A Multi-Gene Signature of Non-Muscle-Invasive Bladder Cancer Identifies Patients Who Respond to Immunotherapies Including Bacillus Calmette-Guérin and Immune Checkpoint Inhibitors.

Approximately 75% of bladder cancer cases originate as non-muscle-invasive bladder cancer (NMIBC). Despite initial diagnosis, NMIBC commonly recurs, with up to 45% advancing to muscle-invasive bladder cancer (MIBC) and metastatic disease. Treatment for high-risk NMIBC typically includes procedures like transurethral resection and, depending on recurrence risk, intravesical chemotherapy or immunotherapy such as Bacillus Calmette-Guérin (BCG). However, persistent shortages of BCG necessitate alternative first-line treatments. We aim to use a multi-gene signature in high-risk NMIBC patients to determine whether patients may benefit from immune checkpoint inhibitors (ICIs) as an alternative to BCG and to evaluate their clinical utility. The multi-gene signature obtained from the three independent NMIBC cohorts was applied to stratify the UROMOL2016 cohort (n = 476) using consensus clustering. Each subtype was distinguished by biological pathway analysis. Validation analysis using a machine learning algorithm was performed in six independent cohorts including the BRS (n = 283) cohort treated with BCG and the IMvigor210 (n = 298) clinical trials treated with PD-L1 inhibitors. Based on consensus cluster analysis, NMIBC patients in the UROMOL2016 cohort were classified into three classes exhibiting distinguished characteristics, including DNA damage repair (DDR). Survival analysis showed that the NMIBC-DDR class had the highest rates of disease progression (progression-free survival, p = 0.002 by log-rank test) in the UROMOL cohort and benefited from BCG and ICIs (respectively, p = 0.02 and p = 0.03 by log-rank test). This study suggests that the multi-gene signature may have a role in identifying high-risk NMIBC patients and improving the responsiveness of ICIs. Additionally, we propose immunotherapy as a new first-line treatment for patients with high-risk NMIBC because of the shortage of BCG supply. It is important to help more patients prioritize cancer immunotherapy.

Metagenomic sequencing expedites diagnosis of disseminated BCG in an infant with BRAFV600E mutation.

INTRODUCTION: Disseminated bacillus Calmette-Guérin (BCG) disease is a rare but serious BCG complication in children. Early diagnosis and timely interventions are essential to improve prognosis. However, its manifestations can closely mimic those of Langerhans cell histiocytosis (LCH), which usually leads to a high rate of misdiagnoses. Herein we report the first case of successful application of biopsy tissue metagenomic next-generation sequencing (mNGS) in the differential diagnosis of disseminated BCG disease and LCH. CASE STUDY: A 5-month-old female infant was transferred to our center for the treatment of paroxysmal cough, intermittent hematochezia and trunk rash. Examination on admission showed moderate anemia, erythropenia, thrombocytopenia and hepatosplenomegaly. The immunohistochemistry of her intestinal biopsy samples showed CD1a (+) and Langerin (+). Genetic testing of both peripheral blood and bone marrow samples suggested BRAFV600E mutation. Hence, she was initially diagnosed with LCH. However, no improvement was observed after a course of systemic chemotherapy. The left axillary lymph node and colonic mucosal biopsy specimens were sent for mNGS which resulted in sequence reads of Mycobacterium bovis-BCG. Triple antimycobacterial therapy was started according to the diagnosis. RESULTS: The diagnosis of this case was corrected as disseminated BCG disease by mNGS. Currently, she is doing well clinically and continues to follow-up at our outpatient clinic. CONCLUSIONS: This case suggests that mNGS is a valuable tool in the differential diagnosis of disseminated BCG disease and LCH, which can improve the early diagnosis rate of disseminated BCG disease.

Construction of rBCG carrying the IL-2-BZLF1 fusion gene and its immunological function.

