Saliva microbiome in relation to SARS-CoV-2 infection in a prospective cohort of healthy US adults.

BACKGROUND: The clinical outcomes of SARS-CoV-2 infection vary in severity, potentially influenced by the resident human microbiota. There is limited consensus on conserved microbiome changes in response to SARS-CoV-2 infection, with many studies focusing on severely ill individuals. This study aimed to assess the variation in the upper respiratory tract microbiome using saliva specimens in a cohort of individuals with primarily mild to moderate disease. METHODS: In early 2020, a cohort of 831 adults without known SARS-CoV-2 infection was followed over a six-month period to assess the occurrence and natural history of SARS-CoV-2 infection. From this cohort, 81 participants with a SARS-CoV-2 infection, along with 57 unexposed counterparts were selected with a total of 748 serial saliva samples were collected for analysis. Total bacterial abundance, composition, population structure, and gene function of the salivary microbiome were measured using 16S rRNA gene and shotgun metagenomic sequencing. FINDINGS: The salivary microbiome remained stable in unexposed individuals over the six-month study period, as evidenced by all measured metrics. Similarly, participants with mild to moderate SARS-CoV-2 infection showed microbiome stability throughout and after their infection. However, there were significant reductions in microbiome diversity among SARS-CoV-2-positive participants with severe symptoms early after infection. Over time, the microbiome diversity in these participants showed signs of recovery. INTERPRETATION: These findings demonstrate the resilience of the salivary microbiome in relation to SARS-CoV-2 infection. Mild to moderate infections did not significantly disrupt the stability of the salivary microbiome, suggesting its ability to maintain its composition and function. However, severe SARS-CoV-2 infection was associated with temporary reductions in microbiome diversity, indicating the limits of microbiome resilience in the face of severe infection. FUNDING: This project was supported in part by Danone North America and grants from the National Institutes of Health, United States.

Elevated inflammatory fecal immune factors in men who have sex with men with HIV associate with microbiome composition and gut barrier function.

Introduction: People living with HIV infection (PLWH) exhibit elevated levels of gastrointestinal inflammation. Potential causes of this inflammation include HIV infection and associated immune dysfunction, sexual behaviors among men who have sex with men (MSM) and gut microbiome composition. Methods: To better understand the etiology of gastrointestinal inflammation we examined levels of 28 fecal soluble immune factors (sIFs) and the fecal microbiome in well-defined cohorts of HIV seronegative MSM (MSM-SN), MSM with untreated HIV infection (MSM-HIV) and MSM with HIV on anti-retroviral treatment (MSMART). Additionally, fecal solutes from these participants were used to stimulate T-84 colonic epithelial cells to assess barrier function. Results: Both MSM cohorts with HIV had elevated levels of fecal calprotectin, a clinically relevant marker of GI inflammation, and nine inflammatory fecal sIFs (GM-CSF, ICAM-1, IL-1ß, IL-12/23, IL-15, IL-16, TNF-ß, VCAM-1, and VEGF). Interestingly, four sIFs (GM-CSF, ICAM-1, IL-7 and IL-12/23) were significantly elevated in MSM-SN compared to seronegative male non-MSM. Conversely, IL-22 and IL-13, cytokines beneficial to gut health, were decreased in all MSM with HIV and MSM-SN respectively. Importantly, all of these sIFs significantly correlated with calprotectin, suggesting they play a role in GI inflammation. Principal coordinate analysis revealed clustering of fecal sIFs by MSM status and significant associations with microbiome composition. Additionally, fecal solutes from participants in the MSM-HIV cohort significantly decreased colonic transcellular fluid transport in vitro, compared to non-MSM-SN, and this decrease associated with overall sIF composition and increased concentrations of eight inflammatory sIFs in participants with HIV. Lastly, elevated levels of plasma, sCD14 and sCD163, directly correlated with decreased transcellular transport and microbiome composition respectively, indicating that sIFs and the gut microbiome are associated with, and potentially contribute to, bacterial translocation. Conclusion: Taken together, these data demonstrate that inflammatory sIFs are elevated in MSM, regardless of HIV infection status, and are associated with the gut microbiome and intestinal barrier function.

Assessing saliva microbiome collection and processing methods.

