Monday, December 26, 2016

Break announcement- 4

Greetings

Fig 1: My Blog Statistics (Page hits) till date.
As has been the recent custom in this blog, am taking a official break from blogging. I would consider that I have successfully completed 5 years of blogging, and the end of this year has seen a steady rise in readership base. Including this post, I have posted a total of 54 posts for the year 2016. Thats not bad. That almost one post a week. I encourage you to go back and read my earlier posts that have been missed.

Wish all my readers, a happy festive time, vaccation and a happy new year in advance. I will see you back in 2017, with exciting new stories of microbiology.

Wednesday, December 21, 2016

Mycobacterium Tuberculosis CdnP hijacks Immune response

Greetings

M tuberculosis is a topic that has come up several times in this blog. Tuberculosis is rampant in many developing and underdeveloped countries. The lead problem with combating tuberculosis has been difficulty in laboratory diagnosis, which has been now addressed with new generation cheaper tests which are available worldwide. An equally enormous challenge is treatment, which is due to its long treatment time often ranging to several months. That makes vaccination and exceptionally important tool. Though a universal vaccination campaign is in working order (BCG vaccine), TB is something that has not been eradicated or even controlled.

TB-immunology is a deeply researched topic. As I have explained in multiple earlier blog posts, it is wrongly conveyed by many that TB doesn’t cause an immune response. TB infection does elicit a strong Type IV immune response. This is evident from tests such as Mantoux positivity and granulomatous histopathological findings. What is not achieved is, the immune response is not directed enough to induce pathogen clearance. There are several factors at an independent level which coordinates the immune resistant ability- Intracellular localisation being one among them.

So, give this question a deep thought. Does all intracellular pathogen (Keep the question limited to bacterial pathogens for simplicity) cause chronic infection via resistance to immune attack? The answer is definitely “No”. So mere sitting inside a cell doesn’t help, since intracellular location in itself isn’t a very good defence. Cytosolic surveillance pathway such cGAS-STING pathway can recognise intracellular pathogens and stimulate an innate immune response. These pathways have been well studied for viral infections, but not for bacterial infections. See my earlier post on STING response pathway (Link)

A simple explanation goes something like this. When M tuberculosis infects and internalises into a cell such as a macrophage, the bacterial DNA and c-di-AMP signal is captured in the human cell as the bacterial entry. In response, a molecule called cGAMP is made which signals innate response. In reality, Dey et al;2016 (Paper published in Nature chemical biology) have identified that this signalling never quite happens.
Fig 1: Graphical abstract of the study. Source
The findings of the study are schematically represented in Fig 1. Bacterial cyclic dinucleotide phosphodiesterase (CdnP) action reduces immune detection of bacterial derived c-di-AMP and c-di-GMP and host-derived 2′3′-cGAMP by degrading them into non immunogenic nucleotides. These molecules being danger signals, non detection of these signals leads to numbing of innate response pathways. Host cells possess a control system called ENPP1, which degrades the 2′3′-cGAMP thereby controlling the inflammation. The researchers show that ENPP1 can also degrade bacterial cyclic di-nucleotides. To further look into the evidence, researchers created TB mutants for CdnP and infected a mouse model through aerosol route.

Fig 2: PDEi tested for CdnP inhibitory activity. Source
They showed that indeed there was a significant difference in survival and lack of CdnP was associated with increased survival. This is an indication that CdnP is a possible drug target. So the group tested several phosphodiesterase inhibitors (PDEi) including some FDA approved drugs- Tadalafil, Sildenafil and Cilomilast and Cilostazol. Fig 2 shows commercially available PDEi tested for their ability to inhibit CdnP activity. FDA-approved drugs are marked as *. Though the study shows possible PDEi the authors caution that they are not optimal for development as drugs owing to potential membrane-permeation liability. Further, these PDEi may cross react with ENPP1 which may further impact the outcome.