In this research, a recombinant Bacillus Calmette Guerin (rBCG) vector vaccine carrying a human IL-2 and EBV BZLF1 fusion gene (IL-2-BZLF1-rBCG) was constructed. The IL-2-BZLF1-rBCG construct was successfully generated and stably expressed the IL-2 and BZLF1 proteins. IL-2-BZLF1-rBCG activated the immune system and promoted the secretion of IFN-γ and TNF-α by CD4+ and CD8+ T cells. IL-2-BZLF1-rBCG activated lymphocytes to effectively kill EBV-positive NPC cells in vitro. Additionally, IL-2-BZLF1-rBCG stimulated the proliferation of NK cells and lymphocytes in vivo, activated related immune responses, and effectively treated EBV-positive NPC. The immune response to and pharmacological effect of IL-2-BZLF1-rBCG were explored in vitro and in vivo to provide a theoretical and experimental basis for the prevention and treatment of EBV-positive tumors with an rBCG vector vaccine. KEY POINTS: • rBCG with human IL-2 and BZLF1 of EB virus was constructed • The IL-2-BZLF1 fusion gene was stably expressed with rBCG • rBCG with IL-2-BZLF1 has an obvious immune response in vitro and in vivo.

Exploring the genetic factors behind the discrepancy in resistance to bovine tuberculosis between African zebu cattle and European taurine cattle.

Caused by the pathogenic agent Mycobacterium bovis, bovine tuberculosis (bTB) is a major concern in cattle breeding due to both its zoonotic potential and economic impact. Greater resistance to this disease has been reported in certain African zebu breeds compared to European taurine breeds. However the genetic basis for the lower susceptibility to bTB infection observed in zebu cattle remains poorly explored. This study was conducted on whole genome sequencing data of three bTB infection-resistant African zebu breeds and two bTB infection-susceptible taurine breeds to decipher the genetic background. A set of four selection signature statistics based on linkage disequilibrium, site frequency spectrum, and population differentiation were used on SNPs whereas between population variance based VST and t-test were used on CNVs. As a complement, genes from previous literature reported as candidate genes for bTB resistance were also inspected to identify genetic variations. Interestingly, the resulting nine candidate genes had deleterious missense variants (SHC3, IFNGR1, TLR2, TLR6, IL1A, LRRK2, EP300 and IRAK4) or a CNV difference (CD48) segregating between the groups. The genes found in the study play a role in immune pathways activated during Mycobacterium infection, contributing to the proliferation of immune cells and the granuloma formation, ultimately modulating the outcome of the infectious event. In particular, a deleterious variant in the LRRK2 gene, whose deficiency has been linked to improved prognosis upon tuberculosis infection, was found in the bTB infection-resistant zebu breeds. Therefore, these genes constitute credible candidates in explaining the discrepancy in Mycobacterium bovis infection susceptibility among different breed.

A secreted form of chorismate mutase (Rv1885c) in Mycobacterium bovis BCG contributes to pathogenesis by inhibiting mitochondria-mediated apoptotic cell death of macrophages.

BACKGROUND: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), and its pathogenicity is associated with its ability to evade the host defense system. The secretory form of the chorismate mutase of M. tuberculosis (TBCM, encoded by Rv1885c) is assumed to play a key role in the pathogenesis of TB; however, the mechanism remains unknown. METHODS: A tbcm deletion mutant (B∆tbcm) was generated by targeted gene knockout in BCG to investigate the pathogenic role of TBCM in mice or macrophages. We compared the pathogenesis of B∆tbcm and wild-type BCG in vivo by measuring the bacterial clearance rate and the degree of apoptosis. Promotion of the intrinsic apoptotic pathway was evaluated in infected bone marrow-derived macrophages (BMDMs) by measuring apoptotic cell death, loss of mitochondrial membrane potential and translocation of pore-forming proteins. Immunocytochemistry, western blotting and real-time PCR were also performed to assess the related protein expression levels after infection. Furthermore, these findings were validated by complementation of tbcm in BCG. RESULTS: Deletion of the tbcm gene in BCG leads to reduced pathogenesis in a mouse model, compared to wild type BCG, by promoting apoptotic cell death and bacterial clearance. Based on these findings, we found that intrinsic apoptosis and mitochondrial impairment were promoted in B∆tbcm-infected BMDMs. B∆tbcm down-regulates the expression of Bcl-2, which leads to mitochondrial outer membrane permeabilization (MOMP), culminating in cytochrome c release from mitochondria. Consistent with this, transcriptome profiling also indicated that B∆tbcm infection is more closely related to altered mitochondrial-related gene expression than wild-type BCG infection, suggesting an inhibitory role of TBCM in mitochondrial dysfunction. Moreover, genetic complementation of B∆tbcm (C∆tbcm) restored its capacity to inhibit mitochondria-mediated apoptotic cell death. CONCLUSIONS: Our findings demonstrate the contribution of TBCM to bacterial survival, inhibiting intrinsic apoptotic cell death of macrophages as a virulence factor of M. tuberculosis complex (MTBC) strains, which could be a potential target for the development of TB therapy.

