Biology subjects

Krych, L.

Publications and source records attributed to Krych, L..

15 recordsLinked to original sources

bioRxiv2026

Multilayer analysis reveals extensive hidden population complexity in a dairy starter microbiome comprising few species

The genomic and epigenomic complexity of microbiomes with low species richness remains poorly characterized. Here, we integrated 111 non-redundant isolate genomes with deep long-read metagenomics to investigate an undefined mesophilic dairy starter. Closely related isolates harboured distinct combinations of features associated with fermentation traits and phage defence, including surface polysaccharide genotypes, plasmids, prophages, defence systems, and methylation profiles. Metagenomics revealed additional elements and inferred host associations. However, the limited overlap between approaches across core-genome lineages and accessory repertoires underscored complementary but incomplete recovery, to the extent that the dominant metagenomic cell wall polysaccharide genotype (29.6% abundance) was absent from isolates. Metagenomic methylation signatures supported 36 species-level plasmid-host assignments, corroborated by isolate genomes in nine of ten testable cases. Our findings revealed extensive combinatorial diversity hidden from either approach alone, providing a plausible molecular basis for phage resilience, while likely representing only a lower bound on population complexity.

microbiology↗

bioRxiv2026

Ultra-High Multiplexing Enables Near-Full-Length 16S rRNA Gene Amplicon Sequencing of Over 1,200 Gut Microbiome Samples on a Single Nanopore Flow Cell

Next-generation sequencing (NGS) of the prokaryotic 16S rRNA gene revolutionized gut microbiome research two decades ago. However, short read lengths remain an inherent limitation of platforms such as the widely used Illumina platforms (2 x 150-300 bp). Recent advances in Oxford Nanopore Technologies (ONT) flow cell chemistry (R10.4.1) have substantially improved sequencing accuracy. Combined with a custom multiple-primer strategy that comprehensively targets 16S rRNA gene variants to generate near-full-length amplicons, this approach enables read-by-read taxonomic classification, a feature not feasible with short-read sequencing platforms. Although our multiple-primer strategy could enable parallel sequencing of more than 18,000 samples (192 x 96), current flow cell capacity offers sufficient sequencing depth for approximately 1,000-1,500 samples. To validate the scalability and our per-read classification pipeline, we show that more than a thousand human fecal microbiome samples spiked with two bacterial strains (Imtechella halotolerans and Allobacillus halotolerans), not otherwise present in human fecal samples, can be successfully sequenced on a single flow cell, achieving a per-molecule error rate sufficient for direct per-read classification and at an adequate read depth for downstream analysis. This level of scalability significantly reduces per-sample costs, making the approach more accessible to a broader research community. To embrace these advancements, we have developed RubyRed, a pipeline that processes raw sequencing data and assigns taxonomic classifications on a per-read basis. Using spike-in references (I. halotolerans and A. halotolerans), we demonstrate high mean single-read sequencing accuracy (99% and 98.9%, respectively), with the majority of reads exceeding the canonical threshold required for species-level taxonomic classification based on the 16S rRNA gene.

microbiology↗

bioRxiv2025

Characterization of a virulent bacteriophage consortium targeting Enterobacteriaceae from inflamed preterm gut mucosa

Preterm infants have a high risk of intestinal inflammation which can progress to necrotizing enterocolitis (NEC). The gut microbial colonization commencing at birth is essential for proper intestinal maturation, but this process is often disrupted in preterm infants, leading to dysbiosis and increased risk of developing NEC. Bacteriophages (phages), viruses that specifically infect bacteria, are an important constituent of the gut microbiome and protects the gut epithelium against invading bacteria. This study aimed to isolate and characterize phages for use as a preventive measure against NEC-associated bacteria. We initially cultured Enterobacteriaceae from ileal mucosa of preterm piglets that exhibited severe NEC-like pathology. We then screened 23 donor fecal samples for inhibition of bacterial growth and isolated a collection of unique phages to use further. The phages were characterized by whole genome sequencing, host receptor binding determination, and immune cell activation in vitro. The final phage collection consisted of ten virulent phages within five genera, representing myovirus, podovirus and siphovirus morphologies. All phages in the collection induced expression of both pro-and anti-inflammatory genes in co-culture with macrophage-like THP-1 cells, but to different extents than Escherichia coli. Ultimately, we selected one phage for a high-dose oral administration to newborn piglets and assessed its infectivity and presence in different gut segments. This intervention did not result in any direct side effects, while both infective phages and signatures of phage DNA were detected in the intestinal content and mucosa. Having characterized a set of rationally selected virulent phages, we support the advancement of phage therapy as a potential protection against NEC.

