Strict Anaerobes Research Articles

Oxygen exposure decreases the yield of high-molecular-weight DNA from some anaerobic bacteria and bacterial communities during DNA extraction

ObjectivesThe central challenge in third-generation sequencing lies in meeting the requirements for DNA quality (integrity and purity) and quantity. Therefore, novel improvements in DNA extraction methods are needed to satisfy these requirements. We reasoned that in anaerobic microbial communities, the presence of certain strict anaerobes containing oxygen-activated DNase activity might contribute substantially to the poor integrity of extracted metagenomic DNA (or genomic DNA from some pure cultures) if exposed to air. MethodsTo test this hypothesis, we developed an enhanced genomic and metagenomic DNA isolation technique that we applied to a specifically chosen set of both strict and aerotolerant anaerobes, as well as to the hindgut microbiota of a herbivorous marine fish. ResultsConsidering the quality (or degradation) of extracted DNA obtained under anaerobic versus aerobic conditions, we found that DNA extracted aerobically from cells of some strict anaerobes showed more degradation of high molecular weight DNA than analogous preparations under anaerobic conditions. In contrast, with the selected aerotolerant anaerobes, no discernible difference was found between the molecular sizes of DNA extracted aerobically and anaerobically. Metagenomic DNA extracted from the fish hindgut microbiota showed higher yields and better quality under anaerobic conditions compared to aerobic conditions. ConclusionOur study effectively demonstrates the advantages of our improved extraction protocol in anaerobic conditions. This is evident through the improved quality of extracted DNA. Such findings may be valuable for studies, especially metagenomic studies, where the quality and quantity of DNA are crucial for downstream analysis.

Microbiology and Epidemiology of Orbital Cellulitis in Pediatric and Young Adult Patients.

Using data from the large and geographically diverse Military Health System (MHS) beneficiary population, we aimed to characterize and update the epidemiology and microbiology of pediatric orbital cellulitis given previous data are limited to small, single-center studies. Following institutional review board approval, we performed a retrospective analysis using the Military Health System admissions, microbiology, and pharmacy data between June 2009 and September 2019. Patients less than 22 years of age with radiological confirmation of orbital cellulitis were included. Demographic data, presence of sinusitis, advanced imaging reports, blood and wound culture results with antibiotic susceptibilities, and antibiotic prescriptions were collected. Descriptive statistics were used to summarize demographic characteristics. Imaging findings were grouped by Chandler's stage (CS), an imaging-based measure of the progressive severity of orbital involvement. A Cochran-Armitage trend test was used to evaluate the relationship between CS and likelihood of positive confirmatory culture. There was a male predominance (66.9%) and 55.5% of subjects had comorbid sinusitis. Of the 130 subjects included, 33.8% had one or more positive cultures, 30.8% had a positive wound culture, and 4.6% had a positive blood culture. The most identified organism was coagulase-negative staphylococci (23.3%), followed by Staphylococcus aureus (18.9%), Streptococcus intermedius (17.8%), and strict anaerobes as a group (13.3%). Gram-negative organisms were rare. Twenty-five percent of S. aureus were methicillin-resistant. Clindamycin resistance was identified in 9% of all S. aureus, 50% of coagulase-negative staphylococci, and 25% of S. intermedius. Clindamycin plus ceftriaxone was the most prescribed empiric antibiotic regimen (36.2%). Likelihood of a positive culture significantly increased with advancing CS. Orbital cellulitis occurs most frequently in males with sinusitis. Likelihood of positive wound culture is increased with a more advanced CS. Staphylococcus and Streptococcus spp. and anaerobes are the most identified pathogens in orbital cellulitis, while gram-negative organisms are rare. Empiric antibiotic selection should include an anti-methicillin-resistant S. aureus agent combined with a broad-spectrum beta-lactam and anaerobic coverage.

Draft genome sequence of rhamnolipid-producing bacteria Thermoanaerobacter thermocopriae strain CM-CNRG TB177 isolated from an oil reservoir in Mexico.

