Mycobacterium tuberculosis promotes genomic instability in macrophages

BACKGROUND Mycobacterium tuberculosis is an intracellular pathogen, which may either block cellular defensive mechanisms and survive inside the host cell or induce cell death. Several studies are still exploring the mechanisms involved in these processes. OBJECTIVES To evaluate the genomic instability of M. tuberculosis-infected macrophages and compare it with that of uninfected macrophages. METHODS We analysed the possible variations in the genomic instability of Mycobacterium-infected macrophages using the DNA breakage detection fluorescence in situ hybridisation (DBD-FISH) technique with a whole human genome DNA probe. FINDINGS Quantitative image analyses showed a significant increase in DNA damage in infected macrophages as compared with uninfected cells. DNA breaks were localised in nuclear membrane blebs, as confirmed with DNA fragmentation assay. Furthermore, a significant increase in micronuclei and nuclear abnormalities were observed in infected macrophages versus uninfected cells. MAIN CONCLUSIONS Genomic instability occurs during mycobacterial infection and these data may be seminal for future research on host cell DNA damage in M. tuberculosis infection.

Bacterial immunostat: mycobacterium tuberculosis lipids and their role in the host immune response

Abstract: The lipid-rich cell wall of Mycobacterium tuberculosis is a dynamic structure that is involved in the regulation of the transport of nutrients, toxic host-cell effector molecules, and anti-tuberculosis drugs. It is therefore postulated to contribute to the long-term bacterial survival in an infected human host. Accumulating evidence suggests that M. tuberculosis remodels the lipid composition of the cell wall as an adaptive mechanism against host-imposed stress. Some of these lipid species (trehalose dimycolate, diacylated sulphoglycolipid, and mannan-based lipoglycans) trigger an immunopathologic response, whereas others (phthiocerol dimycocerosate, mycolic acids, sulpholipid-1, and di-and polyacyltrehalose) appear to dampen the immune responses. These lipids appear to be coordinately expressed in the cell wall of M. tuberculosis during different phases of infection, ultimately determining the clinical fate of the infection. This review summarizes the current state of knowledge on the metabolism, transport, and homeostatic or immunostatic regulation of the cell wall lipids, and their orchestrated interaction with host immune responses that results in bacterial clearance, persistence, or tuberculosis.

Evaluation of the role of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer of epithelial cells

Abstract This study was undertaken in order to assess the involvement of Mycobacterium tuberculosis pili (MTP) as an adhesin, invasin, and cytokine inducer in the M. tuberculosis-epithelial cell interaction. A MTP-deficient strain of M. tuberculosis demonstrated a significant reduction of 69.39% (p = 0.047) and 56.20% (p = 0.033) in its ability to adhere to and invade A549 pulmonary epithelial cells, respectively, in comparison with the wild-type strain. Complementation of the MTP-deficient mutant restored its adhesion and invasion capacity back to the wild-type levels. Overall, it was found that similar concentrations of IL-1β, IL-4, IL-6, IL-8, G-CSF, IFN-γ, MCP-1, and TNF-α were induced in A549 cells infected with the MTP-proficient and MTP-deficient strains. However, at 48 h post-infection, the MTP-deficient mutant induced significantly lower levels of TNF-α than the wild-type strain (p = 0.033). Furthermore, at 72 h post-infection, the mutant induced significantly higher levels of IL-8 than the wild-type (p = 0.005). We conclude that MTP is an adhesin/invasin of epithelial cells and, while playing a role in M. tuberculosis entry, they do not appear to largely influence the epithelial cell cytokine response.

Challenging Mycobacterium tuberculosi s dormancy mechanisms and their immunodiagnostic potential

ABSTRACT Mycobacterium tuberculosis is the etiologic agent of tuberculosis, one of the world's greatest cause of morbidity and mortality due to infectious disease. Many evolutionary mechanisms have contributed to its high level of adaptation as a host pathogen. Prior to become dormant, a group of about 50 genes related to metabolic changes are transcribed by the DosR regulon, one of the most complex and important systems of host-pathogen interaction. This genetic mechanism allows the mycobacteria to persist during long time periods, establishing the so-called latent infection. Even in the presence of a competent immune response, the host cannot eliminate the pathogen, only managing to keep it surrounded by an unfavorable microenvironment for its growth. However, conditions such as immunosuppression may reestablish optimal conditions for bacterial growth, culminating in the onset of active disease. The interactions between the pathogen and its host are still not completely elucidated. Nonetheless, many studies are being carried out in order to clarify this complex relationship, thus creating new possibilities for patient approach and laboratory screening.

