Pathogen Clostridium Difficile Research Articles

Comparative prebiotic potential of galacto- and fructo-oligosaccharides, native inulin, and acacia gum in Kenyan infant gut microbiota during iron supplementation.

Iron fortification to prevent anemia in African infants increases colonic iron levels, favoring the growth of enteropathogens. The use of prebiotics may be an effective strategy to reduce these detrimental effects. Using the African infant PolyFermS gut model, we compared the effect of the prebiotics short-chain galacto- with long-chain fructo-oligosaccharides (scGOS/lcFOS) and native inulin, and the emerging prebiotic acacia gum, a branched-polysaccharide-protein complex consisting of arabinose and galactose, during iron supplementation on four Kenyan infant gut microbiota. Iron supplementation did not alter the microbiota but promoted Clostridioides difficile in one microbiota. The prebiotic effect of scGOS/lcFOS and inulin was confirmed during iron supplementation in all investigated Kenyan infant gut microbiota, leading to higher abundance of bifidobacteria, increased production of acetate, propionate, and butyrate, and a significant shift in microbiota composition compared to non-supplemented microbiota. The abundance of the pathogens Clostridium difficile and Clostridium perfringens was also inhibited upon addition of the prebiotic fibers. Acacia gum had no effect on any of the microbiota. In conclusion, scGOS/lcFOS and inulin, but not acacia gum, showed a donor-independent strong prebiotic potential in Kenyan infant gut microbiota. This study demonstrates the relevance of comparing fibers in vitro prior to clinical studies.

Wastewater treatment plants, an "escape gate" for ESCAPE pathogens.

Antibiotics are an essential tool of modern medicine, contributing to significantly decreasing mortality and morbidity rates from infectious diseases. However, persistent misuse of these drugs has accelerated the evolution of antibiotic resistance, negatively impacting clinical practice. The environment contributes to both the evolution and transmission of resistance. From all anthropically polluted aquatic environments, wastewater treatment plants (WWTPs) are probably the main reservoirs of resistant pathogens. They should be regarded as critical control points for preventing or reducing the release of antibiotics, antibiotic-resistant bacteria (ARB), and antibiotic-resistance genes (ARGs) into the natural environment. This review focuses on the fate of the pathogens Enterococcus faecium, Staphylococcus aureus, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae spp. (ESCAPE) in WWTPs. All ESCAPE pathogen species, including high-risk clones and resistance determinants to last-resort antibiotics such as carbapenems, colistin, and multi-drug resistance platforms, were detected in wastewater. The whole genome sequencing studies demonstrate the clonal relationships and dissemination of Gram-negative ESCAPE species into the wastewater via hospital effluents and the enrichment of virulence and resistance determinants of S. aureus and enterococci in WWTPs. Therefore, the efficiency of different wastewater treatment processes regarding the removal of clinically relevant ARB species and ARGs, as well as the influence of water quality factors on their performance, should be explored and monitored, along with the development of more effective treatments and appropriate indicators (ESCAPE bacteria and/or ARGs). This knowledge will allow the development of quality standards for point sources and effluents to consolidate the WWTP barrier role against the environmental and public health AR threats.

Characterization of the glmS Ribozymes from Listeria Monocytogenes and Clostridium Difficile.

The glmS ribozyme regulates the expression of the essential GlmS enzyme being involved in cell wall biosynthesis. While >450 variants of the glmS ribozyme were identified by in silico approaches and homology searches, only a few have yet been experimentally investigated. Herein, we validate and characterize the glmS ribozymes of the human pathogens Clostridium difficile and Listeria monocytogenes. Both ribozymes, as their previous characterized homologs rely on glucosamine-6-phosphate as co-factor and the presence of divalent cations for exerting the cleavage reaction. The observed EC50 values in turn were found to be in the submicromolar range, at least an order of magnitude lower than observed for glmS ribozymes from other bacteria. The glmS ribozyme of L. monocytogenes was further shown to bear unique properties. It discriminates between co-factors very stringently and other than the glmS ribozyme of C. difficile retains activity at low temperatures. This finding illustrates that albeit being highly conserved, glmS ribozymes have unique characteristics.