The double-sided effects of Mycobacterium bovis bacillus Calmette-Guérin vaccine on helminthic infections ­ current data and future prospects.

Bacillus Calmette-Guérin (BCG), a live attenuated strain derived from an isolate of Mycobacterium bovis, is one of the childhood vaccinations widely used against tuberculosis (TB). In addition to its effects on mycobacterial diseases, the information has shown the protection effect of BCG in helminthic diseases. In the current review, the role of BCG vaccine in non-specific protection helminthic infection is reviewed. In human alveolar echinococcosis (AE), treatment with BCG enhances host's innate immune response against the parasite via the number and activation of monocytes. In cysticercosis, despite the enhancement of Th1-biased immune responses by coadministration of rcC1 plus BCG-DNA, the level of induced protection did not increase compared to immunization with rcC1 antigen alone. Also, pretreatment of mice with live BCG vaccine induced a high level of protection against subsequent parasite infection with Taenia taeniaeformis. The reduction of the parasite burden in mice infected with Mesocestoides corti that received two doses of BCG post-infection demonstrated the therapeutic effect of BCG. The protective potential of the schistosomula/BCG vaccine against Schistosoma japonicum in sheep study showed a reduction in the number of adult worms and mean faecal egg counts post-challenge. In trichinellosis, BCG can induce hyperplasia of the reticuloendothelial system and activation of macrophages in mice. Therefore, these data revealed that BCG vaccination can exert non-specific protective effects for the prevention of diseases other than tuberculosis. Medicinal doses of BCG may be considered a new approach to the treatment of helminth infections.

Differences in responses to the intracellular macrophage environment between Mycobacterium bovis BCG vaccine strains Moreau and Pasteur.

BACKGROUND: The Bacille Calmette-Guérin (BCG) vaccine comprises a family of strains with variable protective efficacy against pulmonary tuberculosis (TB) and leprosy, partly due to genetic differences between strains. OBJECTIVES: Previous data highlighting differences between the genomes and proteomic profiles of BCG strains Moreau and Pasteur led us to evaluate their behaviour in the macrophage microenvironment, capable of stimulating molecular responses that can impact the protective effect of the vaccine. METHODS: Strain infectivity, viability, co-localisation with acidified vesicles, macrophage secretion of IL-1 and MCP-1 and lipid droplet biogenesis were evaluated after infection. FINDINGS: We found that BCG Moreau is internalised more efficiently, with significantly better intracellular survival up to 96 h p.i., whereas more BCG Pasteur bacilli were found co-localised in acidified vesicles up to 6 h p.i. IL-1ß and MCP-1 secretion and lipid droplet biogenesis by infected macrophages were more prominent in response to BCG Pasteur. MAIN CONCLUSION: Overall, our results show that, compared to Pasteur, BCG Moreau has increased fitness and better endurance in the harsh intracellular environment, also regulating anti-microbial responses (lower IL-1b and MCP-1). These findings contribute to the understanding of the physiology of BCG Moreau and Pasteur in response to the intraphagosomal environment in a THP-1 macrophage model.

Mmu-miR-25-3p promotes macrophage autophagy by targeting DUSP10 to reduce mycobacteria survival.