Exposure to cetyl pyridinium chloride and loss of integrity of cell wall of mycobacteria.

Background: Cetyl pyridinium chloride (CPC) liquefied sputum was shown to reduce AFB smear positivity presumably damaging cell wall of M. tuberculosis. Settings: National Institute for Research in Tuberculosis, Chennai, (Tamil Nadu). Objective: To assess the cell wall damage of mycobacteria in CPC liquefied sputum, by Transmission Electron Microscopy (TEM) and mycobacteriophage adsorption studies. Methods: Pooled sputum sample from smear positive pulmonary TB patients was homogenized and liquefied with CPC. It was examined in TEM daily for four days, to assess cell wall damage of M. tuberculosis, and photomicrographs were taken. M. smegmatis mc2155, treated with CPC, was infected with mycobacteriophage (phAE129) to study phage adsorption on cell wall and plaque formation. CPC untreated sputum and M. smegmatis formed controls. Results: Photomicrographs showed that cell wall of M. tuberculosis was intact in controls and damaged in CPC preserved sputum for 96 hours. Plaque formation was seen and absent respectively in CPC untreated and treated M. smegmatis cells. Conclusion: Exposure to CPC damaged the cell wall of M. tuberculosis within 96 hours. Mycobacteriophage failed to form plaques after M. smegmatis mc2155 was treated with CPC implying inhibition of phage adsorption on damaged cell wall and thus providing a clue for poor staining and smear positivity in microscopy.

Use of the Ogawa-Kudoh method to isolate mycobacteria in a tuberculosis reference laboratory in northwestern Paraná, Brazil

Culturing is the gold standard method for confirming a diagnosis of tuberculosis (TB). The Brazilian Ministry of Health recently proposed the use of the Ogawa-Kudoh method for sputa cultures to detect Mycobacterium tuberculosis. The aim of the present study was to evaluate 8 years of using the Ogawa-Kudoh method in a TB reference laboratory in northwestern Paraná, Brazil. The present study consisted of a retrospective analysis of sputa cultures records for the detection of mycobacteria using the Ogawa-Kudoh method in the Laboratory of Medical Bacteriology, Laboratory of Teaching and Research in Clinical Analysis (LEPAC), State University of Maringá, from July 2003 to September 2011. The following variables were analyzed: Ziehl Neelsen (Z-N) smears and cultures results and the age and gender of the patients. Sputa samples from 3,231 patients with suspected TB were analyzed. Of these, 67.17% were male with an average age of 45.58 years. Of the total number of Z-N-negative samples (n=2,949), 42 (1.42%) were positive for M. tuberculosis (p >0.05). The Ogawa-Kudoh method is an excellent tool for diagnosing pulmonary TB. It is easy to perform, requires less biosafety equipment than the Petroff method, has a low cost, and has good sensitivity for detecting of M. tuberculosis.

A cultura é o método padrão ouro para confirmação da tuberculose (TB). O Ministério da Saúde Brasileiro propôs, recentemente, a utilização do método de Ogawa-Kudoh para cultura de escarro na detecção de Mycobacterium tuberculosis. O objetivo deste estudo foi avaliar oito anos de utilização do método de Ogawa-Kudoh na rotina de um laboratório de referência na região noroeste do Paraná, Brasil. Realizou-se estudo retrospectivo dos registros das culturas de escarro para a detecção de micobactérias, usando o método Ogawa-Kudoh conduzido no Laboratório de Bacteriologia Médica, Laboratório de ensino e pesquisa em Análises Clínicas (LEPAC) da Universidade Estadual de Maringá (UEM), de Julho de 2003 a Setembro de 2011. As seguintes variáveis foram analisadas: esfregaço Ziehl Neelsen (Z-N), cultura, idade e sexo do paciente. Analisaram-se 3.231 amostras de escarro de pacientes com suspeita de tuberculose. Destes, 67,17% eram do sexo masculino com idade média de 45,58 anos. Do total de amostras Z-N negativas (n=2.949), 42 amostras (42/2949, 1,42%) apresentaram cultura positiva para M. tuberculosis (p>0,05). A utilização do método Ogawa-Kudoh representa excelente ferramenta para o diagnóstico precoce da TB pulmonar. É de fácil execução, requer menos equipamentos de biossegurança do que o método de Petroff, apresenta baixo custo e boa sensibilidade para detecção de M. tuberculosis.

