Tuberculoma caseous necrosis-derived bacteria (Corynebacterium and Staphylococcus) enhance granuloma formation in vitro and stimulate mycobacterial biofilm development

Granuloma formation represents one of the key stages in tuberculosis pathogenesis, characterized by the development of specialized cellular structures surrounding Mycobacterium tuberculosis within foci. These structures are presented by complex, multicomponent assemblies that both detain the pathogen and simultaneously serve as a niche for its persistence. It has been firmly established that the mycobacterial cord factor is an important driver of granuloma induction, being involved in intercellular interactions and the organization of granuloma three-dimensional scaffold. However, the influence of the resident microbiota on this process, as well as its contribution to the formation of mycobacterial biofilms, remains understudied. This work investigates an effect of Corynebacterium kefirresidentii and Staphylococcus epidermidis lysates (isolated from tuberculoma-related caseous necrosis) on in vitro granuloma formation using C57BL/6 mouse peritoneal macrophages and splenocytes. Additionally, an influence of C. kefirresidentii on vaccine strain M. bovis BCG growth and biofilm development was examined. The study demonstrates that C. kefirresidentii induces the formation of a rapidly growing M. bovis BCG biofilm with characteristic cord-like architecture and a prominent extracellular matrix. This effect was accompanied by a statistically significantly expanded area under the growth curve compared to the control (p < 0.0001). Co-cultivation experiments with the clinical strain M. tuberculosis Beijing B0/W148 and bacterial lysates revealed limited antimycobacterial activity but demonstrated a pronounced stimulatory effect on the formation of granuloma-like structures in the M. tuberculosis-induced in vitro granulemogenesis model. C. kefirresidentii and S. epidermidis lysates significantly propagated granuloma formation as evidenced by markedly increased both cellular aggregate number and size (p < 0.01), including the formation of large clusters containing more than 50 cells. Taken together, these findings suggest that satellite pathobiota derived from caseous necrosis of tuberculous foci might be involved in modulating key mechanisms of infection pathogenesis and point at its potential contribution to unfavorable outcomes in pulmonary tuberculosis.