The predominant lactic acid bacteria and yeasts involved in the spontaneous fermentation of millet during the production of the traditional porridge Hausa koko in Ghana.

Spontaneous fermentation of cereals like millet involves a diverse population of microbes from various sources, including raw materials, processing equipment, fermenting receptacles, and the environment. Here, we present data on the predominant microbial species and their succession at each stage of the Hausa koko production process from five regions of Ghana. The isolates were enumerated using selective media, purified, and phenotypically characterised. The LAB isolates were further characterised by 16S rRNA Sanger sequencing, typed using (GTG)5 repetitive-PCR, and whole genome sequencing, while 28S rRNA Sanger sequencing was performed for yeast identification. The pH of the millet grains ranged from mean values of 6.02-6.53 to 3.51-3.99 in the final product, depending on the processors. The mean LAB and yeast counts increased during fermentation then fell to final counts of log 2.77-3.95 CFU/g for LAB and log 2.10-2.98 CFU/g for yeast in Hausa koko samples. At the various processing stages, the counts of LAB and yeast revealed significant variations (p < 0.0001). The species of LAB identified in this study were Limosilactobacillus pontis, Pediococcus acidilactici, Limosilactobacillus fermentum, Limosilactobacillus reuteri, Pediococcus pentosaceus, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Schleiferilactobacillus harbinensis, and Weissella confusa. The yeasts were Saccharomyces cf. cerevisiae/paradoxus, Saccharomyces cerevisiae, Pichia kudriavzevii, Clavispora lusitaniae and Candida tropicalis. The identification and sequencing of these novel isolates and how they change during the fermentation process will pave the way for future controlled fermentation, safer starter cultures, and identifying optimal stages for starter culture addition or nutritional interventions. These LAB and yeast species are linked to many indigenous African fermented foods, potentially acting as probiotics in some cases. This result serves as the basis for further studies into the technological and probiotic potential of these Hausa koko microorganisms.

Desulfovibrio diazotrophicus sp. nov., a sulfate-reducing bacterium from the human gut capable of nitrogen fixation.

Sulfate-reducing bacteria (SRB) are widespread in human guts, yet their expansion has been linked to colonic diseases. We report the isolation, sequencing and physiological characterization of strain QI0027T , a novel SRB species belonging to the class Desulfovibrionia. Metagenomic sequencing of stool samples from 45 Chinese individuals, and comparison with 1690 Desulfovibrionaceae metagenome-assembled genomes recovered from humans of diverse geographic locations, revealed the presence of QI0027T in 22 further individuals. QI0027T encoded nitrogen fixation genes and based on the acetylene reduction assay, actively fixed nitrogen. Transcriptomics revealed that QI0027T overexpressed 42 genes in nitrogen-limiting conditions compared to cultures supplemented with ammonia, including genes encoding nitrogenases, a urea uptake system and the urease complex. Reanalyses of 835 public stool metatranscriptomes showed that nitrogenase genes from Desulfovibrio bacteria were expressed in six samples suggesting that nitrogen fixation might be active in the gut environment. Although frequently thought of as a nutrient-rich environment, nitrogen fixation can occur in the human gut. Animals are often nitrogen limited and have evolved diverse strategies to capture biologically active nitrogen, ranging from amino acid transporters to stable associations with beneficial microbes that provide fixed nitrogen. QI0027T is the first Desulfovibrio human isolate for which nitrogen fixation has been demonstrated, suggesting that some sulfate-reducing bacteria could also play a role in the availability of nitrogen in the gut.

An optimised protocol for detection of SARS-CoV-2 in stool.

