Transcriptomic analysis of polysaccharide utilization loci reveals substrate preferences in ruminal generalists Segatella bryantii TF1-3 and Xylanibacter ruminicola KHP1.

Bacteria of the genera Xylanibacter and Segatella are among the most dominant groups in the rumen microbiota. They are characterized by the ability to utilize different hemicelluloses and pectin of plant cell-wall as well as plant energy storage polysaccharides. The degradation is possible with the use of cell envelope bound multiprotein apparatuses coded in polysaccharide utilization loci (PULs), which have been shown to be substrate specific. The knowledge of PUL presence in rumen Xylanibacter and Segatella based on bioinformatic analyses is already established and transcriptomic and genetic approaches confirmed predicted PULs for a limited number of substrates. In this study, we transcriptomically identified additional different PULs in Xylanibacter ruminicola KHP1 and Segatella bryantii TF1-3. We also identified substrate preferences and found that specific growth rate and extent of growth impacted the choice of substrates preferentially used for degradation. These preferred substrates were used by both strains simultaneously as judged by their PUL upregulation. Lastly, ß-glucan and xyloglucan were used by these strains in the absence of bioinformatically and transcriptomically identifiable PUL systems.

Type IV pili are widespread among non-pathogenic Gram-positive gut bacteria with diverse carbohydrate utilization patterns.

Type IV pili (T4P) are bacterial surface-exposed appendages that have been extensively studied in Gram-negative pathogenic bacteria. Despite recent sequencing efforts, little is known regarding these structures in non-pathogenic anaerobic Gram-positive species, particularly commensals of the mammalian gut. Early studies revealed that T4P in two ruminal Gram-positive species are associated with growth on cellulose, suggesting possible associations of T4P with substrate utilization patterns. In the present study, genome sequences of 118 taxonomically diverse, mainly Gram-positive, bacterial strains isolated from anaerobic (gastrointestinal) environments, have been analysed. The genes likely to be associated with T4P biogenesis were analysed and grouped according to T4P genetic organization. In parallel, consortia of Carbohydrate Active enZYmes (CAZymes) were also analysed and used to predict carbohydrate utilization abilities of selected strains. The predictive power of this approach was additionally confirmed by experimental assessment of substrate-related growth patterns of selected strains. Our analysis revealed that T4P systems with diverse genetic organization are widespread among Gram-positive anaerobic non-pathogenic bacteria isolated from different environments, belonging to two phylogenetically distantly related phyla: Firmicutes and Actinobacteria.

Non-oral Prevotella stepping into the spotlight.

Species now affiliated to genus Prevotella have been known for decades as an integral part of human oral cavity microbiota. They were frequently isolated from patients with periodontitis or from dental root canals but also from healthy subjects. With the exception of Prevotella intermedia, they were considered opportunistic pathogens, as they were isolated also from various bacterial abscesses from the head, neck, breast, skin and various other body sites. Consequently, Prevotella were not in the focus of research activities. On the other hand, the four species found in the rumen never caused any disease and seemed early on to be numerous and important part of the rumen ecosystem indicating this genus harbored bacteria with enormously diverse habitats and lifestyles. The purpose of this review is to illustrate the main research themes performed in Prevotella on a path from less noted oral bacteria and from hard to cultivate and study rumen organisms to important mutualistic bacteria in guts of various mammals warranting major research efforts.

In Silico Selection Approach to Develop DNA Aptamers for a Stem-like Cell Subpopulation of Non-small Lung Cancer Adenocarcinoma Cell Line A549.

