Wednesday, June 17, 2015

Blogger's Desk #7: Guest post: Being a microbiologist: Roles and Responsibilities


I have had a great time being a blogger. Often at times I have received excellent questions and occasionally requests on topic that I might be willing to post about. More recently people have asked me if I could talk about what being a microbiologist actually means. There is actually not much posted about it in the blogosphere on this topic. Many aspiring microbiologists have no idea on what the subject is going to offer them. And there is a list of those who have the degree but has no idea why.

So I requested Dr. Sridhar Rao, who blogs frequently and has a personal webpage that has a lot of microbiology in it. He had put it so neat, much better than I could dream of writing. Here is what he posted.
In the mid-nineties, people suggested that microbiology was a promising subject with good career prospects. A decade later, the same was said about bioinformatics and biotechnology. Sadly, none of them took off as expected. Not that people haven’t made good career out of these, but the sector itself failed to make any breakthrough. Microbiology is a vastly diverse field with multiple branches, medical microbiology being one of them.

What is medical microbiology?

It is a branch of microbiology that deals with the study of microbes that concerns human well-being. It involves the study of microbes that participate in human health and sickness. One has to understand the natural habitat of these organisms, their physiological properties, factors that make them pathogenic and the process by which they initiate infections. Understanding these is crucial to make the right diagnosis and therefore appropriate treatment as well as prevention.

Who should study it?

Since this branch of microbiology is “medical”, the subject is often taught in medical curriculum at both graduate and postgraduate levels. It is an integral part (along with immunology) in the second year of MBBS syllabus. Post graduate course in microbiology is offered in medical colleges as MD or M.Sc degree. 

What is the scope of this subject?

Human ailments due to infectious diseases is a continuous concern, which requires participation by medical microbiologists. A qualified microbiologists has several roles to play in this filed.

a. As a consultant/specialist

Not all clinical conditions can be diagnosed accurately based on clinical presentations; the use of laboratory tests become crucial in many cases. The process begins with selection of appropriate laboratory test, which depends on several factors, such as the patient condition, age and the stage of illness. The physician and the microbiologists must consult each other before ordering a laboratory test. Ideally, a microbiologist must join the physician in the wards or be available on-call while seeking laboratory test. The channel of communication between the hospital and the laboratory must never break down; in many cases, the two behave as a disconnected islands. The physician must ensure that the requisition form is filled appropriately and sufficient information on the presumptive diagnosis, nature of clinical specimen and the timing of collection is communicated properly. The laboratory too must be able to reach out to the physician for additional information. 

Microbiologist must inspect the quality of the specimen and must be able to reject them if they fail minimum standards. The laboratory must communicate with the patient as well as the physician citing reasons for rejection and advice on proper specimen collection. A microbiologist must hold one’s ground as there is no point performing a test on unsuitable specimen. It would serve no purpose! The report issued by the laboratory must be informative and must help the physician in selecting proper treatment. A comment from the microbiologist goes a long way in helping the physician interpret the report. The role of a microbiologist is not limited to reporting the requested test; if the microbiologist believes that certain additional tests may be performed to arrive at accurate diagnosis, the same must be communicated to the physician. Apart from suggesting appropriate antibiotic based on susceptibility test report, a microbiologist must participate actively in formulating hospital antibiotic usage/prescription policy.

b. As a scientist

Microbiology is a science; the skills and techniques must be honed repeatedly by participating in the bench work. Many believe that by obtaining the masters’ degree one reaches the pinnacle and therefore should quit bench work. In my opinion, this is regressive. There are technicians for the bench work, but whenever possible, one must take part in bench work to keep the skill intact or refine it. Science evolves only when people research. Newer discoveries help understanding the issues better. Newer techniques make the diagnostic processes quicker and efficient. A microbiologist must evolve along with the subject. One must keep pace with the newer concepts and technologies. A good microbiologist must observe patterns in the results and apply analytical mind. Whenever possible, the microbiologist must engage in research. If engagement in bench work is impossible, the person must be able to formulate research strategies, analyze results and publish findings. As a researcher, one must have basic knowledge of research methodology, including statistical analysis.

c. As a teacher

Once a person is deemed to be a professional microbiologist, it becomes his or her responsibility to train the subsequent generation. Knowledge must be disseminated. The microbiologist must be able to inspire new batch of students, make them understand and question the principles. The microbiologist must be able to pass on the skills to the students and help them become efficient. A microbiologist must be a role model for subsequent batch of students. With changing scenario, the microbiologist must be able to alter the pattern in teaching, which includes setting syllabus and evaluation of students.

