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Many pathogenic microbial ecosystems are polymicrobial, and community function can be shaped by interbacterial interactions. Little is known, however, regarding the genetic determinants required for fitness in heterotypic community environments. In periodontal diseases, Porphyromonas gingivalis is a primary pathogen, but only within polymicrobial communities. Here, we used a transposon sequencing (Tn-Seq) library of P. gingivalis to screen for genes that influence fitness of the organism in a coinfection murine abscess model with the oral partner species Streptococcus gordonii and Fusobacterium nucleatum. Genes impacting fitness with either organism were involved in diverse processes, including metabolism and energy production, along with cell wall and membrane biogenesis. Despite the overall similarity of function, the majority of identified genes were specific to the partner species, indicating that synergistic mechanisms of P. gingivalis vary to a large extent according to community composition. Only two genes were identified as essential for P. gingivalis fitness in abscess development with both S. gordonii and F. nucleatum: ptk1, encoding a tyrosine kinase, and inlJ, encoding an internalin family surface protein. Ptk1, but not InlJ, is required for community development with S. gordonii, and we found that the action of this kinase is similarly required for P. gingivalis to accumulate in a community with F. nucleatum. A limited number of P. gingivalis genes are therefore required for species-independent synergy, and the Ptk1 tyrosine kinase network may integrate and coordinate input from multiple organisms.

Patients with cystic fibrosis (CF) experience lifelong respiratory infections which are a significant cause of morbidity and mortality. These infections are polymicrobial in nature, and the predominant bacterial species undergo a predictable series of changes as patients age. Young patients have populations dominated by opportunists that are typically found within the microbiome of the human nasopharynx, such as nontypeable Haemophilus influenzae (NTHi); these are eventually supplanted and the population within the CF lung is later dominated by pathogens such as Pseudomonas aeruginosa (Pa). In this study, we investigated how initial colonization with NTHi impacts colonization and persistence of Pa in the respiratory tract.Analysis of polymicrobial biofilms in vitro by confocal microscopy revealed that NTHi promoted greater levels of Pa biofilm volume and diffusion. However, sequential respiratory infection of mice with NTHi followed by Pa resulted in significantly lower Pa as compared to infection with Pa alone. Coinfected mice also had reduced airway tissue damage and lower levels of inflammatory cytokines as compared with Pa infected mice. Similar results were observed after instillation of heat-inactivated NTHi bacteria or purified NTHi lipooligosaccharide (LOS) endotoxin prior to Pa introduction. Based on these results, we conclude that NTHi significantly reduces susceptibility to subsequent Pa infection, most likely due to priming of host innate immunity rather than a direct competitive interaction between species. These findings have potential significance with regard to therapeutic management of early life infections in patients with CF.

Zebrafish (Danio rerio) is an attractive model organism to use for an array of scientific studies, including host-microbe interactions. Zebrafish contain a core (i.e., consistently detected) intestinal microbiome consisting primarily of Proteobacteria. Furthermore, this core intestinal microbiome is plastic and can be significantly altered due to external factors. Zebrafish are particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae. As an intestinal pathogen, V. cholerae must colonize the intestine of an exposed host for pathogenicity to occur. Members of the resident intestinal microbial community likely must be reduced or eliminated by V. cholerae for colonization, and subsequent disease, to occur. Many studies have explored a variety of aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms but few have researched how a V. cholerae infection changes the resident intestinal microbiome. In this study, 16S rRNA gene sequencing was used to examine how five genetically diverse V. cholerae strains alter the intestinal microbiome following an infection. We found that V. cholerae colonization induced significant changes in the zebrafish intestinal microbiome. Notably, changes in the microbial profile were significantly different from each other, based on the particular strain of V. cholerae used to infect zebrafish hosts. We conclude that V. cholerae significantly modulates the zebrafish intestinal microbiota to enable colonization and that specific microbes that are targeted depend on the V. cholerae genotype.

