Bacterium Clostridium Difficile Research Articles

A comparative assessment of microbiocenosis of saliva and oropharynx in patients with migraine

To identify changes in the microbiome of saliva and to compare it with the microbiome of the oropharynx of patients with migraine. Sixty patients with migraine (21-56 years old), were examined using a headache diary, MIDAS and VAS. A microbiological examination of saliva and smear from the mucosa of the posterior wall of the oropharynx with evaluation by the method of mass spectrometry of microbial markers (MSMM) with the determination of 57 microorganisms was performed. All patients had comorbid chronic diseases of the gastrointestinal tract and upper respiratory tract (URT), according to anamnestic data and examination by specialists. A significant increase in the content of markers of resident (conditionally pathogenic) microorganisms characteristic of chronic diseases of URT (strepto- and staphylococci); markers of transient microorganisms characteristic of intestinal microflora (clostridia, gram-negative rods, anaerobes) that are normally absent; viral markers of cytomegaloviruses and herpes groups; a decrease in the content of fungi were identified in saliva. A comparative analysis of the microbiome of saliva and oropharynx showed: 1) a significant decrease in the concentration of coccal flora Enterococcus spp., Streptococcus mutans, Staphylococcus aureus, anaerobic bacteria Clostridium difficile and Clostridium perfringens in saliva; enterobacteria Helicobacter pylori; gram-negative rods Kingella spp., fungi and Epstein-Barr virus; 2) an increase in salivary concentrations of Staphylococcus epidermidis, anaerobic Clostridium ramosum and Fusobacterium spp./Haemophilus spp. and gram-negative bacilli Porphyromonas spp. A comparative assessment of the microbiota of a smear from the posterior wall of the oropharynx and saliva using MMSM showed the presence of dysbiosis both in the oropharynx and in the saliva of patients with migraine. However, there were fewer deviations from the norm in saliva, therefore, for diagnostic purposes, a smear from the posterior wall of the oropharynx is more significant as a biomarker for patients with migraine.

CHANGE CHARACTERISTICS IN SALIVA AND FECES MICROBIOTA OF A DESMINOPATHY T341P PATIENT

Background. A rare muscular disease, desminopathy, is caused by mutations in the DES gene. At present, information about changes in the microbiota of biological media present in such patients is very scarce.
 Purpose. The aim of the study is to study changes in the saliva and feces microbiota of patients with desminopathy T341P in a heterozygous state.
 Materials and methods. The retrospective investigation comprised observation of the observation of a proband with the family form of desminopathy T341P. 56 clinically significant microorganisms were numerically analyzed immediately in the obtained biological material by gas chromatography-mass spectrometry method.
 Results. The emergence of Epstein-Barr viruses, Cytomegalovirus, Herpes spp and gram-negative rods was noted in the proband’s biological media under investigation during the desminopathy progression. Excessive bacterial growth of fecal microbiota was observed along with a decrease in saliva microorganisms. There is an excess of the norm in the total number of microorganisms and an increase in their species diversity. Propionibacterium jensenii, Eubacterium spp and Eggerthella lenta predominated in the feces and Clostridium ramosum – in the saliva. An increase in fecal microbiota transient with Peptostreptococcus anaerobius 18623 dominance was observed along with the emergence and rapid 441-time growth of potentially dangerous bacterium Clostridium difficile. The total level of endotoxin in the proband’s saliva and fecal microbiota increases to exceed the norm in 13.7 and 81.8 times, respectively. At the same time, a low level of plasmalogen was noted.
 Conclusion. The investigation results can be useful for developing complex intervention tactics.

