Anaerobic Spore-forming Bacteria Research Articles

Unraveling Cecal Alterations in Clostridioides difficile Colonized Mice through Comprehensive Metabolic Profiling.

The disruption of gut microbiota caused by antibiotics favors the intestinal colonization of Clostridioides difficile - a Gram-positive, spore-forming anaerobic bacterium that causes potentially fatal gastrointestinal infections. In an endeavor to elucidate the complexities of the gut-brain axis in the context of Clostridium difficile infection (CDI), a murine model has been used to investigate the potential effects of antibiotic administration and subsequent colonization by C. difficile, as well as the impact of three different 10-day treatments (metronidazole, probiotics, and fecal microbiota transplantation), on the cecal metabolome for the first time. This follows our previous research which highlighted the metabolic effect of CDI and these treatments in the brain and employs the same four different metabolomics-based methods (targeted GC-MS/MS, targeted HILIC-MS/MS, untargeted RP-LC-HRMS/MS and untargeted GC-MS). A total of 286 unique metabolites have been identified in the mouse cecal profiles and statistical analysis revealed that CDI, as well as the subsequent treatments, significantly alters cecal metabolites and lipids implicated in various biochemical pathways centered around amino acid metabolism, glycerophospholipid metabolism, and central carbon metabolism. To our knowledge, this study represents the first exploration of the effects of C. difficile-induced colitis and potential treatments on the cecal tissue metabolome.

Microbiological Quality Assessment of Some Commercially Available Breads.

Bread is a staple, energy-rich food for people of all ages, so quality is important to consumers. In our region, most of the commercially available bread, whether packaged or unpackaged, is produced by local bakeries, so monitoring microbial levels and the types of microbes present on bread can help to draw attention to protect the final product. It can also help to ensure the food safety, quality, and shelf life of bread. The freshly baked product is microbiologically sterile. Post-process contamination affects the microbial load of bread. In this study, the microbial load of 30 different commercial bread crumbs and crusts was determined. The different types of bread with different compositions were analyzed for total viable bacteria, Escherichia coli, Staphylococcus aureus, aerobic and anaerobic spore-forming bacteria, and culturable microscopic fungi. The K-means clustering algorithm was used to cluster the different types of bread based on the number of aerobic mesophilic bacteria. Significant differences (p < 0.05) were found in the total viable bacterial count for bread crusts and crumbs. The bacterial count of bread varied between 10.00 ± 0.00-395.00 ± 52.4 CFU/g for bread crusts and 10.00 ± 0.0-310.67 ± 94 CFU/g for bread crumbs. The results of 16S rDNA sequence analysis showed that the most frequently occurring bacterial species belonged to the genus Bacillus, but species of the genus Staphylococcus were also present. Chryseobacterium spp. predominated on multigrain bread, Marinilactobacillus spp. on rustic potato bread, and Staphylococcus warneri on sliced brown potato bread. The results contribute to a better understanding of the microbial dynamics in locally produced breads from the Eastern Carpathians of Transylvania, with the aim of improving food safety, quality control, and consumer protection.

Genetic diversity, biofilm formation, and Vancomycin resistance of clinical Clostridium innocuum isolates

