Characterization of Chicken IgY Specific to Clostridium difficile R20291 Spores and the Effect of Oral Administration in Mouse Models of Initiation and Recurrent Disease

Indexación: Web of Science; Scopus.Clostridium difficile infection (CDI) are the leading cause of world-wide nosocomial acquired diarrhea. The current main clinical challenge in CDI is the elevated rate of infection recurrence that may reach up to 30% of the patients, which has been associated to the formation of dormant spores during the infection. We sought to characterize the effects of oral administration of specific anti-spore IgY in mouse models of CDI and recurrent CDI. The specificity of anti-spore IgY was evaluated in vitro. In both, initiation mouse model and recurrence mouse model, we evaluated the prophylactic and therapeutic effect of anti-spore IgY, respectively. Our results demonstrate that anti-spore IgY exhibited high specificity and titers against C. difficile spores and reduced spore adherence to intestinal cells in vitro. Administration of anti-spore IgY to C57BL/6 mice prior and during CDI delayed the appearance of the diarrhea by 1.5 day, and spore adherence to the colonic mucosa by 90%. Notably, in the recurrence model, co-administration of anti-spore IgY coupled with vancomycin delayed the appearance of recurrent diarrhea by a median of 2 days. Collectively, these observations suggest that anti-spore IgY antibodies may be used as a novel prophylactic treatment for CDI, or in combination with antibiotics to treat CDI and prevent recurrence of the infection.https://www.frontiersin.org/articles/10.3389/fcimb.2017.00365/ful

Characterization of the Adherence of Clostridium difficile Spores: The Integrity of the Outermost Layer Affects Adherence Properties of Spores of the Epidemic Strain R20291 to Components of the Intestinal Mucosa

Indexación: Web of Science.Clostridium difficile is the causative agent of the most frequently reported nosocomial diarrhea worldwide. The high incidence of recurrent infection is the main clinical challenge of C. difficile infections (CBI). Formation of C. difficile spores of the epidemic strain R20291 has been shown to be essential for recurrent infection and transmission of the disease in a mouse model. However, the underlying mechanisms of how these spores persist in the colonic environment remains unclear. In this work, we characterized the adherence properties of epidemic R20291 spores to components of the intestinal mucosa, and we assessed the role of the exosporium integrity in the adherence properties by using cdeC mutant spores with a defective exosporium layer. Our results showed that spores and vegetative cells of the epidemic R20291 strain adhered at high levels to monolayers of Caco-2 cells and mucin. Transmission electron micrographs of Caco-2 cells demonstrated that the hair-like projections on the surface of R20291 spores are in close proximity with the plasma membrane and microvilli of undifferentiated and differentiated monolayers of Caco-2 cells. Competitive-binding assay in differentiated Caco-2 cells suggests that spore-adherence is mediated by specific binding sites. By using spores of a cdeC mutant we demonstrated that the integrity of the exosporium layer determines the affinity of adherence of C. difficile spores to Caco-2 cells and mucin. Binding of fibronectin and vitronectin to the spore surface was concentration-dependent, and depending on the concentration, spore-adherence to Caco-2 cells was enhanced. In the presence of an aberrantly-assembled exosporium (cdeC spores), binding of fibronectin, but not vitronectin, was increased. Notably, independent of the exosporium integrity, only a fraction of the spores had fibronectin and vitronectin molecules binding to their surface. Collectively, these results demonstrate that the integrity of the exosporium layer of strain R20291 contributes to selective spore adherence to components of the intestinal mucosa.http://journal.frontiersin.org/article/10.3389/fcimb.2016.00099/ful

Clostridium difficile spores and its relevance in the persistence and transmission of the infection

