size and lifetime antimicrobial use in children. In this thesis, all in-house 16S amplicon assays produced similar salivary microbiota profiles for the individual samples, i.e. there was no superior protocol. Salivary microbiota profiles of Finnish children were gender-specific in terms of alpha- and beta-diversity and relative abundances of bacteria. A prominent finding was the decrease in the core bacteria in overweight and obese children. Lifetime AM exposure to saliva microbiota showed that Azithromycin use was associated with alpha-diversity in all children, and in girls. Microbiota dissimilarities were significant between children with low, medium and high number of AM user groups in all children with all AMs combined. Similar trend was significant with Azithromycin use, whereas Amoxicillin use affected the dissimilarity only in boys. This thesis suggests that the saliva microbiota is significantly associated with body size, antimicrobial use and gender in Finnish children. Thus, saliva microbiota profiles open new possibilities to study the potential roles of microbiota in weight development and management in children. In addition, the involuntary consequences of lifetime AM use are a concern and the importance of microbiota in the development of new therapeutic strategies should be emphasized in order to limit the use of AMs wisely. Studies have shown that the saliva microbiota is more resilient and stable than gut microbiota when exposed to antibiotics. Thus, the saliva-based screening of microbial biomarkers in health surveillance, and the associations with oral and general health status, may be considered feasible, simple, economical and easy to collect with high compliance for all age groups compared to faecal samples. However, further research on metabolic and functional potential of saliva microbiota is needed to fully understand the saliva microbiota – host relationship.Ihmisen iholla ja limakalvoilla elävä normaalimikrobisto auttaa ylläpitämään terveyttä ja torjumaan sairauksia. Elämäntapatekijät, kuten ruokavalio ja fyysinen aktiivisuus sekä ympäristö ja altistus antibiooteille todennäköisesti muokkaavat bakteeristoa keskeisellä tavalla. Suolistobakteerit näyttävät olevan yhteydessä ylipainoon ja lihavuuteen, mutta syljen bakteeriston ja painoindeksin (BMI) välisiä yhteyksiä on vielä toistaiseksi tutkittu niukasti. Lihavuus on maailmanlaajuinen epidemia, ja vuonna 2016 ylipainoisia ja lihavia oli jo 1,9 miljardia. Lapsuudenaikainen ylipaino ja lihavuus todennäköisesti jatkuu aikuisuuteen ja on yhteydessä suurentuneeseen sairausriskiin varhaisemmalla iällä. Siksi bakteerien ja painon välinen yhteys on tärkeää selvittää. Antibioottien käyttö ja samalla antibioottiresistenssi on lisääntynyt. Resistenssi johtaa siihen, että monen sairauden hoitomahdollisuudet kutistuvat. Altistuminen antimikrobilääkkeille (AM-lääkkeet) vaikuttaa suoliston bakteeriston monimuotoisuuteen ja koostumukseen, mutta niiden vaikutuksista syljen bakteeristoon tiedetään toistaiseksi vähän. Lapset käyttävät AM-lääkkeitä usein, joten syljen bakteeriston ja käytön välisiä yhteyksiä on tärkeää tutkia. Tämän väitöskirjan tavoite oli kehittää kustannustehokas menetelmä, jolla syljen bakteeristoprofiili voidaan määrittää laajoissa väestöaineistoissa, sekä lisäksi käyttää tätä uutta menetelmää syljen bakteeriston ja painon sekä elinaikaisen AM-lääkkeiden käytön välisten yhteyksien määrittämiseksi lapsilla. Tulokset osoittivat, että kaikkien testattujen 16S rRNA -geenin monistukseen ja sekvensointiin käytettyjen menetelmien tuottamat syljen bakteeristoprofiilit olivat keskenään samankaltaisia, ts. mikään menetelmistä ei ollut selvästi muita parempi. Syljen bakteeristoprofiilit olivat sukupuolelle ominaisia alfa- ja betadiversiteetin sekä tiettyjen bakteerien suhteellisen runsauden suhteen. Merkittävä löydös oli ylipainoisilla ja lihavilla lapsilla näkynyt ydinbakteeriston väheneminen. Elinaikaisen AM-lääkealtistuksen ja syljen bakteeriston analyysi osoitti, että atsitromysiinin käyttö oli yhteydessä alentuneeseen alfadiversiteettiin kaikilla lapsilla sekä erikseen tytöillä. Syljen bakteeriston koostumus erosi merkitsevästi matalan, keskitason ja korkean AM-lääkkeiden käytön ryhmien välillä kaikilla lapsilla. Tarkasteltaessa eri antibiootteja erikseen atsitromysiinin käytön suhteen löydettiin samanlainen suuntaus, mutta amoksisilliinin kohdalla bakteeriston koostumus erosi merkitsevästi ainoastaan pojilla. Tämä väitöskirjatyö esittää, että syljen bakteeristo on merkitsevästi yhteydessä painoon, AM-lääkkeiden käyttöön ja sukupuoleen suomalaislapsilla. Syljen bakteeriston profiloinnilla on mahdollista kehittää uusia tapoja tutkia bakteerien mahdollista roolia lasten painonkehityksessä. Vaikutukset normaalibakteeristoon tulisi ottaa huomioon myös kehitettäessä uusia hoitomenetelmiä, jotta AM-lääkkeiden käyttöä saataisiin rajoitettua. Aiemmat tutkimukset ovat osoittaneet, että syljen bakteeristo on vastustuskykyisempi ja vakaampi antibiootteja vastaan kuin suoliston bakteeristo, joten mikrobimarkkereiden seulonta syljestä ulostenäytteen sijaan on helppo, taloudellinen ja kaikille ikäryhmille soveltuva vaihtoehto käytettäväksi terveydenhuollossa. Bakteeriston metabolisia ja toiminnallisia piirteitä on kuitenkin tutkittava lisää, jotta syljen bakteerien ja isännän välisiä vuorovaikutuksia ja niiden merkitystä terveydelle voidaan ymmärtää paremmin

