Tuesday, September 27, 2016

BtB#10- Probiotics

Greetings

I'm very much sure that you have at least heard of the term "Probiotics" though many are not quite familiar with what it actually is. The concept of Probiotic was proposed by Elie Metchnikoff (1907) where he theorized the advantages of using a host-friendly bacteria in health improvement. The credit of introducing the term (1953) goes to Werner Georg Kollath, a German bacteriologist, hygienist and food scientist. Digging back into the history, the term Probiotic has had different definitions. The first definition (1965) was, a substance secreted by one microbe that enhances or stimulates the growth of another. This has changed to, live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.

There are many different probiotic formulations available in the market. The exact composition varies but almost all of the validated products have Lactobacillus and Bifidobacterium species incorporated into it. A common question asked is, if regular consumption of Probiotic good and do these microbes impact improvement in health? Though it has been debated, most experts agree that commercial probiotics are not something you want in your regular diet. Probiotics works in several ways. For example, Lactobacillus species (also found in yogurt and curd) are known to produce vitamins of interest and boost immune functioning. Bifidobacterium species are known to regulate other commensals in the gut. 

Fig 1: Prebiotic vs Probiotic.
Source
Probiotics are often recommended when there is a microbial imbalance. Currently, probiotics mainly target gut microbiome. In cases where a patient is on antibacterial therapy, where gut microbiome is bound to be influenced, probiotics are recommended. One question is if the probiotic bacteria themselves have a resistance pattern. Studies have shown that probiotic bacteria are resistant to many different drugs (depending on the species involved in the formulation). This may be of indirect help since, probiotic microbe shouldn't be attacked by the antibiotic so that they are available to do their work.

A second not so common but the related term is "Prebiotics". Prebiotics are a non-digestible food ingredient that promotes the growth of beneficial microorganisms. The important differences between  probiotic and prebiotic are shown in Fig 1.

Table 1: Effects of Probiotics as an adjuvant. Source 
Considering that there is a lot of research in recent times indicating the importance of microbiome, certain experts have opinioned that probiotics could be used as an influencer to modulate the biome. For example, Several candidate rotavirus vaccines had low efficacy in clinical trials which were peculiar to a subpopulation. Apart from genetic factors, certain studies have indicated possibility of microbiome as an important determinant of efficacy. Also considering that microbiome is a known factor in immune development. So does the probiotic qualify as a vaccine in its own right? Well, that could be debated. Probiotics are also being researched as a vaccine adjuvant (See Table 1 for some examples), Psychobiotics etc. 

It is a wrong idea that probiotics are only for the gut. The concept of probiotics can be used in any context where a microbiome is involved. For example, skin is highly covered by microbiome. There is a substantial research evidence that probiotics can play a significant role in skin heath, such as improvement in atopic dermatitis, healing of scars and improving skin’s innate immunity.

It is already a recommended practice according to many guidelines, to take probiotics as an adjunct to antibiotics. In future (Im slightly overblowing the conept), I anticipate it will be a custom to profile a person's microbiome and then give them a probiotic configuration accordingly to help in the treatment of multiple conditions. 

Monday, September 26, 2016

BtB#9: Bacterial growth curve

Greetings,

Most of the readers are quite familiar with the textbook concept of doubling time. Perhaps the best known example is E coli with a generation time of about 20 min. But how far a bacteria can push. Can it be anymore faster? Scientists have worked out the thermodynamic limitations of a bacterial cell division (Link). The model basically implies that theoretically, E coli has a potential to push its doubling time further but it is already in its best possible practical limits.

Fig 1: Comparison of Generation time of various bacteria.
In a recently published blog post by Elio, which does a great job of explaining bacterial generation time, there is a mention of E coli strain which can double at about 12.5 min. Another bacterium Vibrio natriegens, is probably the real fast one with a generation time of less than 10 min. The strain DSMZ 759 takes approximately 9.8 minutes. There is also mention of 7 min as a time under particular conditions for this species. That is an incredible speed. See Fig 1 for a general comparison of doubling time in different well-studied species (The comparison is generalised for a species, since strain variations are known). On the other side of the spectrum, there is M leprae with the longest known generation time of about 14 days (Compare that with Mycobacterium tuberculosis for which it is about 18 hours)

Before I go further down the discussion, let me first talk about basics of Bacterial growth curve. Everyone is familiar with the curve shown in Fig 2, so let me be very brief. 