Staphylococcus aureus is associated with the development of persistent and severe inflammatory diseases of the upper airways. Yet, S. aureus is also carried asymptomatically in the sinonasal cavity of ∼50% of healthy adults. The causes of this duality and host and microbial factors that tip the balance between S. aureus pathogenesis and commensalism are poorly understood. We have shown that by degrading mucins, anaerobic microbiota support the growth of airway pathogens by liberating metabolites that are otherwise unavailable. Given the widely reported culture-based detection of anaerobes from individuals with chronic rhinosinusitis (CRS), here we tested our hypothesis that CRS microbiota is characterized by a mucin-degrading phenotype that alters S. aureus physiology. Using 16S rRNA gene sequencing, we indeed observed an increased prevalence and abundance of anaerobes in CRS relative to non-CRS controls. PICRUSt2-based functional predictions suggested increased mucin degradation potential among CRS microbiota that was confirmed by direct enrichment culture. Prevotella, Fusobacterium, and Streptococcus comprised a core mucin-degrading community across CRS subjects that generated a nutrient pool that augmented S. aureus growth on mucin as a carbon source. Finally, using transcriptome sequencing (RNA-seq), we observed that S. aureus transcription is profoundly altered in the presence of mucin-derived metabolites, though expression of several key metabolism- and virulence-associated pathways varied between CRS-derived bacterial communities. Together, these data support a model in which S. aureus metabolism and virulence in the upper airways are dependent upon the composition of cocolonizing microbiota and the metabolites they exchange.

Zebrafish (Danio rerio) are an attractive model organism for a variety of scientific studies, including host-microbe interactions. The organism is particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae, an intestinal pathogen. V. cholerae must colonize the intestine of an exposed host for pathogenicity to occur. While numerous studies have explored various aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms, few, if any, have examined how a V. cholerae infection alters the resident intestinal microbiome and the role of the type six secretion system (T6SS) in that process. In this study, 16S rRNA gene sequencing was utilized to investigate how strains of V. cholerae both with and without the T6SS alter the aforementioned microbial profiles following an infection. V. cholerae infection induced significant changes in the zebrafish intestinal microbiome, and while not necessary for colonization, the T6SS was important for inducing mucin secretion, a marker for diarrhea. Additional salient differences to the microbiome were observed based on the presence or absence of the T6SS in the V. cholerae utilized for challenging the zebrafish hosts. We conclude that V. cholerae significantly modulates the zebrafish intestinal microbiome to enable colonization and that the T6SS is important for pathogenesis induced by the examined V. cholerae strains. Furthermore, the presence or absence of T6SS differentially and significantly affected the composition and structure of the intestinal microbiome, with an increased abundance of other Vibrio bacteria observed in the absence of V. cholerae T6SS.

The ability to predict invasive fungal infections (IFI) in patients with hematological malignancies is fundamental for successful therapy. Although gut dysbiosis is known to occur in hematological patients, whether airway dysbiosis also contributes to the risk of IFI has not been investigated. Nasal and oropharyngeal swabs were collected for functional microbiota characterization in 173 patients with hematological malignancies recruited in a multicenter, prospective, observational study and stratified according to the risk of developing IFI. A lower microbial richness and evenness were found in the pharyngeal microbiota of high-risk patients that were associated with a distinct taxonomic and metabolic profile. A murine model of IFI provided biologic plausibility for the finding that loss of protective anaerobes, such as Clostridiales and Bacteroidetes, along with an apparent restricted availability of tryptophan, is causally linked to the risk of IFI in hematologic patients and indicates avenues for antimicrobial stewardship and metabolic reequilibrium in IFI. This work was supported by FunMeta Project (ERC-2011-AdG 293714), MicroTher(ERC-2018-PoC-813099), and Gilead (IN-IT-131-4525-518872.9) to L.R. and the GrantAgency of the Czech Republic (GACR No 17-24592Y) and the Czech Ministry ofEducation, Youth and Sports (CETOCOEN PLUS CZ.02.1.01/0.0/0.0/15_003/0000469;LM2015051 and CETOCOEN EXCELLENCE Teaming 2 project CZ.02.1.01/0.0/0.0/18_046/0015975; and Horizon2020 project 857560) to Z.S.