BACKGROUND: SARS-CoV-2 has been detected in stool samples of COVID-19 patients, with potential implications for faecal-oral transmission. Compared to nasopharyngeal swab samples, the complexity of the stool matrix poses a challenge in the detection of the virus that has not yet been solved. However, robust and reliable methods are needed to estimate the prevalence and persistence of SARS-CoV-2 in the gut and to ensure the safety of microbiome-based procedures such as faecal microbiota transplant (FMT). The aim of this study was to establish a sensitive and reliable method for detecting SARS-CoV-2 in stool samples. RESULTS: Stool samples from individuals free of SARS-CoV-2 were homogenised in saline buffer and spiked with a known titre of inactivated virus ranging from 50 to 750 viral particles per 100 mg stool. Viral particles were concentrated by ultrafiltration, RNA was extracted, and SARS-CoV-2 was detected via real-time reverse-transcription polymerase chain reaction (RT-qPCR) using the CDC primers and probes. The RNA extraction procedure we used allowed for the detection of SARS-CoV-2 via RT-qPCR in most of the stool samples tested. We could detect as few as 50 viral particles per 100 mg of stool. However, high variability was observed across samples at low viral titres. The primer set targeting the N1 region provided more reliable and precise results and for this primer set our method had a limit of detection of 1 viral particle per mg of stool. CONCLUSIONS: Here we describe a sensitive method for detecting SARS-CoV-2 in stool samples. This method can be used to establish the persistence of SARS-CoV-2 in stool and ensure the safety of clinical practices such as FMT.

Lactobacillus garii sp. nov., isolated from a fermented cassava product.

A novel Gram-positive, catalase negative, rod-shaped strain, FI11369T, was isolated from gari, a traditional West African fermented food derived from cassava. Based on 16S rRNA gene sequence similarity, the closest type strains were Lactobacillus xiangfangensis LMG 26013T (99.4 % similarity), Lactobacillus plajomi NBRC 107333T (99.1 %), Lactobacillus paraplantarum DSM 10667T (99.1 %), Lactobacillus pentosus DSM 20314T (99.0 %), Lactobacillus plantarum subsp. plantarum ATCC 14917T (99.0 %), Lactobacillus modestisalitolerans NBRC 107235T (98.9 %), Lactobacillus plantarum subsp. argentoratensis DSM 16365T (98.9 %) and Lactobacillus daowaiensis NCIMB 15183T (98.8 %). The genome of strain FI11369T was sequenced and the average nucleotide identity (ANI) was compared with its closest relatives. ANI analysis showed that the closest relative, L. xiangfangensis DSM 27103T, had only a 82.4 % similarity. The main fatty acids of FI11369T were saturated C16 : 0 (18.2 %), unsaturated C18 : 1 ω9c (43.8 %) and cyclopropane C19 : 0 cyclo (ω10c and/or ω6; 22.5 %). Based on the genotypic and phenotypic data obtained in this study, a novel Lactobacillus species, Lactobacillus garii sp. nov., with the type strain FI11369T (=NCIMB 15148=DSM 108249), is proposed.

Deep-sea mussels from a hybrid zone on the Mid-Atlantic Ridge host genetically indistinguishable symbionts.

The composition and diversity of animal microbiomes is shaped by a variety of factors, many of them interacting, such as host traits, the environment, and biogeography. Hybrid zones, in which the ranges of two host species meet and hybrids are found, provide natural experiments for determining the drivers of microbiome communities, but have not been well studied in marine environments. Here, we analysed the composition of the symbiont community in two deep-sea, Bathymodiolus mussel species along their known distribution range at hydrothermal vents on the Mid-Atlantic Ridge, with a focus on the hybrid zone where they interbreed. In-depth metagenomic analyses of the sulphur-oxidising symbionts of 30 mussels from the hybrid zone, at a resolution of single nucleotide polymorphism analyses of ~2500 orthologous genes, revealed that parental and hybrid mussels (F2-F4 generation) have genetically indistinguishable symbionts. While host genetics does not appear to affect symbiont composition in these mussels, redundancy analyses showed that geographic location of the mussels on the Mid-Atlantic Ridge explained most of the symbiont genetic variability compared to the other factors. We hypothesise that geographic structuring of the free-living symbiont population plays a major role in driving the composition of the microbiome in these deep-sea mussels.

Genomic analysis of a rare recurrent Listeria monocytogenes prosthetic joint infection indicates a protected niche within biofilm on prosthetic materials.