BACKGROUND: Detection of circulating lung cancer cells with cancer-stem like characteristics would represent an improved tool for disease prognosis. However, current antibodies based methods have some disadvantages and therefore cell SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was used to develop DNA aptamers, recognizing cell surface markers of non-small lung carcinoma (NSLC) cells. MATERIALS AND METHODS: The human adenocarcinoma cell line A549 was used for selection in seven cell SELEX cycles. We used human blood leukocytes for negative selection, and lung stem cell protein marker CD90 antibody binding A549 cells for positive selection. RESULTS: The obtained oligonucleotide sequences after the seventh SELEX cycle were subjected to in silico selection analysis based on three independent types of bioinformatics approaches, selecting two closely related aptamer candidates in terms of consensus sequences, structural motifs, binding affinity (Kd) and stability (ΔG). We selected and identified the aptamer A155_18 with very good binding characteristics to A459 cells, selected for CD90 antibody binding. The calculated phylogenetic tree showed that aptamers A155_18 and the known A549 cell aptamer S6 have a close structural relationship. MEME sequence analysis showed that they share two unique motifs, not present in other sequences. CONCLUSIONS: The novel aptamer A155_18 has strong binding affinity for A549 lung carcinoma cell line subpopulation that is expressing stem cell marker CD90, indicating a possible stemness, characteristic for the A459 line, or a subpopulation present within this cell line. This aptamer can be applied as diagnostic tool, identifying NSLC circulating cells.

Development of narrow-spectrum topoisomerase-targeting antibacterials against mycobacteria.

New 2-pyrrolamidobenzothiazole-based inhibitors of mycobacterial DNA gyrase were discovered. Among these, compounds 49 and 51, show excellent antibacterial activity against Mycobacterium tuberculosis and Mycobacterium abscessus with a notable preference for mycobacteria. Both compounds can penetrate infected macrophages and reduce intracellular M. tuberculosis load. Compound 51 is a potent inhibitor of DNA gyrase (M. tuberculosis DNA gyrase IC50 = 4.1 nM, Escherichia coli DNA gyrase IC50 of <10 nM), selective for bacterial topoisomerases. It displays low MIC90 values (M. tuberculosis: 0.63 µM; M. abscessus: 2.5 µM), showing specificity for mycobacteria, and no apparent toxicity. Compound 49 not only displays potent antimycobacterial activity (MIC90 values of 2.5 µM for M. tuberculosis and 0.63 µM for M. abscessus) and selectivity for mycobacteria but also exhibits favorable solubility (kinetic solubility = 55 µM) and plasma protein binding (with a fraction unbound of 2.9 % for human and 4.7 % for mouse). These findings underscore the potential of fine-tuning molecular properties to develop DNA gyrase B inhibitors that specifically target the mycobacterial chemical space, mitigating the risk of resistance development in non-target pathogens and minimizing harm to the microbiome.

Genomic insights into antibiotic resistance and mobilome of lactic acid bacteria and bifidobacteria.

Lactic acid bacteria (LAB) and Bifidobacterium sp. (bifidobacteria) can carry antimicrobial resistance genes (ARGs), yet data on resistance mechanisms in these bacteria are limited. The aim of our study was to identify the underlying genetic mechanisms of phenotypic resistance in 103 LAB and bifidobacteria using whole-genome sequencing. Sequencing data not only confirmed the presence of 36 acquired ARGs in genomes of 18 strains, but also revealed wide dissemination of intrinsic ARGs. The presence of acquired ARGs on known and novel mobile genetic elements raises the possibility of their horizontal spread. In addition, our data suggest that mutations may be a common mechanism of resistance. Several novel candidate resistance mechanisms were uncovered, providing a basis for further in vitro studies. Overall, 1,314 minimum inhibitory concentrations matched with genotypes in 92.4% of the cases; however, prediction of phenotype based on genotypic data was only partially efficient, especially with respect to aminoglycosides and chloramphenicol. Our study sheds light on resistance mechanisms and their transferability potential in LAB and bifidobacteria, which will be useful for risk assessment analysis.

Expanding the rumen Prevotella collection: The description of Prevotella communis, sp. nov. of ovine origin.