Syphilis is a bacterial infection caused by Treponema pallidum subsp. pallidum. Infection with T. pallidum subsp. pallidum and its dissemination lead to the synthesis of proinflammatory cytokines triggered by the interaction of bacterial lipoproteins with Toll-like receptor 2 (TLR2). TLR2 contains several nonsynonymous single-nucleotide polymorphisms that may impact the activation of its signaling cascade and alter the responsiveness to, or the course of, various infectious diseases, including those caused by pathogenic spirochetes. To investigate whether TLR2 polymorphism may influence susceptibility to syphilis, 221 healthy individuals with no history of syphilis (controls) and 137 patients diagnosed with syphilis (cases) were screened for the presence of the Arg753Gln polymorphism in the TLR2 gene (2258G→A; rs5743708). The Arg753Gln variant occurs at a significantly lower frequency in syphilis patients (4 of 137 [3%]) than in controls (24 of 221 [10.9%]). These data suggest that TLR2 Arg753Gln may protect from the development of syphilis due to reduced signaling.

Mucosal surfaces like those present in the lung, gut, and mouth interface with distinct external environments. These mucosal gateways are not only portals of entry for potential pathogens but also homes to microbial communities that impact host health. Secretory immunoglobulin A (SIgA) is the single most abundant acquired immune component secreted onto mucosal surfaces and, via the process of immune exclusion, shapes the architecture of these microbiomes. Not all microorganisms at mucosal surfaces are targeted by SIgA; therefore, a better understanding of the SIgA-coated fraction may identify the microbial constituents that stimulate host immune responses in the context of health and disease. Chronic diseases like type 2 diabetes are associated with altered microbial communities (dysbiosis) that in turn affect immune-mediated homeostasis. 16S rRNA gene sequencing of SIgA-coated/uncoated bacteria (IgA-Biome) was conducted on stool and saliva samples of normoglycemic participants and individuals with prediabetes or diabetes (n = 8/group). These analyses demonstrated shifts in relative abundance in the IgA-Biome profiles between normoglycemic, prediabetic, or diabetic samples distinct from that of the overall microbiome. Differences in IgA-Biome alpha diversity were apparent for both stool and saliva, while overarching bacterial community differences (beta diversity) were also observed in saliva. These data suggest that IgA-Biome analyses can be used to identify novel microbial signatures associated with diabetes and support the need for further studies exploring these communities. Ultimately, an understanding of the IgA-Biome may promote the development of novel strategies to restructure the microbiome as a means of preventing or treating diseases associated with dysbiosis at mucosal surfaces.

Gastroesophageal reflux is a common gastrointestinal issue that can lead to aspiration and contribute to respiratory problems. Little is known about how reflux can alter the respiratory microenvironment. We aimed to determine if the presence of gastric pepsinogen in the trachea was associated with changes in the microbial and inflammatory microenvironment. A pediatric cohort at high risk of reflux aspiration was prospectively recruited, and the tracheal microenvironment was examined. Pepsinogen A3 (PGA3) and cytokines were measured. The microbiome (bacterial and fungal) was profiled using 16S rRNA and internal transcribed spacer 2 (ITS2) amplicon sequencing. Increased bacterial richness and an altered composition driven by an enrichment of Prevotella correlated with high PGA3 levels. Fungal richness increased with PGA3, with higher Candida relative abundances observed in a subset of samples with high PGA3 levels. Source tracking of tracheal microbial taxa against taxa from matched oral and gastric samples revealed a significantly greater contribution of oral than of gastric taxa with higher PGA3 levels. Tracheal cytokines were differentially produced when stratified according to PGA3, with higher levels of interleukin-1 (IL-1)-related cytokines and IL-8 being associated with high PGA3 levels. Network analysis across cytokine and microbiome measures identified relationships between IL-1-related proteins and microbial taxa, with the presence of respiratory issues associated with higher levels of IL-1β, IP-10, and Prevotella. In conclusion, PGA3 levels in the trachea are correlated with increases in specific microbial taxa and inflammatory molecules, with an increase in oral microbes with increasing PGA3.

