MicrobiologyBytes

Mycobacterium tuberculosis can persist in the host for decades after infection before reactivating to cause disease. The bacterial and host factors that contribute towards latent TB infection and reactivation disease have long remained enigmatic. However, there is considerable circumstantial evidence to suggest that the persisting organisms may include bacteria in physiological states that are characterized by impaired culturability (i.e. colony-forming ability). These observations suggest a plausible link between an intrinsic microbiological property of M. tuberculosis the ability to enter into a state of dormancy from which culturability can be restored and the clinically defined phenomenon of latent infection.
M. tuberculosis contains five resuscitation-promoting factor (Rpf)-like proteins, RpfA-E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA-E genes were shown to be collectively dispensable for growth of the organism in broth culture. The defect in resuscitation of multiple mutants from a non-culturable state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild-type organisms confirming that the phenotype was associated with rpf-like gene loss and that the non-culturable cells of the mutant strains were viable. Other phenotypes uncovered by mutagenesis revealed a functional differentiation within this protein family.
Researchers have yet to unravel the complexities which underpin the in vivo phenotypes and relate them to the various in vitro phenotypes associated with rpf-like gene loss. The fact that some Rpfs interact with other proteins in the cell to form protein complexes that may cleave distinct forms of peptidoglycan further adds to the complexity of Rpf function and regulation. However, the collection of mutant strains reported in this and earlier studies are an important resource for future biochemical, microbiological and physiological studies on this fascinating family of proteins.

The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro. Molecular Microbiology, 21 Dec 2007

2007 has been a record breaking year for MicrobiologyBytes, so here’s a look back at some of the highlights:

We started January off with noroviruses and ancient plague, then relaxed a bit by playing with Lego and brewing beer.

In February, we looked at yaws and Mimivirus, then went green by reducing our carbon footprint with microdiesel.

And in March we marked World Tuberculosis Day by looking at new drugs for an old foe.

April started off with an exploration of whether viruses evolve to protect their hosts, then we took out first look at colony collapse disorder affecting bees.

May was dominated by news about extreme drug resistant tuberculosis (XDR-TB) and chikungunya, then later looked at probiotics.

In June we looked at the origins of yellow fever and quorum sensing in Serratia (quorum sensing remains one of the most popular topics on MicrobiologyBytes).

July began with flesh eating bacteria and finished up with prions and Alzheimers disease.

In August, most people took a holiday and this was the quietest month of the year in terms of visitors, but we still managed to fit in Hendra, chikungunya and Marburg viruses.

September brought lots of bad news for UK farmers, so we looked at the biology of the bluetongue and foot and mouth disease virus outbreaks in the UK.

In October we covered the bacterial SOS system and debated the strategy for HPV vaccination in the UK.

November started with the terrorist threat posed by glanders and melioidosis then considered the dangers of Chlamydia infection and the opportunities presented by DNA microarrays.

We finished up the year with bacteriocins and bacterial morphology.

Phew. Overall, the most popular posts of the year were:

1. Hepatitis C Virus: a mountain to climb
2. Fungal Infections and All About Fungi
3. Infectobesity
4. Toll-Like Receptors
5. DNA microarrays

See you next year!

In this article in Microbiology Today, Hazel Dockrell describes the role of gamma interferon in the fight against TB and predicts a complex future.

Mycobacterium tuberculosis is an intracellular pathogen, choosing to live within macrophages, where it inhibits antibacterial processes such as phagosome-lysosome fusion. It also expresses haemolysin-like molecules that might, like Listeria, enable its escape into the cytoplasm, although confirmed evidence of this is still lacking. It induces granuloma formation within the lungs, which can progress to causing necrosis, enabling its spread by coughing, and resulting in the destruction of lung tissue. The classic test for infection, the Mantoux skin test, measures recruitment and activation of antigen-specific T cells in a delayed-type hypersensitivity test. This focus on cell-mediated immunity has led to a major interest in the role of gamma interferon.