Genomic Analysis Reveals Versatile Organisms for Quorum Quenching Enzymes: Acyl-Homoserine Lactone-Acylase and -Lactonase

Microbial virulence and their resistance to multiple drugs have obliged researchers to look for novel drug targets. Virulence of pathogenic microbes is regulated by signal molecules such as acylated homoserine lactone (AHL) produced during a cell density dependent phenomenon of quorum sensing (QS). In contrast, certain microbes produce AHL-lactonases and -acylases to degrade QS signals, also termed as quorum quenching. Mining sequenced genome databases has revealed organisms possessing conserved domains for AHL-lactonases and –acylases: i) Streptomyces (Actinobacteria), ii) Deinococcus (Deinococcus-Thermus), iii) Hyphomonas (α-Proteobacteria), iv) Ralstonia (β-Proteobacteria), v) Photorhabdus (γ-Proteobacteria), and certain marine gamma proteobacterium. Presence of genes for both the enzymes within an organism was observed in the following: i) Deinococcus radiodurans R1, ii) Hyphomonas neptunium ATCC 15444 and iii) Photorhabdus luminescens subsp. laumondii TTO1. These observations are supported by the presence motifs for lactonase and acylase in these strains. Phylogenetic analysis and multiple sequence alignment of the gene sequences for AHL-lactonases and –acylases have revealed consensus sequences which can be used to design primers for amplifying these genes even among mixed cultures and metagenomes. Quorum quenching can be exploited to prevent food spoilage, bacterial infections and bioremediation

High abundance of sugar metabolisers in saliva of children with caries

Dental caries is a biofilm-mediated, dynamic disease with early onset. A balanced salivary microbiota is a foundation of oral health, while dysbiosis causes tooth decay. We compared the saliva microbiota profiles in children with and without caries. The study consisted of 617 children aged 9-12 years from the Finnish Health in Teens (Fin-HIT) study with available register data on oral health. Caries status was summarised based on Decayed, Missing, and Filled Teeth (DMFT) index in permanent dentition. The children were then classified into the following two groups: DMFT value >= 1 was considered as cavitated caries lesions (hereafter called 'caries') (n=208) and DMFT=0 as 'cavity free' (n=409). Bacterial 16S rRNA gene (V3-V4 regions) was amplified using PCR and sequenced by Illumina HiSeq. The mean age (SD) of the children was 11.7 (0.4) years and 56% were girls. The children had relatively good dental health with mean DMFT of 0.86 (1.97). Since sex was the key determinant of microbiota composition (p=0.014), we focused on sex-stratified analysis. Alpha diversity indexes did not differ between caries and cavity free groups in either sexes (Shannon: p=0.40 and 0.58; Inverse Simpson: p=0.51 and 0.60, in boys and girls, respectively); neither did the composition differ between the groups (p=0.070 for boys and p=0.230 for girls). At the genus level, Paludibacter and Labrenzia had higher abundances in the caries group compared to cavity free group in both sexes (pPeer reviewe

Saliva microbiota differs between children with low and high sedentary screen times

Publisher Copyright: © 2021 The Author(s)This study examined whether the diversity, composition and functional capacity of the saliva microbiota differed between children with low and high sedentary screen times. We analyzed the saliva microbiota using 16S rRNA (V3–V4) sequencing from 193 children with low and 183 children with high TV/screen viewing times while sitting. Microbiota diversity was higher among children with low screen times compared to children with high screen times. Furthermore, microbiota composition differed between the screen time groups. In addition, we identified ten differentially abundant taxonomic groups, including Veillonella, Prevotella and Streptococcus, and five differentially present metabolic pathways between the screen time groups. Children with high screen times exhibited a higher capacity to synthesize the fatigue- and activity-related amino acids ornithine and arginine. To conclude, children with high sedentary screen (sitting) times exhibited a lower diversity and a different composition and functionality of the microbiota compared to children with low screen times.Peer reviewe

