An Intestinal Organ Culture System Uncovers a Role for the Nervous System in Microbe-Immune Crosstalk.

Investigation of host-environment interactions in the gut would benefit from a culture system that maintained tissue architecture yet allowed tight experimental control. We devised a microfabricated organ culture system that viably preserves the normal multicellular composition of the mouse intestine, with luminal flow to control perturbations (e.g., microbes, drugs). It enables studying short-term responses of diverse gut components (immune, neuronal, etc.). We focused on the early response to bacteria that induce either Th17 or RORg+ T-regulatory (Treg) cells in vivo. Transcriptional responses partially reproduced in vivo signatures, but these microbes elicited diametrically opposite changes in expression of a neuronal-specific gene set, notably nociceptive neuropeptides. We demonstrated activation of sensory neurons by microbes, correlating with RORg+ Treg induction. Colonic RORg+ Treg frequencies increased in mice lacking TAC1 neuropeptide precursor and decreased in capsaicin-diet fed mice. Thus, differential engagement of the enteric nervous system may partake in bifurcating pro- or anti-inflammatory responses to microbes.

Unveiling Microbial Diversity: Raman Spectroscopy's Discrimination of Clostridium and Related Genera.

In the clinical environment, the identification of phylogenetic closely related genera and species like Clostridium and Bacillus spp. is challenging. Both genera contain representatives of pathogenic and nonpathogenic species that need to be distinguished for a proper diagnostic read-out. Therefore, reliable and accurate detection methods must be employed for the correct identification of these genera and species. Despite their high pathogenicity, clostridial infections and food contaminations present significant challenges due to their unique cultivation conditions and developmental needs. Therefore, in many diagnostic protocols, the toxins are used for microbiological documentation. However, the applied laboratory methods suffer in accuracy, sometimes require large bacterial loads to provide reliable results, and cannot differentiate pathogenic from nonpathogenic strains. Here, Raman spectroscopy was employed to create an extensive Raman database consisting of pathogenic and nonpathogenic Bacillus and Clostridium species. These genera, as well as representatives of Paraclostridium and Clostridioides were specifically selected for their phylogenetic relation, cultivation conditions, and metabolic activity. A chemometric evaluation of the Raman spectra of single vegetative cells revealed a high discriminating power at the genus level. However, bacilli are considerably easier to classify at the species level than clostridia. The discrimination between the genera and species was based on their phylogeny and not their aerobic and anaerobic cultivation conditions. These encouraging results demonstrated that Raman spectroscopy coupled with chemometrics is a robust and helpful method for differentiating Clostridium species from Bacillus, Clostridioides, and Paraclostridium species. This approach has the potential to be a valuable tool in clinical diagnostics.

Identification of volatile metabolites produced from levodopa metabolism by different bacteria strains of the gut microbiome.

Interspecies pathways in the gut microbiome have been shown to metabolize levodopa, the primary treatment for Parkinson's disease, and reduce its bioavailability. While the enzymatic reactions have been identified, the ability to establish the resulting macromolecules as biomarkers of microbial metabolism remains technically challenging. In this study, we leveraged an untargeted mass spectrometry-based approach to investigate volatile organic compounds (VOCs) produced during levodopa metabolism by Enterococcus faecalis, Clostridium sporogenes, and Eggerthella lenta. We cultured these organisms with and without their respective bioactive metabolites and detected levodopa-induced shifts in VOC profiles. We then utilized bioinformatics to identify significant differences in 2,6-dimethylpyrazine, 4,6-dimethylpyrimidine, and 4,5-dimethylpyrimidine associated with its biotransformation. Supplementing cultures with inhibitors of levodopa-metabolizing enzymes revealed specific modulation of levodopa-associated diazines, verifying their relationship to its metabolism. Furthermore, functional group analysis depicts strain-specific VOC profiles that reflect interspecies differences in metabolic activity that can be leveraged to assess microbiome functionality in individual patients. Collectively, this work identifies previously uncharacterized metabolites of microbe-mediated levodopa metabolism to determine potential indicators of this activity and further elucidate the metabolic capabilities of different gut bacteria.