Indexación: Web of Science; Scielo.Clostridium difficile es un patógeno anaerobio, formador de esporas y el agente etiológico más importante de las diarreas asociadas a antimicrobianos, tanto nosocomiales como adquiridas en la comunidad. Las infecciones asociadas a C. difficile poseen una elevada tasa de morbilidad en países desarrollados y en vías de desarrollo. Los dos factores de virulencia principales son TcdA y TcdB, toxinas que causan la remodelación del citoesqueleto lo cual desencadena los síntomas clínicos asociados a esta enfermedad infecciosa. A pesar que las esporas de C. difficile son el principal vehículo de infección, persistencia en el hospedero y de transmisión, pocos estudios se han enfocado sobre este clave aspecto. Es altamente probable que la espora juegue roles esenciales en los episodios de recurrencia y de transmisión horizontal de la infección por este microorganismo. Estudios recientes han revelado características únicas de las esporas de C. difficile que las hacen capaces de ser altamente transmisibles y persistir dentro del hospedero. Más aún, algunas de estas propiedades están relacionadas con la resistencia de sus esporas a los desinfectantes más comúnmente usados en los recintos hospitalarios. La presente revisión resume los conocimientos más relevantes en la biología de las esporas de C. difficile, con un énfasis en aquellos aspectos con implicancias clínicas, incluido el control de infecciones en el ambiente hospitalario.C. difficile is an anaerobic spore former pathogen and the most important etiologic agent of nosocomial and community acquired antibiotics associated diarrheas. C. difficile infections (CDI) are responsible for an elevated rate of morbidity in developed and developing countries. Although the major virulence factors responsible for clinical symptoms of CDI are the two toxins TcdA and TcdB, C. difficile spores are the main vehicle of infection, persistence and transmission of CDI. Recent work has unrevealed unique properties of C. difficile spores that make them remarkable morphotypes of persistence and transmission in the host, including their resistance to antibiotics, the host immune response and disinfectants. The present review summarizes relevant aspects of C. difficile spore biology that have major implications from a clinical and medical perspective.http://ref.scielo.org/3xfbk

Analysis of the Germination of Individual Clostridium sporogenes Spores with and without Germinant Receptors and Cortex-Lytic Enzymes

The Gram-positive spore-forming anaerobe Clostridium sporogenes is a significant cause of food spoilage, and it is also used as a surrogate for C. botulinum spores for testing the efficacy of commercial sterilization. C. sporogenes spores have also been proposed as a vector to deliver drugs to tumor cells for cancer treatments. Such an application of C. sporogenes spores requires their germination and return to life. In this study, Raman spectroscopy and differential interference contrast (DIC) microscopy were used to analyze the germination kinetics of multiple individual C. sporogenes wild-type and germination mutant spores. Most individual C. sporogenes spores germinated with L-alanine began slow leakage of ∼5% of their large Ca-dipicolinic acid (CaDPA) depot at T1, all transitioned to rapid CaDPA release at Tlag1, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlys. T1, Tlag1, Trelease, and Tlys times for individual spores were heterogeneous, but ΔTrelease (Trelease – Tlag1) periods were relatively constant. However, variability in T1 (or Tlag1) times appeared to be the major reason for the heterogeneity between individual spores in their germination times. After Trelease, some spores also displayed another lag in rate of change in DIC image intensity before the start of a second obvious DIC image intensity decline of 25–30% at Tlag2 prior to Tlys. This has not been seen with spores of other species. Almost all C. sporogenes spores lacking the cortex-lytic enzyme (CLE) CwlJ spores exhibited a Tlag2 in L-alanine germination. Sublethal heat treatment potentiated C. sporogenes spore germination with L-alanine, primarily by shortening T1 times. Spores without the CLEs SleB or CwlJ exhibited greatly slowed germination with L-alanine, but spores lacking all germinant receptor proteins did not germinate with L-alanine. The absence of these various germination proteins also decreased but did not abolish germination with the non-GR-dependent germinants dodecylamine and CaDPA, but spores without CwlJ exhibited no germination with CaDPA. Finally, C. sporogenes spores displayed commitment in germination, but memory in GR-dependent germination was small, and less than the memory in Bacillus spore germination