The infection-induced suicide of host cells (apoptosis) following invasion by intracellular pathogens is an ancient defense mechanism observed in multicellular organisms of both the animal and plant kingdoms. It is therefore not surprising that persistent pathogens of viral, bacterial, and protozoal origin have evolved to inhibit the induction of host cell death. Mycobacterium tuberculosis, the etiological agent of tuberculosis, has latently infected about one third of the world’s population and can persist for decades in the lungs of infected, asymptomatic individuals.
The survival and persistence of M. tuberculosis depends on its capacity to manipulate multiple host defense pathways, including the ability to actively inhibit the death by apoptosis of infected host cells. The genetic basis for this anti-apoptotic activity and its implication for mycobacterial virulence have not previously been determined. Using a novel gain-of-function genetic screen, a recent paper demonstrated that inhibition of infection-induced apoptosis of macrophages is controlled by multiple genetic loci in M. tuberculosis. Characterization of one of these loci in detail revealed that the anti-apoptosis activity was attributable to the type I NADH-dehydrogenase of M. tuberculosis, and was mainly due to the subunit of this multicomponent complex encoded by the nuoG gene. A mutant of M. tuberculosis in which nuoG was deleted triggered a marked increase in apoptosis by infected macrophages, and subsequent analysis of this mutant in the mouse tuberculosis model provided direct evidence for a causal link between the capacity to inhibit apoptosis and bacterial virulence. The discovery of anti-apoptosis genes in M. tuberculosis could provide a powerful approach to the generation of better attenuated vaccine strains, and may also identify a new group of drug targets for improved chemotherapy.

Mycobacterium tuberculosis nuoG Is a Virulence Gene That Inhibits Apoptosis of Infected Host Cells.
2007 PLoS Pathog 3 (7): e110

Multi-drug resistant TB (MDR-TB), is caused by strains of Mycobacterium tuberculosis which are resistant to at least two of the main first-line TB drugs, is already a growing concern. XDR-TB is defined as strains which are resistant to all the current the front-line drugs, but also three or more of the six classes of second-line drugs. In a few rare cases, a strain resistant to all currently available drugs have been seen.
Since WHO tuberculosis experts convened in South Africa last year to discuss how to address the problem, XDR-TB has slowly continued making its way around the world, recently being detected in India where it could account for 8% of those who suffer from TB, although there is no official data on the prevalence of XDR-TB in India and it is nearly impossible to determine the true prevalence of this strain in India.
The latest news is that a U.S. citizen – currently in hospital isolation in Georgia after becoming the first person quarantined in the U.S. by the government for more than 40 years – put air passengers at risk by boarding transatlantic flights knowing he was infected with XDR-TB when he traveled to Europe for his wedding and honeymoon.
Enforced quarantine is likely to become a more frequent choice for health care authorities and governments as XDR-TB continues its inexorable spread.

Related:

• XDR-TB
• World Tuberculosis Day: new drugs for an old foe
• A single dose of vitamin D may prevent tuberculosis
• Latest news on XDR-TB

Vitamin D was used to treat tuberculosis in the pre-antibiotic era. Prospective studies to evaluate the effect of vitamin D supplementation on antimycobacterial immunity have not previously been performed. A double-blind randomized controlled trial was conducted in 192 healthy adult tuberculosis contacts in London, UK. Participants were randomized to receive a single oral dose of 2.5 mg vitamin D or placebo and followed up at 6 weeks. A single oral dose of 2.5 mg vitamin D significantly enhanced the ability of participants’ blood to restrict the growth of recombinant reporter mycobacteria in vitro without affecting antigen-stimulated interferon-gamma responses. Clinical trials should be performed to determine whether vitamin D supplementation prevents reactivation of latent tuberculosis infection.