27 strains representing eight new Prevotella species were isolated from rumen of a single sheep in eight weeks interval. One of the putative species encompassing the highest number of isolated strains which also exhibited some genetic variability in preliminary data, was then selected for description of a novel species. We examined six strains in genomic and phenotypic detail, two of which may actually be the same strain isolated nearly three weeks apart. Other strains formed clearly diverged intraspecies lineages as evidenced by core genome phylogeny and phenotypic differences. Strains of the proposed new Prevotella species are strictly saccharolytic as is usual for rumen Prevotella, and use plant cell-wall xylans and pectins for growth. However, the range of cell-wall polysaccharides utilised for growth is rather limited compared to rumen generalists such as Prevotella bryantii or Prevotella ruminicola and this extends also to the inability to utilise starch, which is unexpected for the members of the genus Prevotella. Based on the data obtained, we propose Prevotella communis sp. nov. to accommodate strain E1-9T as well as other strains with the similar properties. The proposed species is widespread: two other strains were previously isolated from sheep in Japan and is also common in metagenomic data of cattle and sheep rumen samples from Scotland and New Zealand. It was also found in a collection of metagenome-assembled genomes originating from cattle in Scotland. Thus, it is a ubiquitous bacterium of domesticated ruminants specialising in degradation of a somewhat restricted set of plant cell wall components.

Broad host range may be a key to long-term persistence of bacteriophages infecting intestinal Bacteroidaceae species.

The longitudinal studies have found that the human gut microbiota is stable over time with some major bacterial lineages or even strains persisting for years. This was recently extended to gut bacteriophages using the metagenomic data. Here, we focused on cultivation of the major Bacteroidetes of human gut, the Bacteroides and Phocaeicola strains, and their bacteriophages from two healthy donors. The persistence of Bacteroides and Phocaeicola species and strains was confirmed. We isolated 28 genetically different phages grouped into seven distinct clusters, two of these were new. Moreover, the bacteriophages from several groups, although being genetically quite homogeneous, had the ability to infect the strains belonging to different species isolated from several sampling time-points and different donors. We propose that the ability to infect several host species, which differ in their nutritional niches, may promote long-term persistence of dominant gut bacteriophage groups.

Lactic acid bacteria and bifidobacteria deliberately introduced into the agro-food chain do not significantly increase the antimicrobial resistance gene pool.

Lactic acid bacteria (LAB) and bifidobacteria may serve as reservoirs of antimicrobial resistance, but the risk posed by strains intentionally introduced into the agro-food chain has not yet been thoroughly investigated. The aim of our study was to evaluate whether probiotics, starter and protective cultures, and feed additives represent a risk to human health. In addition to commercial strains of LAB and bifidobacteria, isolates from human milk or colostrum, intestinal mucosa or feces, and fermented products were analyzed. Phenotypic susceptibility data of 474 strains showed that antimicrobial resistance was more common in intestinal isolates than in commercial strains. Antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) were characterized in the whole genome sequences of 1114 strains using comparative genomics. Intrinsic ARGs were abundant in enterococci, bifidobacteria, and lactococci but were considered non-risky due to the absence of MGEs. The results revealed that 13.8% of commercial strains contained acquired ARGs, most frequently for tetracycline. We associated 75.5% of the acquired ARGs with known or novel MGEs, and their potential for transmission was assessed by examining metagenomic sequences. We confirmed that ARGs and MGEs were not as abundant or diverse in commercial strains as in human intestinal isolates or isolates from human milk, suggesting that strains intentionally introduced into the agro-food chain do not pose a significant threat. However, attention should be paid especially to individual probiotic strains containing elements that have been shown to have high potential for transferability in the gut microbiota.Abbreviations: ARG, antimicrobial resistance gene; ICE, integrative and conjugative element; IME, integrative and mobilizable element; LAB, lactic acid bacteria; MDR, multidrug resistance; MIC, minimum inhibitory concentration; MGE, mobile genetic element; TRRPP, tetracycline-resistant ribosomal protection protein; WGS, whole genome sequences.

How Can We Advance Integrative Biology Research in Animal Science in 21st Century? Experience at University of Ljubljana from 2002 to 2022.

In this perspective analysis, we strive to answer the following question: how can we advance integrative biology research in the 21st century with lessons from animal science? At the University of Ljubljana, Biotechnical Faculty, Department of Animal Science, we share here our three lessons learned in the two decades from 2002 to 2022 that we believe could inform integrative biology, systems science, and animal science scholarship in other countries and geographies. Cultivating multiomics knowledge through a conceptual lens of integrative biology is crucial for life sciences research that can stand the test of diverse biological, clinical, and ecological contexts. Moreover, in an era of the current COVID-19 pandemic, animal nutrition and animal science, and the study of their interactions with human health (and vice versa) through integrative biology approaches hold enormous prospects and significance for systems medicine and ecosystem health.