New Generation Dielectrophoretic-Based Microfluidic Device for Multi-Type Cell Separation

This study introduces a new generation of dielectrophoretic-based microfluidic device for the precise separation of multiple particle/cell types. The device features two sets of 3D electrodes, namely cylindrical and sidewall electrodes. The main channel of the device terminates with three outlets: one in the middle for particles that sense negative dielectrophoresis force and two others at the right and left sides for particles that sense positive dielectrophoresis force. To evaluate the device performance, we used red blood cells (RBCs), T-cells, U937-MC cells, and Clostridium difficile bacteria as our test subjects. Our results demonstrate that the proposed microfluidic device could accurately separate bioparticles in two steps, with sidewall electrodes of 200 µm proving optimal for efficient separation. Applying different voltages for each separation step, we found that the device performed most effectively at 6 Vp-p applied to the 3D electrodes, and at 20 Vp-p and 11 Vp-p applied to the sidewall electrodes for separating RBCs from bacteria and T-cells from U937-MC cells, respectively. Notably, the device's maximum electric fields remained below the cell electroporation threshold, and we achieved a separation efficiency of 95.5% for multi-type particle separation. Our findings proved the device's capacity for separating multiple particle types with high accuracy, without limitation for particle variety.

Zoonotic Risks of Sleeping with Pets.

Background: Pets are increasingly becoming part of the family and interactions between pets and their owners is changing. This results in extended and more intimate contact between owners and their pets, which give rise to zoonotic risks. Objective: To establish the presence of potential zoonotic pathogens in pets that sleep with their owner. Methods: As a pilot study, a group of 28 healthy dogs and 22 healthy cats were monitored for the presence of the zoonotic parasites Cheyletiella, Ctenocephalides spp. and Toxocara spp., the dermatophyte Microsporum canis, and the bacteria Clostridium difficile, Salmonella spp., Campylobacter jejuni and Enterobacteriaceae. This was investigated by taking samples from the fur, the footpads and the animal bed. The owners filled in a questionnaire. Results: In total, 29 of the 50 pets (58%) slept on the bed, of which 15 pets (30%) slept in the bed (under the blankets). A total of 19/22 dogs (86%) and 7/22 cats (32%) tested positive for Enterobacteriaceae on the fur or footpads. Fleas were found in 5/22 of the cats’ (23%) and 2/28 of the dogs’ (7%) favourite sleeping spots. High levels of aerobic colonies were found, up to 216 colony forming units/cm2. Other pathogens were not found in this study. Conclusions: The results of this preliminary study confirm literature reports that pets may constitute a potential risk in the transmission of zoonotic pathogens to their owner, especially during direct contact when sleeping in the same bed. Owners should therefore be informed about these risks and educated to interact with their pets in a more responsible way.

Acute Responses to Infectious and Inflammatory Disorders of the Gastrointestinal System

Infectious and inflammatory disorders of the gastrointestinal system are life-threatening and occur frequently in hospitalized adults. Inflammatory and inflammation-related diseases of the gastrointestinal tract seen in the acutely and critically ill have numerous causes. In acute pancreatitis and toxic infections caused by the bacterium Clostridium difficile, where severe infections can develop, inflammation plays a causative and crucial role. Severe acute pancreatitis puts a patient at risk for infected necrosis, which can result in septicemia and shock. Similarly, patients treated with antibiotics are at risk for C difficile colitis, which can progress to toxic megacolon. These conditions require volume resuscitation and interventions supported by current evidence. Percutaneous or surgical interventions are often undertaken at a critical point in these illnesses. Patients who require surgery for these diagnoses present challenges for the interprofessional team. Inflammatory and infectious disorders often can lead to complications of systemic inflammatory response syndrome, sepsis, and multiorgan failure. New strategies are on the horizon to prevent the onset of and improve care for patients with severe acute pancreatitis, fulminant C difficile infection, and megacolon.

