Biology subjects

Read, T. D.

Publications and source records attributed to Read, T. D..

At least 19 recordsLinked to original sources

bioRxiv2026

Protein-level mapping reveals novel Immune State Types (ISTs) associated with Chlamydia trachomatis infection, inflammatory remodeling, and scarring in trachoma-hyperendemic Amhara Ethiopia

ABSTRACT Background: Trachoma remains the leading infectious cause of blindness. Over 100M people are at risk, and 1.9M already live with visual impairment or blindness. Ethiopia bears the greatest burden of disease with ~61M or 59% of the known affected global population. Although Chlamydia trachomatis (Ct) infection initiates disease, infection alone fails to explain why inflammation resolves in some but progresses to irreversible scarring and vision loss in others. Previous studies have largely examined gene expression or limited protein panels, leaving the conjunctival immune protein environment poorly characterized. We hypothesized that distinct conjunctival immune states capture these pathogenic processes and could reveal biomarkers for selecting treatment and surveillance strategies. Methods and Findings: We conducted a cross-sectional study of 667 participants residing in the hyperendemic Amhara region of Ethiopia at least one year after the most recent azithromycin mass drug administration to treat Ct. The upper tarsal conjunctivae was graded for TF and TI, trachomatous inflammation-follicular or intense, respectively; I-TS/TT, inflammation with scarring with or without trichiasis, defined as one or more eyelashes touching the globe of the eye; and TS and TT, scarring with or without trichiasis but no inflammation. T0 represented no trachoma. The protein concentrations of 41 immune mediators were quantified using Meso Scale Discovery electrochemiluminescence immunoassays. Associations with infection and grade were evaluated using covariate-adjusted models with false discovery rate correction. Bernoulli mixture modeling (BMM) revealed four distinct Immune State Types (ISTs). Both approaches supported a shared biological framework characterized by selective immune activation in Ct infection, broad inflammation in TF/TI, and chemokine remodeling versus immune quiescence in the scarred conjunctiva. Ct infection was associated with a focused IFN-{gamma}-centered response. IST1 captured this immune response where 51.9% of the participants had Ct; we therefore termed IST1 Quiescent Infection. In contrast, TF/TI was associated with higher concentrations of proinflammatory, Th1/Th17, chemokine, regulatory, and remodeling pathways mediators. This profile fit well within IST2, termed Broad Inflammation, where 72.4% of the participants had TF/TI. TS/TT retained considerable chemokine activity, whereas I-TS/TT showed a narrower, IL-17A-dominant inflammatory profile. BMM separated scarring into two states characterized by distinct profiles: IST3, Conjunctival Remodeling, with significantly stronger chemokine and T-cell maintenance signals; and IST4, Quiescent Scarring, with the majority of chemokines being significantly downregulated. This suggests that scarred conjunctiva can retain substantial immune activity or shift toward a more attenuated state. Conclusions: This study establishes the first IST framework for trachoma, revealing biologically distinct immune states with different potential clinical implications. IST1 indicates the need for antibiotic treatment, whereas IST2 suggests that novel immunotherapeutics might be effective in thwarting disease progression. The upregulated chemokines in IST3 may indicate risk of progression to TT necessitating prospective studies and potential novel antifibrotic interventions. Together, our findings provide a strong biological foundation for stratifying patients by immune state and matching them to preventive and therapeutic care. Prospective validation and streamlined biomarker panels will support the translation of this framework into field settings to help avert disease progression and blindness.

microbiology↗

bioRxiv2026

Detecting and typing Chlamydia trachomatis strains in metagenomes using the MetaChlam pipeline

The Gram negative bacteria Chlamydia trachomatis (Ct), an obligate intracellular human pathogen, is a predominant cause of sexually transmitted infections and ocular trachoma globally, exerting a significant impact on public health. Ct "strains" (major lineages within the species) are known to have different tissue tropisms and be associated with different disease outcomes. Metagenome samples from typical sites where Ct infects (e.g., endocervix, conjunctiva, rectum) rarely contain enough reads for traditional genotyping methods such as Multi-Locus Sequence Typing (MLST) or ompA genotyping. To overcome these limitations, we implemented an ensemble tool called MetaChlam that can accurately classify Ct strains with as few as 250 Ct reads. Using 109 publicly available Ct genomes from naturally circulating strains, we established that an ANI-based threshold of 99.75% was capable of distinguishing Ct strains from each other. We implemented metagenome-based typing using the previously developed LINtax, Strainscan, StrainGE, and Sourmash softwares. MetaChlam integrated the four tools along with custom databases into an automated nextflow pipeline. Using simulated metagenomic reads, we found that our pipeline accurately identified the correct strains in both single strain and multi-strain mixtures of samples. Finally, we showed that MetaChlam had higher specificity for the true presence of Ct reads in NCBI SRA metagenomic datasets than NCBI PebbleScout software. A surprising finding of these analyses was that reads from Ct, an obligate human intracellular pathogen, can be found as contaminants in samples from sites where the organism is almost certainly not present. Overall, our study enhances the characterization and classification of Ct strains and provides protocols for identification and typing of Ct in shotgun metagenome data. The MetaChlam pipeline is available on Github: https://github.com/parul-sharma/MetaChlam.

