Rodent Gut Bacteria Coexisting with an Insect Gut Virus in Tapeworm Parasitic Cysts: Metagenomic Evidence of Microbial Selection in Extra-Intestinal Clinical Niches.

In medicine, parasitic cysts (e.g., brain cysticerci) are believed to be sterile, and are primarily treated with antiparasitic medications, not antibiotics, which could prevent abscess formation and localized inflammation. This study quantified the microbial composition of parasitic cysts in a wild rodent, using multi-kingdom metagenomics to comprehensively assess if parasitic cysts are sterile, and further understand gut microbial translocation and adaptation in wildlife confined environments, outside the gut. Analysis was conducted on DNA from two hepatic parasitic cysts from a feline tapeworm, Hydatigera (Taenia) taeniaeformis, affecting a wild vole mouse (Microtus pennsylvanicus), and from feces, liver and peritoneal fluid of this and two other concurrent individual wild voles trapped during pest control in one of our university research vegetable gardens. Bacterial metagenomics revealed the presence of gut commensal/opportunistic species, Parabacteroides distasonis, Bacteroides (Bacteroidota); Klebsiella variicola, E. coli (Enterobacteriaceae); Enterococcus faecium and Lactobacillus acidophilus (Bacillota) inhabiting the cysts, and peritoneal fluid. Remarkably, viral metagenomics revealed various murine viral species, and unexpectedly, a virus from the insect armyworm moth (Pseudaletia/Mythimna unipuncta), known as Mythimna unipuncta granulovirus A (MyunGV-A), in both cysts, and in one fecal and one peritoneal sample from the other non-cyst voles, indicating the survival and adaption potential of the insect virus in voles. Metagenomics also revealed a significantly lower probability of fungal detection in cysts compared to that in peritoneal fluid/feces (p < 0.05), with single taxon detection in each cyst (Malassezia and Pseudophaeomoniella oleicola). The peritoneal fluid had the highest probability for fungi. In conclusion, metagenomics revealed that bacteria/viruses/fungi coexist within parasitic cysts supporting the potential therapeutic benefits of antibiotics in cystic diseases, and in inflammatory microniches of chronic diseases, such as Crohn's disease gut wall cavitating micropathologies, from which we recently isolated similar synergistic pathogenic Bacteroidota and Enterobacteriaceae, and Bacillota.

Antigenic operon fragmentation and diversification mechanism in Bacteroidota impacts gut metagenomics and pathobionts in Crohn's disease microlesions.

Comensal Bacteroidota (Bacteroidota) and Enterobacteriacea are often linked to gut inflammation. However, the causes for variability of pro-inflammatory surface antigens that affect gut commensal/opportunistic dualism in Bacteroidota remain unclear. By using the classical lipopolysaccharide/O-antigen 'rfb operon' in Enterobacteriaceae as a surface antigen model (5-rfb-gene-cluster rfbABCDX), and a recent rfbA-typing strategy for strain classification, we characterized the integrity and conservancy of the entire rfb operon in Bacteroidota. Through exploratory analysis of complete genomes and metagenomes, we discovered that most Bacteroidota have the rfb operon fragmented into nonrandom patterns of gene-singlets and doublets/triplets, termed 'rfb-gene-clusters', or rfb-'minioperons' if predicted as transcriptional. To reflect global operon integrity, contiguity, duplication, and fragmentation principles, we propose a six-category (infra/supra-numerary) cataloging system and a Global Operon Profiling System for bacteria. Mechanistically, genomic sequence analyses revealed that operon fragmentation is driven by intra-operon insertions of predominantly Bacteroides-DNA (thetaiotaomicron/fragilis) and likely natural selection in gut-wall specific micro-niches or micropathologies. Bacteroides-insertions, also detected in other antigenic operons (fimbriae), but not in operons deemed essential (ribosomal), could explain why Bacteroidota have fewer KEGG-pathways despite large genomes. DNA insertions, overrepresenting DNA-exchange-avid (Bacteroides) species, impact our interpretation of functional metagenomics data by inflating by inflating gene-based pathway inference and by overestimating 'extra-species' abundance. Of disease relevance, Bacteroidota species isolated from cavitating/cavernous fistulous tract (CavFT) microlesions in Crohn's Disease have supra-numerary fragmented operons, stimulate TNF-alpha from macrophages with low potency, and do not induce hyperacute peritonitis in mice compared to CavFT Enterobacteriaceae. The impact of 'foreign-DNA' insertions on pro-inflammatory operons, metagenomics, and commensalism/opportunism requires further studies to elucidate their potential for novel diagnostics and therapeutics, and to elucidate the role of co-existing pathobionts in Crohn's disease microlesions.

