Biology subjects

Joseph, S. J.

Publications and source records attributed to Joseph, S. J..

5 recordsLinked to original sources

bioRxiv2026

neissflow: Streamlining Genomic Epidemiology of Neisseria gonorrhoeae with Nextflow

Antimicrobial-resistant Neisseria gonorrhoeae (Ng) poses a growing global public health threat. Existing tools available for Ng genome analysis carry notable limitations, including incomplete resistance marker coverage, absence of species identification, and lack of phylogenetic capability. We developed neiss-flow, a highly parallelized Nextflow pipeline for Ng genome analysis that integrates five subworkflows: read preprocessing, species identification, de novo assembly, antimicrobial resistance (AMR) profiling, and recombination-aware phylogenetic analysis with outbreak detection. neissflow performs extensive quality control on reads, assemblies, variant calls, and phylogenetic results. Validation was performed on two datasets: a mixed-species dataset (n=158; 105 Ng and 53 non-gonococcal species) for sensitivity/specificity assessment, and a reproducibility dataset (n=283 replicate sequences from 17 reference strains) for consistency and phylogenetic validation. neissflow achieved 100% sensitivity and specificity for Ng species identification compared with MALDI-TOF and PubMLST methods. All nine AMR and typing analytes demonstrated [≥]98.1% concordance with PubMLST genotype calls. Genotype-phenotype validation confirmed perfect concordance for key resistance determinants including gyrA mutations with ciprofloxacin resistance, 23S rRNA mutations with high-level azithromycin resistance, and tetM plasmid gene with high-level tetracycline resistance. Reproducibility analysis demonstrated 99.97% concordance across 3,093 analyte calls. Phylogenetic validation demonstrated 100% accuracy for both strain-level and intra-MLST clustering. neissflow is a robust, accessible, and standardized pipeline, positioning it as a valuable tool for public health laboratories engaged in Ng AMR monitoring and outbreak investigations. ImportanceNeisseria gonorrhoeae (Ng) is the second most common reported bacterial sexually transmitted infection and has developed resistance to all clinically relevant antibiotics. Surveillance of Ng resistance informs clinical recommendations for treatment of gonococcal infections. Whole genome sequencing (WGS) offers powerful insights into resistance mechanisms and transmission dynamics. However, many public health laboratories lack the resources needed to analyze these data effectively. We developed neissflow as an end-to-end, accessible Ng WGS analysis pipeline. neissflow demonstrated exceptional accuracy and reproducibility across diverse reference datasets. neissflow enables broader adoption of whole-genome sequencing-based surveillance and supports timely public health responses to emerging antimicrobial resistance in Ng by lowering technical barriers.

bioinformatics↗

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↗

bioRxiv2021

Global emergence and dissemination of Neisseria gonorrhoeae ST-9363 isolates with reduced susceptibility to azithromycin

Neisseria gonorrhoeae multi-locus sequence type (ST) 9363 genogroup isolates have been associated with reduced azithromycin susceptibility (AZMrs) and show evidence of clonal expansion in the U.S. Here we analyze a global collection of ST-9363 genogroup genomes to shed light on the emergence and dissemination of this strain. The global population structure of ST-9363 genogroup falls into three lineages: Basal, European, and North American; with 32 clades within all lineages. Although, ST-9363 genogroup is inferred to have originated from Asia in the mid-19th century; we estimate the three modern lineages emerged from Europe in the late 1970s to early 1980s. The European lineage appears to have emerged and expanded from around 1986 to 1998, spreading into North America and Oceania in the mid-2000s with multiple introductions, along with multiple secondary reintroductions into Europe. Our results suggest two separate acquisition events of mosaic mtrR and mtrR promoter alleles: first during 2009-2011 and again during the 2012-2013 time, facilitating the clonal expansion of this genogroup with AZMrs in the U.S. By tracking phylodynamic evolutionary trajectories of clades that share distinct demography as well as population-based genomic statistics, we demonstrate how recombination and selective pressures in the mtrCDE efflux operon granted a fitness advantage to establish ST-9363 as a successful gonococcal lineage in the U.S. and elsewhere. Although it is difficult to pinpoint the exact timing and emergence of this young genogroup, it remains critically important to continue monitoring it, as it could acquire additional resistance markers.

microbiology↗

bioRxiv2021

Selective whole genome amplification as a tool to enrich specimens with low Treponema pallidum genomic DNA copies for whole genome sequencing

Downstream next generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome, and the capture and removal of CpG-methylated host DNA using the NEBNext(R) Microbiome DNA Enrichment Kit followed by MDA with the REPLI-g Single Cell Kit as enrichment methods to improve the yields of T. pallidum DNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93-98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit propagated isolates, containing >14 T. pallidum genomic copies/l of sample for SWGA and >129 genomic copies/l for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens, showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which have been challenging until now. ImportanceSyphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogen Treponema pallidum subspecies pallidum. Primary syphilis typically presents as single or multiple mucocutaneous lesions, and if left untreated, can progress through multiple stages with varied clinical manifestations. Molecular studies often rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past two decades, WGS data is needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of a SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers of T. pallidum.

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↗