Acetan and Acetan-Like Polysaccharides: Genetics, Biosynthesis, Structure, and Viscoelasticity.

Bacteria produce a variety of multifunctional polysaccharides, including structural, intracellular, and extracellular polysaccharides. They are attractive for the industrial sector due to their natural origin, sustainability, biodegradability, low toxicity, stability, unique viscoelastic properties, stable cost, and supply. When incorporated into different matrices, they may control emulsification, stabilization, crystallization, water release, and encapsulation. Acetan is an important extracellular water-soluble polysaccharide produced mainly by bacterial species of the genera Komagataeibacter and Acetobacter. Since its original description in Komagataeibacter xylinus, acetan-like polysaccharides have also been described in other species of acetic acid bacteria. Our knowledge on chemical composition of different acetan-like polysaccharides, their viscoelasticity, and the genetic basis for their production has expanded during the last years. Here, we review data on acetan biosynthesis, its molecular structure, genetic organization, and mechanical properties. In addition, we have performed an extended bioinformatic analysis on acetan-like polysaccharide genetic clusters in the genomes of Komagataeibacter and Acetobacter species. The analysis revealed for the first time a second acetan-like polysaccharide genetic cluster, that is widespread in both genera. All species of the Komagataeibacter possess at least one acetan genetic cluster, while it is present in only one third of the Acetobacter species surveyed.

Novel Siphoviridae Bacteriophages Infecting Bacteroides uniformis Contain Diversity Generating Retroelement.

Intestinal phages are abundant and important components of gut microbiota, yet the isolated and characterized representatives that infect abundant gut bacteria are sparse. Here we describe the isolation of human intestinal phages infecting Bacteroidesuniformis. Bacteroides is one of the most common bacterial groups in the global human gut microbiota; however, to date not many Bacteroides specific phages are known. Phages isolated in this study belong to a novel viral genus, Bacuni, within the Siphoviridae family. Their genomes encode diversity-generating retroelements (DGR), which were shown in other bacteriophages to promote phage adaptation to rapidly changing environmental conditions and to broaden their host range. Three isolated phages showed 99.83% genome identity but one of them infected a distinct B. uniformis strain. The tropism of Bacuni phages appeared to be dependent on the interplay of DGR mediated sequence variations of gene encoding putative phage fimbrial tip proteins and mutations in host genes coding for outer-membrane proteins. We found prophages with up to 85% amino acid similarity over two-thirds of the Bacuni phage genome in the B. acidifaciens and Prevotella sp. genomes. Despite the abundance of Bacteroides within the human microbiome, we found Bacuni phages only in a limited subset of published gut metagenomes.

Characterization of antimicrobial resistance in lactobacilli and bifidobacteria used as probiotics or starter cultures based on integration of phenotypic and in silico data.

Lactic acid bacteria and bifidobacteria deliberately introduced into the food chain may act as a reservoir of antimicrobial resistance genes (ARGs), which is considered a safety concern. In the present study, resistance to antimicrobials of commercial probiotic strains, probiotic candidate strains, and starter cultures (n = 20) was characterised based on integration of phenotypic and in silico data. Minimum inhibitory concentrations (MICs) of 16 antimicrobials were determined for lactobacilli and bifidobacteria that were isolated from pharmaceutical products or obtained from the manufacturers or culture collections. Using different databases and bioinformatic tools, we predicted ARGs, mutations, genomic islands, and mobile genetic elements (MGEs) in their whole genome sequences. In addition, a comprehensive in silico analysis of the prevalence of the tetW gene and its genetic environment across lactobacilli and bifidobacteria (n = 1423) was conducted. Several strains exhibited phenotypic resistance to kanamycin, tetracycline, chloramphenicol, quinupristin-dalfopristin, ciprofloxacin, or neomycin. These resistances, however, did not always correspond to the presence of ARGs and vice versa. We detected an acquired tetW gene in four commercial strains of Bifidobacterium animalis subsp. lactis, whereas homologs of antimicrobial resistance (AR) proteins were predicted in all 20 proteomes. The prevalence of the tetW gene, which was often flanked by MGEs, was higher in analysed bifidobacteria (31.9%) than lactobacilli (6.3%). In addition, sequences flanking tetW were associated with putative genomic islands and were conserved in several strains, including potential pathogens. Our findings provide an insight into AR of probiotics, probiotic candidates, and starter cultures with an emphasis on tetracycline and into the safety of these strains in the context of AR.