Tuberculosis: una dinámica continua entre el pasado y el presente, para imaginar el futuro / Tuberculosis: steady dynamics between past and present to imagine the future

Progress in understanding the biological processes that allow Mycobacterium tuberculosis to be a successful parasite have accelerated in the last twenty years. This progress has been stimulated by the return of tuberculosis (TB) as an important disease in industrialized countries, by its increase in emergent nations in the tail of population increases and poverty and by the spread of multiple drug resistant (MDR) and extensively drug resistant (XDR) M. tuberculosis as a result of treatment failures. Progress on M. tuberculosis biology has also been fueled by advances in microbiology and molecular biology, including molecular genetics, genomics, proteomics and in vitro and in vivo models of infection. The study of latency or dormancy, a phenomenon central to understanding the persistence of M. tuberculosis and the development of TB in individuals, its spread in human populations and the emergence of antibiotic-resistant/tolerant organisms, has been preferred targets for investigators in this area. In this manner, factors that trigger M. tuberculosis latency (e. g, hypoxia, nutrient starvation, NO exposure) have been characterized and the metabolic shifts to host lipid utilization, tolerance to antimicrobials and resistance to host immune mechanisms involved in latency have been determined. Similarly, genetic changes and the resulting antimicrobial mechanisms mediating the MDR and XDR states have been characterized and potential new vaccines that avoid reactivation from latency and infection are being developed. Despite this progress, and given the fact that effective anti tuberculosis therapy was developed and first introduced clinically at the end of the 1940s, there are now more cases of latent and active TB worldwide than ever before. This reinforces the concept of TB as a bacterial disease with strong social and economical! determinants which are presently stimulating increased transmission in many human groups, undermining diagnostics, treatment and prevention. It suggests that in a scenario of global economical crisis the struggle against TB will be weakened, unless efforts are included to alleviate poverty, decrease economic inequality, improve public health and allow democracy and political organization.

Aspectos biológicos, clínicos y epidemiológicos de la tuberculosis latente: [revision] / Biological, clinical and epidemiological aspects of latent tuberculosis: [review]

Mycobacterium tuberculosis afecta a la humanidad desde hace más de 20 000 años. Su morbimortalidad es elevada, por lo que repercute económicamente en los países en desarrollo. La infección latente, caracterizada por la presencia de bacilos vivos en tejidos del huésped, con ausencia de signos y síntomas clínicos, es una característica de esta enfermedad, ya que la micobacteria puede adaptar su metabolismo para mantenerse viva con baja o nula replicación, dificultando su eliminación de los tejidos por los fármacos antituberculosos y permaneciendo inadvertida al reconocimiento y eliminación por el sistema inmunológico. Varias son las interrogantes de esta forma de tuberculosis (TB): la falta de conocimiento del metabolismo del bacilo en estado durmiente, su relación con la inmunidad del hospedero y la identificación de antígenos como marcadores diagnósticos de infección subclínica durante la latencia. Este artículo resume los aspectos biológicos, clínicos y epidemiológicos más importantes de esta forma de tuberculosis.

Mycobacterium tuberculosis, the causal agent of tuberculosis, has affected humankind for approximately 20 000 years. Tuberculosis is a devastating disease, particularly in developing countries. One of its most notable characteristics is latent infection, in which live bacilli persist in the host tissues without clinical manifestations. Thus, the tuberculous bacilli adapt their metabolism to remain viable with low or no replication, avoiding their elimination by the immune system or conventional chemotherapy. Among the several problems that are particularly important to the understanding of this form of tuberculosis, and are not well-known, are the key metabolic steps that allow mycobacteria to remain in a dormant state and its interaction with host immunity. This article reviews some of the most significant biological, clinical and epidemiological aspects of this form of tuberculosis.

Comparative growth pattern of multi drug resistance versus susceptible isolates of Mycobacterium tuberculosis in mice lungs.