Molecular mechanism of germination of Clostridium perfringens spores

Clostridium perfringens is the causative agent of a wide variety of diseases in animals and humans. C. perfringens can produce more than 15 toxins. However, individual strains produce a subset of these toxins. Although a small percentage of C. perfringens isolates (mostly belonging to type A) produce C. perfringens enterotoxin (CPE), these are very important human gastrointestinal (GI) pathogens, causing C. perfringens type A food poisoning (FP) and nonfood-borne GI diseases (NFBGID). Due to its anaerobic nature and the ability to form extremely resistant spores found ubiquitously in the environment, to cause the wide array of C. perfringens-associated diseases (CPAD), these C. perfringens spores must germinate, release the nascent cell, grow and produce their toxins. Therefore, germination of C. perfringens spores is the initial and perhaps most important step for the progression of diseases in animals and humans. Although extensive research has been conducted on the mechanism of spore germination of Bacillus species, very few studies of spore germination have been conducted in Clostridium species mainly due to the lack of molecular genetic tools. Genomic comparisons reveal significant differences in the backbone of the germination apparatus between Bacillus and Clostridium species. Consequently, a detail understanding of the molecular mechanism of germination of C. perfringens spores is essential for the development of novel preventive strategies for CPAD as well as diseases caused by other pathogenic Clostridium species. The first focus of this work was to identify and characterize the germinants and the receptors involved in C. perfringens spore germination. Result from these studies found differential germination requirements between spores of FP and NFBGID isolates in that: (i) while a mixture of L-asparagine and KCl was a good germinant for spores of FP and NFBGID isolates, KCl and, to a lesser extent, L-asparagine triggered spore germination in FP isolates only; and ii) L-alanine and L-valine induced significant germination of spores of NFBGID but not FP isolates. In contrast to B. subtilis, C. perfrinegns genomes sequenced to date possess no tricistronic gerA-like operon, but has a monocistronic gerAA that is far from a gerK locus. The gerK locus contains a bicistronic gerKA-gerKC operon and a monocistronic gerKB upstream and in the opposite orientation to gerKA-gerKC. Consequently, through the construction of mutations into strain SM101, a C. perfringens FP isolate, the role of gerAA, gerKA-gerKC and gerKB genes in C. perfringens spore germination were investigated. Results indicated that KCl, L-asparagine and Ca-DPA required GerKA and/or GerKC receptors, while GerAA and GerKB played an auxiliary role in germination. Lack of GerKA and/or GerKC, and GerKB significantly reduced spores colony forming efficiency, indicating a role in spore viability. The fact that C. perfringens spores lacking the main germinant receptor(s) proteins, GerKA and/or GerKC, are still able to germinate albeit poorly compared to wild-type, and that C. perfringens spores germinate with K+ ions alone, raises the hypothesis that GrmA-like antiporters might also play some role in C. perfringens spore germination. Two putative GrmA-like antiporters (i.e., GerO and GerQ) are encoded in the genome of all C. perfringens sequenced to date. This study shows that gerO and gerQ genes are expressed uniquely during sporulation and the mother cell compartment of the sporulating cell. Complementation studies of K+ uptake and Na+ sensitive E. coli mutants indicate that while GerO is capable of translocating K+ and Na+, GerQ is only capable of translocating, to a small extent, Na+ ions. Spores lacking GerO had defective germination in rich medium, KCl, L-asparagine, and Ca-DPA, but not with dodecylamine, defect that might be prior to DPA release during germination. In contrast, loss of GerQ had a much smaller effect on spore germination. Two adjacent Asp residues, important in ion transcloation of the E. coli Na+/H+ antiporter NhaA were also present in GerO, but not GerQ, and replacement of these residues for Asn reduced the protein’s ability to complement gerO spores. Although results from this study indicate that putative antiporters have some role on C. perfringens spore germination, it is unclear whether their role is direct or during spore formation. C. perfringens type A FP