ABSTRACT
Treatment with antibiotics is a major risk factor for Clostridioides difficile infection, likely due to depletion of the gastrointestinal microbiota. Two microbiota-mediated mechanisms thought to limit C. difficile colonization include conversion of conjugated primary bile salts into secondary bile salts toxic to C. difficile growth, and competition between the microbiota and C. difficile for limiting nutrients. Using a continuous flow model of the distal colon, we investigated how treatment with six clinically-used antibiotics influenced susceptibility to C. difficile infection in 12 different microbial communities cultivated from healthy individuals. Antibiotic treatment reduced microbial richness; disruption varied by antibiotic class and microbiota composition, but did not correlate with C. difficile susceptibility. Antibiotic treatment also disrupted microbial bile salt metabolism, increasing levels of the primary bile salt, cholate, and decreasing levels of the secondary bile salt, deoxycholate. However, decreased levels of deoxycholate did not correlate with increased C. difficile susceptibility. Further, bile salts were not required to inhibit C. difficile colonization. We tested whether amino acid fermentation contributed to persistence of C. difficile in antibiotic-treated communities. C. difficile mutants unable to use proline as an electron acceptor in Stickland fermentation due to disruption of proline reductase (ΔprdB) had significantly lower levels of colonization than wild-type strains in four of six antibiotic-treated communities tested. This data provides further support for the importance of bile salt-independent mechanisms in regulating colonization of C. difficile.
ABSTRACT
Mechanistic investigation of host-microbe interactions in the human gut are hindered by difficulty of co-culturing microbes with intestinal epithelial cells. On one hand the gut bacteria are a mix of facultative, aerotolerant or obligate anaerobes, while the intestinal epithelium requires oxygen for growth and function. Thus, a coculture system that can recreate these contrasting oxygen requirements is critical step towards our understanding microbial-host interactions in the human gut. Here, we demonstrate Intestinal Organoid Physoxic Coculture (IOPC) system, a simple and cost-effective method for coculturing anaerobic intestinal bacteria with human intestinal organoids (HIOs). Using commensal anaerobes with varying degrees of oxygen tolerance, such as nano-aerobe Bacteroides thetaiotaomicron and strict anaerobe Blautia sp., we demonstrate that IOPC can successfully support 24-48 hours HIO-microbe coculture. The IOPC recapitulates the contrasting oxygen conditions across the intestinal epithelium seen in vivo. The IOPC cultured HIOs showed increased barrier integrity, and induced expression of immunomodulatory genes. A transcriptomic analysis suggests that HIOs from different donors show differences in the magnitude of their response to coculture with anaerobic bacteria. Thus, the IOPC system provides a robust coculture setup for investigating host-microbe interactions in complex, patient-derived intestinal tissues, that can facilitate the study of mechanisms underlying the role of the microbiome in health and disease.
Subject(s)
Coculture Techniques , Intestinal Mucosa , Organoids , Oxygen , Humans , Organoids/microbiology , Organoids/metabolism , Oxygen/metabolism , Coculture Techniques/methods , Intestinal Mucosa/microbiology , Intestinal Mucosa/metabolism , Intestinal Mucosa/cytology , Gastrointestinal Microbiome , Host Microbial Interactions , Bacteria, Anaerobic/growth & development , Bacteria, Anaerobic/metabolism , Intestines/microbiology , Intestines/cytology , Bacteroides thetaiotaomicron/metabolismABSTRACT
IMPORTANCE: Clostridioides difficile is one of the leading causes of hospital-acquired infections worldwide and presents challenges in treatment due to recurrent gastrointestinal disease after treatment with antimicrobials. The mechanisms by which C. difficile colonizes the gut represent a key gap in knowledge, including its association with host cells and mucosa. Our results show the importance of flagellin for specific adhesion to mucosal hydrogels and can help to explain prior observations of adhesive defects in flagellin and pilin mutants.
