Biology subjects

Ksiezarek, M.

Publications and source records attributed to Ksiezarek, M..

7 recordsLinked to original sources

bioRxiv2026

Critical assessment of intratumor and low-biomass microbiome using long-read sequencing

The detection of low-biomass microbial DNA in human tissues is often confounded by contamination, as demonstrated in the debates over the existence of microbiomes in the placenta, brain, blood, and tumors. Here we show that genomic DNA fragment length serves as a discriminator: while genuine microbiome genomes have long genomic DNA fragments, contaminant DNA is typically short. Using germ-free mouse tissues with bacterial spike-ins and human cell lines, we developed a metric that normalizes microbial read length to host read length. Across multiple human tumor and normal tissues, we found genuine microbiome signals are largely limited to tissues with natural microbial exposure (e.g., gastrointestinal tract, cervix, vagina, skin), while other tissues (e.g. kidney, brain, blood, and placenta) showed no evidence of resident microbiome. These findings support DNA fragment length as a metric for quality controlling low-biomass microbiome profiling, clarifying the debates and strengthen future studies of resident microbiome in tissues.

genomics↗

bioRxiv2025

Epigenetic phase variation in the gut microbiome enhances bacterial adaptation

The human gut microbiome within the gastrointestinal tract continuously adapts to variations in diet, medications, and host physiology. A strategy for bacterial genetic adaptation is epigenetic phase variation (ePV) mediated by bacterial DNA methylation, which can regulate gene expression, enhance clonal heterogeneity, and enable a single bacterial strain to exhibit variable phenotypic states. Genome-wide and site-specific ePVs have been characterized in human pathogens antigenic variation and virulence factor production. However, the role of ePV in facilitating adaptation within the human microbiome remains poorly understood. Here, we comprehensively cataloged genome-wide and site-specific ePV in human infant and adult gut microbiomes. First, using long-read metagenomic sequencing, we detected genome-wide ePV mediated by complex structural variations of DNA methyltransferases, highlighting those associated with antibiotics or fecal microbiota transplantation. Second, we analyzed a collection of public short-read metagenomic sequencing datasets, uncovering a great prevalence of genome-wide ePV in the human gut microbiome. Third, we quantitatively detected site-specific ePVs using single-molecule methylation analysis to identify dynamic variation associated with antibiotic treatment or probiotic engraftment. Finally, we performed an in-depth assessment of an Akkermansia muciniphila isolate from an infant, highlighting that ePVs can regulate gene expression and enhance the bacterial adaptive capacity by employing a bet-hedging strategy to increase tolerance to differing antibiotics. Our findings indicate that epigenetic modifications are a common strategy used by gut bacteria to adapt to the fluctuating environment.

microbiology↗

bioRxiv2024

Long-read metagenomics empowers precise tracking of bacterial strains and their genomic changes after fecal microbiota transplantation

Fecal microbiota transplantation (FMT) has revolutionized the treatment of recurrent Clostridioides difficile infection (rCDI) and is being evaluated across other diseases. Accurate tracking of bacterial strains that stably engraft in recipients is critical for understanding the determinants of strain engraftment, evaluating their correlation with clinical outcomes, and guiding the development of therapeutic bacterial consortia. While short-read sequencing has advanced FMT research, it faces challenges in strain-level de novo metagenomic assembly. In this study, we described a novel framework, LongTrack, which uses long-read metagenomic assemblies and rigorous informatics tailored for FMT strain tracking. We highlighted LongTracks advantage over short-read approaches especially when multiple strains co-exist in the same sample. We showed LongTrack uncovered hundreds of engrafted strains across six FMT cases of rCDI and inflammatory bowel disease patients. Furthermore, long reads also allowed us to assess the genomic and epigenomic stability of engrafted strains during the 5-year follow-ups, revealing structural variations that may be associated with strain adaptation in a new host environment. Combined, our study advocates the use of long-read metagenomics and LongTrack to enhance strain tracking in future FMT studies, paving the way for the development of more effective defined biotherapeutic as an alternative to FMT.

microbiology↗

bioRxiv2024

Predictive power of different Akkermansia phylogroups in clinical response to PD-1 blockade against non-small cell lung cancer

Immune checkpoint blockade has emerged as a promising form of cancer therapy. However, only some patients respond to checkpoint inhibitors, while a significant proportion of patients do not, calling for the discovery of reliable biomarkers. Recent studies reported the importance of the gut microbiome in the clinical response to PD-1 blockade against non-small cell lung cancer (NSCLC), highlighting Akkermansia muciniphila as a candidate biomarker. Motivated by the genomic and phenotypic differences across Akkermansia muciniphila strains and Akkermansia (Akk) phylogroups (AmIa, AmIb, AmII, AmIII and AmIV), we analyzed fecal metagenomic sequencing data from four publicly available NSCLC cohorts (n = 575). Encouragingly, we found that patients responses to PD-1 blockade are significantly different across different Akk phylogroups, highlighting the relatively stronger association between AmIa and positive responses than the other phylogroups. Importantly, we also highlight the importance of considering the clinical heterogeneity among independent cohorts in across validation analysis. We built a machine learning model based on Akk gene profiles, which shed light on a group of Akk genes that may be associated with the response to PD-1 blockade. In summary, our study underlines the benefits of high-resolution analysis of Akk genomes in the search for biomarkers that may improve the prediction of patients responses to cancer immunotherapy.