bioinformatics↗

bioRxiv2026

Ocular community state types reveal distinct microbial compositions among microbiomes with implications for trachoma control

Trachoma, a chronic ocular disease caused by Chlamydia trachomatis (Ct), is the leading infectious cause of blindness worldwide. Despite WHOs SAFE (Surgery, Antibiotics, Facial cleanliness, Environmental improvement) strategy, [~]100M are at risk of blindness. Using metagenomic shotgun sequencing, we characterized the ocular microbiome of 680 villagers in Amhara Ethiopia, identifying 10 Community State Types (CSTs) associated with different population characteristics. Children with the highest prevalence of inflammatory trachoma and Ct were in CST10, dominated by Haemophilus influenzae and four other Haemophilus spp. Adults with the highest prevalence of scarring trachoma were in CST3 and CST6, dominated by Corynebacterium macginleyi. CST5, dominated by Mesomycoplasma hyorhinis and Staphylococcus aureus, had the lowest prevalence of Ct and trachoma, and was the only CST without zoonotic Chlamydia spp. Both M. hyorhinis, a zoonotic porcine bacterium, and S. aureus are capable of forming biofilms, which may competitively prevent/down-regulate chlamydial infections. Other CSTs were dominated by environmental species like Vibrio. This is the first microbiome study to develop CSTs for trachoma. Pathogenic and potentially protective microbes showed distinct associations with demographic, clinical, and chlamydial characteristics, which will guide the design of microbial therapeutics as alternatives to antibiotics and strategies for WHOs global elimination of blinding trachoma.

microbiology↗

bioRxiv2025

Comparative genomics of Sympodiorosea identifies genome evolution mediated through selective pressure on the metabolic gene repertoire

Biological interactions involving host-associated fungi are manifestations of chemistry shaped over evolutionary time. For antagonistic fungi, specialization to host fungi is often facilitated through the acquisition of genes encoding novel secretions, including proteins and specialized metabolites that shape interactions with host defenses, as well the acquisition of nutrients. Alternatively, novel function may arise from diversifying selection eliciting evolutionary innovation in inert secretions. Through specialization, fungal metabolic capacity can serve as an imprint of the selective forces imposed on the antagonist and provide insights into its natural history and occupied niche. Here, we conduct a phylogenomic investigation of antagonistic ascomycetes in the genus Sympodiorosea, which are commonly found in basal fungus-growing ant gardens. Sympodiorosea and closely related genera of Escovopsis, Luteomyces, and Escovopsioides are canonically referenced as virulent mycoparasites, however, recent work has illuminated the possibility for diverse, non-virulent species to emerge within these attine-associated fungi. We explored genomic variation in Sympodiorosea to gain insight into the genomic potential for alternative lifestyles, focusing on diversity and evolution of metabolic genes. Our study revealed a constrained selective landscape across the Sympodiorosea genome. However, outcomes of in vitro interactions with host fungi are diverse and predictable based on the antagonists ant-species-of-origin, suggesting functional diversification. Phylogenomics reveals that gain/loss events of genes involved in secretions (secretome) are potential drivers of diversification. Additionally, we demonstrate that purifying selection acts more intensely on secretome-related genes relative to other genes. In contrast, few genes from the secretome experience diversifying selection, suggesting a mechanistic role for driving both functional differences between strains and host-specialization. Comparative genomics including other fungi within the family Hypocreales reveals that Sympodiorosea has experienced expansions and contractions in proteases genes that are discordant from expectations under a strictly mycoparasitic lifestyle, indicating either the potential for an alternative lifestyle within ant gardens, or that Sympodiorosea recently evolved from other niches. These results provide novel insight into genomic evolution of these fungi and inform future experimental studies of the ecology and chemistry of interactions within the complex fungus-growing ant symbiosis.

evolutionary biology↗

bioRxiv2025

Putative azithromycin-resistance mutations in Chlamydia trachomatis are globally distributed but arose before azithromycin was discovered