Background & objectives: Rise in prevalence of multi-drug resistance (MDR) in tubercle bacilli is a serious cause of concern. As mutations with two house keeping genes rpoB and katG are associated with resistance to two important anti-tubercular drugs rifampicin and isoniazid respectively, there is a need to understand the growth kinetics of organisms with such mutated genes in experimental animals. This study was undertaken to study the growth kinetics of susceptible as well multi-drug resistance Mycobacterium tuberculosis isolates in mice. Methods: Two MDR (having mutations in rpoB and catG) and two drug susceptible isolates of M. tuberculosis along with H37Rv were grown in mice after aerogenic infection. Results: The MDR isolates grew slowly up to 3 wk though the growth was significantly different from sensitive strains. However, after 3 wk, the growth in sensitive as well MDR strains was similar, suggesting that even the mutations in the MDR strains did not have any impact on the growth kinetics. Interpretation & conclusions: The effect of mutations in other parts of these genes need to be studied. Retention of property of MDR strains to establish infection after aerogenic infection has epidemiological significance in terms of the transmission of MDR tuberculosis.

Patterns of tuberculosis in the Americas: how can modern biomedicine inform the ancient past?

Tuberculosis (TB) is an infectious disease that continues to take its toll on human lives. Paleopathological research indicates that it has been a significant cause of death among humans for at least five thousand years. Because of the devastating consequences to human health, social systems, and endangered primate species, TB has been the subject of many and varied research efforts throughout the world, efforts that are amassing an enormous amount of data concerning the causative agent Mycobacterium tuberculosis. Despite sequencing of the M. tuberculosis genome and numerous molecular epidemiological studies, many questions remain regarding the origin, evolution, and future co-evolutionary trajectory of M. tuberculosis and humans. Indeed, the origin of pre-Columbian New World TB has been and remains hotly debated, and resolution of this controversy will likely only come with integration of data and theory from multiple disciplines. In this paper, we discuss the pre-Columbian TB controversy, and then use research from biological and biomedical sciences to help inform paleopathological and archaeological studies of this ubiquitous disease that plagued our ancient forbears.

Nuevas herramientas para la detección de la tuberculosis latente

La tuberculosis es un problema serio de salud pública en el mundo, especialmente en países en vía de desarrollo, inclusive Colombia. En nuestro país estamos enfocados en el manejo clínico de los pacientes con tuberculosis activa, pero no hay campañas efectivas que identifiquen y provean terapia a los individuos con las formas latentes de la infección y con alto riesgo de progresar hacia la enfermedad. En esta revisión se plantea la importancia de realizar dichas campañas para evitar la diseminación de la infección en la comunidad. Esto incluye la búsqueda activa y el tratamiento profiláctico de los contactos de casos recientes, así como la de individuos con tuberculosis latente con alto riesgo de desarrollar la enfermedad. En ausencia de una prueba de oro para detectar la tuberculosis latente, la prueba cutánea de la tuberculina se ha utilizado por más de 100 años para dicho fin, a pesar de sus limitaciones de sensibilidad y especificidad. En esta revisión se evalúan las ventajas y desventajas de una nueva generación de inmunoensayos que incluye la prueba comercial Quantiferon y el desarrollo experimental del ELISPOT. Ambas se basan en la detección de IFN ©secretado por linfocitos de sangre periférica cuando se incuban con antígenos específicos del bacilo tuberculoso. Finalmente, se plantea la importancia de desarrollar pruebas moleculares enfocadas en detectar el ADN de la micobacteria como posible complemento a los inmunoensayos descritos.

New tools for detection of latent tuberculosis Tuberculosis is a serious public health problem worldwide, particularly in developing countries. In Colombia, the focus is on the clinical management of patients with active disease, but not on preventive programs that identify and treat individuals with a latent tuberculosis infection. This review emphasized the importance of preventative programs and their critical role in the curtailment of infection dissemination in the community. An effective program includes chemoprophylactic treatment of household contacts and detection of individuals with latent tuberculosis infection and with high risk of reactivation of disease. The tuberculin skin test has been used effectively for more than 100 years, despite inherent sensitivity and specificity limitations. Herein the advances provided by a new generation of immunoassays are reviewed, includingthe commercially-available Quantiferon and the experimental development of ELISPOT. Both are based on the detection of IFN....secretion by peripheral T cells upon incubation with Mycobacterium tuberculosis antigens. Finally, the importance of molecular techniques aimed at detecting DNA from the mycobacterium is discussed as a possible complement to the described immunoassays. New tools for detection of latent tuberculosis Tuberculosis is a serious public health problem worldwide, particularly in developing countries. In Colombia, the focus is on the clinical management of patients with active disease, but not on preventive programs that identify and treat individuals with a latent tuberculosis infection. This review emphasized the importance of preventative programs and their critical role in the curtailment of infection dissemination in the community. An effective program includes chemoprophylactic treatment of household contacts and detection of individuals with latent tuberculosis infection and with high risk of reactivation of disease. The tuberculin skin test has been used effectively for more than 100 years, despite inherent sensitivity and specificity limitations. Herein the advances provided by a new generation of immunoassays are reviewed, includingthe commercially-available Quantiferon and the experimental development of ELISPOT. Both are based on the detection of IFN....secretion by peripheral T cells upon incubation with Mycobacterium tuberculosis antigens. Finally, the importance of molecular techniques aimed at detecting DNA from the mycobacterium is discussed as a possible complement to the described immunoassays.

