The human microbiome is the name given to the collected communities of bacteria that live on and in the human body. The oral microbiome is one of the most diverse (1
) of any human-associated microbial community (2
). The oral microbiome is a causative factor in conditions such as dental caries (3
), periodontal disease (4
), and halitosis (5
) and has also been implicated as a reservoir for infection at other body sites (2
) and in the pathogenesis of nonoral diseases, such as inflammatory bowel disease (6
). Strictly speaking there is no single “oral microbiome,” as its composition is highly heterogeneous at different sites in the mouth (7
), but the term is commonly used to encompass all of these sites. Site-specific microbiomes can be observed in the periodontal sulcus, dental plaque, tongue, buccal mucosa, and saliva (9
). The salivary microbiome exhibits long-term stability and can be considered an important reservoir that contains microorganisms from all distinct ecological niches of the oral cavity. Characterizing and understanding the factors defining the composition of the salivary microbiome are thus crucial to understanding the oral microbiome (10
Some factors that are thought to influence the human microbiome include environment, diet, disease status, and host genetics (12
). The relative importance of these factors for the oral microbiome is still under debate, with the majority of previous studies focusing on the gut microbiome (7–9
), although it seems reasonable to assume some potential interaction between the salivary microbiome and microbial communities in other parts of the human body, including the intestinal tract (10
There is evidence that genetically related individuals tend to share more gut microbes than unrelated individuals do, whether or not they are living in the same house at the time of sampling (13
). However, the levels of covariation are similar in monozygotic and dizygotic twins, suggesting that a shared early environment may be a more important factor than genetics (13
). The effect of cohabitation with direct and frequent contact is greatest when considering the skin microbiome, with a less-evident effect on the gut and salivary microbiomes (11
There is also evidence that genetic variation is linked to microbiome composition across other body sites, including the mouth (12
), with a recent genome-wide association study (GWAS) identifying several human loci associated (P
< 5 × 10−8
) with microbial taxonomies in the gut microbiome (16
). However, no study thus far has incorporated both genetic relatedness (as a continuous variable) and shared environment into the same analysis of the salivary microbiome.
Despite high diversity between individuals, the salivary microbiome appears to have little geographical structure at the genus level at the global scale (17
). Nevertheless, at smaller geographical scales, it appears that the environment plays a role in the oral microbiome. Song et al. studied 60 household units and found that the bacterial composition of dorsal tongue bacterial samples was more similar between cohabiting family members than for individuals from different households, with partners and mother-child pairs having significantly more similar communities (18
). However, this did not include information on genetic relatedness in addition to family relationships. It appears that household-level differences in the salivary microbiome may also apply to genetically unrelated individuals and nonpartners, with a similar pattern observed in analysis of 24 household pairs of genetically unrelated individuals, only half of whom were considered romantic couples at the time of sampling (19
The establishment of the oral microbiome appears to proceed rapidly in the first few years of life, with a notable increase in diversity from 0 to 3 years (18
), especially after the eruption of teeth (20
). The plaque microbiome also appears stable within adult individuals over a period of at least 3 months, with a unique “fingerprint” of oligotypes discernible even within a single bacterial genus (21
). Another study indicates that the salivary microbiome is relatively stable over a year, despite measurable effects of interventions like flossing (22
). Taken together, these findings suggest the intriguing hypothesis that once a particular oral microbiome is established earlier in life, it can potentially persist for months and perhaps even years, particularly if external factors such as diet remain fixed. If this were true, shared upbringing effects would continue to be detected in the salivary microbiome even after individuals are no longer living in the same household (15
A recently described large Ashkenazi Jewish family (23
) offers an opportunity to investigate the effect of both environment and genetics in closely related individuals. The availability of host genetic data for this cohort means that we can calculate similarity between individuals based on single nucleotide polymorphisms (SNPs), rather than using measures of relatedness from pedigrees that do not precisely correspond to shared genetic content (24
). We hypothesized that using this more accurate measure of host genetic similarity could lead to different conclusions about the proportion of shared microbiome composition attributable to genetics compared to previous studies. While, like other studies, we lack information on potential confounders such as diet and lifestyle (17
), due to shared cultural practices between members of the ultraorthodox Ashkenazi Jewish community (25
), we believe that confounding factors are likely to be more controlled for in this cohort than in others. For this reason, this cohort represents a unique opportunity to compare the salivary microbiome within a large number of individuals living in separate locations but nevertheless sharing a similar diet, lifestyle, and genetic background and to investigate the long-term effect of shared upbringing on salivary microbiome composition.
We have conducted, to our knowledge, the first simultaneous investigation of the role of environment and host genetics in shaping the human salivary microbiome in a cohort of closely related individuals within a large Ashkenazi Jewish family. We found a weak correlation between host kinship and salivary microbiome dissimilarity before taking shared household into account and an apparent small but significant effect of genetics when using kinships based on the family pedigree as proxies for genetic similarity. However, when using kinship estimates based on genome-wide SNPs between individuals and simultaneously controlling for shared household with a permutational analysis of variance, we find no support for any clear effect of human genetics, suggesting that shared environment has a much larger effect than genetics and is the dominant factor affecting the salivary microbiome. Typically shared household had an order of magnitude greater effect compared with other significant variables. For example, in our analysis where city was also used as an environmental variable, the variance explained was as follows: household (18.3%), age (3.8%), and sequencing plate (2.9%) (Table 2
We also observed that younger children living in the same household shared subtle variations in phylotype abundance within genera with their parents (Fig. 3
). However, despite a persistence of household effects, it would be wrong to conclude that the salivary microbiome is completely fixed once established, as it clearly has aspects that can change over time. For example, shared household explained more variation for spousal pairs (likely due to frequent contact between them) and that phylotypes observed in younger children and their parents were not seen in older children (likely due to less frequent contact between them). Taken together, these observations support the view that human genetics does not play a major role in shaping the salivary microbiome, at least not in individuals of the same ethnicity, compared to the environment and contact with other individuals.
