Tuesday, May 29, 2012

Staphylococcus below a cover glass- Makes the difference


              In the modern day of Laboratory diagnostics, people rely more on hifi techniques like PCR, DNA sequencing or even a microarray analysis for even the most common pathogen. The fact is it doesn't have to be so. Even to this date despite robotics and digtalization of most of the laboratory set up, a simple gram stain microscopy and simple biochemical tests such as catalase and oxidase is invaluable. And i don't think anyone would counter state it.

            This blog is about a paper published in "Journal of Microbiological Methods". The article is entitled  "A simple and cost-effective cover-glass test for the differentiation between staphylococci and micrococci in clinical laboratory" by  Oto Melter etal (Link to the article).


      Staphylococcus is a well studied bacterial pathogen. Rosenbach provided the first taxonomic description of Staphylococcus in 1884 when he divided the genus into Staphylococcus aureus and Staphylococcus albus. The genus Staphylococcus now belongs to the broad Bacillus–Lactobacillus–Streptococcus cluster consisting of Gram-positive bacteria that have a low G + C content of DNA. The closest relatives of staphylococci are the macrococci. They are also related to salinicocci, enterococci, planococci, bacilli and listerias on the basis of partial oligonucleotide sequencing of 16S rRNA and rDNA. Taxonomically, the genus Staphylococcus is in the bacterial family Staphylococcaceae, which includes three lesser known genera, Gamella, Macrococcus and Salinicoccus.

          Staphylococcus aureus has a large set of virulence factors in its armory. The most important adhesion factors involved in colonizing and subsequent skin infections are- Fibronectin-binding proteins A and B, Collagen-binding protein (Cna), Biofilm associated protein (Bap), Elastin-binding protein (EbpS), Map/Eap, Extracellular matrix-binding protein homologue (Ehb) and many more. It also produces exotoxins such as secrets several cytolytic toxins such as α-hemolysin, β-hemolysin, γ-hemolysin, leukocidin, and Panton-Valentine leukocidin (PVL). It can also produce additional group of exotoxins, which include the toxic shock syndrome toxin-1 (TSST-1), the staphylococcal enterotoxins (SEA, SEB, SECn, SED, SEE, SEG, SEH, and SEI) and the exfoliative toxins (ETA and ETB). (Click here for a reference). In addition to all the above, SpA is shown to be an important central virulence determinant. (S. Bronner et al; click here)

Clinical conditions:

         Impetigo (It is also known as school sores) is a superficial bacterial skin infection which is most common among pre-school children. It is of 2 types- Impetigo contagiosa and bullous impetigo. The former type is seen as a result of infection and the latter is usually an immunological complication. Cellulitis is a diffuse inflammation of connective tissue with severe inflammation of dermal and subcutaneous layers of the skin. Erysipelas is the term used for a more superficial infection of the dermis and upper subcutaneous layer that presents clinically with a well defined edge. Erysipelas and cellulitis often coexist, so it is often difficult to make a distinction between the two. Folliculitis refers to the infection and its inflammation. Boil (or furuncle) is a skin disease caused by the infection of hair follicles, resulting in the localized accumulation of pus and dead tissue. Suggested risk factors for impetigo include: household crowding, inadequate access to water, heat and humidity, lack of education, and inadequate implementation of adequate personal hygiene.

Photo 1: Gram-positive Staphylococcus aureus, from a laboratory culture (Source), and Staphylococcus aureus in culture (Source)

Previously we studied Community acquired Staphylococcus aureus (unpublished data), and its antibiotic sensitivity pattern was as follows.

1. Methicillin
2. Cefazolin
3. Cefuroxime
4. Erythromycin
5. Clindamycin

Micrococcus species:

            Micrococcus are genus of bacteria in the Micrococcaceae family, often present as commensals in the skin flora. They are usually recovered in skin culture along with other bacteria. The commonly encountered species include M. luteus; M. lylae; M. roseus etc. They are differentiated from the staphylococcus species by modified oxidase test, susceptibility to furazolidone and lysostaphin, glucose fermentation, acid production from glycerol and growth in Mannitol salt agar. (For details click here).