Description of Komagataeibacter melaceti sp. nov. and Komagataeibacter melomenusus sp. nov. Isolated from Apple Cider Vinegar.

Two novel strains AV382 and AV436 were isolated from a submerged industrial bioreactor for production of apple cider vinegar in Kopivnik (Slovenia). Both strains showed very high (≥98.2%) 16S rRNA gene sequence similarities with Komagataeibacter species, but lower 16S-23S rRNA gene internal transcribed spacer (ITS). The highest similarity of the 16S-23S rRNA gene ITS of AV382 was to Komagataeibacter kakiaceti LMG 26206T (91.6%), of AV436 to Komagataeibacter xylinus LMG 1515T (93.9%). The analysis of genome sequences confirmed that AV382 is the most closely related to K. kakiaceti (ANIb 88.2%) and AV436 to K. xylinus (ANIb 91.6%). Genome to genome distance calculations exhibit for both strains ≤47.3% similarity to all type strains of the genus Komagataeibacter. The strain AV382 can be differentiated from its closest relatives K. kakiaceti and Komagataeibacter saccharivorans by its ability to form 2-keto and 5-keto-D-gluconic acids from glucose, incapability to grow in the presence of 30% glucose, formation of C19:0 cyclo ω8c fatty acid and tolerance of up to 5% acetic acid in the presence of ethanol. The strain AV436 can be differentiated from its closest relatives K. xylinus, Komagataeibacter sucrofermentans, and Komagataeibacter nataicola by its ability to form 5-keto-D-gluconic acid, growth on 1-propanol, efficient synthesis of cellulose, and tolerance to up to 5% acetic acid in the presence ethanol. The major fatty acid of both strains is C18:1ω7c. Based on a combination of phenotypic, chemotaxonomic and phylogenetic features, the strains AV382T and AV436T represent novel species of the genus Komagataeibacter, for which the names Komagataeibactermelaceti sp. nov. and Komagataeibacter melomenusus are proposed, respectively. The type strain of Komagataeibacter melaceti is AV382T (= ZIM B1054T = LMG 31303T = CCM 8958T) and of Komagataeibacter melomenusus AV436T (= ZIM B1056T = LMG 31304T = CCM 8959T).

Comparative genomics of Clostridioides difficile toxinotypes identifies module-based toxin gene evolution.

Clostridioides difficile is a common cause of nosocomial diarrhoea. Toxins TcdA and TcdB are considered to be the main virulence factors and are encoded by the PaLoc region, while the binary toxin encoded in the CdtLoc region also contributes to pathogenicity. Variant toxinotypes reflect the genetic diversity of a key toxin-encoding 19 kb genetic element (the PaLoc). Here, we present analysis of a comprehensive collection of all known major C. difficile toxinotypes to address the evolutionary relationships of the toxin gene variants, the mechanisms underlying the origin and development of variability in toxin genes and the PaLoc, and the relationship between structure and function in TcdB variants. The structure of both toxin genes is modular, composed of interspersed blocks of sequences corresponding to functional domains and having different evolutionary histories, as shown by the distribution of mutations along the toxin genes and by incongruences of domain phylogenies compared to overall C. difficile cluster organization. In TcdB protein, four mutation patterns could be differentiated, which correlated very well with the type of TcdB cytopathic effect (CPE) on cultured cells. Mapping these mutations to the three-dimensional structure of the TcdB showed that the majority of the variation occurs in surface residues and that point mutation at residue 449 in alpha helix 16 differentiated strains with different types of CPE. In contrast to the PaLoc, phylogenetic trees of the CdtLoc were more consistent with the core genome phylogenies, but there were clues that CdtLoc can also be exchanged between strains.

