A tuberculose é considerada um problema emergente de saúde pública. Este estudo tem como objetivo avaliar a associação da patogenicidade/virulência e propriedades imunomoduladoras de isolados clínicos de Mbv (cepas B2 e MP287/03) e Mtb (cepa Beijing 1471), e da cepa de Mtb H37Rv, como referência de virulência. Os isolados, MP287/03 e Beijing 1471, apresentaram maior virulência em relação às demais cepas, levando os camundongos à morte ainda na fase aguda de infecção. Foi verificada baixa produção de mediadores pró-inflamatórios nos animais infectados com o isolado MP287/03, enquanto nos infectados com o isolado Beijing 1471 os níveis destes mediadores foram exacerbados. O desbalanço na produção destes mediadores pode ter contribuído para morte precoce dos animais. Baseado nesse estudo, nós podemos concluir que as propriedades que conferem hipervirulência aos isolados clínicos de Mbv e Mtb estão principalmente relacionadas à alta capacidade de crescimento intracelular das bactérias, que parece ser pouco alterada pela presença de citocinas pró-inflamatórias. Sendo assim, as infecções por isolados hipervirulentos podem acarretar consequências semelhantes, mesmo quando associadas a diferentes padrões de modulação da resposta imune. / Tuberculosis is an emergent problem of public health. This study aimed to evaluate the association between pathogenicity/virulence and immunemodulatory ability of Mbv (B2 and MP287/03) and Mtb (Beijing 1471) clinical isolates, using H37Rv strain as reference of virulence. The virulence was assessed in C57BL/6 mice infected with a low dose of bacilli (~100 bacteria) via intratracheal route. MP287/03 and Beijing 1471 isolates showed higher virulence than all others strains, leading to mice death during the acute phase. It was verified low production of pro-inflammatory mediators in mice infected by MP287/03 bacteria, whereas in mice infected by Beijing 1471 bacteria were observed exacerbated levels of pro-inflammatory mediators. The disbalance of these mediators may have contributed to the early mouse death. Based on this study, we concluded that the properties that confer hypervirulence to Mbv and Mtb clinical isolates are primarily related to the high intracellular growth capacity of the bacteria, which seems to be marginally affected by the presence of pro-inflammatory cytokines. Therefore, the infection by hypervirulent isolates can lead to similar outcomes, even when associated to different patterns of modulation of the immune response.

No description available.

For centuries medicinal plants have been used all over the world for the treatment and prevention of various ailments, particularly in developing countries where infectious diseases are endemic and modern health facilities and services are inadequate. In recent years the use of and search for drugs derived from plants have been accelerated. Ethnopharmacologists, botanists, microbiologists, and natural-product chemists are trying to discover phytochemicals and “leads” which could be developed for the treatment of infectious diseases. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimicrobial properties. The evaluation of these plants for biological activity is necessary, both to substantiate their use by communities, and also to discover possible new drug or herbal preparations. Twenty five plants selected through ethno-botanical surveys in Mozambique which are used to treat respiratory diseases, wounds, viruses, stomach ailments and etc., were collected and investigated for antimicrobial activity. Acetone extracts of selected plants were tested for antibacterial, antimycobacterial and anti-HIV-1 activity. Antibacterial activity was evaluated using the agar diffusion method. Five Gram positive (Bacillus cereus, Bacillus pumilus, Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis) and five Gram negative (Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens) bacterial species were used in this study. The extracts of each plant were tested at concentrations ranging from 0.125 to 5.0 mg/ ml. Most of the plant extracts inhibited the growth of the Gram-positive microorganisms. The minimum inhibitory concentration of eight plants (Cassia abbreviata, Elephanthorrhiza elephantina, Hemizygia bracteosa, Hoslundia opposita, Momordica balsamina, Rhoicissus tomentosa and Salvadora australis) against Gram-positive bacteria was found to be 0.5 mg/ml. Gram-positive bacteria were found to be susceptible to extracts of Lippia javanica at concentration of 0.125 mg/ml. Among