Biology subjects

Read, T.

Publications and source records attributed to Read, T..

13 recordsLinked to original sources

bioRxiv2025

Effect of Azithromycin treatment on the microbial composition, functional dynamics and resistomes of endocervical, vaginal and rectal microbiomes of women in Fiji with Chlamydia trachomatis infection

Antibiotics disrupt mucosal microbial communities, yet the effects on microbiomes with Chlamydia trachomatis (Ct) infection remain poorly understood. Some data exist on vaginal microbiomes pre- and post-treatment, but none are available for the endocervix or rectum that are primary sites of infection. We applied metagenomic shotgun sequencing to vaginal, endocervical and rectal samples from women who, overtime, had Ct persistence, clearance, or no infection to evaluate azithromycin-induced changes in microbial composition, function, and the resistome. Our results show a shift in composition and function that support Ct post-treatment with azithromycin resistance mutations in the Ct rplV gene and significant endocervical enrichment of azithromycin resistance genes in Lactobacillus iners and Gardnerella vaginalis, the strains of which have moderate/high potential for biofilm formation. These findings highlight the unintended ecological consequences of azithromycin, including resistance gene propagation and microbiome shifts that support persistent/recurrent Ct, emphasizing the need for novel treatment and microbiome- preserving strategies.

microbiology↗

bioRxiv2024

Intraspecific Diversity of Staphylococcus aureus Populations Isolated from Cystic Fibrosis Respiratory Infections

Chronic bacterial infections are often polymicrobial, comprising multiple bacterial species or variants of the same species. Because chronic infections may last for decades, they have the potential to generate high levels of intraspecific variation through within-host diversification over time, and the potential for superinfections to occur through the introduction of multiple pathogen populations to the ongoing infection. Traditional methods for identifying infective agents generally involve isolating one single colony from a given sample, usually after selecting for a specific pathogen or antibiotic resistance profile. Isolating a recognized virulent or difficult to treat pathogen is an important part of informing clinical treatment and correlative research; however, these reductive methods alone, do not provide researchers or healthcare providers with the potentially important perspective on the true pathogen population structure and dynamics over time. To begin to address this limitation, in this study, we compare findings on Staphylococcus aureus single colonies versus and pools of colonies taken from fresh sputum samples from three patients with cystic fibrosis to isolates collected from the same sputum samples and processed by the clinical microbiology laboratory. Phenotypic and genotypic analysis of isolated S. aureus populations revealed coexisting lineages in two of three sputum samples as well as population structures that were not reflected in the single colony isolates. Altogether, our observations presented here demonstrate that clinically relevant diversity can be missed with standard sampling methods when assessing chronic infections. More broadly, this work outlines the potential impact that comprehensive population-level sampling may have for both research efforts and more effective treatment practices. Data SummaryThe authors confirm all supporting data, code and protocols have been provided within the article.

microbiology↗

bioRxiv2024

Host and antibiotic jointly select for greater virulence in Staphylococcus aureus

Widespread antibiotic usage has resulted in the rapid evolution of drug-resistant bacterial pathogens and poses significant threats to public health. Resolving how pathogens respond to antibiotics under different contexts is critical for understanding disease emergence and evolution going forward. The impact of antibiotics has been demonstrated most directly through in vitro pathogen passaging experiments. Independent from antibiotic selection, interactions with hosts have also altered the evolutionary trajectories and fitness landscapes of pathogens, shaping infectious disease outcomes. However, it is unclear how interactions between hosts and antibiotics impact the evolution of pathogen virulence. Here, we evolved and re-sequenced Staphylococcus aureus, a major bacterial pathogen, varying exposure to host and antibiotics to tease apart the contributions of these selective pressures on pathogen adaptation. After 12 passages, S. aureus evolving in Caenorhabditis elegans nematodes exposed to a sub-minimum inhibitory concentration of antibiotic (oxacillin) became highly virulent, regardless of whether the ancestral pathogen was methicillin-resistant (MRSA) or methicillin-sensitive (MSSA). Host and antibiotic exposure selected for reduced drug susceptibility in MSSA lineages while increasing MRSA total growth outside hosts. We identified mutations in genes involved in complex regulatory networks linking virulence and metabolism, including codY, agr, and gdpP, suggesting that rapid adaptation to infect hosts may have pleiotropic effects. In particular, MSSA populations under selection from host and antibiotic accumulated mutations in the global regulator gene codY, which controls biofilm formation in S. aureus. These populations had indeed evolved more robust biofilms--a trait linked to both virulence and antibiotic resistance--suggesting evolution of one trait can confer multiple adaptive benefits. Mutations that arose in these genes were also enriched in clinical isolates associated with systemic infections in humans. Despite evolving in similar environments, MRSA and MSSA populations--differing only in the presence of an intact accessory gene (mecA)--proceeded on divergent evolutionary paths, with MSSA populations exhibiting more similarities across replicate populations. Our results underscore the importance of considering the host context as a critical driver of pathogen traits like virulence and antibiotic resistance.