Background: The present study aimed to investigate the regulation of miR-25-3p on macrophage autophagy and its effect on macrophage clearance of intracellular Mycobacterium bovis Bacillus Calmette-Guerin (BCG) retention based on the previous findings on the differential expression of exosomal miRNA in macrophages infected with BCG. Methods: Through enrichment analysis and Hub gene analysis, key differentially expressed miRNA and its target genes were selected. The targeted binding ability of the screened mmu-miR-25-3p and its predicted target gene DUSP10 was determined through the TargetScan database, and this was further verified by dual luciferase reporter gene assay. mmu-miR-25-3p mimics, mmu-miR-25-3p inhibitor, si-DUSP10, miR-NC,si-NC and PD98059 (ERK Inhibitor) were used to intervene macrophages Raw264.7. Rt-qPCR was used to detect the expression levels of mmu-miR-25-3p and DUSP10 mRNA. Western blot was used to detect the expression levels of DUSP10, LC3-II, p-ERK1/2, beclin1, Atg5 and Atg7. The autophagy flux of macrophage Raw264.7 in each group was observed by confocal laser microscopy, and the expression distribution of DUSP10 and the structure of autophagosomes were observed by transmission electron microscopy. Finally, the intracellular BCG load of macrophage Raw264.7 was evaluated by colony-forming unit (CFU) assay. Results: Bioinformatics analysis filtered and identified the differentially expressed exosomal miRNAs. As a result, mmu-miR-25-3p expression was significantly increased, and dual specificity phosphatase 10 (DUSP10) was predicted as its target gene that was predominantly involved in autophagy regulation. The dual luciferase reporter gene activity assay showed that mmu-miR-25-3p was targeted to the 3'-untranslated region (UTR) of DUSP10. The infection of BCG induced the upregulation of mmu-miR-25-3p and downregulation of DUSP10 in RAW264.7 cells, which further increased the expression of LC3-II and promoted autophagy. Upregulated mmu-miR-25-3p expression decreased the level of DUSP10 and enhanced the phosphorylation of ERK1/2, which in turn upregulated the expression of LC3-II, Atg5, Atg7, and Beclin1. Immuno-electron microscopy, transmission electron microscopy, and autophagic flux analysis further confirmed that the upregulation of mmu-miR-25-3p promotes the autophagy of macrophages after BCG infection. The CFU number indicated that upregulated mmu-miR-25-3p expression decreased the mycobacterial load and accelerated residual mycobacteria clearance. Conclusion: mmu-miR-25-3p promotes the phosphorylation of ERK1/2 by inhibiting the expression of DUSP10, thus enhancing the BCG-induced autophagy of macrophages. These phenomena reduce the bacterial load of intracellular Mycobacterium and facilitate the clearance of residual mycobacteria. mmu-miR-25-3p has great potential as a target for anti-tuberculosis immunotherapy and can be the optimal miRNA loaded into exosomal drug delivery system in future studies.

Direct detection of Mycobacterium bovis by a dry loop-mediated isothermal amplification assay in cattle samples collected during routine abattoir examination in Malawi.

The lack of quick, accurate, and low-cost detection methods has hindered the active control strategies for bovine tuberculosis (bTB) in resource-limited countries with a high burden of disease. We developed a dry loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Mycobacterium bovis, the principal causative agent of bTB, and evaluated the efficacy of the assay using suspected bTB samples collected during routine meat inspection at major regional abattoirs in Malawi. Template genomic DNA was extracted directly from the granulomatous bTB-like lesion (crude extracted DNA), as well as growth from the incubated mycobacterial growth indicator tubes (MGIT). Field results were visualized by the naked eye within 40 min following a color change of the amplified products. The sensitivity and specificity of the dry LAMP assay while using 152 DNA samples extracted from MGIT with confirmed M. bovis results were 98% and 88%, respectively. When 43 randomly selected crude DNA samples from lesions were used, the sensitivity and specificity of the dry LAMP assay were 100% and 75%, respectively. Our LAMP assay offers the potential to meet the demands for a low-cost and rapid field detection tool for bTB in resource-limited countries in which bTB is endemic.

Redox Cycling Dioxonaphthoimidazoliums Disrupt Iron Homeostasis in Mycobacterium bovis Bacillus Calmette-Guérin.