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

BACKGROUND: Clostridium 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. METHODS: Fifty-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. RESULTS: Of 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. CONCLUSIONS: This 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.

Clostridium aquiflavi sp. nov., isolated from yellow water of Nongxiangxing baijiu in the Yibin region of China.

A Gram-stain-positive, strictly anaerobic, endospore-forming and rod-shaped (0.6-0.8×2.7-13.1 µm) bacterium, designated as 5 N-1T, was isolated from a yellow water sample collected from the manufacturing process of Nongxiangxing baijiu in the Yibin region of Sichuan, PR China. Growth occurred at 15-40 °C (optimum growth at 37 °C), at pH 6.0-9.0 (optimum growth at pH 7.0) and in NaCl concentrations of 0-1 % (w/v) and ethanol concentrations of 0-2 % (v/v). The major fatty acids in strain 5 N-1T were C16 : 0, C18 : 0 and C14 : 0. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four unidentified aminophospholipids and one unidentified lipid. Phylogenetic analysis of its 16S rRNA gene sequence indicated that strain 5 N-1T was most closely related to Clostridium weizhouense YB-6T (97.70 %) and Clostridium uliginosum DSM 12992T (97.56 %). The average nucleotide identity and digital DNA‒DNA hybridization values between strain 5 N-1T and the above two type strains were 80.89 and 80.05 % and 25.80 and 25.30 %, respectively, which were all below the species thresholds. The genome size of strain 5 N-1T was 3.5 Mbp and the DNA G+C content was 27.5 mol%. Based on the results of phenotypic and genotypic analyses, strain 5 N-1T represents a novel species of the genus Clostridium, for which the name Clostridium aquiflavi sp. nov. is proposed. The type strain is Clostridium aquiflavi 5 N-1T (=CICC 24886T=JCM 35355T).

Clostridium omnivorum sp. nov., isolated from anoxic soil under the treatment of reductive soil disinfestation.

Reductive soil disinfestation (RSD), also known as biological soil disinfestation, is a bioremediation method used to suppress soil-borne plant pathogens by stimulating the activity of indigenous anaerobic bacteria in the soil. An anaerobic bacterial strain (E14T) was isolated from an anoxic soil sample subjected to RSD treatment and then comprehensively characterized. Cells of the strain were Gram-stain-positive, curved to sigmoid, and spore-forming rods. Cells were motile with a polar flagellum. Strain E14T grew in peptone-yeast extract broth, indicating that it utilized proteinous compounds. Strain E14T was also saccharolytic and produced acetate, isobutyrate, butyrate, isovalerate and gases (H2 and CO2) as fermentation products. The strain did not decompose any of examined polysaccharides except for starch. The major cellular fatty acids of strain E14T were iso-C15:0 and iso-C15:0 DMA. The closest relative to strain E14T, based on 16S rRNA gene sequences, was Clostridium thermarum SYSU GA15002T (96.2 %) in the Clostridiaceae. Whole-genome analysis of strain E14T showed that its genome was 4.66 Mb long with a genomic DNA G+C content of 32.5 mol%. The average nucleotide identity (ANIb) between strain E14T and C. thermarum SYSU GA15002T was 69.0 %. The presence of the genes encoding glycolysis and butyrate production via the acetyl-CoA pathway was confirmed through genome analysis. Based on the obtained phylogenetic, genomic and phenotypic data, we propose that strain E14T should be assigned to the genus Clostridium in the family Clostridiaceae as Clostridium omnivorum sp. nov. The type strain is E14T (=NBRC 115133T=DSM 114974T).

Clostridium tanneri sp. nov., isolated from the faecal material of an alpaca.