the 22 acetone extracts tested, two were found to have activity against Gram-negative bacteria at a concentration of 5.0 mg/ml (Adenia gummifera and Momordica balsamina). Rhoicissus revoilli inhibited E. cloacae, a Gram-negative strain, at a concentration of 2.5 mg/ml. To evaluate antimycobacterium activity ten plants species were tested against H37Rv, a drug-sensitive strain of Mycobacterium tuberculosis at concentrations ranging from 0.5 to 5.0 mg/ml using BACTEC radiometric method. Four of the plant species tested (Cassia abbreviata, Hemizigya bracteosa, Lippia javanica and Melia azedarach) were observed to be active against the H37Rv. (ATCC 27294) strain of TB at a concentration of 0.5 mg/ml which was the lowest concentration used in this study. Seventeen plant species, were screened for anti-HIV bioactivity in order to identify their ability to inhibit the enzymes glycohydrolase (á -glucosidase and â- glucuronidase) and eleven species were further tested against Reverse transcriptase. It was found that 8 plant species (Cassia abbreviata, Elephantorrhiza elephantina, Rhoicissus tomentosa, Pseudolachnostylis maprouneifolia, Lippia javanica, Litogyne gariepina, Maerua junceae and Momordica balsamina) showed inhibitory effects against á-glucosidase and â-glucuronidase at a concentration of 200 ìg/ml. The results of the tests revealed that the plant extracts of Melia azedarach and Rhoicissus tomentosa appeared to be active, showing 49 and 40% inhibition of the enzyme activity respectively. Lippia javanica was found to have the best activity exhibiting a minimum inhibitory concentration of 0.125 mg/ml. The extracts also showed positive activity against Mycobacterium tuberculosis at concentration of 0.5 mg/ml and HIV-enzyme glycohydrolase was (á-glucosidase and â-glucuronidase) inhibited by 62 % and 73 % respectively. Considering its medicinal use local for HIV and various infections, it was therefore, selected for identifying its bioactive constituents. In the initial screening of plants used in Mozambique Hoslundia oppositademonstrated good antitubercular activity. It was therefore, selected to identify its bioactive constituents. A Phytochemical investigation of L. javanica led to the isolation of eight compounds, 4-ethyl-nonacosane (1), (E)-2(3)-tagetenone epoxide (2), myrcenone (3), piperitenone (4), apigenin (5), cirsimaritin (6), 6-methoxyluteolin 4'-methyl ether (7), 6-methoxyluteolin and 3',4',7-trimethyl ether (8). Three known compounds, 5,7-dimethoxy-6-methylflavone (9), hoslunddiol (10) and euscaphic acid (11) were isolated from H. opposita. This is the first report of compounds (1), (2), (5-8) from L. javanica and of compound (9) from H. opposita. The compounds were tested against Mycobacterium tuberculosis and HIV-1 reverse transcriptase for bioactivity. It was found that compounds (2), (4) and (9) inhibited the HIV-1 Reverse transcriptase enzyme by 91%, 53% and 52% respectively at 100 ìg/ml. Of all the compounds tested against a drug-sensitive strain of Mycobacterium tuberculosis, euscaphic acid (11) was found to exhibit a minimum inhibitory concentration of 50 ìg/ml against this strain. The present study has validated scientifically the traditional use of L. javanica and H. opposita and a few other Mozambican medicinal plants to some extent. / Thesis (PhD)--University of Pretoria, 2010. / Plant Science / unrestricted

Tuberculosis (TB) has re-emerged as a major worldwide public health hazard with increasing incidence among adults and children. Although cases among children represent a small percentage of all TB cases, they are a reservoir from which many adult cases will arise. Estimates indicate that 9 million people develop TB annually, out of which 1 million (11 percent) occur in children less than 15 years old. Childhood tuberculosis is on the increase worldwide because of persisting inability to conform the diagnosis, leading to a large number of children dying of undiagnosed tuberculosis. Diagnosis of pulmonary tuberculosis has depended on bacteriological examination of sputum. In most of the developing countries sputum smear microscopy has been used as it has been found to be cheap and relative efficient. As a result of the high TB burden, there is an urgent need for improved methods of laboratory diagnosis of TB. This is especially needed in children were diagnosis is more challenging as mycobacteria is being detected in fewer than 50 percent of the cases. Children cannot produce adequate sputum samples for examination. Their