Unveiling the pathogenic mechanisms of Clostridium perfringens toxins and virulence factors.

Clostridium perfringens causes multiple diseases in humans and animals. Its pathogenic effect is supported by a broad and heterogeneous arsenal of toxins and other virulence factors associated with a specific host tropism. Molecular approaches have indicated that most C. perfringens toxins produce membrane pores, leading to osmotic cell disruption and apoptosis. However, identifying mechanisms involved in cell tropism and selective toxicity effects should be studied more. The differential presence and polymorphisms of toxin-encoding genes and genes encoding other virulence factors suggest that molecular mechanisms might exist associated with host preference, receptor binding, and impact on the host; however, this information has not been reviewed in detail. Therefore, this review aims to clarify the current state of knowledge on the structural features and mechanisms of action of the major toxins and virulence factors of C. perfringens and discuss the impact of genetic diversity of toxinotypes in tropism for several hosts.

Modulating the early-life gut microbiota using pro-, pre-, and synbiotics to improve gut health, child development, and growth.

In children exposed to poor hygiene and sanitation, invasion of the gut by pathogenic microbes can result in a subclinical enteropathy termed "environmental enteric dysfunction" (EED) that contributes to undernutrition, growth faltering, and impaired organ development. EED may already be present by age 6-12 weeks; therefore, interventions that can be started early in life, and used alongside breastfeeding, are needed to prevent or ameliorate EED. A healthy gut microbiota is critical for intestinal development and repair, nutrient digestion and absorption, and resisting colonization or overgrowth by pathogens. However, its development can be impaired by several environmental factors. Dietary supplementation with pro-, pre-, or synbiotics may be a pragmatic and safe means of building the resilience of the developing gut microbiota against adverse environmental factors, thereby preventing EED.

The role of human milk fats in shaping neonatal development and the early life gut microbiota.

Human breast milk (HBM) is the main source of nutrition for neonates across the critical early-life developmental period. The highest demand for energy is due to rapid neurophysiological expansion post-delivery, which is largely met by human milk lipids (HMLs). These HMLs also play a prebiotic role and potentially promote the growth of certain commensal bacteria, which, via HML digestion, supports the additional transfer of energy to the infant. In tandem, HMLs can also exert bactericidal effects against a variety of opportunistic pathogens, which contributes to overall colonisation resistance. Such interactions are pivotal for sustaining homeostatic relationships between microorganisms and their hosts. However, the underlying molecular mechanisms governing these interactions remain poorly understood. This review will explore the current research landscape with respect to HMLs, including compositional considerations and impact on the early life gut microbiota. Recent papers in this field will also be discussed, including a final perspective on current knowledge gaps and potential next research steps for these important but understudied breast milk components.

A Scoping Review of Gut Microbiome and Bifidobacterium Research in Zimbabwe: Implications for Future Studies.

Background: Gut microbiota play a key role in host health, with certain Bifidobacterium strains critical for immune development. The healthy gut of breastfed infants is dominated by these pioneer microbes, especially the strains that feed on human milk oligosaccharides. Objective: This is a scoping review of gut microbiome research from Zimbabwe. It focuses on distribution and dynamic changes of bifidobacteria, and milk components that promote growth of microbes in infants, together with the distribution of associated gut microbes in adults. Design: Online databases were searched for publications from 2000 to 2023. Results and Analysis: Fourteen publications on microbiota of infants and adults were included in this scoping review. Most were cross-sectional, while three were clinical trials/cohort protocols. Publications focused on pediatrics (78.5%), pregnant women (14.3%), and men (7.2%). Zimbabwe has a high burden of HIV; hence 35.7% of study populations were delineated by HIV status. The laboratory methods used included shotgun metagenomics (62%) or 16S rRNA gene amplicon sequencing. Almost 85% of the studies focused on total microbiome profiles and rarely reported the distribution of different Bifidobacterium species and variants. None of the papers studied human breast milk composition. There were reports of reduced abundance of beneficial genera in pregnant women, children, and adolescents living with HIV. Additionally, gut microbiota was reported to be poorly predictive of child growth and vaccine response, though this was not conclusive. Conclusion: There are few studies that characterize the gut microbiome by Zimbabwe-based researchers. However, studies on strain level diversity of Bifidobacterium and other key microbes, and their role in health during and beyond infancy, lag behind in Zimbabwe and other low- and middle-income countries. Such cohorts are needed to inform future mechanistic studies and downstream translational work such as next-generation probiotics and prebiotics.

