The successful culture of Leptospira spp. from the environment is challenging. Here, we optimized the isolation of Leptospira spp. from water samples spiked with different species and initial concentrations of this organism. The time periods between water sampling and the isolation process were varied (0, 2, and 4 weeks). Bacterial cultures were observed under a microscope, and cultures were graded for cell density, weekly, for 12 weeks. Most pathogenic Leptospira spp. were difficult to culture under all conditions. All conditions of water samples spiked with novel species of Leptospira subclade P1 were culture positive within 2 weeks. For Leptospira subclade P2, storing samples for 2 weeks prior to isolation resulted in more successful isolation compared with isolation after other storage conditions. For subclade S1, all samples with initial bacterial concentrations of more than 10³ colonies/mL, under all storage conditions, were successfully cultured. These results suggest that storing contaminated water samples for 2 to 4 weeks in the dark at an ambient temperature prior to culturing can improve the isolation of Leptospira spp. from the samples. We implemented this protocol and collected water samples from natural sources accessed by both humans and animals. Leptospira spp. was identified in 32% (35/109) of water samples. The animal species using a water source influenced the likelihood of water samples being contaminated with Leptospira spp. Cultures of Leptospira spp. from environmental samples can provide useful information for understanding the complex interactions between humans, animals and the environment in the transmission of leptospirosis.

Tuberculosis disease (TB), caused by Mycobacterium tuberculosis, is a major public health issue in Thailand. The high prevalence of modern Beijing (Lineage 2.2.1) strains has been associated with multi- and extensively-drug resistant infections (MDR-, XDR-TB), complicating disease control. The impact of rarer proto-Beijing (L2.1) strains is less clear. In our study of thirty-seven L2.1 clinical isolates spanning thirteen years, we found a high prevalence of XDR-TB cases (32.4%). With ≤ 12 pairwise SNP distances, 43.2% of L2.1 patients belong to MDR-TB or XDR-TB transmission clusters suggesting a high level of clonal expansion across four Thai provinces. All XDR-TB (100%) were likely due to transmission rather than inadequate treatment. We found a 47 mutation signature and a partial deletion of the fadD14 gene in the circulating XDR-TB cluster, which can be used for surveillance of this rare and resilient M. tuberculosis strain-type that is causing increasing health burden. We also detected three novel deletion positions, a deletion of 1,285 bp within desA3 (Rv3230c), large deletions in the plcB, plcA, and ppe38 gene which may play a role in the virulence, pathogenesis or evolution of the L2.1 strain-type.

Tuberculosis (TB) remains a major global health challenge, particularly in resource-limited settings where access to rapid and reliable diagnostics is limited. Conventional diagnostic methods, such as smear microscopy and culture, are either time-consuming or lack adequate sensitivity. This study optimized recombinase polymerase amplification (RPA) using 16 primer combinations targeting IS6110 highly specific to the Mycobacterium tuberculosis complex (MTC). A novel naked-eye assay, TB-GoldDx, was developed by integrating RPA combined with gold nanoparticles (AuNPs), enabling equipment-free diagnostics. TB-GoldDx demonstrated a detection limit of 0.001 ng of MTB H37Rv DNA (~210 bacilli) per 25 µL reaction. Among 100 bacterial strains, it achieved 95.83% sensitivity and 100% specificity among 100 bacterial strains, comprising 72 MTB isolates and 28 nontuberculous bacterial species. In 140 sputum samples, the assay showed 81.43% sensitivity and 58.57% specificity versus acid-fast bacilli (AFB) smear microscopy, with sensitivity improving to 95.45% in high-load AFB 3+ specimens. Compared to a commercial line probe assay (LPA), TB-GoldDx exhibited slightly higher sensitivity (84.78% vs. 82.61%) but lower specificity (54.05% vs. 78.38%). Delivering rapid, visual results in under an hour, TB-GoldDx offers a low-cost, easily deployable solution for point-of-care tuberculosis detection, especially in underserved regions, reinforcing global End TB efforts.

Drug-resistant tuberculosis remains a major challenge to tuberculosis control, particularly in settings with limited access to conventional molecular testing. This study developed and preliminarily evaluated RR-TB GoldDx and Hr-TB GoldDx, rapid visual assays for detecting rifampicin- and isoniazid-resistance-associated mutations in Mycobacterium tuberculosis. The assays combine recombinase polymerase amplification with unmodified gold nanoparticles (AuNPs) for amplification confirmation and thiol-probe-modified AuNPs for detecting resistance-associated mutations at rpoB codons 516, 526, and 531, katG codon 315, and position −15 of the fabG1–inhA promoter. Evaluation using DNA from 50 M. tuberculosis isolates yielded positive internal-control results for all samples. RR-TB GoldDx achieved accuracies of 84% and 86% against phenotypic drug-susceptibility testing and DNA sequencing, respectively, with Cohen’s kappa values of 0.68 and 0.72. Hr-TB GoldDx achieved 80% accuracy and a kappa value of 0.60 against phenotypic testing, while the inhA promoter and katG assays achieved sequencing-based accuracies of 84% and 86%, respectively. Results were available within 1 h at an estimated reagent cost of THB 500–550 (approximately USD 15–16) per test without conventional thermocycling, real-time fluorescence detection, or gel electrophoresis. These instrument-minimal assays show potential for rapid drug-resistance screening but require validation in larger, genetically diverse cohorts and direct respiratory specimens.

Tuberculosis (TB), caused by Mycobacterium tuberculosis , is one of the world’s deadliest diseases, currently responsible for ~1.5 million deaths per year and rising. Recently, rifampicin-resistant M. tuberculosis was designated as a critical priority pathogen status by the World Health Organization. However, few controlled laboratory studies are available that systematically assess the molecular drivers of antibiotic resistance development in TB. In this study, we paired laboratory-directed evolution and population-level deep-sequencing approaches to map the evolutionary pathways taken by M. tuberculosis to develop resistance to first- and last-line therapies (rifampicin and linezolid) and then characterized de novo resistance mutation occurrence over time. We demonstrated that the majority of M. tuberculosis populations readily acquire mutations in genes commonly found in rifampicin- and linezolid-resistant clinical isolates ( rpoB and rplC ). However, we also identified mutations in six genes, mostly present in subpopulations (17–41%) and not previously linked to rifampicin or linezolid resistance, including four associated with rifampicin resistance ( Rv0052 , ppsD , ppsE and mptC ) and two associated with linezolid resistance ( glpK and echA12 ). The ppsD , glpK and mptC mutations were also identified in published individual sequencing reads of antibiotic-resistant clinical isolates. Further investigation of the identified resistance determinants ppsD / E established that mutations in these genes appear to mediate resistance across multiple species, with an Escherichia coli mutant of the ortholog ( fabF ), representing a shared domain featured in PpsD and PpsE, phenotypically displaying increased antibiotic tolerance to low-level rifampicin. This study highlights the power of using controlled laboratory studies to uncover minority variants in populations of M. tuberculosis . These outcomes will lead to improved diagnosis of antibiotic resistance emergence in TB, to optimize management and treatment of TB infections, and ultimately to minimize patient deaths.