Clinical case of severe pseudomembranous colitis caused by pathogenic infection <i>Clostridium Difficile</i>

Objective. Literature review of pseudomembranous colitis caused by pathogenic Clostridium difficile infection and description of a clinical case of pseudomembranous colitis.
 Materials and methods. A clinical case of a patient with pseudomembranous colitis is presented.
 Results. In a patient with multiple injuries and grade II shock, whose condition required numerous surgical interventions and intensive antibiotic therapy, after the completion of antibiotic therapy there appeared signs of pseudomembranous colitis loose stools (up to 5 times a day), fever up to 39 C, signs of endogenous intoxication, A C. difficile toxin was found in faeces. Antibiotic-associated colitis was diagnosed; metronidazole 500 mg 3 times a day, activated charcoal, infusion therapy were prescribed on the 12th day, the therapy led to recovery. During the subsequent reconstructive operation, lincomycin 600 mg was administered by infusion for prophylactic purposes, after which, 6 hours later, the patient developed a clinical picture of severe pseudomembranous colitis. A laboratory study of faeces again revealed toxin A C. difficile. The recurrence of the disease required a long-term treatment in the gastroenterology department.
 Conclusions. Pseudomembranous colitis is certainly a complex pathology that is a serious hospital-acquired infection. Accurate diagnosis requires correct assessment of the patients condition through clinical and laboratory diagnostic methods, correct prescription of conservative treatment and choice of surgical intervention tactics.

Protein Dynamics and Substrate Protonation States Mediate the Catalytic Action of trans-4-Hydroxy-l-Proline Dehydratase.

The enzyme trans-4-hydroxy-l-proline (Hyp) dehydratase (HypD) is among the most abundant glycyl radical enzymes (GREs) in the healthy human gut microbiome and is considered a promising antibiotic target for the prominent antibiotic-resistant pathogen Clostridium difficile. Although an enzymatic mechanism has been proposed, the role of the greater HypD protein environment in mediating radical reactivity is not well understood. To fill this gap in understanding, we investigate HypD across multiple time- and length-scales using electronic structure modeling and classical molecular dynamics. We observe that the Hyp substrate protonation state significantly alters both its enzyme-free reactivity and its dynamics within the enzyme active site. Accurate coupled-cluster modeling suggests the deprotonated form of Hyp to be the most reactive protonation state for C5-Hpro-S activation. In the protein environment, hydrophobic interactions modulate the positioning of the Cys434 radical to enhance the reactivity of C5-Hpro-S abstraction. Long-time dynamics reveal that changing Hyp protonation states triggers the switching of a Leu643-gated water tunnel, a functional feature that has not yet been observed for members of the GRE superfamily.

Spatial distribution of LTi-like cells in intestinal mucosa regulates type 3 innate immunity

Lymphoid tissue inducer (LTi)-like cells are tissue resident innate lymphocytes that rapidly secrete cytokines that promote gut epithelial integrity and protect against extracellular bacterial infections.Here, we report that the retention of LTi-like cells in conventional solitary intestinal lymphoid tissue (SILT) is essential for controlling LTi-like cell function and is maintained by expression of the chemokine receptor CXCR5. Deletion of Cxcr5 functionally unleashed LTi-like cells in a cell intrinsic manner, leading to uncontrolled IL-17 and IL-22 production. The elevated production of IL-22 in Cxcr5-deficient mice improved gut barrier integrity and protected mice during infection with the opportunistic pathogen Clostridium difficile Interestingly, Cxcr5-/- mice developed LTi-like cell aggregates that were displaced from their typical niche at the intestinal crypt, and LTi-like cell hyperresponsiveness was associated with the local formation of this unconventional SILT. Thus, LTi-like cell positioning within mucosa controls their activity via niche-specific signals that temper cytokine production during homeostasis.