Rodent Gut Bacteria Coexisting with an Insect Gut Virus in Parasitic Cysts: Metagenomic Evidence of Microbial Translocation and Co-adaptation in Spatially-Confined Niches.

In medicine, parasitic cysts or cysticerci (fluid-filled cysts, larval stage of tapeworms) are believed to be sterile (no bacteria), and therein, the treatment of cysticerci infestations of deep extra-intestinal tissues (e.g., brain) relies almost exclusively on the use of antiparasitic medications, and rarely antibiotics. To date, however, it is unclear why common post-treatment complications include abscessation. This study quantified the microbial composition of parasitic cyst contents in a higher-order rodent host, using multi-kingdom shotgun metagenomics, to improve our understanding of gut microbial translocation and adaptation strategies in wild environments. Analysis was conducted on DNA from two hepatic parasitic cysts (Hydatigera (Taeenia) taeniaeformis) in an adult vole mouse (Microtus arvalis), and from feces, liver, and peritoneal fluid of three other vole family members living in a vegetable garden in Ohio, USA. Bacterial metagenomics revealed the presence of gut commensal/opportunistic species, including Parabacteroides distasonis, Klebsiella variicola, Enterococcus faecium, and Lactobacillus acidophilus, inhabiting the cysts. Parabacteroides distasonis and other species were also present outside the cyst in the peritoneal fluid. Remarkably, viral metagenomics revealed various murine viral species, but unexpectedly, it detected an insect-origin virus from the army moth (Pseudaletia/Mythimna unipuncta) known as Mythimna unipuncta granulovirus A (MyunGV-A) in both cysts, and in one fecal and one peritoneal sample from two different voles, indicating survival of the insect virus and adaption in voles. Metagenomics also revealed a significantly lower probability of fungal detection in the cysts compared to other samples (peritoneal fluid, p<0.05; and feces p<0.05), with single taxon detection in each cyst for Malassezia and Pseudophaeomoniella oleicola. The samples with a higher probability of fungi were the peritoneal fluid. In conclusion, commensal/pathobiont bacterial species can inhabit parasitic tapeworm cysts, which needs to be considered during therapeutic decisions of cysticerci or other chronic disease scenarios where immune privileged and spatially restricted ecosystems with limited nutrients and minimal presence of immune cells could facilitate microbial adaptation, such as within gut wall cavitating micropathologies in Crohn's disease.

Clonal Parabacteroides from Gut Microfistulous Tracts as Transmissible Cytotoxic Succinate-Commensal Model of Crohn's Disease Complications.

Crohn's disease (CD) has been traditionally viewed as a chronic inflammatory disease that cause gut wall thickening and complications, including fistulas, by mechanisms not understood. By focusing on Parabacteroides distasonis (presumed modern succinate-producing commensal probiotic), recovered from intestinal microfistulous tracts (cavernous fistulous micropathologies CavFT proposed as intermediate between 'mucosal fissures' and 'fistulas') in two patients that required surgery to remove CD-damaged ilea, we demonstrate that such isolates exert pathogenic/pathobiont roles in mouse models of CD. Our isolates are clonally-related; potentially emerging as transmissible in the community and mice; proinflammatory and adapted to the ileum of germ-free mice prone to CD-like ileitis (SAMP1/YitFc) but not healthy mice (C57BL/6J), and cytotoxic/ATP-depleting to HoxB8-immortalized bone marrow derived myeloid cells from SAMP1/YitFc mice when concurrently exposed to succinate and extracts from CavFT-derived E. coli , but not to cells from healthy mice. With unique genomic features supporting recent genetic exchange with Bacteroides fragilis -BGF539, evidence of international presence in primarily human metagenome databases, these CavFT Pdis isolates could represent to a new opportunistic Parabacteroides species, or subspecies (' cavitamuralis' ) adapted to microfistulous niches in CD.