Fig 2: Typical bacterial growth curve. Source
Growth has a different meaning in different context. Growth can mean an increase in cell size or cell numbers. Historically for studies on bacteria, it was not convenient to investigate the growth and reproduction of individual microorganisms because of their small size. The concept is an average value for the population, and therefore we deal with the total population number. So it's important to note that the demarcations are not absolute. Not every cell is in “Lag” in lag phase and not every cell is replicating in log phase. You have to take it as an average of the majority.

Before I go down further, I want to digress on a point which I think is an important distinction to make. What is the distinction between binary fission and eukaryotic cell division? In Binary fission, other than the fact that there is no true nucleus for “nuclear events” of division, the cell division ensures equal distribution of chromosome contents only. Binary fission in bacteria doesn't control for cytoplasmic contents to be equally divided between daughter cells. For example, one of the daughters may or may not receive all the plasmid copies from its parent cells. In sharp contrast, in eukaryotic cell division every component of the cell (nucleus and cytoplasmic contents), is equally distributed between the daughter cells.

The bacterial growth curve is a concept of pure batch culture and not continuous culture. In a batch culture (also known as a closed culture), the bacteria are grown in a medium and condition best suitable for its growth, without replenishment. There is a limitation for nutrients. In contrast, bacteria growing in continuous culture are continuously replenished with media and toxic contents removed regularly and hence the cells don’t shift to stationary phase.

Bacterial growth curve has the following set of assumptions built into it
  • Pure batch culture
  • Nutrients are provided in excess, to begin with, and there are no inhibitors
  • Cells are given the best possible environment for growth
  • There is a very low number of the organism in the seed inoculum.
In a batch culture, nutrient concentrations decline and concentrations of wastes increase. The growth of microorganisms reproducing by binary fission is plotted as the logarithm of the number of viable cells versus the incubation time. The resulting curve has four distinct phases
  1. Lag Phase
  2. Exponential (Log) Phase
  3. Stationary Phase
  4. Decline (Death) phase
Lag Phase:

Fig 3: Lag Phase difference in
2 different cultures.
When a bacterium is inoculated into a fresh culture medium there is no immediate cell division and an increase in cell number. The bacterium senses the presence of a large number of nutrients and potential to multiply. The time period is used to synthesise materials for building the cell. At this phase, there is no increase in cell number but the cell tends to increase in size and metabolism slowly adjusts to the new environment. The time period of lag phase is variable and depends on multiple factors including the culture medium in use, where is the inoculum obtained from.

If the bacterial inoculum is obtained from the previous culture where the medium was similar and when it was in a log phase the lag phase will be very short (or may even be absent) since the cells are already adjusted and ready to replicate (Culture 1 in Fig 3). But if it's taken from a fragile state and the cells are damaged it will take some time for the cell to adjust and the lag phase will be longer (Culture 2 in Fig 3).

Exponential phase:
Fig 4: Growth rate depends on
Nutrient concentration.
Source: Prescott
Microbiology Textbook (5th Ed)
.
Once the cells are ready for replication they start dividing rapidly. At this time, the cells have no limitations placed and are primed for the division at the fastest rate possible. The peak performance of every cell is at the best possible rate and hence most cells are uniform in terms of their physiology and biochemistry.

All biochemical assays, antibiotic sensitivity assays performed in routine diagnostics is tested in this phase. This phase represents the best possible doubling time for a bacterial cell and doubling time is calculated in this phase. By increasing the concentration of nutrients we can increase the doubling rate. If we plot a graph (Nutrient concentration vs growth rate) we can see that it’s a hyperbolic curve (See Fig 4). At a very high nutrient level, the transport systems are saturated, and the growth rate just cannot rise any further.

I want to make an interesting example of E coli to illustrate how the speed of replication is enhanced. In E. coli, DNA replication starts at a unique site (called as OriC) and replicates through a mechanism called as theta replication. The E coli genome is roughly 4.8 × 106 bp genome. It is theoretically estimated that one round of complete DNA replication requires roughly 36-38 minutes to complete in E coli. But E coli can double in less than 20 min. The trick is that as the daughter DNA strand is synthesised a granddaughter DNA is synthesised simultaneously (by using daughter DNA as a template) which is catching up. By the time the E coli cells divide, their DNA is almost doubled and ready for the division again.

