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ABSTRACT We assessed the performance of a novel real-time PCR-based transrenal DNA (trDNA) assay for the specific detection of Mycobacterium tuberculosis as a candidate marker of the early anti-tuberculosis therapy response. The study was performed on 288 urine samples from 72 tuberculosis patients collected at baseline and days 3, 7, and 14 of treatment with amoxicillin-clavulanic acid alone or in combination with meropenem, ertapenem, optimized-dose rifampicin, or standard treatment control in South Africa. trDNA was detected in one-third of the samples. The highest proportion of positive PCR results (cycle threshold < 36) was observed on days 3 and 7, reflecting the point in time when maximum bacterial killing and disintegration are expected. When analyzed by study arms, the trend was observed in groups treated with active antibiotics affecting cell wall integrity (meropenem, control) but not in inactive drugs (ertapenem, amoxicillin/clavulanic acid alone) or active drugs not affecting the cell wall (rifampicin). Overall, however, the trDNA assay did not correlate well with sputum culture-based decline of viable bacteria. This is possibly due to trDNA reflecting the killing of both culturable and non-culturable bacteria and should be explored further. IMPORTANCE This study presents the results of the evaluation of a novel method for the detection of Mycobacterium tuberculosis, the causative agent of tuberculosis, in urine. Detecting parts of the mycobacteria in urine is of particular interest as it allows us to use a sample that is easy to obtain and that does not require uncomfortable procedures or safety precautions like obtaining sputum for culture, which is the most commonly used sample in the diagnosis of tuberculosis. In certain groups of individuals who cannot produce sputum, for example, children, non-sputum-based methods have particular importance. We found that the method tested was able to detect bacterial killing by active antibiotics that disrupt the cell wall and lead to fragmentation of bacteria. However, the assay can't detect inactive bacteria or bacteria that are active with an intact cell wall.

ABSTRACT Shorter and more effective treatment regimens as well as new drugs are urgent priorities for reducing the immense global burden of tuberculosis (TB). As treatment of TB currently requires multiple antibiotics with diverse mechanisms of action, any new drug lead requires assessment of potential interactions with existing TB antibiotics. We previously described the discovery of wollamides, a new class of Streptomyces-derived cyclic hexapeptides with antimycobacterial activity. To further assess the value of the wollamide pharmacophore as an antimycobacterial lead, we determined wollamide interactions with first- and second-line TB antibiotics by determining fractional inhibitory combination index and zero interaction potency scores. In vitro two-way and multiway interaction analyses revealed that wollamide B1 synergizes with ethambutol, pretomanid, delamanid, and para-aminosalicylic acid in inhibiting the replication and promoting the killing of phylogenetically diverse clinical and reference strains of the Mycobacterium tuberculosis complex (MTBC). Wollamide B1 antimycobacterial activity was not compromised in multi- and extensively drug-resistant MTBC strains. Moreover, growth-inhibitory antimycobacterial activity of the combination of bedaquiline/pretomanid/linezolid was further enhanced by wollamide B1, and wollamide B1 did not compromise the antimycobacterial activity of the isoniazid/rifampicin/ethambutol combination. Collectively, these findings add new dimensions to the desirable characteristics of the wollamide pharmacophore as an antimycobacterial lead compound. IMPORTANCE Tuberculosis (TB) is an infectious disease that affects millions of people globally, with 1.6 million deaths annually. TB treatment requires combinations of multiple different antibiotics for many months, and toxic side effects can occur. Therefore, shorter, safer, more effective TB therapies are required, and these should ideally also be effective against drug-resistant strains of the bacteria that cause TB. This study shows that wollamide B1, a chemically optimized member of a new class of antibacterial compounds, inhibits the growth of drug-sensitive as well as multidrug-resistant Mycobacterium tuberculosis isolated from TB patients. In combination with TB antibiotics, wollamide B1 synergistically enhances the activity of several antibiotics, including complex drug combinations that are currently used for TB treatment. These new insights expand the catalogue of the desirable characteristics of wollamide B1 as an antimycobacterial lead compound that might inspire the development of improved TB treatments.