Negative interactions determine Clostridioides difficile growth in synthetic human gut communities

Understanding the principles of colonization resistance of the gut microbiome to the pathogen Clostridioides difficile will enable the design of defined bacterial therapeutics. We investigate the ecological principles of community resistance to C. difficile using a synthetic human gut microbiome. Using a dynamic computational model, we demonstrate that C. difficile receives the largest number and magnitude of incoming negative interactions. Our results show that C. difficile is in a unique class of species that display a strong negative dependence between growth and species richness. We identify molecular mechanisms of inhibition including acidification of the environment and competition over resources. We demonstrate that Clostridium hiranonis strongly inhibits C. difficile partially via resource competition. Increasing the initial density of C. difficile can increase its abundance in the assembled community, but community context determines the maximum achievable C. difficile abundance. Our work suggests that the C. difficile inhibitory potential of defined bacterial therapeutics can be optimized by designing communities featuring a combination of mechanisms including species richness, environment acidification, and resource competition.     

Gut Microbiota Patterns Associated with Colonization of Different Clostridium difficile Ribotypes

C. difficile infection is associated with disturbed gut microbiota and changes in relative frequencies and abundance of individual bacterial taxons have been described. In this study we have analysed bacterial, fungal and archaeal microbiota by denaturing high pressure liquid chromatography (DHPLC) and with machine learning methods in 208 faecal samples from healthy volunteers and in routine samples with requested C. difficile testing. The latter were further divided according to stool consistency, C. difficile presence or absence and C. difficile ribotype (027 or non-027). Lower microbiota diversity was a common trait of all routine samples and not necessarily connected only to C. difficile colonisation. Differences between the healthy donors and C. difficile positive routine samples were detected in bacterial, fungal and archaeal components. Bifidobacterium longum was the single most important species associated with C. difficile negative samples. However, by machine learning approaches we have identified patterns of microbiota composition predictive for C. difficile colonization. Those patterns also differed between samples with C. difficile ribotype 027 and other C. difficile ribotypes. The results indicate that not only the presence of a single species/group is important but that certain combinations of gut microbes are associated with C. difficile carriage and that some ribotypes (027) might be associated with more disturbed microbiota than the others.     

Surface-Layer Protein A (SlpA) Is a Major Contributor to Host-Cell Adherence of Clostridium difficile

Clostridium difficile is a leading cause of antibiotic-associated diarrhea, and a significant etiologic agent of healthcare-associated infections. The mechanisms of attachment and host colonization of C. difficile are not well defined. We hypothesize that non-toxin bacterial factors, especially those facilitating the interaction of C. difficile with the host gut, contribute to the initiation of C. difficile infection. In this work, we optimized a completely anaerobic, quantitative, epithelial-cell adherence assay for vegetative C. difficile cells, determined adherence proficiency under multiple conditions, and investigated C. difficile surface protein variation via immunological and DNA sequencing approaches focused on Surface-Layer Protein A (SlpA). In total, thirty-six epidemic-associated and non-epidemic associated C. difficile clinical isolates were tested in this study, and displayed intra- and inter-clade differences in attachment that were unrelated to toxin production. SlpA was a major contributor to bacterial adherence, and individual subunits of the protein (varying in sequence between strains) mediated host-cell attachment to different extents. Pre-treatment of host cells with crude or purified SlpA subunits, or incubation of vegetative bacteria with anti-SlpA antisera significantly reduced C. difficile attachment. SlpA-mediated adherence-interference correlated with the attachment efficiency of the strain from which the protein was derived, with maximal blockage observed when SlpA was derived from highly adherent strains. In addition, SlpA-containing preparations from a non-toxigenic strain effectively blocked adherence of a phylogenetically distant, epidemic-associated strain, and vice-versa. Taken together, these results suggest that SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.     