Herman O Sintim, one of the authors in the paper comments, “The host cGAMP never gets to a high enough concentration to activate the immune response. This is a very effective strategy the bacteria have developed to suppress an immune response.”

  Dey R, Dey B, Zheng Y, Cheung L, Zhou J, Sayre D et al. Inhibition of innate immune cytosolic surveillance by an M. tuberculosis phosphodiesterase. Nature Chemical Biology. 2016. doi:10.1038/nchembio.2254

Wednesday, December 14, 2016

Microbiome and the Parkinson's

Photo 1: An essay on the shaking palsy.
Greetings

The concept of microbiome being involved with clinical conditions is now a heavily discussed concept. I have written several posts about this topic and readers are referred to my earlier posts for details. Most recently, a paper published in Cell has explored the connection between Microbiome and Parkinson's disease, which is making a lot of news. Certain findings of the paper have been hyped in the media and so I decided to write a detailed post about this paper.

Parkinson's disease (PD) is a chronic, progressive movement disorder (a subtype of motor system disorders) caused due to a loss of dopamine-producing brain cells. The condition was long known but gained its name and importance following a publication "An Essay on the Shaking Palsy" by a London doctor James Parkinson in 1817. PD is a type of Synucleniopathy (or rather called as α-Synucleinopathy). Synucleinopathies are neurodegenerative diseases characterised by the abnormal accumulation of aggregates of α-synuclein protein in neurons, nerve fibres or glial cells. In PD, it is postulated that the aggregates accumulate in dopaminergic neurons leading to death of the cells. However, there is no single explanation for the pathogenesis and biological pathway leading to PD.

The concept of gut microbiota and its link with Central nervous system is a theme I have talked about several times in this blog. Association of microbiome with neurodegenerative diseases is not a new publication, neither is the association with Parkinsons. A paper published in 2014 by Filip Scheperjans and colleagues, showed by fecal microbiome study from 72 patient vs 72 control subjects that there is a significant drop in abundance of Prevotellaceae in feces of PD patients by as much as 77.6%. In fact, Prevotella is also negatively assocaited with autism. Prevotella species is known to influence higher levels of neuroactive short chain fatty acids (SCFA) and a high capacity for biosynthesis of thiamine and folate, which is also in agreement with findings of lowered levels of short chain fatty acids in PD feces sample. Here is an interesting observation. Using intestinal biopsy of PD patients before onset, α-synuclein has been shown to be positive by immunostaining but negative for healthy controls. It has been shown that that newly diagnosed PD patients had increased intestinal permeability and abnormal accumulation of α-synuclein in enteric neurons. This also correlates with a significant number of papers showing that more than 70% of the PD patients on average have some gastrointestinal abnormalities, which precedes the appearance of motor symptoms.

In the latest paper in cell by Sampson et al; 2016, they looked into if microbiome can influence the Synucleinopathy. As stated by the senior author Mazmanian, "Because GI problems often precede the motor symptoms by many years, and because most PD cases are caused by environmental factors, we hypothesized that bacteria in the gut may contribute to PD."

Fig 1: Graphical abstact of the paper.
Source
The experiments were conducted using mice engineered to overexpress the protein α-synuclein. In the first experiment, 3 groups of mice was prepared- 2 groups had complex intestinal microbiome and one group was germ free (GF). Experiments measuring motor skills showed that germ-free performed much better. They also showed that GF mice treated with microbially produced SCFA activated microglia.

In the next set of experiment, GF mice were transplanted with fecal samples from human patients with and without Parkinson's disease. The germ-free mice who received transplants from PD patients showed a significant increase in PD symptoms. See Fig 1, for a summary.

Sarkis Mazmanian comments, "Our findings provide a completely new paradigm for how environmental factors may contribute to Parkinson's disease and possibly other neurodegenerative disorders. The notion that these diseases may be impacted by pathology in the gut and not only in the brain is a radical departure from conventional research in neuroscience. Parkinson's disease is complex and there are several genetic predispositions and environmental risks that play a role, but we believe our findings shed light on a previously unrecognized and potentially important part of this puzzle."