microbiology↗

bioRxiv2024

A single strand-based library preparation method for unbiased virome characterization

The gut virome is an integral component of the gut microbiome, playing a crucial role in maintaining gut health. However, accurately depicting the entire gut virome is challenging due to the inherent limitations and biases associated with current sequencing library preparation methods. To overcome these problems, we repurposed the ligation-based single-stranded library (SSLR) preparation method for virome studies. We demonstrate that the SSLR method exhibits exceptional efficiency in quantifying viral DNA genomes (both dsDNA and ssDNA) and outperforms existing double-stranded (Nextera) and single-stranded (xGen, MDA+Nextera) library preparation approaches in terms of minimal amplification bias, evenness of coverage, and integrity of assembling viral genomes. The SSLR method can be utilized for the simultaneous library preparation of both DNA and RNA viral genomes. Furthermore, the SSLR method showed its ability to capture highly modified phage genomes, which were often lost using other library preparation approaches.

microbiology↗

bioRxiv2024

Matching Excellence: ONT's Rise to Parity with PacBio in Genome Reconstruction of Non-Model Bacterium with High GC Content

Reconstruction of complete bacterial genomes is a vital aspect of microbial research, as it provides complex information about genetic content, gene ontology, and regulation. It has become a domain of 3rd generation, long-read sequencing platforms, as short-read technologies can deliver mainly fragmented genomes. PacBio platform can provide high-quality complete genomes, yet remains one of the most expensive sequencing strategies. Oxford Nanopore Technology (ONT) offers the advantage of producing the longest reads, being at the same time the most cost-effective option in terms of platform costs, as well as library preparation, and sequencing. However, ONTs error rate, although significantly reduced lately, still holds a certain level of distrust in the scientific community. In recent years, hybrid assembly of Nanopore and Illumina data has been used to solve ONTs issue with error rate and has yielded the best results in terms of genome completeness, quality, and price. However, the latest advancements in Nanopore technology, including new flow cells (R10.4.1), new library preparation chemistry (V14) and duplex-mode, updated basecallers (Dorado v0.4.1), and the realization that sequencing in dark mode results in significantly increased throughput, have had a significant impact on the quality of generated data and, thus, the recovery of complete genomes by ONT sequencing alone. In this study, we compared the data generated by ONT using three sequencing strategies (Native barcoding, RAPID barcoding, and custom-developed: BARSEQ) against PacBio and Illumina (NextSeq) as well as Illumina-ONT hybrid data. For this purpose, we employed three strains of the actinobacteria Propionibacterium freudenreichii, whose genomes have been proven difficult to reconstruct due to high GC content, regions of repeated sequences and massive genome rearrangements. Our data indicate that DNA libraries prepared with the native barcoding kit, sequenced with V14 chemistry on R10.4.1 flow cell, and assembled with Flye resulted in the reconstruction of complete genomes of overall quality highly similar to that of genomes reconstructed with PacBio. The highest level of quality can be achieved by hybrid assembly of data from the Native barcoding kit complemented with data from custom-developed BARSEQ, both sequenced on R10.4.1 flow cell. In conclusion, our results demonstrate that ONT can be used as a cost-effective sequencing strategy, without the need for complementing with other sequencing technologies, for the reconstruction of complete genomes of the highest quality.

genomics↗

bioRxiv2023

De novo clustering of long-read amplicons improves phylogenetic insight into microbiome data

Long-read amplicon profiling through read classification limits phylogenetic analysis of amplicons while community analysis of multicopy genes, relying on unique molecular identifier (UMI) corrections, often demands deep sequencing. To address this, we present a long amplicon consensus analysis (LACA) workflow employing multiple de novo clustering approaches based on sequence dissimilarity. LACA controls the average error rate of corrected sequences below 1% for the Oxford Nanopore Technologies (ONT) R9.4.1 and ONT R10.3 data, 0.2% for ONT R10.4.1, and 0.1% for high-accuracy ONT Duplex and Pacific Biosciences (PacBio) circular consensus sequencing (CCS) data in both simulated 16S rRNA and real 16-23S rRNA amplicon datasets. In high-accuracy PacBio CCS data, the clustering-based correction matched UMI correction, while outperforming 4xUMI correction in noisy ONT R10.3 and R9.4.1 data. Notably, LACA preserved phylogenetic fidelity in long operational taxonomic units and enhanced microbiome-wide phenotype characterization for synthetic mock communities and human vaginal samples.

bioinformatics↗

bioRxiv2023

Investigating the Rescue Potential of Fecal Microbiota Transplants in Anorexia Nervosa using Antibiotic-treated Mice in a Split Group Cross-Over Study

