Cancerous tissue is a largely unexplored microbial niche that provides a unique environment for the colonization and growth of specific bacterial communities, and with it, the opportunity to identify novel bacterial species. Here, we report distinct features of a novel Fusobacterium species, F. sphaericum sp. nov. (Fs), isolated from primary colon adenocarcinoma tissue. We acquire the complete closed genome and associated methylome of this organism and phylogenetically confirm its classification into the Fusobacterium genus, with F. perfoetens as its closest neighbor. Fs is phenotypically and genetically distinct, with morphological analysis revealing its coccoid shape, that while similar to F. perfoetens is rare for most Fusobacterium members. Fs displays a metabolic profile and antibiotic resistance repertoire consistent with other Fusobacterium species. In vitro, Fs has adherent and immunomodulatory capabilities, as it intimately associates with human colon cancer epithelial cells and promotes IL-8 secretion. An analysis of the prevalence and abundance of Fs in > 20,000 human metagenomic samples shows that it is a rarely detected member within human stool with variable relative abundance, found in both healthy controls and patients with colorectal cancer (CRC). Our study sheds light on a novel bacterial species isolated directly from the human CRC tumor niche and given its in vitro interaction with cancer epithelial cells suggests that its role in human health and disease warrants further investigation.

The gut microbiota of bats is vital for their roles in health and the ecosystem, yet studies on hibernating bats in southwest China, particularly in the unique karst landscape of Guizhou, are limited. We captured three hibernating bat species-Pipistrellus (PB), Rhinolophus (RB), and Myotis (MB)-in Liping County, collecting rectal samples for 16S rRNA amplicon sequencing. Data processing involved Trimmomatic, Flash, and Qiime2 for operational taxonomic unit (OTU) standardization and species annotation via the Greengenes database. Differential abundance was analyzed using LEfSe, and diversity metrics were assessed through alpha and beta diversity analyses. The RB group was predominantly composed of Proteobacteria (80.99%), while MB and PB exhibited diverse compositions with significant OTU richness (729 in MB). Notable genera included Hafnia and Yersinia in RB and Cosenzaea myxofaciens in MB. High proportions of unclassified taxa were observed, particularly in RB (83.81%). Functional predictions indicated metabolic pathways, with a significant representation of human diseases in PB. Culturomics revealed the successful cultivation of Huaxiibacter chinensis and Enterobacter chengduensis from bats for the first time and appears to have identified a new bacterium that is likely closely related to Clostridium paraputrificum.IMPORTANCEOur research reveals significant differences in the composition and diversity of the gut microbiota among three bat groups (PB, MB, and RB) from Guizhou. While Proteobacteria predominates in all groups, its abundance varies. Notably, the high richness of operational taxonomic units (OTUs) in the MB group suggests a more diverse microbial ecosystem, underscoring the complex interactions between species diversity, diet, gut microbiota, and overall ecological dynamics in bats. Furthermore, the substantial presence of unknown bacterial species in their intestines highlights the critical importance of cultivation-based approaches. The presence of specific taxa may have potential health implications for both bats and humans. These findings emphasize the need for further investigations into the functional roles of these microbiota and their contributions to host health. Future research should focus on longitudinal studies to elucidate these intricate interactions.

The culture performance of the fastidious anaerobe agar (FAA-HB) was evaluated for fecal samples and candidate-species for EUCAST antimicrobial susceptibility testing (AST). Thirty-six anaerobic species were cultured from fecal samples and most EUCAST candidate-species exhibited confluent growth within 16-20 hours. The FAA-HB performed excellent for both culture and AST of a broad spectrum of anaerobic bacteria.

Research on Bacillus thuringiensis (Bt)-pest interactions has prioritized Cry toxin receptors, with limited attention to gut bacteria's role in modulating Bt sensitivity. This study identified two Enterobacter strains in Plutella xylostella with opposing effects on Cry1Ac susceptibility. Enterobacter hormaechei (PxG15) degraded Cry1Ac protoxin and activated the proPO-AS and JNK pathway, which reduced Cry1Ac's damage to the midgut while limiting the invasion of gut bacteria into the hemolymph. Although Enterobacter asburiae (PxG1) rapidly activated the proPO-AS, the JNK pathway was activated in a much slower and weaker mode when compared to PxG15, which attenuated the repairing efficiency of the midgut under the treatment of Cry1Ac, leading to death resulting from sepsis from the quick invasion of gut bacteria into the hemolymph. This study illustrates the intricate interrelationships among Cry1Ac, the pest's midgut bacteria, and its immune system, offering novel insights into how gut bacteria shape pest survival following Cry1Ac exposure.

Due to the heterogeneity of the human gut environment, the gut microbiota is complex and diverse, and has been insufficiently explored. In this study, one fresh fecal sample was cultured using 12 commercial or modified media and incubation of culture plates anaerobically and aerobically, the conventional experienced colony picking (ECP) was first used to isolate the colonies and obtain pure culture strains. On this basis, all the colonies grown on the culture plates were collected for culture-enriched metagenomic sequencing (CEMS), and the original sample was also subjected to direct culture-independent metagenomic sequencing (CIMS), the study compared the effects of three methods for analyzing the microbiota contained in the sample. It was found that compared with CEMS, conventional ECP failed to detect a large proportion of strains grown in culture media, resulting in missed detection of culturable microorganisms in the gut. Microbes identified by CEMS and CIMS showed a low degree of overlap (18% of species), whereas species identified by CEMS and CIMS alone accounted for 36.5% and 45.5%, respectively. It suggests that both culture-dependent and culture-independent approaches are essential in revealing gut microbial diversity. Moreover, based on the CEMS results, growth rate index (GRiD) values for various strains on different media were calculated to predict the optimal medium for bacterial growth; this method can be used to design new media for intestinal microbial isolation, promote the recovery of specific microbiota, and obtain new insights into the human microbiome diversity. This is among the first studies on CEMS of the human gut microbiota.

A brief history of techniques in anaerobic microbiology are presented leading up to the incorporation of several improvements we have used over the years to improve our culture of anaerobic microorganisms of environmental, industrial and clinical importance. Two overriding aspects from our combined 90 years of experience here are: the better one's control of anaerobic conditions and gas phases, the better results are obtained; techniques can and should be targeted for individual microorganisms and accompanying experiments. Continued improvements in anaerobic microbiology are expected and encouraged for the future.