Our results confirm the seemingly paradoxical situation that the salivary microbiome is largely consistent across global geographical scales but can show large variation between households in the same city. Previous studies have also found evidence of small variations in salivary microbiome composition comparing samples across a global scale (17
). As noted previously, this variation could be influenced by differences in environmental or cultural factors, in which case controlling for these differences would decrease the amount of geographical variation. All individuals in our study follow a traditional Ashkenazi Jewish lifestyle and subsequently are thought to share a similar diet and lifestyle regardless of geographical location (25
), which may reduce the variation attributable to city-level differences.
The establishment of the oral microbiome early in life may lead to the persistence of a similar composition over several years. The microbial composition of sites within the mouth has been previously observed to be persistent within individuals over periods of months (21
) to a year (22
), and we see similar strain-level variation between spouses and their young children as observed between individuals by Utter et al. (21
) (Fig. 3
). Our results indicate the persistence of household effects in individuals no longer cohabiting, suggesting that the salivary microbiome composition established early in life via shared upbringing is able to persist for at least several years. It has been observed that monozygotic twins do not have significantly more similar gut microbiomes than dizygotic twins (13
). Stahringer et al. observed the same effect in the salivary microbiome and also found that the salivary microbiomes of twins became less similar as they grew older and ceased cohabiting, concluding that “nurture trumps nature” in the salivary microbiome (15
). Our findings from a large number of related individuals rather than twins support this view, including the persistence of shared upbringing effects. Shared upbringing appears to be the dominant factor affecting microbiome composition in both the gut and the mouth, rather than genetic similarity. This may have implications for understanding the familial aggregation of diseases such as inflammatory bowel disease, which has been suggested to have an environmental component (28
The salivary microbiome appears to be far more resilient to perturbation than the gut microbiome is, with a rapid return to baseline composition after a short course of antibiotics (29
). While this could be because of the pharmacokinetics of the antibiotics involved, Zaura et al. speculate that this difference may be due to the salivary microbial ecosystem’s higher intrinsic resilience to stress, as the mouth is subject to more frequent perturbation (30
). Our work supporting the dominant role of the environment in affecting salivary microbiome composition suggests that another important factor in long-term persistence may be the regular reseeding of the ecosystem with bacteria from the external environment.
The fact that we reached our conclusion about the lack of effect of genetics only after including kinship based on genome-wide SNP markers casts doubt on the reliability of pedigrees for calculating relatedness. There are several possible reasons for a discrepancy between kinship estimates from pedigrees and allele sharing (24
). One possibility is errors in the pedigree, most likely due to extrapair paternities, although this explanation can be ruled out in this data set. More importantly, inherent stochasticity in the Mendelian process of inheritance means that although parents always pass on 50% of their genes to their offspring, SNPs are inherited together in blocks (i.e., haplotypes), meaning that the relatedness between two offspring in a family can be substantially different from 50%. Finally, and most importantly for this closely related population, shallow pedigrees cannot fully capture complex inbreeding patterns. Thus, while pedigrees are a good model for host relatedness in microbiome studies of large randomly mating populations, they should be used with caution in closely related large families like this one.
Because all individuals in our main cohort were members of the same extended Ashkenazi Jewish family, the genetic variation in our data set is therefore much lower than between individuals from a wider population. It is conceivable that host genetics between more distantly related individuals may play a significant role in affecting salivary microbiome composition. However, we note that a recent study of the nasopharyngeal microbiome among Hutterite individuals (a founder population in North America) detected associations between host variation and microbial composition with a similar cohort size (31
), demonstrating that limited genetic variation can be associated with the composition of other microbiomes; it may simply be that the salivary microbiome is relatively unaffected by such variation.
Furthermore, our study looked at only overall genetic similarity, assessed using community comparison metrics based on taxon abundances. They therefore do not preclude the existence of fine-scale links between particular microbial taxa and individual genetic loci, particularly in immune-sensing genes such as those identified in the gut microbiome by Bonder et al. using a much larger cohort (32
), although our study was not designed or have the statistical power to detect such associations.
Additionally, we lack detailed information on diet and lifestyle factors of individuals in this study. However, the shared cultural practices within this ultraorthodox Ashkenazi Jewish family mean that it is not unreasonable to assume that they share similar lifestyles and diet despite living in different locations around the world (25
The apparent persistence of shared upbringing could be confounded by the fact that individuals may continue living near the household where they grew up. If this were the case, then our observation could instead be due to the persistence of a shared environment beyond the household at a level intermediate between household and city, rather than the persistence of a stable salivary microbiome following environmental change. Finally, our samples represent only a single cross-sectional snapshot in time. More long-term longitudinal studies like the work of Stahringer et al. on twins (15
) are necessary to investigate the persistence of the salivary microbiome after its establishment early in life in a variety of relatedness settings.
In summary, our results incorporating a measure of genetic relatedness using SNPs demonstrate that the overall composition of the human salivary microbiome in a large Ashkenazi Jewish family is largely influenced by shared environment rather than host genetics. An apparent significant effect of host genetics using pedigree-based estimates disappears when using genetic markers instead, which shows that in future microbiome research, the use of pedigree relatedness as a proxy for host genetic similarity should be done with caution. Geographical structuring occurs to a greater extent at the household level within cities than between cities on different continents. Living in the same household is associated with a more similar salivary microbiome, and this effect persists after individuals have left the household. This is consistent with the long-term persistence of the salivary microbiome composition established earlier in life due to shared upbringing.