A strictly anaerobic, Gram-stain-negative rod-shaped bacterium, designated A1-XYC3T, was isolated from the faeces of an alpaca (Lama pacos). On the basis of the results of a comparative 16S rRNA gene sequence analysis, the isolate was assigned to the genus Clostridium with the highest sequence similarities to Clostridium magnum DSM 2767T (96.8 %), Clostridium carboxidivorans P7T (96.3 %) and Clostridium aciditolerans JW/YJL-B3T (96.1 %). The average nucleotide identity between A1-XYC3T, C. magnum, C. carboxidivorans and C. aciditolerans was 77.4, 76.1 and 76.6  %, respectively. The predominant components of the cellular fatty acids of A1-XYC3T were C14 : 0, C16 : 0 and summed feature 10, containing C18:0/C17:0 cyclo. The DNA G+C content was 32.4 mol%. On the basis of biochemical, phylogenetic, genotypic and chemotaxonomic criteria, this isolate represents a novel species within Clostridium sensu stricto for which the name Clostridium tanneri sp. nov. is proposed. The type strain of this species is strain A1-XYC3T (=CCM 9376T=NRRL B-65691T).

Exploiting the Thermotolerance of Clostridium Strain M1NH for Efficient Caproic Acid Fermentation from Ethanol and Acetic Acid.

A novel thermotolerant caproic acid-producing bacterial strain, Clostridium M1NH, was successfully isolated from sewage sludge. Ethanol and acetic acid at a molar ratio of 4:1 proved to be the optimal substrates, yielding a maximum caproic acid production of 3.5 g/L. Clostridium M1NH exhibited remarkable tolerance to high concentrations of ethanol (up to 5% v/v), acetic acid (up to 5% w/v), and caproic acid (up to 2% w/v). The strain also demonstrated a wide pH tolerance range (pH 5.5-7.5) and an elevated temperature optimum between 35 and 40 °C. Phylogenetic analysis based on 16S rRNA gene sequences revealed that Clostridium M1NH shares a 98% similarity with Clostridium luticellarii DSM 29923 T. The robustness of strain M1NH and its efficient caproic acid production from low-cost substrates highlight its potential for sustainable bio-based chemical production. The maximum caproic acid yield achieved by Clostridium M1NH was 1.6-fold higher than that reported for C. kluyveri under similar fermentation conditions. This study opens new avenues for valorizing waste streams and advancing a circular economy model in the chemical industry.

Approaching toxigenic Clostridia from a One Health perspective.

Spore-forming pathogens have a unique capacity to thrive in diverse environments, and with temporal persistence afforded through their ability to sporulate. Their prevalence in diverse ecosystems requires a One Health approach to identify critical reservoirs and outbreak-associated transmission chains, given their capacity to freely move across soils, waterways, foodstuffs and as commensals or infecting pathogens in human and animal populations. Among anaerobic spore-formers, genomic resources for pathogens including C. botulinum, C. difficile, and C. perfringens enable our capacity to identify common and unique factors that support their persistence in diverse reservoirs and capacity to cause disease. Publicly available genomic resources for spore-forming pathogens at NCBI's Pathogen Detection program aid outbreak investigations and longitudinal monitoring in national and international programs in public health and food safety, as well as for local healthcare systems. These tools also enable research to derive new knowledge regarding disease pathogenesis, and to inform strategies in disease prevention and treatment. As global community resources, the continued sharing of strain genomic data and phenotypes further enhances international resources and means to develop impactful applications. We present examples showing use of these resources in surveillance, including capacity to assess linkages among clinical, environmental, and foodborne reservoirs and to further research investigations into factors promoting their persistence and virulence in different settings.

Clinical and microbiological characteristics of anaerobic bacteremia during 1994-2019: A Danish population-based cohort study.