C. difficile exploits a host metabolite produced during toxin-mediated disease.

Several enteric pathogens can gain specific metabolic advantages over other members of the microbiota by inducing host pathology and inflammation. The pathogen Clostridium difficile is responsible for a toxin-mediated colitis that causes 450,000 infections and 15,000 deaths in the United States each year1; however, the molecular mechanisms by which C. difficile benefits from this pathology remain unclear. To understand how the metabolism of C. difficile adapts to the inflammatory conditions that its toxins induce, here we use RNA sequencing to define, in a mouse model, the metabolic states of wild-type C. difficile and of an isogenic mutant that lacks toxins. By combining bacterial and mouse genetics, we demonstrate that C. difficile uses sorbitol derived from both diet and host. Host-derived sorbitol is produced by the enzyme aldose reductase, which is expressed by diverse immune cells and is upregulated during inflammation-including during toxin-mediated disease induced by C. difficile. This work highlights a mechanism by which C. difficile can use a host-derived nutrient that is generated during toxin-induced disease by an enzyme that has not previously been associated with infection.

Network analysis of nitrate-sensitive oral microbiome reveals interactions with cognitive function and cardiovascular health across dietary interventions

Many oral bacteria reduce inorganic nitrate, a natural part of a vegetable-rich diet, into nitrite that acts as a precursor to nitric oxide, a regulator of vascular tone and neurotransmission. Aging is hallmarked by reduced nitric oxide production with associated detriments to cardiovascular and cognitive function. This study applied a systems-level bacterial co-occurrence network analysis across 10-day dietary nitrate and placebo interventions to test the stability of relationships between physiological and cognitive traits and clusters of co-occurring oral bacteria in older people. Relative abundances of Proteobacteria increased, while Bacteroidetes, Firmicutes and Fusobacteria decreased after nitrate supplementation. Two distinct microbiome modules of co-occurring bacteria, that were sensitive to nitrate supplementation, showed stable relationships with cardiovascular (Rothia-Streptococcus) and cognitive (Neisseria-Haemophilus) indices of health across both dietary conditions. A microbiome module (Prevotella-Veillonella) that has been associated with pro-inflammatory metabolism was diminished after nitrate supplementation, including a decrease in relative abundance of pathogenic Clostridium difficile. These nitrate-sensitive oral microbiome modules are proposed as potential pre- and probiotic targets to ameliorate age-induced impairments in cardiovascular and cognitive health.

S2841 SARS-CoV-2 Associated Gastroenteritis Under Microscope

INTRODUCTION: SARS-CoV-2 most commonly presents with respiratory symptoms and fever. A significant proportion of patients are also experiencing gastrointestinal (GI) symptoms with loss of appetite, nausea, vomiting and diarrhea (variable, up to 10%); however, there is limited data about the associated histologic findings in the GI tract. CASE DESCRIPTION/METHODS: A 71 year old male presented with fever for past five days after going on a cruise two days prior. Antibiotics were initiated along with self-quarantine. Over the next few days, he developed cough, shortness of breath, fever and myalgia. SARS-CoV-2 infection was confirmed on nasopharyngeal swab. He was intubated for ARDS and started on Remdesivir, which was stopped due to acute kidney injury. Over the next month he developed septic shock, arrythmias, spontaneous venous bleeds, acalculous cholecystitis, ventilator associated pneumonia and profuse diarrhea. GI pathogen panel and Clostridium difficile testing were negative. CT of the abdomen showed colonic and distal small bowel wall thickening with adjacent inflammatory changes. EGD and colonoscopy demonstrated ulceration of the duodenum, splenic flexure, sigmoid colon and rectum (Figure 1). Biopsies from the terminal ileum showed increased apoptotic bodies (>10 apoptosis per 10 crypts), mild villous blunting, patchy crypt dropout and crypt abscess (Figure 2). Colon biopsies had similar features along with mucosal erosion and focal crypt abscess formation. No significant increase in intra-epithelial lymphocytes was identified. Viral stains for CMV and Adenovirus were negative. DISCUSSION: Limited data on GI specimens from SARS-CoV-2 positive patients have showed no significant damage with few infiltrating plasma cells and lymphocytes in lamina propria along with interstitial edema. In our case, we observed extensive apoptotic injury with acute inflammation. Although confirmatory stool testing was not performed, SARS CoV-2 was favored as the etiology due a long gap between the administration of Remdesivir and lack of any other identifiable causes. Further studies are needed to characterize SARS-CoV-2 associated gastrointestinal disease.Figure 1.: Sigmoid colon with focal ulceration.Figure 2.: Terminal ileum with increased apoptotic bodies, patchy crypt dropout and crypt abscess (H&E, 200X).

