Non-tuberculous mycobacteria (NTM) infections are increasing in incidence and associated mortality. NTM are naturally resistant to a variety of antibiotics, complicating treatment. We conducted a literature assessment on the efficacy of bedaquiline in treating NTM species in vitro and in vivo (animal models and humans); meta-analyses were performed where possible. Four databases were searched using specific terms. Publications were included according to predefined criteria. Bedaquiline’s impact on NTM in vitro, MICs and epidemiological cut-off (ECOFF) values were evaluated. A meta-analysis of bedaquiline efficacy against NTM infections in animal models was performed. Culture conversion, cure and/or relapse-free cure were used to evaluate the efficacy of bedaquiline in treating NTM infection in humans. Fifty studies met the inclusion criteria: 33 assessed bedaquiline’s impact on NTM in vitro, 9 in animal models and 8 in humans. Three studies assessed bedaquiline’s efficacy both in vitro and in vivo. Due to data paucity, an ECOFF value of 0.5 mg/mL was estimated for Mycobacterium abscessus only. Meta-analysis of animal studies showed a 1.86× reduction in bacterial load in bedaquiline-treated versus no treatment within 30 days. In humans, bedaquiline-including regimens were effective in treating NTM extrapulmonary infection but not pulmonary infection. Bedaquiline demonstrated strong antibacterial activity against various NTM species and is a promising drug to treat NTM infections. However, data on the genomic mutations associated with bedaquiline resistance were scarce, preventing statistical analyses for most mutations and NTM species. Further studies are urgently needed to better inform treatment strategies.

Background: The emergence of drug-resistant Mycobacterium tuberculosis (M. tb) strains remains a threat to tuberculosis (TB) prevention and care. Understanding the drug resistance profiles of circulating strains is crucial for effective TB control. This study aimed to describe the genetic diversity of rifampicin-resistant M. tb strains circulating in Botswana using whole genome sequencing (WGS).Methods: This study included 202 stored M. tb isolates from people diagnosed with rifampicin-resistant TB (RR-TB) between January 2016 and June 2023. Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method. Library preparation was performed using the Illumina DNA prep kit following the manufacturer's instructions. Sequencing was done on Illumina NextSeq2000. TBProfiler software was used to identify known M. tb lineages and drug resistance profiles. Statistical analyses were performed on STATA version 18.Results: WGS analysis revealed multidrug resistance (57.9%: 95% CI; 50.7–64.8), Pre-XDR (16.8%, 95% CI: 11.9–22.7), RR-TB (20.2%: 95% CI: 14.98–26.5), and HR-TB (0.5%, 95% CI; 0.01–2.7). We identified a high genetic diversity with three predominant lineages: lineage 4 (60.9%, 95% CI; 53.8–67.7), lineage 1 (22.8%: 95% CI; 17.2–29.2), and lineage 2 (13.9%, 95% CI: 9.4–19.4). The most frequently observed drug resistance mutations for rifampicin, isoniazid, ethambutol, streptomycin, pyrazinamide, and fluoroquinolones were rpoB S450L (28.6%), katG S315T (60.5%), embA_c.-29_-28delCT, embB Q497R (31.7%), rrs_n.517C>T (47.1%), pncA_c.375_389delCGATGAGGTCGATGT (36.0%) and gyrA A90V (79.4%), respectively. No bedaquiline and delamanid resistance-associated mutations were detected.Conclusions: This study highlights the high genetic diversity of M. tb strains, with a predominance of lineage 4 among people with RR-TB in Botswana. It provides valuable insights into the genetic diversity of rifampicin-resistant M. tb strains circulating in Botswana.

Background: Extensively drug-resistant tuberculosis (XDR-TB) is a global health threat, being expensive and difficult to treat, with high mortality rates. The Western Cape Province (WCP), South Africa, has a particularly high burden of XDR-TB (>800 cases in the past ten years). Drug resistance genotypes and transmission present substantial regional variability. Thus, a better understanding of genetic diversity, clustering and the factors related to transmission can aid in prioritising resources to effectively target high-risk populations and regions that are disproportionately affected. We describe genetic diversity, drug resistance profiles and identify potential factors associated with the spread of XDR-TB strains collected in the WCP.Methods: We included 729 XDR-TB samples (one per patient), identified through routine diagnosis spanning 2010 to 2019, from six healthcare districts (HCDs) in the WCP. Genomic DNA from cultured isolates was sequenced using the Illumina platform. Sequences were analysed for strain type, drug resistance mutations, and genomic clustering using the TBProfiler and MTBseq pipelines. We conducted logistic regression analysis to identify potential factors associated with genomic traits related to the spread of XDR-TB strains.Results: Of the 729 XDR-TB strains, sublineage 2.2.2 (Atypical Beijing: n=378, 58.79%) strains were predominant, followed by Sublineage 2.2.1 (Typical Beijing: n=260, 40.43%). Atypical Beijing strains were more likely to cluster than Typical Beijing strains. Most of the clusters were small, with a few large and very large clusters, and the strains within very large clusters (primarily Atypical Beijing) were more likely to be found within Cape Town Metropole, Cape Winelands and Garden Route HCDs. Certain Atypical Beijing strains were found resistant to new and repurposed drugs recently introduced in the WHO treatment guidelines and clustered, indicating potential transmission.Conclusions: Near-untreatable Atypical Beijing strains are prevalent in the WCP. Hence, hotspot areas for clustering in Cape Town Metropole, Cape Winelands and Garden Route HCDs should be prioritised for targeted intervention to prevent ongoing XDR-TB transmission.