OBJECTIVES: Bacteremia with anaerobic bacteria is generally a marker of severe prognosis. However, population-based data is lacking. Our aim was to describe the epidemiology and the 30-day mortality rate of anaerobic bacteremia in a Danish population-based setting. METHODS: In this population-based cohort study, all first-time episodes of anaerobic bacteremia from the North Denmark Bacteremia Research Database during 1994-2019 were identified. Information on comorbidities, discharge diagnoses, and mortality was retrieved. 30-day mortality rates were calculated and a multivariate logistic regression analysis to identify risk factors for death was performed. RESULTS: 1750 episodes with anaerobic bacteremia were identified, corresponding to an incidence rate of 12.5 per 100,000 inhabitants (increasing from 11.2 in 1994-2014 to 17.7 in 2015-2019). Of these episodes, a third were polymicrobial, and the majority (70 %) of patients had one or more comorbid conditions. Abdominal infection was the source of bacteremia in 61 % of patients, while it was unknown for 15 %. The most frequently isolated genera were Bacteroides (45 %), Clostridium (20 %) and Fusobacterium (6 %). The overall crude 30-day mortality rate was 27 %, but rates were even higher for patients of high age, with liver disease, and solid tumors. The odds ratio (OR) for 30-day mortality was 1.32 for Clostridium species, and 1.27 for polymicrobial bacteremia with aerobic bacteria. CONCLUSIONS: The incidence rate of anaerobic bacteremia increased, and the 30-day mortality rate remained high during the study period. Multiple factors influence 30-day mortality rates, including high age, liver disease, solid tumor, polymicrobial bacteremia, and bacteremia with Clostridium species.

Clostridium thailandense sp. nov., a novel CO2-reducing acetogenic bacterium isolated from peatland soil.

Some species of the genus Clostridium are efficient acetate producers and have been deemed useful for upgrading industrial biogas. An acetogenic, strictly anaerobic, Gram-stain-positive, subterminal endospore-forming bacterium designated strain PL3T was isolated from peatland soil enrichments with H2 and CO2. Cells of strain PL3T were 0.8-1.0×4.0-10.0 µm in size and rod-shaped. Growth of strain PL3T occurred at pH 6.0-7.5 (optimum, pH 7.0), at 20-40 °C (optimum, 30 °C) and with 0-1.5 % (w/v) NaCl (optimum, 0.5%). Biochemical analyses revealed that strain PL3T metabolized lactose, maltose, raffinose, rhamnose, lactic acid, sorbitol, arabinose and glycerol. Acetic acid was the predominant metabolite under anaerobic respiration with H2/CO2. The major cellular fatty acids were C16 : 0, C16 : 1 cis 9 and C17 : 0 cyc. The main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, aminolipid and aminophospholipid. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PL3T belongs to the genus Clostridium with the highest sequence similarity to Clostridium aciditolerans DSM 17425T (98.6 %) followed by Clostridium nitrophenolicum (97.8 %). The genomic DNA G+C content of strain PL3T was 31.1 mol%.The genomic in silico DNA-DNA hybridization value between strain PL3T and C. aciditolerans DSM 17425T was 25.1 %, with an average nucleotide identity of 80.2 %. Based on phenotypic, chemotaxonomic and phylogenetic differences, strain PL3T was suggested to represent a novel species of the genus Clostridium, for which the name Clostridium thailandense sp. nov. is proposed. The type strain is PL3T (=DSM 111812T=TISTR 2984T).

Phenotypic and genomic analysis of isopropanol and 1,3-propanediol producer Clostridium diolis DSM 15410.

Clostridium diolis DSM 15410 is a type strain of solventogenic clostridium capable of conducting isopropanol-butanol-ethanol fermentation. By studying its growth on different carbohydrates, we verified its ability to utilize glycerol and produce 1,3-propanediol and discovered its ability to produced isopropanol. Complete genome sequencing showed that its genome is a single circular chromosome and belongs to the cluster I (sensu scricto) of the genus Clostridium. By cultivation analysis we highlighted its specific behavior in comparison to two selected closely related strains. Despite the fact that several CRISPR loci were found, 16 putative prophages showed the ability to receive foreign DNA. Thus, the strain has the necessary features for future engineering of its 1,3-propanediol biosynthetic pathway and for the possible industrial utilization in the production of biofuels.