It must be noted that this study uses a mouse model overexpressing α-Synuclein. It is unilkely that human PD can be treated with fecal microbiome transfer or use of probiotics. But what is more likely is use of probiotc may alleivate the PD symptoms.

References

1. Scheperjans F, Aho V, Pereira P, Koskinen K, Paulin L, Pekkonen E et al. Gut microbiota are related to Parkinson's disease and clinical phenotype. Movement Disorders. 2014;30(3):350-358. 

2. Shannon K, Keshavarzian A, Dodiya H, Jakate S, Kordower J. Is alpha-synuclein in the colon a biomarker for premotor Parkinson's Disease? Evidence from 3 cases. Movement Disorders. 2012;27(6):716-719. 

3. Sampson T, Debelius J, Thron T, Janssen S, Shastri G, Ilhan Z et al. Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson’s Disease. Cell. 2016;167(6):1469-1480.e12.

Wednesday, December 07, 2016

Blogger's Desk#10: Top 10 My Picks of Microbiology Blogs

Greetings

I have been busy recently and haven't been able to post regularly in the last couple of days. I have a couple of things that I wanted to write about and more recently I have been getting a lot of mail requests to write more basic stuff, that is useful fro grad students. The "Back to basics" and "Laboratory series" section is being especially liked by many. In future, I would sure look into it more in the future.

There are several microbiology blogs out there on the web and many have a great readership base. As 2016 is nearing to an end, I want to post my picks of microbiology blogs worth reading. Please be aware that this is my top 10 and I have my personal bias built into this list. If you think I should have included one (which is not on the list),  please add a comment.

Let us start with a reverse countdown of my choices.

10. The Genome factory

Tagline: Bioinformatics tips, tricks, tools and commentary with a microbial genomics bent.
Contributor: Torsten Seemann
http://thegenomefactory.blogspot.com

Microbiologists by default, have a problem in understanding computational biology though most of us are working on this concept. The blog has some really good posts, but posts are very rare. There are hardly 2-3 posts a year, since 2011. So if you want to know something about the Bioinformatics, have a look at this page once.

9. Aetiology

Tagline: Discussing causes, origins, evolution, and other implications of disease and other phenomena.
Contributor: Taca C. Smith
http://scienceblogs.com/aetiology/

She has been writing since around 2005, and posts appear once in a month (skipped once in a while). The posts are true to the tagline with a good explanation. There is a wide range of topics in this post, and worth the time spent reading.

8. Infectious thoughts:

Tagline: The blog of Dr. Siouxsie Wiles, a microbiologist who knows a lot about bacteria, viruses and infectious diseases.
Contributor: Siouxsie Wiles
http://sciblogs.co.nz/infectious-thoughts/

The page has a lot of science information and written so well, no wonder there is a big reader base for this blog. The blog is not specifically focussed on microbiology and there are a lot of posts on other scientifically important stuff.

7. Living in a microbial world

Tagline: -
Contributor: Nicola Fawcett
https://livinginamicrobialworld.wordpress.com/about/

A blog that focuses a lot on microbial stuff and antibiotics. The blog is up and running but the posts are not very frequent and appear approximately once in a month. This blog is up since around 2015 and its lucid style makes it something of an interesting approach.

6. Microbiology Info

Tagline: Online microbiology notes
Contributor: Sagar Aryal
http://www.microbiologyinfo.com

This page is all about teaching basics to the undergrads who are looking for some basic information. There is no particular periodicity of publication, but he writes when he wants to. The pages are pretty much up to date and most of the information that is being sought by a learner can be found here. The page also has many other interesting things, such as an app for bacterial identification.

5. Prophage

Tagline: The Blog for Bacteria, Phages, Computers, and Science
Contributor: Geoffrey Hannigan
http://prophage.blogspot.com

The blog has a very broad focus on Prophage, and there are quite a good number of important updates about research and other related, interesting topics. Active since 2013, has a good number of posts.