Stationary phase:

The cell numbers have divided rapidly and reached a peak at this stage. The cell number has usually increased by about 6-9 fold (Depending on conditions and strain). At this phase, the total number of viable cells roughly remains the same throughout. This is maybe because the cells cease to divide or number of cells dying is roughly equal to the number of cells being formed. In stationary phase, bacterial cells are unusually resistant to many different chemicals. The bacterium often shows shrinkage and nucleoid condensation. Bacteria also produce proteins called as starvation proteins which make the cell much more resistant to damage. For example, The Dps (DNA-binding protein from starved cells) protein protects DNA. There is some evidence that certain pathogens at least (such as S Typhimurium) express more virulent genes in stationary phase.

Death Phase:

The bacteria have virtually exhausted everything that medium had and the culture medium has become unfavourable for growth. The cells start dying at this phase. Interestingly, the phase is not a steep curve but rather follows a complex log scale. A majority of the microbial population dies in a logarithmic fashion, the death rate decreases after the population has been drastically reduced. This is most probably due to the resistance offered by persistent cells. In bacteria with the ability for spore formation, spore formation begins with the end of the stationary phase and in the decline phase, is mostly left with spores.

Now coming to some real world questions.

What phase are bacteria in when it is in a host? The answer is not straightforward for a couple of reasons. The host represents an unlimited nutrition supply at the bacterial scale, but host also fights the bacteria and hence there are significant inhibitions to achieving a log phase. It has often been quoted that T pallidum pallidum has a doubling time of about 33 hours. However, this is based on growth in Rabbit testes (Not ideal since the host is involved in the dynamics) and I would want to cast my doubt on how accurate that is.

Second question, What is the limit of bacterial doubling time in terms of speed? In other words, how fast could a bacteria divide? There are theoretical projections that we could design something to be about 4 min max. I'm not sure of if any lab has achieved this. One proposed way of doing it is to artificially engineer a cell with as much reduced genome as possible. Craig Venters Lab, has already created an artificial cell called as Syn3.0. Syn3 contains a synthetic genome inserted into Mycoplasma which on its own was able to grow and live like a normal cell. The cell carries a total of 473 genes (of which function of 149 essential genes is unknown). Syn3 has a doubling time of roughly 3 hours in ideal conditions which is far high for an organism with just so few DNA. Very large genome organisms can have a quick replication time. So it is not logical to conclude that DNA length is the rate limiting step. A more logical idea is to include DNA polymerases that are known to be having faster addition rate are included in the genome with more origins of replication included, that would do the trick.

For now, 7 min appears to be the fastest.

Friday, September 16, 2016

Influenza D Virus

Greetings

Influenza is a topic about which I have written a good number of posts (Link). Influenza virus is a member of Orthomyxoviridae characterised by a single-stranded segmented RNA genome. The influenza viruses are classified into types A, B, C and D on the basis of their core proteins. Influenza A and B contains 8 genome segments whereas C and D contain 7. The evidence that Influenza D is significantly different from C comes from 2 lines- Genetic studies showing large differences and hemagglutination inhibition assays, showing a lack of cross-reactivity. Till recently, the name "Influenza D" has been tentatively used. Recently the executive committee of ICTV has officially approved the nomenclature.

Fig 1: Structure of Influenza D Virion. Source
Structurally, Influenza D virus (IDV) is an enveloped, rounded virus measuring 80-120 nm in diameter. The genome is segmented ssRNA(-) linear genome, encapsidated by nucleoprotein (NP). Contains 7 segments coding for 7 proteins. Viral RNA polymerase (PB1, PB2 and PA) transcribes one mRNA from each genome segment. Transcription is primed by cap snatching. See Fig 1. A major distinguishing feature of IDV is the hemagglutinin-esterase (HE) fusion protein, which is responsible for receptor recognition, viral fusion, and destruction of the host receptor. IDV has approx 50% amino acid identity to human influenza C virus (ICV). The segments of IVC and IVD that contain PB1 share a 72% identity, while the segment containing HE has diverged further to 53%. IDV, (similar to ICV) uses 9-O-acetylated sialic acid as its receptor.

IDV is primarily an infection of the cattle. Research has conclusively established that IDV can transmit between cattle but it is not clear as to what is the risk of human disease. The virus can clearly infect human cells in cell culture models, and transmission in ferrets has been documented. Serological positivity has been documented against IDV from people with occupational contacts with cattle.

Pigs and cattle are the primary natural reservoir. The IDV can be isolated and cultured in swine testicle at 37 C. IDV has been isolated from pigs in Oklahoma and from cattle in China, France, and the United States, including the states of Minnesota, Kansas, Nebraska, Oklahoma, and Texas. I did a literature search but couldn't find any reported human IDV cases.