Breastfeeding may have a long-term effect on oral microbiota : results from the Fin-HIT cohort

Background Breastfeeding contributes to gastrointestinal microbiota colonization in early life, but its long-term impact is inconclusive. We aimed to evaluate whether the type of feeding during the first six months of life was associated with oral microbiota in adolescence. Methods This is a cross-sectional sub-study using baseline information of 423 adolescents from the Finnish Health in Teens (Fin-HIT) cohort. Type of feeding was recalled by parents and dichotomized as (i) No infant formula; (ii) Infant formula (breastmilk + formula or only formula). Saliva microbiota was analysed using 16S rRNA (V3-V4) sequencing. Alpha diversity and beta diversity were compared between feeding type groups using ANCOVA and PERMANOVA, respectively. Differential bacteria abundance was tested using appropriate general linear models. Results Mean age and body mass index were 11.7 years and 18.0 kg/m(2), respectively. The No formula group contained 41% of the participants. Firmicutes (51.0%), Bacteroidetes (19.1%), and Proteobacteria (16.3%) were the most abundant phyla among all participants. Alpha and beta diversity indices did not differ between the two feeding groups. Three Operational Taxonomic Units (OTUs) belonging to Eubacteria and Veillonella genera (phylum Firmicutes) were more abundant in the No formula than in the Infant formula group (log2fold changes/ p - values - 0.920/ <0.001, - 0.328/ 0.001, - 0.577/ 0.004). Conclusion Differences exist in abundances of some OTUs in adolescence according to feeding type during the first six months of life, but our findings do not support diversity and overall oral microbiota composition in adolescents being affected by early feeding type.Peer reviewe

Reproducibility and repeatability of six high-throughput 16S rDNA sequencing protocols for microbiota profiling

Culture-independent molecular techniques and advances in next generation sequencing (NGS) technologies make large-scale epidemiological studies on microbiota feasible. A challenge using NGS is to obtain high reproducibility and repeatability, which is mostly attained through robust amplification. We aimed to assess the reproducibility of saliva microbiota by comparing triplicate samples. The microbiota was produced with simplified in-house 16S amplicon assays taking advantage of large number of barcodes. The assays included primers with Truseq (TS-tailed) or Nextera (NX-tailed) adapters and either with dual index or dual index plus a 6-nt internal index. All amplification protocols produced consistent microbial profiles for the same samples. Although, in our study, reproducibility was highest for the TS-tailed method. Five replicates of a single sample, prepared with the TS-tailed 1-step protocol without internal index sequenced on the HiSeq platform provided high alpha-diversity and low standard deviation (mean Shannon and Inverse Simpson diversity was 3.19 +/- 0.097 and 13.56 +/- 1.634 respectively). Large-scale profiling of microbiota can consistently be produced by all 16S amplicon assays. The TS-tailed-1S dual index protocol is preferred since it provides repeatable profiles on the HiSeq platform and are less labour intensive.Peer reviewe

Gender-Specific Associations Between Saliva Microbiota and Body Size

Objective: The human intestinal microbiota likely play an important role in the development of overweight and obesity. However, the associations between saliva microbiota and body mass index (BMI) have been sparsely studied. The aim of this study was to identify the associations between saliva microbiota and body size in Finnish children. Methods: The saliva microbiota of 900 Finnish children, aged 11-14 years with measured height and weight, was characterized using 16S rRNA (V3-V4) sequencing. Results: The core saliva microbiota consisted of 14 genera that were present in more than 95% of the Finnish children. The saliva microbiota profiles were gender-specific with higher alpha-diversity in boys than girls and significant differences between the genders in community composition and abundances. Alpha-diversity differed between normal weight and overweight girls and between normal weight and obese boys. The composition was dissimilar between normal weight and obese girls, but not in boys. The relative abundance profiles differed according to body size. Decrease in commensal saliva bacteria were observed in all the body sizes when compared to normal weight children. Notably, the relative abundance of bacteria related to, Veillonella, Prevotella, Selenomonas, and Streptococcus was reduced in obese children. Conclusion: Saliva microbiota diversity and composition were significantly associated with body size and gender in Finnish children. Body size-specific saliva microbiota profiles open new avenues for studying the potential roles of microbiota in weight development and management.Peer reviewe