genomics↗

bioRxiv2022

mEnrich-seq: Methylation-guided enrichment sequencing of bacterial taxa of interest from microbiome

Metagenomics has enabled the comprehensive study of microbiomes. However, many applications would benefit from a method that can sequence specific bacterial taxa of interest (pathogens, beneficial microbes, or low-abundance taxa), but not the vast background of other taxa in a microbiome sample. To address this need, we developed mEnrich-seq, a method that can enrich taxa of interest from metagenomic DNA before sequencing. The core idea is to exploit the self vs. non-self genome differentiation provided by natural bacterial DNA methylation and rationally choose methylation-sensitive restriction enzymes (REs), individually or in combination, to deplete host DNA and most background microbial DNA while enriching bacterial taxa of interest. This core idea is integrated with library preparation procedures in a way that only non-digested DNA libraries are sequenced. We performed in-depth evaluations of mEnrich-seq and demonstrated its use in several applications to enrich (up to 117-fold) genomic DNA of pathogenic or beneficial bacteria from human urine and fecal samples, including several species that are hard to culture or of low abundance. We also assessed the broad applicability of mEnrich-seq and found that 3130 (68.03%) of the 4601 strains with mapped methylomes to date can be targeted by at least one commercially available RE, representing 54.78% of the species examined in this analysis. mEnrich-seq provides microbiome researchers with a versatile and cost-effective approach for selective sequencing of diverse taxa of interest directly from the microbiome.

genomics↗

bioRxiv2022

Extended bacterial diversity of the urinary microbiome of reproductive-age healthy European women captured by culturomics and long-read amplicon sequencing

The recognition of microbiome inhabiting the healthy female bladder engendered the need for comprehensive characterization of the female urinary microbiome (FUM) in health and disease. Although previous studies reported FUM composition at different taxonomic levels, progress towards reliable identification at species level is highly required. The aim of this study was to comprehensively characterize bacterial species of FUM of healthy reproductive-age European women by two complementary methodologies i.e., extended culturomics and long-read third generation sequencing of near full-length 16S rRNA gene. A wide diversity of bacterial species was captured (297 species) with a median of 53 species/sample, including 16 putative uropathogens. Clustering FUM into community structure types revealed high inter-individual differences. Notably, there was not a single species common to all samples, although the Lactobacillus genus was detected in all samples. Lactobacillus crispatus, Lactobacillus iners and Lactobacillus mulieris were observed in high relative abundance in several samples as well as other species (e.g., Streptococcus agalactiae, Atopobium vaginae, Gardnerella vaginalis, Gardnerella swidsinskii), while more prevalent species were often low abundant members (e.g., Finegoldia magna). We captured remarkable richness within Corynebacterium spp. (25 species) and Lactobacillaceae (4 genera, 14 species). While amplicon sequencing allowed detection of more anaerobic species (e.g., 11 Peptoniphilus spp.), culturomics enabled the identification of recently recognized Gardnerella species and putative novel Corynebacterium species. This study provided fine-grained FUM profiling at species level and revealed detailed FUM structure, which is critical to unveil the potential relationship between specific microbiome members and urinary diseases/disorders. IMPORTANCEDespite evidence of the resident microbial community in the female lower urinary tract, bacterial species diversity and abundance in healthy women is still unclear. This study demonstrated that complementarity between optimized culture-dependent and -independent approaches is highly beneficial for comprehensive FUM species profiling by detecting higher FUM species diversity than previously reported, including identification of unreported Lactobacillaceae species and putative novel Corynebacterium species. Although some particular species were present in high relative abundance, low-abundant members were more prevalent. FUM classification into community structure types demonstrated high inter-individual differences in urinary microbiome composition among healthy women. We also report moderate correlation between culture-dependent and -independent derived data highlighting drawbacks resulting from each methodological approach. Our findings suggest that FUM bacterial diversity reported from previous studies may be underestimated. Finally, our results contribute to the fundamental knowledge of healthy FUM required for further exploration of the urinary microbiome role in urinary tract diseases.

microbiology↗

bioRxiv2021

The darkest place is under the candlestick - healthy urogenital tract as a source of UTI-related Escherichia coli lineages

Since the discovery of the urinary microbiome, including identification of Escherichia coli in healthy hosts, its involvement in UTI development is a subject of high interest. We explored population diversity and antimicrobial resistance of E. coli from urogenital microbiome of asymptomatic and recurrent UTI (rUTI) women. We also evaluated the genomic relationship between extraintestinal pathogenic E. coli (ExPEC) strains from healthy and diseased hosts, particularly of the ST131 lineage. E. coli was highly prevalent in asymptomatic women (48%) with slightly higher prevalence in vaginal samples comparing to urine, and occasionally with multiclonal population in the same individual. B2 was the most frequent phylogenetic group, with most strains classified as ExPEC. We demonstrated that virulence associated genes profile does not allow to distinguish strains isolated from healthy and rUTI host. We identified E. coli widespread lineages e.g., sequence types (ST) 127, ST131 (asymptomatic cohort) and ST73, ST131 (rUTI), frequently resistant to at least one antibiotic. Phylogenomics of ST131 and other ExPEC lineages revealed close relatedness between healthy and diseased host. These findings demonstrate that healthy urogenital microbiome is a source of potentially pathogenic and antibiotic resistant E. coli strains, including globally spread E. coli lineages causing UTI including ST131.

microbiology↗