The draft genome of Thermoanaerobacter thermocopriae CM-CNRG TB177 isolated from an oil reservoir in Mexico was determined and annotated. The organism is a thermophilic and strict anaerobe bacterium that produces rhamnolipids, using glucose as a carbon source. The predicted genome size is 2,496,169 bp and 2,550 genes.

Coronal and Root Canal Microbiota in Apical Periodontitis with Different PAI.

Apical periodontitis is an inflammatory disease triggered by oral pathogens invading necrotic root canals. The aim of this study was to evaluate the coronal and root canal bacterial community profiles in primary endodontic infections with different periapical (PAI) indices in comparison to oral mucosa controls. A total of 31 patients with primary apical periodontitis, 14 with PAI-1 and 17 with PAI-3 were recruited. Microbial specimens from mucosa (control samples) and endodontic necrotic tissues were collected in each patient. Microbiota composition was studied through 16S ribosomal RNA gene amplicon sequencing analysis. Overall, 2953 taxa from 168 different genera of 451 various microbial species were retrieved in the controls and PAI-1 and PAI-3 groups. Firmicutes is the predominant phylum in the oral controls (34.5%) and PAI-1 (44.4%) groups, while Bacteroidetes is predominant in PAI-3 (38.6%). The Proteobacteria (21.5%) and Fusobacteria (12.5%) relative abundance is higher in oral controls while that of Synergistetes is higher in the PAI-1 (3.5%) and PAI-3 (2.5%) groups, being almost absent in controls (less than 0.1%). Most of the increased bacterial species found in the PAI groups were strict anaerobes. A diminished microbial diversity was found in apical periodontitis with higher PAI. These samples were also characterized by an increase in bacteria belonging to phyla and genera with an increased anaerobic character.

Clostridioides difficile superoxide reductase mitigates oxygen sensitivity.

Clostridioides difficile causes a serious diarrheal disease and is a common healthcare-associated bacterial pathogen. Although it has a major impact on human health, the mechanistic details of C. difficile intestinal colonization remain undefined. C. difficile is highly sensitive to oxygen and requires anaerobic conditions for in vitro growth. However, the mammalian gut is not devoid of oxygen, and C. difficile tolerates moderate oxidative stress in vivo. The C. difficile genome encodes several antioxidant proteins, including a predicted superoxide reductase (SOR) that is upregulated upon exposure to antimicrobial peptides. The goal of this study was to establish SOR enzymatic activity and assess its role in protecting C. difficile against oxygen exposure. Insertional inactivation of sor rendered C. difficile more sensitive to superoxide, indicating that SOR contributes to antioxidant defense. Heterologous C. difficile sor expression in Escherichia coli conferred protection against superoxide-dependent growth inhibition, and the corresponding cell lysates showed superoxide scavenging activity. Finally, a C. difficile SOR mutant exhibited global proteome changes under oxygen stress when compared to the parent strain. Collectively, our data establish the enzymatic activity of C. difficile SOR, confirm its role in protection against oxidative stress, and demonstrate SOR's broader impacts on the C. difficile vegetative cell proteome.IMPORTANCEClostridioides difficile is an important pathogen strongly associated with healthcare settings and capable of causing severe diarrheal disease. While considered a strict anaerobe in vitro, C. difficile has been shown to tolerate low levels of oxygen in the mammalian host. Among other well-characterized antioxidant proteins, the C. difficile genome encodes a predicted superoxide reductase (SOR), an understudied component of antioxidant defense in pathogens. The significance of the research reported herein is the characterization of SOR's enzymatic activity, including confirmation of its role in protecting C. difficile against oxidative stress. This furthers our understanding of C. difficile pathogenesis and presents a potential new avenue for targeted therapies.