It appears that Influenza D is not as big as a threat as some people have projected it to be for a couple of possible reasons. First, IDV appears to be a very stable clone and hence antigenic variability is very low. In contrast with Influenza A, this means that herd immunity is easy to achieve. Second, IDV cases are not reported, though serological positivity is documented. This implies that probably the virus can cause subclinical infection but is not a significant health threat.

References:

Ferguson L, Olivier A, Genova S, Epperson W, Smith D, Schneider L et al. Pathogenesis of Influenza D Virus in Cattle. J Virol. 2016;90(12):5636-5642. 

White S, Ma W, McDaniel C, Gray G, Lednicky J. Serologic evidence of exposure to influenza D virus among persons with occupational contact with cattle. Journal of Clinical Virology. 2016;81:31-33.

Song H, Qi J, Khedri Z, Diaz S, Yu H, Chen X et al. An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism. PLoS Pathog. 2016;12(1):e1005411.

Saturday, September 10, 2016

Guest Post- Vibrio parahaemolyticus

Greetings,

I'm introducing Dr Pendru Ragunath (Associate Professor , School of Medicine, Texila American University Guyana, South America) whom I had the privilege of talking to. He has published a lot on V parahemolyticus and so, I requested him if he could please share some of his knowledge. He has happily sent me his thoughts which I'm posting here.

--------------------------------------------------------------------------------------------------------------------------

Global warming might increase the number of Vibrio parahaemolyticus outbreaks

Vibrio parahaemolyticus is a gram negative, halophilic bacterium that occurs in estuarine environments worldwide. It was first identified as a foodborne pathogen in Japan in 1950. By the late 1960s and early 1970s, V. parahaemolyticus was recognised as a cause of diarrhoeal disease worldwide, although most common in Asia and the United States of America. Seventy percent of the seafood-borne gastroenteritis cases in Japan are due to V. parahaemolyticus and in India, this organism accounts for about 10% of gastroenteritis cases admitted to the Infectious Diseases Hospital in Kolkata. With the emergence of a pandemic clone of V. parahaemolyticus, this organism has assumed significance in recent years. Vibrios concentrate in the gut of filter-feeding molluscan shellfish, such as oysters, clams and mussels, where they multiply and cohere. Although thorough cooking destroys these organisms, oysters are often eaten raw are the most common food associated with V. parahaemolyticus infection. 

However, not all strains of V. parahaemolyticus are pathogenic. Although the mechanism by which this bacterium causes enteric disease is not fully understood, clinical isolates most often produce either the thermo stable direct hemolysin (TDH) or TDH related hemolysin (TRH) encoded by tdh and trh genes, respectively. TDH and TRH are considered major virulence factors in V. parahaemolyticus. The occurrence of tdh and/or trh genes among environmental V parahaemolyticus isolates typically 1–10%, but this depends on location, sample, source and detection method. Detection of tdh bearing V. parahaemolyticus is conventionally studied by their ability to produce β-haemolysis on a high-salt blood agar called Wagatsuma agar. The reaction is called Kanagawa phenomenon (KP), which requires fresh human or rabbit blood and tends to give false positive reactions and 16% of KP-negative strains as studied on Wagatsuma agar were found to carry the tdh gene. There is no commercially available detection method for TRH. Therefore, it would be important to detect the virulence genes of V. parahaemolyticus in clinical samples as well as in seafood by DNA-based molecular techniques such as polymerase chain reaction (PCR) and colony hybridization.

Both genes, trh and tdh share approximately 70% homology. Similar to TDH, TRH also activates cl− channels resulting in altered ion flux. Although TDH and TRH correlate with pathogenic strains, they do not fully account for V. parahaemolyticus pathogenicity. Several studies have reported that some of the clinical strains do not contain tdh and/or trh. Even in the absence of these hemolysins, V. parahaemolyticus remains pathogenic indicating other virulence factors exist.

The atmosphere affects oceans, and oceans influence the atmosphere. Because of global warming, the temperature of the air rises, oceans absorb some of this heat and also become warmer. Overall, the world's oceans are warmer now than at any point in the last 50 years. The change is most obvious in the top layer of the ocean, which has grown much warmer since the late 1800s. This top layer is now getting warmer at a rate of 0.2°F per decade. Oceans are expected to continue getting warmer, both in the top layer and in deeper waters. Even if people stop adding extra greenhouse gases to the atmosphere now, oceans will continue to get warmer for many years as they slowly absorb extra heat from the atmosphere. 