Mulberry bacterial blight (MBB) is a complex and one of the devastating diseases of mulberry that causes serious reduction in the yield and quality of mulberry. In recent years, the transformation of sericulture industry, mulberry production system, and increasing seedling trade have resulted in the spread of MBB to different parts of China, posing a major economic threat to the farmers and industry. This study investigated the occurrence of MBB in eight provinces of China during years 2023 and 2024. The MBB disease samples were collected and the composition of the MBB pathogenic microbiome was analyzed by combining culturomics and metagenomic sequencing methods. A total of 498 bacterial strains were isolated and identified through culturomics, and then 109 suspected pathogen strains were preliminarily screened based on metagenomic sequencing data. Finally, 10 pathogens including, Pseudomonas syringae, P. fulva, P. fluorescens, Pantoea ananatis, Pectobacterium parvum, P. carotovorum, Flavobacterium fluviale, Citrobacter portucalensis, Klebsiella grimontii, Stenotrophomonas maltophilia, were identified through Koch's postulates. Based on the distribution pattern of pathogens and the changes in the microbiome community of mulberry following infection with P. syringae, we infer that P. syringae, and P. fulva are important pathogens of MBB. In addition, based on the analysis of meteorological data, different bacteria showed adaptability to different environments, leading to differences in the pathogens of MBB under different climate conditions and latitudes. The data presented herein provides a foundation for understanding the occurrence, spatial distribution and pathogenic mechanism of MBB and its major pathogens.

Inflammatory bowel disease (IBD) is characterized by disruptions in the gut microbiome. While most studies on gut dysbiosis in IBD rely on sequencing-based methods, we employed a streamlined culturomics approach to obtain a more comprehensive understanding of gut microbiota imbalance in patients with IBD that may not be captured by sequencing alone. A total of 367 bacteria were identified at the species level, including 211 species from ulcerative colitis patients, 164 species from Crohn's disease (CD) patients, and 263 species from healthy individuals. Consistent with our 16S rRNA gene amplicon sequencing results, a significant decrease in microbial diversity and a severe imbalance, especially in CD patients, were also observed in the culture-based analysis. Our culturomics approach provided additional insights, highlighting dysbiosis in unique anaerobic and Gram-negative species in CD patients. Moreover, species-level findings for Bifidobacterium and Enterobacterales emphasized specific species expansions in IBD patients. Notably, Mediterraneibacter gnavus, Thomasclavelia ramosa, Parabacteroides merdae, and Collinsella aerofaciens are of particular clinical interest due to their correlation with inflammatory biomarkers. This comprehensive analysis underscores the value of integrating a culture-based approach with a genome-based approach to provide complementary insights and therapeutic targets in IBD.

The fundamental step in microbiological detection is the preparation of culture medium. The reliability and accuracy of microbiological assay heavily relies on this step. Currently in China, the most recognized standard method for such medium evaluation is ISO 11133-2014. However, this method requires highly complicated biosafety management, detailed standard strains record-keeping and substantial working time.

Bacterial tablet is considered to be a novel strategy for bacteria culture medium evaluation. The filter membrane as a carrier ensures uniform cell dispersion, forming a singular colony that can facilitate counting. We examined the viability and storage durability of vacuum freeze-dried bacterial tablets using a filter membrane as a carrier and utilized the results to evaluate culture medium. We found that the recipe GBSS (Glycerinum, Brain Heart Infusion, Sucrose, Sodium Glutamate) showed the highest survival rate for Escherichia coli in vacuum freeze-drying. As a qualified medium, the productivity of target bacterium should be greater than 0.7. A comparison of freeze-dried bacterial tablet method with ISO 11133-2014 quantitative method showed the sensitivity and specificity of this novel method were 94.1% and 88.9% respectively. The results suggested that vacuum freeze-dried bacterial tablet method had high conformity when compared to ISO 11133-2014 quantitative method (χ 2 = 0.25, p > 0.05; Kappa = 0.75).

Hence, vacuum freeze-drying method is an integral part of preservation of bacterial strains and the preparation of related biological products. In conclusion, we have developed a novel and effective disposable product for estimating efficiency of the culture medium.

The gut microbiota has been increasingly recognised as a critical determinant of human health, influencing a wide range of physiological processes. A healthy gut microbiota is essential for maintaining metabolic, immune, and gastrointestinal homeostasis, contributing to overall well-being. Alterations in its composition and functionality, often referred to as microbial dysbiosis, are strongly associated with the development of gut-related and systemic diseases. The gut microbiota synthesises several components and interacts with epithelial cell receptors, influencing processes that extend beyond nutritional status to the pathogenesis of diseases such as obesity, which extend beyond their known contribution to nutritional status. Therefore, this state-of-the-art review synthesises findings from recent studies on the composition, functions, and influencing factors of the gut microbiota, with a focus on its role in obesity. A systematic search of peer-reviewed literature was conducted to ensure comprehensive coverage, while expert insights are incorporated to discuss emerging research directions and future perspectives in the field.

Antimicrobial resistance (AMR) is one of our greatest public health challenges. Targeted use of antibiotics (ABs) can reduce the occurrence and spread of AMR and boost the effectiveness of treatment. This requires knowledge of the AB susceptibility of the pathogens involved in the disease. Therapeutic recommendations based on classical AB susceptibility testing (AST) are based on the analysis of only a fraction of the bacteria present in the disease process. Next and third generation sequencing technologies allow the identification of antimicrobial resistance genes (ARGs) present in a bacterial community. Using this metagenomic approach, we can map the antimicrobial resistance potential (AMRP) of a complex, multi-bacterial microbial sample. To understand the interpretiveness of AMRP, the concordance between phenotypic AMR properties and ARGs was investigated by analyzing data from 574 Escherichia coli strains of five different studies. The overall results show that for 44% of the studied ABs, phenotypically resistant strains are genotypically associated with a 90% probability of resistance, while for 92% of the ABs, the phenotypically susceptible strains are genotypically susceptible with a 90% probability. ARG detection showed a phenotypic prediction with at least 90% confidence in 67% of ABs. The probability of detecting a phenotypically susceptible strain as resistant based on genotype is below 5% for 92% of ABs. While the probability of detecting a phenotypically resistant strain as susceptible based on genotype is below 5% for 44% of ABs. We can assume that these strain-by-strain concordance results are also true for bacteria in complex microbial samples, and conclude that AMRP obtained from metagenomic ARG analysis can help choose efficient ABs. This is illustrated using AMRP by a canine external otitis sample.