Taxonomic and phenotypic analysis of bifidobacteria isolated from IBD patients as potential probiotic strains

BackgroundInflammatory Bowel Diseases (IBD) are a major public health issue with unclear aetiology. Changes in the composition and functionality of the intestinal microbiota are associated with these pathologies, including the depletion of strict anaerobes such as Feacalibacterium prausnitzii. Less evidence is observed for depletion in other anaerobes, among which bifidobacteria. This study characterized the taxonomic and functional diversity of bifidobacteria isolated from the human intestinal microbiota in active and non-active IBD patients by a culturomics approach and evaluated if these bifidobacteria might be used as probiotics for gut health.ResultsA total of 341 bifidobacteria were isolated from the intestinal microbiota of IBD patients (52 Crohn’s disease and 26 ulcerative colitis patients), with a high proportion of Bifidobacterium dentium strains (28% of isolated bifidobacteria). In ulcerative colitis, the major species identified was B. dentium (39% of isolated bifidobacteria), in active and non-active ulcerative colitis. In Crohn’s disease, B. adolescentis was the major species isolated from non-active patients (40%), while similar amounts of B. dentium and B. adolescentis were found in active Crohn’s disease patients. The relative abundance of B. dentium was increased with age, both in Crohn’s disease and ulcerative colitis and active and non-active IBD patients. Antibacterial capacities of bifidobacteria isolated from non-active ulcerative colitis against Escherichia coli LF82 and Salmonella enterica ATCC 14028 were observed more often compared to strains isolated from active ulcerative colitis. Finally, B. longum were retained as strains with the highest probiotic potential as they were the major strains presenting exopolysaccharide synthesis, antibacterial activity, and anti-inflammatory capacities. Antimicrobial activity and EPS synthesis were further correlated to the presence of antimicrobial and EPS gene clusters by in silico analysis.ConclusionsDifferent bifidobacterial taxonomic profiles were identified in the microbiota of IBD patients. The most abundant species were B. dentium, mainly associated to the microbiota of ulcerative colitis patients and B. adolescentis, in the intestinal microbiota of Crohn’s disease patients. Additionally, the relative abundance of B. dentium significantly increased with age. Furthermore, this study evidenced that bifidobacteria with probiotic potential (antipathogenic activity, exopolysaccharide production and anti-inflammatory activity), especially B. longum strains, can be isolated from the intestinal microbiota of both active and non-active Crohn’s disease and ulcerative colitis patients.

Conditional essentiality of the 11-subunit complex I-like enzyme in strict anaerobes: the case of Desulfitobacterium hafniense strain DCB-2.

In oxidative phosphorylation, respiratory complex I serves as an entry point in the electron transport chain for electrons generated in catabolic processes in the form of NADH. An ancestral version of the complex, lacking the NADH-oxidising module, is encoded in a significant number of bacterial genomes. Amongst them is Desulfitobacterium hafniense, a strict anaerobe capable of conserving energy via organohalide respiration. This study investigates the role of the complex I-like enzyme in D. hafniense energy metabolism using rotenone as a specific complex I inhibitor under different growth conditions. The investigation revealed that the complex I-like enzyme was essential for growth with lactate and pyruvate but not in conditions involving H2 as an electron donor. In addition, a previously published proteomic dataset of strain DCB-2 was analysed to reveal the predominance of the complex under different growth conditions and to identify potential redox partners. This approach revealed seven candidates with expression patterns similar to Nuo homologues, suggesting the use of diverse electron sources. Based on these results, we propose a model where the complex I-like enzyme serves as an electron entry point into the respiratory chain for substrates delivering electrons within the cytoplasm, such as lactate or pyruvate, with ferredoxins shuttling electrons to the complex.