evolutionary biology↗

bioRxiv2023

Incomplete immunity in a natural animal-microbiota interaction boosts pathogen virulence

Strong partial immunity in recovered hosts is predicted to favour more virulent pathogens upon re-infection in the population. We present empirical evidence that the incomplete immunity generated by commensal host microbiota can similarly select for higher pathogen virulence. We tracked the evolutionary trajectories of a widespread pathogen (Pseudomonas aeruginosa) experimentally passaged through populations of nematode hosts which had been immune-primed by a natural commensal. Immune protection selected for pathogens more than twice as likely to kill the nematode as those evolved in non-primed or immune-compromised animals. Despite the higher virulence that emerged, pathogen molecular evolution in immune-primed hosts was slower and more constrained compared to evolution in immune-compromised hosts, where substantial genetic differentiation was exhibited. These findings directly attribute the partial protective immunity provided by host-microbiome interactions as a significant selective force shaping the virulence and evolutionary dynamics of novel infectious diseases.

evolutionary biology↗

bioRxiv2023

Integrative analysis of microbial 16S gene and shotgun metagenomic sequencing data improves statistical efficiency

The most widely used technologies for profiling microbial communities are 16S marker-gene sequencing and shotgun metagenomic sequencing. Interestingly, many microbiome studies have performed both sequencing experiments on the same cohort of samples. The two sequencing datasets often reveal consistent patterns of microbial signatures, highlighting the potential for an integrative analysis to improve power of testing these signatures. However, differential experimental biases, partially overlapping samples, and differential library sizes pose tremendous challenges when combining the two datasets. Currently, researchers either discard one dataset entirely or use different datasets for different objectives. In this article, we introduce the first method of this kind, named Com-2seq, that combines the two sequencing datasets for the objective of testing differential abundance at the genus and community levels while overcoming these difficulties. We demonstrate that Com-2seq substantially improves statistical efficiency over analysis of either dataset alone and works better than two ad hoc approaches.

bioinformatics↗

bioRxiv2023

The unique microbial diversity, community composition and networks of Pacific Islander endocervical and vaginal microbiomes in the presence or absence of Chlamydia trachomatis infection using metagenomic shotgun sequencing