Subject(s)
Clostridioides difficile , Clostridium Infections , Gastrointestinal Diseases , Humans , Flagellin/genetics , Clostridioides difficile/genetics , Clostridioides , Mucous MembraneABSTRACT
Introduction: Kombucha is a popular fermented tea that has attracted considerable attention due, in part, to its suggested health benefits. Previous results from animal models led us to hypothesize kombucha may reduce blood sugar levels in humans with diabetes. The objective of this pilot clinical study was to evaluate kombucha for its anti-hyperglycemic activities in adults with diabetes mellitus type II. Methods: The study was organized as a prospective randomized double-blinded crossover study at a single-center urban hospital system. Participants (n = 12) were instructed to consume either a kombucha product or a placebo control (each 240 mL) for 4 weeks. After an 8-week washout period, participants consumed the alternate product. Fasting blood glucose levels were self-determined at baseline and at 1 and 4 weeks during each treatment period. Secondary health outcomes, including overall health, insulin requirement, gut health, skin health, mental health, and vulvovaginal health were measured by questionnaire at the same time points. The kombucha microbiota was assessed by selective culturing and 16S rRNA gene (bacteria) and ITS (fungi) sequencing. Fermentation end products were assessed by HPLC. Statistical significance of changes in fasting blood glucose was determined using paired, two-tailed student's t-tests. Results: Kombucha lowered average fasting blood glucose levels at 4 weeks compared to baseline (164 vs. 116 mg/dL, p = 0.035), whereas the placebo did not (162 vs. 141 mg/dL, p = 0.078). The kombucha microbiota, as assessed by cultural enumeration, was mainly comprised of lactic acid bacteria, acetic acid bacteria, and yeast, with each group present at about 106 colony forming units (CFU)/mL. Likewise, 16S rRNA gene sequencing confirmed that lactic acid and acetic acid bacteria were the most abundant bacteria, and ITS sequencing showed Dekkera was the most abundant yeast. The primary fermentation end products were lactic and acetic acids, both less than 1%. Ethanol was present at 1.5%. Discussion: Although this pilot study was limited by a small sample size, kombucha was associated with reduced blood glucose levels in humans with diabetes. Larger follow-up studies are warranted. Clinical trial registration: ClinicalTrials.gov, identifier NCT04107207.
ABSTRACT
Cadmium (Cd) is a heavy metal toxic to the gut microbiome. In this study, we cultivated two human gut microbiomes (A and B) in bioreactors with Cd at 0 and 20 ppm for 7 days to investigate effects of Cd on the gut microbiome and to isolate Cd-tolerant bacteria autochthonous to the gut. Cd showed profound toxicity, abolishing butyrate production, depleting microbes in microbiome B, and simplifying microbiome A to a small Cd-tolerant community after 2 d of incubation. When spiked into the Cd-sensitive microbiome B, the Cd-tolerant community from microbiome A and isolates from that community worked synergistically with microbiome B to enhance butyrate production and maintained this synergism at Cd concentrations up to 5 ppm. Bacteria isolated from this Cd-tolerant community included Enterococcus faecium, Enterobacter cloacae, Lactococcus lactis, and Lactobacillus taiwanensis species. This work demonstrates a straightforward method for identifying Cd-tolerant bacteria autochthonous to the human gut that synergize with the microbiome to protect against Cd-related loss of butyrate production.
Subject(s)
Cadmium , Gastrointestinal Microbiome , Hazardous Substances , Humans , Bacteria , Butyrates , Cadmium/toxicity , Gastrointestinal Microbiome/drug effects , Bioreactors , Hazardous Substances/toxicityABSTRACT
Mucins are glycoproteins which can be found in host cell membranes and as a gelatinous surface formed from secreted mucins. Mucosal surfaces in mammals form a barrier to invasive microbes, particularly bacteria, but are a point of attachment for others. Clostridioides difficile is anaerobic bacterium which colonizes the mammalian GI tract and is a common cause of acute GI inflammation leading to a variety of negative outcomes. Although C. difficile toxicity stems from secreted toxins, colonization is a prerequisite for C. difficile disease. While C. difficile is known to associate with the mucus layer and underlying epithelium, the mechanisms underlying these interactions that facilitate colonization are less well-understood. To understand the molecular mechanisms by which C. difficile interacts with mucins, we used ex vivo mucosal surfaces to test the ability of C. difficile to bind to mucins from different mammalian tissues. We found significant differences in C. difficile adhesion based upon the source of mucins, with highest levels of binding observed to mucins purified from the human colonic adenocarcinoma line LS174T and lowest levels of binding to porcine gastric mucin. We also observed that defects in adhesion by mutants deficient in flagella, but not type IV pili. These results imply that interactions between host mucins and C. difficile flagella facilitate the initial host attachment of C. difficile to host cells and secreted mucus.