4. Pharmaceutical Microbiology

Tagline: Microbiology, pharmaceuticals, healthcare and contamination control news and discussion site
Contributor: Tim Sandle
http://www.pharmamicroresources.com

Almost everyone who reads microbiology news knows Tim. Just google his name and you will get a huge list of articles he contributed as a science communicator. I'm not sure if his web page should be called a blog, but it is updated regularly and has a lot of content that a microbiologist should be mandatory aware of.

3. Contagion

Tagline: Contagions is place to collect some thoughts on history, infectious disease and science in general
Contributor: Michelle Ziegler
https://contagions.wordpress.com

A biologist with interests in public health that's how the author defines himself. The tagline is well justified by his posts. The way things are written makes it a never boring read.

2. Virology blog

Tagline: -
Contributor: Vincent Racaniello
http://www.virology.ws

A great deal of global population listens to his microbiology podcasts and his blog is full of interesting stuff. If you haven't read his posts, well you really haven't read a good blog. A must in blog roll.

1. Small things considered

Tagline- http://schaechter.asmblog.org
Contributor- Multiple
http://schaechter.asmblog.org

This is a blog that I have never missed to read in years. I have read every post in it and what inspired me to start blogging. If I recall it right, this blog is up since about 2006 and clearly is the best, professionally maintained. Quoting from their website, "The purpose of this blog is to share our appreciation for the width and depth of the microbial activities on this planet. We will emphasize the unusual and the unexpected phenomena for which we have a special fascination". Needn't highlight that they are part of the ASM, with high quality of information.


Tuesday, November 22, 2016

BtB# 11- Bacterial secretion systems

Greetings

A given individual bacteria has to compete and interact with multiple factors outside the cell which means communication is an important factor. Most of the molecules that are involved in such a process is a macromolecule protein (such as toxins and sensor molecules). The release of these molecules is subject to tight regulation. As already known, a bacterial cell structure is very highly organised with multiple layers. The cell envelope of Gram negative bacteria is composed of two membrane layers and a periplasmic space in between, whereas the envelope of Gram-positive bacteria consists of a cytoplasmic membrane followed by a very thick peptidoglycan layer.

The transport of molecules within such intact cellular structure is managed by specialised machinery called as secretion systems. The secretion system spans across the cell wall from the inner membrane to outer membrane. Currently, 7 types of secretion systems have been defined, each labelled as Type I- Type VII. For purpose of simplicity remember that T1SS (Type 1 secretion system) to T6SS is seen in Gram negative bacteria (which is further classified into Sec-dependent and Sec-independent) and T7SS is seen in Gram positive bacteria.

Bacterial secretion systems are highly diverse and each type has multiple subtypes which vary by its structural and regulatory details. An individual bacterial species can have more than one type of system for accomplishing multiple roles. In an old post on Toll-like receptors, I used the concept that instead of writing pages and pages of data I summarised it all into one single table, so it becomes easy to learn. I'm doing the same here.

Table 1: Bacterial secretion system.







Monday, November 14, 2016

Introducing mosquitos into wild to protect against mosquitos

Greetings

A long time ago, I had talked about the vector control program that can be used to intervene with mosquito vectored infectious disease. Perhaps there is no another species group that we hate so much than the mosquito. Of them, Aedes aegypti is responsible for several thousand infections a year globally since they aid in the transmission of Yellow fever virus, Dengue virus, Zika Virus etc. Biological vector control is a tricky business and not everyone understands what Oxitec is trying to do with genetically modified mosquitoes (GMM).