Gampsocleis gratiosa Brunner von Wattenwyl, 1862, is a type of omnivorous chirping insect with a long history of artificial breeding. It has high economic value and is also an excellent orthopteran model organism. In this study, 12 types of culture media combined with 16S rRNA sequencing were employed to isolate 838 bacterial strains from the gut of G. gratiosa. After sequence comparison, a total of 98 species of bacteria were identified, belonging to 3 phyla, 5 classes, 11 orders, 20 families, and 45 genera. Firmicutes and Proteobacteria accounted for the majority (92.86%). At the order level, Enterobacteriaceae, Bacillales, and Lactobacillales predominated (79.59%). At the genus level, Klebsiella (11.22%) and Enterococcus (7.14%) predominated. This study also enumerated the strain morphological, physiological and biochemical properties of 98 species of bacteria, including colony morphology, Gram staining, bacterial motility test, temperature gradient growth, pH gradient growth, citrate utilization test, temperature oxidase test, contact enzyme test, methyl red test, V-P test, indole test, gelatin liquefaction test, nitrate reduction test, hydrogen sulfide test, starch hydrolysis test, cellulose decomposition test, esterase (corn oil) test and antibiotic susceptibility testing. Additionally, 16 antibiotics were utilized to test the bacterial susceptibility of the strains. This study explored the types and community structure of some culturable microorganisms in the intestinal tract of G. gratiosa and recorded their physiological characteristics. These data reflect the physiological functions of the intestinal microorganisms of G. gratiosa and provide support for subsequent research on the interaction mechanism between microorganisms and their hosts.

Mammalian milk contains a variety of complex bioactive and nutritional components and microorganisms. These microorganisms have diverse compositions and functional roles that impact host health and disease pathophysiology, especially mastitis. The advent and use of high throughput omics technologies, including metagenomics, metatranscriptomics, metaproteomics, metametabolomics, as well as culturomics in milk microbiome studies suggest strong relationships between host phenotype and milk microbiome signatures in mastitis. While single omics studies have undoubtedly contributed to our current understanding of milk microbiome and mastitis, they often provide limited information, targeting only a single biological viewpoint which is insufficient to provide system-wide information necessary for elucidating the biological footprints and molecular mechanisms driving mastitis and milk microbiome dysbiosis. Therefore, integrating a multi-omics approach in milk microbiome research could generate new knowledge, improve the current understanding of the functional and structural signatures of the milk ecosystem, and provide insights for sustainable mastitis control and microbiome management.

The gut microbiome significantly impacts human health, yet cultivation challenges hinder its exploration. Here, we combine deep whole-metagenome sequencing with culturomics to selectively enrich for taxa and functional capabilities of interest. Using a modified commercial base medium, 50 growth modifications were evaluated, spanning antibiotics, physico-chemical conditions, and bioactive compounds. Whole-metagenome sequencing identified medium additives, like caffeine, that enhance taxa often associated with healthier subjects (e.g., Lachnospiraceae, Oscillospiraceae, Ruminococcaceae). We also explore the impact of modifications on the composition of cultured communities and establish a link between medium preference and microbial phylogeny. Leveraging these insights, we demonstrate that combinations of media modifications can further enhance the targeted enrichment of taxa and metabolic functions, such as Collinsella aerofaciens, or strains harboring biochemical pathways involved in dopamine metabolism. This streamlined, scalable approach unlocks the potential for selective enrichment, advancing microbiome research by understanding the impact of different cultivation parameters on gut microbes.

In the context of increasing pollution pressure on aquatic ecosystems, it is essential to improve our knowledge of habitat quality and its suitability for organisms. It is particularly relevant to better integrate early life stages of fish into pollution biomonitoring programs, as they are reliable indicators of ecosystem integrity and because of their high sensitivity to pollutants. To avoid the influence of environmental parameters on their development, a lab-on-field approach, called the ex-situ exposure method, was developed. Aquatic organisms were exposed to a continuous flux of water under semi-controlled temperature, oxygen, and photoperiod conditions to avoid the influence of these confounding factors when interpreting the results. To investigate the potential role of water contamination, this active biomonitoring method was applied to the Garonne River (Southwest France), where migratory fish populations have declined. Two model species from different taxa were used: embryos of the Japanese medaka (Oryzias latipes) and adults of the crustacean Gammarus fossarum. The results showed a significant impact of water quality on embryo mortality and early hatching in two separate experiments on Japanese medaka. In addition, an induction of feeding rate was observed in exposed gammarids, but no impact on their embryo survival, suggesting differences in sensitivity between the two species selected. Chemical and biological analyses did not identify trace metals, pesticides, or microorganisms as potential sources of toxicity in medaka embryos or G. fossarum. These results raise concerns about the quality of the water in the Garonne River and its toxicity to aquatic organisms.

In general, periprosthetic joint infection (PJI) is regarded as one of the most common complications of total joint arthroplasty (TJA) and may lead to surgical failure, revision surgery, amputation or death. Nowadays, PJI has become a global health concern, which brings a great burden to public healthcare. In addition, there are still obstacles to achieve high success rates in the prevention, diagnosis and treatment of PJI. However, promising studies are also available with the advancements in biotechnology. This article will present an overview of the current methods used in the prevention, diagnosis and management of PJI while underlining the new technologies utilized.