A Single Dose of Vitamin D Enhances Immunity to Mycobacteria.
Am. J. Respir. Crit. Care Med. 2007

A new report claims the “virtual extermination” of badgers in the Republic of Ireland has failed to stop the spread of bovine tuberculosis. Badgers have been blamed for spreading bovine tuberculosis (Mycobacterium bovis) to cattle. Although so many badgers have been killed that they are extinct in many areas of Ireland, the level of TB in cattle is twice as high as in Britain. In the UK, the government-backed Randomised Badger Culling Trial (also known as the Krebs Trial), which ended in 2003, showed that culling could make the TB problem worse.
Relevant:

• Bovine TB, the hepatitis C virus receptor and antibiotics
• Culling badgers a low priority for curbing cattle tuberculosis

World Tuberculosis Day is observed on March 24 each year and commemorates the date in 1882 when Robert Koch announced the discovery of Mycobacterium tuberculosis, the bacterium which causes TB. Worldwide, tuberculosis remains one of the leading causes of death from infectious disease. An estimated 2 billion people (one third of the world’s population) are infected with M. tuberculosis. Each year, approximately 9 million people become ill with TB and nearly 2 million die from the disease, with new infections occurring at a rate of one every second. Tuberculosis has plagued humans for as long as we’ve been on this planet, probably evolving alongside us. In the UK, the Health Protection Agency has just announced that tuberculosis has increased another 2% to over 8000 cases in 2006. Since the late 1980s the number of people in the UK diagnosed with TB has risen every year, although in the USA CDC has just reported a 3.2% decline in cases in 2006.

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Tuberculosis is spread by aerosol droplets expelled by people with infected lungs when they cough, sneeze, speak, or spit. The disease most commonly affects the lungs (pulmonary TB), but can also affect the central nervous system, most other organs, bones, and even the skin. Worldwide, TB is the leading cause of death in terms of curable infectious diseases.

Although there are several drug-resistant forms of the organism, such as multi-drug resistant or MDR-TB (strains which are resistant to at least two of the main first-line TB drugs such as isoniazid and rifampin) and the virtually untreatable extreme drug resistant XDR-TB (strains which are resistant to all the current the front-line drugs, and three or more of the six classes of second-line drugs), the majority of TB strains are not resistant to drugs in the way that other bacteria such as MRSA are resistant to most antibiotics.

Patients with TB typically have to take four different antibiotics for two months and then continue with two of these antibiotics for an additional four months. DOTS (Directly Observed Treatment Short-course), the WHO-recommended TB control strategy includes directly observed treatment for at least the first two months in order to ensure compliance. Poor adherence to therapy results in the emergence of MDR and XDR strains, hence the need for new drugs to shorten treatment of drug-sensitive TB, and for effective treatment of MDR- and XDR-TB.

Why is such long treatment needed? Traditionally the answer was thought to lie in the fact that Mycobacterium tuberculosis enters a dormant or non-replicating state in an infected person. Because most of antibiotics act only on replicating bacteria, the dormant state of TB was thought to render it resistant to treatment. But that notion has now been challenged by a paper in PLoS Medicine (Why is long-term therapy required to cure tuberculosis? 2007 PLoS Med 4: e120).

Soon after the discovery of streptomycin, the first effective drug against TB, it became clear that while many patients treated with this antibiotic initially improved dramatically, most subsequently developed streptomycin-resistant strains so that there was little improvement in mortality over untreated patients. The development of new antibiotics led to the realization that there were two requisites for an effective cure: treatment with multiple antibiotics and a long course of therapy.

TB displays multiple mechanisms of drug resistance when growing in vivo. Some of these are classic genetic mechanisms, but others are phenotypic and reversible, such as growth within enclosed granulomas and in biofilms. In tuberculosis, more than with other bacterial infections, these phenotypic mechanisms seems to be of great importance.

New drugs which target non-replicating bacteria are likely to revolutionize TB therapy. Such agents have the potential not only to treat MDR and XDR strains but also to dramatically shorten the duration of therapy, and shorter treatment times should allow higher patient adherence, reduced transmission of infection, and decreased drug resistance, leading in turn to fewer deaths and the ultimate prospect of controlling one of the human race’s oldest enemies, tuberculosis.

• Wikipedia: Tuberculosis