Draft genome sequence of Bifidobacterium breve DSM 32583, isolated from human milk.

Here, we describe the draft genome sequence of Bifidobacterium breve DSM 32583 isolated from human milk obtained from a healthy mother. Potentially, this B. breve strain could serve as a probiotic.

An update on the current understanding of the infant skin microbiome and research challenges.

Multiple factors contribute to establishment of skin microbial communities in early life, with perturbations in these ecosystems impacting health. This review provides an update on methods used to profile the skin microbiome and how this is helping enhance our understanding of infant skin microbial communities, including factors that influence composition and disease risk. We also provide insights into new interventional studies and treatments in this area. However, it is apparent that there are still research bottlenecks that include overreliance on high-income countries for skin microbiome 'surveys', many studies still focus solely on the bacterial microbiota, and also technical issues related to the lower microbial biomass of skin sites. These points link to pertinent open-research questions, such as how the whole infant skin microbiome interacts and how microbial-associated functions shape infant skin health and immunity.

A hybrid and poly-polish workflow for the complete and accurate assembly of phage genomes: a case study of ten przondoviruses.

Bacteriophages (phages) within the genus Przondovirus are T7-like podoviruses belonging to the subfamily Studiervirinae, within the family Autographiviridae, and have a highly conserved genome organisation. The genomes of these phages range from 37 to 42 kb in size, encode 50-60 genes and are characterised by the presence of direct terminal repeats (DTRs) flanking the linear chromosome. These DTRs are often deleted during short-read-only and hybrid assemblies. Moreover, long-read-only assemblies are often littered with sequencing and/or assembly errors and require additional curation. Here, we present the isolation and characterisation of ten novel przondoviruses targeting Klebsiella spp. We describe HYPPA, a HYbrid and Poly-polish Phage Assembly workflow, which utilises long-read assemblies in combination with short-read sequencing to resolve phage DTRs and correcting errors, negating the need for laborious primer walking and Sanger sequencing validation. Our assembly workflow utilised Oxford Nanopore Technologies for long-read sequencing for its accessibility, making it the more relevant long-read sequencing technology at this time, and Illumina DNA Prep for short-read sequencing, representing the most commonly used technologies globally. Our data demonstrate the importance of careful curation of phage assemblies before publication, and prior to using them for comparative genomics.

Allocoprobacillus halotolerans gen. nov., sp. nov and Coprobacter tertius sp. nov., isolated from human gut microbiota.

Two novel bacterial isolates were cultured from faecal samples of patients attending the Breast Care clinic at the Norwich and Norfolk University Hospital. Strain LH1062T was isolated from a 58-year-old female diagnosed with invasive adenocarcinoma with ductal carcinoma in situ. Strain LH1063T was isolated from a healthy 51-year-old female. Isolate LH1062T was predicted to be a potential novel genus most closely related to Coprobacillus, whilst LH1063T was predicted to be a novel species belonging to Coprobacter. Both strains were characterized by polyphasic approaches including 16S rRNA gene analysis, core-genome analysis, average nucleotide identity (ANI) comparisons and phenotypic analysis. Initial screening of the 16S rRNA gene of LH1062T returned a nucleotide identity of 93.4 % to Longibaculum muris. For LH1063T, nucleotide identity was a 92.6 % to Coprobacter secundus. Further investigations showed that LH1062T had a genome size of 2.9 Mb and G+C content of 31.3 mol %. LH1063T had a genome size of 3.3Mb and G+C content of 39.2 mol %. Digital DNA-DNA hybridization (dDDH) and ANI values of LH1062T with its closest relative, Coprobacillus cateniformis JCM 10604T, were 20.9 and 79.54 %, respectively. For LH1063T, the dDDH and ANI values with its closest relative, Coprobacter secundus 177T, were 19.3 and 77.81 %, respectively. Phenotypic testing confirmed that LH1062T could not be matched to a known validly published isolate in any database; thereby indicating a novel genus for which the name Allocoprobacillus gen. nov. is now proposed with LH1062T (=DSM 114537T=NCTC 14686T) being the type strain of the proposed novel species Allocoprobacillus halotolerans sp. nov. Strain LH1063T (=DSM 114538T=NCTC 14698T) fits within the genus Coprobacter and, it being the third species within this genus, the name Coprobacter tertius sp. nov. is proposed.

