Colonization Of Gastrointestinal Tract Research Articles

Bifidobacterium bifidum and the infant gut microbiota: an intriguing case of microbe-host co-evolution.

Bifidobacterium bifidum is reported to be among the first colonizers of the newborn's gastrointestinal tract due to its ability to metabolize human milk oligosaccharides (HMOs). In order to investigate biological features that allow this bifidobacterial species to colonize a newborn, bifidobacterial internally transcribed spacer profiling of stool samples of 50 mother-infant dyads, as well as corresponding breastmilk samples, was performed. Hierarchical clustering based on bifidobacterial population profiles found in infant faecal samples revealed the presence of four bifidobacterial clusters or the so-called bifidotypes. Bifidobacterium bifidum was shown to be a key member among bifidotypes, in which its presence correlate with several different bifidobacterial species retrieved in infant faecal samples. For this reason, we investigated cross-feeding behaviour facilitated by B. bifidum on a bioreactor model using human milk as growth substrate. Transcriptional profiles of this strain were evaluated when grown on nine specific glycans typically constituting HMOs. Remarkably, these analyses suggest extensive co-evolution with the host and other bifidobacterial species in terms of resource provision and sharing, respectively, activities that appear to support a bifidobacteria-dominant microbiome.

Analysis of the Isotopic Composition of Exhaled Air by the Diode Laser Spectroscopy Method in the 2-μm Region for Diagnosis of Helicobacter pylori-Associated Diseases

To diagnose digestive diseases associated with Helicobacter pylori infection, we applied laser spectroscopic analysis of the 13СО2/12СО2 isotope ratio in the exhaled air, which is based on the use of tunable diode lasers. To analyze the isotopic composition of the exhaled air, we developed a spectrophotometer based on a tunable diode laser. The 13СО2/12СО2 ratio was determined using the spectral range near 2.05 μm, in which the R-branch of the 20013–00001 band of 12СО2 overlaps with the P-branch of the 20012–0001 band of 13СО2. H. pylori infection in the gastrointestinal tract of a group of 309 subjects was diagnosed using the 13C-urea breath test (13C-UBT). The breath test results were compared with the results of morphological analysis of the gastric and duodenal mucosa specimens obtained during fiber gastroduodenoscopy. To compare the results of laser 13C-UBT and histology, we have for the first time proposed to use the total values of the results of the histological analysis of biopsy specimens taken in the body and distal part of the stomach and in the duodenum. The dependence of the results of laser 13C-UBT on the age, nosology, activity and severity of the inflammatory process, and the degree of atrophy and metaplasia, as well as the type of therapy was analyzed. It is shown that the distribution of the results of laser 13C-UBT in patients obtained before and after eradication therapy reflects the epidemiology of gastroduodenal diseases, the incidence of H. pylori infections, the patterns of gastrointestinal tract colonization by H. pylori, the parameters of inflammation, as well as the efficacy of therapy and the characteristics of the recovery of bacterial colonies after unsuccessful therapy.

Shape Follows Function: Gastrointestinal Signals for Enterococcal Colonization

Vancomycin-resistant Enterococcus faecium (VRE) is a major cause of nosocomial infections. A new study by McKenney et al. (Cell Host Microbe 2019;25:695-705.e5) reports that VRE undergo a morphotype switch in response to lithocholic acid (LCA) to facilitate gastrointestinal (GI) tract colonization. This metabolic cue is a potential target to decrease VRE colonization and subsequent transmission of antibiotic resistance.

Molecular Switch Controlling Expression of the Mannose-Specific Adhesin, Msa, in Lactobacillus plantarum.

