MicrobiologyBytes

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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I’m trying to persuade a student to do my final year project next year on badger culling and bovine tuberculosis:

Bovine tuberculosis (bTB) remains an important public health concern worldwide as a result of deficiencies in preventing and/or controlling measures targeting the spread of its causative agent Mycobacterium bovis. While the risk posed by M. bovis to human health is low in most developed countries, the main causes of concern related to M. bovis in industrialized countries are epizootics in domesticated and wild mammal populations. Infection with M. bovis remains a significant livestock zoonosis in the European Union where some member states experience a reemergence of the disease despite significant historical efforts to implement eradication plans. In Great Britain, the disease was eliminated from most cattle herds by 1960, with the exception of infection hotspots in southwest England, after the implementation of a herd testing and slaughter policy. However, efforts to completely eradicate bTB in Great Britain have been hampered by the maintenance of M. bovis in wildlife host populations, acting as reservoirs of infection, in particular badgers (Meles meles). Since 1979, incidence in British cattle has increased and the infection has become more geographically widespread. Over 7 million cattle were tested for bovine bTB in 2009 and one in ten herds experienced bTB-related movement restrictions during the year as a result of at least one member of the herd failing the tuberculin skin test or showing lesions consistent with bTB during the slaughterhouse inspection – an event known as a “herd breakdown”.

Local Cattle and Badger Populations Affect the Risk of Confirmed Tuberculosis in British Cattle Herds. 2011 PLoS ONE 6(3): e18058. doi:10.1371/journal.pone.0018058
Background: The control of bovine tuberculosis (bTB) remains a priority on the public health agenda in Great Britain, after launching in 1998 the Randomised Badger Culling Trial (RBCT) to evaluate the effectiveness of badger (Meles meles) culling as a control strategy. Our study complements previous analyses of the RBCT data (focusing on treatment effects) by presenting analyses of herd-level risks factors associated with the probability of a confirmed bTB breakdown in herds within each treatment: repeated widespread proactive culling, localized reactive culling and no culling (survey-only).
Methodology/Principal Findings: New cases of bTB breakdowns were monitored inside the RBCT areas from the end of the first proactive badger cull to one year after the last proactive cull. The risk of a herd bTB breakdown was modeled using logistic regression and proportional hazard models adjusting for local farm-level risk factors. Inside survey-only and reactive areas, increased numbers of active badger setts and cattle herds within 1500 m of a farm were associated with an increased bTB risk. Inside proactive areas, the number of M. bovis positive badgers initially culled within 1500 m of a farm was the strongest predictor of the risk of a confirmed bTB breakdown.
Conclusions/Significance: The use of herd-based models provide insights into how local cattle and badger populations affect the bTB breakdown risks of individual cattle herds in the absence of and in the presence of badger culling. These measures of local bTB risks could be integrated into a risk-based herd testing programme to improve the targeting of interventions aimed at reducing the risks of bTB transmission.

Related:

• Public health and bovine tuberculosis
• Culling badgers a low priority for curbing cattle tuberculosis
• Irish badger cull ‘futile’ in controlling bovine tuberculosis

Extensively drug-resistant (XDR) tuberculosis (TB) is defined as TB that is resistant to at least rifampin and isoniazid (multidrug resistant [MDR]), as well as to any member of the quinolone family and at least one second-line anti-TB injectable drug: kanamycin, capreomycin, or amikacin. According to the World Health Organization (WHO), XDR-TB has been reported in 57 countries and is a major concern for global health. The WHO Global Task Force on XDR-TB has recommended laboratory-based surveillance to better understand the prevalence of XDR TB in developing countries. However, surveillance data on XDR-TB from China remain scarce. Shandong Province is the second largest province in China, with a population of 94 million. Shandong Provincial Chest Hospital (SPCH) is the only provincial-level hospital specializing in TB clinical service and control. In collaboration with the SPCH TB reference laboratory, researchers retrospectively analyzed the drug-resistance profiles of a group of clinical Mycobacterium tuberculosis isolates to estimate the prevalence of XDR-TB in China.

32% of isolates resistant to more than one first-line drug; 10% isolates were multidrug resistant, and 2% were XDR. XDR-TB is of major concern in China.

Laboratory-based Surveillance of Extensively Drug-Resistant Tuberculosis, China. EID 17(3) March 2011
To estimate the prevalence of extensively drug-resistant tuberculosis (XDR TB) in China, we retrospectively analyzed drug-resistance profiles of 989 clinical Mycobacterium tuberculosis isolates. We found 319 (32.3%) isolates resistant to >1 first-line drugs; 107 (10.8%) isolates were multidrug resistant, of which 20 (18.7%) were XDR. XDR TB is of major concern in China.