spores are capable of germinating with K+ ions, an intrinsic mineral of meats commonly associated with FP. Inorganic phosphate (Pi) is also intrinsically found in meat products. Consequently, we hypothesized that FP spores are capable of germinating in presence of Pi. Results from this study show that spores of the majority of FP, but not NFBGID isolates, are able to germinate in presence of Pi. Pi-induced germination of FP spores is primarily through the GerKA and/or GerKC protein, while GerAA and to a much lesser extent, GerKB, play auxiliary roles. The putative Na+/K+-H+ antiporter, GerO, is also required for normal Pi-induced germination. These results suggest that the differential germination phenotypes between spores of FP and NFGID isolates is tightly regulated by their adaptation to different environmental niches. A second focus of this work was to investigate the mechanism of signal transduction between the germinant receptors and the downstream effectors. In B. subtilis, the SpoVA proteins have been associated with Ca-DPA uptake and subsequent release during sporulation and germination, respectively. In addition, Ca-DPA acts as a signal molecule for cortex hydrolysis during B. subtilis spore germination, activating the cortex lytic enzyme (CLE) CwlJ. Results from this study show that in contrast to B. subtilis spoVA mutants, where spores lyse quickly during purification, C. perfringens spoVA spores were stable and germinated similarly as wild-type spores. These results suggest major differences in the regulation of the germination pathway between C. perfringens and B. subtilis, and suggest that activation of CLEs in C. perfringens might be through a different pathway than the Ca-DPA pathway of B. subtilis. A third focus of this work was to investigate the in vivo role of the CLE involved in peptidoglycan (PG) spore cortex hydrolysis during C. perfringens spore germination. Two C. perfringens CLEs (i.e., SleC and SleM) degrade PG spore cortex hydrolysis in vitro, however, due to lack of genetic tools, their in vivo role in spore germination remains unclear. Results from this study show that C. perfringens sleC spores released their DPA slower than wild-type and were not able to germinate with nutrients and non-nutrient germinants. In contrast, sleM spores germinated similar as wild type in presence of nutrient and non-nutrient germinants, indicating that while SleC is essential for cortex hydrolysis and viability of C. perfringens spores, SleM although can degrade cortex PG in vitro, is not essential. A fourth focus of this work was to investigate the in vivo role of the Csp proteases in the initiation of cortex hydrolysis. In vitro work has shown that Csp proteins process the inactive proSleC into the mature enzyme, SleC. However, the in vivo role of the Csp proteins has not been established. In this study, spores a C. perfringens cspB mutant exhibited significantly less viability than wild-type spores, and were unable to germinate with either rich medium or Ca-DPA. Germination of cspB spores was blocked prior to DPA release and cortex hydrolysis. Results from this study indicate that CspB is essential to generate active SleC and allow cortex hydrolysis early in C. perfringens spore germination. In contrast to B. subtilis, Ca-DPA did not activate the CLEs during spore germination present in cspB spores supporting previous results that Ca-DPA acts trough the GerKA and/or GerKC receptor. A final focus of this work was to develop a strategy to inactivate C. perfringens spores in meat products. C. perfringens spores posses high heat and pressure resistance, however, they loss their resistance properties during early stages of germination. In contrast to B. subtilis spores, germination of C. perfringens spores could not be triggered with low pressures. However, they germinated efficiently when heat activated in presence of L-asparagine and KCl at temperatures lethal for vegetative cells, and these germinated spores were efficiently inactivated by subsequent treatment with pressure assisted thermal processing (586 MPa at 73ºC for 10 min). This study shows the feasibility of a novel strategy to inactivate C. perfringens spores in meat products formulated with germinants. Collectively, the present study contributes to the understanding of the mechanism of spore germination in the pathogenic bacterium C. perfringens, and developed an alternative and novel strategy to inactivate C. perfringens spores in meat products