An efficient method for high molecular weight bacterial DNA extraction suitable for shotgun metagenomics from skin swabs.

The human skin microbiome represents a variety of complex microbial ecosystems that play a key role in host health. Molecular methods to study these communities have been developed but have been largely limited to low-throughput quantification and short amplicon-based sequencing, providing limited functional information about the communities present. Shotgun metagenomic sequencing has emerged as a preferred method for microbiome studies as it provides more comprehensive information about the species/strains present in a niche and the genes they encode. However, the relatively low bacterial biomass of skin, in comparison to other areas such as the gut microbiome, makes obtaining sufficient DNA for shotgun metagenomic sequencing challenging. Here we describe an optimised high-throughput method for extraction of high molecular weight DNA suitable for shotgun metagenomic sequencing. We validated the performance of the extraction method, and analysis pipeline on skin swabs collected from both adults and babies. The pipeline effectively characterised the bacterial skin microbiota with a cost and throughput suitable for larger longitudinal sets of samples. Application of this method will allow greater insights into community compositions and functional capabilities of the skin microbiome.

Noninvasive Fecal Cytokine and Microbiota Profiles Predict Commencement of Necrotizing Enterocolitis in a Proof-of-Concept Study.

Background and Aims: Necrotizing enterocolitis (NEC) is a life-threatening disease and the most common gastrointestinal emergency in premature infants. Accurate early diagnosis is challenging. Modified Bell's staging is routinely used to guide diagnosis, but early diagnostic signs are nonspecific, potentially leading to unobserved disease progression, which is problematic given the often rapid deterioration observed. We investigated fecal cytokine levels, coupled with gut microbiota profiles, as a noninvasive method to discover specific NEC-associated signatures that can be applied as potential diagnostic markers. Methods: Premature babies born below 32 weeks of gestation were admitted to the 2-site neonatal intensive care unit (NICU) of Imperial College hospitals (St. Mary's or Queen Charlotte's & Chelsea) between January 2011 and December 2012. During the NICU stay, expert neonatologists grouped individuals by modified Bell's staging (healthy, NEC1, NEC2/3) and fecal samples from diapers were collected consecutively. Microbiota profiles were assessed by 16S rRNA gene amplicon sequencing and cytokine concentrations were measured by V-Plex multiplex assays. Results: Early evaluation of microbiota profiles revealed only minor differences. However, at later time points, significant changes in microbiota composition were observed for Bacillota (adj. P = .0396), with Enterococcus being the least abundant in Bell stage 2/3 NEC. Evaluation of fecal cytokine levels revealed significantly higher concentrations of IL-1α (P = .045), IL-5 (P = .0074), and IL-10 (P = .032) in Bell stage 1 NEC compared to healthy individuals. Conclusion: Differences in certain fecal cytokine profiles in patients with NEC indicate their potential use as diagnostic biomarkers to facilitate earlier diagnosis. Additionally, associations between microbial and cytokine profiles contribute to improving knowledge about NEC pathogenesis.

Particular genomic and virulence traits associated with preterm infant-derived toxigenic Clostridium perfringens strains.