Temperature has been found to be a major factor in both the seasonal and geographical distribution of V. parahaemolyticus in shellfish-growing areas of the temperate regions. V. parahaemolyticus bacteria grow in seawater and can end up in shellfish like oysters and clams. When water temperatures rise in the summer, the accumulations of the naturally occurring bacteria increase to the point that eating undercooked shellfish can give people nausea, fever and diarrhoea. Based on their results, a group of researchers from Netherlands, derived an empirical formula to predict the Vibrios concentration as a function of temperature. According to that study, for an average temperature increase of 3.7°C, V. parahaemolyticus illness risks were calculated to be two to three times higher than in the current situation. If such extreme situations occur more often during future summers, then the number of V. parahaemolyticus outbreaks might increase.

References

Raghunath, P., Pradeep, B., Karunasagar, I., and Karunasagar, I., 2007. Rapid detection and enumeration of trh-carrying Vibrio parahaemolyticus with the alkaline phosphatase-labeled oligonucleotide probe. Environmental Microbiology 9, 266-270. 

Raghunath, P., Acharya, S., Bhanumathi, A., Karunasagar, I., and Karunasagar, I., 2008. Detection and molecular characterization of Vibrio parahaemolyticus isolated from seafood harvested along the southwest coast of India. Food Microbiology 25, 824-830.

Raghunath, P., Karunasagar, I., and Karunasagar, I., 2009. Improved isolation and detection of pathogenic Vibrio parahaemolyticus from seafood using a new enrichment broth. International Journal of Food Microbiology 129, 200-203.

Kumar, K., Raghunath, P., Devegowda, D., Deekshit, V. K., Venugopal, M. N., Karunasagar, I. and Karunasagar, I. 2011. Development of monoclonal antibody based sandwich ELISA for the rapid detection of pathogenic Vibrio parahaemolyticus in seafood International Journal of Food Microbiology 145, 244-249. 

Raghunath, P., 2015. Roles of thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) in Vibrio parahaemolyticus. Front. Microbiol. 5, 805.

- Pendru Raghunath

If you have any queries, please contact Dr Pendru Raghunath at raghunath_kmc@yahoo.co.in or raghunathreddyp@gmail.com

Saturday, September 03, 2016

Announcing Microbiology Talk- Podcast

Greetings

All those readers who have been reading my blogs for a long time and following the contents might be aware that I had long ago announced that I would be interested in working on a podcast. However, my end of the issue was I was not sure how and didn't have team mates then. Also, my tech knowledge to create the content was (and still is), quite limited.

Podcasting is not a new phenomenon. There are several different high-quality podcasts out there done professionally and appealing to a wide range of audience. I regularly listen to several of them. Interestingly, medical microbiology related posts are less (There are a still a significant few of them). I also realised that there is really no microbiology podcast from the South-east Asian region and that would be something really useful to have.

Blogging has given me a great platform to communicate and work on certain topics, but blogging has its own set of limitations, which can be masked in a podcast platform. So, a team has been gathered. All of them have a great base in blogging and microbiology communication. The team includes-

Sagar Aryal the "techy guy" who made the website and created the technical platform
Saumyadip Sarkar the "communication guy" who does recording, editing and contacting scientists.
Sridhar Rao the "Mentor" who has made sure things are in order
And me...

We have already recorded 3 episodes and 2 are available online. I understand that we are learning how to do this stuff and create a good audio content. Everyone is working on creating better content, and it is getting better with each episode. The first recording was about biofilms, the second on Zika and we are soon going to release a new episode on Leprosy vaccine.

The show will host educational audio content about basic microbiology, interesting recent research findings and will be inviting researchers from the field to talk about their work. We are already working on one (Details are a secret until you listen to the show). Also, if you want someone to be there on the show we will welcome them also. All details are there on the Website. Check out all tabs.

If you are reading this post, you are obviously interested in microbiology and so I believe you will be interested in the audio content (Podcast). Give it a look, download, listen, subscribe and keep listening. For we are going to go big from here on. So, get on to the following links

Subscribe in iTunes: https://itunes.apple.com/us/podcast/microbiology-talk-mitalk/id1146831417

And meanwhile also let us get some idea of what people expect from an educational podcast by answering this questionnaire. It will not take more than 5 minutes of your time.