BackgroundClostridium innocuum, previously considered a commensal microbe, is a spore-forming anaerobic bacterium. C. innocuum displays inherent resistance to vancomycin and is associated with extra-intestinal infections, antibiotic-associated diarrhea, and inflammatory bowel disease. This study seeks to establish a multilocus sequence typing (MLST) scheme to explore the correlation between C. innocuum genotyping and its potential pathogenic phenotypes.MethodsFifty-two C. innocuum isolates from Linkou Chang Gung Memorial Hospital (CGMH) in Taiwan and 60 sequence-available C. innocuum isolates from the National Center for Biotechnolgy Information Genome Database were included. The concentrated sequence of housekeeping genes in C. innocuum was determined by amplicon sequencing and used for MLST and phylogenetic analyses. The biofilm production activity of the C. innocuum isolates was determined by crystal violet staining.ResultsOf the 112 C. innocuum isolates, 58 sequence types were identified. Maximum likelihood estimation categorized 52 CGMH isolates into two phylogenetic clades. These isolates were found to be biofilm producers, with isolates in clade I exhibiting significantly higher biofilm production than isolates in clade II. The sub-inhibitory concentration of vancomycin seemed to minimally influence biofilm production by C. innocuum isolates. Nevertheless, C. innocuum embedded in the biofilm structure demonstrated resistance to vancomycin treatments at a concentration greater than 256 µg/mL.ConclusionsThis study suggests that a specific genetic clade of C. innocuum produces a substantial amount of biofilm. Furthermore, this phenotype assists C. innocuum in resisting high concentrations of vancomycin, which may potentially play undefined roles in C. innocuum pathogenesis.

Clostridial Myonecrosis: A Comprehensive Review of Toxin Pathophysiology and Management Strategies.

Clostridial myonecrosis, commonly known as gas gangrene (GG), is a rapidly progressing and potentially fatal bacterial infection that primarily affects muscle and soft tissue. In the United States, the incidence of GG is roughly 1000 cases per year, while, in developing countries, the incidence is higher. This condition is most often caused by Clostridium perfringens, a Gram-positive, spore-forming anaerobic bacterium widely distributed in the environment, although other Clostridium species have also been reported to cause GG. The CP genome contains over 200 transport-related genes, including ABC transporters, which facilitate the uptake of sugars, amino acids, nucleotides, and ions from the host environment. There are two main subtypes of GG: traumatic GG, resulting from injuries that introduce Clostridium spores into deep tissue, where anaerobic conditions allow for bacterial growth and toxin production, and spontaneous GG, which is rarer and often occurs in immunocompromised patients. Clostridium species produce various toxins (e.g., alpha, theta, beta) that induce specific downstream signaling changes in cellular pathways, causing apoptosis or severe, fatal immunological conditions. For example, the Clostridium perfringens alpha toxin (CPA) targets the host cell's plasma membrane, hydrolyzing sphingomyelin and phosphatidylcholine, which triggers necrosis and apoptosis. The clinical manifestations of clostridial myonecrosis vary. Some patients experience the sudden onset of severe pain, swelling, and muscle tenderness, with the infection progressing rapidly to widespread tissue necrosis, systemic toxicity, and, if untreated, death. Other patients present with discharge, pain, and features of cellulitis. The diagnosis of GG primarily involves clinical evaluation, imaging studies such as X-rays, computer tomography (CT) scans, and culture. The treatment of GG involves surgical exploration, broad-spectrum antibiotics, antitoxin, and hyperbaric oxygen therapy, which is considered an adjunctive treatment to inhibit anaerobic bacterial growth and enhance the antibiotic efficacy. Early recognition and prompt, comprehensive treatment are critical to improving the outcomes for patients affected by this severe and life-threatening condition.

SpoIVA is an essential morphogenetic protein for the formation of heat- and lysozyme-resistant spores in Clostridium sporogenes NBRC 14293.

Clostridium sporogenes is an anaerobic spore-forming bacterium genetically related to Clostridium botulinum but lacks toxin genes. The sporulation mechanism and spore structures of anaerobic bacteria, including C. sporogenes, have not been comprehensively analyzed. Based on 16S rRNA gene analysis, it has been determined that C. sporogenes NBRC 14293 belongs to C. botulinum Group I. Moreover, SpoIVA is highly conserved in Bacillus and Clostridium species. Therefore, the aim of the present study is to investigate the mechanism of spore formation in C. sporogenes by performing a functional analysis of spoIVA encoding SpoIVA, a protein involved in the early development of the spore coat and cortex in Bacillus subtilis. Inactivation of spoIVA in C. sporogenes resulted in the loss of resistance of sporulating cells to lysozyme and heat treatments. Phase-contrast microscopy indicated that the inactivation of spoIVA caused the development of abnormal forespores and production of only a few immature spores. In the spoIVA mutant, abnormal swirl structures were detected in the mother cell using both phase-contrast and transmission electron microscopy. These swirls were stained with auramine O, pararosaniline hydrochloride, and 2-(4-aminophenyl)benzothiazole to examine the surface of mature spores of the wild-type strain. We found that the spore coat and exosporium proteins were misassembled and that they accumulated in the mother cells of the mutant. The results of this study indicate that SpoIVA is a spore morphogenetic protein, providing novel insights into spore morphogenesis in C. sporogenes.