Clostridium difficile toxin B induces morphological changes consistent with autophagy in the human adenocarcinoma cell line (HT-29)

Purpose: Toxin B (TcdB) is a product of the bacteria Clostridium difficile, and can trigger apoptosis or necrosis. In the present manuscript, we proposed that TcdBVPI and TcdB8864 would cause morphological changes in cultured cells that are characteristic of autophagy, a constitutive cellular event closely linked with apoptosis or necrosis.
 Methods: We examined an in vitro model of cultured HT-29 cells to determine the occurrence of autophagy. The cells were incubated separately with TcdBVPI and TcdB8864 for 2 to 24 hours, and then examined using transmission electron microscopy.
 Results: In HT-29 cells, ultrastructural changes were observed following 8 hours of treatment with TcdBVPI and TcdB8864. Upon exposure to both toxins, complex changes occurred that affected the cellular framework, including the dilated regions of the granular endoplasmic reticulum, the Golgi apparatus, as well as electron-lucent and electron-dense autophagosome-like structures. Additionally, lipid droplets were present in the cytoplasm of HT-29 cells.
 Conclusion: Our findings indicate that TcdBVPI and TcdB8864 indeed induce ultrastructural changes in HT-29 cells. The presence of these autophagic structures in the cytoplasm of the HT-29 cells suggests that autophagy may be induced during treatment by both toxins. Therefore, this effect could represent an important protective mechanism for cell survival.

The knowledge of nurses about prevention of infections caused by the bacteria Clostridium difficile

<p>Introduction. Clostridium difficile is the leading cause of nosocomial diarrhea, associated with the use of antibiotics. The most common ways of transmitting the infection in hospitals are contaminated surfaces of the premises and the hands of medical staff. Methods. The study involved 68 nurses/technicians employed at the University Hospital Foca in the departments of surgery and internal medicine. As a research instrument, we used a specially designed questionnaire, created by the authors for the purpose of this research. Results. The research showed that 61.8% of respondents knew that hand washing with warm water and soap was considered the most effective prevention of the spread of infections, and 55.88% meant that they used chlorine-based preparations and hydrogen peroxide as the only effective disinfectant. Nurses with a work experience of less than 5 years showed better knowledge than other groups. Conclusion. The knowledge of nurses about the prevention of C. difficile infection is not at a satisfactory level, which indicates the growing need for education of nurses.</p>

A Pilot Study on the Contamination of Assistance Dogs' Paws and Their Users' Shoe Soles in Relation to Admittance to Hospitals and (In)Visible Disability.

(1) Background: People with disabilities may benefit from an assistance dog (AD). Despite regulations that prohibit the denial of ADs to public places, this still occurs on a regular basis. The main argument for denial of access is that dogs compromise hygiene with their presence, which could cause a health hazard. Meanwhile, people are allowed to walk into and out of public places freely. (2) Objective: As a pilot study, to investigate the number of Enterobacteriaceae and the presence of Clostridium difficile bacteria on the paws of ADs and pet dogs (PDs) as well as the shoe soles of their users and owners. With the results, an assessment can be made as to whether measures are required to reduce environmental contamination (e.g., in hospitals). (3) Methods: In total, 25 ADs, 25 PDs, and their 50 users/owners participated in the study. Each participant walked their dog for 15–30 min prior to the sampling of the front paws. Each PD owner or AD user filled out a general questionnaire about the care of their dogs, and AD users were asked to fill out an additional questionnaire on their experiences regarding the admittance of their ADs to public places (in particular, hospitals). Dutch hospitals were questioned on their protocols regarding the admittance of ADs and their visitor numbers, including the percentage of AD users, to put these numbers into perspective. (4) Results: Dog paws were more often negative for Enterobacteriaceae compared to shoe soles (72% and 42%, respectively) and also had significantly lower bacterial counts (mean of 3.54log10 and 5.03log10 colony-forming units (CFUs), respectively; p < 0.05). This was most distinct in the comparison between PDs and their owners (3.75log10 and 5.25log10 CFUs; p < 0.05); the numbers were similar between ADs and their users (3.09log10 and 4.58log10 CFUs; p = 0.2). C. difficile was found on one (4%) AD user’s shoe soles. Moreover, 81% of AD users had been denied access with their current AD once or several times, the main reason being hygiene. The results of the visibly and invisibly disabled were significantly different. The number of AD users as opposed to the total number of hospital visitors was 0.03% in one hospital and is estimated to be 0.02% in the Netherlands. (5) Conclusions: The general hygiene of dogs’ paws is far better than that of shoe soles, mostly demonstrated by the better general hygiene of PD paws compared with their owners’ shoe soles; ADs and their users had comparable levels of general hygiene. In addition, the number of AD users amongst the total number of hospital visitors in the Netherlands is very limited. Thus, hygiene measures to reduce any contamination due to dog paws do not seem necessary.