d. As a student

Learning never ends; when working in a profession that continues to evolve, one must be up-to-date. As research is being conducted round the world and newer discoveries are made, every microbiologist must update oneself. Newer concepts on etiology, pathogenesis, diagnosis and treatment keep coming up and one must be aware of these. As a responsible microbiologist, one must make a habit of reading microbiology journals and participate in continuing medical education programs as often as possible. It is never too late to learn something new; one must grab any opportunity to attend workshops and acquire new skills. 

e. Others

Apart from the roles mentioned above, a microbiologist can have additional responsibilities. A microbiologist is an integral member of infection control committee in hospital. As a part of the team, a microbiologist must be able to identify or track potential sources of infections and suggest ways and means to prevent nosocomial infections. In the laboratory, a microbiologist must be able to come up with Standard Operating Procedures (SOP) for all procedures performed. At the same time, the microbiologist must also be a quality control officer, and be able to monitor and ensure the quality of laboratory tests. A microbiologist can also play a vital role in designing laboratory and management of laboratory and wastes. Microbiologist must also participate in patient/community education programme through various interactions schemes. One can write manuals, textbooks, or even blogs to disseminate the knowledge to the masses. A microbiologist can interact with government and help in identification of deficiencies and formulations of new regulations. As an active member, one must participate in debates and deliberations of regional as well as national level associations of microbiologist at conferences. 

Although possibilities are many, often opportunities are few. Yet, one must make honest attempts to expand the roles a microbiologist can have.

Sridhar Rao...

Saturday, June 13, 2015

Chip makes a quick decision about antibiotics


Let me revisit the topic of antibiotic resistance. Antibiotic misuse is a leading cause of resistance. The misuse arises due to two reasons- unwarranted broad spectrum antibiotic prescription and unregulated use There is a lot of literature reporting the inappropriate use. This stems from having to guess the organism and start therapy before the lab tests are back. Guessing the organism is probably the easier part but predicting the antibiotic is more difficult. This has been eased to an extent by surveillance studies that identify the local antibiogram pattern. However, given the evolving nature of bacteria many a times the best guess is not even near good. There are several important questions and concepts when it comes to use of antibiotics. I recommend you read a blog post by Sridhar Rao

This is a standard clinical problem in most parts of the world. The problem arises from the need to grow the organism for testing. An alternative is genomic or proteomic based testing. These methods are expensive. Though whole genomic sequencing and MALDI TOF instruments are pushing the boundaries its application is still not in wide use. Also use of such methods require that resistance signatures are already known in the database. This has pushed the need to bring in systems that are rapid, reliable and low cost. A new paper published in LabChip by Justin Besant and team looks into just that. As Besant comments, "Guessing can lead to resistance to these broad-spectrum antibiotics, and in the case of serious infections, to much worse outcomes for the patient, we wanted to determine whether bacteria are susceptible to a particular antibiotic, on a timescale of hours, not days."

Fig 2: Representative differential
pulse voltammograms. Source
What exactly is antibiotic resistance? You can get so many definitions based on which guidelines are talking about it (Seriously!!). But perhaps the definition that nails it reads so, "If organism is active metabolically in presence of the inhibiting agent, that's resistance". That means if you can demonstrate metabolism in organism, given sufficient time of exposure we can detect resistance. As mentioned in the paper there are several methods of achieving this under research such as detection of bacterial motion using AFM cantilevers, electrochemical measurements of bacterial growth, optical detection of bacterial growth, and optical detection of redox reporters of bacterial metabolism.

Fig 1: Schematic of in‐well
bacterial capture. Source
In the study the researcher's used a microfluidic chamber, containing microbeads trapping the bacteria from sample. This is trapped in the nano-well, containing antibiotic and resazurin. Resazurin is a proven redox indicator used in detecting bacterial metabolism. By comparing the electrochemical signature of resazurin, the test indirectly measures the metabolism and thus resistance. The test is extremely rapid owing to micro volumes and results are quite fast. Indeed the turn-around time was as short as 30 min (Not even genetic test is that fast

Of course this technique would require some additional tuning and standardization for routine microbiology tests. But I think this technique is going to make it to the market, as a benchtop automated analyzer. As the author puts it, "The electronics for our electro-chemical readout can easily fit in a very small benchtop instrument, and this is something you could see in a doctor's office, for example. The next step would be to create a device that would allow you to test many different antibiotics at many different concentrations, but we're not there yet."
Besant JD, Sargent EH, & Kelley SO (2015). Rapid electrochemical phenotypic profiling of antibiotic-resistant bacteria. Lab on a chip PMID: 26008802

Wednesday, June 10, 2015

MERS COV- Outbreak in Korea


With advances in ability to do high throughput DNA sequencing, it has become easier to trace and study infections that has been previously known. This is evident from the recent literature. A lot of new strains have been shown to cause infections sometimes in small pockets of areas, sometimes making big news. I have tried to avoid talking about such pseudo outbreaks with few cases. However it is difficult to say what constitutes an outbreak.  The standard definition states, ":A sudden increase in number of cases by a specific strain of pathogen". If you analyze the definition fair enough, that's a very sloppy definition. 