Related:

• Extreme drug resistant tuberculosis (XDR-TB)
• XDR tuberculosis – implications for global public health.N Engl J Med. (2007) 356: 656–659

Mycobacterium tuberculosis (Mtb) has the ability to lie dormant in the human body for decades, only progressing to active disease in 5–10% of immunocompetent individuals. The organism is transmitted through aerosols, and enters the pulmonary system through inhalation. Within the lung, the bacillus can take up residence inside an alveolar macrophage triggering the aggregation of immune cells and the formation of a granuloma. During the course of infection, granulomas play a dual role – serving as a niche for the invading bacteria, whilst, protecting the host from active disease. The population of granulomas within the infected host consists of both primary and post-primary lesions. Primary granulomas containing the inhaled founder strain are morphologically different from post-primary granulomas that have developed through disseminated infection. This results in a heterogeneous population of bacilli that are unique to the in vivo experience

Portrait of a Pathogen: The Mycobacterium tuberculosis Proteome In Vivo. (2010) PLoS ONE 5(11): e13938. doi:10.1371/journal.pone.0013938
Background: Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a facultative intracellular pathogen that can persist within the host. The bacteria are thought to be in a state of reduced replication and metabolism as part of the chronic lung infection. Many in vitro studies have dissected the hypothesized environment within the infected lung, defining the bacterial response to pH, starvation and hypoxia. While these experiments have afforded great insight, the picture remains incomplete. The only way to study the combined effects of these environmental factors and the mycobacterial response is to study the bacterial response in vivo.
Methodology/Principal Findings: We used the guinea pig model of tuberculosis to examine the bacterial proteome during the early and chronic stages of disease. Lungs were harvested thirty and ninety days after aerosol challenge with Mtb, and analyzed by liquid chromatography-mass spectrometry. To date, in vivo proteomics of the tubercle bacillus has not been described and this work has generated the first large-scale shotgun proteomic data set, comprising over 500 unique protein identifications. Cell wall and cell wall processes, and intermediary metabolism and respiration were the two major functional classes of proteins represented in the infected lung. These classes of proteins displayed the greatest heterogeneity indicating important biological processes for establishment of a productive bacterial infection and its persistence. Proteins necessary for adaptation throughout infection, such as nitrate/nitrite reduction were found at both time points. The PE-PPE protein class, while not well characterized, represented the third most abundant category and showed the most consistent expression during the infection.
Conclusions/Significance: Cumulatively, the results of this work may provide the basis for rational drug design – identifying numerous Mtb proteins, from essential kinases to products involved in metal regulation and cell wall remodeling, all present throughout the course of infection.

Related:

• Systems biology approaches to tuberculosis
• Do mycobacteria produce endospores?
• Why is TB more common in men than in women?

Mycobacterium tuberculosis is a remarkably successful human pathogen. The interaction with the human host is complex and much remains unknown. Recent advances in systems biology have allowed the integration of data from humans and animal models into computational approaches. For example, mathematical models provide a platform for in silico manipulation of host-pathogen interactions to gain insight into this infection across temporal and biologic scales. This article reviews recent studies on global approaches toward identifying comprehensive responses of both host and bacillus during infection, and the potential for incorporation of these data into many types of useful computational systems. Systems biology approaches provide a unique opportunity to study interventions that may improve therapy and vaccines against this major killer.

Tuberculosis: global approaches to a global disease. Curr Opin Biotechnol. Jul 14 2010

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• Cough!
• Origin and spread of Mycobacterium tuberculosis

Mycobacterium tuberculosis was first isolated more than 125 years ago. Although a huge amount of research has been devoted to it over this time, tuberculosis still represents a major public health threat in many countries. The main hindrances in fighting this disease include a lack of understanding of the human infection, its establishment and progression, as well as the host-pathogen interactions that determine the different outcomes. Furthermore, the treatment regimen of six months administration of up to four drugs has not evolved in more than four decades, and recent years have seen an alarming increase in multi-drug resistant (MDR) and extensively drug-resistant (XDR) strains. It is clear then that novel and imaginative approaches are needed to speed up both basic and translational research in tuberculosis, especially in the areas of vaccine and drug development.

Optimisation of Bioluminescent Reporters for Use with Mycobacteria. 2010 PLoS ONE 5(5): e10777. doi:10.1371/journal.pone.0010777
Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI.
One of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging.
While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.

Related:

• Public health and bovine tuberculosis
• Origin and spread of Mycobacterium tuberculosis
• Tuberculosis – is the white plague winning?