Drug-resistant tuberculosis among prisoners of three prisons in Bangkok and the vicinity.

The purpose of this study was to determine the prevalence of drug-resistant tuberculosis and some factors associated with drug resistance among prisoners of three prisons in Bangkok and the vicinity. Susceptibility testing to four first-line antituberculous drugs was performed on 165 M. tuberculosis strains isolated from prisoners of three prisons including Klongprem Central (KC) prison, Bangkwang Central (BC) prison and the Correctional Institution (CI) for Male Drug Addicts. Of 165 smear positive tuberculosis (TB) cases with drugs susceptibility results, resistance to one or more drugs was 49.7 per cent. Resistance to one, two, three, and four drugs was 20.0, 13.3, 4.2 and 12.1 per cent, respectively. Multidrug resistant tuberculosis (MDR-TB) was 18.8 per cent. Patients classified as primary and acquired drug resistant were 6.7 and 50.0 per cent. The primary drug resistance to one or more drugs among prisoners at KC, BC and CI were 42.5, 36.4 and 53.9 per cent, respectively and MDR-TB were 8.2, 3.0, and 7.7 per cent, respectively. Of several factors analyzed in the present study, only a history of previous TB treatment was significantly associated with drug resistance (p < 0.05). In conclusion, the results indicate the high prevalence of drug-resistant tuberculosis and the seriousness of the TB problem in prisons. The public health sector and prison authorities should work in close collaboration and co-ordination to continue improving TB case detection. Directly Observed Treatment Short course (DOTS) is highly recommended. Moreover, discharged prisoners with tuberculosis should be appropriately referred to hospitals or TB control centers.

Understanding the phenomenon of persistence in mycobacterial infections.

Persistence of live organisms despite chemotherapy for long periods is a significant problem in both leprosy and tuberculosis. The consequence of this persistence is varying rates of relapses which undermine the success of treatment. The mechanisms of the dormancy are ill-understood, and as explanation a switch over to alternate modes of metabolism such as glyoxylate bypass and other shunts has been suggested. This presentation reviews the information available on this aspect. In-depth studies by designing and investigating model system(s) using molecular genetic approaches may help in gaining better understanding of the mechanisms of dormancy and persistence in mycobacterial infections and devising appropriate strategies and tools for the better management of these complications.

New vaccines against tuberculosis

It has proved difficult to vaccinate effectively against tuberculosis with mycobacterial components or even with whole dead mycobacteria;protection was always inferior to that obtained with the live attenuated vaccine known as bacillus Calmette-Guérin (BCG). We have found that this may no longer be the case. Expression of the gene for a single mycobacterial antigen (Mycobacterium leprae hsp 65) in adult BALB/c mice resulted in substantial cell-mediated protection agains challenge with M. tuberculosis, but only when it was generated as an endogenous antigen within antigen-presenting cells. CD4 and CD8 T cells cloned from spleens of immunized mice passively transferred protection to non-immunized mice, and CD8 cells selectively lysed macrophages infected with M. tuberculosis. The ability of the clones to protect recipient mice against challenge infection was most strongly associated with specific cytotoxic capacity and secondarily with IFN-gamma production. Three modes of expressing the gene have been tested: a)expression froma retroviral vector (pZIPNeoSV) in implanted J774 tumor cells, b)expression from the same vector via bone marrow cells transfected in vitro and used to reconstitute irradiated mice, and c)in a preliminary experiment, from cytomegalovirus (CMV) immediate-early and hydroxymethylglutaryl Co-A reductase promotors injected as plasmid DNA into muscle.