Over the past 12 years, culturomics, a high-throughput culture method, has been developed, considerably widening the repertoire of known cultured bacteria. An exhaustive database, including a list of microbes isolated by culture from human skin, was recently established by performing a review of the literature. The aim of the present study was to use the culturomics approach to explore the African skin microbiota. Skin swabs from the palms of human hands were collected between January and December 2016 from healthy subjects from the villages of Dielmo and Ndiop in rural Senegal. Three culture media were selected for the isolation of bacteria in aerobic conditions. Bacterial colonies were subjected to matrix-assisted laser desorption ionization-time of flight mass spectroscopy and the 16 S rRNA gene was sequenced for unidentified colonies. A total of 176 bacterial species were isolated. This increased the repertoire of bacterial species on the skin by 14.0%, by adding 71 bacteria, including seven new species. The culturomics approach characterizing microbial diversity has significantly changed our view of the skin microbiota, raising many important questions about the host-microorganism relationship and its relevance to skin diseases. In particular, the difference between the palm microbiota of these African populations (composed mainly of the genera Staphylococcus, Arthrobacter, Bacillus, and Microbacterium) and that of Western populations, whose main genera are Staphylococcus, Propionibacterium, Micrococcus, Corynebacterium, Enhydrobacter, and Streptococcus. This study demonstrates the need to continue to explore the skin microbiome using the culturomics approach.

A healthy ocular surface is inhabited by microorganisms that constitute the ocular microbiome. The core of the ocular microbiome is still a subject of debate. Numerous culture-dependent and gene sequencing studies have revealed the composition of the ocular microbiome. There was a confirmed correlation between the ocular microbiome and ocular surface homeostasis as well as between ocular dysbiosis and pathologies such as blepharitis, microbial keratitis, and conjunctivitis. However, the role of the ocular microbiome in the pathogenesis and treatment of ocular surface diseases remains unclear. This article reviews available data on the ocular microbiome and microbiota, their role in maintaining ocular homeostasis, and the impact of dysbiosis on several ophthalmic disorders. Moreover, we aimed to discuss potential treatment targets within the ocular microbiota.

Bacterial extracellular vesicles (BEVs) enable communication between bacteria and their natural habitats, including multicellular organisms such as humans. Consequently, the study of BEVs has rapidly gained attention with recent research raising the prospect of developing BEVs as biomarkers and treatments to manage (mal)functioning of natural habitats. Although diverse technologies are available, the composition of their source, their heterogeneity in biophysical and biochemical features, and their multifaceted cargo composition challenges the analysis of BEVs. To map current practices in BEV research, we analyzed 845 publications released in 2015-2021, reporting 3338 BEV-related experiments. The extracted data are accessible via the publicly available EV-TRACK knowledgebase ( https://evtrack.org/ ). We identify the need for transparent reporting, delineate knowledge gaps, outline available best practices and define areas in need of guidance to ensure advances in BEV research and accelerate BEV applications.

Endophytes, microorganisms inhabiting internal plant tissues, play a pivotal role in plant growth and disease resistance. Moreover, previous studies have established that Musa plants derive disease protective functions from their microbiome. Notably, one of the crop wild relatives of banana, the Calcutta 4 variety, exhibits resistance to various phytopathogens such as Pseudocercospora fijiensis (P. fijiensis), while the Williams commercial cultivar (cv.) is highly susceptible. Therefore, this study aims primarily to characterize and compare the endophytic microbiota composition of Calcutta 4 and Williams banana plants when grown sympatrically. Alongside, differences in endophytic microbiome between plant sections (shoot or roots), growth phases (in vitro or greenhouse) and fitness factors such as the addition of plant growth-promoting bacteria Bacillus subtilis EA-CB0575 (T2 treatment) or infection by P. fijiensis (T3 treatment) were examined. Both culture-dependent and -independent techniques were used to evaluate these differences and assess the culturability of banana endophytes under varying conditions. Microbial cultures resulted in 331 isolates distributed across 54 genera when all treatments were evaluated, whereas 16 S sequencing produced 9510 ASVs assigned in 1456 genera. Alpha and beta diversity exhibited significant differences based on plant section, with an increase in phylogenetic diversity observed in plants with pathogen infection (T3) compared to control plants (T1). Additionally, four differentially abundant genera associated with nitrogen metabolism were identified in T3 plants and seven genera showed differential abundance when comparing varieties. When culture-dependent and -independent methods were compared, it was found that isolates represented 3.7 % of the genera detected by culture-independent methods, accounting for 12-41 % of the total data depending on the treatment. These results are crucial for proposing management strategies derived from crop wild relatives to enhance the resilience of susceptible commercial varieties against fitness factors affecting crop development. Additionally, they help to decipher the pathogenic effects of P. fijiensis in banana plants and advance the understanding of how plant domestication influences the endosphere.

Bacterial culturomics is a set of techniques to isolate and identify live bacteria from complex microbial ecosystems. Despite its potential to revolutionize microbiome research, bacterial culturomics has significant challenges when applied to human gut microbiome studies due to its labor-intensive nature. Therefore, we established a streamlined culturomics approach with minimal culture conditions for stool sample preincubation. We evaluated the suitability of non-selective medium candidates for maintaining microbial diversity during a 30-day incubation period based on 16S rRNA gene amplicon analysis. Subsequently, we applied four culture conditions (two preincubation media under an aerobic/anaerobic atmosphere) to isolate gut bacteria on a large scale from eight stool samples of healthy humans. We identified 8141 isolates, classified into 263 bacterial species, including 12 novel species candidates. Our analysis of cultivation efficiency revealed that seven days of aerobic and ten days of anaerobic incubation captured approximately 91% and 95% of the identified species within each condition, respectively, with a synergistic effect confirmed when selected preincubation media were combined. Moreover, our culturomics findings expanded the coverage of gut microbial diversity compared to 16S rRNA gene amplicon sequencing results. In conclusion, this study demonstrated the potential of a streamlined culturomics approach for the efficient isolation of gut bacteria from human stool samples. This approach might pave the way for the broader adoption of culturomics in human gut microbiome studies, ultimately leading to a more comprehensive understanding of this complex microbial ecosystem.

The biotechnological industry faces a crucial demand for novel bioactive compounds, particularly antimicrobial agents, to address the rising challenge of bacterial resistance to current available antibiotics. Traditional strategies for cultivating naturally occurring microorganisms often limit the discovery of novel antimicrobial producers. This study presents a protocol for targeted selection of bacterial strains using the supernatant of Paenibacillus elgii, which produces abundant signal molecules and antimicrobial peptides. Soil samples were inoculated in these enriched culture media to selectively cultivate bacteria resistant to the supernatant, indicating their potential to produce similar compounds. The bacterial strains isolated through this method were assessed for their antibacterial activity. In addition, the functional annotation of the genome of one of these strains revealed several gene clusters of biotechnological interest. This study highlights the effectiveness of using this approach for selective cultivation of microorganisms with potential for biotechnological applications.

