Publications

Mycobacterium brumae is a rapid-growing, non-pathogenic Mycobacterium species, originally isolated from environmental and human samples in Barcelona, Spain. Mycobacterium brumae is not pathogenic and it's in vitro phenotype and immunogenic properties have been well characterized. However, the knowledge of its underlying genetic composition is still incomplete. In this study, we first describe the 4 Mb genome of the M. brumae type strain ATCC 51384T assembling PacBio reads, and second, we assess the low intraspecies variability by comparing the type strain with Illumina reads from three additional strains. Mycobacterium brumae genome is composed of a circular chromosome with a high GC content of 69.2% and containing 3,791 CDSs, 97 pseudogenes, one prophage and no CRISPR loci. Mycobacterium brumae has shown no pathogenic potential in in vivo experiments, and our genomic analysis confirms its phylogenetic position with other non-pathogenic and rapid growing mycobacteria. Accordingly, we determined the absence of virulence-related genes, such as ESX-1 locus and most PE/PPE genes, among others. Although the immunogenic potential of M. brumae was proved to be as high as Mycobacterium bovis BCG, the only mycobacteria licensed to treat cancer, the genomic content of M. tuberculosis T cell and B cell antigens in M. brumae genome is considerably lower than those antigens present in M. bovis BCG genome. Overall, this work provides relevant genomic data on one of the species of the mycobacterial genus with high therapeutic potential.Copyright © 2023 Renau-Mínguez, Herrero-Abadía, Ruiz-Rodriguez, Sentandreu, Torrents, Chiner-Oms, Torres-Puente, Comas, Julián and Coscolla.

JTD Keywords: antimicrobial susceptibility, bcg, identification, immunogenic, non-pathogenic, nontuberculous mycobacteria, resistance mechanisms, strain, therapeutic, Diversity, Immunogenic, Non-pathogenic, Nontuberculous mycobacteria, Therapeutic

The mechanism of action of intravesical Mycobacterium bovis BCG immunotherapy treatment for bladder cancer is not completely known, leading to misinterpretation of BCG-unresponsive patients, who have scarce further therapeutic options. BCG is grown under diverse culture conditions worldwide, which can impact the antitumor effect of BCG strains and could be a key parameter of treatment success. Here, BCG and the nonpathogenic Mycobacterium brumae were grown in four culture media currently used by research laboratories and BCG manufacturers: Sauton-A60, -G15 and -G60 and Middlebrook 7H10, and used as therapies in the orthotopic murine BC model. Our data reveal that each mycobacterium requires specific culture conditions to induce an effective antitumor response. since higher survival rates of tumor-bearing mice were achieved using M. brumae-A60 and BCG-G15 than the rest of the treatments. M. brumae-A60 was the most efficacious among all tested treatments in terms of mouse survival, cytotoxic activity of splenocytes against tumor cells, higher systemic production of IL-17 and IFN-gamma, and bladder infiltration of selected immune cells such as ILCs and CD4(TEM). BCG-G15 triggered an antitumor activity based on a massive infiltration of immune cells, mainly CD3(+) (CD4(+) and CD8(+)) T cells, together with high systemic IFN-gamma release. Finally, a reduced variety of lipids was strikingly observed in the outermost layer of M. brumae-A60 and BCG-G15 compared to the rest of the cultures, suggesting an influence on the antitumor immune response triggered. These findings contribute to understand how mycobacteria create an adequate niche to help the host subvert immunosuppressive tumor actions.

JTD Keywords: bcg, innate immune response, innate-lymphoid cells, lipid, non-muscle invasive, Bcg, Calmette-guerin bcg, Glycerol, Identification, Immune-response, Innate immune response, Innate-lymphoid cells, Lipid, Lipids, Mycolic acids, Neutral-red, Non-muscle invasive, Phenolic glycolipids, Tuberculosis, Tumor microenvironment, Virulence

Mycobacterium bovis bacillus Calmette-Guérin (BCG) efficacy as an immunotherapy tool can be influenced by the genetic background or immune status of the treated population and by the BCG substrain used. BCG comprises several substrains with genetic differences that elicit diverse phenotypic characteristics. Moreover, modifications of phenotypic characteristics can be influenced by culture conditions. However, several culture media formulations are used worldwide to produce BCG. To elucidate the influence of growth conditions on BCG characteristics, five different substrains were grown on two culture media, and the lipidic profile and physico-chemical properties were evaluated. Our results show that each BCG substrain displays a variety of lipidic profiles on the outermost surface depending on the growth conditions. These modifications lead to a breadth of hydrophobicity patterns and a different ability to reduce neutral red dye within the same BCG substrain, suggesting the influence of BCG growth conditions on the interaction between BCG cells and host cells.