Performance of prognostic index in severe Clostridium difficile-associated infection. Retrospective analysis in a university hospital

Indexación: ScieloIntroducción: Por consenso, la infección asociada a Clostridium difficile (IACD) grave es aquella que resulta en hospitalización en unidad de cuidados intensivos, colectomía o muerte dentro de 30 días. Múltiples índices pronósticos (IP) intentan predecir estos eventos adversos. Objetivo: evaluar el rendimiento de cuatro IP en la predicción de IACD grave. Metodología: pacientes hospitalizados ≥ 18 años con IACD fueron evaluados retrospectivamente. Se excluyeron pacientes con infección recurrente o cáncer hematológico. Se evaluaron cuatro IP: UPMC versión 1, Calgary versión 1, Hines VA y Calgary versión 2. Resultados: Siete de 81 pacientes (8,1%) presentaron una IACD grave. El valor predictor positivo (VPP) y valor predictor negativo (VPN) de los IP varió entre 20-75% y 91,3-95,7%, respectivamente. Sólo el índice de Hines VA tuvo un índice Kappa satisfactorio (0,74;IC 95% 0,46-1) con un VPP de 75% y un VPN de 95,7%. Sin embargo, por las variables incluidas en este IP, sólo pudo ser calculado en 32,6% de los pacientes. Conclusión: El índice de Hines VA presenta el mejor valor predictor y concordancia para descartar una IACD grave. Como otros IP, tiene la limitación de incluir variables difícilmente evaluables en todos los pacientes y tiende a sobreestimar un curso desfavorable.Introduction: By consensus severe, Clostridium difficile-associated infection (CDAI) is one that results in hospitalization in ICU, colectomy or death within 30 days. Multiple prognostic indices (IP) attempt to predict these adverse events. Objective: To evaluate the performance of 4 PI in predicting severe CDI. Methods: Hospitalized patients ≥ 18 years old with ICD were retrospectively evaluated. Patients with recurrent infection or hematological cancer were excluded. Four PI were evaluated: UPMC version 1, Calgary version 1, Hines VA and Calgary version 2. Results: Seven of 81 patients (8.1%) met the definition of severe CDI. Positive predicted value (PPV) and negative predicted value (NPV) of PI ranged from 20-75% and 91.3-95.7%, respectively. Only Hines VA index had a satisfactory Kappa index (0.74; 95% CI 0.41-1) with a PPV of 75% and NPV of 95,7%. However, because of the variables included, this PI could be calculated only in 32.6% of patients. Conclusion: Hines VA index has the best predicted value and agreement to rule out a severe CDI. Like others PI it has the limitation of including difficult variables to assess in all patients and tends to overestimate an unfavorable course.http://www.scielo.cl/pdf/rci/v31n6/art03.pd

Further Characterization of Clostridium perfringens Small Acid Soluble Protein-4 (Ssp4) Properties and Expression

Background: Clostridium perfringens type A food poisoning (FP) is usually caused by C. perfringens type A strains that carry a chromosomal enterotoxin gene (cpe) and produce spores with exceptional resistance against heat and nitrites. Previous studies showed that the extreme resistance of spores made by most FP strains is mediated, in large part, by a variant of small acid soluble protein 4 (Ssp4) that has Asp at residue 36; in contrast, the sensitive spores made by other C. perfringens type A isolates contain an Ssp4 variant with Gly at residue 36. Methodology/Principal Findings: The current study has further characterized Ssp4 properties and expression. Spores made by cpe-positive type C and D strains were found to contain the Ssp4 variant with Gly at residue 36 and were shown to be heat- and nitrite-sensitive; this finding may help to explain why cpe-positive type C and D isolates rarely cause food poisoning. Saturation mutagenesis indicated that both amino acid size and charge at Ssp4 residue 36 are important for DNA binding and for spore resistance. C. perfringens Ssp2 was shown to bind preferentially to GC-rich DNA on gel-shift assays, while Ssp4 preferred binding to AT-rich DNA sequences. Maximal spore heat and nitrite resistance required production of all four C. perfringens Ssps, indicating that these Ssps act cooperatively to protect the spore's DNA, perhaps by binding to different chromosomal sequences. The Ssp4 variant with Asp at residue 36 was also shown to facilitate exceptional spore survival at freezer and refrigerator temperatures. Finally, Ssp4 expression was shown to be dependent upon Spo0A, a master regulator. Conclusions/Significance: Collectively, these results provide additional support for the importance of Ssps, particularly the Ssp4 variant with Asp at residue 36, for the extreme spore resistance phenotype that likely contributes to C. perfringens type A food poisoning transmission. © 2009 Li et al

Association of Fidaxomicin with C. difficile spores: Effects of Persistence on Subsequent Spore Recovery, Outgrowth and Toxin Production.