sputum samples, if produced, has a low bacterial yield and making detection of mycobacteria by using the smear microscopy difficult. Therefore, gastric lavages from children are being recommended as the best specimen for culture. In this study, gastric lavages from 408 children suspected of having tuberculosis were examined for the recovery of mycobacteria. Recovery was optimized by the use of the relatively new non-radiometric fully automated BACTEC MGIT 960. BACTEC MGIT 960 produced a positivity rate of 27.2 percent against 17.2 percent that of Lowenstein-Jensen (L-J) media, which is a conventional culture method used widely. The direct microscopy which is the cheapest traditional method used in diagnosis of tuberculosis (TB) yielded a 5.6 percent positive rate. The BACTEC MGIT 960 had also a very high isolate detection rate of 98.2 percent compared to that of L-J media of 61.9 percent, and only 20.4 percent were detected with the direct microscopy. On time taken to detection or mean time to detection (TTD) of v isolates, the BACTEC MGIT 960 technique had a shorter mean time to detection, 12.5 days as compared to 34.3 days shown by the L-J media technique. The study showed that children normally get tuberculosis from adult members of the household. A positive TB case was found in the households of 55.4 percent of the suspects. The study has found that 46.4 percent of the children below the age of 4 years developed the disease, compared to 10.5 percent the older children in the age group 10 to 14 years. The study found that tuberculosis in children is mainly caused by Mycobacterium tuberculosis. Out of the 113 isolates detected, 110 (97.3 percent) were M. tuberculosis. The remaining 2.7 percent were the non-tuberculous M. avium complex and M. kansasii. It was inconclusive whether the 2.7 percent of other species were causing tuberculosis and this need to be studied further.

Bovine tuberculosis (BTB) is a chronic infectious disease caused by the bacterium Mycobacterium bovis. It infects animals and can be a source of zoonosis. In the last five years, Canada has faced two outbreaks of bovine tuberculosis in the years 2018 and 2016. BTB is mainly diagnosed using the Tuberculin skin test, a test that detects the cellular immune response to administered purified protein derivative (PPD). A drawback of this test is the high level of false-positive test results caused by the immune response to PPD proteins that are conserved in non-tuberculous mycobacteria. Current serodiagnostic tests can detect the disease especially in the advanced state with, low sensitivity ranging between 9-45%. It is hypothesized that the profiling of the humoral immune responses to selected proteins will lead to the discovery of novel immunogenic protein antigens for improved BTB diagnostic tests. Bioinformatic tools used in this research for the prediction of extracellular or outermembrane proteins from M. bovis genome sequences identified 96 protein candidates. Also, a proteomic study conducted to identify proteins secreted by M. bovis, together with the review of previous proteomic studies of the PPD, led to the identification of an additional 92 protein candidates. A high throughput system was developed to efficiently express and analyze these antigens involving the PCR amplification, in vivo cloning, and in vitro expression of open reading frames (ORFs) coding for selected protein candidates. This was followed by a high throughput recombinant protein purification and a microarray analysis of these purified recombinant proteins with sera from M. bovis-infected animals. The system successfully amplified, cloned, and expressed 159 recombinant proteins. From the microarray screening, 13 antigens exhibited immunological reactions and were able to differentiate the sera of the infected animals from the controls. Out of those 13 antigens, 4 novel antigens Mb2740c, Mb0598c, Mb3469c, and Mb3453c, in addition to two well-known antigens MPB70 and MPB83, gave the highest reactivity; they were selected for further evaluation for diagnostic applications using ELISA and dot blot assays. Two antigens Mb3469c and Mb3453c had a significant ability to differentiate between infected and control cattle as tested in ELISA and dot blot assays, respectively and demonstrated by ROC curve analysis. These two novel antigens could be added to the panel of serodiagnostic antigens for improving BTB serodiagnosis and could be beneficial in the detection of outbreaks caused by certain M. bovis strains.