Nondigestible Oligosaccharides Enhance Bacterial Colonization Resistance against Clostridium difficile In Vitro

Clostridium difficile is the principal etiologic agent of pseudomembranous colitis and is a major cause of nosocomial antibiotic-associated diarrhea. A limited degree of success in controlling C. difficile infection has been achieved by using probiotics; however, prebiotics can also be used to change bacterial community structure and metabolism in the large gut, although the effects of these carbohydrates on suppression of clostridial pathogens have not been well characterized. The aims of this study were to investigate the bifidogenicity of three nondigestible oligosaccharide (NDO) preparations in normal and antibiotic-treated fecal microbiotas in vitro and their abilities to increase barrier resistance against colonization by C. difficile by using cultural and molecular techniques. Fecal cultures from three healthy volunteers were challenged with a toxigenic strain of C. difficile, and molecular probes were used to monitor growth of the pathogen, together with growth of bifidobacterial and bacteroides populations, over a time course. Evidence of colonization resistance was assessed by determining viable bacterial counts, short-chain fatty acid formation, and cytotoxic activity. Chemostat studies were then performed to determine whether there was a direct correlation between bifidobacteria and C. difficile suppression. NDO were shown to stimulate bifidobacterial growth, and there were concomitant reductions in C. difficile populations. However, in the presence of clindamycin, activity against bifidobacteria was augmented in the presence of NDO, resulting in a further loss of colonization resistance. In the absence of clindamycin, NDO enhanced colonization resistance against C. difficile, although this could not be attributed to bifidobacterium-induced inhibitory phenomena.     

Clostridium difficile heterogeneously impacts intestinal community architecture but drives stable metabolome responses

Clostridium difficile-associated diarrhoea (CDAD) is caused by C. difficile toxins A and B and represents a serious emerging health problem. Yet, its progression and functional consequences are unclear. We hypothesised that C. difficile can drive major measurable metabolic changes in the gut microbiota and that a relationship with the production or absence of toxins may be established. We tested this hypothesis by performing metabolic profiling on the gut microbiota of patients with C. difficile that produced (n=6) or did not produce (n=4) toxins and on non-colonised control patients (n=6), all of whom were experiencing diarrhoea. We report a statistically significant separation (P-value <0.05) among the three groups, regardless of patient characteristics, duration of the disease, antibiotic therapy and medical history. This classification is associated with differences in the production of distinct molecules with presumptive global importance in the gut environment, disease progression and inflammation. Moreover, although severe impaired metabolite production and biological deficits were associated with the carriage of C. difficile that did not produce toxins, only previously unrecognised selective features, namely, choline- and acetylputrescine-deficient gut environments, characterised the carriage of toxin-producing C. difficile. Additional results showed that the changes induced by C. difficile become marked at the highest level of the functional hierarchy, namely the metabolic activity exemplified by the gut microbial metabolome regardless of heterogeneities that commonly appear below the functional level (gut bacterial composition). We discuss possible explanations for this effect and suggest that the changes imposed by CDAD are much more defined and predictable than previously thought.     

Bile salt hydrolase-mediated inhibitory effect of <Emphasis Type="Italic">Bacteroides ovatus</Emphasis> on growth of <Emphasis Type="Italic">Clostridium difficile</Emphasis>

Clostridium difficile infection (CDI) is one of the most common nosocomial infections. Dysbiosis of the gut microbiota due to consumption of antibiotics is a major contributor to CDI. Recently, fecal microbiota transplantation (FMT) has been applied to treat CDI. However, FMT has important limitations including uncontrolled exposure to pathogens and standardization issues. Therefore, it is necessary to evaluate alternative treatment methods, such as bacteriotherapy, as well as the mechanism through which beneficial bacteria inhibit the growth of C. difficile. Here, we report bile acid-mediated inhibition of C. difficile by Bacteroides strains which can produce bile salt hydrolase (BSH). Bacteroides strains are not commonly used to treat CDI; however, as they comprise a large proportion of the intestinal microbiota, they can contribute to bile acid-mediated inhibition of C. difficile. The inhibitory effect on C. difficile growth increased with increasing bile acid concentration in the presence of Bacteroides ovatus SNUG 40239. Furthermore, this inhibitory effect on C. difficile growth was significantly attenuated when bile acid availability was reduced by cholestyramine, a bile acid sequestrant. The findings of this study are important due to the discovery of a new bacterial strain that in the presence of available bile acids inhibits growth of C. difficile. These results will facilitate development of novel bacteriotherapy strategies to control CDI.     

