The microbiome of the oral cavity and the digestive tract exists in a delicate homeostasis, and this balance of bacteria plays an important role in the body's immune and inflammatory potency levels [1, 2]. The oral and nasal mucosal membranes are the first entry point for many respiratory viruses, and the microbiome plays a significant role in the pathogenesis of these viral infections [3]. There is certainly precedent for the idea that microbiota, whether digestive (gut/oral) or respiratory, could play a role in the resistance and response to infection by viruses such as the SARS-CoV-2 virus [4], associated with 5.3 million deaths and 272 million cases worldwide by the end of 2021. Salivary microbiota, specifically bacteria, play a significant role in facilitating attachment of incoming microbes into its ecosystem and, from there, breakthrough to the mucosal membranes of the host system. Salivary microbiota have the potential to control viral uptake into cells and whether virus introduction into the host is sufficient for a productive, detectable infection. In addition, these microbiota can create molecules that alter host epithelial barriers [5].

Many studies have identified changes in the gut, oral, and nasopharyngeal bacteria that occur during and post SARS-CoV-2 infection vs. healthy controls [6–11]; however, the mechanism of these changes remains unknown. The SARS-CoV-2 virus has a profound effect on the oral cavity's function, with symptoms including loss of taste, dry mouth, dysgeusia, and opportunist infections that may alter oral microbiota [12]. We can presume that changes in other oral microbiota due to SARS-CoV-2 infection may contribute to these symptoms within the oral cavity. Additional work has attempted to study the changes in oral/gut bacteria that occur during the course of SARS-CoV-2 active infection and its correlation with severity of disease [8, 11, 13–15]. These studies shed light on bacteria that have the potential to increase the morbidity rate with SARS-CoV-2 infection at sites such as the oro-pharynx [16, 17].

The COVID-19 etiological agent, SARS-CoV-2 betacoronavirus, has high spreading capability, mediated by several vehicles, such as oral droplets [18–20]. The oral cavity might be considered a preferred route for SARS-CoV-2 entry, and there is ample evidence that spread primarily via a droplet [2]. These droplets can enter the human body through the mucosal membranes of, primarily, the nasal cavity via the ACE2 + TMPRSS2 + nasal epithelial cells [3]. While there has been more investigation of the nasal-lung axis of infection [4], infection can occur via the oral cavity [1, 2]. This is supported by high expression of both the ACE2 and TMPRSS2 receptors in the salivary glands and the oral cavity mucosa. While oral manifestation of SARS-CoV-2 infection occurs in oral tissues, it is not clear how often it is the primary site of SARS-CoV-2 initial infection [21]. After entry into the oral cavity, a successful infection requires survival of the virus in the saliva or on the mucosa until its adherence to the mucosal surface, followed by penetrance through the ECM binding to the ACE receptor for uptake, and then successful viral reproduction to the point that the level of viral particles exceed the threshold for detection. The question addressed here is whether certain salivary oral microbiota may facilitate attachment and penetration, increasing the risk of a successful infection process.

This was a retrospective study; the purpose was to investigate preexisting oral salivary bacterial diversity and its impact on susceptibility to SARS2-CoV-2 infection. Salivary samples were collected from patients of record, with known pre-pandemic medical and dental history, who were followed through the pandemic. The saliva microbiome is known to be relatively stable over time for individual adults [22, 23], and this method offered the unusual proposition that the salivary microbiome was known prior to infection. This allowed the inquiry into whether there are salivary bacteria taxa profiles associated with susceptibility to successful COVD-19 infections. Based on the location and time of the study in central Illinois, this analysis is of the association between successful infection by alpha, beta, and gamma variants of SARS-CoV-2 and levels of specific oral bacteria taxa (https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-classifications.html).

A small group of oral taxa was shown to be differentially abundant in subjects who went on to be COVID-19 positive. Two of these, Prevotella and Neisseria, are of interest in that they have been tied to COVID-19 in the past. Multivariable analysis (Figure 3) revealed these taxa may be associated in part with differences in age, tobacco usage, and/or numbers of medications required in the groups studied.

A species of the genus Prevotella was observed to be more abundant in the saliva of patients who went on to become SARS-CoV-2 positive (Table 2).