BackgroundPacific Islanders are a vulnerable population with a high prevalence of Chlamydia trachomatis (Ct) sexually transmitted infections (STIs) and remain underrepresented in research. Here, 258 vaginal and 92 paired endocervical samples from women of diverse ethnicities in Fiji were evaluated using metagenomics to characterize microbial relative abundance, composition and networks including associations with Neisseria gonorrhoeae, human papilloma virus (HPV), Mycoplasma genitalium, Candida and bacterial vaginosis (BV). ResultsPacific Islander ethnicities and age <25 years were significantly associated with Ct infection. Using VALENCIA, a sub-community state type (subCST) classifier, 93 (36.04%) vaginal and 38 (41.30%) endocervical microbiomes did not match reference subCSTs. Four unique subCSTs were developed to better classify Pacific Islander microbiomes: IV-D0, dominated by Gardnerella vaginalis; IV-D1, high/moderate G. vaginalis relative abundance with Prevotella spp.; IV-D2, high/moderate G. vaginalis with Lactobacillus iners; and IV-E, moderate Prevotella spp. Ct positive endocervical and paired vaginal microbiomes were significantly more likely to have differential species relative abundance (81.58%) than Ct negative pairs (35.84%; AOR: 7.93; 95% CI: 2.93-21.93; P<0.0001). A significantly higher alpha diversity was found for iTaukei ethnicity, all subCST-IV types, BV, and Ct for vaginal microbiomes. For endocervical microbiomes, higher diversity was significant for subCST-IV-A, subCST-IV-D1, and subCST-IV-E, and high-risk HPV types. Overall, there was a significantly higher diversity for the endocervix in paired microbiomes. Vaginal microbiomes showed significant divergence in community composition as above and for Candida. Endocervical composition varied significantly by subCST type and Ct status. Gut and BV-associated bacterial clusters were present in Ct positive and negative paired endocervical and vaginal microbiome networks but were smaller with fewer bacterial and no Lactobacillus spp. interactions in Ct-infected endocervical networks where G. vaginalis generated polymicrobial biofilms along with Ct likely influence pathogenicity. ConclusionsFijian endocervical and vaginal microbiomes represent divergent microbial abundance and compositions, especially for Pacific Islander ethnicities, with distinct subCSTs compared to other global populations. The higher microbial diversity of the endocervix with prevalent G. vaginalis, L. iners and Prevotella spp. suggest that these microbiomes/networks may predispose to and/or promote chlamydial and HPV pathogenesis. Prospective studies are needed to further define causal associations to develop successful interventions.

microbiology↗

bioRxiv2023

Unraveling Genomic Diversity in Pseudomonas aeruginosa Cystic Fibrosis Lung Infection and Its Impact on Antimicrobial Resistance

Pseudomonas aeruginosa is an opportunistic pathogen which causes chronic, drug-resistant lung infections in cystic fibrosis (CF) patients. In this study, we explore the role of genomic diversification and evolutionary trade-offs in antimicrobial resistance (AMR) diversity within P. aeruginosa populations sourced from CF lung infections. We analyzed 300 clinical isolates from four CF patients (75 per patient), and found that genomic diversity is not a consistent indicator of phenotypic AMR diversity. Remarkably, some genetically less diverse populations showed AMR diversity comparable to those with significantly more genetic variation. We also observed that hypermutator strains frequently exhibited increased sensitivity to antimicrobials, contradicting expectations from their treatment histories. Investigating potential evolutionary trade-offs, we found no substantial evidence of collateral sensitivity among aminoglycoside, beta-lactam, or fluoroquinolone antibiotics, nor did we observe trade-offs between AMR and growth in conditions mimicking CF sputum. Our findings suggest that (i) genomic diversity is not a prerequisite for phenotypic AMR diversity; (ii) hypermutator populations may develop increased antimicrobial sensitivity under selection pressure; (iii) collateral sensitivity is not a prominent feature in CF strains, and (iv) resistance to a single antibiotic does not necessarily lead to significant fitness costs. These insights challenge prevailing assumptions about AMR evolution in chronic infections, emphasizing the complexity of bacterial adaptation during infection. ImportanceUpon infection in the cystic fibrosis (CF) lung, Pseudomonas aeruginosa rapidly acquires genetic mutations, especially in genes involved in antimicrobial resistance (AMR), often resulting in diverse, treatment-resistant populations. However, the role of bacterial population diversity within the context of chronic infection is still poorly understood. In this study, we found that hypermutator strains of P. aeruginosa in the CF lung undergoing treatment with tobramycin evolved increased sensitivity to tobramycin relative to non-hypermutators within the same population. This finding suggests that antimicrobial treatment may only exert weak selection pressure on P. aeruginosa populations in the CF lung. We further found no evidence for collateral sensitivity in these clinical populations, suggesting that collateral sensitivity may not be a robust, naturally occurring phenomenon for this microbe. Preprint servers: This manuscript has been submitted as a preprint to bioRxiv

microbiology↗

bioRxiv2023

Comparison of genomic diversity between single and pooled Staphylococcus aureus colonies isolated from human colonisation cultures.