Using genetic relatedness to understand heterogeneous distributions of urban rat-associated pathogens.

Urban Norway rats (Rattus norvegicus) carry several pathogens transmissible to people. However, pathogen prevalence can vary across fine spatial scales (i.e., by city block). Using a population genomics approach, we sought to describe rat movement patterns across an urban landscape and to evaluate whether these patterns align with pathogen distributions. We genotyped 605 rats from a single neighborhood in Vancouver, Canada, and used 1,495 genome‐wide single nucleotide polymorphisms to identify parent–offspring and sibling relationships using pedigree analysis. We resolved 1,246 pairs of relatives, of which only 1% of pairs were captured in different city blocks. Relatives were primarily caught within 33 meters of each other leading to a highly leptokurtic distribution of dispersal distances. Using binomial generalized linear mixed models, we evaluated whether family relationships influenced rat pathogen status with the bacterial pathogens Leptospira interrogans, Bartonella tribocorum, and Clostridium difficile, and found that an individual's pathogen status was not predicted any better by including disease status of related rats. The spatial clustering of related rats and their pathogens lends support to the hypothesis that spatially restricted movement promotes the heterogeneous patterns of pathogen prevalence evidenced in this population. Our findings also highlight the utility of evolutionary tools to understand movement and rat‐associated health risks in urban landscapes.

The enteric pathogen Clostridium difficile chemotaxes towards mucin glycans during the early colonization phase of infection

BackgroundInfection with the pathogen Clostridium difficile is a major cause of antibiotic‐induced diarrhea and colitis. In the United States, C. difficile infects millions of patients each year and results in healthcare associated costs of over 1 billion dollars. Despite the significant burden of C. difficile infection, the exact mechanisms of C. difficile host colonization remain unknown. It is well known that antibiotic use sets the stage for infection and that toxin production induces symptoms. However, how C. difficile identifies and colonizes its ecological niche are unclear. Similar to other pathogens, flagellum‐mediated motility and the ability to interact with the intestinal mucus layer are likely critical factors for C. difficile colonization. We hypothesized that C. difficile would recognize mucin glycans present on human MUC2 and would chemotax towards these oligosaccharides. We also speculated that MUC2 would influence C. difficile gene expression and promote colonization.Methods & ResultsTo address these gaps in knowledge we fluorescently tagged C. difficile R20291 with CFDA‐SE and measured chemotaxis using a standard capillary assay. We observed that C. difficile was able to chemotax towards purified human MUC2 from a variety of sources, including human goblet cell lines HT29‐MTX, LS174T, T84 as well as human stool. We also found that C. difficile could recognize oligosaccharides present on MUC2 glycans, such as fucose, galactose, mannose, GalNac, GluNAc, and neurominic acid. The greatest accumulation of C. difficile was observed with mannose. To confirm these findings, we live‐imaged C. difficile chemotaxis in IBIDI chemotaxis slides and found that the presence of MUC2 or mannose increased the velocity and distance traveled by C. difficile compared to buffer medium alone. Examination of C. difficile genomes from several ribotypes revealed that C. difficile lacks the glycosyl hydrolases (GH) required for O‐linked mucin glycan degradation, although it did contain GH family 38, which cleaves mannose residues. To assess whether C. difficile could use mucin oligosaccharides as a primary carbon source, we grew C. difficile in a fully defined minimal medium (CDMM) in the absence of glucose supplemented with individual glycan oligosaccharides or MUC2. Consistent with our GH analysis, C. difficile was unable to grow on intact MUC2 alone. However, C. difficile was able to utilize all mucin oligosaccharides for growth. Further analysis of the C. difficile genome confirmed the transporters required for uptake of these sugars are present in all species. Finally, RNAseq demonstrated shifts in gene expression in C. difficile R20291 in response to human MUC2.ConclusionsThese data indicate that C. difficile recognizes and utilizes mucin‐derived oligosaccharides. We also demonstrate that C. difficile does not possess the enzymatic capacity to degrade these glycans on its own. Therefore, it must require the presence of other mucin‐degrading microbes for growth, pointing to the role of the microbial community in supporting C. difficile colonization.