Cwp84, a Surface-associated Cysteine Protease, Plays a Role in the Maturation of the Surface Layer of Clostridium difficile

Clostridium difficile is a major and growing problem as a hospital-associated infection that can cause severe, recurrent diarrhea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood but undoubtedly involves protein components within the surface layer (S-layer), which play a role in adhesion. In C. difficile, the S-layer is composed of two principal components, the high and low molecular weight S-layer proteins, which are formed from the post-translational cleavage of a single precursor, SlpA. In the present study, we demonstrate that a recently characterized cysteine protease, Cwp84 plays a role in maturation of SlpA. Using a gene knock-out approach, we show that inactivation of the Cwp84 gene in C. difficile 630ΔErm results in a bacterial phenotype in which only immature, single chain SlpA comprises the S-layer. The Cwp84 knock-out mutants (CDΔCwp84) displayed significantly different colony morphology compared with the wild-type strain and grew more slowly in liquid medium. SlpA extracted from CDΔCwp84 was readily cleaved into its mature subunits by trypsin treatment. Addition of trypsin to the growth medium also cleaved SlpA on CDΔCwp84 and increased the growth rate of the bacterium in a dose-dependent manner. Using the hamster model for C. difficile infection, CDΔCwp84 was found to be competent at causing disease with a similar pathology to the wild-type strain. The data show that whereas Cwp84 plays a role in the cleavage of SlpA, it is not an essential virulence factor and that bacteria expressing immature SlpA are able to cause disease.     

Mixotrophic and Autotrophic Growth of Thiobacillus acidophilus on Glucose and Thiosulfate

Mixotrophic growth of the facultatively autotrophic acidophile Thiobacillus acidophilus on mixtures of glucose and thiosulfate or tetrathionate was studied in substrate-limited chemostat cultures. Growth yields in mixotrophic cultures were higher than the sum of the heterotrophic and autotrophic growth yields. Pulse experiments with thiosulfate indicated that tetrathionate is an intermediate during thiosulfate oxidation by cell suspensions of T. acidophilus. From mixotrophic growth studies, the energetic value of thiosulfate and tetrathionate redox equivalents was estimated to be 50% of that of redox equivalents derived from glucose oxidation. Ribulose 1,5-bisphosphate carboxylase (RuBPCase) activities in cell extracts and rates of sulfur compound oxidation by cell suspensions increased with increasing thiosulfate/glucose ratios in the influent medium of the mixotrophic cultures. Significant RuBPCase and sulfur compound-oxidizing activities were detected in heterotrophically grown T. acidophilus. Polyhedral inclusion bodies (carboxysomes) could be observed at low frequencies in thin sections of cells grown in heterotrophic, glucose-limited chemostat cultures. Highest RuBPCase activities and carboxysome abundancy were observed in cells from autotrophic, CO₂-limited chemostat cultures. The maximum growth rate at which thiosulfate was still completely oxidized was increased when glucose was utilized simultaneously. This, together with the fact that even during heterotrophic growth the organism exhibited significant activities of enzymes involved in autotrophic metabolism, indicates that T. acidophilus is well adapted to a mixotrophic lifestyle. In this respect, T. acidophilus may have a competitive advantage over autotrophic acidophiles with respect to the sulfur compound oxidation in environments in which organic compounds are present.     