Azithromycin is widely used to treat Chlamydia trachomatis infections, yet the extent of resistance to the drug across the species has not been addressed. We surveyed mutations and substitutions linked to putative azithromycin resistance across 1,354 high-quality C. trachomatis genomes. Mutations in the rplV gene encoding three non-synonymous substitutions, compared to the canonical C. trachomatis reference strain D//TW-3/Cx sequence, were found to be common but largely conserved within lineages causing prevalent urogenital and anorectal infections and lymphogranuloma venereum (LGV). Time-scaled phylogenetic analysis suggested that these mutations predate the clinical introduction of azithromycin. In contrast, no consistent resistance-associated patterns were observed in 23S rRNA or rplD genes. This large-scale genomic surveillance provides critical insights into the evolutionary trends of putative azithromycin resistance in C. trachomatis and underscores the importance of integrating genomic monitoring with phenotypic susceptibility testing to accurately assess and manage antimicrobial resistance.

genomics↗

bioRxiv2025

Transition of Staphylococcus aureus tetracycline resistance plasmid pT181 from independent multicopy replicon to predominantly integrated chromosomal element over 65 years

Mobile genetic elements (MGEs), including plasmids, phages and genome islands, are major sources of bacterial genetic diversity. The small plasmid pT181 confers tetracycline resistance in bacterial pathogen Staphylococcus aureus via an efflux pump, TetK. pT181 was one of the earliest sequenced S. aureus plasmids and has been isolated in both clinical and livestock-associated strains for decades, both as an independent replicon and integrated in the chromosome as part of staphylococcal cassette chromosome mec (SCCmec). Bacterial genome analysis tools and high-quality sequences with metadata are publicly available, but these resources remain underleveraged for examining historical data, especially when studying the spread of MGEs across a species and over time. Using publicly available reads and metadata, we explored the evolution of pT181 over nearly seven decades of samples to identify temporal trends in sequence evolution, copy number changes, and spread across S. aureus and beyond. pT181 was prevalent across S. aureus (found in 9.5% of 83,366 genomes tested), with a conserved sequence outside of three hypervariable regions. The history of pT181 since 1954 was characterized by spread across strains, significant variation in plasmid copy number of the independent replicon, and increasing frequency of integration of the plasmid into the S. aureus chromosome. We identified multiple chromosomal integration locations of the plasmid, including outside of the previously characterized SCCmec. We found that pT181 was transferred across Staphylococcaceae and into a Gram negative species. The repeated integration of pT181 into the chromosome may indicate coevolution of the plasmid and the host, potentially to facilitate increased antibiotic resistance.

genomics↗

bioRxiv2024

Average Nucleotide Identity based Staphylococcus aureus strain grouping allows identification of strain-specific genes in the pangenome

Staphylococcus aureus causes both hospital and community acquired infections in humans worldwide. Due to the high incidence of infection S. aureus is also one of the most sampled and sequenced pathogens today, providing an outstanding resource to understand variation at the bacterial subspecies level. We processed and downsampled 83,383 public S. aureus Illumina whole genome shotgun sequences and 1,263 complete genomes to produce 7,954 representative substrains. Pairwise comparison of core gene Average Nucleotide Identity (ANI) revealed a natural boundary of 99.5% that could be used to define 145 distinct strains within the species. We found that intermediate frequency genes in the pangenome (present in 10-95% of genomes) could be divided into those closely linked to strain background ("strain-concentrated") and those highly variable within strains ("strain-diffuse"). Non-core genes had different patterns of chromosome location; notably, strain-diffuse associated with prophages, strain-concentrated with the vSa{beta} genome island and rare genes (<10% frequency) concentrated near the origin of replication. Antibiotic genes were enriched in the strain-diffuse class, while virulence genes were distributed between strain-diffuse, strain-concentrated, core and rare classes. This study shows how different patterns of gene movement help create strains as distinct subspecies entities and provide insight into the diverse histories of important S. aureus functions. ImportanceWe analyzed the genomic diversity of Staphylococcus aureus, a globally prevalent bacterial species that causes serious infections in humans. Our goal was to build a genetic picture of the different strains of S. aureus and which genes may be associated with them. We used a large public dataset (>84,000 genomes) that was re-processed and subsampled to remove redundancy. We found that individual genomes could be grouped into strains by sharing > 99.5% identical nucleotide sequence of the core part of their genome. We also showed that a portion of genes that are present in intermediate frequency in the species are strongly associated with some strains but completely absent from others, suggesting a role in strain-specificity. This work lays the foundation for understanding individual gene histories of the S. aureus species and also outlines strategies for processing large bacterial genomic datasets.

microbiology↗

bioRxiv2023

Genomic insights into the evolution of secondary metabolism of Escovopsis and its allies, specialized fungal symbionts of fungus-farming ants