Comparative Genomics of Clostridioides difficile.

Clostridioides difficile, a Gram-positive spore-forming anaerobic bacterium, has rapidly emerged as the leading cause of nosocomial diarrhoea in hospitals. The availability of large numbers of genome sequences, mainly due to the use of next-generation sequencing methods, has undoubtedly shown their immense advantages in the determination of C. difficile population structure. The implementation of fine-scale comparative genomic approaches has paved the way for global transmission and recurrence studies, as well as more targeted studies, such as the PaLoc or CRISPR/Cas systems. In this chapter, we provide an overview of recent and significant findings on C. difficile using comparative genomic studies with implications for epidemiology, infection control and understanding of the evolution of C. difficile.

Identification of an anti-CRISPR protein that inhibits the CRISPR-Cas type I-B system in Clostridioides difficile.

Clostridioides difficile is the widespread anaerobic spore-forming bacterium that is a major cause of potentially lethal nosocomial infections associated with antibiotic therapy worldwide. Due to the increase in severe forms associated with a strong inflammatory response and higher recurrence rates, a current imperative is to develop synergistic and alternative treatments for C. difficile infections. In particular, phage therapy is regarded as a potential substitute for existing antimicrobial treatments. However, it faces challenges because C. difficile has highly active CRISPR-Cas immunity, which may be a specific adaptation to phage-rich and highly crowded gut environment. To overcome this defense, C. difficile phages must employ anti-CRISPR mechanisms. Here, we present the first anti-CRISPR protein that inhibits the CRISPR-Cas defense system in this pathogen. Our work offers insights into the interactions between C. difficile and its phages, paving the way for future CRISPR-based applications and development of effective phage therapy strategies combined with the engineering of virulent C. difficile infecting phages.

Identification and Characterisation of Spore-Forming Bacteria in Bovine Raw Milk Collected from Four Dairy Farms in New Zealand

Contamination of milk and dairy products with pathogenic and spoilage bacteria may result in huge economic loss due to recalls of products. This study aimed to identify spore-forming bacteria from raw milk and characterise those for toxin production and their spoilage ability. Bovine raw milk collected from dairy farms in the Manawatu region of the North Island of New Zealand was tested for the presence of both aerobic and anaerobic spore-forming bacteria using standard culture-based techniques, as well as genomic analysis. The spore-forming bacteria were investigated for the presence of toxin genes and their spoilage potential. A low number of aerobic spore-forming bacteria were detected in raw-milk samples collected from the four farms in summer and winter. The 16S rRNA sequence types similar to important food spoilage bacteria like C. beijerinckii, C. sporogenes, B. licheniformis and members of the Paenibacillus genus, as well as potentially toxigenic bacteria such as B. cereus and C. perfringens were isolated. Genes responsible for important toxin production were present in some of the tested spore-forming bacteria. This pilot study highlights the presence of various spoilage and pathogenic spore-forming bacteria in raw milk from these farms. A low number of spore-forming bacteria indicates the implementation of good hygienic farm practices and management to reduce the contamination of raw milk with spore-forming bacteria.

Gut Microbiota Associated with Clostridioides difficile Carriage in Three Clinical Groups (Inflammatory Bowel Disease, C. difficile Infection and Healthcare Workers) in Hospital Field.

