Rapid Gene Silencing Followed by Antimicrobial Susceptibility Testing for Target Validation in Antibiotic Discovery.

Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB)-an ancient yet widespread global infectious disease to which 1.6 million people lost their lives in 2021. Antimicrobial resistance (AMR) has been an ongoing crisis for decades; 4.95 million deaths were associated with antibiotic resistance in 2019. While AMR is a multi-faceted problem, drug discovery is an urgent part of the solution and is at the forefront of modern research.The landscape of drug discovery for TB has undoubtedly been transformed by the development of high-throughput gene-silencing techniques that enable interrogation of every gene in the genome, and their relative contribution to fitness, virulence, and AMR. A recent advance in this area is CRISPR interference (CRISPRi). The application of this technique to antimicrobial susceptibility testing (AST) is the subject of ongoing research in basic science.CRISPRi technology can be used in conjunction with the high-throughput SPOT-culture growth inhibition assay (HT-SPOTi) to rapidly evaluate and assess gene essentiality including non-essential, conditionally essential (by using appropriate culture conditions), and essential genes. In addition, the HT-SPOTi method can develop drug susceptibility and drug resistance profiles.This technology is further useful for drug discovery groups who have designed target-based inhibitors rationally and wish to validate the primary mechanisms of their novel compounds' antibiotic action against the proposed target.

Modified HT-SPOTi: An Antimicrobial Susceptibility Testing to Evaluate Formulated Therapeutic Combinations Against Bacterial Growth and Viability.

Antimicrobial resistance (AMR) poses a serious threat to global health, potentially causing 10 million deaths per year globally by 2050. To tackle AMR, researchers from all around the world have generated a selection of various formulated (viz. nanoparticulate, liposomal) therapeutic combinations to be evaluated for new antimicrobial drug discovery. To meet the urgent need for accelerating new antibacterial drug development, we need rapid but reliable whole-cell assay methods and models to test formulated therapeutic combinations against several pathogens in different in vitro conditions as models of actual infections.Over the past two decades, high-throughput spot-culture growth inhibition assay (HT-SPOTi) has been demonstrated to be a gold-standard drug susceptibility method for evaluating novel chemotherapeutic entities and existing drugs against various microbes of global concern. Our modified HT-SPOTi method serves the purpose of evaluating drug combinations against Gram-positive/negative microorganisms as well as acid-fast bacilli. The newly developed and modified HT-SPOTi assay builds upon the limitations of our previously published method to incorporate antimicrobial susceptibility testing with formulated therapeutic combinations. The modified HT-SPOTi is compared with a range of other antimicrobial susceptibility testing methods and validated using a library of existing antibiotics as well as formulated therapeutic combinations. The modified HT-SPOTi assay can serve as an efficient and reliable high-throughput drug screening platform to discover new potential antimicrobial molecules, including as part of therapeutic formulations.This chapter describes the generation of drug susceptibility profile for formulated therapeutic combinations using modified HT-SPOTi in a semi-automated system.

Rationally Designed Novel Phenyloxazoline Synthase Inhibitors: Chemical Synthesis and Biological Evaluation to Accelerate the Discovery of New Antimycobacterial Antibiotics.

The uncontrolled spread of drug-resistant tuberculosis (DR-TB) clinical cases necessitates the urgent discovery of newer chemotypes with novel mechanisms of action. Here, we report the chemical synthesis of rationally designed novel transition-state analogues (TSAs) by targeting the cyclization (Cy) domain of phenyloxazoline synthase (MbtB), a key enzyme of the conditionally essential siderophore biosynthesis pathway. Following bio-assay-guided evaluation of TSA analogues preferentially in iron-deprived and iron-rich media to understand target preferentiality against a panel of pathogenic and non-pathogenic mycobacteria strains, we identified a hit, i.e., TSA-5. Molecular docking, dynamics, and MMPBSA calculations enabled us to comprehend TSA-5's stable binding at the active site pocket of MbtB_Cy and the results imply that the MbtB_Cy binding pocket has a strong affinity for electron-withdrawing functional groups and contributes to stable polar interactions between enzyme and ligand. Furthermore, enhanced intracellular killing efficacy (8 µg/mL) of TSA-5 against Mycobacterium aurum in infected macrophages is noted in comparison to moderate in vitro antimycobacterial efficacy (64 µg/mL) against M. aurum. TSA-5 also demonstrates whole-cell efflux pump inhibitory activity against Mycobacterium smegmatis. Identification of TSA-5 by focusing on the modular MbtB_Cy domain paves the way for accelerating novel anti-TB antibiotic discoveries.

Immunobiology of tubercle bacilli and prospects of immunomodulatory drugs to tackle tuberculosis (TB) and other non-tubercular mycobacterial infections.

The COVID-19 pandemic has set back progress made on antimicrobial resistance (AMR). Without urgent re-focus, we risk slowing down drug discovery and providing treatment for drug resistant Mycobacterium tuberculosis. Unique in its immune evasion, dormancy and resuscitation, the causal pathogens of tuberculosis (TB) have demonstrated resistance to antibiotics with efflux pumps and the ability to form biofilms. Repurposing drugs is a prospective avenue for finding new anti-TB drugs. There are many advantages to discovering novel targets of an existing drug, as the pharmacokinetic and pharmacodynamic properties have already been established, they are cost-efficient and can be commercially accelerated for the new development. One such group of drugs are non-steroidal anti-inflammatory drugs (NSAIDs) that are originally known for their ability to supress the host proinflammatory responses. In addition to their anti-inflammatory properties, some NSAIDs have been discovered to have antimicrobial modes of action. Of particular interest is Carprofen, identified to inhibit the efflux mechanism and disrupt biofilm formation in mycobacteria. Due to the complexities of host-pathogens interactions in the lung microbiome, inflammatory responses must carefully be controlled alongside the in vivo actions of the prospective anti-infectives. This critical review explores the potential dual role of a selection of NSAIDs, as an anti-inflammatory and anti-tubercular adjunct to reverse the tide of antimicrobial resistance in existing treatments.

Finding common ground? Evaluating an intervention to improve teamwork among primary health-care professionals.

OBJECTIVE: Multidisciplinary care has been shown as the most effective option for chronic disease. The aim of the Team-link study was to assess the effectiveness of an intervention to improve teamwork among general practitioners (GPs), practice staff and allied health professionals (AHPs). This paper describes changes to teamwork using qualitative data collected in the study. DESIGN: Qualitative data about changes in internal and external professional collaboration were collected from facilitators' observations, GPs' reports and responses to a survey of AHPs assessing multidisciplinary teamwork. SETTING: Multidisciplinary teams within general practices and external collaborations with AHPs including dietitians, diabetic educators, exercise physiologists, podiatrists, psychologists and physiotherapists. PARTICIPANTS: GPs, practice nurses, practice staff, AHPs. INTERVENTION: A 6-month intervention consisting of an educational workshop and structured facilitation using specially designed materials, backed up by informal telephone support, was delivered to 26 practices. MAIN OUTCOME MEASURE: Data were analysed thematically using an approach based on identifying actors and associated collaborative actions. RESULTS: New and enhanced communication pathways were observed between GPs, practice staff, patients and AHPs following the intervention. The enhanced information sharing expedited communication and improved interprofessional collaboration within general practices and with AHPs. There was evidence of increased patient participation and empowerment in the care process and improved collaboration by practice staff and allied health providers. CONCLUSION: The Team-link intervention improved professional collaboration among GPs, practice staff, AHPs and patients, increasing understanding and trust and enhancing multidisciplinary teamwork for chronic disease care in primary care settings.