MicrobiologyBytes

Smallpox may have been eradicated 35 years ago, but we are still battling many other major global health scourges. Malaria, for example, kills some 1.2 million people every year, and recent cholera epidemics in Zimbabwe, Somalia and Haiti have killed thousands. The death toll from HIV/AIDS is even higher, with almost 2 million deaths last year, but new drugs and health care delivery mechanisms mean that this number is falling.

But there’s one major disease where the battle is still being badly lost – tuberculosis. Mycobacterium tuberculosis still kills nearly 2 million people in the developing world every year. Even more alarmingly, in 2005 researchers in the province of KwaZulu-Natal in South Africa spotted an outbreak of a strain of M. tuberculosis that was resistant to the four key classes of drugs used to treat the disease. These super drug-resistant bugs have now been found in 58 countries, their spread fuelled by the lethal combination of HIV and TB. The mortality rate worldwide for the victims of this extensively drug resistant TB (XDR-TB) is more than 80%, making diagnosis almost a death sentence.

The scientific and medical challenges are huge. We don’t have an effective vaccine. We lack diagnostics and biomarkers. The current drug regimen – multiple drugs that must be taken reliably for six months – is virtually impossible to administer successfully in the developing world, where rural farmers may have to walk three hours to the nearest clinic. Too often, treatment simply leads to the development of drug resistance. And we have only just begun to study the bacterium’s biology and to learn how it manipulates the human immune system.

To understand TB and its deadly synergy with HIV, and to develop new diagnostics and treatments, we need more research. But the current research model could use some help. This article describes a new approach that involves bringing world-leading basic research to the epicenter of epidemics, rather than trying to fight diseases from laboratories at universities or government agencies thousands of miles away.

Point of view: Basic research at the epicenter of an epidemic. (2013) eLife 2: e00639 doi: http://dx.doi.org/10.7554/eLife.00639

A new tuberculosis vaccine, MVA85A, has recently been found to be less effective than initially thought, prompting widespread consternation in the press. The excellent NHS Choices website reports that “although the stories are based on solid science, the news is actually less worrying than the headlines suggest”. MVA85A is a booster vaccine that researchers hope might help improve the effectiveness of the existing BCG vaccine. Although the BCG is effective in the UK, new vaccines and boosters are needed as it is less effective in other countries. The effectiveness of BCG against tuberculosis is variable and has been found to be less effective in countries such as South Africa, where as many as 1% of the population has TB. An effective booster vaccine would therefore be useful. Although a recent study found the new vaccine is safe, it does not appear to have performed better than the placebo in children who had already had the BCG vaccine. Despite this setback, several further lines of investigation are being pursued by researchers, who now want to look at whether the MVA85A vaccine might work better in other sub-populations, and whether it might improve protection against pulmonary tuberculosis (lung infection) in people who have HIV, for example.

NHS Choices: Disappointing results for new TB vaccine

Another paper just published has found that whole genome sequencing of Mycobacterium tuberculosis provides more accurate information on clustering and longitudinal spread of the pathogen than the standard test (classical genotyping). Importantly, whole genome sequencing revealed that first outbreak isolates were falsely clustered by classical genotyping and do not belong to one recent transmission chain. By using whole genome sequencing, the authors estimated that the genetic material of M. tuberculosis evolved at a rate at 0.4 mutations per genome per year, suggesting that the bacterium grows in its natural host (infected people) with a doubling time of 22 hours, or 400 generations per year. This finding about the evolution of M. tuberculosis indicates how information from whole genome sequencing can be used to help trace future outbreaks. Importantly, as the costs of whole genome sequencing are declining, this test could soon become the standard method for identifying transmission patterns and rates of infectious disease outbreaks.

Whole Genome Sequencing versus Traditional Genotyping for Investigation of a Mycobacterium tuberculosis Outbreak: A Longitudinal Molecular Epidemiological Study. (2013) PLoS Med 10(2): e1001387. doi:10.1371/journal.pmed.1001387

The world is starting to win the war against tuberculosis, but drug-resistant forms pose a new threat.
Nature News: http://goo.gl/Ua9c8

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Tuberculosis (TB) and HIV co-infections place an immense burden on health care systems and pose particular diagnostic and therapeutic challenges. Infection with HIV is the most powerful known risk factor predisposing for Mycobacterium tuberculosis infection and progression to active disease, which increases the risk of latent TB reactivation 20-fold. TB is also the most common cause of AIDS-related death. Thus, M. tuberculosis and HIV act in synergy, accelerating the decline of immunological functions and leading to subsequent death if untreated. The mechanisms behind the breakdown of the immune defense of the co-infected individual are not well known. The aim of this review is to highlight immunological events that may accelerate the development of one of the two diseases in the presence of the co-infecting organism.

Tuberculosis and HIV Co-Infection. (2012) PLoS Pathog 8(2): e1002464. doi:10.1371/journal.ppat.1002464

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Mycobacterium tuberculosis is one of the most life-threatening pathogens of all time. Despite the development of vaccines and antibiotics, this pathogen is still a major public health problem. The HIV epidemic has also had an important impact on the rise of M. tuberculosis infections since immunodeficient people are highly susceptible. Commonly, M. tuberculosis has been thought to reside in a membrane-bound compartment within its host cells during the entire infection cycle from invasion to cell death.

Using a fluorescence-based method, new research provides evidence that M. tuberculosis is able to rupture its membrane-bound compartment and gain access to the host cytosol, where it can cause cell death. Importantly, the researchers were able to track the dynamics of infection to understand the consequences of M. tuberculosis phagosomal rupture. This revealed that phagosomal rupture results in cell toxicity and host cell death involving necrosis. Together, this data provides a new angle in the worldwide fight against tuberculosis and could lead to new approaches in the development of innovative treatments.

Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death. (2012) PLoS Pathog 8(2): e1002507. doi:10.1371/journal.ppat.1002507
Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisΔRD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective.

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Sudan is a large country with a diverse population and history of civil conflict. Poverty levels are high with a gross national income per capita of less than two thousand dollars. The country has a high burden of tuberculosis (TB) with an estimated 50,000 incident cases during 2009, when the estimated prevalence was 209 cases per 100,000 of the population. Few studies have been undertaken on TB in Sudan and the prevalence of drug resistant disease is not known.

In this study Mycobacterium tuberculosis isolates from 235 patients attending three treatment centers in Sudan were screened for susceptibility to isoniazid, rifampicin, ethambutol and streptomycin by the proportion method on Lowenstein Jensen media. 232 isolates were also genotyped by spoligotyping. Demographic details of patients were recorded using a structured questionnaire. Statistical analyses were conducted to examine the associations between drug resistance with risk ratios computed for a set of risk factors (gender, age, case status – new or relapse, geographic origin of the patient, spoligotype, number of people per room, marital status and type of housing).

Multi drug-resistant tuberculosis (MDR-TB), being resistance to at least rifampicin and isoniazid, was found in 5% of new cases and 24% of previously treated patients. Drug resistance was associated with previous treatment with risk ratios of 3.51 for resistance to any drug and 5.23 for MDR-TB. Resistance was also associated with the geographic region of origin of the patient, being most frequently observed in patients from the Northern region and least in the Eastern region with risk ratios of 7.43 and 14.09 for resistance to any drug and MDR-TB.

“We conclude that emergence of drug resistant tuberculosis has the potential to be a serious public health problem in Sudan and that strengthened tuberculosis control and improved monitoring of therapy is needed. Further surveillance is required to fully ascertain the extent of the problem.”

Tuberculosis in Sudan: a study of Mycobacterium tuberculosis strain genotype and susceptibility to anti-tuberculosis drugs. BMC Infectious Diseases 11:219 2011

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Before the genomic era there was already a longstanding interest in understanding the origins of bacterial pathogens and the molecular attributes of virulence. Large-scale genome sequencing has provided a rapid and unbiased means of uncovering the evolution of many pathogens, contributing to both fundamental microbiological insights and the development of new disease-control strategies. For these reasons, the evolution of one of the most devastating human pathogens, Mycobacterium tuberculosis, has captivated researchers since its discovery in 1882. This interest was stimulated not only by the epidemiologic importance of the pathogen but also by the lack of consensus on its origins and its apparent exception to the stereotypes of bacterial evolution (e.g. acquisition of pathogenicity islands). So where did M. tuberculosis come from?

The rise and fall of the Mycobacterium tuberculosis genome. Trends Microbiol. 2011 19(4): 156-161
When studied from the perspective of non-tuberculous mycobacteria (NTM) it is apparent that Mycobacterium tuberculosis has undergone a biphasic evolutionary process involving genome expansion (gene acquisition and duplication) and reductive evolution (deletions). This scheme can instruct descriptive and experimental studies that determine the importance of ancestral events (including horizontal gene transfer) in shaping the present-day pathogen. For example, heterologous complementation in an NTM can test the functional importance of M. tuberculosis-specific genetic insertions. An appreciation of both phases of M. tuberculosis evolution is expected to improve our fundamental understanding of its pathogenicity and facilitate the evaluation of novel diagnostics and vaccines.

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Despite the availability of antibiotics that rapidly kill bacteria in vitro, the treatment of chronic bacterial infections, such as tuberculosis, requires long-term drug therapy. The reasons for this are unclear, but many have hypothesized that the slow replication and concomitantly low metabolic rate of bacteria in the host environment produce an “antibiotic-tolerant” state. Researchers tested this hypothesis by identifying the bacterial pathways responsible for slowing the growth and metabolism of Mycobacterium tuberculosis in response to stress. They found that diverse growth-limiting stresses trigger a common signal transduction pathway that slows bacterial growth by redirecting cellular carbon fluxes away from central metabolic pathways and towards storage. Disruption of this metabolic switch increased the antibiotic sensitivity of the bacterium during infection, verifying that this response significantly contributes to antibiotic tolerance and suggesting new strategies for accelerating therapy.

Metabolic Regulation of Mycobacterial Growth and Antibiotic Sensitivity. (2011) PLoS Biol 9(5): e1001065. doi:10.1371/journal.pbio.1001065
Treatment of chronic bacterial infections, such as tuberculosis (TB), requires a remarkably long course of therapy, despite the availability of drugs that are rapidly bacteriocidal in vitro. This observation has long been attributed to the presence of bacterial populations in the host that are “drug-tolerant” because of their slow replication and low rate of metabolism. However, both the physiologic state of these hypothetical drug-tolerant populations and the bacterial pathways that regulate growth and metabolism in vivo remain obscure. Here we demonstrate that diverse growth-limiting stresses trigger a common signal transduction pathway in Mycobacterium tuberculosis that leads to the induction of triglyceride synthesis. This pathway plays a causal role in reducing growth and antibiotic efficacy by redirecting cellular carbon fluxes away from the tricarboxylic acid cycle. Mutants in which this metabolic switch is disrupted are unable to arrest their growth in response to stress and remain sensitive to antibiotics during infection. Thus, this regulatory pathway contributes to antibiotic tolerance in vivo, and its modulation may represent a novel strategy for accelerating TB treatment.

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UK scientists had an important role to play in the development of the first antibiotics for the treatment of tuberculosis in the mid-20th century. As we enter the second decade of the 21st century, the world is now confronted with the appearance of extremely drug-resistant strains. In this article in Microbiology Today (pdf) Stephen Gillespie asks what are UK scientists doing this time to help combat this serious threat?

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