Biofilm formation by Streptococcus pyogenes (group A streptococcus [GAS]) in model systems mimicking the respiratory tract is poorly documented. Most studies have been conducted on abiotic surfaces, which poorly represent human tissues. We have previously shown that GAS forms mature and antibiotic-resistant biofilms on physiologically relevant epithelial cells. However, the roles of the substratum, extracellular matrix (ECM) components, and GAS virulence factors in biofilm formation and structure are unclear. In this study, biofilm formation was measured on respiratory epithelial cells and keratinocytes by determining biomass and antibiotic resistance, and biofilm morphology was visualized using scanning electron microscopy. All GAS isolates tested formed biofilms that had similar, albeit not identical, biomass and antibiotic resistance for both cell types. Interestingly, functionally mature biofilms formed more rapidly on keratinocytes but were structurally denser and coated with more ECM on respiratory epithelial cells. The ECM was crucial for biofilm integrity, as protein- and DNA-degrading enzymes induced bacterial release from biofilms. Abiotic surfaces supported biofilm formation, but these biofilms were structurally less dense and organized. No major role for M protein, capsule, or streptolysin O was observed in biofilm formation on epithelial cells, although some morphological differences were detected. NAD-glycohydrolase was required for optimal biofilm formation, whereas streptolysin S and cysteine protease SpeB impaired this process. Finally, no correlation was found between cell adherence or autoaggregation and GAS biofilm formation. Combined, these results provide a better understanding of the role of biofilm formation in GAS pathogenesis and can potentially provide novel targets for future treatments against GAS infections.

The cryptic plasmid pCM is critical for chlamydial colonization in the gastrointestinal tract. Nevertheless, orally inoculated plasmid-free Chlamydia sp. was still able to colonize the gut. Surprisingly, orally inoculated Chlamydia sp. deficient in only plasmid-encoded pGP3 was no longer able to colonize the gut. A comparison of live organism recoveries from individual gastrointestinal tissues revealed that pGP3-deficient Chlamydia sp. survived significantly better than plasmid-free Chlamydia sp. in small intestinal tissues. However, the small intestinal pGP3-deficient Chlamydia sp. failed to reach the large intestine, explaining the lack of live pGP3-deficient Chlamydia sp. in rectal swabs following an oral inoculation. Interestingly, pGP3-deficient Chlamydia sp. was able to colonize the colon following an intracolon inoculation, suggesting that pGP3-deficient Chlamydia sp. might be prevented from spreading from the small intestine to the large intestine. This hypothesis is supported by the finding that following an intrajejunal inoculation that bypasses the gastric barrier, pGP3-deficient Chlamydia sp. still failed to reach the large intestine, although similarly inoculated plasmid-free Chlamydia sp. was able to do so. Interestingly, when both types of organisms were intrajejunally coinoculated into the same mouse small intestine, plasmid-free Chlamydia sp. was no longer able to spread to the large intestine, suggesting that pGP3-deficient Chlamydia sp. might be able to activate an intestinal resistance for regulating Chlamydia sp. spreading. Thus, the current study has not only provided evidence for reconciling a previously identified conflicting phenotype but also revealed a potential intestinal resistance to chlamydial spreading. Efforts are under way to further define the mechanism of the putative intestinal resistance.

Stenotrophomonas maltophilia is a Gram-negative bacterium found ubiquitously in the environment that has historically been regarded as nonpathogenic. S. maltophilia is increasingly observed in patient sputa in cystic fibrosis (CF), and while existing epidemiology indicates that patients with S. maltophilia have poorer diagnoses, its clinical significance remains unclear. Moreover, as multidrug resistance is common among S. maltophilia isolates, treatment options for these infections may be limited. Here, we investigated the pathogenicity of S. maltophilia alone and during polymicrobial infection with Pseudomonas aeruginosa. Colonization, persistence, and virulence of S. maltophilia were assessed in experimental respiratory infections of mice. The results of this study indicate that S. maltophilia transiently colonizes the lung accompanied by significant weight loss and immune cell infiltration and the expression of early inflammatory markers, including interleukin 6 (IL-6), IL-1α, and tumor necrosis factor alpha (TNF-α). Importantly, polymicrobial infection with P. aeruginosa elicited significantly higher S. maltophilia counts in bronchoalveolar lavages and lung tissue homogenates. This increase in bacterial load was directly correlated with the density of the P. aeruginosa population and required viable P. aeruginosa bacteria. Microscopic analysis of biofilms formed in vitro revealed that S. maltophilia formed well-integrated biofilms with P. aeruginosa, and these organisms colocalize in the lung during dual-species infection. Based on these results, we conclude that active cellular processes by P. aeruginosa afford a significant benefit to S. maltophilia during polymicrobial infections. Furthermore, these results indicate that S. maltophilia may have clinical significance in respiratory infections.

