Wednesday, February 27, 2013

Some bacteria loves antibiotic


     In a recent post (Link), I had summarized a few facts about the Antibiotic resistance problem. I also made an attempt to given an overview of various factors that can be involved in antibiotic resistance. So far so good. As a sequel to that post i will talk a little bit about antibiotic dependent bacteria.

   Just to get head start, Antibiotics are substances (Primarily biological compounds), that can inhibit the growth or kill a cell. Sop, when we say antibacterial substances (Such as Penicillins, Carbapenems etc) we refer to an antibiotic that is acting against the bacteria. Simple. The best scenario (to us, not bacteria) is that the antibiotic acts against the bacterial providing clinical relief. But more often than not, the bacteria is not responding to many antibiotics. We say, its resistant. But now imagine what happens if the bacteria starts enjoying the antibiotics you are throwing at it, to an extent that the bacteria is now dependent on the antibacterial substance for its growth!!!

Photo 1: Vancomycin-dependent
enterococci (VDE)
    That makes a sufficient introduction to the problem we are looking for. This is by far the greatest and strongest mode of bacterial resistance that we can expect to encounter. These strains are called as "antibiotic dependent bacteria". In a study published in Lancet, it was seen that, after the initial clinical treatment failure, the bacteria (in this case Enterococcus faecalis) was resistant to vancomycin but also needed it for its growth. Eltringham, a clinical microbiologist said "This is the first instance of isolating [the drug-dependent enterocci] in sick patients, where [the bugs] were almost certainly contributing to the infection" For reference and more details go here. Am not sure of what was the first report ever, but upon a literature search (Data digging!!!), I found an article by Dean JL etal to be the first reported case (Link).

  The photo to the right shows a VDE (vancomycin dependent Enterococci). The VDE strain can only grow contiguous to the end of the strip with the highest concentrations. These strains are very difficult to deal with especially in Hospital settings. Reference

   That leaves me thinking. How beneficial is that for the organism. The scenario is most probably something like this. The Clinician begins with an emperic antibiotic coverage till there is Microbiology report. Meanwhile the organism is mutating and acquiring resistance. When there is a report, the "right antibiotics" are given. Now the selection pressure enhances the resistance and an occasional mutant that can not only survive, but can actually utilize the drug, has now a competitive advantage and is selected. The clinician is now presented with a treatment failure, a grave one.

    But then there is a second angle to this drama. The new "superbug" is now leaking to the community or to say a second person, the chances that it will survive is bleak. This is simply because the drug is now an obligation for growth. So unless and until the receiver is on that drug the organism is now selecting for mutants or just doesn't survive. That explains the fact that why this phenomenon is so rare.

   After this incident, here and there a few cases of "Antibiotic dependent superbug" was seen. But then a big blow to this story came, when the phenomenon was reported for a tubercle bacilli. This time it is Tubercle strain dependent on Rifampin. The bacterium was identified in a patient in China, by researchers at the Johns Hopkins Bloomberg School of Public Health.

    Once the organism develops a dependence, the implication of growing it in laboratory is serious. The strain may grow poorly and will be identified only when an enhanced growth is seen in the areas near to the drug. This is much more difficult when it comes to MDR-TB which is often difficult to grow. As Ying Zhang puts it "Rifampin-dependent tuberculosis is an unrecognized and potentially serious treatment issue. Rifampin resistance is ominous. Our study highlights the potential dangers of continued treatment of MDR-TB with rifamycins that occur frequently due to delayed or absent drug susceptibility testing in the field. Further studies are urgently needed to determine how common such rifampin-dependent MDR-TB is in field conditions and if it contributes to the worsening of the disease in MDR patients and treatment failures". Source

    Perhaps such cases are under-reported and rare. But then, these are the strains that are the true superbugs.
Farrag N, Eltringham I, & Liddy H (1996). Vancomycin-dependent Enterococcus faecalis. Lancet, 348 (9041), 1581-2 PMID: 8950890

Tambyah PA, Marx JA, & Maki DG (2004). Nosocomial infection with vancomycin-dependent enterococci. Emerging infectious diseases, 10 (7), 1277-81 PMID: 15324549

Zhong M, Zhang X, Wang Y, Zhang C, Chen G, Hu P, Li M, Zhu B, Zhang W, & Zhang Y (2010). An interesting case of rifampicin-dependent/-enhanced multidrug-resistant tuberculosis. The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease, 14 (1), 40-4 PMID: 20003693

Monday, February 18, 2013

Say Hi to "Methanogens in human flora"


Photo 1: Archaea. Source
   One of my very early posts on this blog was on halocins. The post mainly concentrated on an emerging antibiotic of interest that is extracted from Archaea. After that this is my second post on archaea. The Archaea represent one domain, in the tree of life. They represent a very huge variety of species within it. Most of them are extremophiles and are present in extreme conditions. Considering that they are similar to bacteria and many known mesophilic species are present, there has been no description of an archaea as pathogen (as far as I know), is a bit suspicious.

