Genomic characterization and early macrophage inflammatory responses to an emerging Mycobacterium wolinskyi isolate

Mycobacterium wolinskyi is an emerging rapidly growing nontuberculous mycobacterium increasingly recognized as a cause of skin, soft-tissue, and device-associated infections, including infections in immunocompetent individuals. However, its genomic characteristics and interactions with host immune cells remain poorly understood. In this study, we characterized a clinical M. wolinskyi isolate, designated QL01, recovered from a facial skin infection in an 11-year-old child, by combining antimicrobial susceptibility testing, whole-genome sequencing, comparative genomic analysis, and macrophage infection assays with transcriptomic profiling. QL01 was resistant to clarithromycin, imipenem, and trimethoprim-sulfamethoxazole, while remaining susceptible to amikacin, linezolid, moxifloxacin, minocycline, and doxycycline. Whole-genome sequencing generated a complete circular chromosome and identified antimicrobial resistance-associated homologs and host interaction-associated homologs. Comparative analysis revealed variable conservation of type VII secretion system (T7SS)- and Mce-associated loci among representative rapidly growing mycobacteria. Following uptake by mouse bone marrow-derived macrophages, viable intracellular QL01 remained recoverable after 24 h. RNA sequencing revealed an early innate immune and pro-inflammatory transcriptional response, with enrichment of TNF, NF-κB, Toll-like receptor, and C-type lectin receptor signaling pathways. RT-qPCR further confirmed increased expression of Ccl2 , Tnf , Il1b , Tlr2 , Clec4e , and Tnfaip3 . ELISA confirmed increased secretion of TNF-α, IL-1β, and IFN-β. Inhibition of IKK/NF-κB signaling attenuated QL01-induced phosphorylation of p65 and IκBα and reduced inflammatory cytokine secretion. These findings characterize the genomic features of a clinical M. wolinskyi isolate and describe the early macrophage response to this emerging nontuberculous mycobacterial species.