ABSTRACT
It is well established that the gastrointestinal (GI) microbiota plays a major role in human health. Dietary interventions, and consumption of fermented foods that contain live microbes, in particular, are among the approaches being investigated to modulate the GI microbiota and improve health. However, the persistence of fermented food-associated bacteria (FAB) within the GI tract is typically limited by host factors that limit colonization and competition with autochthonous microbes. In this research, we examined if the addition of prebiotics, dietary substrates that are selectively metabolized by microbes to improve health, would enhance the persistence of FAB. We evaluated the persistence of bacteria from three live microbe-containing fermented foods-kefir, sausage, and sauerkraut-in fecal microbial communities from four healthy adults. Fecal communities were propagated in vitro and were inoculated with fermented food-associated microbes from kefir, sausage, or sauerkraut at ~107 CFU/mL. Communities were diluted 1:100 every 24 h into fresh gut simulation medium to simulate microbial community turnover in the GI tract. We measured the persistence of Lactobacillaceae from fermented foods by quantitative PCR (qPCR) and the persistence of other FAB through 16S rRNA gene sequencing. FAB were unable to persist in vitro, reaching undetectable levels within 96 h. Addition of prebiotics, including xylooligosaccharides and a mixture of fructooligosaccharides and galactooligosaccharides enhanced the persistence of some species of FAB, but the level of persistence varied by fecal donor, fermented food, and prebiotic tested. Addition of prebiotics also increased the relative abundance of Bifidobacterium species, which most likely originated from the fecal microbiota. Collectively, our results support previous in vivo studies demonstrating the transient nature of FAB in the GI tract and indicate that consumption of prebiotics may enhance their persistence.
ABSTRACT
Fungal infections are a major health problem that often begin in the gastrointestinal tract. Gut microbe interactions in early childhood are critical for proper immune responses, yet there is little known about the development of the fungal population from infancy into childhood. Here, as part of the TEDDY (The Environmental Determinants of Diabetes in the Young) study, we examine stool samples of 888 children from 3 to 48 months and find considerable differences between fungi and bacteria. The metagenomic relative abundance of fungi was extremely low but increased while weaning from milk and formula. Overall fungal diversity remained constant over time, in contrast with the increase in bacterial diversity. Fungal profiles had high temporal variation, but there was less variation from month-to-month in an individual than among different children of the same age. Fungal composition varied with geography, diet, and the use of probiotics. Multiple Candida spp. were at higher relative abundance in children than adults, while Malassezia and certain food-associated fungi were lower in children. There were only subtle fungal differences associated with the subset of children that developed islet autoimmunity or type 1 diabetes. Having proper fungal exposures may be crucial for children to establish appropriate responses to fungi and limit the risk of infection: the data here suggests those gastrointestinal exposures are limited and variable.
Subject(s)
Diabetes Mellitus, Type 1 , Probiotics , Adult , Autoimmunity , Bacteria , Candida , Child , Child, Preschool , Fungi , Gastrointestinal Tract/microbiology , HumansABSTRACT
It is widely accepted that the pathogen Clostridioides difficile exploits an intestinal environment with an altered microbiota, but the details of these microbe-microbe interactions are unclear. Adherence and colonization of mucus has been demonstrated for several enteric pathogens and it is possible that mucin-associated microbes may be working in concert with C. difficile. We showed that C. difficile ribotype-027 adheres to MUC2 glycans and using fecal bioreactors, we identified that C. difficile associates with several mucin-degrading microbes. C. difficile was found to chemotax toward intestinal mucus and its glycan components, demonstrating that C. difficile senses the mucus layer. Although C. difficile lacks the glycosyl hydrolases required to degrade mucin glycans, coculturing C. difficile with the mucin-degrading Akkermansia muciniphila, Bacteroides thetaiotaomicron, and Ruminococcus torques allowed C. difficile to grow in media that lacked glucose but contained purified MUC2. Collectively, these studies expand our knowledge on how intestinal microbes support C. difficile.