Clostridioides difficile is an anaerobic spore-forming Gram-positive bacterium. C. difficile carriage and 16S rDNA profiling were studied in three clinical groups at three different sampling times: inflammatory bowel disease (IBD) patients, C. difficile infection (CDI) patients and healthcare workers (HCWs). Diversity analysis was realized in the three clinical groups, the positive and negative C. difficile carriage groups and the three analysis periods. Concerning the three clinical groups, β-diversity tests showed significant differences between them, especially between the HCW group and IBD group and between IBD patients and CDI patients. The Simpson index (evenness) showed a significant difference between two clinical groups (HCWs and IBD). Several genera were significantly different in the IBD patient group (Sutterella, Agathobacter) and in the CDI patient group (Enterococcus, Clostridioides). Concerning the positive and negative C. difficile carriage groups, β-diversity tests showed significant differences. Shannon, Simpson and InvSimpson indexes showed significant differences between the two groups. Several genera had significantly different relative prevalences in the negative group (Agathobacter, Sutterella, Anaerostipes, Oscillospira) and the positive group (Enterococcus, Enterobacteriaceae_ge and Enterobacterales_ge). A microbiota footprint was detected in C. difficile-positive carriers. More experiments are needed to test this microbiota footprint to see its impact on C. difficile infection.

Microbiological Quality of Polish Artisanal Varietal Honeys.

On the basis of routine microbiological tests and selected physicochemical parameters, the quality and food safety of Polish varietal honeys were evaluated. The study included 21 honey samples from 5 varieties (multifloral, honeydew, rapeseed, buckwheat and linden), in which the moisture and extract content, water activity, pH and free acids were determined, and the colony count, the presumptive Bacillus spp., the total fungal count and the presence of anaerobic spore-forming bacilli were examined. More than half (52%, 11/21) of the analyzed honeys contained fewer microorganisms than 10 cfu/g, and in the remaining samples, their numbers ranged from 5 × 101 cfu/g to 4.5 × 102 cfu/g. In all the honeys, the number of presumptive Bacillus spp. in 1 g was less than 10 cfu. In 81% (17/21) of the samples, the total count of fungi in 1 g of honey was less than 10 cfu, and the most contaminated was buckwheat honey (3 samples). The anaerobic spore-forming bacteria was detected in 0.1 g only in one sample of buckwheat honey. The values of the physicochemical parameters did not exceed the accepted limits, which indicated that the honey environment was unfavourable for the development of the tested microbial profile.

Rating-analysis of the sensitivity of biorisk factors to the effects of chemical disinfect-ants in the context of ensuring food safety

The article presents the results of a rating-analysis of the sensitivity of biorisk factors to the effects of chemical disinfectants in the context of ensuring food safety. In the course of research, it was found out that among the group of pathogens with very low resistance to chemical disinfectants, the main share is occupied by risk factors of bacterial etiology. Among the group of pathogens with high resistance, the main share is occupied by risk factors of helminthic etiology. The group of pathogens with very high resistance to chemical disinfectants mainly consists of risk factors of helminthic etiology and spore-forming anaerobic bacteria. The results of the study can be used by food production enterprises and agricultural organizations.

Composition and fate of heat-resistant anaerobic spore-formers in the milk powder production line