Cancerous tissue is a largely unexplored microbial niche that provides a unique environment for the colonization and growth of specific bacterial communities, and with it, the opportunity to identify novel bacterial species. Here, we report distinct features of a novel Fusobacterium species, F. sphaericum sp. nov. ( Fs ), isolated from primary colon adenocarcinoma tissue. We acquire the complete closed genome and associated methylome of this organism and phylogenetically confirm its classification into the Fusobacterium genus, with F. perfoetens as its closest neighbor. Fs is phenotypically and genetically distinct, with morphological analysis revealing its coccoid shape, that while similar to F. perfoetens is rare for most Fusobacterium members. Fs displays a metabolic profile and antibiotic resistance repertoire consistent with other Fusobacterium species. In vitro, Fs has adherent and immunomodulatory capabilities, as it intimately associates with human colon cancer epithelial cells and promotes IL-8 secretion. Analysis of the prevalence and abundance of Fs in >20,000 human metagenomic samples shows that it is a low-prevalence member within human stool with variable relative abundance, found in both healthy controls and patients with colorectal cancer (CRC). Our study sheds light on a novel bacterial species isolated directly from the human CRC tumor niche, and given its interaction with cancer epithelial cells suggests that its role in human health and disease warrants further investigation.

Pullorum disease, an intestinal disease in chickens caused by Salmonella enterica serovar pullorum (S. Pullorum), is a significant threat to the poultry industry and results in substantial economic losses. The bacteria's transmission, both vertical and horizontal, makes it difficult to completely eliminate it. Control strategies for pullorum disease primarily involve stringent eradication programs that cull infected birds and employ antibiotics for treatment. However, eradication programs are costly, and antibiotic use is restricted. Therefore, developing alternative control strategies is essential. Increasingly, studies are focusing on modulating the gut microbiota to control intestinal diseases. Modulating the chicken gut microbiota may offer a novel strategy for preventing and controlling pullorum disease in poultry. However, the impact of S. Pullorum on the chicken gut microbiota has not been well established, prompting our exploration of the relationship between S. Pullorum and the chicken gut microbiota in this study. In this study, we initially analyzed the dynamic distribution of the gut microbiota in chickens infected with S. Pullorum. Alpha diversity analysis revealed a decrease in observed OTUs and the Shannon diversity index in the infected group, suggesting a reduction in the richness of the chicken gut microbiota due to S. Pullorum infection. Principal coordinate analysis (PCoA) showed distinct clusters between the gut microbiota of infected and uninfected groups, indicating S. Pullorum infection changed the chicken gut microbiota structure. Specifically, S. Pullorum infection enriched the relative abundance of the genera Escherichia-Shigella (65% in infected vs. 40.6% in uninfected groups) and Enterococcus (10.8% vs. 3.7%) while reducing the abundance of Lactobacillus (9.9% vs. 32%) in the chicken microbiota. Additionally, based on the observed changes in the chicken gut microbiota, we isolated microorganisms, including Bifidobacterium pseudolongum, Streptococcus equi and Lacticaseibacillus paracasei (L. paracasei), which were decreased by S. Pullorum infection. Notably, the L. paracasei Lp02 strain was found to effectively inhibit S. Pullorum proliferation in vitro and alleviate its infection in vivo. We found that S. Pullorum infection reduced the richness of the chicken gut microbiota and enriched the relative abundance of the genera Escherichia-Shigella and Enterococcus while decreasing the abundance of the anaerobic genus Lactobacillus. Furthermore, microbiota analysis enabled the isolation of several antimicrobial microorganisms from healthy chicken feces, with a L. paracasei strain notably inhibiting S. Pullorum proliferation in vitro and alleviating its infection in vivo. Overall, this research enhances our understanding of the interaction between gut microbiota and pathogen infection, as well as offers new perspectives and strategies for modulating the chicken gut microbiota to control pullorum disease.

Some lactic acid bacteria (LAB) can provide significant health benefits, which are critically important for the conservation of endangered animals, such as giant pandas. However, little is known about the diversity and culturability of LAB in the giant panda gut microbiota. To understand the roles of LAB in giant panda conservation, it is critical to culture bacterial strains of interest. In this study, we established a pipeline to culture bacterial strains using enrichment of target bacteria with different liquid media and growth conditions. Then, the strains were isolated in solid media to study their functions. Using 210 samples from the culture enrichment method and 138 culture-independent samples, we obtained 1120 amplicon sequencing variants (ASVs) belonging to Lactobacillales. Out of the 1120 ASVs, 812 ASVs from the culture enrichment approach were twofold more diverse than 336 ASVs from the culture-independent approach. Many ASVs of interest were not detected in the culture-independent approach. Using this pipeline, we isolated many relevant bacterial strains and established a giant panda gut bacteria strain collection that included strains with low-abundance in culture-independent samples and included most of the giant panda LAB described by other researchers. The strain collection consisted of 60 strains representing 35 species of 12 genera. Thus, our pipeline is powerful and provides guidance in culturing gut microbiota of interest in hosts such as the giant panda.IMPORTANCECultivation is necessary to screen strains to experimentally investigate microbial traits, and to confirm the activities of novel genes through functional characterization studies. In the long-term, such work can aid in the identification of potential health benefits conferred by bacteria and this could aid in the identification of bacterial candidate strains that can be applied as probiotics. In this study, we developed a pipeline with low-cost and user-friendly culture enrichment to reveal the diversity of LAB in giant pandas. We compared the difference between culture-independent and culture enrichment methods, screened strains of interest that produced high concentrations of short-chain fatty acids (SCFAs), and we investigated the catalog of virulence factors, antibiotic resistance, butyrate and lactate synthesis genes of the strains at a genomic level. This study will provide guidance for microbiota cultivation and a foundation for future research aiming to understand the functions of specific strains.