Subject(s)
Clostridioides difficile , Clostridioides , Clostridiales , Humans , Monosaccharides , Mucins , MucusABSTRACT
BACKGROUND & AIMS: Although Clostridioides difficile infection (CDI) is known to involve the disruption of the gut microbiota, little is understood regarding how mucus-associated microbes interact with C difficile. We hypothesized that select mucus-associated bacteria would promote C difficile colonization and biofilm formation. METHODS: To create a model of the human intestinal mucus layer and gut microbiota, we used bioreactors inoculated with healthy human feces, treated with clindamycin and infected with C difficile with the addition of human MUC2-coated coverslips. RESULTS: C difficile was found to colonize and form biofilms on MUC2-coated coverslips, and 16S rRNA sequencing showed a unique biofilm profile with substantial cocolonization with Fusobacterium species. Consistent with our bioreactor data, publicly available data sets and patient stool samples showed that a subset of patients with C difficile infection harbored high levels of Fusobacterium species. We observed colocalization of C difficile and F nucleatum in an aggregation assay using adult patients and stool of pediatric patients with inflammatory bowel disease and in tissue sections of patients with CDI. C difficile strains were found to coaggregate with F nucleatum subspecies in vitro; an effect that was inhibited by blocking or mutating the adhesin RadD on Fusobacterium and removal of flagella on C difficile. Aggregation was shown to be unique between F nucleatum and C difficile, because other gut commensals did not aggregate with C difficile. Addition of F nucleatum also enhanced C difficile biofilm formation and extracellular polysaccharide production. CONCLUSIONS: Collectively, these data show a unique interaction of between pathogenic C difficile and F nucleatum in the intestinal mucus layer.
Subject(s)
Adhesins, Bacterial/metabolism , Clostridioides difficile/pathogenicity , Clostridium Infections/immunology , Fusobacterium nucleatum/immunology , Gastrointestinal Microbiome/immunology , Adhesins, Bacterial/genetics , Bacterial Adhesion/immunology , Biofilms , Bioreactors/microbiology , Clostridioides difficile/genetics , Clostridioides difficile/immunology , Clostridioides difficile/metabolism , Clostridium Infections/microbiology , Feces/microbiology , Flagella/genetics , Flagella/metabolism , Fusobacterium nucleatum/metabolism , HT29 Cells , Humans , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Intestinal Mucosa/pathology , Mucin-2/metabolismABSTRACT
Recently, an opportunity to perform a broad ruggedness assessment of our liquid chromatography-tandem mass spectrometry (LC-MS/MS) system presented itself during the analytical planning phase of a large-scale human fecal microbiome study. The specific aim of this project was to study the microbial-mediated metabolism of a targeted set of bile acids/salts by mixed bacterial communities cultured from the feces of 12 healthy volunteers when grown in a custom growth medium and following exposure to different clinically-relevant antibiotics. The magnitude of this study offered a rare opportunity to significantly stress procedures and LC-MS/MS system components comprised in our bile acid/salt targeted metabolomics method. With this second specific aim in mind, we modified the sample analysis plan to include a series of figure-of-merit (FoM)-based tests that are commonly used in regulated bioanalytical labs to assess LC and MS system ruggedness for a specific assay - these FoM-based testing parameters were monitored continuously over the course of sample analysis and the results are presented in this report. In total, the assessment included 1206 sequential injections (180 calibration standards, 136 blank-internal standard samples, and 890 diluted medium samples) that took place over 8-days. Completion of the 8-days of non-stop sample analysis revealed no critical hardware or software failures, and the analysis of the FoM-based tests indicated no observable degradation of system performance over the number of samples and time tested. The FoM-based test metrics presented may be used as a template to assess the ruggedness of any LC-MS/MS-based targeted metabolomics workflow.