The metabolic intimacy of symbiosis often demands the work of specialists. Natural products and defensive secondary metabolites can drive specificity by ensuring infection and propagation across host generations. But in contrast to bacteria, little is known about the diversity and distribution of natural product biosynthetic pathways among fungi and how they evolve to facilitate symbiosis and adaptation to their host environment. In this study, we define the secondary metabolism of Escovopsis and closely related genera, members of which are specialized, diverse ascomycete fungi best known as mycoparasites of the fungal cultivars grown by fungus-growing ants. We ask how the gain and loss of various biosynthetic pathways corresponds to divergent lifestyles. Long-read sequencing allowed us to define the chromosomal features of representative Escovopsis strains, revealing highly reduced genomes (21.4-38.3 Mb) composed of 7-8 chromosomes. Escovopsis genomes are highly co-linear, with genes localizing not only in the same chromosome, but also in the same order. Macrosynteny is high within Escovopsis clades, and decreases with increasing phylogenetic distance, while maintaining a high degree of mesosynteny. To explore the evolutionary history of biosynthetic pathways in this group of symbionts relative to their encoding lineages, we performed an ancestral state reconstruction analysis, which revealed that, while many secondary metabolites are shared with non-ant associated sordariomycetes, 56 pathways are unique to the symbiotic genera. Reflecting adaptation to diverging ant agricultural systems, we observe that the stepwise acquisition of these pathways mirrors the ecological radiations of attine ants and the dynamic recruitment and replacement of their fungal cultivars. As different clades encode characteristic combinations of biosynthetic gene clusters, these delineating profiles provide important insights into the possible mechanisms underlying specificity between these symbionts and their hosts. Collectively, our findings shed light on the evolutionary dynamic nature of secondary metabolism in Escovopsis and its allies, reflecting adaptation of the symbionts to an ancient agricultural system.

evolutionary biology↗

bioRxiv2023

Experimentally Evolved Staphylococcus aureus Survives in the Presence of Pseudomonas aeruginosa by Acquiring Mutations in the Amino Acid Transporter, GltT

Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacterial pathogens isolated from cystic fibrosis (CF) related lung infections. When both of these opportunistic pathogens are found in a coinfection, CF patients tend to have higher rates of pulmonary exacerbations and experience a more rapid decrease in lung function. When cultured together under standard laboratory conditions, it is often observed that P. aeruginosa effectively inhibits S. aureus growth. Previous work from our group revealed that S. aureus from CF infections have isolate-specific survival capabilities when cocultured with P. aeruginosa. In this study, we designed a serial transfer evolution experiment to identify mutations that allow S. aureus to adapt to the presence of P. aeruginosa. Using S. aureus USA300 JE2 as our ancestral strain, populations of S. aureus were repeatedly cocultured with fresh P. aeruginosa strain, PAO1. After 8 coculture periods, S. aureus populations that survived better in the presence of PAO1 were observed. We found two independent mutations in the highly conserved S. aureus aspartate transporter, gltT, that were unique to evolved P. aeruginosa-tolerant isolates. Subsequent phenotypic testing demonstrated that gltT mutants have reduced uptake of glutamate and outcompete wild-type S. aureus when glutamate is absent from chemically-defined media. These findings together demonstrate that the presence of P. aeruginosa exerts selective pressure on S. aureus to alter its uptake and metabolism of key amino acids when the two bacteria are cultured together. ImportanceStaphylococcus aureus and Pseudomonas aeruginosa are the two most common bacterial pathogens that infect people with the genetic disease, cystic fibrosis (CF). They are often found together in CF-associated polymicrobial infections that are associated with worse patient prognosis. Understanding how these very different opportunistic pathogens influence each other in a shared environment is pertinent to improving the treatment of polymicrobial infections. While much attention has been brought to the interspecific interactions between S. aureus and P. aeruginosa, few studies have used experimental evolution methods to identify determinants of their competition and coexistence. Here, we use a serial transfer experimental evolution approach and identified a single genetic change associated with improved survival of S. aureus in the presence of P. aeruginosa. Our findings implicate metabolism of shared resources as an important factor in S. aureuss ability to survive in the presence of P. aeruginosa.

microbiology↗

bioRxiv2023

Patterns of within-host spread of Chlamydia trachomatis between vagina, endocervix and rectum revealed by comparative genomic analysis