Comparison of the penile microbiome in infant male circumcision: Mogen clamp versus Shangring

BackgroundThis study aimed to characterise the infant penile (coronal sulcus) microbiome and the effects of early infant male circumcision (EIMC), following a standard surgical method (Mogen Clamp) and a non-surgical alternative (ShangRing). MethodsWe collected coronal sulcus swabs at baseline and on days 7 and 14 post-circumcision from infants assigned to receive EIMC by Mogen Clamp (n = 15) or ShangRing (n = 15), in a randomised trial in Rakai and Kakuuto, Uganda. We used 16S rRNA gene-based sequencing and broad-coverage qPCR to characterise the infant penile microbiome and assess the effects of EIMC in both study arms. FindingsPrior to EIMC, the infant penile microbiome had a mixture of facultative and strict anaerobes. In both study arms, EIMC caused penile microbiome proportional abundance changes characterised by decreases in penile anaerobes [ShangRing Prevotella: -15.0%, (SD=19.1); Mogen clamp Prevotella: -3.6% (11.2); ShangRing Veillonella: -11.3% (17.2); Mogen clamp Veillonella: -2.6% (11.8)] and increases in skin-associated facultative anaerobes [ShangRing Corynebacterium: 24.9%, (22.4); Mogen clamp Corynebacterium: 4.7% (21.3); ShangRing Staphylococcus: 21.1% (20.5); Mogen clamp Staphylococcus: 18.1% (20.1)]. Clostridium tetani was not detected during the study. InterpretationMogen Clamp and ShangRing EIMC both changed the composition of the infant penile microbiome by reducing the proportional abundances of anaerobes and uropathogens, which is consistent with medical male circumcision findings in adults. C. tetani was not increased by either EIMC method. FundingBill and Melinda Gates Foundation.

Vaginal microbes alter epithelial transcriptome and induce epigenomic modifications providing insight into mechanisms for susceptibility to adverse reproductive outcomes.

The cervicovaginal microbiome is highly associated with women's health, with microbial communities dominated by Lactobacillus species considered optimal. Conversely, a lack of lactobacilli and a high abundance of strict and facultative anaerobes, including Gardnerella vaginalis, have been associated with adverse reproductive outcomes. However, how host-microbial interactions alter specific molecular pathways and impact cervical and vaginal epithelial function remains unclear. Using RNA-sequencing, we characterized the in vitro cervicovaginal epithelial transcriptional response to different vaginal bacteria and their culture supernatants. We showed that G. vaginalis upregulates genes associated with an activated innate immune response. Unexpectedly, G. vaginalis specifically induced inflammasome pathways through activation of NLRP3-mediated increases in caspase-1, IL-1β and cell death, while live L. crispatus had minimal transcriptomic changes on epithelial cells. L. crispatus culture supernatants resulted in a shift in the epigenomic landscape of cervical epithelial cells that was confirmed by ATAC-sequencing showing reduced chromatin accessibility. This study reveals new insights into host-microbe interactions in the lower reproductive tract and suggests potential therapeutic strategies leveraging the vaginal microbiome to improve reproductive health.

Functional characterization of Fur from the strict anaerobe Clostridioides difficile provides insight into its redox-driven regulatory capacity.

Clostridioides (formerly Clostridium) difficile is a leading cause of infectious diarrhea associated with antibiotic therapy. The ability of this anaerobic pathogen to acquire enough iron to proliferate under iron limitation conditions imposed by the host largely determines its pathogenicity. However, since high intracellular iron catalyzes formation of deleterious reactive hydroxyl radicals, iron uptake is tightly regulated at the transcriptional level by the ferric uptake regulator Fur. Several studies relate lacking a functional fur gene in C. difficile cells to higher oxidative stress sensitivity, colonization defect and less toxigenicity, although Fur does not appear to directly regulate either oxidative stress response genes or pathogenesis genes. In this work, we report the functional characterization of C. difficile Fur and describe an additional oxidation sensing Fur-mediated mechanism independent of iron, which affects Fur DNA-binding. Using electrophoretic mobility shift assays, we show that Fur binding to the promoters of fur, feoA and fldX genes, identified as iron and Fur-regulated genes in vivo, is specific and does not require co-regulator metal under reducing conditions. Fur treatment with H2O2 produces dose-dependent soluble high molecular weight species unable to bind to target promoters. Moreover, Fur oligomers are dithiotreitol sensitive, highlighting the importance of some interchain disulfide bond(s) for Fur oligomerization, and hence for activity. Additionally, the physiological electron transport chain NADPH-thioredoxin reductase/thioredoxin from Escherichia coli reduces inactive oligomerized C. difficile Fur that recovers activity. In conjunction with available transcriptomic data, these results suggest a previously underappreciated complexity in the control of some members of the Fur regulon that is based on Fur redox properties and might be fundamental for the adaptive response of C. difficile during infection.