Subject(s)
Bacteriological Techniques/methods , Chromatography, Liquid/methods , Microbiota , Tandem Mass Spectrometry/methods , Bacteria , Bile Acids and Salts , Calibration , Feces , Humans , Metabolomics , Sensitivity and SpecificityABSTRACT
Due to the physicochemical properties of bile acids/salts (i.e., hydrophobic and ionizable), the application of reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods are ideally suited for the measurement of these compounds in a host of microbiologically-relevant matrices. Here, we provide a detailed bioanalytical protocol that contains several modifications of a method previously described by Wegner et al. [1]. Briefly, this modified method exhibits the following advantages for the measurement of cholic acid (CA), taurocholic acid (TCA), and deoxycholic acid (DCA) in microbiome-relevant sample matrices: i) fecal sample processing has been streamlined by the elimination of lyophilization and manual homogenization steps; ii) the Sciex 6500 QTRAP hybrid triple-quadrupole/linear ion trap mass spectrometer has sufficient sensitivity to perform the measurement of bile acids/salts in negative ion mode - ammonium adducts of bile acids/salts are not required for detection; and, iii) assay throughput has been boosted by more than 5-fold by shortening the chromatographic duty cycle of a single sample injection from 45 min to 8.4 min. Recently, the method was used to perform 508 sequential injections (72 calibration standards, 52 blank-internal standard sample, and 368 MiniBioReactor Array (MBRA)-derived samples) from four separate batches over a 4-day time period.
ABSTRACT
The gastrointestinal microbiome plays an important role in limiting susceptibility to infection with Clostridioides difficile To better understand the ecology of bacteria important for C. difficile colonization resistance, we developed an experimental platform to simplify complex communities of fecal bacteria through dilution and rapidly screen for their ability to resist C. difficile colonization after challenge, as measured by >100-fold reduction in levels of C. difficile in challenged communities. We screened 76 simplified communities diluted from cultures of six fecal donors and identified 24 simplified communities that inhibited C. difficile colonization in vitro Sequencing revealed that simplified communities were composed of 19 to 67 operational taxonomic units (OTUs) and could be partitioned into four distinct community types. One simplified community could be further simplified from 56 to 28 OTUs through dilution and retain the ability to inhibit C. difficile We tested the efficacy of seven simplified communities in a humanized microbiota mouse model. We found that four communities were able to significantly reduce the severity of the initial C. difficile infection and limit susceptibility to disease relapse. Analysis of fecal microbiomes from treated mice demonstrated that simplified communities accelerated recovery of indigenous bacteria and led to stable engraftment of 19 to 22 OTUs from simplified communities. Overall, the insights gained through the identification and characterization of these simplified communities increase our understanding of the microbial dynamics of C. difficile infection and recovery.IMPORTANCEClostridioides difficile is the leading cause of antibiotic-associated diarrhea and a significant health care burden. Fecal microbiota transplantation is highly effective at treating recurrent C. difficile disease; however, uncertainties about the undefined composition of fecal material and potential long-term unintended health consequences remain. These concerns have motivated studies to identify new communities of microbes with a simpler composition that will be effective at treating disease. This work describes a platform for rapidly identifying and screening new simplified communities for efficacy in treating C. difficile infection. Four new simplified communities of microbes with potential for development of new therapies to treat C. difficile disease are identified. While this platform was developed and validated to model infection with C. difficile, the underlying principles described in the paper could be easily modified to develop therapeutics to treat other gastrointestinal diseases.