So, what is this paper about?

The paper is about a simple cost effective differentiation methodology, between pathogenic Staphylococcus and Micrococcus (and related genera such as Dermacoccus, Kytococcus, and Kocuria). The cover glass test, is carried out by using sterile cover-glass which is placed with a pair of sterile tweezers onto a heavily inoculated area of the Columbia blood agar plate with bacterial culture. After overnight incubation at 37 °C, the plate is checked for pigmented colonies or bacterial biomass underneath the cover glass.

Photo 2: Cover-glass (Source)

The growth of staphylococcal colonies was interpreted as weakly positive (+), moderately positive (++) or strongly positive (+++) depending on bacterial growth alteration. (Considered strongly positive if it is an unaffected growth). A negative result will mean that the strain is not able to grow beneath the coverslip.

The test was found to be positive for the staphylococcus (with a few exceptions) and negative for Micrococcus. The way this test makes sense is because staphylococci are facultatively anaerobic and Micrococci are aerobic.

As per i see it, the test is seriously simple enough to perform and cost effective. Many such tests can be carried in a single culture plate. But, I still would recommend that the Modified oxidase test which gives the same result with similar accuracy should be a better bet, because the results are much faster. However, this test can be of academic interest.

*(I didn't use any pictures or tables from the article, owing to copyright issues.) For a full length detail you have to read the article which actually is behind a pay-wall.

ResearchBlogging.orgMelter O, Tkadlec J, & Sedláček I (2012). A simple and cost-effective cover-glass test for the differentiation between staphylococci and micrococci in clinical laboratory. Journal of microbiological methods, 89 (3), 213-5 PMID: 22446099

Further Reading:

1. Staphylococcus Aureus Diagnosis. (Link)

Monday, May 21, 2012

Interpreting Science data- Don't be mislead.


There is always a lot of things happening in Medical science.  I often spend long time (Whenever i can of course) reading research articles. Many a time i pick up the article from pages like science daily or from podcasts or sometimes even by chance. Though this blog is truly dedicated to write about Microbiology only, i felt this is worth making an exception.

Let me try telling you a story (A small part of it is fabricated to give you the right glimpse) to get you my point.

A study was initiated by a group of Industrial researchers on the use of a toothpaste containing an additive that is supposed to reduce bleeding of gums. As study population, certain poor people were selected. (Ah you know, they happily agree for a free toothpaste). So a 100 people were randomized and assessed for dental hygiene and asked to brush with test paste. The 2nd group of people was given a placebo.

The investigators came back after an year and found there is no significant difference in terms of oral hygiene in 2 populations. Years of research, millions of money is a loss for the industry. The industry which made it decided to look deeper into the matter. Least to say, their preliminary test results indicated things other way round.

So a second team of experts went around collecting data from the same population and came up with a paradoxical result- "Inconclusive evidence". Boy, same study population, same toothpaste and 2 ambiguous result? Before reading further i encourage you to come up with your own possibilities on what might be the reason.

Ok. So what happened precisely is, the people who were given the toothpaste were selling them in the loose to other people. So the study population didn't really use the material at al. When the second group came across this fact, they declared that the findings were totally inconclusive.

Another quick scenario. A scientist decided to look into a problem of particular disease in a village. He found that every person who drank milk was infected with this strange disease and concluded that the milk had something to do (Statistical co relation is a player) with this disease. However, much latter they found that its not the case. The fact was, the milkman was a carrier of that disease, though milk itself had nothing to do with it.

In both case there is an obvious poor data collection. But you wouldn't know it until a deeper study was done. If a proper study was done in the first place, it would have saved additional work up. So what am i trying to tell you? Simple, a lot of data will be generated when we do studies, but the meaning of the data may be nothing but bogus. We should be careful when we analyze a simple observational study and relying on the published material.
This cartoon is taken from "Scientific Misconduct Blog" (Click here for Source)

            Coming to my second argument. Suppose a study for variable Y event is  Z and you gather data on expression of Z in response to Y and say "yes, Z is expressed in response to Y" or "no". The first thing you need to understand is that the answer in science is often dependent on the question you ask (I was listening to a podcast in "Brain science" an stuck with this idea! Sorry but i don't remember the episode number). Its turns out that if you were to ask some other question, you would be possibly arrive at some other solution for the same problem or question.