Chlamydia trachomatis, a gram-negative obligate intracellular bacterium, commonly causes sexually transmitted infections (STIs). Little is known about C. trachomatis transmission within the host, which is important for understanding disease epidemiology and progression. We used RNA-bait enrichment and whole-genome sequencing to compare rectal, vaginal and endocervical samples collected at the same time from 26 study participants who attended Fijian Ministry of Health and Medical Services clinics and tested positive for C. trachomatis at each anatomic site. The 78 C. trachomatis genomes from participants were from two major clades of the C. trachomatis phylogeny (the "prevalent urogenital and anorecta"l clade and "non-prevalent urogenital and anorectal" clade). For 21 participants, genome sequences were almost identical in each anatomic site. For the other five participants, two distinct C. trachomatis strains were present in different sites; in two cases, the vaginal sample was a mixture of strains. The absence of large numbers of fixed SNPs between C. trachomatis strains within many of the participants could indicate recent acquisition of infection prior to the clinic visit without sufficient time to accumulate significant variation in the different body sites. This model suggests that many C. trachomatis infections may be resolved relatively quickly in the Fijian population, possibly reflecting common prescription or over-the-counter antibiotics usage. ImportanceChlamydia trachomatis is a bacterial pathogen that causes millions of sexually transmitted infections (STIs) annually across the globe. Because C. trachomatis lives inside human cells, it has historically been hard to study. We know little about how the bacterium spreads between body sites. Here, samples from 26 study participants who had simultaneous infections in their vagina, rectum and endocervix were genetically analyzed using an improved method to extract C. trachomatis DNA directly from clinical samples for genome sequencing. By analyzing patterns of mutations in the genomes, we found that 21 participants shared very similar C. trachomatis strains in all three anatomic sites, suggesting recent infection and spread. For five participants two C. trachomatis strains were evident, indicating multiple infections. This study is significant in that improved enrichment methods for genome sequencing provides robust data to genetically trace patterns of C. trachomatis infection and transmission within an individual for epidemiologic and pathogenesis interrogations.

microbiology↗

bioRxiv2022

Development of an amplicon nanopore sequencing strategy for detection of mutations conferring intermediate resistance to vancomycin in Staphylococcus aureus strains

Staphylococcus aureus is a major cause of bacteremia and other hospital-acquired infections. The cell-wall active antibiotic vancomycin is commonly used to treat both methicillin-resistant (MRSA) and sensitive (MSSA) infections, but vancomycin intermediate S. aureus (VISA) variants can arise through de novo mutations. Here we performed pilot experiments to develop a combined PCR/long-read sequencing-based method for detection of previously known VISA-causing mutations. We amplified 16 genes (walR, walK, rpoB, graR, graS, vraF, vraG, stpI, vraR, vraS, agrA, sarA, clpP, ccpA, prsA, and yvqF) known from prior studies to be associated with mutations responsible for VISA as 10 amplicons and sequenced amplicon pools as long-reads with Oxford Nanopore adapter ligation on Flongle flow cells. We then detected mutations by mapping reads against a parental consensus or known reference sequence and comparing called variants against a database of known VISA mutations from laboratory selection. There was high (>1000x) coverage of each amplicon in the pool, no relationship between amplicon length and coverage, and the ability to detect the causative mutation (walK 646C>G) in a VISA mutant derived from the USA300 strain (N384-3 from parental strain N384). Mixing mutant (N384-3) and parental (N384) DNA at various ratios from 0 to 1 mutant suggested a mutation detection threshold of roughly the average minor allele frequency of 6.5% at 95% confidence (two standard errors above mean mutation frequency). The study lays the groundwork for direct S. aureus antibiotic phenotype inference using rapid nanopore sequencing from clinical samples. ImportanceBacteremia mortality is known to increase rapidly with time after infection, making rapid diagnostics and treatment necessary. Successful treatment depends on correct administration of antibiotics based on knowledge of strain antibiotic susceptibility. Staphylococcus aureus is a major causative agent of bacteremia and is also increasingly antibiotic resistant. In this work, we develop a method to improve detection of a complex, polygenic antibiotic resistance phenotype in S. aureus, vancomycin-intermediate resistance (VISA) through long-read genomic sequencing of amplicons representing genes most commonly mutated in VISA selection. This method both speeds up VISA determination relative to purely culture-based detection and incorporates the most comprehensive database of VISA genetic determinants known to date.

microbiology↗

bioRxiv2022

Detecting patterns of accessory genome coevolution in bacterial species using data from thousands of bacterial genomes