JTD Keywords: cell wall, efficacy, glycerol, hydrophobicity, lipid, neutral red, pdim, pgl, protein, strains, viability, virulence, Acylglycerol, Albumin, Article, Asparagine, Bacterial cell wall, Bacterial gene, Bacterium culture, Bcg vaccine, Catalase, Cell wall, Chloroform, Controlled study, Escherichia coli, Gene expression, Genomic dna, Glycerol, Glycerol monomycolate, Hexadecane, Housekeeping gene, Hydrophobicity, Immune response, Immunogenicity, Immunotherapy, Lipid, Lipid fingerprinting, Magnesium sulfate, Mercaptoethanol, Methanol, Methylglyoxal, Molybdatophosphoric acid, Mycobacterium bovis bcg, Neutral red, Nonhuman, Pdim, Petroleum ether, Pgl, Phenotype, Physical chemistry, Real time reverse transcription polymerase chain reaction, Rna 16s, Rna extraction, Rv0577, Staining, Thin layer chromatography, Unclassified drug

Intravesical Mycobacterium bovis Bacillus Calmette–Guérin (BCG) immunotherapy remains the gold-standard treatment for non-muscle-invasive bladder cancer patients, even though half of the patients develop adverse events to this therapy. On exploring BCG-alternative therapies, Mycolicibacterium brumae, a nontuberculous mycobacterium, has shown outstanding anti-tumor and immunomodulatory capabilities. As no infections due to M. brumae in humans, animals, or plants have been described, the safety and/or toxicity of this mycobacterium have not been previously addressed. In the present study, an analysis was made of M. brumae- and BCG-intravenously-infected severe combined immunodeficient (SCID) mice, M. brumae-intravesically-treated BALB/c mice, and intrahemacoelic-infected-Galleria mellonella larvae. Organs from infected mice and the hemolymph from larvae were processed to count bacterial burden. Blood samples from mice were also taken, and a wide range of hematological and biochemical parameters were analyzed. Finally, histopathological alterations in mouse tissues were evaluated. Our results demonstrate the safety and non-toxic profile of M. brumae. Differences were observed in the biochemical, hematological and histopathological analysis between M. brumae and BCG-infected mice, as well as survival curves rates and colony forming units (CFU) counts in both animal models. M. brumae constitutes a safe therapeutic biological agent, overcoming the safety and toxicity disadvantages presented by BCG in both mice and G. mellonella animal models.

JTD Keywords: Bladder cancer, Nontuberculous mycobacteria, BCG, Safety, Galleria mellonella, Mice

Purpose γ Irradiated Mycobacterium bovis bacillus Calmette-Guérin has shown in vitro and ex vivo antitumor activity. However, to our knowledge the potential antitumor capacity has not been demonstrated in vivo. We studied the in vivo potential of γ irradiated bacillus Calmette-Guérin and γ irradiated M. brumae, a saprophytic mycobacterium that was recently described as an immunotherapeutic agent. Materials and Methods The antitumor capacity of γ irradiated M. brumae was first investigated by analyzing the in vitro inhibition of bladder tumor cell proliferation and the ex vivo cytotoxic effect of M. brumae activated peripheral blood cells. The effect of γ irradiated M. brumae or bacillus Calmette-Guérin intravesical treatment was then compared to treatment with live mycobacteria in the orthotopic murine model of bladder cancer. Results Nonviable M. brumae showed a capacity to inhibit in vitro bladder cancer cell lines similar to that of live mycobacteria. However, its capacity to induce cytokine production was decreased compared to that of live M. brumae. γ Irradiated M. brumae could activate immune cells to inhibit tumor cell growth, although to a lesser extent than live mycobacteria. Finally, intravesical treatment with γ irradiated M. brumae or bacillus Calmette-Guérin significantly increased survival with respect to that of nontreated tumor bearing mice. Both γ irradiated mycobacteria showed lower survival rates than those of live mycobacteria but the minor efficacy of γ irradiated vs live mycobacteria was only significant for bacillus Calmette-Guérin. Conclusions Our results show that although γ irradiated mycobacteria is less efficacious than live mycobacteria, it induces an antitumor effect in vivo, avoiding the possibility of further mycobacterial infections.

JTD Keywords: BCG vaccine, Gamma rays, Immunotherapy, Mycobacterium, Urinary bladder neoplasms