Novel Dietary Proteins Selectively Affect Intestinal Health In Vitro after Clostridium difficile-Secreted Toxin A Exposure.

Bacterial gastroenteritis forms a burden on a global scale, both socially and economically. The Gram-positive bacterium Clostridium difficile is an inducer of gastrointestinal bacterial infections, often triggered following disruption of the microbiota by broad-spectrum antibiotics to treat other conditions. The clinical manifestatiaons, e.g., diarrhea, are driven by its toxins secretion, toxin A (TcdA) and toxin B (TcdB). Current therapies are focused on discontinuing patient medication, including antibiotics. However, relapse rates upon therapy are high (20–25%). Here, eighteen dietary proteins were evaluated for their capacity to restore gut health upon C. difficile-derived TcdA exposure. We used bioengineered intestinal tubules to assess proteins for their beneficial effects by examining the epithelial barrier, cell viability, brush-border enzyme activity, IL-6 secretion, IL-8 secretion and nitric oxide (NO) levels upon TcdA challenge. TcdA effectively disrupted the epithelial barrier, increased mitochondrial activity, but did not affect alkaline phosphatase activity, IL-6, IL-8 and NO levels. Intervention with dietary proteins did not show a protective effect on epithelial barrier integrity or mitochondrial activity. However, bovine plasma and potato protein increased alkaline phosphatase activity, egg-white protein increased IL-6 and IL-8 release and wheat, lesser mealworm and yeast protein increased NO levels after TcdA exposure. Hence, dietary proteins can influence parameters involved in intestinal physiology and immune activation suggesting that supplementation with specific dietary proteins may be of benefit during C. difficile infections.

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

Clostridial infections in the world and in Slovakia in the patient safety context

The analysis of the scientific literature found that Infection with Gram-positive sporulating bacteria Clostridium difficile(CDI) is currently a serious health problem worldwide due to its increasing incidence and mortality and it is the most commoncause of diarrhea in hospitalized patients. In this work we analyze preventive measures, which include contact isolation, handhygiene and reorganization/coordination of nursing care and of the ward. Preventive measures are time-consuming andsignificantly increase the workload, which can affect the perception of the patient's safety level by nurses and the ability toconcentrate their efforts to adhere to good practices for preventing the transmission of the infection. We also emphasize thecrucial role of nurses in increasing patient safety due to their constant presence at patient's bedside. Knowing the patient'severyday clinical condition, nurses can effectively engage and help reduce Clostridium difficile (CDI) infection in the hospital.

Development of an ADP-ribosylation assay for residual toxicity in C. difficile binary toxin CDTa using automated capillary western blot

CDTa, an actin ADP-ribosylation transferase, is a binary toxin produced by the bacterium Clostridium difficile which is commonly associated with the hypervirulent strain present in Clostridium difficile infections. The mutated form of CDTa, 4mCDTa, is one of the components in the tetravalent Clostridium difficile vaccine in which the residual toxicity of the ADP-ribosylation activity needs to be monitored for safety reasons. There are several ADP- ribosylation activity methods employing techniques such as ELISA, manual Western blot, or SDS PAGE, but all these methods are usually time consuming and labor intensive. Here we describe the development of new quantitative capillary based western for monitoring the presence of ADP-ribosylation activity in CDTa and 4mCDTa using novel, automated Simple Western™ technology. Furthermore, we have measured for the first time the enzyme’s kinetic parameters, KM (NAD) and kcat for native CDTa using this new quantitative capillary western technology.