The cryptic plasmid is important for chlamydial colonization in the gastrointestinal tract. We used a combination of intragastric, intrajejunal, and intracolon inoculations to reveal the impact of the plasmid on chlamydial colonization in distinct regions of gastrointestinal tract. Following an intragastric inoculation, the plasmid significantly improved chlamydial colonization. At the tissue level, plasmid-positive Chlamydia produced infectious progenies throughout gastrointestinal tract. However, to our surprise, plasmid-deficient Chlamydia failed to produce infectious progenies in small intestine, although infectious progenies were eventually detected in large intestine, indicating a critical role of the plasmid in chlamydial differentiation into infectious particles in small intestine. The noninfectious status may represent persistent infection, since Chlamydia genomes proliferated in the same tissues. Following an intrajejunal inoculation that bypasses the gastric barrier, plasmid-deficient Chlamydia produced infectious progenies in small intestine but was 530-fold less infectious than plasmid-positive Chlamydia, suggesting that (i) the noninfectious status developed after intragastric inoculation might be induced by a combination of gastric and intestinal effectors and (ii) chlamydial colonization in small intestine was highly dependent on plasmid. Finally, following an intracolon inoculation, the dependence of chlamydial colonization on plasmid increased over time. Thus, we have demonstrated that the plasmid may be able to improve chlamydial fitness in different gut regions via different mechanisms, which has laid a foundation to further reveal the specific mechanisms.

Food allergy is a life-threatening response to specific foods, and microbiota imbalance (dysbiosis) in gut is considered a cause of this disease. Meanwhile, the host immune response also plays an important role in the disease. Notably, interleukin 33 (IL-33) released from damaged or necrotic intestinal epithelial cells facilitates IL-2-producing CD4 helper T (Th2) responses. However, causal relationships between the gut and oral dysbiosis and food allergy remain unknown., Food allergy is a life-threatening response to specific foods, and microbiota imbalance (dysbiosis) in gut is considered a cause of this disease. Meanwhile, the host immune response also plays an important role in the disease. Notably, interleukin 33 (IL-33) released from damaged or necrotic intestinal epithelial cells facilitates IL-2-producing CD4 helper T (Th2) responses. However, causal relationships between the gut and oral dysbiosis and food allergy remain unknown. In this study, we analyzed effects of gut and oral dysbiosis on development of food allergy. A murine model of food allergy was established via ovalbumin (OVA) injection in BALB/c mice. Viable fecal bacteria were identified using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). il33 expression in colon-26 mouse colon cells stimulated by isolated fecal bacteria was quantified by real-time PCR. Intestinal T cells from the mice were analyzed by flow cytometry. Salivary IgA levels were quantified by enzyme-linked immunosorbent assay (ELISA), and IgA-bound oral bacteria were detected by flow cytometry. Among fecal bacteria, the abundance of Citrobacter sp. increased in the feces of allergic mice and induced il33 expression in colon-26 cells. Orally administered Citrobacter koseri JCM1658 exacerbated systemic allergic symptoms and reduced intestinal Th17 cells. Salivary IgA and IgA-bound oral bacteria increased in the allergic mice. Based on the results described above, food allergy induced both gut and oral dysbiosis. Citrobacter sp. aggravated allergy symptoms by inducing IL-33 release from intestinal epithelial cells.

The symbiotic relationship between the gut microbiome and the host provides a nutrient-rich environment for gut microbes and has beneficial effects on host health. Although the composition of the gut microbiome is known to be influenced by both host genetics and environmental factors, host effects on the activities and functions of the gut microbial communities remain poorly understood. Intestinal epithelial cells exert front-line responses to gut microbes and contribute to maintaining a healthy intestinal homeostasis. Here, seeking to elucidate whether intestinal epithelial cells modulate Lactobacillus rhamnosus GG (LGG) functions, we examined the production of p40, an LGG-derived secretory protein that protects intestinal epithelial cells against inflammation. We found that growth medium conditioned with colonic epithelial cell-derived components promotes p40 protein synthesis and secretion by LGG and enhances LGG-stimulated protective responses in intestinal epithelial cells. Furthermore, when LGG was cultured with the colonic luminal contents from healthy mice, p40 production was upregulated but was attenuated with luminal contents from mice with intestinal inflammation. Importantly, the colonic epithelial cell-derived components potentiated LGG-produced p40 levels in a mouse model of colitis and enhanced LGG-mediated amelioration of intestinal inflammation in this model. Notably, we found that colonic epithelial cell-secreted extracellular vesicles participate in communicating with LGG and that heat shock protein 90 (HSP90) in these vesicles might mediate the promotion of p40 production. These results reveal a previously unrecognized mechanism by which the anti-inflammatory effect of LGG is reinforced by intestinal epithelial cells and thereby maintains intestinal health.