Memoria para optar al Título Profesional de Médico Veterinario / La tuberculosis bovina (TBB) es una enfermedad crónica y zoonótica, producida por Mycobacterium bovis. El costo de la enfermedad se relaciona a la disminución en la productividad de los animales severamente infectados, al control de movimiento animal, pruebas de diagnóstico para la detección y eliminación de animales positivos para evitar la propagación del agente, los decomisos y el menor valor de la leche. Se considera como una enfermedad ocupacional, ya que las personas que tienen mayor riesgo de contagiarse con M. bovis son quienes pasan largos periodos en contacto cercano con el ganado. La TBB en Chile se encuentra presente, y para apoyar el plan para su control y erradicación, se está estudiando el uso de la vacuna con cepa BCG de M. bovis en el ganado bovino. Fue desarrollada por Calmette y Guérin, quienes informaron la inducción de protección en el ganado contra la exposición a la bacteria. Como esta vacuna corresponde a una cepa atenuada de M. bovis, el objetivo de este trabajo es evaluar la interferencia que tiene su aplicación, con el diagnóstico tradicional de la infección en planteles lecheros de la Región Metropolitana. Para ello, 56 vaquillas fueron vacunadas y 48 fueron inyectadas con NaCl 0,9% a modo de controles. A todas se les tomó muestra de sangre tres y seis meses después de la vacunación las que luego fueron estimuladas con distintos antígenos y se dejaron incubar. Una vez finalizado el tiempo de incubación, se cosecharon los plasmas y finalmente se procedió a realizar el ensayo diagnóstico, ELISA IFN-γ con antígenos DIVA añadidos. Los antígenos DIVA corresponden a antígenos que fueron eliminados de la cepa vacunal. Los resultados demostraron un aumento en la cantidad de individuos falsos positivos al diagnóstico tradicional de TBB en los primeros seis meses después de la vacunación. Esto quiere decir, que fueron positivos a la tuberculina bovina, pero negativos a la prueba DIVA (Detecting Infected among Vaccinated Animals), debido a la reacción con la vacuna. Se concluye que se hace necesario utilizar una prueba complementaria para el diagnóstico de la tuberculosis en el ganado, en este caso, la prueba DIVA, durante los primeros meses después de vacunar con la cepa M. bovis BCG / Bovine tuberculosis is a chronic and zoonotic disease, caused by Mycobacterium bovis. The cost of the disease is associated to the decrease in the productivity of severely infected animals, the restrictions for the animal movement, the diagnostic tests for the detection and elimination of positive animals to avoid the spread of the agent, confiscations and the lower price of milk. It is considered an occupational disease, since people who have the highest risk for M. bovis zoonotic infection are those who spend long time in close contact with livestock. Bovine tuberculosis in Chile is an endemic disease, and the plan for the control and eradication of the disease is being supported by the study of the vaccination with the M. bovis BCG strain in cattle. It was developed by Calmette and Guerin, who reported the induction of protection in cattle against exposure with M. bovis. As this vaccine corresponds to a strain of M. bovis, the objective of this work is to evaluate the interference that has its application, with the traditional diagnosis of infection in dairy farms from the Metropolitan Region. For this, 56 heifers were vaccinated and 48 were injected with NaCl 0,9%. Blood samples were taken at three and six months after the vaccination. Once at the lab, samples were stimulated with different antigens and, after an incubation period, plasmas were harvested and IFN-γ detected by carrying out an ELISA diagnostic assay.-- The results showed an increase in the number of false positive individuals to the traditional diagnosis of bovine tuberculosis in the first six months after vaccination. This means that they were positive to bovine tuberculin, but negative to the DIVA (Detecting Infected among Vaccinated Animals) test, due to the reaction with the vaccine. It is concluded that it is necessary to use a complementary test for the diagnosis of tuberculosis in cattle, in this case, the DIVA test, during the first months after vaccination with the M. bovis BCG strain / Convenio SAG-FAVET