The dioxonaphthoimidazolium scaffold is a novel, highly bactericidal redox cycling antituberculosis chemotype that is reliant on the respiratory enzyme Type II NADH dehydrogenase (NDH2) for the generation of reactive oxygen species (ROS). Here, we employed Mycobacterium bovis Bacillus Calmette-Guérin (M. bovis BCG) reporter strains to show that ROS generated by the redox cycler SA23 simulated an iron deficient state in the bacteria, which led to a compensatory increase in the expression of the iron acquisition mbtB gene while collaterally reducing the expression of the iron storage bfrB gene. Exacerbating the iron deficiency via the inclusion of an iron chelator or aggravating oxidative stress by deploying a catalase (KatG) loss-of-function mutant strain enhanced the activity of SA23, whereas a combined approach of treating the katG mutant strain with an iron chelator led to even greater gains in activity. Our results support the notion that the activity of SA23 pivots on a vicious cycle of events that involve the derailment of iron homeostasis toward greater acquisition of the metal, overwhelmed oxidative stress defenses due to enhanced Fenton reactivity, and, ultimately, self-inflicted death. Hence, we posit that redox cyclers that concurrently perturb the iron equilibrium and cellular respiration are well-positioned to be potent next-generation anti-tubercular drugs. IMPORTANCE Cellular respiration in mycobacteria is a potentially rich target space for the discovery of novel drug entities. Here, we show that a redox cycling bactericidal small molecule that selectively activates a respiratory complex in mycobacteria has the surprising effect of disrupting iron homeostasis. Our results support the notion that the disruption of cellular respiration is a potent driver of reactive oxygen species (ROS) generation by the redox cycling molecule. Mycobacteria respond by acquiring iron to restore the levels depleted by the prevailing oxidizing conditions, which inadvertently trigger the compensatory acquisition of the metal. This leads to overwhelmed oxidative stress defenses and yet more iron depletion. For organisms that are unable to break out of this pernicious cycle of events, cell death is the inevitable outcome. Hence, aberrant ROS production by a redox cycling bactericidal agent inflicts a plethora of damaging effects on mycobacteria, including the derailment of iron homeostasis.

Gene Silencing of Toll-like Receptor 2 Gene Expression as a Tactic to Control Mycobacterium Tuberculosis and Granuloma Formation.

Mycobacterium tuberculosis (MT) is the causative agent of tuberculosis (TB) in humans. Tuberculosis is one of the top 10 causes of mortality worldwide, resulting in 1.8 million deaths and 10.4 million new cases in 2016. Understanding the fundamental features of MT biology is critical to the eradication of MT in the future. Due to the increasing frequency of antimicrobial treatment resistance and problems in vaccine development, the pathogenesis of TB for its survival and growth is highly dependent on host lipids and stimulated-lipid droplets formation. Toll-like receptor 2 (TLR2) forms heterophilic dimers with TLR1 and TLR6, therefore, recognizing many MT components. Both of these receptors identify the invading antigen and activate downstream protein kinases. Some studies demonstrated that the cyclooxygenase-2 (COX-2) promoter-driven gene expression includes connecting sites for transcription factors, such as nuclear factor-kappa B, CREB, NFAT, and c/EBPß. The current study aimed to investigate the role of the TLR2 receptor in positively regulating prostaglandin E2 production in M. bovis (BCG) infected macrophages in vivo using a human monocytic cell line THP-1. Our results revealed that MT infection triggers a time-dependent increase in COX-2 expression via pathways involving TLR2 receptor activation and enhances COX-2 expression, leading to an increase in lipid droplet formation and suppression of macrophage activation.

Generalized tuberculosis due to Mycobacterium caprae in a red fox phylogenetically related to livestock breakdowns.

BACKGROUND: Tuberculosis (TB) due to Mycobacterium caprae is endemic in goat herds and free-ranging wild boars in Spain, causing infections in other livestock or wild animals to a lesser extent. TB infection in foxes is infrequently reported and they are usually considered spillover hosts of TB. CASE PRESENTATION: A blind, depressed and severely emaciated red fox (Vulpes vulpes) was admitted to a rehabilitation center. After clinical examination it was humanely sacrificed. At necropsy, generalized TB lesions were observed that were subsequently confirmed by histopathology along with a co-infection with canine distemper virus. M. caprae was isolated from mycobacterial culture and spoligotype SB0415 was identified. Whole genome sequencing (WGS) of the isolated M. caprae was carried out and single nucleotide polymorphisms (SNP) were compared with other sequences of M. caprae isolated from livestock and wildlife of the same area throughout the last decade. CONCLUSIONS: This is the first reported case of TB due to M. caprae in a fox in the Iberian Peninsula. WGS and SNP analysis, together with spatial-temporal investigations, associated this case with recent M. caprae outbreaks in cattle and goat herds of the area. The results indicated transmission of M. caprae between livestock and the fox, suggesting that this species may occasionally play a role in the epidemiology of animal TB.