The oral microbiome has been connected with lung health and may be of significance in the progression of SARS-CoV-2 infection. Saliva-based SARS-CoV-2 tests provide the opportunity to leverage stored samples for assessing the oral microbiome. However, these collection kits have not been tested for their accuracy in measuring the oral microbiome. Saliva is highly enriched with human DNA and reducing it prior to shotgun sequencing may increase the depth of bacterial reads. We examined both the effect of saliva collection method and sequence processing on measurement of microbiome depth and diversity by 16S rRNA gene amplicon and shotgun metagenomics. We collected 56 samples from 22 subjects. Each subject provided saliva samples with and without preservative, and a subset provided a second set of samples the following day. 16S rRNA gene (V4) sequencing was performed on all samples, and shotgun metagenomics was performed on a subset of samples collected with preservative with and without human DNA depletion before sequencing. We observed that the beta diversity distances within subjects over time was smaller than between unrelated subjects, and distances within subjects were smaller in samples collected with preservative. Samples collected with preservative had higher alpha diversity measuring both richness and evenness. Human DNA depletion before extraction and shotgun sequencing yielded higher total and relative reads mapping to bacterial sequences. We conclude that collecting saliva with preservative may provide more consistent measures of the oral microbiome and depleting human DNA increases yield of bacterial sequences.

Maternal cecal microbiota transfer rescues early-life antibiotic-induced enhancement of type 1 diabetes in mice.

Early-life antibiotic exposure perturbs the intestinal microbiota and accelerates type 1 diabetes (T1D) development in the NOD mouse model. Here, we found that maternal cecal microbiota transfer (CMT) to NOD mice after early-life antibiotic perturbation largely rescued the induced T1D enhancement. Restoration of the intestinal microbiome was significant and persistent, remediating the antibiotic-depleted diversity, relative abundance of particular taxa, and metabolic pathways. CMT also protected against perturbed metabolites and normalized innate and adaptive immune effectors. CMT restored major patterns of ileal microRNA and histone regulation of gene expression. Further experiments suggest a gut-microbiota-regulated T1D protection mechanism centered on Reg3γ, in an innate intestinal immune network involving CD44, TLR2, and Reg3γ. This regulation affects downstream immunological tone, which may lead to protection against tissue-specific T1D injury.

Systems Analysis of Gut Microbiome Influence on Metabolic Disease in HIV-Positive and High-Risk Populations.

Poor metabolic health, characterized by insulin resistance and dyslipidemia, is higher in people living with HIV and has been linked with inflammation, antiretroviral therapy (ART) drugs, and ART-associated lipodystrophy (LD). Metabolic disease is associated with gut microbiome composition outside the context of HIV but has not been deeply explored in HIV infection or in high-risk men who have sex with men (HR-MSM), who have a highly altered gut microbiome composition. Furthermore, the contribution of increased bacterial translocation and associated systemic inflammation that has been described in HIV-positive and HR-MSM individuals has not been explored. We used a multiomic approach to explore relationships between impaired metabolic health, defined using fasting blood markers, gut microbes, immune phenotypes, and diet. Our cohort included ART-treated HIV-positive MSM with or without LD, untreated HIV-positive MSM, and HR-MSM. For HIV-positive MSM on ART, we further explored associations with the plasma metabolome. We found that elevated plasma lipopolysaccharide binding protein (LBP) was the most important predictor of impaired metabolic health and network analysis showed that LBP formed a hub joining correlated microbial and immune predictors of metabolic disease. Taken together, our results suggest the role of inflammatory processes linked with bacterial translocation and interaction with the gut microbiome in metabolic disease among HIV-positive and -negative MSM.IMPORTANCE The gut microbiome in people living with HIV (PLWH) is of interest since chronic infection often results in long-term comorbidities. Metabolic disease is prevalent in PLWH even in well-controlled infection and has been linked with the gut microbiome in previous studies, but little attention has been given to PLWH. Furthermore, integrated analyses that consider gut microbiome, together with diet, systemic immune activation, metabolites, and demographics, have been lacking. In a systems-level analysis of predictors of metabolic disease in PLWH and men who are at high risk of acquiring HIV, we found that increased lipopolysaccharide-binding protein, an inflammatory marker indicative of compromised intestinal barrier function, was associated with worse metabolic health. We also found impaired metabolic health associated with specific dietary components, gut microbes, and host and microbial metabolites. This study lays the framework for mechanistic studies aimed at targeting the microbiome to prevent or treat metabolic endotoxemia in HIV-infected individuals.

Correction to: An exploration of Prevotella-rich microbiomes in HIV and men who have sex with men.

Following publication of the original article [1], the authors reported an error in Fig. 2. The original Fig. 2 has been incorrectly replaced with the Supplementary Fig. 2. The correct Fig. 2 is presented here.

Can gut microbiota of men who have sex with men influence HIV transmission?