The most common approach to sampling the bacterial populations within an infected or colonised host is to sequence genomes from a single colony obtained from a culture plate. However, it is recognized that this method does not capture the genetic diversity in the population. An alternative is to sequence a mixture containing multiple colonies ("pool-seq"), but this has the disadvantage that it is a non-homogeneous sample, making it difficult to perform specific experiments. We compared differences in measures of genetic diversity between eight single-colony isolates (singles) and pool-seq on a set of 2286 S. aureus culture samples. The samples were obtained by swabbing three body sites on 85 human participants quarterly for a year, who initially presented with a methicillin-resistant S. aureus skin and soft-tissue infection (SSTI). We compared parameters such as sequence quality, contamination, allele frequency, nucleotide diversity and pangenome diversity in each pool to the corresponding singles. Comparing singles from the same culture plate, we found that 18% of sample collections contained mixtures of multiple Multilocus sequence types (MLSTs or STs). We showed that pool-seq data alone could predict the presence of multi-ST populations with 95% accuracy. We also showed that pool-seq could be used to estimate the number of polymorphic sites in the population. Additionally, we found that the pool may contain clinically relevant genes such as antimicrobial resistance markers that may be missed when only examining singles. These results highlight the potential advantage of analysing genome sequences of total populations obtained from clinical cultures rather than single colonies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/544959v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1920f45org.highwire.dtl.DTLVardef@289262org.highwire.dtl.DTLVardef@1a2890borg.highwire.dtl.DTLVardef@1d33d4c_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Pre-epidemic evolution of the USA300 clade and a molecular key for classification

USA300 has remained the dominant community and healthcare associated methicillin-resistant Staphylococcus aureus (MRSA) clone in the United States and in northern South America for at least the past 20 years. In this time, it has experienced epidemic spread in both of these locations. However, its pre-epidemic evolutionary history and origins are incompletely understood. Large sequencing databases, such as NCBI, PATRIC, and Staphopia, contain clues to the early evolution of USA300 in the form of sequenced genomes of USA300 isolates that are representative of lineages that diverged prior to the establishment of the South American (SAE) and North American (NAE) epidemics. In addition, historical isolates collected prior to the emergence of epidemics can help reconstruct early events in the history of this lineage. Here, we take advantage of the accrued, publicly available data, as well as two newly sequenced pre-epidemic historical isolates from 1996, and a very early diverging ACME-negative NAE genome to understand the pre-epidemic evolution of USA300. We use database mining techniques to emphasize genomes similar to pre-epidemic isolates, with the goal of reconstructing the early molecular evolution of the USA300 lineage. Phylogenetic analysis with these genomes confirms that the North American Epidemic and South American Epidemic USA300 lineages diverged from a most recent common ancestor around 1970 with high confidence, and it also pinpoints the independent acquisition events of the of the ACME and COMER loci with greater precision than in previous studies. We solidify evidence for a North American origin of the USA300 lineage and identify multiple introductions of USA300 into South America from North America. Notably, we describe a third major USA300 clade (the pre-epidemic branching clade; PEB1) consisting of both MSSA and MRSA isolates circulating around the world that diverged from the USA300 lineage prior to the establishment of the South American and North American epidemics. We present a detailed analysis of specific sequence characteristics of each of the major clades, and present diagnostic positions that can be used to classify new genomes.

genomics↗

bioRxiv2020

Genes influencing phage host range in Staphylococcus aureus on a species-wide scale

Staphylococcus aureus is a human pathogen that causes serious diseases ranging from skin infections to septic shock. Bacteriophages ("phages") are both natural killers of S. aureus, offering therapeutic possibilities, as well as important vectors of horizontal gene transfer in the species. Here, we used high-throughput approaches to understand the genetic basis of strain-to-strain variation in sensitivity to phages, which defines the host range. We screened 259 diverse S. aureus strains covering more than 40 sequence types for sensitivity to eight phages, which were representatives of the three phage classes that infect the species. The phages were variable in host range, each infecting between 73 and 257 strains. Using genome-wide association approaches, we identified putative loci that affect host range and validated their function using USA300 transposon knockouts. In addition to rediscovering known host range determinants, we found several previously unreported genes affecting bacterial growth during phage infection, including trpA, phoR, isdB, sodM, fmtC, and relA. We used the data from our host range matrix to develop predictive models that achieved between 40 and 95% accuracy. This work illustrates the complexity of the genetic basis for phage susceptibility in S. aureus but also shows that with more data, we may be able to understand much of the variation. With a knowledge of host range determination, we can rationally design phage therapy cocktails that target the broadest host range of S. aureus strains and address basic questions regarding phage-host interactions, such as the impact of phage on S. aureus evolution. ImportanceStaphylococcus aureus is a widespread, hospital- and community-acquired pathogen, many strains of which are antibiotic resistant. It causes diverse diseases ranging from local to systemic infection and affects both the skin and many internal organs, including the heart, lungs, bones, and brain. Its ubiquity, antibiotic resistance, and disease burden make new therapies urgent. One alternative therapy to antibiotics is phage therapy, in which viruses specific to infecting bacteria clear infection. In this work, we identified and validated S. aureus genes that influence phage host range - the number of strains a phage can infect and kill - by testing strains representative of the diversity of the S. aureus species for phage host range and associating strains genome sequences with host range. These findings together improved our understanding of how phage therapy works in the bacterium and improve prediction of phage therapy efficacy based on the infecting strains predicted host range.