Microbial communities inhabiting sedimentary environments in river source regions serve as pivotal indicators of pristine river ecosystems. While the correlation between antibiotic resistome and pathogenicity with core gut bacteria in humans is well established, there exists a significant knowledge gap concerning the interaction of antibiotic resistance genes (ARGs) and human pathogenic bacteria (HPB) with specific microbes in river source basins, often referred to as "terrestrial gut". Understanding the microbial composition, including bacteria and resident genetic elements such as ARGs, HPB, Mobile Genetic Elements (MGEs), and Virulence Factors (VFs), within natural habitats against the backdrop of global change, is imperative. To address this gap, an enrichment-based culturomics complementary along with metagenomics was conducted in this study to characterize the microbial biobank and provide preliminary ecological insights into profiling the dissemination of ARGs in the Lancang River Source Basin. Based on our findings, in the main stream of the Lancang River Source Basin, 674 strains of bacteria, comprising 540 strains under anaerobic conditions and 124 under aerobic conditions, were successfully isolated. Among these, 98 species were identified as known species, while 4 were potential novel species. Of these 98 species, 30 were HPB relevant to human health. Additionally, bacA and bacitracin emerged as the most abundant ARGs and antibiotics in this river, respectively. Furthermore, the risk assessment of ARGs predominantly indicated the lowest risk rank (Rank Ⅳ) in terms of endangering human health. In summary, enrichment-based culturomics proved effective in isolating rare and unknown bacteria, particularly under anaerobic conditions. The emergence of ARGs showed limited correlation with MGEs, indicating minimal threats to human health within the main stream of the Lancang River Source Basin.

Recent and continuing advances in gut microbiome research have pointed out the role of the gut microbiota as an unexplored source of potentially beneficial probiotic microbes. Along the lines of these advances, both public awareness and acceptance of probiotics are increasing. That's why; academic and industrial research is dedicated to identifying and investigating new microbial strains for the development of next-generation probiotics (NGPs). At this time, there is a growing interest in NGPs as biotherapeutics that alter the gut microbiome and affect various diseases development. In this work, we have focused on some emergent and promising NGPs, specifically Eubacterium hallii, Faecalibacterium prausnitzii, Roseburia spp., Akkermansia muciniphila, and Bacteroides fragilis, as their presence in the gut can have an impact on the development of various diseases. Emerging studies point out the beneficial roles of these NGPs and open up novel promising therapeutic options. Interestingly, these NGPs were found to enhance gastrointestinal immunity, enhance immunotherapy efficacy in cancer patients, retain the intestinal barrier integrity, generate valuable metabolites, especially short-chain fatty acids, and decrease complications of chemotherapy and radiotherapy. Although many of these NGPs are considered promising for the prevention and treatment of several chronic diseases, research on humans is still lacking. Therefore, approval of these microbes from regulatory agencies is rare. Besides, some issues limit their wide use in the market, such as suitable methods for the culture and storage of these oxygen-sensitive microbes. The present review goes over the main points related to NGPs and gives a viewpoint on the key issues that still hinder their wide application. Furthermore, we have focused on the advancement in NGPs and human healthiness investigations by clarifying the limitations of traditional probiotic microorganisms, discussing the characteristics of emerging NGPs and defining their role in the management of certain ailments. Future research should emphasize the isolation, mechanisms of action of these probiotics, safety, and clinical efficacy in humans.

Recent studies have emphasized the role of gut microbiota in the onset and progression of osteomyelitis. However, the exact types of gut microbiota and their mechanisms of action remain unclear. Additionally, there is a lack of theoretical support for treatments that improve osteomyelitis by altering the gut microbiota.

In our study, we utilized the largest genome-wide association study (GWAS) meta-analysis to date from the MiBioGen consortium, involving 13,400 participants. The GWAS data for osteomyelitis were sourced from the UK Biobank, which included 4,836 osteomyelitis cases and 486,484 controls. We employed a two-sample Mendelian randomization framework for a detailed investigation into the causal relationship between gut microbiota and osteomyelitis. Our methods included inverse variance weighting, MR-Egger, weighted median, and weighted mode approaches. Additionally, we applied Cochran's Q statistic to assess the heterogeneity of the instrumental variable.

At the class level, Bacilli and Bacteroidia were positively correlated with the risk of osteomyelitis. At the order level, only Bacteroidales showed a positive association with osteomyelitis. At the genus level, an increased abundance of Butyricimonas, Coprococcus3, and Tyzzerella3 was positively associated with the risk of osteomyelitis, whereas Lachnospira was negatively associated. Sensitivity analyses showed no evidence of heterogeneity or pleiotropy.

This study reveals that classes Bacilli and Bacteroidia, order Bacteroidales, and genera Butyricimonas, Coprococcus3, and Tyzzerella3 are implicated in increasing the risk of osteomyelitis, while the genus Lachnospira is associated with a reduced risk. Future investigations are warranted to elucidate the precise mechanisms through which these specific bacterial groups influence the pathophysiology of osteomyelitis.

Cumulative xenobiotic exposure has an environmental and human health impact which is currently assessed under the One Health approach. Bisphenol A (BPA) exposure and its potential link with childhood obesity that has parallelly increased during the last decades deserve special attention. It stands during prenatal or early life and could trigger comorbidities and non-communicable diseases along life. Accumulation in the nature of synthetic chemicals supports the "environmental obesogen" hypothesis, such as BPA. This estrogen-mimicking xenobiotic has shown endocrine disruptive and obesogenic effects accompanied by gut microbiota misbalance that is not yet well elucidated. This study aimed to investigate specific microbiota taxa isolated and selected by direct BPA exposure and reveal its role on the overall children microbiota community and dynamics, driving toward specific obesity dysbiosis. A total of 333 BPA-resistant isolated species obtained through culturing after several exposure conditions were evaluated for their role and interplay with the global microbial community. The selected BPA-cultured taxa biomarkers showed a significant impact on alpha diversity. Specifically, Clostridium and Romboutsia were positively associated promoting the richness of microbiota communities, while Intestinibacter, Escherichia-Shigella, Bifidobacterium, and Lactobacillus were negatively associated. Microbial community dynamics and networks analyses showed differences according to the study groups. The normal-weight children group exhibited a more enriched, structured, and connected taxa network compared to overweight and obese groups, which could represent a more resilient community to xenobiotic substances. In this sense, subnetwork analysis generated with the BPA-cultured genera showed a correlation between taxa connectivity and more diverse potential enzymatic BPA degradation capacities.IMPORTANCEOur findings indicate how gut microbiota taxa with the capacity to grow in BPA were differentially represented within differential body mass index children study groups and how these taxa affected the overall dynamics toward patterns of diversity generally recognized in dysbiosis. Community network and subnetwork analyses corroborated the better connectedness and stability profiles for normal-weight group compared to the overweight and obese groups.