Anaerobic spore-forming bacteria are a continuous threat to the dairy industry due to their ability to withstand processing conditions, such as those during heat treatment. These ubiquitous microorganisms have ample opportunity for multiple entry points into the milk chain, creating food quality and safety issues. Certain spore-formers, namely bacilli and clostridia, are more problematic due to their ability to spoil dairy products and pathogenicity. In this study, we investigated how milk treatment and milk powder production influenced the composition and survival of anaerobic spore-formers. Samples were obtained on three different days (replicate blocks) during the production of dairy powders and examined in a culture-dependent manner using the most probable number method coupled with 16S rRNA amplicon sequencing and metagenomic analysis of the enriched samples. Results revealed that the milk separation greatly affected the spore-former presence and composition which were detected along the entire production line from raw material to milk powders. Throughout the various points of the production line, the occurrence of species belonging to the Bacillus cereus sensu lato was higher than that of clostridia. Sequence variants (SVs) belonging to the anaerobic spore-forming genus Clostridium were taxonomically assigned to two SVs groups and were detected in all three replicate blocks. A total of 19 metagenome-assembled genomes were recovered from nine enrichments. Four near-complete and two medium-quality genomes were found in raw milk/milk powder samples and further assigned as Clostridium tyrobutyricum and Clostridium diolis, which may constitute a problem in the finished dairy product. In conclusion, our findings highlight spore-formers' importance on dairy quality and may aid in their intervention and control in the dairy production line.

Standardisation of traditional rennet produced from sheep abomasum

Traditional rennet (TR) from small ruminants is required to produce many cheeses, but can be a source of technological and safety problems due to low milk-clotting activity (MCA) and microbiological quality. TR samples were obtained from producers of Erzincan tulum cheese in the Gölyurt plateaus (Turkey). Total aerobic mesophilic bacteria, yeast and moulds, total coliforms, Escherichia coli, Staphylococcus aureus, and anaerobic spore-forming bacteria counts in the TRs were in the ranges of <1 to 8.07 log cfu mL−1, <2 to 7.33 log cfu mL−1, <1 to 4.32 log cfu mL−1, <1 to 3.19 log cfu mL−1, <1 to 3.46 log cfu mL−1, and <1 log cfu mL−1, respectively. In standardising the TRs, microfiltration decreased the number of microorganisms (<1–2 log cfu mL−1); evaporation increased the MCA by 5 times and reached 1:806–1:5336 MCU mL−1. As a result, increased microbiological safety and MCA of standardised rennet and improved sensory properties of cheese were obtained by a practical and reproducible process.

Inhibitory effects of Clostridium butyricum culture and supernatant on inflammatory colorectal cancer in mice.

Clostridium butyricum (CB) is a spore-forming, gram-positive and obligate anaerobic rod bacterium. CB can modulate the composition of the gut microbiome and promote the growth of beneficial microbes in the intestine by generating short-chain fatty acids (SCFAs), which in turn protect against colitis and prevents the formation of inflammatory-associated colorectal cancer (CRC) by ameliorating colon inflammatory processes. Yet, it remains unclear whether the culture and supernatant of CB could directly influence inflammatory CRC in mice. In this study, azoxymethane (AOM)+dextran sodium sulphate (DSS) was used to induce CRC model in C57BL/6 mice. Next, the serum levels of inflammatory cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10), and cytokines TNF-α, were measured and the pathohistological examination of the large intestine was performed. Both CB culture and supernatant were found to have anti-inflammatory properties. Subsequently, Western blot and Real-Time Quantitative PCR (RT-qPCR) revealed that CB and supernatant regulate the NF-κB/p65 pathway to inhibit the development and progression of inflammatory CRC in AOM+DSS-treated mice, which could be due to the high levels of butyric acid in the supernatant.

Discovery of novel virulence mechanisms in Clostridium botulinum type A3 using genome-wide analysis