Some lactic acid bacteria, especially Lactobacillus spp., possess adhesive properties enabling colonization of the human gastrointestinal tract. Two probiotic Lactobacillus plantarum strains, WCSF1 and 299v, display highly different mannose-specific adhesion, with L. plantarum 299v being superior to L. plantarum WCFS1 based on a yeast agglutination assay. A straightforward correlation between the mannose adhesion capacity and domain composition of the mannose-specific adhesin (Msa) in the two strains has not been demonstrated previously. In this study, we analyzed the promoter regions upstream of the msa gene encoding a mannose-specific adhesin in these two strains. The promoter region was mapped by primer extension and DNA sequence analysis, and only a single nucleotide change was identified between the two strains. However, Northern blot analysis showed a stronger msa transcript band in 299v than in WCFS1 correlating with the different adhesion capacities. During the establishment of a high-throughput yeast agglutination assay, we isolated variants of WCFS1 that displayed a very strong mannose-specific adhesion phenotype. The region upstream of the msa gene in these variants showed an inversion of a 104-bp fragment located between two perfectly inverted repeats present in the untranslated leader region. The inversion disrupts a strong hairpin structure that otherwise most likely would terminate the msa transcript. In addition, the ribosome binding site upstream of the msa gene, which is also masked within this hairpin structure, becomes accessible upon inversion, thereby increasing the frequency of translation initiation in the variant strains. Furthermore, Northern blot analysis showed a higher abundance of the msa transcript in the variants than in the wild type, correlating with a strong-Msa phenotype.IMPORTANCE Probiotic strains possess adhesive properties enabling colonization of the human intestinal tract through interactions between molecules present on the probiotic bacteria and components of the epithelial surface. In Lactobacillus plantarum, interaction is mediated through bacterial surface proteins like Msa, which binds to mannose residues present on the intestinal cells. Such interactions are believed to be important for the health-promoting effects of probiotics, including displacement of pathogens, immunomodulation, and protective effects on the intestinal barrier function. In this study, we have identified a new molecular switch controlling expression of the msa gene in L. plantarum strain WCFS1. Strains with increased msa expression could be valuable in the development and manufacture of improved probiotic products.

Clinical and microbiological pathogenetic parallels with necrotizing enterocolitis in preterm infants Evidence-based prevention opportunities

There are summarized data of domestic and foreign publications on the issue of necrotizing enterocolitis (NEC) in children born prematurely. There is presented the trend of the prevalence of this pathology in the last decade, especially epidemiology and significance of NEC in the structure of perinatal morbidity and mortality. There were analyzed new concepts on etiology and pathogenesis of the disease. In recent years, there was descripted the key role of some risk factors for the development of NEC - immatureness of intestinal barrier functions, local and systemic immunity, reduced tolerance to enteral nutrition (especially in the absence of breast-feeding), inadequate microbial colonization of the immature gastrointestinal tract. Modern research methods allow us to decipher subtle mechanisms of activation of the cascade of the intestinal inflammation and breakdown of the immune defense mechanisms. There are established cellular and humoral compartments of the immune response in NEC; there were revealed typical deviations in intestinal microbiota. The investigation of etiopathogenetical NEC factors is the basis for the development of targeted methods of the prevention and treatment of this severe disease in premature infants, including with the use of probiotics. There are presented data that the use of probiotics significantly reduces the incidence of severe NEC and mortality risk from NEC.

Sortase-Dependent Proteins Promote Gastrointestinal Colonization by Enterococci.

The human gastrointestinal tract (GIT) is inhabited by a dense microbial community of symbionts. Enterococci are among the earliest members of this community and remain core members of the GIT microbiota throughout life. Enterococci have also recently emerged as opportunistic pathogens and major causes of nosocomial infections. Although recognized as a prerequisite for infection, colonization of the GIT by enterococci remains poorly understood. One way that bacteria adapt to dynamic ecosystems like the GIT is through the use of their surface proteins to sense and interact with components of their immediate environment. In Gram-positive bacteria, a subset of surface proteins relies on an enzyme called sortase for covalent attachment to the cell wall. Here, we show that the housekeeping sortase A (SrtA) enzyme promotes intestinal colonization by enterococci. Furthermore, we show that the enzymatic activity of SrtA is key to the ability of Enterococcus faecalis to bind mucin (a major component of the GIT mucus). We also report the GIT colonization phenotypes of E. faecalis mutants lacking selected sortase-dependent proteins (SDPs). Further examination of the mucin binding ability of these mutants suggests that adhesion to mucin contributes to intestinal colonization by E. faecalis.