Virulence determinants of M. tuberculosis remain largely unknown. Of key interest has been the ability of the bacterium to survive intracellularly within its host cell, the macrophage, and its ability to cause extensive tissue necrosis. Exported proteases are commonly associated with virulence in bacterial pathogens, yet their role in Mycobacterium tuberculosis has virtually not been studied. Preliminary experiments showed M. tuberculosis culture filtrates contained a proteolytic activity inhibited by mixed serine/cysteine protease inhibitors and activated by Ca²⁺, features typical of some serine proteases, notably subtilisins, and possibly metalloproteases. Purification attempts were unsuccessful. A family of five genes that encode putative, secreted, serine proteases has recently been described in M. tuberculosis. These proteases share 36-47% sequence identity and are all encoded with putative signal peptides, suggesting that they are translocated across the cytoplasmic membrane. One member, mycP1, was selected for further study. The gene product, mycosin-1, was 30-35% identical to bacterial subtilisin-like serine proteases and contained the classic catalytic triad and oxyanion hole. Mycosin-1 also contained a typical signal peptide, a likely propeptide, and a Cterminal hydrophobic sequence with a high transmembrane potential. Topology analyses predicted mycosin-1 to be a type I ectoprotein. Consistent with this, expression of mycosin-1 in M. tuberculosis and in Mycobacterium smegmatis transformed with mycP1 (M. smegmatis-P1) was limited strictly to the cell envelope, as seen by Western blotting, and immunogold electron microscopy. Only full-length, 50-kDa mycosin-1 was observed by Western blotting in broth-grown M. tuberculosis and M. smegmatis-P1 lysates, whereas a 40-kDa species was detected in 6-week M. tuberculosis culture filtrates. A similar 40-kDa immunoreactive band was also observed in lysates of macrophages infected with M. tuberculosis, consistent with robust transcription of the mycP 1 gene during growth in macrophages. Since putative mature mycosin-1 has a molecular weight of 38.6 kDa, the 40-kDa protein may represent activated mycosin-1 after propeptide cleavage. In conclusion, mycosin-1 is an exported, cell envelopeassociated subtilisin homolog that is expressed during growth of M. tuberculosis in vitro and in macrophages.

Includes bibliographical references / Tuberculosis (TB) is one of the major infectious diseases and epidemics in the world. It is responsible for severe morbidity and mortality rates, especially in poor and resource-deficient countries. According to the World Health Organization 2014 report, about one third of the world's population is infected with tuberculosis and about 10-15% is co-infected with HIV, which further complicates the TB epidemic. Tuberculosis claims 2-3 million lives every year and is one of the biggest social and financial burdens on many countries. The disease is treatable but has been hampered by the emergence of drug resistance in the causative bacterium, Mycobacterium tuberculosis (Mtb). Resistant strains of Mtb counter the efficacy of various anti-TB drugs via mechanisms that help it overcome the toxic and inhibitory effects of these drugs. These mechanisms include mutation, enzymatic drug degradation, target modification and drug efflux. Drug efflux by efflux pumps (EPs) is one of the major mechanisms responsible for the development of drug tolerance leading to the emergence of drug resistance. These efflux pumps are regulated by the house keeping proteins present in the cell membrane of Mtb and perform a pre-existing role of rescuing the Mtb from toxic agents. These EPs extrude structurally unrelated compounds from the cell including anti-TB drugs and reduce the drug concentration to sub-inhibitory levels and aid Mtb in developing resistance. Therefore, development of antimycobacterials that target EPs and reduce their activity can be a viable strategy to reduce the global TB burden and counter the emergence of resistance. Many strategies have been used to counter the EP-mediated resistance in Mtb. In this study, two strategies were employed: (i) the development of efflux pump inhibitors (EPIs) via structural modification of a known efflux pump inhibitor, verapamil (VER), and the development of hybrid efflux pump inhibitors (HEPIs) incorporating a VER motif; and (ii) the development of antimycobacterial agents based on covalent linking or attachment of efflux pump inhibitor moieties to an anti-TB drug. These agents are termed reversed anti-TB agents and are based on isoniazid for this study.