Listeria monocytogenes is a rare cause of prosthetic joint infections (PJI). In this study, we describe a case of recurrent L. monocytogenes infections, 39 months apart, following debridement and retention of a prosthetic hip. Despite numerous studies reporting persistent L. monocytogenes in human infections, the genomic and phenotypic changes that clinically relevant strains undergo in the host are poorly understood. Improved knowledge of how PJI occurs is needed to improve the management of prosthetic infections. We used a combination of long- and short-read sequencing to identify any potential genomic differences between two L. monocytogenes isolates that occurred over 39-month incubation in the host. The isolates, QI0054 and QI0055, showed three single nucleotide polymorphisms and three insertions or deletions, suggesting that the recurrent infection was caused by the same strain. To identify potential differences in the capacity for persistence of these isolates, their biofilm-forming ability and potential to colonize prosthesis-relevant materials was investigated both in microtitre plates and on prosthetic material titanium, stainless steel 316 and ultra-high molecular weight polyethylene. Whilst the L. monocytogenes isolate from the most recent infection (QI0055) was able to form higher biofilm in microtitre plates, this did not lead to an increase in biomass on prosthetic joint materials compared to the initial isolate (QI0054). Both clinical isolates were able to form significantly more biofilm on the two metal prosthetic materials than on the ultra-high molecular weight polyethylene, in contrast to reference strain Scott A. Transcriptomics revealed 41 genes overexpressed in biofilm state and 643 in planktonic state. Moreover, genes with mutations were actively expressed in both isolates. We conclude the isolates are derived from the same strain and hypothesize that L. monocytogenes formed biofilm on the prosthetic joint materials, with minimal exposure to stresses, which permitted their survival and growth.

Comparative proteomics of related symbiotic mussel species reveals high variability of host-symbiont interactions.

Deep-sea Bathymodiolus mussels and their chemoautotrophic symbionts are well-studied representatives of mutualistic host-microbe associations. However, how host-symbiont interactions vary on the molecular level between related host and symbiont species remains unclear. Therefore, we compared the host and symbiont metaproteomes of Pacific B. thermophilus, hosting a thiotrophic symbiont, and Atlantic B. azoricus, containing two symbionts, a thiotroph and a methanotroph. We identified common strategies of metabolic support between hosts and symbionts, such as the oxidation of sulfide by the host, which provides a thiosulfate reservoir for the thiotrophic symbionts, and a cycling mechanism that could supply the host with symbiont-derived amino acids. However, expression levels of these processes differed substantially between both symbioses. Backed up by genomic comparisons, our results furthermore revealed an exceptionally large repertoire of attachment-related proteins in the B. thermophilus symbiont. These findings imply that host-microbe interactions can be quite variable, even between closely related systems.

Genotypic and Phenotypic Characterization of Fecal Staphylococcus epidermidis Isolates Suggests Plasticity to Adapt to Different Human Body Sites.

Staphylococcus epidermidis is a commensal species that has been increasingly identified as a nosocomial agent. Despite the interest, little is known about the ability of S. epidermidis isolates to adapt to different ecological niches through comparisons at genotype or phenotype levels. One niche where S. epidermidis has been reported is the human gut. Here, we present three S. epidermidis strains isolated from feces and show that they are not phylogenetically distinct from S. epidermidis isolated from other human body sites. Both gut and skin strains harbored multiple genes associated with biofilm formation and showed similar levels of biofilm formation on abiotic surfaces. High-throughput physiological tests using the BIOLOG technology showed no major metabolic differences between isolates from stool, skin, or cheese, while an isolate from bovine mastitis showed more phenotypic variation. Gut and skin isolates showed the ability to metabolize glycine-conjugated bile acids and to grow in the presence of bile, but the gut isolates exhibited faster anaerobic growth compared to isolates of skin origin.

Functional diversity enables multiple symbiont strains to coexist in deep-sea mussels.

Genetic diversity of closely related free-living microorganisms is widespread and underpins ecosystem functioning, but most evolutionary theories predict that it destabilizes intimate mutualisms. Accordingly, strain diversity is assumed to be highly restricted in intracellular bacteria associated with animals. Here, we sequenced metagenomes and metatranscriptomes of 18 Bathymodiolus mussel individuals from four species, covering their known distribution range at deep-sea hydrothermal vents in the Atlantic. We show that as many as 16 strains of intracellular, sulfur-oxidizing symbionts coexist in individual Bathymodiolus mussels. Co-occurring symbiont strains differed extensively in key functions, such as the use of energy and nutrient sources, electron acceptors and viral defence mechanisms. Most strain-specific genes were expressed, highlighting their potential to affect fitness. We show that fine-scale diversity is pervasive in Bathymodiolus sulfur-oxidizing symbionts, and hypothesize that it may be widespread in low-cost symbioses where the environment, rather than the host, feeds the symbionts.

