Lung infections caused by Mycobacterium abscessus are emerging as a global threat to individuals with cystic fibrosis and to other patient groups. Recent evidence for human-to-human transmission worsens the situation. M. abscessus is an intrinsically multidrug-resistant pathogen showing resistance to even standard antituberculosis drugs, such as rifampin. Here, our objective was to identify existing drugs that may be employed for the treatment of M. abscessus lung disease. A collection of more than 2,700 approved drugs was screened at a single-point concentration against an M. abscessus clinical isolate. Hits were confirmed with fresh solids in dose-response experiments. For the most attractive hit, growth inhibition and bactericidal activities against reference strains of the three M. abscessus subspecies and a collection of clinical isolates were determined. Surprisingly, the rifampin derivative rifabutin had MICs of 3 ± 2 μM (3 μg/ml) against the screening strain, the reference strains M. abscessus subsp. abscessus ATCC 19977, M. abscessus subsp. bolletii CCUG 50184-T, and M. abscessus subsp. massiliense CCUG 48898-T, as well as against a collection of clinical isolates. Furthermore, rifabutin was active against clarithromycin-resistant strains. In conclusion, rifabutin, in contrast to rifampin, is active against the Mycobacterium abscessus complex bacteria in vitro and may be considered for treatment of M. abscessus lung disease.

Boromycin is a boron-containing polyether macrolide antibiotic isolated from Streptomyces antibioticus. It was shown to be active against Gram positive bacteria and to act as an ionophore for potassium ions. The antibiotic is ineffective against Gram negative bacteria where the outer membrane appears to block access of the molecule to the cytoplasmic membrane. Here we asked whether boromycin is active against Mycobacterium tuberculosis which, similar to Gram negative bacteria, possesses an outer membrane. The results show that boromycin is a potent inhibitor of mycobacterial growth (MIC50 = 80 nM) with strong bactericidal activity against growing and non-growing drug tolerant persister bacilli. Exposure to boromycin resulted in a rapid loss of membrane potential, reduction of the intracellular ATP level and leakage of cytoplasmic protein. Consistent with boromycin acting as a potassium ionophore, addition of KCl to the medium blocked its antimycobacterial activity. In contrast to the potent antimycobacterial activities of the polyether macrolide, its cytotoxicity and haemolytic activity were low (CC50 = 30 µM, HC50 = 40 µM) with a selectivity index of more than 300. Spontaneous resistant mutants could not be isolated suggesting a mutation frequency of less than 10-9 / CFU. Taken together, the results suggests that targeting mycobacterial transmembrane ion gradients may be an attractive chemotherapeutic intervention level to kill otherwise drug tolerant persister bacilli, and to slow down the development of genetic antibiotic resistance.

At least three betacoronaviruses have spilled over from bats to humans and caused severe diseases, highlighting the threat of zoonotic transmission. Thus, it is important to enhance surveillance capabilities by developing tools capable of detecting a broad spectrum of bat-borne betacoronaviruses. Three monoclonal antibodies (mAbs) targeting the nucleocapsid (N) protein were generated using recombinant N proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The cross-reactivities of these mAbs were evaluated against a panel of betacoronaviruses. Sandwich ELISAs (sELISAs) were subsequently developed to detect bat-borne betacoronaviruses that have high zoonotic potential. Among the mAbs, 7A7 demonstrated the broadest cross-reactivity, recognizing betacoronaviruses from the Sarbecovirus, Merbecovirus and Hibecovirus subgenera. The first sELISA, based on mAbs 7A7 and 6G10, successfully detected N protein in all clinical swab samples from COVID-19 patients with cycle threshold (Ct) values < 25, achieving 75% positivity overall (12/16). Using this as a reference, a second sELISA was established by pairing mAb 7A7 with mAb 8E2, which binds to multiple merbecoviruses. This assay detected the N protein of two merbecoviruses, namely the human MERS-CoV and bat-borne HKU5-CoV, at high sensitivity and has a limit of detection (LOD) that is comparable to the first sELISA used successfully to detect COVID-19 infection. These broadly reactive mAbs could be further developed into rapid antigen detection kits for surveillance in high-risk populations with close contact with wild bats to facilitate the early detection of potential zoonotic spillover events.

Antibodies against neuraminidase (NA) are an independent correlate of protection and the antigenic relatedness between H1N1pdm and H5N1 NAs suggests that seasonal influenza infection may provide cross-reactive immunity. In this study, recent H1N1pdm infection elicited modest NA-inhibiting (NAI) antibody responses against contemporary A/Victoria/4897/2022 (H1N1pdm) but strong responses against older A/California/7/2009 (H1N1pdm). Convalescent sera exhibited significantly higher cross-reactive NAI against clade 2.3.4.4b H5N1 (A/Texas/37/2024) than non-flu A patients, whereas H3N2 infection did not elicit such cross-reactivity. NAI titers were comparable between N1-California and N1-Texas but lower against N1-Victoria, indicating greater antigenic similarity between N1-California and N1-Texas. This was supported by stronger inhibition of N1-Texas by N1-California sheep antiserum compared with N1-Victoria sheep antiserum. Surprisingly, H1N1pdm-infected patients exhibited very low NAI against clade 2.3.2.1a H5N1 (N1-Bangladesh; GMT of 171 vs GMT 1159 for N1-Texas). Mouse antisera demonstrated reduced reciprocal inhibition between N1-Texas and N1-Bangladesh, consistent with their antigenic divergence. When four residues in N1-Bangladesh were substituted with their corresponding residues in N1-Texas, N1-Bangladesh antiserum showed reduced NAI, while N1-Texas antisera showed increased inhibition compared with wild-type N1-Bangladesh. This suggests that these amino acid differences are partially responsible for their antigenic divergence. Importantly, longitudinal analysis revealed that boosted cross-reactive NAI responses waned by day 90 post-infection, highlighting their limited durability. Together, these findings demonstrate that seasonal H1N1pdm infection can transiently boost cross-reactive NAI antibodies against clade 2.3.4.4b H5N1, but antigenic divergence in clade 2.3.2.1a limits cross-reactivity. As such, the impact of pre-existing antibody during an H5N1 outbreak is dependent on the infecting clade.