ObjectiveClostridium botulinum type A is a neurotoxin-producing, spore-forming anaerobic bacterium that causes botulism in humans. The evolutionary genomic context of this organism is not yet known to understand its molecular virulence mechanisms in the human intestinal tract. Hence, this study aimed to investigate the mechanisms underlying virulence and pathogenesis by comparing the genomic contexts across species, serotypes, and subtypes. MethodsA comparative genomic approach was used to analyze evolutionary genomic relationships, intergenomic distances, syntenic blocks, replication origins, and gene abundance with phylogenomic neighbors. ResultsType A strains have shown genomic proximity to group I strains with distinct accessory genes and vary even within subtypes. Phylogenomic data showed that type C and D strains were distantly related to a group I and group II strains. Synthetic plots indicated that orthologous genes might have evolved from Clostridial ancestry to subtype A3 strains, whereas syntonic out-paralogs might have emerged between subtypes A3 and A1 through α-events. Gene abundance analysis revealed the key roles of genes involved in biofilm formation, cell–cell communication, human diseases, and drug resistance compared to the pathogenic Clostridia. Moreover, we identified 43 unique genes in the type A3 genome, of which 29 were involved in the pathophysiological processes and other genes contributed to amino acid metabolism. The C. botulinum type A3 genome contains 14 new virulence proteins that can provide the ability to confer antibiotic resistance, virulence exertion and adherence to host cells, the host immune system, and mobility of extrachromosomal genetic elements. ConclusionThe results of our study provide insight into the understanding of new virulence mechanisms to discover new therapeutics for the treatment of human diseases caused by type A3 strains.

Bacterial species-specific antimicrobial efficacies of three different body embalming solutions for anatomical studies.

While body decompensation is mainly facilitated by bacteria, investigating the antimicrobial properties of body preservation methods is still a neglected research area. We performed microbiological sampling for potentially pathogenic bacteria species of brain, lung, liver, colon, and subcutis samples obtained from bodies perfused with embalming solutions of variable composition with emphasis on variable formaldehyde concentrations. We, thereby, identified spore-forming aerobic and anaerobic bacteria mainly in the samples obtained from the colon of ethanol- and lower-concentrated formaldehyde formulation embalmed bodies. Moreover, we could identify Enterococcus species in bodies preserved with the latter method. Tissue samples of the subcutis remained sterile. Long-term incubation of special mycobacteria growth indicator tubes revealed no growth of mycobacteria in all 60 samples analyzed. Overall, we show survival of bacterial genera known to be especially environmentally resistant but also include potentially pathogenic members. Knowledge of bactericidal capacities of embalming solutions are therefore critical to assess risk and apply appropriate disinfection routines while working with human bodies. Moreover, new formulations to reduce potentially toxic substances for embalming needs to be evaluated regarding their bactericidal capacities.

Assessment of the resistance of alimentary-related risk factors to the effects of chemical disinfectants

Abstract. Currently, much attention is paid to the monitoring of new biopathogens, which are formed as a result of targeted genetic manipulations resulting from human activity, and natural variability. The sensitivity of mutated organisms to various disinfectants can differ significantly from the sensitivity of the original varieties. Thus, the assessment of alimentary-caused biological risk factors in terms of resistance to the effects of chemical disinfectants is an important area of scientific research. The aim of the work is to assess the nutritionally determined biological risk factors in terms of resistance to the effects of chemical disinfectants. The research methods were general scientific methods of cognition, as well as the analysis of alimentary-caused biological risk factors in terms of resistance to chemical disinfectants, which was carried out by us, based on the standardized methodology for the epidemiological assessment of Dubyansky-Maletskaya. The analysis covered a three-year period from 2019 to 2021, and was carried out by us on the materials of the Ryazan region provided by the Center for Hygiene and Epidemiology of the Ryazan Region and the Main Directorate of Veterinary Medicine of the Ryazan Region. The scientific novelty of the study lies in identifying the features of the manifestation of the sensitivity of alimentary-conditioned biological risk factors to various groups of chemical disinfectants. Results. Our study allowed us to draw the following conclusions: the resistance of alimentary-caused biological risk factors to chemical disinfectants is largely due to their etiological characteristics; among the group of pathogens with very low resistance to chemical disinfectants, the main share is occupied by risk factors of bacterial etiology; among the group of pathogens with medium resistance to chemical disinfectants, the main share is occupied by risk factors of viral etiology; among the group of pathogens with high resistance to chemical disinfectants, the main share is occupied by risk factors of helminthic etiology; a group of pathogens with a very high resistance to chemical disinfectants mainly consists of risk factors of helminthic etiology and spore-forming anaerobic bacteria.