Bacterial genomes exhibit widespread horizontal gene transfer, resulting in highly variable genome content that complicates the inference of genetic interactions. In this study, we develop a method for detecting coevolving genes from large datasets of bacterial genomes that we call a "coevolution score". The method is based on pairwise comparisons of closely related individuals, analogous to a pedigree study in eukaryotic populations. This approach avoids the need for an accurate phylogenetic tree and allows very large datasets to be analyzed for signatures of recent coevolution. We apply our method to all of the more than 3 million pairs of genes from the entire annotated Staphylococcus aureus accessory genome of 2,756 annotated genes using a database of over 40,000 whole genomes. We find many pairs of genes that that appear to be gained or lost in a coordinated manner, as well as pairs where the gain of one gene is associated with the loss of the other. These pairs form networks of dozens of rapidly coevolving genes, primarily consisting of genes involved in metal resistance, virulence, mechanisms of horizontal gene transfer, and antibiotic resistance, particularly the SCCmec complex. Our results reflect the fact that the evolution of many bacterial pathogens since the middle of the twentieth century has largely been driven by antibiotic resistance gene gain, and in the case of S. aureus the SCCmec complex is the most prominent of these elements driving the evolution of resistance. The frequent coincidence of these gene gain or loss events suggests that S. aureus switch between antibiotic-resistant niches and antibiotic-susceptible ones. While we focus on gene gain and loss, our method can also detect genes which tend to acquire substitutions in tandem or, in datasets that include phenotypic information, genotype-phenotype or phenotype-phenotype coevolution.

genomics↗

bioRxiv2022

Benchmarking software to predict antibiotic resistance phenotypes in shotgun metagenomes using simulated data

BackgroundThe use of shotgun metagenomics for AMR detection is appealing because data can be generated from clinical samples with minimal processing. Detecting antimicrobial resistance (AMR) in clinical genomic data is an important epidemiological task, yet a complex bioinformatic process. Many software tools exist to detect AMR genes, but they have mostly been tested in their detection of genotypic resistance in individual bacterial strains. Further, these tools use different databases, or even different versions of the same databases. Understanding the comparative performance of these bioinformatics tools for AMR gene detection in shotgun metagenomic data is important because this data type is increasingly used in public health and clinical settings. MethodsWe developed a software pipeline, hAMRoaster (Harmonized AMR Output compAriSon Tool ER; https://github.com/ewissel/hAMRoaster), for assessing accuracy of prediction of antibiotic resistance phenotypes. For evaluation purposes, we simulated a highly resistant mock community and several low resistance metagenomic short read (Illumina) samples based on sequenced strains with known phenotypes. We benchmarked nine open source bioinformatics tools for detecting AMR genes that 1) were conda or Docker installable, 2) had been actively maintained, 3) had an open source license, and 4) took FASTA or FASTQ files as input. hAMRoaster calculated sensitivity, specificity, precision, and accuracy for each tool, comparing detected AMR genes to susceptibility testing. ConclusionOverall, all tools were precise and accurate at all genome coverage levels tested (5x, 50x, 100x sequenced bases / genome length) in the highly resistant mock community with more variability in the low resistance community (1x coverage). This study demonstrated that different bioinformatic tools and pipelines yield differences in AMR gene identification across drug classes, and that these differences become important if researchers are interested in resistance to specific drug classes. SignificanceSoftware selection for metagenomic AMR prediction should be driven by the context of the clinical/research questions and tolerance for true and false negative results. The ability to assess which bioinformatics tool best fits a particular dataset prior to beginning a large-scale project allows for more efficient processing and analysis using optimal tools for a particular research question. As prediction software and databases are in a state of constant refinement, the approach used here--creating synthetic communities containing taxa and phenotypes of interest along with using hAMRoaster to assess performance of candidate software--offers a template to aid researchers in selecting the most appropriate strategy at the time of analysis.

bioinformatics↗

bioRxiv2021

Species-scale genomic analysis of S. aureus genes influencing phage host range and their relationships to virulence and antibiotic resistance genes

Phage therapy has been proposed as a possible alternative treatment for infections caused by the ubiquitous bacterial pathogen Staphylococcus aureus. However, successful phage therapy requires knowing both host and phage genetic factors influencing host range for rational cocktail formulation. To further our understanding of host range, we searched 40,000+ public S. aureus genome sequences for previously identified phage resistance genes. We found that phage adsorption targets and genes that block phage assembly were significantly more conserved than genes targeting phage biosynthesis. Core phage resistance genes had similar nucleotide diversity, ratio of non-synonymous to synonymous substitutions, and functionality (measured by delta-bitscore) to other core genes in a set of 380 non-redundant S. aureus genomes (each from a different MLST sequence type). Non-core phage resistance genes were significantly less consistent with the core genome phylogeny than all non-core genes in this set. Only superinfection immunity genes correlated with empirically determined temperate phage resistance, accessory genome content, and numbers of accessory antibiotic resistance or virulence genes encoded per strain. Taken together, these results suggested that, while phage adsorption genes are heavily conserved in the S. aureus species, they are not undergoing positive selection, arms race dynamics. They also suggested genes classified as involved in assembly are least phylogenetically constrained and superinfection immunity genes best predict both empirical phage resistance and levels of phage-mediated HGT. ImportanceStaphylococcus aureus is a widespread, hospital- and community-acquired pathogen that is commonly antibiotic resistant. It causes diverse diseases affecting both the skin and internal organs. Its ubiquity, antibiotic resistance, and disease burden make new therapies urgent, such as phage therapy, in which viruses specific to infecting bacteria clear infection. S. aureus phage host range not only determines whether phage therapy will be successful by killing bacteria but also horizontal gene transfer through transduction of host genetic material by phages. In this work, we comprehensively reviewed existing literature to build a list of S. aureus phage resistance genes and searched our database of almost 43,000 S. aureus genomes for these genes to understand their patterns of evolution, finding that prophages superinfection immunity correlates best with phage resistance and HGT. These findings improved our understanding of the relationship between known phage resistance genes and phage host range in the species.

