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Background: NIAID has a programme for testing drug candidates against biodefense and emerging bacterial pathogens that uses defined strain panels consisting of standard laboratory reference strains and strains of clinical origin., Objectives: The current studies were performed to assess the activity of standard-of-care drugs, determine benchmark criteria for new investigational antibacterial candidate prioritization and identify reduced non-redundant strain panels for candidate performance classification., Methods: The susceptibilities of each strain in the screening panels to 40 standard-of-care drugs and clinical drug combinations were determined by percentage growth inhibition using multiple concentrations, a method commonly used in efficient high-throughput screening efforts. The drug susceptibility of each strain was categorized based on interpretive criteria to benchmark the activity of each standard-of-care drug and drug combination, followed by confirmation of select active drugs. Exact match and clustering analyses defined focused non-redundant species and pan-species screening panels., Results: This process revealed a broad spectrum of susceptibilities among strains in each species, with important differences between the standard laboratory reference strains and strains of clinical origin. Exact match and clustering analyses identified subsets of non-redundant strains that can more efficiently classify drug activity resulting in individual species screening panels, a pan-species screening panel and a pan-species maximum resistance panel., Conclusions: This study resulted in improved non-redundant species screening panels for benchmarking the performance of new investigational antibacterial candidates with the greatest potential for efficacy against clinically relevant Category A and B priority and emerging pathogens., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Background: Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularaemia in mammals and is classified as a Category A priority pathogen., Methods: We utilized a systematic analysis of antibacterial potency, extent of dissemination by analysis of bacterial burden in a secondary vital organ, and survival rates to assess the efficacy of a novel rifampicin derivative, TPR1. The efficacy of TPR1 was evaluated alone and in combination with the standard of care drug, doxycycline, against type A F. tularensis Schu S4 using a lethal pulmonary model of infection in mice., Results: TPR1 has an MIC value range of 0.125-4 mg/L against reference laboratory strain Schu S4 and a panel of clinical strains. TPR1 alone reduced the bacterial burden in the lungs and spleen at 40 mg/kg and 80 mg/kg, and no antagonism was observed when co-administered with doxycycline. Dosing at 40 mg/kg doxycycline reduced the bacterial burden by 1 log 10  cfu in the lungs and 4 log 10  cfu in the spleen in comparison to untreated controls. Co-administration of TPR1 and doxycycline demonstrated efficacy upon treatment withdrawal after 4 days of treatment, and 100% survival., Conclusions: Significantly, TPR1 demonstrated efficacy when delivered alone and in combination with doxycycline, which provides compelling evidence of a superior treatment strategy that would normally rely on a single chemotherapeutic for efficacy. In addition, this work substantiates the use of rifampicin derivatives as a platform for the development of novel treatments to other bacterial agents in addition to tularaemia., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

There has been a significant reduction in annual tuberculosis incidence since the World Health Organization declared tuberculosis a global health threat. However, treatment of M. tuberculosis infections requires lengthy multidrug therapeutic regimens to achieve a durable cure. The development of new drugs that are active against resistant strains and phenotypically diverse organisms continues to present the greatest challenge in the future. Numerous phylogenomic analyses have revealed that the Mtb genome encodes a significantly expanded repertoire of toxin-antitoxin (TA) loci that makes up the Mtb TA system. A TA loci is a two-gene operon encoding a 'toxin' protein that inhibits bacterial growth and an interacting 'antitoxin' partner that neutralizes the inhibitory activity of the toxin. The presence of multiple chromosomally encoded TA loci in Mtb raises important questions in regard to expansion, regulation and function. Thus, the functional roles of TA loci in Mtb pathogenesis have received considerable attention over the last decade. The cumulative results indicate that they are involved in regulating adaptive responses to stresses associated with the host environment and drug treatment. Here we review the TA families encoded in Mtb, discuss the duplication of TA loci in Mtb, regulatory mechanism of TA loci, and phenotypic heterogeneity and pathogenesis.