Subject(s)
Clostridioides difficile/classification , Clostridioides difficile/physiology , Feces/microbiology , Gastrointestinal Microbiome , Microbiological Techniques/methods , Adult , Animals , Fecal Microbiota Transplantation , Female , Humans , Male , Mice , Middle AgedABSTRACT
While the knowledge on gut microbiota - C. difficile interactions has improved over the years, the understanding of the underlying mechanisms providing colonization resistance as well as preventative measures against the infection remain incomplete. In this study the antibiotic clindamycin and polyphenol extracts from pomegranate and blueberries were used individually and in combination to modulate fecal microbial communities in minibioreactor arrays (MBRA). Modulated communities were inoculated with C. difficile (ribotype 027). Subsequent 7-day periodical monitoring included evaluation of C. difficile growth and activity of toxins TcdA and TcdB as well as analysis of MBRA bacterial community structure (V3V4 16 S metagenomics). Polyphenols affected multiple commensal bacterial groups and showed different synergistic and antagonistic effects in combination with clindamycin. Exposure to either clindamycin or polyphenols led to the loss of colonization resistance against C. difficile. The successful growth of C. difficile was most significantly correlated with the decrease in Collinsella and Lachnospiraceae. Additionally, we demonstrated that Clostridium sporogenes decreased the activity of both C. difficile toxins TcdA and TcdB. The feature was shown to be common among distinct C. sporogenes strains and could potentially be applicable as a non-antibiotic agent for the alleviation of C. difficile infection.
Subject(s)
Bacterial Toxins/toxicity , Clostridium Infections/prevention & control , Disease Resistance/drug effects , Gastrointestinal Microbiome/immunology , Polyphenols/pharmacology , Actinobacteria/immunology , Bacterial Toxins/metabolism , Biological Assay , Bioreactors/microbiology , Clindamycin/adverse effects , Clostridioides difficile/growth & development , Clostridioides difficile/metabolism , Clostridium/immunology , Clostridium Infections/immunology , Clostridium Infections/microbiology , Disease Resistance/immunology , Feces/microbiology , Gastrointestinal Microbiome/drug effects , HumansABSTRACT
Shewanella baltica was the dominant culturable nitrate-reducing bacterium in the eutrophic and strongly stratified Baltic Sea in the 1980s, where it primarily inhabited the oxic-anoxic transition zone. The genomic structures of 46 of these isolates were investigated through comparative genomic hybridization (CGH), which revealed a gradient of genomic similarity, ranging from 65% to as high as 99%. The core genome of the S. baltica species was enriched in anaerobic respiration-associated genes. Auxiliary genes, most of which locate within a few genomic islands (GIs), were nonuniformly distributed among the isolates. Specifically, hypothetical and mobile genetic element (MGE)-associated genes dominated intraclade gene content differences, whereas gain/loss of functional genes drove gene content differences among less related strains. Among the major S. baltica clades, gene signatures related to specific redox-driven and spatial niches within the water column were identified. For instance, genes involved in anaerobic respiration of sulfur compounds may provide key adaptive advantages for clade A strains in anoxic waters where sulfur-containing electron acceptors are present. Genes involved in cell motility, in particular, a secondary flagellar biosynthesis system, may be associated with the free-living lifestyle by clade E strains. Collectively, this study revealed characteristics of genome variations present in the water column and active speciation of S. baltica strains, driven by niche partitioning and horizontal gene transfer (HGT).IMPORTANCE Speciation in nature is a fundamental process driving the formation of the vast microbial diversity on Earth. In the central Baltic Sea, the long-term stratification of water led to formation of a large-scale vertical redoxcline that provided a gradient of environmental niches with respect to the availability of electron acceptors and donors. The region was home to Shewanella baltica populations, which composed the dominant culturable nitrate-reducing bacteria, particularly in the oxic-anoxic transition zone. Using the collection of S. baltica isolates as a model system, genomic variations showed contrasting gene-sharing patterns within versus among S. baltica clades and revealed genomic signatures of S. baltica clades related to redox niche specialization as well as particle association. This study provides important insights into genomic mechanisms underlying bacterial speciation within this unique natural redoxcline.