Vancomycin-resistant Enterococcus faecium (VRE) is a leading cause of hospital-acquired infections. This is particularly true in immunocompromised patients, where the damage to the microbiota caused by antibiotics can lead to VRE domination of the intestine, increasing a patient’s risk for bloodstream infection., Vancomycin-resistant Enterococcus faecium (VRE) is a leading cause of hospital-acquired infections. This is particularly true in immunocompromised patients, where the damage to the microbiota caused by antibiotics can lead to VRE domination of the intestine, increasing a patient’s risk for bloodstream infection. In previous studies we observed that the intestinal domination by VRE of patients hospitalized to receive allogeneic bone marrow transplantation can persist for weeks, but little is known about subspecies diversification and evolution during prolonged domination. Here we combined a longitudinal analysis of patient data and in vivo experiments to reveal previously unappreciated subspecies dynamics during VRE domination that appeared to be stable from 16S rRNA microbiota analyses. Whole-genome sequencing of isolates obtained from sequential stool samples provided by VRE-dominated patients revealed an unanticipated level of VRE population complexity that evolved over time. In experiments with ampicillin-treated mice colonized with a single CFU, VRE rapidly diversified and expanded into distinct lineages that competed for dominance. Mathematical modeling shows that in vivo evolution follows mostly a parabolic fitness landscape, where each new mutation provides diminishing returns and, in the setting of continuous ampicillin treatment, reveals a fitness advantage for mutations in penicillin-binding protein 5 (pbp5) that increase resistance to ampicillin. Our results reveal the rapid diversification of host-colonizing VRE populations, with implications for epidemiologic tracking of in-hospital VRE transmission and susceptibility to antibiotic treatment.

This investigation compared the microbiomes colonizing teeth during the initiation, progression, and resolution of periodontitis in nonhuman primates (Macaca mulatta) at different ages. Subgingival plaque samples were collected at baseline; 0.5, 1, and 3 months following ligature-induced periodontitis; and following naturally occurring disease resolution at 5 months. Samples were analyzed using 16S amplicon sequencing to identify bacterial profiles across age groups: young (

Enterococcus faecalis is a human intestinal pathobiont with intrinsic and acquired resistance to many antibiotics, including vancomycin. Nature provides a diverse and virtually untapped repertoire of bacterial viruses, or bacteriophages (phages), that could be harnessed to combat multidrug-resistant enterococcal infections. Bacterial phage resistance represents a potential barrier to the implementation of phage therapy, emphasizing the importance of investigating the molecular mechanisms underlying the emergence of phage resistance. Using a cohort of 19 environmental lytic phages with tropism against E. faecalis, we found that these phages require the enterococcal polysaccharide antigen (Epa) for productive infection. Epa is a surface-exposed heteroglycan synthesized by enzymes encoded by both conserved and strain-specific genes. We discovered that exposure to phage selective pressure favors mutation in nonconserved epa genes both in culture and in a mouse model of intestinal colonization. Despite gaining phage resistance, epa mutant strains exhibited a loss of resistance to cell wall-targeting antibiotics. Finally, we show that an E. faecalisepa mutant strain is deficient in intestinal colonization, cannot expand its population upon antibiotic-driven intestinal dysbiosis, and fails to be efficiently transmitted to juvenile mice following birth. This study demonstrates that phage therapy could be used in combination with antibiotics to target enterococci within a dysbiotic microbiota. Enterococci that evade phage therapy by developing resistance may be less fit at colonizing the intestine and sensitized to vancomycin, preventing their overgrowth during antibiotic treatment.