Anorexia nervosa (AN) is a complex and serious mental disorder, which may affect individuals of all ages and sex, but primarily affecting young women. The disease is characterized by a disturbed body image, restrictive eating behavior, and a lack of acknowledgment of low body weight. The underlying causes of AN remain largely unknown, and current treatment options are limited to psychotherapy and nutritional support. This paper investigates the impact of Fecal Microbiota Transplants (FMT) from AN patients on food intake, body weight, behavior, and gut microbiota in antibiotic-treated mice. Two rounds of FMT were performed using AN and control (CO) donors. During the second round of FMT, a subset of mice received FMT from a different donor type. This split-group cross-over design was chosen to demonstrate any recovery effect of FMT from a healthy donor. The first FMT, from AN donors, resulted in lower food intake in mice without affecting body weight. After FMT2, serum analysis revealed higher levels of appetite-influencing hormones (PYY and leptin) in mice receiving AN-GM. Gut microbiota analysis showed significant differences between AN and CO mice after FMT1, before cross-over. Specific bacterial genera and families Ruminococcaceae, Lachnospiraceae, and Faecalibacterium showed different abundances in AN and CO receiving mice. Behavioral tests showed decreased locomotor activity in AN mice after FMT1. Overall, the results suggest that AN-GM may contribute to altered food intake and appetite regulation, which can be ameliorated with FMT from a healthy donor potentially offering FMT as a supportive treatment for AN.

microbiology↗

bioRxiv2022

Dietary non-starch polysaccharides impair immunity to enteric nematode infection

The influence of diet on immune function and resistance to enteric infection and disease is becoming ever more established. Highly processed, refined diets can lead to inflammation and gut microbiome dysbiosis, whilst health-promoting dietary components such as phytonutrients and fermentable fibres are thought to promote a healthy microbiome and balanced mucosal immunity. Chicory (Cichorium intybus) is a leafy green vegetable rich in fibres and bioactive compounds that may promote gut health. Unexpectedly, we here show that incorporation of chicory into semisynthetic AIN93G diets renders mice susceptible to infection with enteric helminths. Mice fed a high level of chicory leaves (10% dry matter) had a more diverse gut microbiota, but a diminished type-2 immune response to infection with the intestinal roundworm Heligmosomoides polygyrus. Furthermore, the chicory-supplemented diet significantly increased burdens of the caecum-dwelling whipworm Trichuris muris, concomitant with a highly skewed type-1 immune environment in caecal tissue. The chicory-supplemented diet was rich in non-starch polysaccharides, particularly uronic acids (the monomeric constituents of pectin). In accordance, mice fed pectin-supplemented AIN93G diets had higher T. muris burdens and reduced IgE production and expression of genes involved in type-2 immunity. Importantly, treatment of pectin-fed mice with exogenous IL-25 restored type-2 responses and was sufficient to allow T. muris expulsion. Collectively, our data suggest that increasing levels of fermentable, non-starch polysaccharides in refined diets compromises immunity to helminth infection in mice. This diet-infection interaction may inform new strategies for manipulating the gut environment to promote resistance to enteric parasites.

immunology↗

bioRxiv2021

Dietary Proanthocyanidins Exert Localized Immunomodulatory Effects in Porcine Pulmonary and Gastrointestinal Tissues during Ascaris suum-induced Type 2 inflammation

Bioactive dietary components may considerably influence intestinal health and resistance to enteric disease. Proanthocyanidins (PAC) are dietary polyphenols with putative health-promoting activity that have been increasingly studied for their anti-inflammatory and immunomodulatory effects. However, whether dietary PAC can regulate type-2 immune function and inflammation at mucosal surfaces remains unclear. Here, we investigated whether diets supplemented with purified PAC modulated pulmonary and intestinal mucosal immune responses during infection with the helminth parasite Ascaris suum in pigs. A. suum infection induced a type 2-biased immune response in lung and intestinal tissues, characterized by pulmonary granulocytosis, increased Th2/Th1 T cell ratios in tracheal-bronchial lymph nodes, intestinal eosinophilia, and modulation of genes involved in mucosal barrier function and immunity. We observed that PAC had only minor effects on pulmonary immune responses, regardless of concurrent A. suum infection. However, RNA-sequencing of intestinal tissues revealed that dietary PAC significantly enhanced transcriptional responses related to immune function, antioxidant responses, and cellular stress activity, both in uninfected and A. suum-infected animals. A. suum infection and dietary PAC both induced distinct changes in gut microbiota composition, primarily in the jejunum and colon, respectively. Notably, PAC substantially increased Limosilactobacillus reuteri abundance in the colon of both naive and A. suum-infected animals. Thus, dietary PAC may have distinct beneficial effects on intestinal health during infection with mucosal pathogens, whilst having limited activity to modulate naturally-induced type-2 pulmonary inflammation. Our results shed further light on the mechanisms underlying the health-promoting properties of PAC-rich foods, and may aid in the design of novel dietary supplements to regulate mucosal inflammatory responses in the gastrointestinal tract.