microbiology↗

bioRxiv2020

Genome-wide binding analysis of 195 DNA Binding Proteins reveals "reservoir" promoters and human specific SVA-repeat family regulation

A key aspect in defining cell state is the complex choreography of DNA binding events in a given cell type, which in turn establishes a cell-specific gene-expression program. In the past two decades since the sequencing of the human genome there has been a deluge of genome-wide experiments which have measured gene-expression and DNA binding events across numerous cell-types and tissues. Here we re-analyze ENCODE data in a highly reproducible manner by utilizing standardized analysis pipelines, containerization, and literate programming with Rmarkdown. Our approach validated many findings from previous independent studies, underscoring the importance of ENCODEs goals in providing these reproducible data resources. This approach also revealed several new findings: (i) 1,362 promoters, termed reservoirs, have up to 111 different DNA binding-proteins localized on one promoter yet do not have any expression of steady-state RNA (ii) The human specific SVA repeat element may have been co-opted for enhancer regulation. Collectively, this study performed by the students of a CU Boulder computational biology class (BCHM 5631 - Spring 2020) demonstrates the value of reproducible findings and how resources like ENCODE that prioritize data standards can foster new findings with existing data in a didactic environment.

genomics↗

bioRxiv2019

Suppression of p53 response by targeting p53-Mediator binding with a stapled peptide

DNA-binding transcription factors (TFs) remain challenging to target with molecular probes. Many TFs function in part through interaction with Mediator; we sought to block p53 function by disrupting the p53-Mediator interaction. Through rational design and activity-based screening, we characterized a stapled peptide, with functional mimics of both p53 activation domains, that selectively inhibited p53- and Mediator-dependent transcription in vitro. This \"bivalent peptide\" also suppressed p53 transcriptional response in human cancer cells. Our strategy circumvents the TF and instead targets the TF-Mediator interface, with desired transcriptional outcomes. Different TFs target Mediator through different subunits, suggesting this strategy could be broadly applied.

molecular biology↗

bioRxiv2019

The whale shark genome reveals patterns of vertebrate gene family evolution

Chondrichthyes (cartilaginous fishes) are fundamental for understanding vertebrate evolution, yet their genomes are understudied. We report long-read sequencing of the whale shark genome to generate the best gapless chondrichthyan genome assembly yet with higher contig contiguity than all other cartilaginous fish genomes, and studied vertebrate genomic evolution of ancestral gene families, immunity, and gigantism. We found a major increase in gene families at the origin of gnathostomes (jawed vertebrates) independent of their genome duplication. We studied vertebrate pathogen recognition receptors (PRRs), which are key in initiating innate immune defense, and found diverse patterns of gene family evolution, demonstrating that adaptive immunity in gnathostomes did not fully displace germline-encoded PRR innovation. We also discovered a new Toll-like receptor (TLR29) and three NOD1 copies in the whale shark. We found chondrichthyan and giant vertebrate genomes had decreased substitution rates compared to other vertebrates, but gene family expansion rates varied among vertebrate giants, suggesting substitution and expansion rates of gene families are decoupled in vertebrate genomes. Finally, we found gene families that shifted in expansion rate in vertebrate giants were enriched for human cancer-related genes, consistent with gigantism requiring adaptations to suppress cancer.

genomics↗