Bacterial microbiota shifts in vacuum-packed beef during storage at different temperatures: Impacts on blown pack spoilage

We aimed to evaluate the bacterial growth and diversity in vacuum-packed beef bags stored at different temperatures and to monitor blown-pack spoilage. We used culture-based methods and high-throughput sequencing to study the development of the main bacterial groups naturally present in beef stored at 4 and 15 °C for 28 days. The growth of sulfite-reducing clostridium (SRC) was impaired in beef bags stored at 4 °C; significant differences among SRC counts were observed in beef bags stored at 4 and 15 °C on days 14, 21, and 28 (P = 0.001). Blown pack was observed in most beef bags stored at 15 °C, from day 14 to day 28, but not in beef bags stored at 4 °C. A storage temperature of 4 °C was able to maintain a stable bacterial microbiota (most prevalent: Photobacterium, Hafnia-Obesumbacterium, and Lactococcus). Remarkable changes in microbial abundance occurred at 15 °C from day 14 to day 28, with a predominance of strict anaerobes (Bacteroides) and the presence of Clostridium spp. The relative frequencies of strict anaerobes and Clostridium were statistically higher in the beef bags stored at 15 °C (P < 0.001 and P = 0.004, respectively). The temperature influenced the microbial counts and relative abundance of spoilage bacteria, leading to blown pack spoilage.

Improved microalgae growth and lipid production in anaerobic digestate with ultraviolet radiation pretreatment

Inappropriate sterilization strategies inhibit microalgal growth when culturing microalgae with anaerobic digestate. This study aimed to scientifically select a low-cost disinfection pretreatment of anaerobic digestate for large-scale microalgae cultivations. In this work, three different methods, including autoclaving, ultraviolet or NaClO treatments, were employed to sterilize the municipal anaerobic digestate. Scenedesmus quadricauda was then cultured in diluted liquid digestate for the simultaneous lipid production and nutrient removal. The results indicated that the growth of S. quadricauda was inhibited after NaClO treatment due to the residual free chlorine. The 15-min ultraviolet effectively mitigated microbial contamination and increasing nutrient availability, enhancing the electron transport of microalgal photosynthesis. After 6-days cultivation, the microalgal biomass concentration of the ultraviolet group was 1.09 g/L, comparable to that of the autoclaving group (1.15 g/L). High nutrient removal efficiency was observed: COD (93.30 %), NH4+-N (92.56 %), TN (85.82 %) and TP (95.12 %). Moreover, S. quadricauda outcompeted the indigenous microorganisms, contributing to its dominance in the culture system of ultraviolet group. The facultative anaerobe Comamonadaceae and aerobes Moraxellaceae, rather than strict anaerobe Paludibacteraceae and Bacteroidetes_vadinHA17, played vital roles in synergistic removal of contaminants by bacteria and algae. The potential competition for nitrogen and phosphorus by bacteria contributed to the ultraviolet group having the greatest lipid content (48.19 %). Therefore, this work suggested using 15-min ultraviolet treatment for anaerobic digestate in large-scale microalgae cultivation.

A Versatile Aldehyde: Ferredoxin Oxidoreductase from the Organic Acid Reducing Thermoanaerobacter sp. Strain X514.