           Last part of my argument. A lot of studies is done at a molecular level looking for variables in an event. Let me borrow an idea from Matt Ridley (From his famous book "Agile genes"). If there are N number of variables effecting a phenomenon, then the effect of individual variable is usually low from a single stand point. But when we suppress all other variables, (in terms of looking only at one, or finding controls that negates other variables effect statistically) the variable in question looks significantly important. Many a times this is what happens in molecular studies. We many a times over estimate the function of a variable. This leads to problems when we take the entire scenario into consideration.

         I would like to make a strong note here. In no way, am advocating that research is misleading. Instead am advocating the way we handle data in science. Many a times the interpretation of the data is a subjective matter. Only when the whole study makes a prediction of events, which is true on testing and multiple such tests prove the same, do we accept a study and its theory. And very honestly i must say, because of publishing pressure, i see many papers that just throws the data without making meaningful predictions and proving it with a set of tests.

       This blog is my response to many questions, by many of my students on how to interpret the data in a Journal. All i have done is put the known facts am aware of in a single basket and try to tell you, why we come up with subjective opinions at times when we read an article. Sometimes, we are also mislead because we haven't looked enough, or data is not presented in the right way.

I surely want to conclude with a lay statement that i have often heard "Believe science. Not scientists"

Further reading

1.  Validomics: How do we ensure biologically relevant data? (Link)
2.  Ethics of qualitative research: are there special issues for health services research? (Link)

Monday, May 07, 2012

The Influenza Hush-Bush

          In the last few blogs I was talking about some basic facts of microbiology avoiding the hifi research. Though am still left with some basic blog-posts, this time let me post on one of the hot topics in microbiology, a debate which started from August 2011. For the convenience of understanding to the audience, let me divide the entire post into 3 sections. First a few words on general characters of influenza, 2nd what was the controversy and last on what was the main lessons when the paper was out to public. 

         Every person who knows Medical virology has given a very large chunk of credit to 3 virus- HIV, Hepatitis B and influenza. I don’t mean to make a statement that the rest are not focused on, but the others are definitely less studied compared to the trio mentioned. 

Influenza virus in a nutshell: 
 Fig 1: Orthomyxovirus structure (For source, click here)

          Influenza virus belongs to the family Orthomyxoviridae, within the negative-sense RNA virus order Mononegavirales. The virus is enveloped with segmented RNA (8 RNA segments to be precise). The viral RNA is closely associated with the nucleoprotein (NP) to form the Ribonuclear protein (RNP). The NP is a type-specific antigen. It occurs in one of three antigenic forms into 3 types A, B and C (Basis for the classification of influenza viruses). And yes, the most important point, unlike many other RNA virus, this replicates in nucleus. The replication uses a RNA-dependent RNA polymerase complex, consisting of three proteins, referred as polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2) and polymerase acid (PA) protein.

Fig 2: Replication cycle of Influenza virus (Gary R. Whittaker)

Table 1: Pandemics caused by Influenza A (For source, click here)

Of all the proteins that is isolated and studied- Hemagglutinin (HA) and Neuraminidase (N) has captured the interest because of its involvement in the binding kinetics.


          The HA is a trimer with identical subunits made up of 2 polypeptides. Each monomer is designated with a receptor-binding site at its membrane-distal tip, which means the sequence at this region has a lot to speak for itself. Sialic acid is bound similarly in all HAs examined by hydrophobic interactions and by hydrogen bonds with the 130- and 220-loops and conserved amino acids in the base of the site. It is being proposed that avian and equine viruses has affinity to sialic acid in α2,3-linkage to galactose while the human viruses prefer α2,6-linked sialic acid, and swine viruses appear to bind sialic acid in both linkages.