PaeR, a Multiple Antibiotic Resistance Regulator Homolog from Clostridium difficile is a Sequence‐Specific DNA Binding Protein that Responds to Small Molecule Effectors

Proteins belonging to the multiple antibiotic resistance regulator (MarR) family are ubiquitous amongst species in the bacterial and archaeal domains. These proteins function as transcriptional regulators of a wide variety of cellular processes including catabolic pathways, oxidative stress response mechanisms, efflux or degradation of toxins, and virulence factor expression. DNA binding by MarR proteins is mediated by a characteristic winged helix‐turn‐helix (wHTH) motif which makes sequence‐specific contacts with both the DNA major and minor grooves. For a number of MarR homologs, DNA binding is abrogated by ligand binding. The natural ligands of MarR proteins are structurally diverse but generally fall into three main categories: small aromatic compounds, metals and reactive oxygen species. In this study, we report the first biochemical investigation of a MarR homolog from the pathogenic bacterium Clostridium difficile which we have named PaeR (putative antibiotic efflux regulator). Biophysical investigations of the structural properties of PaeR indicate that this protein exists predominantly as a homodimer in solution and unfolds in a manner consistent with a simple, two‐state transition. PaeR binds at two sites in its own promoter/operator region, suggesting autoregulation. Analysis of binding at a single site indicates a high‐affinity interaction with a microscopic dissociation constant of 3.3 ±0.4 nM. Competitive electrophoretic mobility shift assays (EMASs) indicate that PaeR interacts with DNA in a highly sequence‐specific manner. Ligand binding studies indicate that PaeR binding to its cognate DNA binding sequence is attenuated by small aromatic compounds. These findings, in conjunction with the genetic context of paeR in the C. difficile genome, suggests a possible role of this MarR homolog in regulating antibiotic resistance in this pathogen.Support or Funding InformationBill and Linda Frost Fund

Toxin A and B genes expression of Clostridium difficile in the sub-minimum inhibitory concentration of clindamycin, vancomycin and in combination with ceftazidime