This study compared the EXS 2600 system with the MALDI Biotyper for identifying microorganisms in dairy samples. Of the 196 bacterial isolates from milk, whey, buttermilk, cream, and dairy wastewater, the species and genus consistent identification between two systems showed 74% and 99%, respectively. However, the level of species identification rate exhibited a difference, which was higher in Zybio than in Bruker-76.0% and 66.8%, respectively. Notably, the EXS 2600 system performed better with certain yeast species and H. alvei, while the Biotyper excelled with Pseudomonas bacteria. Unique microbial compositions were found in 85% of dairy samples, with whey and buttermilk having the highest diversity. This research highlights the EXS 2600's potential as a reliable dairy microbial identification tool and underscores the need for a more diverse and comprehensive spectral database, despite the database's focus on clinical applications (as announced).

Viable microorganisms and a diverse microbial ecosystem found in human milk play a crucial role in promoting healthy immune system and shaping the microbial community in the infant's gut. Culturomics is a method to obtain a comprehensive repertoire of human milk microbiota. However, culturomics is an onerous procedure, and needs expertise, making it difficult to be widely implemented. Currently, there is no efficient and feasible culturomics method specifically designed for human milk microbiota yet. Therefore, the aim of this study was to develop a more efficient and feasible culturomics method specifically designed for human milk microbiota. We obtained fresh samples of human milk from healthy Chinese mothers and conducted a 27-day enrichment process using blood culture bottles. Bacterial isolates were harvested at different time intervals and cultured on four different types of media. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, we identified a total of 6601 colonies and successfully obtained 865 strains, representing 4 phyla, 21 genera, and 54 species. By combining CBA and MRS media, we were able to cultivate over 94.4% of bacterial species with high diversity, including species-specific microorganisms. Prolonged pre-incubation in blood culture bottles significantly increased the number of bacterial species by about 33% and improved the isolation efficiency of beneficial bacteria with low abundance in human milk. After optimization, we reduced the pre-incubation time in blood culture bottles and selected optimal picking time-points (0, 3, and 6 days) at 37°C. By testing 6601 colonies using MALDI-TOF MS, we estimated that this new protocol could obtain more than 90% of bacterial species, reducing the workload by 57.0%. In conclusion, our new culturomics strategy, which involves the combination of CBA and MRS media, extended pre-incubation enrichment, and optimized picking time-points, is a feasible method for studying the human milk microbiota. This protocol significantly improves the efficiency of culturomics and allows for the establishment of a comprehensive repertoire of bacterial species and strains in human milk.

Quickly identifying and characterizing isolates from extreme environments is currently challenging while very important to explore the Earth's biodiversity. As these isolates may, in principle, be distantly related to known species, techniques are needed to reliably identify the branch of life to which they belong. Proteotyping these environmental isolates by tandem mass spectrometry offers a rapid and cost-effective option for their identification using their peptide profiles. In this study, we document the first high-throughput proteotyping approach for environmental extremophilic and halophilic isolates. Microorganisms were isolated from samples originating from high-altitude Andean lakes (3700-4300 m a.s.l.) in the Chilean Altiplano, which represent environments on Earth that resemble conditions on other planets. A total of 66 microorganisms were cultivated and identified by proteotyping and 16S rRNA gene amplicon sequencing. Both the approaches revealed the same genus identification for all isolates except for three isolates possibly representing not yet taxonomically characterized organisms based on their peptidomes. Proteotyping was able to indicate the presence of two potentially new genera from the families of Paracoccaceae and Chromatiaceae/Alteromonadaceae, which have been overlooked by 16S rRNA amplicon sequencing approach only. The paper highlights that proteotyping has the potential to discover undescribed microorganisms from extreme environments.

The strains Marseille-Q7072T (= CSUR Q7072T = CECT 30604 T) and Marseille-Q7826T (= CSUR Q7826T = CECT 30727 T) were isolated from vaginal samples. As MALDI-TOF mass spectrometry failed to identify them, their genomes were directly sequenced to determine their taxogenomic identities. Both strains are anaerobic without any oxidase and catalase activity. C16:0 is the most abundant fatty acid for both strains. Strain Marseille-Q7072T is non-spore-forming, non-motile, Gram-stain-positive, and coccus-shaped, while strain Marseille-Q7826T is non-spore-forming, motile, Gram-stain-variable, and curved rod-shaped. The genomic comparison of the Marseille-Q7072T and Marseille-Q7826T strains showed that all digital DNA-DNA hybridisation (dDDH) and mean orthologous nucleotide identity (OrthoANI) values were below published species thresholds (70% and 95-96%, respectively) with other closely related species with standing in nomenclature. Thus, we conclude that both strains are new bacterial species. Strain Marseille-Q7072T is a new member of the Bacillota phylum, for which the name Peptoniphilus genitalis sp. nov. is proposed, while the Marseille-Q7826T strain is a new member of the Actinomycetota phylum, for which the name Mobiluncus massiliensis sp. nov. is proposed.

Gut microbiota is known to have a significant impact on nonalcoholic fatty liver disease (NAFLD), particularly in children with obesity. However, the specific functions of microbiota at the strain level in this population have not been fully elucidated. In this study, we successfully isolated and identified several commensal gut bacterial strains that were dominant in children with obesity and NAFLD. Among these, four novel isolates were found to have significant lipogenic effects in vitro. These strains exhibited a potential link to hepatocyte steatosis by regulating the expression of genes involved in lipid metabolism and inflammation. Moreover, a larger cohort analysis confirmed that these identified bacterial strains were enriched in the NAFLD group. The integrated analysis of these strains effectively distinguished NASH from NAFL. These four strains might serve as potential biomarkers in children with NAFLD. These findings provided new insights into the exploration of therapeutic targets for NAFLD.