In vitro safety evaluation of Bacillus subtilis species complex isolated from Vietnam and their additional beneficial properties

Bacillus genus are Gram-positive, rod shaped, spore-forming, aerobic or facultative anaerobic bacteria, which have the ability to produce a wide variety of enzymes, antimicrobial compounds, vitamins, and carotenoids. Nowadays, Bacillus species have been increasingly proposed for use as probiotics or feed additives as B. amyloliquefaciens, B. licheniformis, B. subtilis, and B. pumilus because of their advantages such as heat-stability of the spores, storage capacity at ambient temperature, and beneficial properties for health. Since microbes are used for customers and livestock, it is critical to substantiate not only the health benefits and efficacy of unique strains but also their safety for the hosts. This study aims to evaluate the in vitro safety and some beneficial properties of Bacillus strains to select potential strains for use in humans or animals. A total of 76 Bacillus strains belonged to the B. amyloliquefaciens and B. subtilis groups were preliminary evaluated for their safety via hemolytic activity. Six non-hemolytic strains were identified and their antibiotic susceptibility, cytotoxicity on the growth of HT29 and Vero cells, extracellular enzyme production, antimicrobial activity, and identification based on 16S rRNA and rpoB gene sequences were studied. Two strains, B. subtilis VTCC 10963 and B. subtilis VTCC 11039, should be considered safe. In addition, these two strains exhibited good extracellular enzyme production (amylase, cellulase, and protease) and strong antibacterial activity against Listeria monocytogenes. Our results support the use of B. subtilis VTCC 10963 and B. subtilis VTCC 11039 for the development of probiotic products and feed additives in the future.

Biological Hydrogen Production by Dark Fermentation in a Stirred Tank Reactor and Its Correlation with the pH Time Evolution

Dark fermentation is a hydrogen generating process carried out by anaerobic spore-forming bacteria that metabolize carbon sources producing gas and short-chain acids. The process can be controlled, and the hydrogen harvested if bacteria are grown in a reactor with favorable conditions. In this work, bacteria selected from natural sources were grown with a defined culture media, while pH was monitored, with the aim of relating the amount of generated hydrogen to the increase in hydron ion concentration. Therefore, a model based on the acid-base species mass balance is proposed and solved to estimate the lag phase time and measure the hydrogen production efficiency and kinetics. Hydrogen production in a stirred batch reactor was performed for 150–200 h, at given operating conditions using a previously defined growth media, to validate the model. Using the proposed model, the cumulated moles of produced hydrogen correlate well with those predicted from the pH curve. Hence, the modified Gompertz model parameters, largely used for describing the hydrogen generation kinetics by dark fermentation, were estimated from the pH curve and from the experimentally measured generated hydrogen. Satisfactory agreement was found, thus, validating the method.

Impact of environmental conditions and gut microbiota on the in vitro germination and growth of Clostridioides difficile.

Clostridioides difficile is a spore-forming anaerobic Gram-positive bacterium responsible for a broad spectrum of intestinal symptoms and healthcare-associated diarrhoea. The hypothesis of this work was that different in vitro conditions, notably pH and human faecal microbiota composition, impact the germination and/or the growth of C. difficile. This study aimed to correlate growth kinetics of the bacterium with these two physiochemical parameters by using a static in vitro model. To better understand the initial gut colonisation, several growth curve assays were carried out to monitor the behaviour of the spores and vegetative forms of C. difficile strain 078 under different conditions mimicking the gut environment. When the faeces were added, no spore germination or growth was observed, but C. difficile spores germinated in vitro when the pH was maintained between 6.6 and 6.9 for four different faeces donors. The evolution of microbiota studied by 16S rDNA profiling showed high proportions of Enterobacteriaceae and E. coli/Shigella when C. difficile grew, regardless of the inoculated faeces. This model helped us to understand that the germination and growth of C. difficile are strongly pH dependent, and further research is needed to evaluate the potential impact of the gut microbiota composition on C. difficile.