Succession in the Gut Microbiome following Antibiotic and Antibody Therapies for Clostridium difficile

Antibiotic disruption of the intestinal microbiota may cause susceptibility to pathogens that is resolved by progressive bacterial outgrowth and colonization. Succession is central to ecological theory but not widely documented in studies of the vertebrate microbiome. Here, we study succession in the hamster gut after treatment with antibiotics and exposure to Clostridium difficile. C. difficile infection is typically lethal in hamsters, but protection can be conferred with neutralizing antibodies against the A and B toxins. We compare treatment with neutralizing monoclonal antibodies (mAb) to treatment with vancomycin, which prolongs the lives of animals but ultimately fails to protect them from death. We carried out longitudinal deep sequencing analysis and found distinctive waves of succession associated with each form of treatment. Clindamycin sensitization prior to infection was associated with the temporary suppression of the previously dominant Bacteroidales and the fungus Saccinobaculus in favor of Proteobacteria. In mAb-treated animals, C. difficile proliferated before joining Proteobacteria in giving way to re-expanding Bacteroidales and the fungus Wickerhamomyces. However, the Bacteroidales lineages returning by day 7 were different from those that were present initially, and they persisted for the duration of the experiment. Animals treated with vancomycin showed a different set of late-stage lineages that were dominated by Proteobacteria as well as increased disparity between the tissue-associated and luminal cecal communities. The control animals showed no change in their gut microbiota. These data thus suggest different patterns of ecological succession following antibiotic treatment and C. difficile infection.     

Thawing permafrost increases old soil and autotrophic respiration in tundra: Partitioning ecosystem respiration using δ13C and ∆14C

Ecosystem respiration (R_eco) is one of the largest terrestrial carbon (C) fluxes. The effect of climate change on R_eco depends on the responses of its autotrophic and heterotrophic components. How autotrophic and heterotrophic respiration sources respond to climate change is especially important in ecosystems underlain by permafrost. Permafrost ecosystems contain vast stores of soil C (1672 Pg) and are located in northern latitudes where climate change is accelerated. Warming will cause a positive feedback to climate change if heterotrophic respiration increases without corresponding increases in primary production. We quantified the response of autotrophic and heterotrophic respiration to permafrost thaw across the 2008 and 2009 growing seasons. We partitioned R_eco using Δ¹⁴C and δ¹³C into four sources–two autotrophic (above – and belowground plant structures) and two heterotrophic (young and old soil). We sampled the Δ¹⁴C and δ¹³C of sources using incubations and the Δ¹⁴C and δ¹³C of R_eco using field measurements. We then used a Bayesian mixing model to solve for the most likely contributions of each source to R_eco. Autotrophic respiration ranged from 40 to 70% of R_eco and was greatest at the height of the growing season. Old soil heterotrophic respiration ranged from 6 to 18% of R_eco and was greatest where permafrost thaw was deepest. Overall, growing season fluxes of autotrophic and old soil heterotrophic respiration increased as permafrost thaw deepened. Areas with greater thaw also had the greatest primary production. Warming in permafrost ecosystems therefore leads to increased plant and old soil respiration that is initially compensated by increased net primary productivity. However, barring large shifts in plant community composition, future increases in old soil respiration will likely outpace productivity, resulting in a positive feedback to climate change.     

Diarylacylhydrazones: Clostridium-selective antibacterials with activity against stationary-phase cells

Current antibiotics for treating Clostridium difficile infections (CDI), that is, metronidazole, vancomycin and more recently fidaxomicin, are mostly effective but treatment failure and disease relapse remain as significant clinical problems. The shortcomings of these agents are attributed to their low selectivity for C. difficile over normal gut microflora and their ineffectiveness against C. difficile spores. This Letter reports that certain diarylacylhydrazones identified during a high-throughput screening/counter-screening campaign show selective activity against two Clostridium species (C. difficile and Clostridium perfringens) over common gut commensals. Representative examples are shown to possess activity similar to vancomycin against clinical C. difficile strains and to kill stationary-phase C. difficile cells, which are responsible for spore production. Structure–activity relationships with additional synthesised analogues suggested a protonophoric mechanism may play a role in the observed activity/selectivity and this was supported by the well-known protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) showing selective anti-Clostridium effects and activity similar to diarylacylhydrazones against stationary-phase C. difficile cells. Two diarylacylhydrazones were shown to be non-toxic towards human FaDu and Hep G2 cells indicating that further studies with the class are warranted towards new drugs for CDI.     