Meal Regularity Plays a Role in Shaping the Saliva Microbiota

Background Diet may influence health directly or indirectly via the human microbiota, emphasizing the need to unravel these complex relationships for future health benefits. Associations between eating habits and gut microbiota have been shown, but less is known about the association between eating habits and saliva microbiota. Objective The aim of this study was to investigate if eating habits and meal patterns are associated with the saliva microbiota. Methods In total, 842 adolescents, aged 11-14 years, from the Finnish Health in Teens (Fin-HIT) study cohort were included in this study. Eating habits and breakfast and dinner patterns were derived from a web-based questionnaire answered in school. Three major eating habit groups were identified: fruit and vegetable avoiders (FV avoiders), healthy and unhealthy. Microbiota profiles were produced from 16S rRNA gene (V3-V4) sequencing of DNA from the saliva samples. Statistical models were adjusted for gender, age, parental language, body mass index (BMI) categories, and sequencing depth. Results Regular breakfast eaters had a higher alpha diversity (Shannon index with mean (standard error of means) 2.27 (0.03) vs. 2.22 (0.03), p = 0.06, inverse Simpson's index with 6.27 (0.17) vs. 5.80 (0.02), p = 0.01), and slight differences in bacterial composition (PERMANOVA: p = 0.001) compared with irregular breakfast eaters. A similar trend in alpha diversity was observed between regular and irregular dinner eaters (Shannon index with 2.27 (0.03) vs. 2.22 (0.03), p = 0.054, inverse Simpson's index with 6.23 (0.17) vs. 6.04 (0.22), p = 0.28), while no difference was found in composition (PERMANOVA: p = 0.08). No differences were identified between eating habit groups and saliva microbiota diversity (Shannon index p = 0.77, inverse Simpson's index p = 0.94) or composition (PERMANOVA: p = 0.13). FV avoiders, irregular breakfast eaters and irregular dinner eaters had high abundances of Prevotella. Conclusion Regularity of eating, especially breakfast eating, was associated with more diverse saliva microbiota and different composition compared with irregular eaters. However, the dissimilarities in composition were small between regular and irregular breakfast eaters. Our results suggest that Prevotella abundances in saliva were common in FV avoiders and meal skippers. However, the clinical implications of these findings need to be evaluated in future studies.Peer reviewe

Antimicrobial drug use in the first decade of life influences saliva microbiota diversity and composition

Background: The human microbiota contributes to health and well-being. Antimicrobials (AM) have an immediate effect on microbial diversity and composition in the gut, but next to nothing is known about their long-term contribution to saliva microbiota. Our objectives were to investigate the long-term impact of AM use on saliva microbiota diversity and composition in preadolescents. We compared the lifetime effects by gender and AMs. We used data from 808 randomly selected children in the Finnish Health In Teens (Fin-HIT) cohort with register-based data on AM purchases from the Social Insurance Institution of Finland. Saliva microbiota was assessed with 16S rRNA (V3-V4) sequencing. The sequences were aligned to the SILVA ribosomal RNA database and classified and counted using the mothur pipeline. Associations between AM use and alpha-diversity (Shannon index) were identified with linear regression, while associations between beta-diversity (Bray-Curtis dissimilarity) and low, medium or high AM use were identified with PERMANOVA. Results: Of the children, 53.6% were girls and their mean age was 11.7 (0.4) years. On average, the children had 7.4 (ranging from 0 to 41) AM prescriptions during their lifespan. The four most commonly used AMs were amoxicillin (n= 2622, 43.7%), azithromycin (n= 1495, 24.9%), amoxicillin-clavulanate (n= 1123, 18.7%) and phenoxymethylpenicillin (n= 408, 6.8%). A linear inverse association was observed between the use of azithromycin and Shannon index (b- 0.015,pvalue = 0.002) in all children, the effect was driven by girls (b- 0.032,pvalue = 0.001), while not present in boys. Dissimilarities were marked between high, medium and low users of all AMs combined, in azithromycin users specifically, and in boys with amoxicillin use. Amoxicillin and amoxicillin-clavulanate use was associated with the largest decrease in abundance ofRikenellaceae. AM use in general and phenoxymethylpenicillin specifically were associated with a decrease ofPaludibacterand pathways related to amino acid degradations differed in proportion between high and low AM users. Conclusions: A systematic approach utilising reliable registry data on lifetime use of AMs demonstrated long-term effects on saliva microbiota diversity and composition. These effects are gender- and AM-dependent. We found that frequent lifelong use of AMs shifts bacterial profiles years later, which might have unforeseen health impacts in the future. Our findings emphasise a concern for high azithromycin use, which substantially decreases bacterial diversity and affects composition as well. Further studies are needed to determine the clinical implications of our findings.Peer reviewe

Pseudomonas putida CSV86: A Candidate Genome for Genetic Bioaugmentation

Peer reviewe