There are several members of Aedes species, and some of them are important in context as vectors of infectious diseases. It is simply meaningless to write in detail about all the important one's (That's not the point of this whole post anyway). See Table 1 for details

Table 1: Features of common Aedes species involved in infection transmission. Global distribution maps are obtained from Hay et al; 2015
Aedes aegypti is a holometabolous insect with 4 stages in life cycle including egg, larvae, pupae, and adult stage. Their life cycle happens in 2 phases- aquatic phase (larvae, pupae) and a terrestrial phase (eggs, adults). The number of days required for life cycle ranges depending on the environmental condition and roughly ranges from 10- 21 days. Mosquito survives on feeding on plant materials and blood is required only by the female. The female produces nearly 100 to 200 eggs per batch (Maximum of five batches of eggs during a lifetime). The number of eggs dependent on the size of the blood meal. A rough calculation is that for every mg of human blood about 40 eggs can be laid (the protein for building the egg comes from blood digest).

Fig 2: Self-limiting gene strategy used by Oxitec. Source
Modified mosquitoes is not an entirely new phenomenon. Other than the Oxitec genetic modification approach, use of Wolbachia has been a popular approach tried by others such as MosquitoMate and EliminateDengue.

What has Oxitec done differently? They have engineered a strain of male aedes aegypti mosquitoes code named OX513A. It basically contains a genetic insert of a gene called tTAV. The expression of tTAV protein is transcriptionally controlled using tetracycline antibiotic. In the presence of the antibiotic, essential proteins for development of mosquito are made available and hence an offspring can be formed. However in wild lack of this control leads to inability to express the essential proteins and hence offsprings are not formed which is passed from the genes in sterile male. For all other reasons, such a genetic sterile male is fit and can compete with wild males thus effectively reducing the population. See Fig 1  for overall details.

A couple of quick clarifications here. Males don't bite for blood and hence such a release doesn't increase any infection transmission. The males are sorted from the females in its pupal stage which has a greater than 99.9% sorting efficiency. Considering that 1000's of mosquito has to be released thrice a week, a few females may be released, but the statistics are highly insignificant. The released mosquito is viable for about a week. Approximately 70 million mosquitoes have been released to date without any negative impact being reported.

The next real question why tetracycline gene? The answer is tetracycline is a compound that is not widely found in the environment. It is estimated that nearly 2000-3000 times more concentration of tetracycline is required in the environment to enable rescue of the phenotype.

Fig 3: RIDL technology used by Oxitech. Source
In a summary, this is how the system works. When tetracycline is present, it binds to the tTA protein so tTA can’t bind to the genetic regulatory element tetO. Larvae with the RIFL transgene thrive when tetracycline is present. If tetracycline isn’t present, the protein produced by the tTA gene causes more expression of tTA by binding to tetO. Then, tTa accumulates and that is toxic to larvae. The gene is transferred to offspring by the male and bingo. The larva fails to develop. See Fig 3 

Trials in Brazil, Panama and the Cayman Islands, shows releases of Oxitec’s mosquitoes reduces the wild population of Aedes aegypti by more than 90%. The tight target which is highly species-specific is superior to insecticides, which has been already tried.

In India, Oxitec is working with Gangabishan Bhikulal Investment and Trading Limited (GBIT) and preliminary evaluation was done against A aegypti local strains from Delhi and Aurangabad. I'm not able to gather if a further trial has been conducted, but I gather that permission is being sought from the Review Committee for Genetic Manipulation (RCGM) for contained laboratory based studies in October 2013.

The tech definitely is promising and many parts of the world inc WHO is looking to implement this program. There is some resistance to release of mosquito by public especially since the technology is not clearly undertood and some fear that releasing more mosquito is a danger. But it is really interesting to see that mosquito can be used to combat mosquito population, something that is really needed at this time for combating vectored infections.