Tuberculosis (TB), caused by Mycobacterium tuberculosis , remains a global health burden due to the pathogen’s ability to develop resistance to current treatment options. Consequently, drug discovery studies are essential for identifying new antimycobacterial agents with novel mechanisms of action. This study investigated the antimycobacterial activity of crude extracts derived from mixed culturable bacteria isolated from intertidal marine sediments. The bacterial diversity of the bioactive mixed cultures was characterized using 16S rRNA gene-based metagenomic analysis. Their pathogen-targeted effects were evaluated against Mycobacterium smegmatis mc ² 155 and M. tuberculosis H37Rv, and THP-1-derived macrophages infected with M. smegmatis mc ² 155. Of the 48 mixed bacterial crude extracts derived from 17 intertidal marine sediments, five-PPB1, GCR1, BB1, PPB2, and CR1-demonstrated strong antimycobacterial activity against M. smegmatis mc ² 155 and M. tuberculosis H37Rv with minimum inhibitory concentrations ranging from 31.25 to 62.50 μg/mL and 7.8125 to 15.625 μg/mL, respectively. At 62.50 μg/mL, CR1 significantly reduced the intracellular M. smegmatis mc ² 155 burden in THP-1-derived macrophages, resulting in 28.08 ± 4.25% mean decrease in bacterial survival ( p < 0.0001) and 94.4% ± 1.14 mean growth inhibition. From the CR1 mixed cultures, nine axenic bacterial isolates were cultivated, and their resulting crude extracts were evaluated for bioactivity. The identified isolates included Marinobacter maritimus , Psychrobacter celer , Pseudomonas benzenivor , Bacillus altitudinis , Bacillus aerius , Bacillus stratosphericus , and Paenibacillus glucanolyticus . Metabolite profiling of axenic crude extracts identified several compounds, including tenacibactin B, maremycin D1, and tubercidine. These findings suggest that South African intertidal marine sediments host diverse microbial communities capable of producing novel antimycobacterial agents.

Whole-genome sequencing (WGS) holds promise for accurate and comprehensive diagnosis of drug resistance in Mycobacterium tuberculosis and identification of transmission events. ‘Early positive cultures’ (EPC) are increasingly used when WGS is implemented to guide clinical care to reduce the turnaround time. We performed a systematic literature review to compare methods used for EPC-based WGS and performed an individual sample data meta-analysis to identify variables associated with bioinformatic quality measures. Of 423 studies identified, 15 met eligibility criteria. We analysed 1,065 FASTQ files from 11 studies using Illumina sequencing; 96.1% passed all quality control thresholds. Median genome coverage was 65× (IQR, 63–82), with a pooled mapping percentage of 91.2%. The meta-analysis showed that the number of sequencing cycles was significantly associated with improved sequencing quality, while other laboratory variables had no consistent effect. Based on these findings, we suggest replacing the term EPC with ‘clinical primary culture’ and propose a standardized workflow and reporting checklist for WGS on primary Mycobacteria Growth Indicator Tube (MGIT) cultures.

Undetected rifampicin resistance is a threat to global tuberculosis (TB) control efforts by delaying effective treatment. In different studies, non-canonical rpoB mutations outside the rifampicin resistance-determining region have been reported at varying prevalences by country. Here, we report cases of rifampicin resistance in Botswana that were missed by the routine molecular diagnostic assays. Individuals were tested under routine programme conditions, in accordance with national guidelines, at four designated drug-resistant TB clinics from 2017 to 2022. Initial testing at the facilities included GeneXpert MTB/RIF ultra and later phenotypic drug susceptibility testing (pDST), as well as the Hain MTBDRsl line probe assay, at the National Tuberculosis Reference Laboratory. A total of nine isolates were subsequently sequenced on the Illumina NextSeq 2000 instrument. At the point of care, routine molecular tests classified all nine individuals as susceptible to rifampicin. Subsequent culture and phenotypic drug susceptibility testing confirmed rifampicin resistance. Whole-genome sequencing identified non-canonical rpoB mutations outside the rifampicin resistance-determining region I49F and V170F, which are associated with low-level rifampicin resistance. Of the nine isolates sequenced, 4 (44%) harboured the rpoB V170F mutation, while 5 (56%) harboured the rpoB I491F mutation. These results highlight a diagnostic gap within the current algorithms and show the value of sequencing-based approaches for accurately detecting drug resistance. Incorporating sequencing into routine clinical practice could help guide the selection of TB treatment and improve treatment outcomes in patients who do not respond to first-line therapy.