Generation and robustness of Boolean networks to model Clostridium difficile infection

One of the more common healthcare associated infection is Chronic diarrhea. This disease is caused by the bacterium Clostridium difficile which alters the normal composition of the human gut flora. The most successful therapy against this infection is the fecal microbial transplant (FMT). They displace C. difficile and contribute to gut microbiome resilience, stability and prevent further episodes of diarrhea. The microorganisms in the FMT their interactions and inner dynamics reshape the gut microbiome to a healthy state. Even though microbial interactions play a key role in the development of the disease, currently, little is known about their dynamics and properties. In this context, a Boolean network model for C. difficile infection (CDI) describing one set of possible interactions was recently presented. To further explore the space of possible microbial interactions, we propose the construction of a neutral space conformed by a set of models that differ in their interactions, but share the final community states of the gut microbiome under antibiotic perturbation and CDI. To begin with the analysis, we use the previously described Boolean network model and we demonstrate that this model is in fact a threshold Boolean network (TBN). Once the TBN model is set, we generate and use an evolutionary algorithm to explore to identify alternative TBNs. We organize the resulting TBNs into clusters that share similar dynamic behaviors. For each cluster, the associated neutral graph is constructed and the most relevant interactions are identified. Finally, we discuss how these interactions can either affect or prevent CDI.

Obolevanje i umiranje od infekcija uzrokovanih bakterijom clostridium difficile na teritoriji Beograda

Introduction/Aim: The aim of the study was to show the characteristics of infections caused by the bacteria Clostridium difficile (C. difficile) originating in the territory of Belgrade in the period between 2013 and 2017. Methods: Data for this report was collected from the reports of infectious diseases and hospital infections, as well as surveys of the patients, medical documentation, and from annual reports on the prevention, treatment and elimination of infectious diseases. Results of epidemiological and laboratory testing were also included. Incidence rates, mortality rates and proportions were used in analysis. Results: In the observed 5 year period the incidence rate was between 34.0/100,000 (2013) and 75.1/100,000 (2016). The mortality rate ranged between 1.1/100,000 (2013) and 2.1/100,000 (2016). The highest average incidence rates were observed in the elderly. Most cases of mortality belonged to the age category 70 and more years of age (11.3/100,000) and were the smallest in the age group 30-39 years (0.1/100,000). Infections with C. difficile were most common in the month of March (608 affected, with an incidence rate of 11.3/100,000). In the observed period there were 12 registered epidemics with 51 cases. Conclusion: The effective prevention and control of the C. difficile infection is predicated upon the continuation of active implementation of disease management in accordance with the incidence rate.

Infections Associated With Hospitalization Over One Year Period

Abstract The aim of this study was to identify the bacterial species circulating in various wards of the Emergency Clinical Hospital Constanta, involved in infections associated with hospitalization. Clinical specimens were obtained during 2016 year period. Identification of species and sensitivity tests were performed on VITEK 2 System. There were isolated 163 bacterial strains suspected to be responsible for infections associated with hospitalization. Surgery wards had the most important number of cases, 49% of all. The second most affected ward was neonatology (11%), as many as gynecology (5%) and pediatrics (6 %) together. According to the site of the infection, digestive are majoritary (42.94%), followed by surgical wounds (16.56%) and respiratory infections (15.33%). Septicemia cases (11.04%) are important due to the severity. Distribution on months varies between a minimum number of cases in January (5) and a maximum of cases in April (19).Gram-negative bacilli were the most frequently involved (49%). The most frequent species was Acinetobacter baumanii (28 cases), followed by Pseudomonas aeruginosa (15) and E.coli (7). Anaerobic bacteria (Clostridium difficile) are the second most important group (32% of the cases), followed by Gram positive cocci (15%) mostly staphylococci. Multidrug resistance was diagnosed in most Acinetobacter baumanii strains. A special attention must be given to associations of bacteria, that even though are in small number (2% of the cases), are more difficult to be treated.

Structure-Activity Relationship of Flavin Analogues That Target the Flavin Mononucleotide Riboswitch.