Aldehyde:ferredoxin oxidoreductases (AORs) have been isolated and biochemically-characterized from a handful of anaerobic or facultative aerobic archaea and bacteria. They catalyze the ferredoxin (Fd)-dependent oxidation of aldehydes to acids. Recently, the involvement of AOR in the reduction of organic acids to alcohols with electrons derived from sugar or synthesis gas was demonstrated, with alcohol dehydrogenases (ADHs) carrying out the reduction of the aldehyde to the alcohol (AOR-ADH pathway). Here, we describe the biochemical characterization of an AOR of the thermophilic fermentative bacterium Thermoanaerobacter sp. strain X514 (AORX514). The putative aor gene (Teth514_1380) including a 6x-His-tag was introduced into the genome of the genetically-accessible, related species Thermoanaerobacter kivui. The protein was purified to apparent homogeneity, and indeed revealed AOR activity, as measured by acetaldehyde-dependent ferredoxin reduction. AORX514 was active over a wide temperature (10 to 95 °C) and pH (5.5 to 11.5) range, utilized a wide variety of aldehydes (short and branched-chained, aliphatic, aromatic) and resembles archaeal sensu stricto AORs, as the protein is active in a homodimeric form. The successful, recombinant production of AORX514 in a related, well-characterized and likewise strict anaerobe paves the road towards structure-function analyses of this enzyme and possibly similar oxygen-sensitive or W/Mo-dependent proteins in the future.

Advances in equol production: Sustainable strategies for unlocking soy isoflavone benefits

Daidzein is an abundant polyphenol present in soy and other legumes. It accumulates mostly in seed and seed pod during the plant development in response to biotic and abiotic stress. Due to the high consumption of soy and soy derivative food, daidzein is one of the most abundant polyphenols in human and cattle diets. Similar to other phytochemicals, daidzein is not absorbed or metabolized by human cells, but transformed by enzymatic cascades of the gut microbiome. The product of daidzein “fermentation” is equol and related molecules. Equol [7-hydroxy-3-(4'-hydroxyphenyl)-chroman] is considered the most active metabolite of all soybean isoflavones, presenting a high level of antioxidant and estrogenic activity. This bioactive molecule presents estrogen-like activity by specifically binding to 5α-dihydrotestosterone and inhibiting its binding to the androgen receptor. Biotransformation of daidzein into equol requires enzymes that are not widespread along all microbiome taxa but restricted to a relatively small number of enzymes from the family Eggerthellaceae (phylum Actinomycetota, formerly Actinobacteria), strict anaerobes that inhabit the colon of humans and anoxic areas of the intestinal tract of other mammals. The isolation and characterization of equol-producing bacteria from the human gut resulted in the identification of bacterial species that, due to their metabolic capacity, were named Adlercreutzia equolifaciens, Slackia equolifaciens, Slackia isoflavoniconvertens, among others. Combining genomic and biochemical analysis, the puzzle of diadzein biotransformation was decoded. It includes an initial step catalyzed by β-glucosidases, enzymes that are commonly found in the intestinal tract and deglycosylate the natural form of daidzein in the plant, followed by the action of bacterial enzymes daidzein reductase, dihydrodaidzein reductase, tetrahydrodaidzein reductase, and dihydrodaidzein racemase. Undoubtedly, improving daidzein production from soy products or enhancing daidzein biotransformation by the human gut microbiome is of notorious biotechnological and biomedical interest. In addition, the chemistry catalyzed by the equol biosynthetic enzymes is of interest in the green chemistry field. However, the extreme oxygen sensitivity of the microbes and enzymes that participate in these pathways still impose a challenge for translating the knowledge obtained with recombinant enzymes and model organisms into the food industry. In this manuscript we review the process of equol biosynthesis and the effort in synthetic biology for production of equol into industrial or biomedical setups.

Alternatives to β-Lactams as Agents for the Management of Dentoalveolar Abscess.