Antibiotics prescribed for infections have diverse effects on microbiota and the pathogen Clostridium difficile (C. difficile) as the most important antibiotic-associated diarrhea. This study aims to determine the gene expression of toxins A and B at the transcription level in the sub-MIC of vancomycin (VAN), clindamycin (CLI), and ceftazidime (CAZ) alone and in combination. The MIC and fractional inhibitory concentration (FIC) of two C. difficile samples (a clinical isolate and ATCC 9689) were determined by microdilution and checkerboard microdilution methods, respectively. The total RNA was extracted from the medium inoculated with ∼106 CFU/mL of fresh bacteria in the pre-reduced medium containing ½ MIC of antibiotics alone and ½ FIC of antibiotics in combination. Real-time PCR was performed by sybrGreen methods in triplicate, and the data were analyzed by the comparative ΔΔCT method. All antibiotics except CAZ (alone and in combination) decreased the gene expression of toxins A and B within 24 hours. VAN and CLI reduced toxin gene expression within 24 and 48 hours. However, CAZ alone and in combination with VAN as well as CLI increased the gene expression of toxins A and B. The results confirmed toxin gene transcription and toxin production are associated with the type of isolates and antibiotics, as well as the combined form of antibiotics. This could be the reason which can explain the occurrence of C. difficile infection among patients who were treated with the third generation of cephalosporins alone and in combination with another antibiotic in the form of combinational therapy.

Clostridium difficile clade 3 (RT023) have a modified cell surface and contain a large transposable island with novel cargo

The major global pathogen Clostridium difficile (recently renamed Clostridioides difficile) has large genetic diversity including multiple mobile genetic elements. In this study, whole genome sequencing of 86 strains from the poorly characterised clade 3, predominantly PCR ribotype (RT)023, of C. difficile revealed distinctive surface architecture characteristics and a large mobile genetic island. These strains have a unique sortase substrate phenotype compared with well-characterised strains of C. difficile, and loss of the phage protection protein CwpV. A large genetic insertion (023_CTnT) comprised of three smaller elements (023_CTn1-3) is present in 80/86 strains analysed in this study, with genes common among other bacterial strains in the gut microbiome. Novel cargo regions of 023_CTnT include genes encoding a sortase, putative sortase substrates, lantibiotic ABC transporters and a putative siderophore biosynthetic cluster. We demonstrate the excision of 023_CTnT and sub-elements 023_CTn2 and 023_CTn3 from the genome of RT023 reference strain CD305 and the transfer of 023_CTn3 to a non-toxigenic C. difficile strain, which may have implications for the use of non-toxigenic C. difficile strains as live attenuated vaccines. Finally, we show that the genes within the island are expressed in a regulated manner in C. difficile RT023 strains conferring a distinct “niche adaptation”.

Quantification of Multiple Bacteria in Calcified Structural Valvular Heart Disease

Genome studies of heart valve tissue (HVT) in patients with structural valvular heart disease (sVHD) and acute infective endocarditis (aIE) showed polymicrobial infections. Subject of this study is the quantification of bacterial DNA in HVT of sVHD in comparison to aIE. It will be examined whether the bacterial DNA concentration can be used as surrogate marker to differentiate chronic and acute infections. DNA was isolated from HVT of 100 patients with sVHD and 23 microbiologically positively tested patients with aIE. Selected pathogens (Cutibacterium acnes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae, Clostridium difficile, and Klebsiella pneumoniae) were quantified using TaqMan-qPCR. Polymicrobial infiltration of HVT was investigated by immunohistologic methods. Of 100 sVHD patients, 94 tested positive for bacteria by 16S-rDNA and 72 by TaqMan-qPCR. In 29% of the sVHD cohort and in 70% of the aIE cohort, a coinfection with more than 2 bacteria was observed as indication of a polymicrobial infection. The most common pathogens in the sVHD patients were C. acnes (59%; 5-4074 pg/mL), E. faecalis (16%, 174-2781 pg/mL), and S. aureus (15%, 8-105 pg/mL). The DNA concentration of E. faecalis (P = 0.0285) and S. aureus (P = 0.0149) is significantly lower in the sVHD cohort than in the aIE cohort. sVHD is associated with bacterial infection and infiltration of the HVT in a majority of cases. TaqMan-qPCR is a valid instrument for the specific detection of bacteria in HVT and allows discrimination between sVHD and aIE for E. faecalis and S. aureus.