Fig 1: Number of cases of MERS
Data from WHO; June 10, 2015
The world is talking about, what looks like a possible outbreak of MERS virus infection. I have previously had an in depth discussion of MERS virus (Link). As of today, a total of 108 cases have been confirmed with 9 deaths (Link). The index case was notified on 20 May by WHO. The case was a 68-year-old with a history of travel to 4 countries in the Middle East. The case was non symptomatic during return on May 4. Subsequently, after a week the case developed symptoms and sought medical care at 2 outpatient clinics thereby had sufficient time for potential spread. The contact tracing identified 25 confirmed cases and the numbers have been slowly growing since.

Fig 2: Age of people- MERS fatality.
I have received a couple of queries asking how fatal or worried should people be about this infection. So I looked into what I can ind from literature about this current outbreak. I couldn't find anything solid, but had an impression that the virus seems to be fatal only in cases where there is increased risk. Age and co-morbid conditions play an important role. So I gathered the statistics, from around the web and found that all were aged. The minimum age was 57 years, female. I have given the stats in Figure 2. I have tried my best to keep the source as reliable as possible. A case of teenager has surprisingly been recorded to be severely infected, but reports indicate the case is stable.

I was also interested with the distribution of cases, but found Wikipedia already has a updated information. See Figure 3. Looking at the graph, a natural question that was asked is the genetic status of virus. I have seen a couple of internet pages already arguing evolving status. The genetic sequence has already been made available and it hasn't varied enough from past sequences from the Middle East.

South Korea and China are currently under high alert situation and have quarantined several contacts with known confirmed cases. There is scare in public about the virus. With more than 2800 people under quarantine, 108 confirmed cases of which there is 9 fatal the actual fatality rate appears to be quite low. Here's the punch point. In a recently published data, 0·15% of people of healthy people were seropositive for MERS-COV antibodies from Saudi Arabia. This makes me think that probably lot less people are even exposed to this virus. Perhaps those who are in contact with camels. There appears to be a significant risk of virus infection, but a negligible probability of clinical significant serious case. But at this point the data is not enough to make a strong case. Further the dynamics of immune response is not well known for MERS infection and hence interpreting serological studies needs a caution. With publication of a more realistic animal model for studying MERS, some insights are bound to come.

Photo 1: MERS scare in S Korea. Source
TWiV has devoted a significant amount of time in its show discussing the outbreak (Link). The impact of the outbreak is summed in this wedding photo. Hilariously, surgical mask is not a good protection against virus. It needs to be at least a N90 grade to be really useful.
Butler, D. (2015). South Korean MERS outbreak is not a global threat Nature DOI: 10.1038/nature.2015.17709

Butler, D. (2015). South Korean MERS outbreak spotlights lack of research Nature, 522 (7555), 139-140 DOI: 10.1038/522139a

Butler, D. (2015). How to make sense of MERS outbreak data Nature DOI: 10.1038/nature.2015.17729

Müller MA, Meyer B, Corman VM, Al-Masri M, Turkestani A, Ritz D, Sieberg A, Aldabbagh S, Bosch BJ, Lattwein E, Alhakeem RF, Assiri AM, Albarrak AM, Al-Shangiti AM, Al-Tawfiq JA, Wikramaratna P, Alrabeeah AA, Drosten C, & Memish ZA (2015). Presence of Middle East respiratory syndrome coronavirus antibodies in Saudi Arabia: a nationwide, cross-sectional, serological study. The Lancet. Infectious diseases, 15 (5), 559-64 PMID: 25863564

Tuesday, June 02, 2015

Predatory Bacteria- Alternative to Antibiotics??


Antibiotics and Antibiotic resistance never seems to be an out of news topic. I have posted several posts on this theme and probably doesn't need a fresh introduction. Antibiotic resistance is something which we can not eliminate (Link). With much less incentive to work on new antibiotics and reports of antibiotic resistance in more and more strains, alternatives are sought to fight the infectious disease Some of the approaches that have already been worked on (Link).