References:

1. Oxitec. How the technology works. Link
2. Patil et al. (2014). Mating competitiveness and life-table comparisons between transgenic and Indian wild-type Aedes aegypti L. Pest Management Science, DOI 10.1002/ps.3873.
3. Julie Steenhuysen. US One Step Closer to Releasing Engineered Mosquito to Fight Zika. Scientific American. Link

Thursday, October 27, 2016

ETX2514: A new Beta Lactamase Inhibitor

Greetings

Antibiotic resistance is a scary topic to talk about. I have discussed in detail why we could never have an antibiotic for which resistance couldn't develop (Link). The emergence of MCR-1 colistin resistant gene is something that we really didn't want to see. Hospital-associated infection is a common topic discussed everywhere because a lot of hospitals is a place containing all the nasty drug resistant pathogens. There is a global movement to invent new antibiotics and there is political support in some countries to fast track development of new antibiotics. Currently new generation antibiotic is really needed against ESKAPE pathogens. In this group, the gram negatives are the first priority targets, with Acinetobacter topping the list, especially in a hospital setting.

Fig 1: Pipeline of Entasis Therapeutics.
Source
Entasis Therapeutics (AstraZeneca spinout) is one such company interested in developing antibiotics, has secured $50 million to progress its pipeline of drugs. It has announced the initiation of Phase 1 clinical study of ETX2514. The study will evaluate the safety, tolerability and pharmacokinetics of ETX2514 in healthy volunteers. The clinical trial will be conducted in Australia (124 volunteers) and is expected to be completed in the first half of 2017.

Robin Isaacs Chief Medical Officer of Entasis Therapeutics comments, “We are very enthusiastic about the initiation of this clinical study, which will begin to establish the safety, tolerability and administration profile of ETX2514 in the clinic. This study builds on our extensive research in preclinical infection models which indicate that the administration of sulbactam in combination with ETX2514 holds great promise against drug-resistant A baumannii infections." The company is also currently advancing an oral drug to treat gonorrhoea through mid-stage clinical trials. It is also involved in developing intravenous and oral drugs for pneumonia, blood infections, urinary tract infections, and infections following surgery, though those are all in preclinical stages of development.

To digress, there are a few compounds that are in clinical testing against ESKAPE pathogens and many of them are yet to clinical trials. I found an interesting list of drug candidates that are currently in testing phase with possible potential.

Table 1: Some antibiotics against ESKAPE pathogens in Phase 3 testing. Source
As I have mentioned several times in my previous posts (See my posts here, here and here), it is easier to invent something that will make the existing drugs sensitive rather than invent something that is totally new.


Fig 2: Structure of ETX2514.
Source
ETX2514 a β-lactamase inhibitor. Chemically, it is diazabicyclooctenone. Because it is potent against multiple classes of β-lactamase enzymes, ETX2514 expands the spectrum of gram-negative, drug-resistant bacteria. I couldn't find any formal publication about this drug and hence for digging the details am relying on a poster presented about the drug in Microbe 2016 ASM conference.

I gain from the details presented, the company first performed a whole genome sequencing of 132 A baumanii isolates and found a variety of resistance encoding bla genes (Molecular class A, C and D). There is a very small group of strains having molecular class B bla genes. Most of them possessed Class D. Class D is same as functional group 2d. They are poorly inhibited by clavulanic acid, "bla genes" are β-lactamase encoding genes. Thus it makes sense to attack these products. Based on structure-based design and quantum mechanics calculations series of diazabicyclooctenones was identified. ETX2514 is an improved chemistry design from the original design. Interestingly, ETX2514 covalently binds to the catalytic Ser-90 of AmpC and displays a similar conformation to avibactam. It can strongly bind to Penicillin-binding proteins (PBP).

Fig 3: Main conclusions of the presentation on ETX2514.
Source
The results from the  study state as follows 

"MIC90 of any BL combined with ETX2514 was ≤ 0.12 mg/L against both K pneumoniae and E coli. Imipenem was the most effective BL partner for ETX2514 against P aeruginosa (MIC90 = 2 mg/L) while sulbactam was the most potent partner against A baumannii (MIC90 = 4 mg/L)". Other conclusions from the study are shown in Fig 3.

