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Drug-resistance (DR) in many bacterial pathogens often arises from the repetitive formation of drug-tolerant bacilli, known as persisters. However, it is unclear whether Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), undergoes a similar phenotypic transition. Recent metabolomics studies have identified that a change in trehalose metabolism is necessary for Mtb to develop persisters and plays a crucial role in metabolic networks of DR-TB strains. The present study used Mtb mutants lacking the trehalose catalytic shift and showed that the mutants exhibited a significantly lower frequency of the emergence of DR mutants compared to wildtype, due to reduced persister formation. The trehalose catalytic shift enables Mtb persisters to survive under bactericidal antibiotics by increasing metabolic heterogeneity and drug tolerance, ultimately leading to development of DR. Intriguingly, rifampicin (RIF)-resistant bacilli exhibit cross-resistance to a second antibiotic, due to a high trehalose catalytic shift activity. This phenomenon explains how the development of multidrug resistance (MDR) is facilitated by the acquisition of RIF resistance. In this context, the heightened risk of MDR-TB in the lineage 4 HN878 W-Beijing strain can be attributed to its greater trehalose catalytic shift. Genetic and pharmacological inactivation of the trehalose catalytic shift significantly reduced persister formation, subsequently decreasing the incidence of MDR-TB in HN878 W-Beijing strain. Collectively, the trehalose catalytic shift serves as an intrinsic factor of Mtb responsible for persister formation, cross-resistance to multiple antibiotics, and the emergence of MDR-TB. This study aids in the discovery of new TB therapeutics by targeting the trehalose catalytic shift of Mtb.

Introduction: Schistosomiasis is a neglected tropical disease that puts over 200 million people at risk, and prevention options are sparse with no approved vaccine. Our vaccine candidate, SchistoShield ® , is based on an approximately 87 kDa large subunit of calcium activated neutral protease - termed Sm-p80 - combined with a potent TLR4 agonist-based adjuvant. SchistoShield ® has been shown to prevent disease throughout the parasitic life cycle - including egg, juvenile, and adult worm stages - in numerous animal models up to and including baboons. SchistoShield ® has been shown safe in both preclinical toxicology studies in rabbits and in a Phase 1 clinical trial in the USA. A Phase 1b trial was initiated in 2023 in endemic regions of Africa, and to date no serious safety signals have been reported., Methods: In preparation for large-scale Phase 2 clinical trials and eventual vaccine deployment, the Sm-p80 antigen production process has been transferred to a manufacturing organization, Quratis Corporation in South Korea, which specializes in preparation of vaccines for large-scale European and African trials. The process of scaling from our current production level of ~2000 vaccine doses, to a process that will generate more than 100 million doses has required multiple improvement steps in the process including fermentation, downstream purification of the protein antigen, lyophilization, and fill and finish., Results: In this study, we detail the large-scale production process of the SchistoShield ® protein product by Quratis. In addition, an effort was made to analyze and compare the Quratis-made lot of Sm-p80, referred to as QTP-105, to the cGMP lot of Sm-p80 which is in use in human trials in the USA and Africa, referred to as Sm-p80 DP (made in USA). We show that QTP-105 demonstrates excellent potency, purity, identity, and endotoxin levels compared to our Phase 1 Sm-p80 DP and is suitable for use in Phase 2 studies and beyond., Competing Interests: Conflict of interest JK, JD, SG, and DC are employees of PAI Life Sciences, a for-profit corporation which is involved in the development and implementation of the SchistoShield® vaccine product. JL, SC, KY, JY, SB, JS, BK are employees of Quratis Corp., a for-profit corporation involved in the manufacture of the eventual SchistoShield® product. DC and AS hold patents regarding Sm-p80 vaccine components. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Background: The current conventional drug susceptibility test (DST) for Mycobacterium tuberculosis (Mtb) takes several weeks of incubation to obtain results. As a rapid method, molecular DST requires only a few days to get the results but does not fully cover the phenotypic resistance. A new rapid method based on the ability of viable Mtb bacilli to hydrolyze fluorescein diacetate to free fluorescein with detection of fluorescent mycobacteria by flow cytometric analysis, was recently developed., Methods: To evaluate this cytometric method, we tested 39 clinical isolates which were susceptible or resistant to isoniazid (INH) or rifampin (RIF), or ethambutol (EMB) by phenotypic or molecular DST methods and compared the results., Results: The susceptibility was determined by measuring the viability rate of Mtb and all the isolates which were tested with INH, RIF, and EMB showed susceptibility results concordant with those by the phenotypic solid and liquid media methods. The isolates having no mutations in the molecular DST but resistance in the conventional phenotypic DST were also resistant in this cytometric method. These results suggest that the flow cytometric DST method is faster than conventional agar phenotypic DST and may complement the results of molecular DST., Conclusion: In conclusion, the cytometric method could provide quick and more accurate information that would help clinicians to choose more effective drugs.