Mycobacterium tuberculosis (Mtb) is the leading cause of death from an infectious disease. The success of the pathogen lies in its ability to subvert hostile intracellular macrophage environment. We performed genome-wide transcriptional deep sequencing on total RNA in murine bone marrow-derived macrophages (BMDM) infected with hypervirulent Beijing strain (HN878) in an extensive time kinetic manner using single molecule sequencer and cap analysis gene expression (CAGE) technique. CAGE analysis revealed nearly 36000 unique RNA transcripts with approximately 16000 are not unannotated to a specific gene. This thesis addressed global changes in RNA expression levels in macrophages infected with Mtb in a time kinetic manner to pinpoint novel host protection and immune evasion genes and elucidate the role of these genes in vitro macrophage assays and in vivo knockout mouse studies. The data in this thesis showed that basic leucine zipper transcription factor 2 (Batf2) was an important factor that regulates inflammatory responses in Mtb infection. Deletion of Batf2 led to the survival of mice with reduced lung inflammation and histopathology due to reduced recruitment of inflammatory macrophages. We also showed that Batf2 was highly expressed in peripheral blood from adolescents who progressed from infection to tuberculosis disease and a predictive human biomarker for tuberculosis disease. In contrast to Batf2, we showed that Protein Kinase C-delta (PKC-δ) deficient mice are highly susceptible to tuberculosis and human lung proteomics dataset revealed that PKC-δ was highly upregulated in the necrotic and cavitory regions of human granulomas in multi-drug resistant subjects. PKC-δ deficient mice had a significant reduction in alveolar macrophages and dendritic cells, reduced accumulation of lipid bodies and serum fatty acids. In vitro experiments showed that PKCδ was required for optimal killing effector functions which were independent of phagosome maturation and autophagy in primary murine macrophages. Our studies suggested that these novel genes play a role in the immune response to Mtb and should be studied more thoroughly to evaluate their potential in possible TB interventions.

<p><i>Mycobacterium abscessus </i>is a nontuberculous mycobacterium found in the environment that is becoming an emerging infectious pathogen capable of causing numerous types of infections. It is more antibiotic resistant than <i>Mycobacterium tuberculosis</i> and is becoming more prevalent in developed nations. Current treatments are not standardized and have poor success records and there is no definitive method in properly treating these infections. We tested an <i>in vitro </i>model that mimics the reducing environment that <i>M. abscessus</i> experiences during an infection by subjecting it to thiol-reductive stress. We observed that thiol reductive stress stabilized biofilms formed by <i>M. abscessus</i>. We found that <i>M. abscessus </i>in biofilms became tolerant to the antibiotics: clarithromycin, amikacin, and streptomycin. We postulated that efflux pumps might be involved in transport of the precursors of lipids associated with biofilm formation. Therefore, we investigated whether the efflux pump inhibitors affected biofilm formation and found that verapamil, CCCP and reserpine inhibited the formation of biofilms. We investigated the biosynthesis of lipids during biofilm formation by metabolic radiolabeling with ¹⁴C-acetate, the building block of fatty acids. We found that the biosynthesis of several phospholipids were elevated during biofilm formation and that the efflux pump inhibitor verapamil and exogenous fatty acids inhibited their biosynthesis. Further studies are needed to understand the roles of these lipids in biofilm formation.</p>