Bacteria have a remarkable ability to survive, persist, and ultimately adapt to environmental challenges. A ubiquitous environmental hazard is DNA damage, and most bacteria have evolved a network of genes to combat genotoxic stress. This network is known as the SOS response and aids in bacterial survival by regulating genes involved in DNA repair and damage tolerance. Recently, the SOS response has been shown to play an important role in bacterial pathogenesis, and yet the role of the SOS response in nonpathogenic organisms and in physiological settings remains underexplored. Using a commensal Escherichia coli strain, MP1, we showed that the SOS response plays a vital role during colonization of the murine gut. In an unperturbed environment, the SOS-off mutant is impaired for stable colonization relative to a wild-type strain, suggesting the presence of genotoxic stress in the mouse gut. We evaluated the possible origins of genotoxic stress in the mouse gut by examining factors associated with the host versus the competing commensal organisms. In a dextran sulfate sodium (DSS) colitis model, the SOS-off colonization defect persisted but was not exacerbated. In contrast, in a germ-free model, the SOS-off mutant colonized with efficiency equal to that seen with the wild-type strain, suggesting that competing commensal organisms might be a significant source of genotoxic stress. This report extends our understanding of the importance of a functional SOS response for bacterial fitness in the context of a complex physiological environment and highlights the SOS response as a possible mechanism that contributes to ongoing genomic changes, including potential antibiotic resistance, in the microbiome of healthy hosts.

Nontypeable Haemophilus influenzae (NTHi) is an extremely common human pathobiont that persists on the airway mucosal surface within biofilm communities, and our previous work has shown that NTHi biofilm maturation is coordinated by the production and uptake of autoinducer 2 (AI-2) quorum signals. To directly test roles for AI-2 in maturation and maintenance of NTHi biofilms, we generated an NTHi 86-028NP mutant in which luxS transcription was under the control of the xylA promoter (NTHi 86-028NP luxS xylA::luxS), rendering AI-2 production inducible by xylose. Comparison of biofilms under inducing and noninducing conditions revealed a biofilm defect in the absence of xylose, whereas biofilm maturation increased following xylose induction. The removal of xylose resulted in the interruption of luxS expression and biofilm dispersal. Measurement of luxS transcript levels by real-time reverse transcription-PCR (RT-PCR) showed that luxS expression peaked as biofilms matured and waned before dispersal. Transcript profiling revealed significant changes following the induction of luxS, including increased transcript levels for a predicted family 8 glycosyltransferase (NTHI1750; designated gstA); this result was confirmed by real-time RT-PCR. An isogenic NTHi 86-028NP gstA mutant had a biofilm defect, including decreased levels of sialylated matrix and significantly altered biofilm structure. In experimental chinchilla infections, we observed a significant decrease in the number of bacteria in the biofilm population (but not in effusions) for NTHi 86-028NP gstA compared to the parental strain. Therefore, we conclude that AI-2 promotes NTHi biofilm maturation and the maintenance of biofilm integrity, due at least in part to the expression of a probable glycosyltransferase that is potentially involved in the synthesis of the biofilm matrix.

Enterococci are Gram-positive commensals of the mammalian intestinal tract and harbor intrinsic resistance to broad-spectrum cephalosporins. Disruption of colonization resistance in humans by antibiotics allows enterococci to proliferate in the gut and cause disseminated infections. In this study, we used Enterococcus faecalis (EF)-colonized mice to study the dynamics of enterococci, commensal microbiota, and the host in response to systemic ceftriaxone administration. We found that the mouse model recapitulates intestinal proliferation and dissemination of enterococci seen in humans. Employing a ceftriaxone-sensitive strain of enterococci (E. faecalis JL308), we showed that increased intestinal abundance is critical for the systemic dissemination of enterococci. Investigation of the impact of ceftriaxone on the mucosal barrier defenses and integrity suggested that translocation of enterococci across the intestinal mucosa was not associated with intestinal pathology or increased permeability. Ceftriaxone-induced alteration of intestinal microbial composition was associated with transient increase in the abundance of multiple bacterial operational taxonomic units (OTUs) in addition to enterococci, for example, lactobacilli, which also disseminated to the extraintestinal organs. Collectively, these results emphasize that ceftriaxone-induced disruption of colonization resistance and alteration of mucosal homeostasis facilitate increased intestinal abundance of a limited number of commensals along with enterococci, allowing their translocation and systemic dissemination in a healthy host.