It is true that sulbactam is used clinically as a β-lactamase inhibitor (BLI). It is known that the chemical also has inherent antibacterial activity against a few such as Neisseria gonorrhoeae, Bacteroides fragilis and Acinetobacter species, which works by binding through PBPs. Sulbactam inhibits PBP1 and PBP3 but not PBP2 in A baumannii.

Table 2: Activity spectrum of ETX2514. Source
A follow up of this study was just presented at ID week. The MICs of nearly 600 isolates of A. baumannii are shown  in Table 2. Most interestingly a triple combination of Imipenem or Meropenem/Sulbactam/ETX2514 brought the MIC90 to less than 0.03 mg/L. Microscopic studies showed that A baumannii became rounded and enlarged in the presence of ETX2514 further confirming the activity against PBP (See Photo 1). Remember, PBP is essential for cell wall and structure maintenance.

Photo 1: Effects of ETX2514 on A baumanii cell structure.
Source
Traditionally, beta lactamase inhibitors have a limited range of a molecular class of β-lactamase that can be inhibited. But ETX2514 is designed to broadly specific and hence has an upper hand. So, will the strains that are resistant to ETX2514 automatically be resistant to a huge range of BL/BLI combination. Since this will be used when the bugs are already resistant to traditional BL/BLI combination does that matter?

To answer that question, we need to look at mutants. Several mutants of A baumanii strains have been recovered while studying ETX2514 activity. The frequency of resistance was 7.6 x 10-10. These strain have been sequenced by WGS. The resistance was mapped to residues S390T, S395F or F548C in PBP3 or to mutations in tRNA synthetase genes (aspS and gltX). The latter is associated with resistance to PBP2 inhibitors in E. coli. Purified mutant PBP3 proteins had reduced affinity for sulbactam and variable affinity for Imipenem and Meropenem.

Now put this whole thing in context. Sulbactam attacks PBP1 and 3 but not 2. There is evidence that ETX2514 attacks PBP2. So when everything combines together, the antimicrobial activity is good. Even in diverse cases since there is a difficulty in having all PBP1-3 in mutated form. So any beta lactam drug that doesn't work on PBP2, but does so with others is a good to go combination. An example would be aztreonam. The activity will not synergistically increase with β-lactamase such as mecillinam which specifically binds PBP2.

This brings in another question as to how this fits into real world scenario. The study reported "BLAST analysis of 1,537 whole genome sequenced strains of A baumannii showed no variation in PBP3 at S390, S395 or V505. Nine strains were found to have a T511S substitution but no T511A variants were found. This suggests that pre-existing target-mediated resistance to sulbactam-ETX2514 is not a significant resistance mechanism in the clinical setting". But resistance will appear once the drug becomes widely used.

References

Penwell W, Shapiro A, Giacobbe R, Gu R, Gao N, Thresher J et al. Molecular Mechanisms of Sulbactam Antibacterial Activity and Resistance Determinants in Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy. 2015;59(3):1680-1689.

Shapiro et al. ETX2514, a Novel, Rationally Designed Inhibitor of Class A, C and D b-lactamases, for the Treatment of Gram-negative Infections. Poster number LB-024. ASM Microbe; June 16-20, 2016.

Mcleod et al. Sulbactam combined with the Novel β-lactamase Inhibitor ETX2514 for the Treatment of Multidrug-resistant Acinetobacter baumannii Infections. Poster number 2246. ID week 2016. October 26- 30, 2016.

Monday, October 24, 2016

Lab series# 15: Biochemical tests for identification of bacterial isolates

Classic clinical microbiology techniques such as culture and phenotypic analysis form the major chunk of microbial identification, especially so for the bacterial isolates. The most sophisticated microbial laboratories still use the microbial culture techniques and need to isolate the bacteria and biochemically identify the isolate. Many different automated biochemical testing equipment are available which uses the same principle, only that the system is automated. In a majority of the microbial laboratories around the world, molecular tests are not available or feasible and identification is made through classic biochemical tests.