The flavin mononucleotide (FMN) riboswitch is an emerging target for the development of novel RNA-targeting antibiotics. We previously discovered an FMN derivative, 5FDQD, that protects mice against diarrhea-causing Clostridium difficile bacteria. Here, we present the structure-based drug design strategy that led to the discovery of this fluoro-phenyl derivative with antibacterial properties. This approach involved the following stages: (1) structural analysis of all available free and bound FMN riboswitch structures; (2) design, synthesis, and purification of derivatives; (3) in vitro testing for productive binding using two chemical probing methods; (4) in vitro transcription termination assays; and (5) resolution of the crystal structures of the FMN riboswitch in complex with the most mature candidates. In the process, we delineated principles for productive binding to this riboswitch, thereby demonstrating the effectiveness of a coordinated structure-guided approach to designing drugs against RNA.

Facile Solid-Phase Synthesis and Assessment of Nucleoside Analogs as Inhibitors of Bacterial UDP-Sugar Processing Enzymes.

The privileged uptake of nucleosides into cells has generated interest in the development of nucleoside-analog libraries for mining new inhibitors. Of particular interest are applications in the discovery of substrate mimetic inhibitors for the growing number of identified glycan-processing enzymes in bacterial pathogens. However, the high polarity and the need for appropriate protecting group strategies for nucleosides challenges the development of synthetic approaches. Here, we report an accessible, user-friendly synthesis that branches from a common solid phase-immobilized uridinyl-amine intermediate, which can be used as a starting point for diversity-oriented synthesis. We demonstrate the generation of five series of uridinyl nucleoside analogs for investigating inhibitor structure-activity relationships. This library was screened for inhibition of representative enzymes from three functional families including a phosphoglycosyl transferase, a UDP-aminosugar acetyltransferase, and a glycosyltransferase. These candidates were taken from the Gram-negative bacteria Campylobacter concisus and Campylobacter jejuni and the Gram-positive bacterium Clostridium difficile, respectively. Inhibition studies show that specific compound series preferentially inhibit selected enzymes, with IC50 values ranging from 35 ± 7 μM to 174 ± 21 μM. Insights from the screen provide a strong foundation for further structural elaboration, to improve potency, which will be enabled by the same synthetic strategy. The solid-phase strategy was also used to synthesize pseudouridine analogs of lead compounds. Finally, the compounds were found to be nontoxic to mammalian cells, further supporting the opportunities for future development.

New Insights Into Functions and Possible Applications of Clostridium difficile CRISPR-Cas System

Over the last decades the enteric bacterium Clostridium difficile (novel name Clostridioides difficile) – has emerged as an important human nosocomial pathogen. It is a leading cause of hospital-acquired diarrhea and represents a major challenge for healthcare providers. Many aspects of C. difficile pathogenesis and its evolution remain poorly understood. Efficient defense systems against phages and other genetic elements could have contributed to the success of this enteropathogen in the phage-rich gut communities. Recent studies demonstrated the presence of an active CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) subtype I-B system in C. difficile. In this mini-review, we will discuss the recent advances in characterization of original features of the C. difficile CRISPR-Cas system in laboratory and clinical strains, as well as interesting perspectives for our understanding of this defense system function and regulation in this important enteropathogen. This knowledge will pave the way for the development of promising biotechnological and therapeutic tools in the future. Possible applications for the C. difficile strain monitoring and genotyping, as well as for CRISPR-based genome editing and antimicrobials are also discussed.

Clostridium difficile Lipoprotein GerS Is Required for Cortex Modification and Thus Spore Germination.