ABSTRACT
A wide diversity of fungi have been detected in the human gastrointestinal (GI) tract with the potential to provide or influence important functions. However, many of the fungi most commonly detected in stool samples are also present in food or the oral cavity. Therefore, to recognize which gut fungi are likely to have a sustained influence on human health, there is a need to separate transient members of the GI tract from true colonizers. To identify colonizing fungi, the eukaryotic rRNA operon's second internal transcribed spacer (ITS2) was sequenced from the stool, saliva, and food of healthy adults following consumption of different controlled diets. Unlike most bacterial 16S rRNA genes, the only fungal ITS2 operational taxonomic units (OTUs) detected in stool DNA across multiple diets were also present in saliva and/or food. Additional analyses, including culture-based approaches and sequencing of the 18S rRNA gene, ITS2 cDNA, and DNA extracted using alternative methods, failed to detect additional fungi. Two abundant fungi, Saccharomyces cerevisiae and Candida albicans, were examined further in healthy volunteers. Saccharomyces became undetectable in stool when a S. cerevisiae-free diet was consumed, and the levels of C. albicans in stool were dramatically reduced by more frequent cleaning of teeth. Extremely low fungal abundance, the inability of fungi to grow under conditions mimicking the distal gut, and evidence from analysis of other public datasets further support the hypothesis that fungi do not routinely colonize the GI tracts of healthy adults. IMPORTANCE We sought to identify the fungi that colonize healthy GI tracts and that have a sustained influence on the diverse functions of the gut microbiome. Instead, we found that all fungi in the stool of healthy volunteers could be explained by their presence in oral and dietary sources and that our results, together with those from other analyses, support the model that there is little or no gastrointestinal colonization by fungi. This may be due to Westernization, primate evolution, fungal ecology, and/or the strong defenses of a healthy immune system. Importantly, fungal colonization of the GI tract may often be indicative of disease. As fungi can cause serious infections in immunocompromised individuals and are found at increased abundance in multiple disorders of the GI tract, understanding normal fungal colonization is essential for proper treatment and prevention of fungal pathogenesis.
Subject(s)
Fungi/classification , Gastrointestinal Tract/microbiology , Mycobiome/physiology , Adult , Candida albicans/genetics , Candida albicans/isolation & purification , DNA, Ribosomal Spacer/genetics , Diet , Feces/microbiology , Food Microbiology , Fungi/isolation & purification , Gastrointestinal Tract/physiology , Healthy Volunteers , Humans , Mouth/microbiology , RNA, Ribosomal, 18S/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/isolation & purification , Saliva/microbiology , Sequence Analysis, DNAABSTRACT
Niche partitioning and sequence evolution drive genomic and phenotypic divergence, which ultimately leads to bacterial diversification. This study investigated the genomic composition of two Shewanella baltica clades previously identified through multilocus sequencing typing and recovered from the redox transition zone in the central Baltic Sea. Comparative genomic analysis revealed significantly higher interclade than intraclade genomic dissimilarity and that a subset of genes present in clade A were associated with potential adaptation to respiration of sulfur compounds present in the redox transition zone. The transcriptomic divergence between two representative strains of clades A and D, OS185 and OS195, was also characterized and revealed marked regulatory differences. We found that both the transcriptional divergence of shared genes and expression of strain-specific genes led to differences in regulatory patterns between strains that correlate with environmental redox niches. For instance, under anoxic conditions of respiratory nitrate ammonification, OS185-the strain isolated from a nitrate-rich environment-upregulated nearly twice the number of shared genes upregulated by OS195-the strain isolated from an H2S-containing anoxic environment. Conversely, OS195 showed stronger induction of strain-specific genes, especially those associated with sulfur compound respiration, under thiosulfate-reducing conditions. A positive association between the level of transcriptional divergence and the level of sequence divergence for shared genes was also noted. Our results provide further support for the hypothesis that genomic changes impacting transcriptional regulation play an important role in the diversification of ecologically distinct populations.IMPORTANCE This study examined potential mechanisms through which co-occurring Shewanella baltica strains diversified to form ecologically distinct populations. At the time of isolation, the strains studied composed the major fraction of culturable nitrate-reducing communities in the Baltica Sea. Analysis of genomic content of 13 S. baltica strains from two clades representing different ecotypes demonstrated that one clade specifically possesses a number of genes that could favor successful adaptation to respire sulfur compounds in the portion of the water column from which these strains were isolated. In addition, transcriptional profiling of fully sequenced strains representative of these two clades, OS185 and OS195, under oxygen-, nitrate-, and thiosulfate-respiring conditions demonstrated that the strains exhibit relatively similar transcriptional responses during aerobic growth but more-distinct transcriptional responses under nitrate- and thiosulfate-respiring conditions. Results from this study provide insights into how genomic and gene regulatory diversification together impacted the redox specialization of the S. baltica strains.