Анализ изотопического состава выдыхаемого воздуха методами диодной лазерной спектроскопии в районе 2 μm для диагностики Helicobacter pylori-ассоциированных заболеваний-=SUP=-*-=/SUP=-

Spectral analysis of 13СО2/12СО2 isotope ratio in exhaled air based on tunable diode laser spec-troscopy was applied for diagnostics of digestive organ diseases associated with Helicobacter pylori infection. Special spectrophotometer based on tunable diode laser was developed for the analysis of breath isotope content. Spectral range near 2.05 microns where the rotational-vibration R-branch of the 20013-00001 band of 12CO2 interferes with the P-branch of the 20012-00001 band of 13CO2 was used. The 13C-Urea Breath Test (13C-UDT) was used for the H. pylori infection diagnostic in gastrointestinal tract of 309 tested persons. The results of the isotope breath test were compared with the data of morphological analysis of gastric and duodenal mu-cosa obtained by fibrogastroduodenoscopia. For the first time the total value of the histology analysis results for biopsy material taken in the stomach body, in the distal part of stomach and in the duodenum was proposed to be used for the comparative analysis with 13C-UBT data. A relationship of 13C-UBT data with age, nosology, activity and severity of inflammation process, atrophy degree, and type of eradication therapy was analyzed. Distribution of 13C-UBT data ob-tained before and after therapy was demonstrated to reflect epidemiology of gastroduodenal dis-eases, H.pylori infection incidence, specifics of gastrointestinal tract colonization by H.pylori, parameters of inflammation process, as well as therapy effectiveness and peculiarity of the in-fection restoration at failed eradication therapy.

Effect of maternal diet on select fecal bacteria of foals.

Adult horses depend on the microbial community in the hindgut to digest fiber and produce short-chain fatty acids that are use for energy. Colonization of the foal gastrointestinal tract is essential to develop this symbiosis. However, factors affecting colonization are not well understood. The objectives of this study were to evaluate the age-related changes and effects of maternal diet on select fecal bacterial groups in foals from 1 to 28 d of age. Thoroughbred foals (n = 18) were from dams fed forage and one of two concentrates: an oat-based (OB) or corn and wheat middlings-based (CWB) pelleted concentrate. The mares had access to assigned concentrates, along with a mixed hay and cool-season grass pasture, 28 d before and 28 d after parturition. Fecal samples were collected from foals at 1 d (14 to 36 h), 4, 14, and 28 d after birth. Fecal samples were serially diluted with phosphate-buffered saline before inoculation of enriched, selective media to enumerate Lactobacillus spp., amylolytic bacteria, and cellulolytic bacteria. Enumeration data were log-transformed then analyzed with mixed model analysis of variance with repeated measures (SAS 9.3) to test the main effects of maternal diet (OB or CWB), time of sample, and interaction between maternal diet and time. Cellulolytic bacteria first appeared in foal feces between 4 and 14 d of age and increased with age (P < 0.05). Amylolytic bacteria and lactobacilli were abundant at 1 d and then increased with age (P < 0.05). There was an interaction between maternal diet and time for Lactobacillus spp. with OB foals having more lactobacilli than CWB foals at 1 and 4 d (P < 0.05); however, there were no differences observed at 14 d (P > 0.05). Maternal diet did not influence amylolytic or cellulolytic bacteria (P > 0.05). These results indicate that colonization of the hindgut is a sequential process beginning early in the foal’s life and that maternal diet may influence some bacteria in the gastrointestinal tract of foals.

PSXVI-33 Evaluation of the microbiome of mares’ milk through lactation.