Molecular determinants of the mechanism and substrate specificity of Clostridium difficile proline-proline endopeptidase-1

Pro-Pro endopeptidase-1 (PPEP-1) is a secreted metalloprotease from the bacterial pathogen Clostridium difficile that cleaves two endogenous adhesion proteins. PPEP-1 is therefore important for bacterial motility and hence for efficient gut colonization during infection. PPEP-1 exhibits a unique specificity for Pro-Pro peptide bonds within the consensus sequence VNP↓PVP. In this study, we combined information from crystal and NMR structures with mutagenesis and enzyme kinetics to investigate the mechanism and substrate specificity of PPEP-1. Our analyses revealed that the substrate-binding cleft of PPEP-1 is shaped complementarily to the major conformation of the substrate in solution. We found that it possesses features that accept a tertiary amide and help discriminate P1' residues by their amide hydrogen bond-donating potential. We also noted that residues Lys-101, Trp-103, and Glu-184 are crucial for proteolytic activity. Upon substrate binding, these residues position a flexible loop over the substrate-binding cleft and modulate the second coordination sphere of the catalytic zinc ion. On the basis of these findings, we propose an induced-fit model in which prestructured substrates are recognized followed by substrate positioning within the active-site cleft and a concomitant increase in the Lewis acidity of the catalytic Zn2+ ion. In conclusion, our findings provide detailed structural and mechanistic insights into the substrate recognition and specificity of PPEP-1 from the common gut pathogen C. difficile.

Type I Toxin-Antitoxin Systems in Clostridia.

Type I toxin-antitoxin (TA) modules are abundant in both bacterial plasmids and chromosomes and usually encode a small hydrophobic toxic protein and an antisense RNA acting as an antitoxin. The RNA antitoxin neutralizes toxin mRNA by inhibiting its translation and/or promoting its degradation. This review summarizes our current knowledge of the type I TA modules identified in Clostridia species focusing on the recent findings in the human pathogen Clostridium difficile. More than ten functional type I TA modules have been identified in the genome of this emerging enteropathogen that could potentially contribute to its fitness and success inside the host. Despite the absence of sequence homology, the comparison of these newly identified type I TA modules with previously studied systems in other Gram-positive bacteria, i.e., Bacillus subtilis and Staphylococcus aureus, revealed some important common traits. These include the conservation of characteristic sequence features for small hydrophobic toxic proteins, the localization of several type I TA within prophage or prophage-like regions and strong connections with stress response. Potential functions in the stabilization of genome regions, adaptations to stress conditions and interactions with CRISPR-Cas defence system, as well as promising applications of TA for genome-editing and antimicrobial developments are discussed.

Lactobacillus gasseri APC 678 Reduces Shedding of the Pathogen Clostridium difficile in a Murine Model.

Clostridium difficile is a common cause of health-care acquired diarrhea, resulting in a spectrum of disease from mild diarrhea to life-threatening illness. Sixty Lactobacillus strains were screened for anti-C. difficile activity using a co-culture method. Based on their ability to inhibit C. difficile, L. gasseri APC 678 and L. rhamnosus DPC 6111 were selected for study in a murine model of C. difficile infection. L. gasseri ATCC 33323, was included as a control. It was established that, relative to control mice not fed Lactobacillus, feeding with L. gasseri APC 678 resulted in a significant reduction by day 7 (8-fold, p = 0.017) of viable C. difficile VPI 10463 in the feces of mice. In contrast, neither L. rhamnosus DPC 6111 nor L. gasseri ATCC 33323 significantly reduced fecal C. difficile shedding. Sequencing of the cecal microbiota showed that in mice fed L. gasseri APC 678 there was a significant increase in bacterial diversity across a number of indices when compared to the control or other Lactobacillus-fed groups. There was no significant change in the relative abundance of Firmicutes or Bacteroidetes in the group fed L. gasseri APC 678 relative to the control, while the groups fed L. rhamnosus DPC 6111 or L. gasseri ATCC 33323 showed a significant decrease in the relative abundance of Firmicutes (p = 0.002 and p = 0.019, respectively) and a significant increase in Bacteroidetes (p = 0.002 and p = 0.023, respectively). These results highlight the potential of L. gasseri APC 678 as a live therapeutic agent to target C. difficile infection.