Dentoalveolar abscess are localized infections within the tooth or the surrounding alveolar bone, often resulting from untreated dental caries or dental trauma causing alveolar bone resorption or even loss. Serious consequences arising from the spread of a dental abscess can often lead to significant morbidity and mortality. The acute dentoalveolar abscess is a polymicrobial infection comprising strict anaerobes, such as anaerobic cocci i.e., Prevotella fusobacterium species, and facultative anaerobes i.e., Streptococci viridians and Streptococcus anginosus. Moreover, inappropriately managed dental infections can progress to severe submandibular space infections with associated serious complications, such as sepsis and airway obstruction. An audit of the Hull Royal Infirmary between 1999 and 2004 showed an increase in the number of patients presenting to oral and maxillofacial surgery services with dental sepsis. Thus, the scientific community is forced to focus on treatment strategies for the management of dentoalveolar abscess (DAA) and other related dental problems. The current treatment includes antibiotic therapy, including β-lactams and non-β- lactams drugs, but it leads to the development of resistant microorganisms due to improper and wide usage. Furthermore, the currently used β-lactam therapeutics is non-specific and easily hydrolyzed by the β-lactamase enzymes. Thus, the research focused on the non-β-lactams that can be the potential pharmacophore and helpful in the management of DAA, as the appropriate use and choice of antibiotics in dentistry plays an important role in antibiotic stewardship. The newer target for the choice is NLRP inflammasome, which is the major chemical mediator involved in dental problems. This review focused on pathogenesis and current therapeutics for the treatment of dentoalveolar abscesses.

A novel system to culture human intestinal organoids under physiological oxygen content to study microbial-host interaction.

Mechanistic investigation of host-microbe interactions in the human gut are hindered by difficulty of co-culturing microbes with intestinal epithelial cells. On one hand the gut bacteria are a mix of facultative, aerotolerant or obligate anaerobes, while the intestinal epithelium requires oxygen for growth and function. Thus, a coculture system that can recreate these contrasting oxygen requirements is critical step towards our understanding microbial-host interactions in the human gut. Here, we demonstrate Intestinal Organoid Physoxic Coculture (IOPC) system, a simple and cost-effective method for coculturing anaerobic intestinal bacteria with human intestinal organoids (HIOs). Using commensal anaerobes with varying degrees of oxygen tolerance, such as nano-aerobe Bacteroides thetaiotaomicron and strict anaerobe Blautia sp., we demonstrate that IOPC can successfully support 24-48 hours HIO-microbe coculture. The IOPC recapitulates the contrasting oxygen conditions across the intestinal epithelium seen in vivo. The IOPC cultured HIOs showed increased barrier integrity, and induced expression of immunomodulatory genes. A transcriptomic analysis suggests that HIOs from different donors show differences in the magnitude of their response to coculture with anaerobic bacteria. Thus, the IOPC system provides a robust coculture setup for investigating host-microbe interactions in complex, patient-derived intestinal tissues, that can facilitate the study of mechanisms underlying the role of the microbiome in health and disease.

Probiotic and postbiotic analytical methods: a perspective of available enumeration techniques.

Probiotics are the largest non-herbal/traditional dietary supplements category worldwide. To be effective, a probiotic strain must be delivered viable at an adequate dose proven to deliver a health benefit. The objective of this article is to provide an overview of the various technologies available for probiotic enumeration, including a general description of each technology, their advantages and limitations, and their potential for the future of the probiotics industry. The current "gold standard" for analytical quantification of probiotics in the probiotic industry is the Plate Count method (PC). PC measures the bacterial cell's ability to proliferate into detectable colonies, thus PC relies on cultivability as a measure of viability. Although viability has widely been measured by cultivability, there has been agreement that the definition of viability is not limited to cultivability. For example, bacterial cells may exist in a state known as viable but not culturable (VBNC) where the cells lose cultivability but can maintain some of the characteristics of viable cells as well as probiotic properties. This led to questioning the association between viability and cultivability and the accuracy of PC in enumerating all the viable cells in probiotic products. PC has always been an estimate of the number of viable cells and not a true cell count. Additionally, newer probiotic categories such as Next Generation Probiotics (NGPs) are difficult to culture in routine laboratories as NGPs are often strict anaerobes with extreme sensitivity to atmospheric oxygen. Thus, accurate quantification using culture-based techniques will be complicated. Another emerging category of biotics is postbiotics, which are inanimate microorganisms, also often referred to as tyndallized or heat-killed bacteria. Obviously, culture dependent methods are not suitable for these products, and alternative methods are needed for their quantification. Different methodologies provide a more complete picture of a heterogeneous bacterial population versus PC focusing exclusively on the eventual multiplication of the cells. Alternative culture-independent techniques including real-time PCR, digital PCR and flow cytometry are discussed. These methods can measure viability beyond cultivability (i.e., by measuring cellular enzymatic activity, membrane integrity or membrane potential), and depending on how they are designed they can achieve strain-specific enumeration.