Gaining a complete understanding of transmission risk factors will assist in efforts to reduce new HIV infections, especially within the disproportionally affected population of men who have sex with men (MSM). We recently reported that the fecal microbiota of MSM elevates immune activation in gnotobiotic mice and enhances HIV infection in vitro over that of fecal microbiota from men who have sex with women. We also demonstrated elevation of the gut homing marker CD103 (integrin αE) on CD4+ T cells by MSM-microbiota. Here we provide additional evidence that the gut microbiota is a risk factor for HIV transmission in MSM by showing elevated frequencies of the HIV co-receptor CCR5 on CD4+ T cells in human rectosigmoid colon biopsies. We discuss our interest in specific MSM-associated bacteria and propose the influx of CD103+ and CCR5+ CD4+ T cells into the colon as a potential link between the MSM microbiota and HIV transmission.

Gut microbiota from high-risk men who have sex with men drive immune activation in gnotobiotic mice and in vitro HIV infection.

Men who have sex with men (MSM) have differences in immune activation and gut microbiome composition compared with men who have sex with women (MSW), even in the absence of HIV infection. Gut microbiome differences associated with HIV itself when controlling for MSM, as assessed by 16S rRNA sequencing, are relatively subtle. Understanding whether gut microbiome composition impacts immune activation in HIV-negative and HIV-positive MSM has important implications since immune activation has been associated with HIV acquisition risk and disease progression. To investigate the effects of MSM and HIV-associated gut microbiota on immune activation, we transplanted feces from HIV-negative MSW, HIV-negative MSM, and HIV-positive untreated MSM to gnotobiotic mice. Following transplant, 16S rRNA gene sequencing determined that the microbiomes of MSM and MSW maintained distinct compositions in mice and that specific microbial differences between MSM and MSW were replicated. Immunologically, HIV-negative MSM donors had higher frequencies of blood CD38+ HLADR+ and CD103+ T cells and their fecal recipients had higher frequencies of gut CD69+ and CD103+ T cells, compared with HIV-negative MSW donors and recipients, respectively. Significant microbiome differences were not detected between HIV-negative and HIV-positive MSM in this small donor cohort, and immune differences between their recipients were trending but not statistically significant. A larger donor cohort may therefore be needed to detect immune-modulating microbes associated with HIV. To investigate whether our findings in mice could have implications for HIV replication, we infected primary human lamina propria cells stimulated with isolated fecal microbiota, and found that microbiota from MSM stimulated higher frequencies of HIV-infected cells than microbiota from MSW. Finally, we identified several microbes that correlated with immune readouts in both fecal recipients and donors, and with in vitro HIV infection, which suggests a role for gut microbiota in immune activation and potentially HIV acquisition in MSM.

An exploration of Prevotella-rich microbiomes in HIV and men who have sex with men.

BACKGROUND: Gut microbiome characteristics associated with HIV infection are of intense research interest but a deep understanding has been challenged by confounding factors across studied populations. Notably, a Prevotella-rich microbiome described in HIV-infected populations is now understood to be common in men who have sex with men (MSM) regardless of HIV status, but driving factors and potential health implications are unknown. RESULTS: Here, we further define the MSM-associated gut microbiome and describe compositional differences between the fecal microbiomes of Prevotella-rich MSM and non-MSM that may underlie observed pro-inflammatory properties. Furthermore, we show relatively subtle gut microbiome changes in HIV infection in MSM and women that include an increase in potential pathogens that is ameliorated with antiretroviral therapy (ART). Lastly, using a longitudinal cohort, we describe microbiome changes that happen after ART initiation. CONCLUSIONS: This study provides an in-depth characterization of microbiome differences that occur in a US population infected with HIV and demonstrates the degree to which these differences may be driven by lifestyle factors, ART, and HIV infection itself. Understanding microbiome compositions that occur with sexual behaviors that are high risk for acquiring HIV and untreated and ART-treated HIV infection will guide the investigation of immune and metabolic functional implications to ultimately target the microbiome therapeutically.

Microbiome and metabolome data integration provides insight into health and disease.

For much of our history, the most basic information about the microbial world has evaded characterization. Next-generation sequencing has led to a rapid increase in understanding of the structure and function of host-associated microbial communities in diverse diseases ranging from obesity to autism. Through experimental systems such as gnotobiotic mice only colonized with known microbes, a causal relationship between microbial communities and disease phenotypes has been supported. Now, microbiome research must move beyond correlations and general demonstration of causality to develop mechanistic understandings of microbial influence, including through their metabolic activities. Similar to the microbiome field, advances in technologies for cataloguing small molecules have broadened our understanding of the metabolites that populate our bodies. Integration of microbial and metabolomics data paired with experimental validation has promise for identifying microbial influence on host physiology through production, modification, or degradation of bioactive metabolites. Realization of microbial metabolic activities that affect health is hampered by gaps in our understanding of (1) biological properties of microbes and metabolites, (2) which microbial enzymes/pathways produce which metabolites, and (3) the effects of metabolites on hosts. Capitalizing on known mechanistic relationships and filling gaps in our understanding has the potential to enable translational microbiome research across disease contexts.