Objectives: The increasing number of clinical strains resistant to one or more of the front-line TB drugs complicates the management of this disease. To develop next-generation benzimidazole-based FtsZ inhibitors with improved efficacy, we employed iterative optimization strategies based on whole bacteria potency, bactericidal activity, plasma and metabolic stability and in vivo efficacy studies., Methods: Candidate benzimidazoles were evaluated for potency against Mycobacterium tuberculosis H37Rv and select clinical strains, toxicity against Vero cells and compound stability in plasma and liver microsomes. The efficacy of lead compounds was assessed in the acute murine M. tuberculosis infection model via intraperitoneal and oral routes., Results: MICs of SB-P17G-A33, SB-P17G-A38 and SB-P17G-A42 for M. tuberculosis H37Rv and select clinical strains were 0.18-0.39 mg/L. SB-P17G-A38 and SB-P17G-A42 delivered at 50 mg/kg twice daily intraperitoneally or orally demonstrated efficacy in reducing the bacterial load by 5.7-6.3 log10 cfu in the lungs and 3.9-5.0 log10 cfu in the spleen. SB-P17G-A33 delivered at 50 mg/kg twice daily intraperitoneally or orally also reduced the bacterial load by 1.7-2.1 log10 cfu in the lungs and 2.5-3.4 log10 cfu in the spleen., Conclusions: Next-generation benzimidazoles with excellent potency and efficacy against M. tuberculosis have been developed. This is the first report on benzimidazole-based FtsZ inhibitors showing an equivalent level of efficacy to isoniazid in an acute murine M. tuberculosis infection model., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: As an initial step in developing novel antibacterials against Burkholderia pseudomallei, we have characterized the FabI enoyl-ACP reductase homologues in the type II fatty acid biosynthesis pathway from this organism and performed an initial enzyme inhibition study., Methods: A BLAST analysis identified two FabI enoyl-ACP reductase homologues, bpmFabI-1 and bpmFabI-2, in the B. pseudomallei genome, which were cloned, overexpressed in Escherichia coli and purified. Steady-state kinetics was used to determine the reaction mechanism and the sensitivity of bpmFabI-1 to four diphenyl ether FabI inhibitors. The antibacterial activity of the inhibitors was assessed using a wild-type strain of Burkholderia thailandensis (E264) and an efflux pump mutant (Bt38)., Results: Consistent with its annotation as an enoyl-ACP reductase, bpmFabI-1 catalysed the NADH-dependent reduction of 2-trans-dodecenoyl-CoA via a sequential Bi Bi mechanism. In contrast, bpmFabI-2 was inactive with all substrates tested and only bpmfabI-1 was transcriptionally active under the growth conditions employed. The sensitivity of bpmFabI-1 to four diphenyl ethers was evaluated and in each case the compounds were slow-onset inhibitors with K(i) values of 0.5-2 nM. In addition, triclosan and PT01 had MIC values of 30 and 70 mg/L for B. pseudomallei as well as a wild-type strain of B. thailandensis (E264), but MIC values of <1 mg/L for the efflux pump mutant Bt38. A reduction in MIC values was also observed for the pump mutant strain with the other diphenyl ethers., Conclusions: Provided that efflux can be circumvented, bpmFabI-1 is a suitable target for drug discovery.

Objectives: The National Institute of Allergy and Infectious Disease classifies Francisella tularensis as a Category A priority pathogen. Despite the availability of drugs for treating tularaemia, the mortality in naturally acquired cases can still approach 30%. In addition, the usefulness of existing drugs for treatment in response to exposure or for prophylaxis is limited because of toxicity and delivery concerns. The aim of this study was to assess the efficacy of the lead alkyl-substituted diphenyl ether, SBPT04, in the F. tularensis murine model of infection., Methods: SBPT04 was delivered by intraperitoneal (ip) and oral (po) routes, and mice were monitored for morbidity, mortality and relapse of disease. Pharmacokinetic studies were performed to evaluate bioavailability. Phase I and Phase II metabolism of SBPT04 was assessed in mouse and human microsomes., Results: SBPT04, a potent inhibitor of the enoyl-ACP reductase enzyme ftuFabI, has efficacy against F. tularensis in the murine model of infection when delivered by both ip and po routes. SBPT04 delivered ip cleared infection by day 4 of treatment, and SBPT04 delivered po resulted in delayed dissemination. Importantly, SBPT04 delivered ip or po demonstrated efficacy with no signs of relapse of disease. Pharmacokinetic studies show increased serum concentrations following ip delivery compared with po delivery, which correlates with the observed survival rate of 100%., Conclusions: In addition to being a potent lead, this work substantiates substituted diphenyl ethers as a platform for the development of novel broad-spectrum chemotherapeutics to other bacterial agents in addition to F. tularensis.

Objectives: Universally conserved events in cell division provide the opportunity for the development of novel chemotherapeutics against Mycobacterium tuberculosis. The aim of this study was to use the beta-lactam antimicrobials cefalexin and piperacillin to inhibit FtsI and characterize the morphological changes and global transcriptional activities of genes to identify a signature response to FtsI inactivation., Methods: Cefalexin and piperacillin were used to block cell division, and microscopy was used to evaluate the effects on bacterial morphology and ultrastructure. Global transcriptional analysis was performed to determine the impact of FtsI inhibition on cell cycle processes and to identify molecular markers., Results: Inhibition of FtsI with cefalexin and piperacillin resulted in filamentous cells with multiple concentric rings and occasional branching as visualized by light and electron microscopy. Whole genome microarray-based transcriptional profiling and transcriptional mapping allowed the evaluation of cell cycle processes in response to inhibition of FtsI and characterization of transcriptional response and cell cycle processes., Conclusions: This study substantiated that FtsZ-ring constriction and septal resolution require the transpeptidase activity of FtsI, making FtsI essential for cell division in M. tuberculosis. Therefore, FtsI is a target for drug discovery, and these studies provided a molecular signature of FtsI inactivation that can be applied to screening strategies for novel FtsI inhibitors.