Fueled by methane: deep-sea sponges from asphalt seeps gain their nutrition from methane-oxidizing symbionts.

Sponges host a remarkable diversity of microbial symbionts, however, the benefit their microbes provide is rarely understood. Here, we describe two new sponge species from deep-sea asphalt seeps and show that they live in a nutritional symbiosis with methane-oxidizing (MOX) bacteria. Metagenomics and imaging analyses revealed unusually high amounts of MOX symbionts in hosts from a group previously assumed to have low microbial abundances. These symbionts belonged to the Marine Methylotrophic Group 2 clade. They are host-specific and likely vertically transmitted, based on their presence in sponge embryos and streamlined genomes, which lacked genes typical of related free-living MOX. Moreover, genes known to play a role in host-symbiont interactions, such as those that encode eukaryote-like proteins, were abundant and expressed. Methane assimilation by the symbionts was one of the most highly expressed metabolic pathways in the sponges. Molecular and stable carbon isotope patterns of lipids confirmed that methane-derived carbon was incorporated into the hosts. Our results revealed that two species of sponges, although distantly related, independently established highly specific, nutritional symbioses with two closely related methanotrophs. This convergence in symbiont acquisition underscores the strong selective advantage for these sponges in harboring MOX bacteria in the food-limited deep sea.

Complete Genome Sequence of Ochrobactrum haematophilum FI11154, Isolated from Kunu-Zaki, a Nigerian Millet-Based Fermented Food.

Ochrobactrum haematophilum FI11154 was isolated from kunu-zaki, a Nigerian traditional fermented millet-based food. Here, we present the first complete genome sequence of this species. The genome consists of five replicons and contains genes related to iron uptake and phosphatase activities.

Short-chain alkanes fuel mussel and sponge Cycloclasticus symbionts from deep-sea gas and oil seeps.

Cycloclasticus bacteria are ubiquitous in oil-rich regions of the ocean and are known for their ability to degrade polycyclic aromatic hydrocarbons (PAHs). In this study, we describe Cycloclasticus that have established a symbiosis with Bathymodiolus heckerae mussels and poecilosclerid sponges from asphalt-rich, deep-sea oil seeps at Campeche Knolls in the southern Gulf of Mexico. Genomic and transcriptomic analyses revealed that, in contrast to all previously known Cycloclasticus, the symbiotic Cycloclasticus appears to lack the genes needed for PAH degradation. Instead, these symbionts use propane and other short-chain alkanes such as ethane and butane as carbon and energy sources, thus expanding the limited range of substrates known to power chemosynthetic symbioses. Analyses of short-chain alkanes in the environment of the Campeche Knolls symbioses revealed that these are present at high concentrations (in the µM to mM range). Comparative genomic analyses revealed high similarities between the genes used by the symbiotic Cycloclasticus to degrade short-chain alkanes and those of free-living Cycloclasticus that bloomed during the Deepwater Horizon oil spill. Our results indicate that the metabolic versatility of bacteria within the Cycloclasticus clade is higher than previously assumed, and highlight the expanded role of these keystone species in the degradation of marine hydrocarbons.

Metabolic and physiological interdependencies in the Bathymodiolus azoricus symbiosis.

The hydrothermal vent mussel Bathymodiolus azoricus lives in an intimate symbiosis with two types of chemosynthetic Gammaproteobacteria in its gills: a sulfur oxidizer and a methane oxidizer. Despite numerous investigations over the last decades, the degree of interdependence between the three symbiotic partners, their individual metabolic contributions, as well as the mechanism of carbon transfer from the symbionts to the host are poorly understood. We used a combination of proteomics and genomics to investigate the physiology and metabolism of the individual symbiotic partners. Our study revealed that key metabolic functions are most likely accomplished jointly by B. azoricus and its symbionts: (1) CO2 is pre-concentrated by the host for carbon fixation by the sulfur-oxidizing symbiont, and (2) the host replenishes essential biosynthetic TCA cycle intermediates for the sulfur-oxidizing symbiont. In return (3), the sulfur oxidizer may compensate for the host's putative deficiency in amino acid and cofactor biosynthesis. We also identified numerous 'symbiosis-specific' host proteins by comparing symbiont-containing and symbiont-free host tissues and symbiont fractions. These proteins included a large complement of host digestive enzymes in the gill that are likely involved in symbiont digestion and carbon transfer from the symbionts to the host.