Mixotrophic organisms become more heterotrophic with rising temperature

The metabolic theory of ecology predicts that temperature affects heterotrophic processes more strongly than autotrophic processes. We hypothesized that this differential temperature response may shift mixotrophic organisms towards more heterotrophic nutrition with rising temperature. The hypothesis was tested in experiments with the mixotrophic chrysophyte Ochromonas sp., grown under autotrophic, mixotrophic and heterotrophic conditions. Our results show that (1) grazing rates on bacterial prey increased more strongly with temperature than photosynthetic electron transport rates, (2) heterotrophic growth rates increased exponentially with temperature over the entire range from 13 to 33 °C, while autotrophic growth rates reached a maximum at intermediate temperatures and (3) chlorophyll contents during mixotrophic growth decreased at high temperature. Hence, the contribution of photosynthesis to mixotrophic growth strongly decreased with temperature. These findings support the hypothesis that mixotrophs become more heterotrophic with rising temperature, which alters their functional role in food webs and the carbon cycle.     

Coping with formaldehyde during C1 metabolism of Paracoccus denitrificans

Methylotrophic bacteria are capable of growth using reduced one-carbon (C1) compounds like methanol or methylamine as free energy sources. Paracoccus denitrificans, which is a facultative methylotrophic organism, switches to this type of autotrophic metabolism only when it experiences a shortage of available heterotrophic free energy sources. Since the oxidation of C1 substrates is energetically less favourable than that of the heterotrophic ones, a global regulatory circuit ensures that the enzymes involved in methylotrophic growth are repressed during heterotrophic growth. Once the decision is made to switch to methylotrophic growth, additional regulatory proteins ensure the fine-tuned expression of the participating enzymes such that the steady-state concentration of formaldehyde, the oxidation product of C1 substrates, is kept below cytotoxic levels.     

Metabolic modeling predicts specific gut bacteria as key determinants for Candida albicans colonization levels

Candida albicans is a leading cause of life-threatening hospital-acquired infections and can lead to Candidemia with sepsis-like symptoms and high mortality rates. We reconstructed a genome-scale C. albicans metabolic model to investigate bacterial-fungal metabolic interactions in the gut as determinants of fungal abundance. We optimized the predictive capacity of our model using wild type and mutant C. albicans growth data and used it for in silico metabolic interaction predictions. Our analysis of more than 900 paired fungal–bacterial metabolic models predicted key gut bacterial species modulating C. albicans colonization levels. Among the studied microbes, Alistipes putredinis was predicted to negatively affect C. albicans levels. We confirmed these findings by metagenomic sequencing of stool samples from 24 human subjects and by fungal growth experiments in bacterial spent media. Furthermore, our pairwise simulations guided us to specific metabolites with promoting or inhibitory effect to the fungus when exposed in defined media under carbon and nitrogen limitation. Our study demonstrates that in silico metabolic prediction can lead to the identification of gut microbiome features that can significantly affect potentially harmful levels of C. albicans.Subject terms: Fungi, Infectious diseases, Metagenomics, Gastrointestinal diseases, Microbiome     