microbiology↗

bioRxiv2021

Species-wide phylogenomics of the Staphylococcus aureus agr operon reveals convergent evolution of frameshift mutations

Staphylococcus aureus is a prominent nosocomial pathogen that causes several life-threatening diseases such as pneumonia and bacteremia. S. aureus modulates expression of its arsenal of virulence factors through sensing and integrating responses to environmental signals. The agr (accessory gene regulator) quorum sensing (QS) system is a major regulator of virulence phenotypes in S. aureus. There are four agr specificity groups each with a different autoinducer peptide sequence (encoded by the agrD gene). Though agr is critical for expression of many toxins, paradoxically, S. aureus strains often have non-functional agr activity due to loss-of-function mutations in the four-gene agr operon. To understand patterns in agr variability across S. aureus, we undertook a species-wide genomic investigation. We developed a software tool (AgrVATE; https://github.com/VishnuRaghuram94/AgrVATE) for typing and detecting frameshift mutations in the agr operon. In an analysis of over 40,000 S. aureus genomes, we showed close association between agr type and S. aureus clonal complex. We also found strong linkage between agrBDC alleles (encoding the peptidase, the autoinducing peptide itself, and the peptide sensor respectively) but not agrA (encoding the -response regulator). More than five percent of genomes were found to have frameshift mutations in the agr operon. Though most mutations occur only once in the entire species, we observed a small number of recurring mutations evolving convergently across different clonal lineages. Phylogenetic patterns suggested that strains with agr frameshifts were evolutionary dead ends. Overall, genomic analysis of agr operon suggests evolution through multiple processes with functional consequences that are not fully understood. ImportanceStaphylococcus aureus is a globally pervasive pathogen that produces a plethora of toxic molecules that can degrade, evade, or inhibit the host immune cells. Production of these toxins is mainly controlled by an active agr quorum sensing system, which senses and responds to bacterial cell density. However, there are many reports of S. aureus strains with genetic changes leading to impaired agr activity, often found during chronic bloodstream infections, and may be associated with increased disease severity. We developed an open-source software called AgrVATE to type agr systems and identify putative frameshifts. We used AgrVATE for a species-wide genomic survey of S. aureus, finding that more than 5 % of strains in the public database had non-functional agr systems. We also provide new insights into the evolution of these genetic mutations in the agr system. Overall, this study contributes to our understanding of a common but relatively understudied means of virulence regulation in S. aureus.

genomics↗

bioRxiv2020

Whole-Genome Enrichment and Sequencing of Chlamydia trachomatis Directly from Patient Clinical Vaginal and Rectal Swabs

Chlamydia trachomatis is the most prevalent cause of bacterial sexually transmitted infections (STIs) worldwide. U.S. cases have been steadily increasing for more than a decade in both the urogenital tract and rectum. C. trachomatis is an obligate intracellular bacterium that is not easily cultured, limiting the capacity for genome studies to understand strain diversity and emergence among various patient populations globally. While Agilent SureSelectXT target-enrichment RNA bait libraries have been developed for whole-genome enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival and rectal samples, efficiencies are only 60-80% for [≥]95-100% genome coverage. We therefore re-designed and expanded the RNA bait library to augment enrichment of the organism from clinical samples to improve efficiency. We describe the expanded library, the limit of detection for C. trachomatis genome copy input, and the 100% efficiency and high-resolution of generated genomes where genomic recombination among paired vaginal and rectal specimens from four patients was identified. This workflow provides a robust approach for discerning genomic diversity and advancing our understanding of the molecular epidemiology of contemporary C. trachomatis STIs across sample types, among geographic populations, sexual networks, and outbreaks associated with proctitis/proctocolitis among women and men who have sex with men. ImportanceChlamydia trachomatis is an obligate intracellular bacterium that is not easily cultured, and there is limited information on rectal C. trachomatis transmission and its impact on morbidity. To improve efficiency of previous studies involving whole genome target enrichment and sequencing of C. trachomatis directly from clinical urine, vaginal, conjunctival, and rectal specimens, we expanded the RNA bait library to augment enrichment of the organism from clinical samples. We demonstrate an increased efficiency in the percentage of reads mapping to C. trachomatis. We show the new system is sensitive for near identical genomes of C. trachomatis from two body sites in four women. Further, we provide a robust genomic epidemiologic approach to advance our understanding of C. trachomatis strains causing ocular, urogenital and rectal infections, and to explore geo-sexual networks, outbreaks of colorectal infections among women and men who have sex with men, and the role of these strains in morbidity.