Fig 3: Crystal structures and phylogenetic organization of pandemic HAs (Gamblin and John, link)


           Neuraminidase is a tetramer of identical subunits. Each of the subunits that form the head is made up of a six-bladed propeller-like structure, the blades of which are formed by four antiparallel strands of β-structure. At the stage of invasion the NA is known to be involved with removal of decoy receptors on mucins, cilia, and cellular glycocalix, strong binding to each of which would impede virus access to functional receptors on surface membrane of target cells.NA plays an essential role in release and spread of progeny virions, following the intracellular viral replication cycle.

Fig 4: Phylogenetic organization and crystal structures of NA (Gamblin and John, link)

For more molecular details about HA and NA, the readers are strongly advised to the work by  Steven J. Gamblin and  John J. Skehel. (Link).

The following video will also be of great help, to get your understanding right.

So what was the debate all about?:

           The debate was about to publish or not to publish the study by Japanese–US team headed by Yoshihiro Kawaoka at the University of Wisconsin-Madison who had genetically created a few mutants of influenza from a non human strain (In this case avian type) and looked into possibility of its transmission to humans with ferrets as models. For, a detailed description of use of ferrets as animal models for influenza go here.

Photo: Ferrets, the animal models

          The first positive side of the debate was by the people who had did the work and other scientists who believed that work doesn't possess and public threat or doesn't infringe the national security. The work only provides the basis for information on the probabilities that the virus can be a possibility of pandemic if overlooked. 2 quote form Yoshihiro Kawaoka, "Our study shows that relatively few amino acid mutations are sufficient for a virus with an avian H5 hemagglutinin to acquire the ability to transmit in mammals" and "H5N1 viruses remain a significant threat for humans as a potential pandemic flu strain. We have found that relatively few mutations enable this virus to transmit in mammals. These same mutations have the potential to occur in nature". The group were assessing the possibility and at least according to me thats justified in a scientific community.

        The opposing group was mainly the National Science Advisory Board for Biosecurity (NSABB), which raised concerns over the possibility of use of the artificial mutations and direct it as a biological weapon (to read about biological warfare refer to my previous post). Statements such as "Probably one of the most dangerous viruses you can make" by virologist Ron Fouchier of Erasmus Medical Center and "I can't think of another pathogenic organism that is as scary as this one, I don't think anthrax is scary at all compared to this" by Keim as reported in Science Insider article gave more thrust. Justification was option of bioterrorism. But many people with high scientific status did argue that the experiment is done and the story has created so much of noise that if someone really wants to make it, then just by looking at the story they can. Instead a public access will give more option to look at the problem.

So what happened when the paper got published?

           The paper was published in Nature Journal (Online on 02 May 2012). Nothing really happened, except the fact that the data gave more neat idea and more answers for many questions on influenza biology. Ferrets are not humans and hence the transmissibility in ferrets doesn't necessarily reflect patterns in humans. A visual and a table from this paper had grabbed most of my attention which is shown below.

Fig 5: Localization of amino acid changes identified in this study on the three-dimensional structure of the monomer of VN1203 HA

          The study created some H5 genetic mutations, in globular head of a of A/Vietnam/1203/2004 (H5N1; VN1203) strain. They also replaced the multi-basic HA cleavage sequence with a non-virulent-type cleavage sequence thus allowing them to work at ease and avoid issues of biosafety. Using a sialylglycopolymers absorbed to plates, they also tested for receptor-binding properties of the selected variants. They were able to demonstrate that the N224K/Q226L combination is critical for the shift from Sia α2,3-Gal to Siaα 2,6-Gal recognition (Reflected in Fig 5).

Table 2: Transmission in ferrets inoculated with H5 avian–human reassortant viruses

            In the transmission study, four of the six contact ferrets were positive for virus between 3rd and 7th  day after contact, and all contact animals were seropositive; no animals died in the course of the transmission experiments. (Data shown in Table 2).

          For a detailed look you must read the paper (since its open access no excuse). You could also listen to a short interview with the author of this work through Nature Podcast- 03 May 2012. However, the main take home message drawn from the study was that a very few mutations in a critical region can create a stage for the virus to possibly cross into the humans. There was no fatality registered with the ferrets and hence its seems legible to comment that the use as a biological warfare agent is out of track. So all the hush bush just delayed some insight.