Background: We have previously shown that fidaxomicin instillation prevents spore recovery in an in-vitro gut model, whereas vancomycin does not. The reasons for this are unclear. Here, we have investigated persistence of fidaxomicin and vancomycin on C. difficile spores, and examined post-antibiotic exposure spore recovery, outgrowth and toxin production. Methods: Prevalent UK C. difficile ribotypes (n=10) were incubated with 200mg/L fidaxomicin, vancomycin or a non-antimicrobial containing control for 1 h in faecal filtrate or Phosphate Buffered Saline. Spores were washed three times with faecal filtrate or phosphate buffered saline, and residual spore-associated antimicrobial activity was determined by bioassay. For three ribotypes (027, 078, 015), antimicrobial-exposed, faecal filtrate-washed spores and controls were inoculated into broth. Viable vegetative and spore counts were enumerated on CCEYL agar. Percentage phase bright spores, phase dark spores and vegetative cells were enumerated by phase contrast microscopy at 0, 3, 6, 24 and 48 h post-inoculation. Toxin levels (24 and 48h) were determined by cell cytotoxicity assay. Results: Fidaxomicin, but not vancomycin persisted on spores of all ribotypes following washing in saline (mean=10.1mg/L; range= 4.0-14mg/L) and faecal filtrate (mean =17.4mg/L; 8.4-22.1mg/L). Outgrowth and proliferation rates of vancomycin-exposed spores were similar to controls, whereas fidaxomicin-exposed spores showed no vegetative cell growth after 24 and 48 h. At 48h, toxin levels averaged 3.7 and 3.3 relative units (RU) in control and vancomycin-exposed samples, respectively, but were undetectable in fidaxomicin-exposed samples. Conclusion: Fidaxomicin persists on C. difficile spores, whereas vancomycin does not. This persistence prevents subsequent growth and toxin production in vitro. This may have implications on spore viability, thereby impacting CDI recurrence and transmission rates

Host serum factor triggers germination of Clostridium perfringens spores lacking the cortex hydrolysis machinery

Clostridium perfringens type A is the causative agent of a variety of histotoxic and enteric diseases. The ability of C. perfringens spores to germinate in vivo might be due to the presence of nutrient germinants in the host tissue and blood. In the current study, we investigated the ability of spores of C. perfringens wild-type and mutation strains to germinate in blood. Results indicate that spores of all three surveyed C. perfringens wild-type isolates germinated better in blood than in brain heart infusion (BHI) broth. However, as expected, spores lacking germinant receptor (GR) protein GerAA or GerKB germinated like wild-type spores in BHI broth and blood. Strikingly, while spores lacking GR proteins GerKA and GerKC showed significantly decreased germination in BHI broth, these spores germinated well in blood, suggesting that blood factor(s) can trigger spore germination through a GR-independent pathway. Using C. perfringens spores lacking cortex lytic enzymes (ΔcspB or ΔsleC ΔsleM), we were able to identify a host serum germination factor with peptidoglycan hydrolysing activity that (i) restored the colony-forming efficiencies of ΔcspB and ΔsleC ΔsleM spores up to ~5–20% of that of total colony-forming spores; (ii) increased the number of c.f.u. of decoated ΔcspB and ΔsleC ΔsleM spores to ~99% of that of colony-forming spores; (iii) and finally lost enzymic activity after heat inactivation, consistent with serum germination factor being an enzyme. Further characterization demonstrated that serum germination factor is very likely lysozyme, which can form a stable high molecular mass complex of ~120 kDa in serum. In conclusion, the current study indicates that a host serum germination factor with peptidoglycan hydrolysing activity is capable of triggering germination of C. perfringens spores by directly degrading the spore peptidoglycan cortex. Collectively, this study contributes to our understanding of the mechanism of in vivo germination of spores of C. perfringens

The orphan germinant receptor protein GerXAO (but not GerX3b) is essential for L-alanine induced germination in Clostridium botulinum Group II

Clostridium botulinum is an anaerobic spore forming bacterium that produces the potent botulinum neurotoxin that causes a severe and fatal neuro-paralytic disease of humans and animals (botulism). C. botulinum Group II is a psychrotrophic saccharolytic bacterium that forms spores of moderate heat resistance and is a particular hazard in minimally heated chilled foods. Spore germination is a fundamental process that allows the spore to transition to a vegetative cell and typically involves a germinant receptor (GR) that responds to environmental signals. Analysis of C. botulinum Group II genomes shows they contain a single GR cluster (gerX3b), and an additional single gerA subunit (gerXAO). Spores of C. botulinum Group II strain Eklund 17B germinated in response to the addition of L-alanine, but did not germinate following the addition of exogenous Ca2+-DPA. Insertional inactivation experiments in this strain unexpectedly revealed that the orphan GR GerXAO is essential for L-alanine stimulated germination. GerX3bA and GerX3bC affected the germination rate but were unable to induce germination in the absence of GerXAO. No role could be identified for GerX3bB. This is the first study to identify the functional germination receptor of C. botulinum Group II