Clostridium perfringens is an anaerobic toxin-producing bacterium associated with intestinal diseases, particularly in neonatal humans and animals. Infant gut microbiome studies have recently indicated a link between C. perfringens and the preterm infant disease necrotizing enterocolitis (NEC), with specific NEC cases associated with overabundant C. perfringens termed C. perfringens-associated NEC (CPA-NEC). In the present study, we carried out whole-genome sequencing of 272 C. perfringens isolates from 70 infants across 5 hospitals in the United Kingdom. In this retrospective analysis, we performed in-depth genomic analyses (virulence profiling, strain tracking and plasmid analysis) and experimentally characterized pathogenic traits of 31 strains, including 4 from CPA-NEC patients. We found that the gene encoding toxin perfringolysin O, pfoA, was largely deficient in a human-derived hypovirulent lineage, as well as certain colonization factors, in contrast to typical pfoA-encoding virulent lineages. We determined that infant-associated pfoA+ strains caused significantly more cellular damage than pfoA- strains in vitro, and further confirmed this virulence trait in vivo using an oral-challenge C57BL/6 murine model. These findings suggest both the importance of pfoA+ C. perfringens as a gut pathogen in preterm infants and areas for further investigation, including potential intervention and therapeutic strategies.

Clinical NEC prevention practices drive different microbiome profiles and functional responses in the preterm intestine.

Preterm infants with very low birthweight are at serious risk for necrotizing enterocolitis. To functionally analyse the principles of three successful preventive NEC regimens, we characterize fecal samples of 55 infants (<1500 g, n = 383, female = 22) longitudinally (two weeks) with respect to gut microbiome profiles (bacteria, archaea, fungi, viruses; targeted 16S rRNA gene sequencing and shotgun metagenomics), microbial function, virulence factors, antibiotic resistances and metabolic profiles, including human milk oligosaccharides (HMOs) and short-chain fatty acids (German Registry of Clinical Trials, No.: DRKS00009290). Regimens including probiotic Bifidobacterium longum subsp. infantis NCDO 2203 supplementation affect microbiome development globally, pointing toward the genomic potential to convert HMOs. Engraftment of NCDO 2203 is associated with a substantial reduction of microbiome-associated antibiotic resistance as compared to regimens using probiotic Lactobacillus rhamnosus LCR 35 or no supplementation. Crucially, the beneficial effects of Bifidobacterium longum subsp. infantis NCDO 2203 supplementation depends on simultaneous feeding with HMOs. We demonstrate that preventive regimens have the highest impact on development and maturation of the gastrointestinal microbiome, enabling the establishment of a resilient microbial ecosystem that reduces pathogenic threats in at-risk preterm infants.

Microbial metabolites as modulators of the infant gut microbiome and host-microbial interactions in early life.

The development of infant gut microbiome is a pivotal process affecting the ecology and function of the microbiome, as well as host health. While the establishment of the infant microbiome has been of interest for decades, the focus on gut microbial metabolism and the resulting small molecules (metabolites) has been rather limited. However, technological and computational advances are now enabling researchers to profile the plethora of metabolites in the infant gut, allowing for improved understanding of how gut microbial-derived metabolites drive microbiome community structuring and host-microbial interactions. Here, we review the current knowledge on development of the infant gut microbiota and metabolism within the first year of life, and discuss how these microbial metabolites are key for enhancing our basic understanding of interactions during the early life developmental window.

Bifidobacterium bacteraemia is rare with routine probiotics use in preterm infants: A further case report with literature review.

Prophylactic administration of oral probiotics is associated with significant reductions in the morbidity and mortality of necrotising enterocolitis in preterm infants. We document the first case of Bifidobacterium longum subsp. infantis sub-clinical bacteraemia, in an extremely low birth weight preterm infant, since introduction of routine probiotic treatment at the Norfolk and Norwich University Hospital 10 years ago. Whole genome comparisons confirmed the isolated strain likely originated from the probiotic product.

Draft Genome Sequence of a Preterm Infant-Derived Isolate of Candida parapsilosis.

Candida parapsilosis is a human fungal pathogen of increasing incidence and causes invasive candidiasis, notably in preterm or low-birthweight neonates. Here, we present the genome sequence of C. parapsilosis NCYC 4289, a fecal isolate from a preterm male infant.

Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies.

Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate. Here we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbial ecology, bioinformatics, immunology, clinical microbiology and gnotobiology, and assess possible mechanisms by which the fetus might interact with microorganisms. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples or during DNA extraction and DNA sequencing. Furthermore, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology and the derivation of germ-free mammals. These conclusions are important to our understanding of human immune development and illustrate common pitfalls in the microbial analyses of many other low-biomass environments. The pursuit of a fetal microbiome serves as a cautionary example of the challenges of sequence-based microbiome studies when biomass is low or absent, and emphasizes the need for a trans-disciplinary approach that goes beyond contamination controls by also incorporating biological, ecological and mechanistic concepts.

Tumour microbiomes and Fusobacterium genomics in Vietnamese colorectal cancer patients.

Perturbations in the gut microbiome have been associated with colorectal cancer (CRC), with the colonic overabundance of Fusobacterium nucleatum shown as the most consistent marker. Despite its significance in the promotion of CRC, genomic studies of Fusobacterium is limited. We enrolled 43 Vietnamese CRC patients and 25 participants with non-cancerous colorectal polyps to study the colonic microbiomes and genomic diversity of Fusobacterium in this population, using a combination of 16S rRNA gene profiling, anaerobic microbiology, and whole genome analysis. Oral bacteria, including F. nucleatum and Leptotrichia, were significantly more abundant in the tumour microbiomes. We obtained 53 Fusobacterium genomes, representing 26 strains, from the saliva, tumour and non-tumour tissues of six CRC patients. Isolates from the gut belonged to diverse F. nucleatum subspecies (nucleatum, animalis, vincentii, polymorphum) and a potential new subspecies of Fusobacterium periodonticum. The Fusobacterium population within each individual was distinct and in some cases diverse, with minimal intra-clonal variation. Phylogenetic analyses showed that within four individuals, tumour-associated Fusobacterium were clonal to those isolated from non-tumour tissues. Genes encoding major virulence factors (Fap2 and RadD) showed evidence of horizontal gene transfer. Our work provides a framework to understand the genomic diversity of Fusobacterium within the CRC patients, which can be exploited for the development of CRC diagnostic and therapeutic options targeting this oncobacterium.

Microbiome-based interventions to modulate gut ecology and the immune system.

The gut microbiome lies at the intersection between the environment and the host, with the ability to modify host responses to disease-relevant exposures and stimuli. This is evident in how enteric microbes interact with the immune system, e.g., supporting immune maturation in early life, affecting drug efficacy via modulation of immune responses, or influencing development of immune cell populations and their mediators. Many factors modulate gut ecosystem dynamics during daily life and we are just beginning to realise the therapeutic and prophylactic potential of microbiome-based interventions. These approaches vary in application, goal, and mechanisms of action. Some modify the entire community, such as nutritional approaches or faecal microbiota transplantation, while others, such as phage therapy, probiotics, and prebiotics, target specific taxa or strains. In this review, we assessed the experimental evidence for microbiome-based interventions, with a particular focus on their clinical relevance, ecological effects, and modulation of the immune system.

The early-life gut microbiome and vaccine efficacy.

Vaccines are one of the greatest successes of public health, preventing millions of cases of disease and death in children each year. However, the efficacy of many vaccines can vary greatly between infants from geographically and socioeconomically distinct locations. Differences in the composition of the intestinal microbiome have emerged as one of the main factors that can account for variations in immunisation outcomes. In this Review, we assess the influence of the gut microbiota upon early life immunity, focusing on two important members of the microbiota with health-promoting and immunomodulatory properties: Bifidobacterium and Bacteroides. Additionally, we discuss their immune stimulatory microbial properties, interactions with the host, and their effect on vaccine responses and efficacy in infants. We also provide an overview of current microbiota-based approaches to enhance vaccine outcomes, and describe novel microbe-derived components that could lead to safer, more effective vaccines and vaccine adjuvants.