Clostridium difficile, also known as Clostridioides difficile, is a Gram-positive, spore-forming bacterium that is a leading cause of antibiotic-associated diarrhea. C.difficile infections begin when its metabolically dormant spores germinate to form toxin-producing vegetative cells. Successful spore germination depends on the degradation of the cortex, a thick layer of modified peptidoglycan that maintains dormancy. Cortex degradation is mediated by the SleC cortex lytic enzyme, which is thought to recognize the cortex-specific modification muramic-δ-lactam. C.difficile cortex degradation also depends on the Peptostreptococcaceae-specific lipoprotein GerS for unknown reasons. In this study, we tested whether GerS regulates production of muramic-δ-lactam and thus controls the ability of SleC to recognize its cortex substrate. By comparing the muropeptide profiles of ΔgerS spores to those of spores lacking either CwlD or PdaA, both of which mediate cortex modification in Bacillus subtilis, we determined that C.difficile GerS, CwlD, and PdaA are all required to generate muramic-δ-lactam. Both GerS and CwlD were needed to cleave the peptide side chains from N-acetylmuramic acid, suggesting that these two factors act in concert. Consistent with this hypothesis, biochemical analyses revealed that GerS and CwlD directly interact and that CwlD modulates GerS incorporation into mature spores. Since ΔgerS, ΔcwlD, and ΔpdaA spores exhibited equivalent germination defects, our results indicate that C.difficile spore germination depends on cortex-specific modifications, reveal GerS as a novel regulator of these processes, and highlight additional differences in the regulation of spore germination in C.difficile relative to B.subtilis and other spore-forming organisms.IMPORTANCE The Gram-positive, spore-forming bacterium Clostridium difficile is a leading cause of antibiotic-associated diarrhea. Because C.difficile is an obligate anaerobe, its aerotolerant spores are essential for transmitting disease, and their germination into toxin-producing cells is necessary for causing disease. Spore germination requires the removal of the cortex, a thick layer of modified peptidoglycan that maintains spore dormancy. Cortex degradation is mediated by the SleC hydrolase, which is thought to recognize cortex-specific modifications. Cortex degradation also requires the GerS lipoprotein for unknown reasons. In our study, we tested whether GerS is required to generate cortex-specific modifications by comparing the cortex composition of ΔgerS spores to the cortex composition of spores lacking two putative cortex-modifying enzymes, CwlD and PdaA. These analyses revealed that GerS, CwlD, and PdaA are all required to generate cortex-specific modifications. Since loss of these modifications in ΔgerS, ΔcwlD, and ΔpdaA mutants resulted in spore germination and heat resistance defects, the SleC cortex lytic enzyme depends on cortex-specific modifications to efficiently degrade this protective layer. Our results further indicate that GerS and CwlD are mutually required for removing peptide chains from spore peptidoglycan and revealed a novel interaction between these proteins. Thus, our findings provide new mechanistic insight into C.difficile spore germination.

Altered intestinal microbiota composition, antibiotic therapy and intestinal inflammation in children and adolescents with cystic fibrosis.

The aim of the present study was to evaluate the effect of cystic fibrosis and antibiotic therapy on intestinal microbiota composition and intestinal inflammation in children and adolescents. A cross-sectional controlled study was conducted with 36 children and adolescents: 19 in the cystic fibrosis group (CFG) and 17 in the control group (CG) matched for age and sex. The CFG was subdivided based on the use of antibiotic therapy (CFAB group) and non-use of antibiotic therapy (CFnAB group). The following data were evaluated: colonization, antibiotic therapy, mutation, breastfeeding, use of infant formula, type of delivery, introduction of solid foods, body mass index, fecal calprotectin and intestinal microbiota composition (fluorescence in situ hybridization). Intestinal inflammation evaluated by fecal calprotectin was significantly higher in the CFG (median: 40.80 µg/g, IQR: 19.80–87.10, p = 0.040) and CFAB group (median: 62.95 µg/g, IQR: 21.80–136.62, p = 0.045) compared to the CG (median: 20.15 µg/g, IQR: 16.20–31.00), and the Bacteroides, Firmicutes, Eubacterium rectale and Faecalibacterium prausnitzii were significantly decreased (p < 0.05) in the CFG compared to the CG, whereas the bacteria Clostridium difficile, Escherichia coli and Pseudomonas aeruginosa were significantly increased in the CFG (p < 0.05). The main differences were found between the CG and CFAB group for Eubacterium rectale (p = 0.006), Bifidobacterium (p = 0.017), Escherichia coli (p = 0.030), Firmicutes (p = 0.002), Pseudomonas aeruginosa (p < 0.001) and Clostridium difficile (p = 0.006). The results of this study confirm intestinal inflammation in patients with CF, which may be related to changes in the composition of the intestinal microbiota.