Microbial communities harbor important biotechnological potential in diverse domains, however, the engineering and propagation of such communities still face both knowledge and know-how gaps. More specifically, culturing tools are needed to propagate and shape microbial communities, to obtain desired properties, and to exploit them. Previous work suggested that micro-confinement and segregation of microorganisms using invert (water-in-oil, w/o) emulsion broth can shape communities during propagation, by alleviating biotic interactions and inducing physiological changes in cultured bacteria. The present work aimed at evaluating invert emulsion and simple broth monophasic cultures for the propagation and shaping of bacterial communities derived from raw milk in a serial propagation design.

The monophasic setup resulted in stable community structures during serial propagation, whereas the invert emulsion system resulted in only transiently stable structures. In addition, different communities with different taxonomic compositions could be obtained from a single inoculum. Furthermore, the implementation of invert emulsion systems has allowed for the enrichment of less abundant microorganisms and consequently facilitated their isolation on culture agar plates.

The monophasic system enables communities to be propagated in a stable manner, whereas the invert emulsion system allowed for the isolation of less abundant microorganisms and the generation of diverse taxonomic compositions from a single inoculum.

The composition of the vaginal microbiota is known to be influenced by various factors and to be associated with several disorders affecting women's health. Although metagenomics is currently a widely used method for studying the human microbiota, it has certain limitations, such as a lack of information on bacterial viability. It is therefore important to use culture-based methods such as culturomics. Here, we used 35 different culture conditions to comprehensively characterize the vaginal bacterial diversity of a single woman's flora. A total of 206 bacterial species, belonging to six phyla (for a little more than half to Firmicutes, followed mainly by Actinobacteria, Bacteroidetes, and Proteobacteria) and 45 families, and 2 fungal species were cultivated. While several species of lactobacilli have been isolated, a wide variety of other bacteria were also separated, including 65 never reported before in vaginal flora, including a new bacterial species, Porphyromonas vaginalis sp. nov. Extensive culture-based methods are essential to establish a comprehensive, evidence-based repertoire of bacterial viability. If combined with molecular methods, they can provide a much more thorough understanding of the vaginal microbiota and fulfil the unknown part of metagenomic studies.

As genetic tools continue to emerge and mature, more information is revealed about the identity and diversity of microbial community members. Genetic tools can also be used to make predictions about the chemistry that bacteria and fungi produce to function and communicate with one another and the host. Ongoing efforts to identify these products and link genetic information to microbiome chemistry rely on analytical tools. This tutorial highlights recent advancements in microbiome studies driven by techniques in mass spectrometry.

Only a few studies dealt with the occurrence of endospore-forming clostridia in the microbiota of infants without obvious health complications.

A methodology pipeline was developed to determine the occurrence of endospore formers in infant feces. Twenty-four fecal samples (FS) were collected from one infant in monthly intervals and were subjected to variable chemical and heat treatment in combination with culture-dependent analysis. Isolates were identified by MALDI-TOF mass spectrometry, 16S rRNA gene sequencing, and characterized with biochemical assays.

More than 800 isolates were obtained, and a total of 21 Eubacteriales taxa belonging to the Clostridiaceae, Lachnospiraceae, Oscillospiraceae, and Peptostreptococcaceae families were detected. Clostridium perfringens, C. paraputrificum, C. tertium, C. symbiosum, C. butyricum, and C. ramosum were the most frequently identified species compared to the rarely detected Enterocloster bolteae, C. baratii, and C. jeddahense. Furthermore, the methodology enabled the subsequent cultivation of less frequently detectable gut taxa such as Flavonifractor plautii, Intestinibacter bartlettii, Eisenbergiella tayi, and Eubacterium tenue. The isolates showed phenotypic variability regarding enzymatic activity, fermentation profiles, and butyrate production.

Taken together, this approach suggests and challenges a cultivation-based pipeline that allows the investigation of the population of endospore formers in complex ecosystems such as the human gastrointestinal tract.

An isolate of a bacterium recovered from an endometrial biopsy failed to be identified by MALDI-TOF mass spectrometry and was subjected to 16S rRNA sequencing. The obtained sequence was compared by BLASTn against the NCBI database, which revealed that the most closely related species was Cellulomonas hominis and Cellulomonas pakistanensis, with 98.85% and 98.45% identity, respectively. Phenotypic characterisation and genome sequencing were performed. The isolate was facultative anaerobic, gram-positive, motile, non-spore forming, and rod-shaped. Cell wall fatty acid profiling revealed that 12-methyl-tetradecanoic acid was the most abundant fatty acid (36%). The genome size was 4.25 Mbp with a G + C content of 74.8 mol%. Genomic comparison of species closely related to this strain showed that all digital DNA-DNA hybridisation (dDDH) and mean orthologous nucleotide identity (OrthoANI) values were below published species thresholds (70% and 95-96%, respectively). Based on these data, we conclude that this isolate represents a new bacterial species belonging to the family Cellulomonadaceae and the phylum Actinomycetota. We propose the name Cellulomonas endometrii sp. nov. The type strain is Marseille-Q7820T (= CSUR Q7820 = CECT 30716).

Microbes are usually present as a specific microbiota, and their classification remains a challenge. MALDI-TOF MS is particularly successful in library-based microbial identification at the species level as it analyzes the molecular weight of peptides and ribosomal proteins. FT-IR allows more accurate classification of bacteria at the subspecies level due to the high sensitivity, specificity and repeatability of FT-IR signals from bacteria, which is not achievable with MALDI-TOF MS. Previous studies have shown that more accurate identification results can be obtained by the fusion of FT-IR and MALDI-TOF MS spectral data. Here, we constructed 20 groups of model microbiota samples and used FT-IR, MALDI-TOF MS, and their fusion data to classify them. Hierarchical clustering analysis (HCA) showed that the classification accuracy of FT-IR, MALDI-TOF MS, and the fusion data was 85%, 90%, and 100%, respectively. These results indicate that both FT-IR and MALDI-TOF MS can effectively classify specific microbiota, and the fusion of their spectral data could improve the classification accuracy. The FT-IR and MALDI-TOF MS data fusion strategy may be a promising technology for specific microbiota classification.