Prevalence of bovine tuberculosis and characterization of the members of the Mycobacterium tuberculosis complex from slaughtered cattle in Rwanda.

BACKGROUND: Bovine tuberculosis (bTB) is an endemic disease in Rwanda, but little is known about its prevalence and causative mycobacterial species. The disease causes tremendous losses in livestock and wildlife and remains a significant threat to public health. MATERIALS AND METHODS: A cross-sectional study employing a systematic random sampling of cattle (n = 300) with the collection of retropharyngeal lymph nodes and tonsils (n = 300) irrespective of granulomatous lesions was carried out in six abattoirs to investigate the prevalence and identify mycobacterial species using culture, acid-fast bacteria staining, polymerase chain reaction, and GeneXpert assay. Individual risk factors and the origin of samples were analysed for association with the prevalence. FINDINGS: Of the 300 sample pools, six were collected with visible TB-like lesions. Our findings demonstrated the presence of Mycobacterium tuberculosis complex (MTBC) in 1.7% (5/300) of sampled slaughtered cattle. Mycobacterium bovis was isolated from 1.3% (4/300) animals while one case was caused by a rifampicin-resistant (RR) M. tuberculosis. Non-tuberculous mycobacteria were identified in 12.0% (36/300) of the sampled cattle. There were no significant associations between the prevalence and abattoir category, age, sex, and breeds of slaughtered cattle. CONCLUSIONS: This study is the first in Rwanda to isolate both M. bovis and RR M. tuberculosis in slaughtered cattle indicating that bTB is present in Rwanda with a low prevalence. The isolation of RR M. tuberculosis from cattle indicates possible zooanthroponotic transmission of M. tuberculosis and close human-cattle contact. To protect humans against occupational zoonotic diseases, it is essential to control bTB in cattle and raise the awareness among all occupational groups as well as reinforce biosafety at the farm level and in the abattoirs.

Genomic, Metabolic, and Immunological Characterization of GMP-Grade Mycobacterium phlei.

Mycobacterium phlei (M. phlei) is an understudied microbe with medical values as an immunomodulating agent. Here, we establish an industrial strain of M. phlei, CUD, and characterize its genomic, metabolic, and immunological profiles. The established strain has been stably passed for more than a decade, indicated by next-generation sequencing of its 5.3 Mb genome. We show that the intramuscular inoculation of heat-inactivated CUD in immunocompetent mice is well tolerated, and can mount immunological responses. Immunophenotyping demonstrates induced innate and adaptive immune responses in peripheral blood, spleen, and inguinal lymph nodes of CUD-treated mice. Using GC-TOF-MS, we find that the metabolomic profiles of different batches are highly concordant. These results demonstrate a highly reproducible production of M. phlei under GMP conditions. IMPORTANCE Heat-inactivated M. phlei demonstrates promising efficacy to treat BCG-unresponsive non-muscle-invasive bladder cancer patients in clinical trials. However, lack of GMP-grade heat-inactivated M. phlei hampers further clinical investigations. Here, we described a GMP-grade, heat-inactivated M. phlei product, and presented initial characterization of its safety and immunomodulating properties. This product will serve as a starting point for further preclinical studies as well as clinical trials such as in combination with immune checkpoint inhibitors to treat bladder cancer.

Plasma Metagenomic Sequencing Expedites Diagnosis of Disseminated BCG in an Infant With IKBKB Mutation.