Stable Carbon Isotope Ratios of Lipid Biomarkers of Sulfate-Reducing Bacteria

We examined the potential use of natural-abundance stable carbon isotope ratios of lipids for determining substrate usage by sulfate-reducing bacteria (SRB). Four SRB were grown under autotrophic, mixotrophic, or heterotrophic growth conditions, and the δ¹³C values of their individual fatty acids (FA) were determined. The FA were usually ¹³C depleted in relation to biomass, with Δδ¹³C_{(FA − biomass)} of −4 to −17‰; the greatest depletion occurred during heterotrophic growth. The exception was Desulfotomaculum acetoxidans, for which substrate limitation resulted in biomass and FA becoming isotopically heavier than the acetate substrate. The δ¹³C values of FA in Desulfotomaculum acetoxidans varied with the position of the double bond in the monounsaturated C₁₆ and C₁₈ FA, with FA becoming progressively more ¹³C depleted as the double bond approached the methyl end. Mixotrophic growth of Desulfovibrio desulfuricans resulted in little depletion of the i17:1 biomarker relative to biomass or acetate, whereas growth with lactate resulted in a higher proportion of i17:1 with a greater depletion in ¹³C. The relative abundances of 10Me16:0 in Desulfobacter hydrogenophilus and Desulfobacterium autotrophicum were not affected by growth conditions, yet the Δδ¹³C_{(FA − substrate)} values of 10Me16:0 were considerably greater during autotrophic growth. These experiments indicate that FA δ¹³C values can be useful for interpreting carbon utilization by SRB in natural environments.     

An in vitro culture model to study the dynamics of colonic microbiota in Syrian golden hamsters and their susceptibility to infection with Clostridium difficile

Clostridium difficile infections (CDI) are caused by colonization and growth of toxigenic strains of C. difficile in individuals whose intestinal microbiota has been perturbed, in most cases following antimicrobial therapy. Determination of the protective commensal gut community members could inform the development of treatments for CDI. Here, we utilized the lethal enterocolitis model in Syrian golden hamsters to analyze the microbiota disruption and recovery along a 20-day period following a single dose of clindamycin on day 0, inducing in vivo susceptibility to C. difficile infection. To determine susceptibility in vitro, spores of strain VPI 10463 were cultured with and without soluble hamster fecal filtrates and growth was quantified by quantitative PCR and toxin immunoassay. Fecal microbial population changes over time were tracked by 16S ribosomal RNA gene analysis via V4 sequencing and the PhyloChip assay. C. difficile culture growth and toxin production were inhibited by the presence of fecal extracts from untreated hamsters but not extracts collected 5 days post-administration of clindamycin. In vitro inhibition was re-established by day 15, which correlated with resistance of animals to lethal challenge. A substantial fecal microbiota shift in hamsters treated with antibiotics was observed, marked by significant changes across multiple phyla including Bacteroidetes and Proteobacteria. An incomplete return towards the baseline microbiome occurred by day 15 correlating with the inhibition of C. difficile growth in vitro and in vivo. These data suggest that soluble factors produced by the gut microbiota may be responsible for the suppression of C. difficile growth and toxin production.     

Systemically Administered IgG Anti-Toxin Antibodies Protect the Colonic Mucosa during Infection with Clostridium difficile in the Piglet Model

The use of anti-toxin human monoclonal antibodies (HMab) as treatment for C. difficile infection has been investigated in animal models and human clinical trials as an alternative to or in combination with traditional antibiotic therapy. While HMab therapy appears to be a promising option, how systemically administered IgG antibodies protect the colonic mucosa during Clostridium difficile infection is unknown. Using the gnotobiotic piglet model of Clostridium difficile infection, we administered a mixture of anti-TcdA and anti-TcdB HMabs systemically to piglets infected with either pathogenic or non-pathogenic C. difficile strains. The HMabs were present throughout the small and large intestinal tissue of both groups, but significant HMabs were present in the lumen of the large intestines only in the pathogenic strain-infected group. Similarly, HMabs measured in the large intestine over a period of 2–4 days following antibody administration were not significantly different over time in the gut mucosa among the groups, but concentrations in the lumen of the large intestine were again consistently higher in the pathogenic strain-infected group. These results indicate that systemically administered HMab IgG reaches the gut mucosa during the course of CDI, protecting the host against systemic intoxication, and that leakage through the damaged colon likely protects the mucosa from further damage, allowing initiation of repair and recovery.     