immunology↗

bioRxiv2021

Parasite-probiotic interactions in the gut: Bacillus sp. and Enterococcus faecium regulate type-2 inflammatory responses and modify the gut microbiota of pigs during helminth infection

Dietary probiotics may enhance gut health by directly competing with pathogenic agents and through immunostimulatory effects. These properties are recognized in the context of bacterial and viral pathogens, but less is known about interactions with eukaryotic pathogens such as parasitic worms (helminths). In this study we investigated whether two probiotic mixtures (comprised of Bacillus amyloliquefaciens, B. subtilis, and Enterococcus faecium [BBE], or Lactobacillus rhamnosus LGG and Bifidobacterium animalis subspecies Lactis Bb12 [LB]) could modulate helminth infection kinetics as well as the gut microbiome and intestinal immune responses in pigs infected with the nodular worm Oesophagostomum dentatum. We observed that neither probiotic mixture influenced helminth infection levels. BBE, and to a lesser extent LB, changed the alpha- and beta-diversity indices of the colon and faecal microbiota, notably including an enrichment of faecal Bifidobacterium spp. by BBE. However, these effects were muted by concurrent O. dentatum infection. BBE (but not LB) significantly attenuated the O. dentatum-induced upregulation of genes involved in type-2 inflammation and restored normal lymphocyte ratios in the ileo-caecal lymph nodes that were altered by infection. Moreover, inflammatory cytokine release from blood mononuclear cells and intestinal lymphocytes was diminished by BBE. Collectively, our data suggest that selected probiotic mixtures can play a role in maintaining immune homeostasis during type 2-biased inflammation. In addition, potentially beneficial changes in the microbiome induced by dietary probiotics may be counteracted by helminths, highlighting the complex inter-relationships that potentially exist between probiotic bacteria and intestinal parasites.

microbiology↗

bioRxiv2021

The phytonutrient cinnamaldehyde limits intestinal inflammation and enteric parasite infection

Phytonutrients such as cinnamaldehyde (CA) have been studied for their effects on metabolic diseases, but their influence on mucosal inflammation and immunity to enteric infection are not well documented. Here, we show that consumption of CA significantly down-regulates transcriptional pathways connected to inflammation in the small intestine of mice. During infection with the enteric helminth Heligomosomoides polygyrus, CA-treated mice displayed higher growth rates and less worms, concomitant with altered T-cell populations in mesenteric lymph nodes. Furthermore, infection-induced changes in gene pathways connected to cell cycle and mitotic activity were counteracted by CA. Mechanically, CA did not appear to exert activity through a prebiotic effect, as CA treatment did not significantly change the composition of the gut microbiota. Instead, in vitro experiments showed that CA directly induced xenobiotic metabolizing pathways in intestinal epithelial cells and suppressed endotoxin-induced inflammatory responses in macrophages. Thus, CA down-regulates inflammatory pathways in the intestinal mucosa and regulates host responses to enteric infection. These properties appear to be largely independent of the gut microbiota and instead connected to CAs ability to induce antioxidant pathways in intestinal cells. Our results encourage further investigation into the use of CA and related phytonutrients as functional food components to promote intestinal health in humans and animals.

immunology↗

bioRxiv2020

Supplementation of a lacto-fermented rapeseed-seaweed blend promotes gut microbial- and gut immune-modulation in weaner piglets

The direct use of medical zinc oxide (ZnO) in feed will be abandoned after 2022 in Europe, leaving an urgent need for substitutes to prevent post-weaning disorders. This study assessed whether rapeseed meal added two brown macroalagae species (Saccharina latissima and Ascophylum nodosum) and fermented using lactic acid bacteria (FRS) could improve piglet performance and gut health. The weaned piglets were fed one of three different feeding regimens (n = 230 each): basal diet, 2.5% and 5% FRS from day 28 of life to day 85. The piglets fed with 2.5% FRS presented superior phenotype with alleviated intraepithelial and stromal lymphocytes infiltration in the gut, enhanced colon mucosa barrier as well as numerically improvements of final body weight. Colon microbiota composition was determined using amplicon sequencing of the V3 and V1 - V8 region of the 16S rRNA gene using Illumina Nextseq and Oxford Nanopore MinION sequencing, respectively. The two amplicon sequencing strategies showed high consistence between the detected bacteria. Both sequencing technologies showed that the FRS fed piglets had a distinctly different microbial composition relative to the basal diet. Compared with piglets fed the basal diet, Prevotella stercorea was verified by both technologies to be more abundant in the FRS piglets, and positively correlated with colon mucosa thickness and negatively correlated with blood levels of leucocytes and IgG. In conclusion, FRS supplementation improved gut health of weaner piglets, and altered their gut microbiota composition. Increasing the dietary inclusion of FRS from 2.5% to 5% did not cause further improvements.