Classically, the nutrients in milk have been studied as critical components to support growth. Recently, other factors have been identified for their role in supporting neonatal nutrition. Once thought to be sterile, milk is known to be populated by numerous microbes, an important factor related to the development of the foals’ gastrointestinal microbiome. It is believed that the initial inoculum comes as the foal passes through the vaginal canal and continues as the foal interacts with its environment, and consumes milk. The objective of this study was to evaluate the bacterial population of mares’ milk. Samples were evaluated and bacterial population data was correlated to the age, BW, and BCS of the mare, days of lactation, offspring gender, and physiological growth measurements of the foal. Eight multiparous Quarter Horse mares foaled naturally and milk samples were obtained aseptically at 36 hr, 70 d, and 136 d post foaling. Mares were offered 8 lb of concentrate (Purina® Ultium Growth®) 2x/d and 18 lb of hay with free choice access to water and salt. Microbial DNA was extracted and PCR amplified prior to sequencing utilizing an Illumina® MiSeq® platform. Bacterial diversities were determined based on OTU assignment between and within samples. Data were analyzed via ANOVA using the MIXED procedure of SAS. 94.57% of reads were mapped to SILVA (V4) database. No differences existed in alpha or beta diversity. The predominant phyla were Firmicutes. Staphylococcus were the predominant genera. The small sample size may have resulted in the lack of observed differences. With the absence of any differences observed when analyzing the metadata, this study provides insights into the bacterial population of mares’ milk through lactation. In the future, this data can be utilized to examine the early colonizers of the foal gastrointestinal tract and identify targets for probiotic or prebiotic supplementation.

Vaccination with the Staphylococcus aureus secreted proteins EapH1 and EapH2 impacts both S. aureus carriage and invasive disease

IntroductionThere is a need for an efficacious vaccine reducing infections due to Staphylococcus aureus, a common cause of community and hospital infection. Infecting organisms originate from S. aureus populations colonising the nares and bowel. Antimicrobials are widely used to transiently reduce S. aureus colonisation prior to surgery, a practice which is selecting for resistant S. aureus isolates. S. aureus secretes multiple proteins, including the protease inhibitors extracellular adhesion protein homologue 1 and 2 (EapH1 and EapH2). MethodsMice were vaccinated intramuscularly or intranasally with Adenovirus serotype 5 and Modified Vaccinia Ankara viral vectors expressing EapH1 and EapH2 proteins, or with control viruses. Using murine S. aureus colonisation models, we monitored S. aureus colonisation by sequential stool sampling. Monitoring of S. aureus invasive disease after intravenous challenge was performed using bacterial load and abscess numbers in the kidney. ResultsIntramuscular vaccination with Adenovirus serotype 5 and Modified Vaccinia Ankara viral vectors expressing EapH1 and EapH2 proteins significantly reduces bacterial recovery in the murine renal abscess model of infection, but the magnitude of the effect is small. A single intranasal vaccination with an adenoviral vaccine expressing these proteins reduced S. aureus gastrointestinal (GI) tract colonisation. ConclusionVaccination against EapH1 / EapH2 proteins may offer an antibiotic independent way to reduce S. aureus colonisation, as well as contributing to protection against S. aureus invasive disease.

Heterogeneous early immune responses to the S. aureus EapH2 antigen induced by gastrointestinal tract colonisation impact the response to subsequent vaccination

IntroductionS. aureus is a pathogen to which individuals are exposed shortly after birth, with immune responses to S. aureus increasing during childhood. There is marked heterogeneity between the anti- S. aureus immune responses of different humans, the basis of which is not fully understood. MethodsTo investigate development of anti-S. aureus immune responses, we studied S. aureus colonised mice under controlled conditions. Mice were either acquired colonised from breeding colonies, or experimentally colonised by exposure to a cage environment which had been sprayed with a S. aureus suspension. Colonisation was monitored by sequential stool sampling, and immunoglobulin levels against both whole fixed S. aureus and individual S. aureus antigens quantified. The immunological impact of colonisation on subsequent vaccination was investigated. ResultsColonised BALB/c and BL/6 mice develop serum anti- S. aureus cell surface IgG1 antibodies. Responses were proportional to the cumulative S. aureus bioburden in the mice, and were higher in BALB/c mice, which have higher colonisation levels, than in C57BL/6 animals. We observed marked variation in the induction of anti-cell surface antibodies, even in genetically identical mice experimentally colonised with the same S. aureus clone. Heterogeneity was also evident when monitoring immune responses to the secreted S. aureus protein EapH2. Approximately 50% of colonised mice developed anti-EapH2 responses (responders); in other mice, responses were not significantly different to those in uncolonised mice (non-responders). Following vaccination with a replication deficient adenovirus expressing EapH2, less anti-EapH2 antibody was generated in non-responder than responder animals. ConclusionsIn genetically identical mice, S. aureus colonisation results in all-or-nothing antibody responses against some antigens, including EapH2. For antigens involved in colonisation success by microbes, apparently stochastic early immune responses may impact both vaccine responses and the establishment of an animal-specific microbiome.