Taxonomic status of the species Clostridium methoxybenzovorans Mechichi et al. 1999.

The taxonomic status of the species Clostridium methoxybenzovorans was assessed. The 16S rRNA gene sequence, whole-genome sequence and phenotypic characterizations suggested that the type strain deposited in the American Type Culture Collection (C. methoxybenzovorans ATCC 700855T) is a member of the species Eubacterium callanderi. Hence, C. methoxybenzovorans ATCC 700855T cannot be used as a reference for taxonomic study. The type strain deposited in the German Collection of Microorganism and Cell Cultures GmbH (DSM 12182T) is no longer listed in its online catalogue. Also, both the 16S rRNA gene and the whole-genome sequences of the original strain SR3T showed high sequence identity with those of Lacrimispora indolis (recently reclassified from Clostridium indolis) as the most closely related species. Analysis of the two genomes showed average nucleotide identity based on blast and digital DNA-DNA hybridization values of 98.3 and 87.9 %, respectively. Based on these results, C. methoxybenzovorans SR3T was considered to be a member of L. indolis.

Clostridium fessum sp. nov., isolated from human faeces.

An obligately anaerobic, Gram-stain-positive and spore-forming strain, SNUG30386T was isolated from a faecal sample of a healthy Korean subject. The strain formed a round ivory-coloured colony and cells were chained rods with tapered ends, approximately 2.0-2.5×0.6-0.8 µm in size. The taxonomic analysis indicated that strain SNUG30386T was within the family Lachnospiraceae. According to the 16S rRNA gene sequence similarity, the closest species to strain SNUG30386T was Clostridium symbiosum (95.6 %), followed by Enterocloster asparagiformis (94.8 %), Enterocloster clostridioformis (94.8 %) and Enterocloster lavalensis (94.6 %). The evolutionary tree based on 16S rRNA gene sequences demonstrated that strain SNUG30386T had split apart at a unique branch point far from other close relatives. Its DNA G+C content was 48.3 mol% calculated from the whole genome sequence. The major cellular fatty acids were C16 : 0 and C14 : 0. Compared to those of the closely related species, strain SNUG30386T showed distinct biochemical activities such as being unable to utilize most of carbon sources except d-glucose and l-arabinose. As a result, based on its unique phylogenetic clade and taxonomic characteristics, we conclude that strain SNUG30386T represents a novel species within the genus Clostridium, for which the name Clostridium fessum sp. nov. is proposed. The type strain of the novel species is SNUG30386T (=KCTC 15633T= JCM 32258T).

Clostridium zeae sp. nov., isolated from corn silage.

A rod-shaped, Gram-stain-negative, strictly anaerobic, catalase-negative and endospore-forming bacterial strain CSC2T was isolated from corn silage preserved in Tochigi, Japan. The strain CSC2T grew at 15-40 °C, at pH 5.0-7.7 and with up to 0.5 % (w/v) NaCl. The main cellular fatty acids were C14 : 0, C16 : 0 and C16 : 0 dimethyl acetal. The cellular polar lipids detected were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, lysophosphatidylethanolamine, phosphatidylserine, lysophosphatidylcholine and two unidentified polar lipids. Phylogenetic analysis of the 16S rRNA gene showed that strain CSC2T was a member of the genus Clostridium and closely related to Clostridium polyendosporum DSM 57272T (95.6 % gene sequence similarity) and Clostridium fallax ATCC 19400T (95.3 %). The genomic DNA G+C content of strain CSC2T was 31.1 mol% (whole genome analysis). The average nucleotide identity based on blast and digital DNA-DNA hybridization values between strain CSC2T and the type strains of phylogenetically related species were below 71 and 24 %, respectively. On the basis of the genotypic, phenotypic and chemotaxonomic characteristics, it is proposed to designate strain CSC2T as representing Clostridium zeae sp. nov. The type strain is CSC2T (=MAFF212476T=JCM 33766T=DSM 111242T).