Contact with adult hens affects the composition of skin and respiratory tract microbiota in newly hatched chicks

Chickens in commercial production are hatched in hatcheries without any contact with their parents and colonization of their skin and respiratory tract is therefore dependent on environmental sources only. However, since chickens evolved to be hatched in nests, in this study we evaluated the importance of contact between hens and chicks for the development of chicken skin and tracheal microbiota. Sequencing of PCR amplified V3/V4 variable regions of the 16S rRNA gene showed that contact with adult hens decreased the abundance of E. coli, Proteus mirabilis and Clostridium perfringens both in skin and the trachea, and Acinetobacter johnsonii and Cutibacterium acnes in skin microbiota only. These species were replaced by Lactobacillus gallinarum, Lactobacillus aviarius, Limosilactobacillus reuteri, and Streptococcus pasterianus in the skin and tracheal microbiota of contact chicks. Lactobacilli can be therefore investigated for their probiotic effect in respiratory tract in the future. Skin and respiratory microbiota of contact chickens was also enriched for Phascolarctobacterium, Succinatimonas, Flavonifractor, Blautia, and [Ruminococcus] torque though, since these are strict anaerobes from the intestinal tract, it is likely that only DNA from nonviable cells was detected for these taxa.

Defining microbial community functions in chronic human infection with metatranscriptomics.

The microbial diversity in polymicrobial infections (PMIs) allows for community members to establish interactions with one another, which can result in enhanced disease outcomes such as increased antibiotic tolerance and chronicity. Chronic PMIs result in large burdens on health systems, as they affect a significant proportion of the population and are expensive and difficult to treat. However, investigations into physiology of microbial communities in actual human infection sites are lacking. Here, we highlight that the predominant functions in chronic PMIs differ, and anaerobes, often described as bystanders, may be significant in the progression of chronic infections. Determining the community structure and functions in PMIs is a critical step toward understanding the molecular mechanisms that increase the virulence potential of the microbial community in these environments.

Exploring oxidative stress pathways in Geobacter sulfurreducens: the redox network between MacA peroxidase and triheme periplasmic cytochromes.

The recent reclassification of the strict anaerobe Geobacter sulfurreducens bacterium as aerotolerant brought attention for oxidative stress protection pathways. Although the electron transfer pathways for oxygen detoxification are not well established, evidence was obtained for the formation of a redox complex between the periplasmic triheme cytochrome PpcA and the diheme cytochrome peroxidase MacA. In the latter, the reduction of the high-potential heme triggers a conformational change that displaces the axial histidine of the low-potential heme with peroxidase activity. More recently, a possible involvement of the triheme periplasmic cytochrome family (PpcA-E) in the protection from oxidative stress in G. sulfurreducens was suggested. To evaluate this hypothesis, we investigated the electron transfer reaction and the biomolecular interaction between each PpcA-E cytochrome and MacA. Using a newly developed method that relies on the different NMR spectral signatures of the heme proteins, we directly monitored the electron transfer reaction from reduced PpcA-E cytochromes to oxidized MacA. The results obtained showed a complete electron transfer from the cytochromes to the high-potential heme of MacA. This highlights PpcA-E cytochromes' efficient role in providing the necessary reducing power to mitigate oxidative stress situations, hence contributing to a better knowledge of oxidative stress protection pathways in G. sulfurreducens.