Transducer-Like Protein in Campylobacter jejuni With a Role in Mediating Chemotaxis to Iron and Phosphate.

Chemotaxis-mediated motility enables Campylobacter jejuni to navigate through complex environmental gradients and colonize diverse niches. C. jejuni is known to possess several methyl accepting chemotaxis proteins (MCPs), also called transducer-like proteins (Tlps). While the role of some of the Tlps in chemotaxis has been identified, their regulation and role in virulence is still not very clear. Here, we investigated the contribution of Tlp2 to C. jejuni chemotaxis, stress survival and colonization of the chicken gastrointestinal tract. The Δtlp2 deletion mutant showed decreased chemotaxis toward aspartate, pyruvate, inorganic phosphate (Pi), and iron (FeSO4). Transcriptional analysis of tlp2 with a promoter fusion reporter assay revealed that the tlp2 promoter (Ptlp2) was induced by Pi and iron, both in the ferrous (Fe2+) and ferric form (Fe3+). RT-PCR analysis using overlapping primers indicated that the phoX gene, located immediately downstream of tlp2, is co-transcribed with tlp2. A transcription start site was identified at 53 bp upstream of the tlp2 start codon. The Δtlp2 mutant showed decreased colonization of the chicken gastrointestinal tract. Collectively, our findings revealed that the tlp2 plays a role in C. jejuni pathogenesis and colonization in the chicken host and its expression is regulated by iron.

Nanonized tetracycline cures deadly diarrheal disease ‘shigellosis’ in mice, caused by multidrug-resistant Shigella flexneri 2a bacterial infection

We reported earlier about nano-formulation of tetracycline through its entrapment within calcium-phosphate nano-particle (CPNP) and about killing of pathogenic bacterium Shigella flexnari 2a, resistant to tetracycline (and 9 other antibiotics), by the nanonized antibiotic (Tet-CPNP). Here, we report on therapeutic role of Tet-CPNP against deadly diarrheal disease ‘shigellosis’ in mice, caused by Shigella infection. Our findings revealed that occurrence of mushy-stool excretion, colon-length shortening, weight-loss and bacterial colonization in gastrointestinal tract of mice due to shigellosis was significantly reduced by Tet-CPNP treatment. Histo- and immuno-logical studies showed that changes in morphology and level of inflammatory cytokines TNF-α, IL-1β and IFN-γ in intestinal tissue of Shigella-infected mice were reverted to almost normal features by Tet-CPNP treatment. Bulk tetracycline had no anti-shigellosis action. Thus, nanonization of tetracycline rejuvenated the old, cheap, broad-spectrum antibiotic from obsolescence (due to resistance generation), making it highly beneficial for diarrhea-prone developing countries with limited health-care budgets.

Semi-Quantification of Total Campylobacter and Salmonella During Egg Incubations Using a Combination of 16S rDNA and Specific Pathogen Primers for qPCR.

Rapid molecular techniques that evaluate eggs for the presence of foodborne pathogens is an essential component to poultry food safety monitoring. Interestingly, it is not just table eggs that contribute to outbreaks of foodborne disease. Broiler layer production actively contributes to sustaining of foodborne pathogens within a flock. The surface contamination of production eggs with invasive pathogens such as Salmonella enterica, Campylobacter jejuni, and Listeria monocytogenes during embryogenesis results in gastrointestinal tract (GIT) colonization. Pathogens that secure a niche within the GIT during embryonic development are nearly impossible to eradicate from the food chain. Therefore, current monitoring paradigms are not comprehensive because they fail to capture the presence of invasive pathogens within the embryonic GIT rapidly. By developing tools to recognize the pathogens’ presence in the GIT during embryogenesis, producers are then able to spot evaluate broiler eggs for their potential risk as carriers of foodborne pathogens. In this study a novel qPCR assay was developed to semi-quantify pathogen load relative to total bacterial burden. Eggs sampled from three independent production broiler flocks of different ages were assayed for S. enterica (invA), C. jejuni (HipO), and L. monocytogenes (HlyA) against total microbial load (16s). The eggs were sampled at 1-day post-set within each flock, 2 weeks post-set, after vaccination (at 2.5 weeks) and 1-day post-hatch. The eggs were washed, and the yolk and embryonic chick GIT were collected. The DNA was extracted and subjected to a qPCR assay. The results confirm a novel technique for pathogen monitoring relative to total bacterial load and a unique method for monitoring the dynamics of foodborne pathogen invasion throughout broiler egg production.

Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A.

ABSTRACTMultidrug-resistant Acinetobacter baumannii is among the most common causes of infectious complications associated with combat-related trauma in military personnel serving overseas. However, little is currently known about its pathogenesis. While the gastrointestinal (GI) tract has been found to be a major reservoir for A. baumannii, as well as to potentially contribute to development of multidrug resistance, no studies have addressed the mechanisms involved in gut colonization. In this study, we address this critical gap in knowledge by first assessing the interaction between secretory IgA (SIgA), the principal humoral immune defense on mucosal surfaces, and the A. baumannii clinical isolate Ci79. Surprisingly, SIgA appeared to enhance A. baumannii GI tract colonization, in a process mediated by bacterial thioredoxin A (TrxA), as evidenced by reduction of bacterial attachment in the presence of TrxA inhibitors. Additionally, a trxA targeted deletion mutant (ΔtrxA) showed reduced bacterial burdens within the GI tract 24 h after oral challenge by in vivo live imaging, along with loss of thiol-reductase activity. Surprisingly, not only was GI tract colonization greatly reduced but the associated 50% lethal dose (LD50) of the ΔtrxA mutant was increased nearly 100-fold in an intraperitoneal sepsis model. These data suggest that TrxA not only mediates A. baumannii GI tract colonization but also may contribute to pathogenesis in A. baumannii sepsis following escape from the GI tract under conditions when the intestinal barrier is compromised, as occurs with cases of severe shock and trauma.

Adaptation of Candida albicans during gastrointestinal tract colonization.

Colonization of the gastrointestinal (GI) tract with Candida albicans (CA), the most common human fungal pathogen, is the first step towards the development of invasive infection. Yet the fungal virulence factors and host factors that modulate CA GI colonization are still poorly understood. In this review, we will review emerging evidence of the importance of select CA genetic determinants and CA's interaction with the host that contribute to its successful adaptation as a pathobiont in the human GI tract. Recent data reveal the importance of 1) CA genetic determinants; 2) host factors; and 3) environmental factors in modulating CA GI colonization in humans. As evidence continues to grow supporting the notion that the GI tract and its resident microbiota are an integral part of the host immune system, it will be critical for studies to interrogate the interaction of CA with the host (including both the host innate and adaptive immune system as well as the endogenous gut microbiota) in order to dissect the mechanisms of CA pathogenesis and thus lay the foundation for novel therapeutic approaches to prevent and/or treat invasive fungal infections.

Epitope mapping of Salmonella flagellar hook-associated protein, FlgK, with mass spectrometry-based immuno-capture proteomics using chicken (Gallus gallus domesticus) sera

Salmonella, a Gram-negative rod, is the leading foodborne pathogen associated with human acute bacterial gastroenteritis worldwide. The Salmonella flagellum is responsible for bacterial movement, colonization and invasion in the host gastrointestinal tract. The flagellum has a complex structure, composed of more than 35 proteins. Among them, we were interested in the flagellar hook-associated protein (FlgK), which is an immunodominant protein in chickens. In this communication, we applied mass spectrometry-based proteomics in conjunction with chicken immunized sera to map the linear immunoepitopes in the FlgK protein, validated the epitopes with peptide ELISA, and determined serum reactivity to the epitopes from commercial chickens. We previously demonstrated the FlgK proteins are highly conserved among Salmonella serovars. The rFlgK protein was produced by the recombinant technique, and was able to induce immune response in chickens. Further, this study identified four peptides (AEG, GAQ, TAD and LEI) in the rFlgK protein that were captured by sera from chickens immunized with the rFlgK protein. These four peptides were also reacted to 64 individual serum samples collected from 44 − 52 weeks old chickens, suggesting that these peptides may represent the shared immuno-epitopes on the FlgK protein. The findings of the specific shared linear immuno-epitopes on the FlgK protein in this study provide a rationale for further evaluation to determine their utility as epitope vaccines covering multiple serotypes for chicken immunization, and subsequently, for providing safer poultry products for human consumption.