BACKGROUND: Infants with inborn errors of immunity (IEI), born in countries where Bacillus-Calmette-Guerin (BCG) vaccination is recommended at birth, are at risk of developing infectious complications following vaccination. A prompt diagnosis of disseminated BCG infection in these infants is essential, as many will require stem cell transplantation (SCT) for the immunologic cure. In patients with IEI, the mortality risk from disseminated mycobacterial infection is high, both before and following SCT. METHODS: A 7-month-old Qatari infant with an IEI, homozygous IKBKB gene mutation, was evaluated at our institution for SCT. He had a history of recurrent pneumonias, but pretransplant evaluation revealed negative cultures from bronchoalveolar fluid, blood and urine. At 8 months of age, the infant developed skin nodules of unclear etiology, prompting additional evaluation. RESULTS: Given his profound immunosuppression and receipt of broad-spectrum antimicrobials, plasma metagenomic next-generation sequencing (mNGS) was obtained and identified Mycobacterium tuberculosis complex within 72 hours. A skin biopsy was performed, and antimycobacterial therapy was initiated. Mycobacterium bovis-BCG was confirmed from cultures 3 weeks later. Treatment was complicated by elevated serum liver transaminases and aminoglycoside-associated high-frequency hearing loss. The infant completed 14 months of treatment from engraftment. Evaluation for active BCG infection after SCT was negative. CONCLUSION: In an infant with a unique IEI, plasma mNGS provided the first diagnosis of disseminated BCG infection. We believe that early initiation of antimycobacterial treatment improved the infant's clinical outcome. Plasma mNGS testing should be considered as a noninvasive screen for infectious pathogens in children with IEIs before SCT.

Computational Mining and Characterization of Hypothetical Proteins of Mycobacterium bovis Toward the Identification of Probable Vaccine Candidates.

A hypothetical protein (HP) is one that is known to exist only on the basis of a corresponding gene but without any function assigned to it. Many HPs have emerged as attractive vaccine candidates against prokaryotic and eukaryotic pathogens as well as against cancers. Mycobacterium bovis is a serious veterinary pathogen of tremendous zoonotic importance. This protocol describes a computational workflow for the identification of the HPs of M. bovis with vaccine potential and their subsequent structural and functional characterization.

Mycobacterium microti Infections in Free-Ranging Red Deer (Cervus elaphus).

Infections with Mycobacterium microti, a member of the M. tuberculosis complex, have been increasingly reported in humans and in domestic and free-ranging wild animals. At postmortem examination, infected animals may display histopathologic lesions indistinguishable from those caused by M. bovis or M. caprae, potentially leading to misidentification of bovine tuberculosis. We report 3 cases of M. microti infections in free-ranging red deer (Cervus elaphus) from western Austria and southern Germany. One diseased animal displayed severe pyogranulomatous pleuropneumonia and multifocal granulomas on the surface of the pericardium. Two other animals showed alterations of the lungs and associated lymph nodes compatible with parasitic infestation. Results of the phylogenetic analysis including multiple animal strains from the study area showed independent infection events, but no host-adapted genotype. Personnel involved in bovine tuberculosis-monitoring programs should be aware of the fastidious nature of M. microti, its pathogenicity in wildlife, and zoonotic potential.

Bovine tuberculosis due to Mycobacterium bovis and other mycobacteria among water buffalo (Bubalus bubalis) from the Brazilian Amazon.

INTRODUCTION: Zoonotic tuberculosis is a disease of public health importance worldwide, especially in developing countries. The present study aimed to investigate the role played by Mycobacterium bovis and other mycobacteria as etiologic agents of bubaline tuberculosis (TB) in the Brazilian Amazon region. METHODOLOGY: Granulomatous lesions suggestive of TB obtained from 109 buffaloes (n =109) during sanitary inspection at slaughter were subjected to histopathological evaluation, immunohistochemical (IHC) detection of Mycobacterium antigens, and to molecular tests (PCR) to detect hsp65, IS6110 and RD4 genes, which are specific to Mycobacterium spp., Mycobacterium tuberculosis Complex (MTBC) and M. bovis, respectively. RESULTS: PCR results indicated Mycobacterium infection in 87.2% of the cases, of which 69.5% were positive for M. bovis, 27.4% belonged to MTBC, and 3.1% were probably non-TB mycobacteria. There was good agreement between the genus-specific molecular technique and the histopathological analysis. This high frequency of TB cases caused by non-M. bovis suggests a diversified scenario of mycobacteria associated with bubaline TB in the Brazilian Amazon region. CONCLUSIONS: The results reinforce the need of discussing the inclusion of more accurate techniques in examinations carried out by Inspection Services in Brazil.