An Integrated Metabolomic and Microbiome Analysis Identified Specific Gut Microbiota Associated with Fecal Cholesterol and Coprostanol in Clostridium difficile Infection

Clostridium difficile infection (CDI) is characterized by dysbiosis of the intestinal microbiota and a profound derangement in the fecal metabolome. However, the contribution of specific gut microbes to fecal metabolites in C. difficile-associated gut microbiome remains poorly understood. Using gas-chromatography mass spectrometry (GC-MS) and 16S rRNA deep sequencing, we analyzed the metabolome and microbiome of fecal samples obtained longitudinally from subjects with Clostridium difficile infection (n = 7) and healthy controls (n = 6). From 155 fecal metabolites, we identified two sterol metabolites at >95% match to cholesterol and coprostanol that significantly discriminated C. difficile-associated gut microbiome from healthy microbiota. By correlating the levels of cholesterol and coprostanol in fecal extracts with 2,395 bacterial operational taxonomic units (OTUs) determined by 16S rRNA sequencing, we identified 63 OTUs associated with high levels of coprostanol and 2 OTUs correlated with low coprostanol levels. Using indicator species analysis (ISA), 31 of the 63 coprostanol-associated bacteria correlated with health, and two Veillonella species were associated with low coprostanol levels that correlated strongly with CDI. These 65 bacterial taxa could be clustered into 12 sub-communities, with each community containing a consortium of organisms that co-occurred with one another. Our studies identified 63 human gut microbes associated with cholesterol-reducing activities. Given the importance of gut bacteria in reducing and eliminating cholesterol from the GI tract, these results support the recent finding that gut microbiome may play an important role in host lipid metabolism.     

Clostridium difficile clinical isolates exhibit variable susceptibility and proteome alterations upon exposure to mammalian cationic antimicrobial peptides

Clostridium difficile is a leading cause of hospital-acquired bacterial infections in the United States, and the increased incidence of recurrent C. difficile infections is particularly problematic. The molecular mechanisms of C. difficile colonization, including its ability to evade host innate immune responses, is poorly understood. We hypothesized that epidemic-associated C. difficile clinical isolates would exhibit increased resistance to mammalian, gut-associated, cationic antimicrobial peptides such as the cathelicidin LL-37. Standardized susceptibility tests as well as comparative proteomic analyses revealed that C. difficile strains varied in their responses to LL-37, with epidemic-associated 027 ribotype isolates displaying greater resistance. Further, exposure of C. difficile strains to sub-lethal concentrations of LL-37 resulted in increased resistance to subsequent peptide challenge, suggesting the presence of inducible resistance mechanisms. Correspondingly, LL-37 exposure altered the C. difficile proteome, with marked changes in abundance of cell wall biosynthesis proteins, surface layer proteins, ABC transporters and lysine metabolism pathway components. Taken together, these results suggest that innate immune avoidance mechanisms could facilitate robust colonization by C. difficile.     

Importance of Glutamate Dehydrogenase (GDH) in Clostridium difficile Colonization In Vivo

Clostridium difficile is the principal cause of antibiotic-associated diarrhea. Major metabolic requirements for colonization and expansion of C. difficile after microbiota disturbance have not been fully determined. In this study, we show that glutamate utilization is important for C. difficile to establish itself in the animal gut. When the gluD gene, which codes for glutamate dehydrogenase (GDH), was disrupted, the mutant C. difficile was unable to colonize and cause disease in a hamster model. Further, from the complementation experiment it appears that extracellular GDH may be playing a role in promoting C. difficile colonization and disease progression. Quantification of free amino acids in the hamster gut during C. difficile infection showed that glutamate is among preferred amino acids utilized by C. difficile during its expansion. This study provides evidence of the importance of glutamate metabolism for C. difficile pathogenesis.