The diverse and extensive plant polysaccharide degradative apparatuses of the rumen and hindgut Prevotella species: A factor in their ubiquity?

Although the Prevotella are commonly observed in high shares in the mammalian hindgut and rumen studies using NGS approach, the knowledge on their actual role, though postulated to lie in soluble fibre degradation, is scarce. Here we analyse in total 23, more than threefold of hitherto known rumen and hindgut Prevotella species and show that rumen/hindgut Prevotella generally possess extensive repertoires of polysaccharide utilization loci (PULs) and carbohydrate active enzymes targeting various plant polysaccharides. These PUL repertoires separate analysed Prevotella into generalists and specialists yet a finer diversity among generalists is evident too, both in range of substrates targeted and in PUL combinations targeting the same broad substrate classes. Upon evaluation of the shares of species analysed in this study in rumen metagenomes we found firstly, that they contributed significantly to total Prevotella abundance though much of rumen Prevotella diversity may still be unknown. Secondly, the hindgut Prevotella species originally isolated in pigs and humans occasionally dominated among the Prevotella with surprisingly high metagenome read shares and were consistently found in rumen metagenome samples from sites as apart as New Zealand and Scotland. This may indicate frequent passage between different hosts and relatively low barriers to their successful establishment in rumen versus the hindgut.

The lytic Myoviridae of Enterobacteriaceae form tight recombining assemblages separated by discontinuities in genome average nucleotide identity and lateral gene flow.

In Bacteria, a working consensus of species circumscription may have been reached and one of the most prominent criteria is high average nucleotide identity (ANI). ANI in effect groups strains that may recombine more or less frequently, depending on their biology, as opposed to rare interspecies gene transfer. For bacteriophages, which show various lifestyles, the nature of the fundamental natural unit, if it exists in a biological sense, is not well understood and defined. The approaches based on dot-plots are useful to group similar bacteriophages, yet are not quantitative and use arbitrarily set cut-offs. Here, we focus on lytic Myoviridae and test the ANI metric for group delineation. We show that ANI-based groups are in agreement with the International Committee on Taxonomy of Viruses (ICTV) classification and already established dot-plot groups, which are occasionally further refined owing to higher resolution of ANI analysis. Furthermore, these groups are separated among themselves by clear ANI discontinuities. Their members readily exchange core genes with each other while they do not with bacteriophages of other ANI groups, not even with the most similar. Thus, ANI-delineated groups may represent the natural units in lytic Myoviridae evolution with features that resemble those encountered in bacterial species.

Complete Genome Sequences of Group III Campylobacter Bacteriophages PC5 and PC14.

Here, we present the whole-genome sequences of bacteriophages PC5 and PC14 specific for Campylobacter jejuni, a leading cause of gastroenteritis in developed countries. Their genomes are syntenic to those of group III Campylobacter bacteriophages and share more than 90% identity at the nucleotide level with members of this group.

Type II restriction modification systems of Prevotella bryantii TC1-1 and Prevotella ruminicola 23 strains and their effect on the efficiency of DNA introduction via electroporation.

The restriction endonucleases PbrTI and Pru2I, isoschizomers of Sau3AI and HaeIII, were partially purified and characterized from anaerobic rumen bacteria Prevotella bryantii TC1-1 and Prevotella ruminicola 23, respectively. These are the first type II restriction endonucleases discovered in strains of the genus Prevotella, and they represent one of the barriers hindering gene transfer in these microorganisms. Heterologous DNA was protected against the action of the PbrTI or Pru2I by incubation in a cell-free extract of the respective strain which contained 20 mM EDTA. This led to the development of a protocol enabling successful electrotransformation of the P. bryantii TC1-1 strain with a pRH3 Bacteroides--Escherichia coli shuttle vector containing up to 7-kb long DNA inserts. Plasmid DNA isolated from the transformed strain facilitated the transfer with further increased efficiency and made possible the introduction of ligation reaction products directly to P. bryantii TC1-1 without passing them first through E. coli.