Earlier studies have noted Prevotella pallens was enriched in saliva samples from patients with COVID-19 with active disease, while Rothia mucilaginosa and some Streptoccoci species were at lower levels [13]. A second study of patients with active disease vs. healthy controls noted oral mucosa were enriched for Haemophilus parainfluenzae, Veillonella infantium, Soonwooa purpurea, Prevotella salivae, Prevotella jejuni, and Capnocytophaga gingivalis, while displaying lower levels of a number of taxa [7]. A third study did not see this higher level of Prevotella at least on the genus level in saliva samples of patients with COVID-19 [33]. Based on the enrichment of Prevotella genes in lung samples from COVID-19 patients with active disease and computational identification of possible roles for Prevotella-encoded proteins in host-viral interactions, there is speculation that this enrichment of Prevotella in some patients could play a fairly direct role in stimulating SARS-CoV2 infection and subsequent disease [34]. Comparison between studies is made difficult by the variety of 16S rRNA amplicons that are examined in the different studies. Nevertheless, the higher level of the single Prevotella species seen in the subjects who became COVID-19 positive (Figure 3; Table 2) may provide some support for that model.

Low levels of the bacteria genus Neisseria in patients with COVID-19 have been proposed to play a role in increased inflammation in some patients [35]. There are several reports of lower levels of Nesseria overall in patients with COVID-19, and Iebba et al. report lower levels, specifically of Neiserria elongata in oral rinses from patients with COVID-19 at FDR <0.11 [7, 8, 15]. As shown in Table 2, Neiserria elongata was at a lower level in the patients who went on to contract COVID-19. One might speculate that this species is lower in COVID-19-positive patients before they get the disease.

While the taxa that differ between the two groups may serve as markers of COVID-19 disease susceptibility, the mechanism is obscure. Differences in resistance to viral establishment could be tied to genetics of the host, inflammatory state, immune function, and the microbial organisms that can affect each other [36]. At the time of the initiation of this work, it was not clear if the oral cavity is a primary site of SARS-CoV-2 infection, and this is still unclear [37, 38]. For that reason, the few differences seen in salivary microbiome in the group that had been COVID-19 positive could be manifestations of lifestyle associated with increased risk anywhere in the body. This is not an unprecedented concept, in that there is ample evidence of oral microbiome differences being linked to differential disease risk throughout the body [39].

This study has several limitations: it was an observational, retrospective, single-center study. Multiple factors can cause minor differences in oral microbiota, so any relationships may be indirect, and the number of COVID-positive subjects limits sensitivity of the study [39, 40]. In addition, it is possible that a small number of COVID-negative subjects had been positive at some time, but this was not known to them [41]. However, the large number of the subjects in the COVID-negative group would minimize that as a source of error. We used V2-V3 16S rRNA analysis, which provides reproducible information on the levels of a large percentage of known oral bacteria taxa, but not all oral bacteria were measured [42]. This study has additional limitations: (1) A lack of functional analysis of microbial genes, (2) Collection of saliva in some cases had occurred over a year prior to the COVID-19 pandemic, (3) The date of COVID-19 positivity was not recorded. (4) The presence of restorations such as dental crowns was not considered, although the patients with implants were excluded. A prospective study with more frequent saliva collection would have addressed some of these limitations.

Given the March 1, 2021 cutoff for previous infection status and the location in the United States, findings may be limited to the alpha and beta SARS2-CoV-2 strains [43]. In addition, salivary properties of viscosity, pH level, and immunoglobulin and inflammatory cytokines levels were not assessed, which may play a role in the protective mechanism against microbial attachment and penetration [44].

These bacteria taxa present in the saliva may be associated with increased risk of early SARS-CoV-2 infection in the unvaccinated population. This study can serve as a prelude to the study of the diverse oral microbiota and its effect on the protective mechanism or facilitation of infection by respiratory viruses.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: BIoproject accession numbers: PRJNA674379 and PRJNA739492.

The studies involving human participants were reviewed and approved by UIC IRB. The patients/participants provided their written informed consent to participate in this study.

NC, MH, TB, GA, and NK contributed to the performed data collection and analysis. NC and GA wrote the first draft of the manuscript. All the authors contributed to the editing and agree with the content of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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