Comparative Study of the Susceptibility to Oxidative Stress between Two Types of Mycobacterium bovis BCG Tokyo 172.

Genomic analysis revealed that the vaccine seed lot of Mycobacterium bovis bacillus Calmette-Guérin (BCG) Tokyo 172 contains two subclones (types I and II), but their phenotypic differences have not been elucidated. In this study, we compared the susceptibility of bacilli types I and II to oxidative stress in vitro and within host cells. Notably, the subclones displayed similar superoxide dismutase activity; however, foam height in the catalase test and lysate catalase/peroxidase activity were higher for type I bacilli than for type II bacilli. Additionally, type I bacilli were less susceptible to hydrogen peroxide (H2O2) than type II bacilli. After exposure to H2O2, antioxidative stress response genes katG, ahpC, sodA, and trxA were more strongly induced in type I bacilli than in type II bacilli. Further, we investigated cell survival in macrophages. Fewer type II bacilli were recovered than type I bacilli. However, in the presence of apocynin, a specific inhibitor of NADPH oxidase, type II recovery was greater than that of type I. The production of interleukin 1ß (IL-1ß), IL-12 p40, and tumor necrosis factor alpha (TNF-α) was higher in type I bacillus-infected macrophages than in type II bacillus-infected macrophages. The proportions of type I and type II bacilli in vaccine lots over 3 years (100 lots) were 97.6% ± 1.5% and 2.4% ± 1.5%, respectively. The study results illustrated that type I bacilli are more resistant to oxidative stress than type II bacilli. Overall, these findings provide important information in terms of the quality control and safety of BCG Tokyo 172 vaccine.IMPORTANCE This study revealed the difference of in vivo and in vitro antioxidative stress properties of BCG Tokyo 172 types I and II as one of the bacteriological characteristics. In particular, the bacilli exhibited differences in catalase/peroxidase activity, which could explain their different protective effects against infection. The differences correlated with survival in the host cell and the production of proinflammatory cytokines to protect against infection by Mycobacterium tuberculosis The proportion of bacilli types I and II in all commercial lots of BCG Tokyo 172 over 3 years (100 lots) was constant. The findings also highlighted the importance of analyzing their content for quality control during vaccine production.

Natural Killer Anti-Tumor Activity Can Be Achieved by In Vitro Incubation With Heat-Killed BCG.

Natural Killer cell receptors allow this heterogeneous immune population to efficiently fight both tumors and infection, so their use as immunotherapy agents is an active field of research. Cytokine activation, particularly by myeloid cell-derived IL15, can induce potent NK anti-tumor responses. While studying the mechanism of action of intravesical instillations of Bacille Calmette-Guérin (BCG) as therapy for patients with high risk non-muscle invasive bladder cancer, we showed that BCG can activate a cytotoxic CD56bright NK cell population which efficiently recognized bladder cancer cells. This pioneer immunotherapy provides an invaluable model to understand the role of different immune populations in tumor elimination. However, during the propagation of BCG worldwide a large number of genetically diverse BCG substrains developed. Here, we investigated the capacity of different BCG substrains to promote NK cell activation and confirmed that they were able to activate lymphocytes. Tice, Connaught and Moreau were the substrains with a stronger NK activation effect as measured by CD56 upregulation. Surprisingly, dead mycobacteria also stimulated PBMC cultures and we further demonstrate here that subcellular fractions of BCG-Tice, in the absence of live mycobacteria, could also induce an NK cell response. Lipids from BCG-Tice, but not from Mycobacterium bovis, stimulated NK cell activation and degranulation, whereas the aqueous fraction of either bacteria did not activate lymphocytes. However, delipidated BCG-Tice bacteria were able to activate effector cells (CD3+CD56+ and NK, CD3-CD56+). These data demonstrate that different components of mycobacteria can stimulate different immune subpopulations resulting in phenotypes suitable for cancer elimination.