Subject(s)
Gene Expression Regulation , Genetic Speciation , Genome, Bacterial/genetics , Shewanella/genetics , Multilocus Sequence Typing , Nucleic Acid Hybridization , SympatryABSTRACT
Each year in the United States, billions of dollars are spent combating almost half a million Clostridium difficile infections (CDIs) and trying to reduce the â¼29,000 patient deaths in which C. difficile has an attributed role. In Europe, disease prevalence varies by country and level of surveillance, though yearly costs are estimated at 3 billion. One factor contributing to the significant health care burden of C. difficile is the relatively high frequency of recurrent CDIs. Recurrent CDI, i.e., a second episode of symptomatic CDI occurring within 8 weeks of successful initial CDI treatment, occurs in â¼25% of patients, with 35 to 65% of these patients experiencing multiple episodes of recurrent disease. Using microbial communities to treat recurrent CDI, either as whole fecal transplants or as defined consortia of bacterial isolates, has shown great success (in the case of fecal transplants) or potential promise (in the case of defined consortia of isolates). This review will briefly summarize the epidemiology and physiology of C. difficile infection, describe our current understanding of how fecal microbiota transplants treat recurrent CDI, and outline potential ways that knowledge can be used to rationally design and test alternative microbe-based therapeutics.
Subject(s)
Clostridioides difficile/physiology , Clostridium Infections/microbiology , Clostridium Infections/therapy , Fecal Microbiota Transplantation , Animals , Clostridioides difficile/genetics , Clostridioides difficile/isolation & purification , Feces/microbiology , Gastrointestinal Microbiome , HumansABSTRACT
Integration of antibiotic and probiotic therapy has the potential to lessen the public health burden of antimicrobial-associated diseases. Clostridium difficile infection (CDI) represents an important example where the rational design of next-generation probiotics is being actively pursued to prevent disease recurrence. Because intrinsic resistance to clinically relevant antibiotics used to treat CDI (vancomycin, metronidazole, and fidaxomicin) is a desired trait in such probiotic species, we screened several bacteria and identified Lactobacillus reuteri to be a promising candidate for adjunct therapy. Human-derived L. reuteri bacteria convert glycerol to the broad-spectrum antimicrobial compound reuterin. When supplemented with glycerol, strains carrying the pocR gene locus were potent reuterin producers, with L. reuteri 17938 inhibiting C. difficile growth at a level on par with the level of growth inhibition by vancomycin. Targeted pocR mutations and complementation studies identified reuterin to be the precursor-induced antimicrobial agent. Pathophysiological relevance was demonstrated when the codelivery of L. reuteri with glycerol was effective against C. difficile colonization in complex human fecal microbial communities, whereas treatment with either glycerol or L. reuteri alone was ineffective. A global unbiased microbiome and metabolomics analysis independently confirmed that glycerol precursor delivery with L. reuteri elicited changes in the composition and function of the human microbial community that preferentially targets C. difficile outgrowth and toxicity, a finding consistent with glycerol fermentation and reuterin production. Antimicrobial resistance has thus been successfully exploited in the natural design of human microbiome evasion of C. difficile, and this method may provide a prototypic precursor-directed probiotic approach. Antibiotic resistance and substrate bioavailability may therefore represent critical new determinants of probiotic efficacy in clinical trials.