Disruption of the healthy intestinal microbiome and homeostasis of the intestinal immune system, which are closely interactive, are two key factors for ulcerative colitis. Here, we show that MI-2, a selective inhibitor of mucosa-associated lymphoid tissue lymphoma translocation-1 (MALT1), alleviated excessive inflammatory responses and was associated with restoration of healthy intestinal microbiome in mice suffering from dextran sulfate sodium (DSS)-induced colitis., Disruption of the healthy intestinal microbiome and homeostasis of the intestinal immune system, which are closely interactive, are two key factors for ulcerative colitis. Here, we show that MI-2, a selective inhibitor of mucosa-associated lymphoid tissue lymphoma translocation-1 (MALT1), alleviated excessive inflammatory responses and was associated with restoration of healthy intestinal microbiome in mice suffering from dextran sulfate sodium (DSS)-induced colitis. We found that the diversity of intestinal microbiome of mice with DSS-induced colitis was significantly lower than that of healthy mice. However, MI-2 treatment in mice with DSS-induced colitis resulted in restored microbially diverse populations. To understand the possibility of the beneficial effect of the restored microbially diverse populations of MI-2-treated mice with DSS-induced colitis, we showed that inserting fecal microbiota from MI-2-treated mice with DSS-induced colitis and healthy control mice into mice with DSS-induced colitis could alleviate symptoms of colitis. The possibility of MI-2 treatment in DSS-induced colitis, associated with restoration of healthy microbially diverse populations in addition to reshaping host immune modulating capacity by reducing inflammatory cytokines (tumor necrosis factor alpha, interleukin-1β [IL-1β], IL-17α, and IL-22), may be considered therapeutic for ulcerative colitis.

Streptococcus gordonii is an early colonizer of the oral cavity. Although a variety of S. gordonii adherence mechanisms have been described, current dogma is that the major receptor for S. gordonii is sialic acid. However, as many bacterial species in the oral cavity produce neuraminidase that can cleave terminal sialic acid, it is unclear whether S. gordonii relies on sialic acid for adherence to oral surfaces or if this species has developed alternative binding strategies. Previous studies have examined adherence to immobilized glycoconjugates and identified binding to additional glycans, but no prior studies have defined the contribution of these different glycan structures in adherence to oral epithelial cells. We determined that the majority of S. gordonii strains tested did not rely on sialic acid for efficient adherence. In fact, adherence of some strains was significantly increased following neuraminidase treatment. Further investigation of representative strains that do not rely on sialic acid for adherence revealed binding not only to sialic acid via the serine-rich repeat protein GspB but also to β-1,4-linked galactose. Adherence to this carbohydrate occurs via an unknown adhesin distinct from those utilized by Streptococcus oralis and Streptococcus pneumoniae . Demonstrating the potential biological relevance of binding to this cryptic receptor, we established that S. oralis increases S. gordonii adherence in a neuraminidase-dependent manner. These data suggest that S. gordonii has evolved to simultaneously utilize both terminal and cryptic receptors in response to the production of neuraminidase by other species in the oral environment.

Abnormalities in the human microbiota are associated with the etiology of allergic diseases. Although disease site-specific microbiota may be associated with disease pathophysiology, the role of the nasal microbiota is unclear. We sought to characterize the microbiota of the site of allergic rhinitis, the inferior turbinate, in subjects with allergic rhinitis ( n = 20) and healthy controls ( n = 12) and to examine the relationship of mucosal microbiota with disease occurrence, sensitized allergen number, and allergen-specific and total IgE levels. Microbial dysbiosis correlated significantly with total IgE levels representing combined allergic responses but not with disease occurrence, the number of sensitized allergens, or house dust mite allergen-specific IgE levels. Compared to the populations in individuals with low total IgE levels (group IgE low ), low microbial biodiversity with a high relative abundance of Firmicutes phylum ( Staphylococcus aureus ) and a low relative abundance of Actinobacteria phylum ( Propionibacterium acnes ) was observed in individuals with high total serum IgE levels (group IgE high ). Phylogeny-based microbial functional potential predicted by the 16S rRNA gene indicated an increase in signal transduction-related genes and a decrease in energy metabolism-related genes in group IgE high as shown in the microbial features with atopic and/or inflammatory diseases. Thus, dysbiosis of the inferior turbinate mucosa microbiota, particularly an increase in S. aureus and a decrease in P. acnes , is linked to high total IgE levels in allergic rhinitis, suggesting that inferior turbinate microbiota may be affected by accumulated allergic responses against sensitized allergens and that site-specific microbial alterations play a potential role in disease pathophysiology.