The Phenomenon of the Formation of Biofilms by Brevibacillus spp. and Bacillus spp. in the Presence of Clinical Strains of Mycobacterium tuberculosis

Abstract

The formation of biofilms by M. tuberculosis on Shkolnikova’s medium (synthetic medium, analogue of Sauton’s medium) has been researched. We studied 150 clinical and 20 laboratory strains of M. tuberculosis. None of the 150 strains isolated from human beings produced biofilms (pellicle), but all yielded abundant planktonic growth. Twenty reference strains of M. tuberculosis produced both biofilms (pellicle) and planktonic growth. The phenomenon of biofilm formation by mixed cultures was observed when inoculating sputum treated with NALC-NaOH from patients with tuberculosis. We obtained 63 mixed biofilms. In 30.2% (19/63) of cases, biofilms contained the DNA of the causative agent of tuberculosis. The RV-PCR method was used to select six samples with the highest concentration of mycobacterial DNA. Molecular cloning and sequencing of a fragment of the 16S rRNA gene from one of the biofilms was carried out. The nucleotide sequence had 99% homology with the Bacillus thermoamylovorans species. From the mixed biofilms obtained, three strains of spore-forming bacilli were isolated. Strains are identified by Sanger’s sequencing of the 16S rRNA gene, one as Bacillus licheniformis, and the other two as Brevibacillus spp. A study of the resistance of isolated strains of spore bacilli against 12 antituberculosis drugs of the first and second series was carried out. All three strains were resistant to maximum concentrations of isoniazid, streptomycin, ethionamide, and ethambutol. Strains of Brevibacillus spp. were additionally resistant to para-aminosalicylic acid (PAS) and kanamycin. In a model experiment, the possibility of cogrowth of clinical strains of M. tuberculosis and B. licheniformis was demonstrated with prolonged co-incubation in Shkolnikova’s medium. In the first few days of growth, B. licheniformis produced a biofilm that remained stable for the entire observation period of 45 days. The hypothesis suggesting the possibility of a short-term persistence of some “saprophytic” species of bacilli in the caseous contents of necrosis foci in the late stages of pulmonary tuberculosis has been postulated.