Genome sequence of the sulfur-oxidizing Bathymodiolus thermophilus gill endosymbiont.

Bathymodiolus thermophilus, a mytilid mussel inhabiting the deep-sea hydrothermal vents of the East Pacific Rise, lives in symbiosis with chemosynthetic Gammaproteobacteria within its gills. The intracellular symbiont population synthesizes nutrients for the bivalve host using the reduced sulfur compounds emanating from the vents as energy source. As the symbiont is uncultured, comprehensive and detailed insights into its metabolism and its interactions with the host can only be obtained from culture-independent approaches such as genomics and proteomics. In this study, we report the first draft genome sequence of the sulfur-oxidizing symbiont of B. thermophilus, here tentatively named Candidatus Thioglobus thermophilus. The draft genome (3.1 Mb) harbors 3045 protein-coding genes. It revealed pathways for the use of sulfide and thiosulfate as energy sources and encodes the Calvin-Benson-Bassham cycle for CO2 fixation. Enzymes required for the synthesis of the tricarboxylic acid cycle intermediates oxaloacetate and succinate were absent, suggesting that these intermediates may be substituted by metabolites from external sources. We also detected a repertoire of genes associated with cell surface adhesion, bacteriotoxicity and phage immunity, which may perform symbiosis-specific roles in the B. thermophilus symbiosis.

Biophysical and Population Genetic Models Predict the Presence of "Phantom" Stepping Stones Connecting Mid-Atlantic Ridge Vent Ecosystems.

Deep-sea hydrothermal vents are patchily distributed ecosystems inhabited by specialized animal populations that are textbook meta-populations. Many vent-associated species have free-swimming, dispersive larvae that can establish connections between remote populations. However, connectivity patterns among hydrothermal vents are still poorly understood because the deep sea is undersampled, the molecular tools used to date are of limited resolution, and larval dispersal is difficult to measure directly. A better knowledge of connectivity is urgently needed to develop sound environmental management plans for deep-sea mining. Here, we investigated larval dispersal and contemporary connectivity of ecologically important vent mussels (Bathymodiolus spp.) from the Mid-Atlantic Ridge by using high-resolution ocean modeling and population genetic methods. Even when assuming a long pelagic larval duration, our physical model of larval drift suggested that arrival at localities more than 150 km from the source site is unlikely and that dispersal between populations requires intermediate habitats ("phantom" stepping stones). Dispersal patterns showed strong spatiotemporal variability, making predictions of population connectivity challenging. The assumption that mussel populations are only connected via additional stepping stones was supported by contemporary migration rates based on neutral genetic markers. Analyses of population structure confirmed the presence of two southern and two hybridizing northern mussel lineages that exhibited a substantial, though incomplete, genetic differentiation. Our study provides insights into how vent animals can disperse between widely separated vent habitats and shows that recolonization of perturbed vent sites will be subject to chance events, unless connectivity is explicitly considered in the selection of conservation areas.

Abundant toxin-related genes in the genomes of beneficial symbionts from deep-sea hydrothermal vent mussels.

Bathymodiolus mussels live in symbiosis with intracellular sulfur-oxidizing (SOX) bacteria that provide them with nutrition. We sequenced the SOX symbiont genomes from two Bathymodiolus species. Comparison of these symbiont genomes with those of their closest relatives revealed that the symbionts have undergone genome rearrangements, and up to 35% of their genes may have been acquired by horizontal gene transfer. Many of the genes specific to the symbionts were homologs of virulence genes. We discovered an abundant and diverse array of genes similar to insecticidal toxins of nematode and aphid symbionts, and toxins of pathogens such as Yersinia and Vibrio. Transcriptomics and proteomics revealed that the SOX symbionts express the toxin-related genes (TRGs) in their hosts. We hypothesize that the symbionts use these TRGs in beneficial interactions with their host, including protection against parasites. This would explain why a mutualistic symbiont would contain such a remarkable 'arsenal' of TRGs.