microbiology↗

bioRxiv2020

Gut Mycobiome Dysbiosis Is Linked to Hypertriglyceridemia among Home Dwelling Elderly Danes

Gut microbial dysbiosis have been linked to frailty in elderly, yet the presence of fungal communities and their possible association with host health are little understood. This study attempts to identify gut microbial fungal associations with the progression of atherogenic dyslipidemia in a population of older adults by investigating the interplay between dietary intake, gut mycobiome composition, plasma and fecal metabolome and anthropometric/body-composition measurements of 99 Danes aged 65 to 81 (69.57 {+/-} 3.64) years. The gut mycobiome composition were determined by high-throughput sequencing of internal transcribed spacer (ITS2) gene amplicons, while the plasma and fecal metabolome was determined by GC-MS. The gut microbiome of the subjects investigated is home to three main eukaryotic phyla, namely Ascomyco-ta, Basidiomycota and Zygomycota, with genera Penicillium, Candida, and Aspergillus being particularly common. Hypertriglyceridemia was associated with fewer observed fungal species, and Bray-Curtis dissimilarity matrix-based analysis showed significant (p < 0.05) clustering according to fasting levels of circulating plasma triglycerides (Tg) and very low-density lipoprotein (VLDL) cholesterol fasting levels, respectively. Higher levels of Tg and VLDL cholesterol significantly associates with increased relative abundance of genus Penicillium, and Saccha:ramyces likely mediated by a higher dietary fatty acids intake (p < 0.05), and Sac-charomyces, Debaryomyces, Candida, Agaricus and Starmerella were moderately associated with SCF As groups. Collectively, these findings suggest that gut mycobiome dysbiosis on older adults is associated with hypertriglyceridemia, a known risk factor for development of cardiovascular disease.

microbiology↗

bioRxiv2019

Physical fitness in community dwelling older adults is linked to dietary intake, gut microbiota and metabolomic signatures

When humans age, changes in body composition arise along with lifestyle-associated disorders influencing fitness and physical decline. Here we provide a comprehensive view of dietary intake, physical activity, gut microbiota (GM) and host metabolome in relation to physical fitness of 207 community dwelling subjects aged +65 years. Stratification on anthropometric/body-composition/physical-performance measurements (ABPm) variables identified two phenotypes (high/low-fitness) clearly linked to dietary intake, physical activity, GM and host metabolome patterns. Strikingly, despite a higher energy intake high-fitness subjects were characterized by leaner bodies and lower fasting proinsulin-C-peptide/blood glucose levels in a mechanism likely driven by higher dietary-fiber intake, physical activity and increased abundance of Bifidobacteriales and Clostridiales species in GM and associated metabolites (i.e. enterolactone). These factors explained 50.1% of the individual variation in physical fitness. We propose that targeting dietary strategies for modulation of GM and host metabolome interactions may allow establishing therapeutic approaches to delay and possibly revert comorbidities of aging.

cell biology↗

bioRxiv2018

Finally, Bulk Typing of Bacterial Species down to Strain Level using ON-rep-seq

Despite the massive developments within culture-independent methods for detection and quantification of microorganisms during the last decade, culture-based methods remain a cornerstone in microbiology. We have developed a new method for bacterial DNA enrichment and tagmentation allowing fast (< 24h) and cost-effective species level identification and strain level differentiation using the MinION portable sequencing platform (ON-rep-seq). DNA library preparation takes less than 5h and ensures highly reproducible distribution of reads that can be used to generate strain level specific read length counts profiles (LCp). We have developed a pipeline that by correcting the random error of reads within peaks of LCp generates a set ([~]10 contigs per sample; 300bp - 3Kb) of high quality (>99%) consensus reads. Whereas, the information from high quality reads is used to retrieve species level taxonomy, comparison of LCp allows for strain level differentiation. With benchmarked 288 isolates identified on a single flow cell and a theoretical throughput to evaluate over 1000 isolates, our method allows for detailed bacterial identification for less than 2$ per sample at very high speed.

microbiology↗