ResearchBlogging.orgImai, M., Watanabe, T., Hatta, M., Das, S., Ozawa, M., Shinya, K., Zhong, G., Hanson, A., Katsura, H., Watanabe, S., Li, C., Kawakami, E., Yamada, S., Kiso, M., Suzuki, Y., Maher, E., Neumann, G., & Kawaoka, Y. (2012). Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets Nature DOI: 10.1038/nature10831

Further Reading:

1.  Publishing risky research; Nature 485, 5 (03 May 2012) doi:10.1038/485005aPublished online 02 May 2012. (Link)

2. Steven J. Gamblin and John J. Skehel. Influenza Hemagglutinin and Neuraminidase Membrane Glycoproteins. September 10, 2010 The Journal of Biological Chemistry, 285, 28403-28409Link

Tuesday, May 01, 2012

Vaccine safety issue

Hello fellows,

         Over a few days, i have been doing some search on vaccines. Like, how they are made, issues related and blah blah blah.... But to my terrifying experience, i found that there is a lot of rumor on the safety of the vaccine issue, which as has been well tackled and explained many times in the weekly podcast- "TWiV".

        I thought, at least as a matter of public interest, I must address this issue. For people who are interested in knowing a little bit about the history of vaccines, click here.

Photo: The arm of Sarah Nelmes, a dairy maid, who had contracted cowpox (Source)

        Ok. Let me get into the point. The question that many people have is "If the vaccines are necessary? What's the risk and benefit? And do I/ my kids need to be vaccinated?". These are some of the fundamental questions addressed here to the best of my knowledge.

         First of all, for dummies, What is vaccines? Vaccines are basically immunity boosters that teaches the immunity that when someone like this is gonna come, you got to act like this (A totally non scientific way of telling, but good for understanding). The vaccines may be live attenuated (Which means its living but no more good enough to cause a pathology), killed vaccines (Self explanatory) and subcomponent vaccines (Only the immunogenic component is used). In any case, the vaccine content is designed to illicit an immune response, but not cause any damage to the maximum possible practical extent.

So,  are vaccines necessary?

          In my (and the large chunk of scientific community) opinion, its YES. For the simple reason that it protects you from various serious conditions simply by training your immune system. The vaccine stimulates a primary immune response and keeps the immune system ready and trained so that when the host encounters the agent again, there is a strong, quick and better response. The basis can be summarized as below.

Fig 1: Primary and Secondary Immunological Responses (For source click here)

            Now, you can easily see what i mean to say. If you are not vaccinated and you encounter the agent, it would take more time and a small response is produced. This gives the pathogen a chance to do more damage. Some argue that why do we need to take as many vaccines as possible if we don't expect to encounter them? The answer is simple. Its more like a Insurance policy. If you are not in trouble, then its a waste of very small (even negligible resource). But if you end up on the wrong side, boy you are in serious no win situation.

What's the risk and benefit?

           I just told about benefit. The risk will be say a 1 in million chances that the live attenuated virus, will revert to its wild form and cause a mild illness Mind you thats a very negligible chance. In fact i would say thats a never to gonna happen chance. Even the antibiotic that we take when we get sick has more chances that it will have a side effect. I happened to read recently that there is more than 10% chance that you will meet with road accidents if you are to drive. Well, if vaccination is hazardous, then i guess we should stop driving. And since most of the modern day vaccines are component vaccines it doesn't carry even the 1 in million chances.

Given the above argument, you should be able to convince any audience and say to then "Hey, don't get you or your near ones into trouble. get them vaccinated." More over there are regulatory bodies that constantly check the safety and efficacy of vaccines.

After all, "Prevention is better than cure"

Further reading:

1. Reports of Health Concerns Following HPV Vaccination (Link)

2. Curlin etal. Integrating Safety and Efficacy Evaluation Throughout Vaccine Research and Development. 
(Click here for link)