Investigating Colonization of the Healthy Adult Gastrointestinal Tract by Fungi.

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.

Identification of New Factors Modulating Adhesion Abilities of the Pioneer Commensal Bacterium Streptococcus salivarius.

Biofilm formation is crucial for bacterial community development and host colonization by Streptococcus salivarius, a pioneer colonizer and commensal bacterium of the human gastrointestinal tract. This ability to form biofilms depends on bacterial adhesion to host surfaces, and on the intercellular aggregation contributing to biofilm cohesiveness. Many S. salivarius isolates auto-aggregate, an adhesion process mediated by cell surface proteins. To gain an insight into the genetic factors of S. salivarius that dictate host adhesion and biofilm formation, we developed a screening method, based on the differential sedimentation of bacteria in semi-liquid conditions according to their auto-aggregation capacity, which allowed us to identify twelve mutations affecting this auto-aggregation phenotype. Mutations targeted genes encoding (i) extracellular components, including the CshA surface-exposed protein, the extracellular BglB glucan-binding protein, the GtfE, GtfG and GtfH glycosyltransferases and enzymes responsible for synthesis of cell wall polysaccharides (CwpB, CwpK), (ii) proteins responsible for the extracellular localization of proteins, such as structural components of the accessory SecA2Y2 system (Asp1, Asp2, SecA2) and the SrtA sortase, and (iii) the LiaR transcriptional response regulator. These mutations also influenced biofilm architecture, revealing that similar cell-to-cell interactions govern assembly of auto-aggregates and biofilm formation. We found that BglB, CshA, GtfH and LiaR were specifically associated with bacterial auto-aggregation, whereas Asp1, Asp2, CwpB, CwpK, GtfE, GtfG, SecA2 and SrtA also contributed to adhesion to host cells and host-derived components, or to interactions with the human pathogen Fusobacterium nucleatum. Our study demonstrates that our screening method could also be used to identify genes implicated in the bacterial interactions of pathogens or probiotics, for which aggregation is either a virulence trait or an advantageous feature, respectively.

Modulators of Enterococcus faecalis Cell Envelope Integrity and Antimicrobial Resistance Influence Stable Colonization of the Mammalian Gastrointestinal Tract.

The Gram-positive bacterium Enterococcus faecalis is both a colonizer of the gastrointestinal tract (GIT) and an agent of serious nosocomial infections. Although it is typically required for pathogenesis, GIT colonization by E. faecalis is poorly understood. E. faecalis tolerates high concentrations of GIT antimicrobials, like cholate and lysozyme, leading us to hypothesize that resistance to intestinal antimicrobials is essential for long-term GIT colonization. Analyses of E. faecalis mutants exhibiting defects in antimicrobial resistance revealed that IreK, a determinant of envelope integrity and antimicrobial resistance, is required for long-term GIT colonization. IreK is a member of the PASTA kinase protein family, bacterial transmembrane signaling proteins implicated in the regulation of cell wall homeostasis. Among several determinants of cholate and lysozyme resistance in E. faecalis, IreK was the only one found to be required for intestinal colonization, emphasizing the importance of this protein to enterococcal adaptation to the GIT. By studying ΔireK suppressor mutants that recovered the ability to colonize the GIT, we identified two conserved enterococcal proteins (OG1RF_11271 and OG1RF_11272) that function antagonistically to IreK and interfere with cell envelope integrity, antimicrobial resistance, and GIT colonization. Our data suggest that IreK, through its kinase activity, inhibits the actions of these proteins. IreK, OG1RF_11271, and OG1RF_11272 are found in all enterococci, suggesting that their effect on GIT colonization is universal across enterococci. Thus, we have defined conserved genes in the enterococcal core genome that influence GIT colonization through their effect on enterococcal envelope integrity and antimicrobial resistance.