    That lead someone to ask "Is there any medically important Archaea?". Let me make the question more blunt first. Is there any archaea of importance to Medical Microbiologists. Most of us, who stick to studying syllabus based would answer "No". For the simple fact that, there is no pathogen of medical importance in this group. But, let me give you a glimpse to "Yes".

      Archaea are unlike bacteria or eukaryotes. They possess unique flagellins and ether-linked lipids and lack murein in their cell walls. They have a different metabolic and genetic configuration. With more and more archaea being genetically sequenced, we have more knowledge of their life kinetics.

Photo 2: M smithii shown by
indirect immunofluorescence. 
    Some archaea are also members of normal flora. Archaea have been found to colonize human flora (Such as oral, vaginal flora). The 2 most famous members among them include Methanobrevibacter smithii and Methanosphaera stadtmanae (Thats a difficult to pronounce name). As the detection methods have become more sensitive and specific, we have come to understand more species is residing within us. If you remember reading from my previous posts, I often stress on the fact that, the normal flora has something to do in our body. Somehow, in some way, they are designed to produce some good effects. That's why they are "Normal flora!!!". Methanognens are good in Methanogenesis, a process confined exclusively to the methanogens, and utilizes substrates such as hydrogen, Carbon-di-oxide, acetate, formate, methanol and methylamines for methane generation.

Photo 3: Capsule formation by M smithii
    With that background, let me now talk about how it becomes medically important to us. In my earlier post on Adenovirus-36 (Link) and Gut microbiome (Link), I emphasized on the importance of Normal flora and how it relates to human health.  For starters on medical archaea, how about Inflammatory bowel disease? IBD is a very complicated problem. The problem is mostly because of subtle changes in the normal flora. In fact, there is a good correlation between, IBD and the loss of methanogen flora. Some interesting links have also been speculated between methanogen population and Crohn's disease, Colorectal cancer, Irritable bowel syndrome etc.

      Let me come to the most interesting proposal. "M smithii has got a good correlation with obesity". I better make some explanations here. 

     The gut flora is well populated (>90%) by Bacteroidetes and the Firmicutes. M smithii, can comprise up to 10% of all anaerobes in the colons of healthy adults. It has been proposed that M. smithii can persist in the distal intestine through multiple mechanisms. This includes production of surface glycans resembling those found in the gut mucosa, regulated expression of adhesin- like proteins, consumption of a variety of fermentation products produced by saccharolytic bacteria, and effective competition for nitrogenous nutrient pools. (Taken from Gordon etal). It is also important for digestion of complex sugars. As I said earlier archaeal membrane lipids, contain ether linkages. Archaeal lipids synthesis uses hydroxymethylglutaryl (HMG)-CoA reductase, which catalyzes the formation of mevalonate, a precursor for membrane (isoprenoid) biosynthesis. Given these facts it is probably guessable how the archaea is linked to obesity. This hasn't been proved. Association is not causation.

     As an additional note (or the internet famous version, PS), Methanobrevibacter smithii ATCC 35061 genome is 1.85 Million bp long and composed of aprox 1837 predicted genes. The chromosomes are circular.

   The purpose of this post is to impress you the fact that archaea in association with humans is an under studied area and maybe they are as important as other members of normal flora in influencing our health condition.
Million M, Maraninchi M, Henry M, Armougom F, Richet H, Carrieri P, Valero R, Raccah D, Vialettes B, & Raoult D (2012). Obesity-associated gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii. International journal of obesity (2005), 36 (6), 817-25 PMID: 21829158

Armougom F, Henry M, Vialettes B, Raccah D, & Raoult D (2009). Monitoring bacterial community of human gut microbiota reveals an increase in Lactobacillus in obese patients and Methanogens in anorexic patients. PloS one, 4 (9) PMID: 19774074

Further Reading:

1. Dridi B, Henry M, El Khéchine A, Raoult D, Drancourt M (2009) High Prevalence of M smithii and Methanosphaera stadtmanae Detected in the Human Gut Using an Improved DNA Detection Protocol. PLoS ONE 4(9): e7063. Link

2. Pauline D Scanlan, Fergus Shanahan and Julian R Marchesi. Human methanogen diversity and incidence in healthy and diseased colonic groups using mcrA gene analysis. BMC Microbiology 2008, 8:79. Link

3. Everly Conway de Macario, Alberto J.L. Macario. Methanogenic archaea in health and disease: A novel paradigm of microbial pathogenesis. International Journal of Medical Microbiology. Volume 299, Issue 2, February 2009, Pages 99–108. Link

Friday, February 15, 2013

Schwan cell programmer- Leprae

     Mycobacterium leprae is one of the renowned pathogens in Microbiology. Of-course, the big share of infection in the group goes to M tuberculosis. Leprae bacilli has not been studied in details compared to other pathogen. The most important hindrance is it has not been cultivable in Culture media (Maybe Culturomics will help in future). Till date, the pathogenesis of leprae bacilli has not been well understood. It was assumed that they somehow the pathogen beats the neuronal cells especially Schwann cells. The assumption isn't completely wrong but (as shown in the paper that am going talk about), not right either. The game changing paper has been difficult to digest, but its a huge leap. Let me start with some basics and then we move on to what the paper reveals.