Metabolic Modeling of Clostridium difficile Associated Dysbiosis of the Gut Microbiota

Recent in vitro experiments have demonstrated the ability of the pathogen Clostridium difficile and commensal gut bacteria to form biofilms on surfaces, and biofilm development in vivo is likely. Various studies have reported that 3%–15% of healthy adults are asymptomatically colonized with C. difficile, with commensal species providing resistance against C. difficile pathogenic colonization. C. difficile infection (CDI) is observed at a higher rate in immunocompromised patients previously treated with broad spectrum antibiotics that disrupt the commensal microbiota and reduce competition for available nutrients, resulting in imbalance among commensal species and dysbiosis conducive to C. difficile propagation. To investigate the metabolic interactions of C. difficile with commensal species from the three dominant phyla in the human gut, we developed a multispecies biofilm model by combining genome-scale metabolic reconstructions of C. difficile, Bacteroides thetaiotaomicron from the phylum Bacteroidetes, Faecalibacterium prausnitzii from the phylum Firmicutes, and Escherichia coli from the phylum Proteobacteria. The biofilm model was used to identify gut nutrient conditions that resulted in C. difficile-associated dysbiosis characterized by large increases in C. difficile and E. coli abundances and large decreases in F. prausnitzii abundance. We tuned the model to produce species abundances and short-chain fatty acid levels consistent with available data for healthy individuals. The model predicted that experimentally-observed host-microbiota perturbations resulting in decreased carbohydrate/increased amino acid levels and/or increased primary bile acid levels would induce large increases in C. difficile abundance and decreases in F. prausnitzii abundance. By adding the experimentally-observed perturbation of increased host nitrate secretion, the model also was able to predict increased E. coli abundance associated with C. difficile dysbiosis. In addition to rationalizing known connections between nutrient levels and disease progression, the model generated hypotheses for future testing and has the capability to support the development of new treatment strategies for C. difficile gut infections.

Proteomic Signatures of Clostridium difficile Stressed with Metronidazole, Vancomycin, or Fidaxomicin.

The anaerobic pathogen Clostridium difficile is of growing significance for the health care system due to its increasing incidence and mortality. As C. difficile infection is both supported and treated by antibiotics, a deeper knowledge on how antimicrobial agents affect the physiology of this important pathogen may help to understand and prevent the development and spreading of antibiotic resistant strains. As the proteomic response of a cell to stress aims at counteracting the harmful effects of this stress, it can be expected that the pattern of a pathogen’s responses to antibiotic treatment will be dependent on the antibiotic mechanism of action. Hence, every antibiotic treatment is expected to result in a specific proteomic signature characterizing its mode of action. In the study presented here, the proteomic response of C. difficile 630∆erm to vancomycin, metronidazole, and fidaxomicin stress was investigated on the level of protein abundance and protein synthesis based on 2D PAGE. The quantification of 425 proteins of C. difficile allowed the deduction of proteomic signatures specific for each drug treatment. Indeed, these proteomic signatures indicate very specific cellular responses to each antibiotic with only little overlap of the responses. Whereas signature proteins for vancomycin stress fulfil various cellular functions, the proteomic signature of metronidazole stress is characterized by alterations of proteins involved in protein biosynthesis and protein degradation as well as in DNA replication, recombination, and repair. In contrast, proteins differentially expressed after fidaxomicin treatment can be assigned to amino acid biosynthesis, transcription, cell motility, and the cell envelope functions. Notably, the data provided by this study hint also at so far unknown antibiotic detoxification mechanisms.