An investigation of the environmental niches of blown pack spoilage causing Clostridium estertheticum and Clostridium gasigenes on New Zealand beef and sheep farms.

The transfer of blown pack spoilage causing Clostridium spores from the farm to the meat plant is of growing concern to the meat industry. This study investigated the environmental niches of these Clostridium spp., specifically Clostridium estertheticum and Clostridium gasigenes in the beef and sheep farm environments in New Zealand. Faecal, soil, grass, drinking water, puddle water and feed (fodder beet, hay, bailage and silage, where available) samples were collected on five beef and sheep farms during Winter and Spring in 2018, in North and South Island, respectively. Beef and sheep farm samples were tested for C. estertheticum and C. gasigenes using enrichment plus PCR, qPCR and direct plating. C. estertheticum was detected in bovine faecal (4%), soil (2-18%) and grass (0-12%) samples at concentration of up to 2.0 log10 cfu/g. C. gasigenes were found in 18-46% of faecal, 16-82% of soil, 12-44% of grass, 0-44.4% of drinking water and 0-58.3% of puddle water samples tested and the direct counts ranged from 2.4 log10 cfu/ml in puddle water to 3.4 log10 cfu/g in soil. C. estertheticum were detected by qPCR in sheep farms in ovine feces (2.3%), soil (2.3%) and fodder beet (10%). All other sample types (grass, drinking water, puddle water, baleage, hay, silage and fodder beet) were negative using direct and enrichment plus PCR methods. In contrast C. gasigenes was detected in of faecal (22.7-38.6%), soil (22.7-84.1%), grass (17.5-34.1%) drinking water (35.7-78.6%), puddle water (33.3-40%), hay baleage (57%), silage (2%) and fodder beet (10%) at concentrations of up to 3.7 log10 cfu/g/ml. It was concluded that C. estertheticum and C. gasigenes were common on beef and sheep farms with the latter having higher incidence and mean concentration.

Comparative genomics of Clostridium species associated with vacuum-packed meat spoilage.

Bacterial species belonging to the genus Clostridium have been recognized as causative agents of blown pack spoilage (BPS) in vacuum packed meat products. Whole-genome sequencing of six New Zealand psychrotolerant clostridia isolates derived from three meat production animal types and their environments was performed to examine their roles in BPS. Comparative genome analyses have provided insight into the genomic diversity and physiology of these bacteria and divides clostridia into two separate species clusters. BPS-associated clostridia encode a large and diverse spectrum of degradative carbohydrate-active enzymes (CAZymes) that enable them to utilize the intramuscular carbohydrate stores and facilitate sporulation. In total, 516 glycoside hydrolases (GHs), 93 carbohydrate esterases (CEs), 21 polysaccharide lyases (PLs), 434 glycosyl transferases (GTs) and 211 carbohydrate-binding protein modules (CBM) with predicted activities involved in the breakdown and transport of carbohydrates were identified. Clostridia genomes have different patterns of CAZyme families and vary greatly in the number of genes within each CAZy category, suggesting some level of functional redundancy. These results suggest that BPS-associated clostridia occupy similar environmental niches but apply different carbohydrate metabolism strategies to be able to co-exist and cause meat spoilage.

Stunted childhood growth is associated with decompartmentalization of the gastrointestinal tract and overgrowth of oropharyngeal taxa.