I was reading a nature news piece (Link), and it stuck me that there are actually a good set of options that can be worked upon. Other than newer antibiotics and some new strategies of antibiotic designs (such as Antimicrobial peptides, Quorum inhibitors etc), Phage therapy (Link), Engineered bacteria (Link) all about which I have written already. However, I never knew that naturally occurring bacteria such as Bdellovibrio bacteriovorus, which can kill other bacteria for survival. That actually makes a good topic to talk about.

Photo 1: Micavibrio preying
on Pseudomonas.
These interesting set of bacteria called as Predatory bacteria. There is a very little research on predatory bacteria and its usefulness as a therapeutic. Predatory bacteria have been studied as early as the 60's. Some of the earliest known members include Vampirococcus, Daptobacter and Bdellovibrio. Based on the mode of attachment of the predator to the victim bacteria, 3 types are recognized- Epibiotic, cytoplasmic, and periplasmic. These predatory bacteria are usually chemoheterotrophs which steal nutrients from other bacteria by attaching themselves to other bacteria. Perhaps the most interesting among them are the epibiots. They simply attach to the cell wall and then start growing into the cell killing the prey bacteria.

A organism called Micavibrio aeruginosavorus preys on Pseudomonas aeruginosa, Burkholderia cepacia, Klebsiella pneumoniae, and numerous clinical isolates of these species. Unlike antibiotics, these organisms can prey successfully even in the presence of biofilms. Micavibrio is usually found in soil environment and especially in waste water areas where you had expect organisms like Pseudomonas. Micavibrio can breakdown a population of P. aeruginosa biofilms by a tenfold margin. In laboratory tests published by Kadouri etal; it killed 104 isolates out of 120 strains isolated from patients. That is pretty convincing, as to its potential value. As it appears in a page in live science, "It is possible that a living antibiotic such as M. aeruginosavorus because it so specifically targets certain pathogens could potentially reduce our dependence on traditional antibiotics and help mitigate the drug-resistance problem we are now facing". There are a growing list of these organisms. For example Bdellovibrio can attack Escherichia coli and Salmonella. Basically M aeruginosavorus sucks out innards of prey, and Bdellovibrio bacteriovorus, burrows and reproduces within its prey.

Fig 1: Micavibrio predatory cycle.
It is interesting to think about why certain bacteria are predatory and host specific. At least in case of Micavibrio, it senses the prey bacteria through quorum sensing which triggers a set of genes thereby building a flagella. It then moves to the prey and switches its genes to another phase triggering a different set of genes. The quorum sensing to an extent explains the ability to have a narrow host range. Further, Micavibrio doesn't have any genes for amino acid transporters, which means it has to directly pull it out from host machinery thereby giving it a phage like behavior which comes with invasiveness.

Despite the straightforward looking aspects, it is not yet ready for clinical applications for a variety of reasons. The complete host range of predator bacteria is not known and hence its effects on host microbiome is not known. Additionally, in lab conditions the predator evolves at a steady pace allowing it to attack more diverse bacteria. This forms the greatest concern as to what its effect would be. The second concern is how we would respond to these organisms, especially in terms of immunity and toxicity of predatory bacteria to host if any.

The follow up experiments by Kadouri etal, probably answers the second question to an extent. They tested ability of Micavibrio and Bdellovibrio to attack clinical Pseudomonas aeruginosa and Serratia marcescens isolates from ocular infections. Also the study assessed effects of predators on human corneal-limbal epithelial and found no significant impact. The authors do caution that these maybe applicable to only surface level infections such as in the case of wounds and burn infections.

However, let me put a warning sign. The reason why we seek alternates to antibiotics is the resistance factor. In all the published studies there were at least a few clinical strains that were resistant to attack. That gives a clue that resistance is possible and already exists. So, when a lot of people are arguing that acquiring resistance is difficult in this case, I'm really skeptic about such conclusions. But this is an exciting addition to combat infectious organisms especially MDRs. And with US$16 million in research grants approved to study in this field a lot of papers data is going to be generated.
Kadouri D, Venzon NC, & O'Toole GA (2007). Vulnerability of pathogenic biofilms to Micavibrio aeruginosavorus. Applied and environmental microbiology, 73 (2), 605-14 PMID: 17098913

Wang Z, Kadouri DE, & Wu M (2011). Genomic insights into an obligate epibiotic bacterial predator: Micavibrio aeruginosavorus ARL-13. BMC genomics, 12 PMID: 21936919

Shanks RM, Davra VR, Romanowski EG, Brothers KM, Stella NA, Godboley D, & Kadouri DE (2013). An Eye to a Kill: Using Predatory Bacteria to Control Gram-Negative Pathogens Associated with Ocular Infections. PloS one, 8 (6) PMID: 23824756