The Weissella Genus: Clinically Treatable Bacteria with Antimicrobial/Probiotic Effects on Inflammation and Cancer.

Weissella is a genus earlier considered a member of the family Leuconostocaceae, which was reclassified into the family Lactobacillaceae in 1993. Recently, there have been studies emphasizing the probiotic and anti-inflammatory potential of various species of Weissella, of which W. confusa and W. cibaria are the most representative. Other species within this genus include: W. paramesenteroides, W. viridescens, W. halotolerans, W. minor, W. kandleri, W. soli, W. ghanensis, W. hellenica, W. thailandensis, W. fabalis, W. cryptocerci, W. koreensis, W. beninensis, W. fabaria, W. oryzae, W. ceti, W. uvarum, W. bombi, W. sagaensis, W. kimchi, W. muntiaci, W. jogaejeotgali, W. coleopterorum, W. hanii, W. salipiscis, and W. diestrammenae. Weissella confusa, W. paramesenteroides, W. koreensis, and W. cibaria are among the few species that have been isolated from human samples, although the identification of these and other species is possible using metagenomics, as we have shown for inflammatory bowel disease (IBD) and healthy controls. We were able to isolate Weissella in gut-associated bacteria (post 24 h food deprivation and laxatives). Other sources of isolation include fermented food, soil, and skin/gut/saliva of insects/animals. With the potential for hospital and industrial applications, there is a concern about possible infections. Herein, we present the current applications of Weissella on its antimicrobial and anti-inflammatory mechanistic effects, the predisposing factors (e.g., vancomycin) for pathogenicity in humans, and the antimicrobials used in patients. To address the medical concerns, we examined 28 case reports focused on W. confusa and found that 78.5% of infections were bacteremia (of which 7 were fatal; 1 for lack of treatment), 8 were associated with underlying malignancies, and 8 with gastrointestinal procedures/diseases of which 2 were Crohn's disease patients. In cases of a successful resolution, commonly administered antibiotics included: cephalosporin, ampicillin, piperacillin-tazobactam, and daptomycin. Despite reports of Weissella-related infections, the evolving mechanistic findings suggest that Weissella are clinically treatable bacteria with emerging antimicrobial and probiotic benefits ranging from oral health, skin care, obesity, and inflammatory diseases to cancer.

Distribution and diversity of glycocin biosynthesis gene clusters beyond Firmicutes.

Glycocins are the ribosomally synthesized glycosylated bacteriocins discovered and characterized in Firmicutes, only. These peptides have antimicrobial activity against several pathogenic bacteria, including Streptococcus pyogenes , methicillin-resistant Staphylococcus aureus and food-spoilage bacteria Listeria monocytogenes. Glycocins exhibit immunostimulatory properties and make a promising source of new antibiotics and food preservatives akin to Nisin. Biochemical studies of Sublancin, Glycocin F, Pallidocin and ASM1 prove that the nested disulfide-bonds are essential for their bioactivities. Using in silico approach of genome mining coupled with manual curation, here we identify 220 new putative glycocin biosynthesis gene clusters (PGBCs) spread across 153 bacterial species belonging to seven different bacterial phyla. Based on gene composition, we have grouped these PGBCs into five distinct conserved cluster Types I-V. All experimentally identified glycocins belong to Type I PGBCs. From protein sequence based phylograms, tanglegrams, global similarity heat-maps and cumulative mutual information analysis, it appears that glycocins may have originated from closely related bacteriocins, whereas recruitment of cognate glycosyltransferases (GTs) might be an independent event. Analysis further suggests that GTs may have coevolved with glycocins in cluster-specific manner to define distinctive donor specificities of GTs or to contribute to glycocin diversity across these clusters. We further identify 162 hitherto unreported PGBCs wherein the corresponding product glycocins have three or less than three cysteines. Secondary structure predictions suggest that these putative glycocins may not form di-nested disulfide-bonds. Therefore, production of such glycocins in heterologous host Escherichia coli is feasible and may provide novel antimicrobial spectrum and or mechanism of action for varied applications.