Linear growth delay (stunting) affects roughly 155 million children under the age of 5 years worldwide. Treatment has been limited by a lack of understanding of the underlying pathophysiological mechanisms. Stunting is most likely associated with changes in the microbial community of the small intestine, a compartment vital for digestion and nutrient absorption. Efforts to better understand the pathophysiology have been hampered by difficulty of access to small intestinal fluids. Here, we describe the microbial community found in the upper gastrointestinal tract of stunted children aged 2-5 y living in sub-Saharan Africa. We studied 46 duodenal and 57 gastric samples from stunted children, as well as 404 fecal samples from stunted and nonstunted children living in Bangui, Central African Republic, and in Antananarivo, Madagascar, using 16S Illumina Amplicon sequencing and semiquantitative culture methods. The vast majority of the stunted children showed small intestinal bacterial overgrowth dominated by bacteria that normally reside in the oropharyngeal cavity. There was an overrepresentation of oral bacteria in fecal samples of stunted children, opening the way for developing noninvasive diagnostic markers. In addition, Escherichia coli/Shigella sp. and Campylobacter sp. were found to be more prevalent in stunted children, while Clostridia, well-known butyrate producers, were reduced. Our data suggest that stunting is associated with a microbiome "decompartmentalization" of the gastrointestinal tract characterized by an increased presence of oropharyngeal bacteria from the stomach to the colon, hence challenging the current view of stunting arising solely as a consequence of small intestine overstimulation through recurrent infections by enteric pathogens.

Microbiological features, epidemiology, and clinical presentation of Clostridioidesdifficile strains from MLST Clade 2: A narrative review.

Clostridioides difficile is an emerging One Health pathogen and a common etiologic agent of diarrhea, both in healthcare settings and the community. This bacterial species is highly diverse, and its global population has been classified in eight clades by multilocus sequence typing (MLST). The C. difficile MLST Clade 2 includes the NAP1/RT027/ST01 strain, which is highly recognized due to its epidemicity and association with severe disease presentation and mortality. By contrast, the remaining 83 sequence types (STs) that compose this clade have received much less attention. In response to this shortcoming, we reviewed articles published in English between 1999 and 2020 and collected information for 27 Clade 2 STs, with an emphasis on STs 01, 67, 41 and 188/231/365. Our analysis provides evidence of large phenotypic differences that preclude support of the rather widespread notion that ST01 and Clade 2 strains are "hypervirulent". Moreover, it revealed a profound lack of (meta)data for nearly 70% of the Clade 2 STs that have been identified in surveillance efforts. Targeted studies aiming to relate wet-lab and bioinformatics results to patient and clinical parameters should be performed to gain a more in-depth insight into the biology of this intriguing group of C. difficile isolates.

Identification of novel autoinducer-2 receptors in Clostridia reveals plasticity in the binding site of the LsrB receptor family.

Autoinducer-2 (AI-2) is unique among quorum-sensing signaling molecules, as it is produced and recognized by a wide variety of bacteria and thus facilitates interspecies communication. To date, two classes of AI-2 receptors have been identified: the LuxP-type, present in the Vibrionales, and the LsrB-type, found in a number of phylogenetically distinct bacterial families. Recently, AI-2 was shown to affect the colonization levels of a variety of bacteria in the microbiome of the mouse gut, including members of the genus Clostridium, but no AI-2 receptor had been identified in this genus. Here, we identify a noncanonical, functional LsrB-type receptor in Clostridium saccharobutylicum. This novel LsrB-like receptor is the first one reported with variations in the binding-site amino acid residues that interact with AI-2. The crystal structure of the C. saccharobutylicum receptor determined at 1.35 Å resolution revealed that it binds the same form of AI-2 as the other known LsrB-type receptors, and isothermal titration calorimetry (ITC) assays showed that binding of AI-2 occurs at a submicromolar concentration. Using phylogenetic analysis, we inferred that the newly identified noncanonical LsrB receptor shares a common ancestor with known LsrB receptors and that noncanonical receptors are present in bacteria from different phyla. This led us to identify putative AI-2 receptors in bacterial species in which no receptors were known, as in bacteria belonging to the Spirochaetes and Actinobacteria phyla. Thus, this work represents a significant step toward understanding how AI-2-mediated quorum sensing influences bacterial interactions in complex biological niches.