Photo 1: Leprae Bacilli. Source
    Mycobacterium leprae (Casually known as Hansen's Bacilli, causing Hansen's disease or Leprosy), is a member of Mycobacterium. The infection is considered as the major factor in crippling disease of infectious origin. Their elegance comes from the point that, they infect the nerves (Not other bacteria can do that, though there are viral examples!!!), and they infect only Peripheral nerves (Not the Central). The effected site ceases to show signs of sensation. The condition is basically active neuritis. Of all the cells that are effected by Leprae, Schwann cell involvement is distinct.

Fig 1: Schwann cells. Source
     Schwann cells or neurolemmocytes (named after physiologist Theodor Schwann), part of peripheral nerve structure. They are considered as equivalent to Oligodendrocytes present in the CNS. The function of Schwann cells is to produce a myelin sheath, and repair damaged neurons. Myelin sheath provides an insulation to nerve conduction. This arrangement helps in faster conduction of nerve impulses. This can be explained by the fact the nerve conduction is slower in non myelinated neurons. In many different demyelinating neuropathies, this insulation is lost. This causes a lack of nerve transmission. Schwann cells may be damaged by autoimmune disorders or toxic attack such as in Guillain-Barré syndrome and diphtheria. Diphtheria toxin causes loss of myelin by interfering with the production of proteins by the Schwann cells that produce and maintain myelin in the PNS. Triethyltin (A biocide chemical) which interrupts the myelin sheath around peripheral nerves is also known to induce demyelination.


Fig 2: Mycobacterium leprae binds to the ErbB2 receptor to induce Schwann cell demyelination and proliferation. Source

        So what we had known earlier? As I said, the M leprae was known to damage schwann cells. In fact that fits well with explanation. Much of the nerve damage caused by the infection is the result of M. leprae triggering extensive demyelination of schwann cells in peripheral nerves. This causes a loss of nerve conduction functions and leads to what is the classical numbness (Loss of sensation) in the disease. Looking into the details of demyelination, lead to the understanding that demyelination can be induced by a Ras- Raf pathway (Ras–Raf–MEK–ERK). The explanation is shown below in Fig 2.

          The most common ultra-structural finding of infection, is infection of epineurial vascular and lymphatic endothelial cells. The receptor for schwann cell is the PGL-1 (Phenolic glycolipid-1) or LBP21 receptor on M. leprae which binds to the α-2 side chain of laminin-2 as well as the related α-dystroglycan receptor.The entry most probably is via complement receptor 3-mediated phagocytosis.

Fig 3: A model for the binding interactions of individual α2LG modules of the G domain with PGL-1 and ML-LBP21 in the M. leprae cell wall. Source

     With this background, it is obvious that schwann cell has an important role to play in the pathogenesis of leprosy. But it is also unclear as to what happens.

Fig 4: Schwann cell reprograming and implications. Source
      So this paper under discussion, by Masaki etal provides some insight. The paper mainly shows that the leprosy bacteria is able to induce a stem cell like condition into the schwann cells. The mechanism of how this is achieved is they turn off the specific factors that keep the cell in schwann cell lineage and then turn on the set of genes (embryonic and developmental genes, most importantly the HOX gene) that is expressed in a more pluripotent state. This plasticity makes the cell more of a "mesodermal like" condition that allows the pathogen dissociation more easily. The study also shows that such transformation can aid in the macrophage Granuloma formation.

    A second related paper by  Wegner etal. has some more to say. SOX-10 is an essential determinant of Schwann cell identity and is the prime candidate to be inactivated that cause lineage de-differentiaition. On prolonged incubation of in mesenchymal stem cell medium the bacteria- containing stem like cells, become strongly proliferative and gains migratory properties. This possibly is the cause for pathogen dissemination.

     What is so striking about this understanding is that after all shwann cells are not destroyed. Rather, they are converted into stem cell like lineage. The mechanism first of all provides me with something to think about as a model system for stem cell inducing properties, and also clarifies leprae molecular infection. Take home message is "Leprae bacilli is Schwann cell gene programmer".
Masaki T, Qu J, Cholewa-Waclaw J, Burr K, Raaum R, & Rambukkana A (2013). Reprogramming Adult Schwann Cells to Stem Cell-like Cells by Leprosy Bacilli Promotes Dissemination of Infection. Cell, 152 (1-2), 51-67 PMID: 23332746

Wegner M (2013). Mighty bugs: leprosy bacteria turn schwann cells into stem cells. Cell, 152 (1-2), 15-6 PMID: 23332743

Further Reading:

1. Luke A. Noon,Alison C. Lloyd. Treating leprosy: an Erb-al remedy.(2007). Trends in Pharmocological science. ; 28(3); 103–105. Link

2. Tabouret G, Astarie-Dequeker C, Demangel C, Malaga W, Constant P, et al. (2010) Mycobacterium leprae Phenolglycolipid-1 Expressed by Engineered M. bovis BCG Modulates Early Interaction with Human Phagocytes. PLoS Pathog 6(10): e1001159. Link