genomics↗

bioRxiv2020

Bactopia: a flexible pipeline for complete analysis of bacterial genomes

Sequencing of bacterial genomes using Illumina technology has become such a standard procedure that often data are generated faster than can be conveniently analyzed. We created a new series of pipelines called Bactopia, built using Nextflow workflow software, to provide efficient comparative genomic analyses for bacterial species or genera. Bactopia consists of a dataset setup step (Bactopia Datasets; BaDs) where a series of customizable datasets are created for the species of interest; the Bactopia Analysis Pipeline (BaAP), which performs quality control, genome assembly and several other functions based on the available datasets and outputs the processed data to a structured directory format; and a series of Bactopia Tools (BaTs) that perform specific post-processing on some or all of the processed data. BaTs include pan-genome analysis, computing average nucleotide identity between samples, extracting and profiling the 16S genes and taxonomic classification using highly conserved genes. It is expected that the number of BaTs will increase to fill specific applications in the future. As a demonstration, we performed an analysis of 1,664 public Lactobacillus genomes, focusing on L. crispatus, a species that is a common part of the human vaginal microbiome. Bactopia is an open source system that can scale from projects as small as one bacterial genome to thousands that allows for great flexibility in choosing comparison datasets and options for downstream analysis. Bactopia code can be accessed at https://www.github.com/bactopia/bactopia.

bioinformatics↗

bioRxiv2019

Genotypic and phenotypic diversity of Staphylococcus aureus from cystic fibrosis lung infections and their interactions with Pseudomonas aeruginosa

Pseudomonas aeruginosa and Staphylococcus aureus are the most common bacteria that infect the respiratory tract of individuals with the genetic disease cystic fibrosis (CF); in fact, S. aureus has recently overtaken P. aeruginosa to become the most common. Substantial research has been performed on the epidemiology of S. aureus in CF; however, there appears to be a gap in knowledge in regard to the pathogenesis of S. aureus in the context of CF lung infections. Most studies have focused on a few S. aureus isolates, often exclusively laboratory adapted strains, and how they are killed by P. aeruginosa. Because of this, little is known about the diversity of S. aureus CF lung isolates in both virulence and interaction with P. aeruginosa. To begin to address this gap in knowledge, we recently sequenced 65 clinical S. aureus isolates from the Emory CF Biospecimen Registry and Boston Childrens Hospital, including the reference isolate JE2, a USA300 strain. Here, we analyzed antibiotic resistance genotypes, sequence type, clonal complex, spa type, and agr type of these isolates. We hypothesized that major virulence phenotypes of S. aureus that may be associated with CF lung infections, namely toxin production and mucoid phenotype, would be retained in these isolates. To test our hypothesis, we plated on specific agars and found that most isolates can hemolyze both rabbit and sheep blood (67.7%) and produce polysaccharide (69.2%), consistent with virulence retention in CF lung isolates. We also identified three distinct phenotypic groups of S. aureus based on their survival in the presence of nonmucoid P. aeruginosa laboratory strain PAO1 and its mucoid derivative. Altogether, our work provides greater insight into the diversity of S. aureus CF isolates, specifically the distribution of important virulence factors and their interaction with P. aeruginosa, all of which have implications in patient health.\n\nAuthor SummaryStaphylococcus aureus is now the most frequently detected pathogen in the lungs of individuals who have cystic fibrosis (CF), followed closely by Pseudomonas aeruginosa. When these two pathogens are found to coinfect the CF lung, patients have a significantly worse prognosis. While P. aeruginosa has been rigorously studied in the context of bacterial pathogenesis in CF, less is known about S. aureus. Here we present an in-depth study of 64 S. aureus CF clinical isolates where we investigated genetic diversity utilizing whole genome sequencing, virulence phenotypes, and interactions with P. aeruginosa. We have found that S. aureus isolated from the CF lung are phylogenetically diverse, most retain known virulence factors, and they vary in interactions with P. aeruginosa from highly sensitive to completely tolerant. Deepening our understanding of how S. aureus responds to its environment and other microbes in the CF lung will enable future development of effective treatments and preventative measures against these formidable infections.

microbiology↗