There are a large number of biochemical tests at the disposal of a microbiologist, but the choice of the panel of tests is based on preliminary findings such as gram staining pattern and growth characters which hint to what the list of organisms can be. For example, finding a gram negative bacilli growing in McConkey agar from stool sample would hint looking at Enterobacteriaceae group and tests like coagulase (which is used for identification of coagulase producing staphylococcus species) would of be no use. In most common scenario less than 15 biochemical tests are required for reliable identification of a bacteria to species level. Having more biochemical tests can increase the confidence in identification, but performing every possible biochemical test is counter productive.

Phenotypic-biochemical tests can be classified into 3 groups

1. Universal

These tests are done for almost any isolate and guide the microbiologist to a possible set of biochemical tests that needs to be done to get a reliable identification.
Examples: Hemolysis pattern, Motility test, Catalase test, Oxidase test

2. Differential

These are a common set of tests that are done to identify the isolate up to species level. The identification is made based on the results from a combination of tests and individual results by themselves are not sufficiently informative to make an identification.
Examples: IMViC tests, Triple Sugar Iron test (TSI), Sugar utilisation tests

3. Specific

These are tests that are specific to a particular set of species or for sub-typing a species. These tests are usually performed to confirm or identify at the subspecies level. The individual tests are informative by themselves in this case.
Example: γ-Glutamyl aminopeptidase test

It should be noted that certain tests are combinatorial in nature and there is more than one test to assess the same phenotypic parameter. For example, Mannitol Motility test is a combination test. The test can assess motility and the ability to utilise Mannitol. Motility can also be tested by using hanging drop technique and mannitol can be assessed using sugar utilisation tests.

In earlier days, most tests were done in large volumes typically using more than 5ml of a medium which takes more time to produce a visible result. Let us take an example case of lactose utilisation. If I’m to put an E coli into 5ml of a lactose-containing medium, it would take the isolate more time to break down lactose and give a pH change sufficient enough to give a reading in comparison to doing the same in a 0.1 ml medium. The simplest way to minimise the time involved in reading it to reduce the total reaction volume, which is one of the key concept in the most automated system allowing faster reading of results. Fig 1, is a hypothetical graph showing the time required to give a positive result in different conditions (The exact details will change depending on the test and other conditions).

So let us talk about few tests of interest in detail. The procedure for these tests can be found anywhere and hence the preparation of the medium and SOP for testing is not discussed. I want to focus on understanding the test principle.

1. Hemolysis Pattern on Blood agar:

Table 1: Hemolysis patterns that are
seen in blood agar.
In a typical scenario, Hemolysis is not considered as a biochemical test and many microbiologists would resist calling it so. For most of the cases, hemolysis pattern in blood agar significantly narrows down the identification. There are 4 types of hemolysis pattern that can be seen in a blood agar.

It should be noted that hemolysis is dependent on the enzyme involved and variations are seen for the same species depending on conditions. A study by Vancanneyt et al which tested multiple strains of E faecium found the following. beta-hemolysis was observed on both sheep and human blood for only 1 strain, 4 strains showed beta-hemolysis on human blood, but not on sheep blood, and rest of the strains studied showed no hemolysis on either medium. Of course, beta hemolysis is not a common feature for Enterococcus group.

Photo 1: Blood agar Plate. Source
For the preparation of Blood agar, sheep blood is the recommended reagent. Most commonly, the obtained sheep blood is defibrinated (using sterile glass beads) and added onto the basal medium to get blood agar. In my experience, washing the blood sample with normal saline and subsequent use of washed sheep RBC gives slightly better results. Certain labs also use horse/ bovine blood without significant deviations in the result.

It has been noted in certain labs that human blood is used for the preparation of blood agar. Standards recommend that this should not be practised since the human blood may contain bloodborne pathogens which form a risk to the technical staff preparing the agar and also the blood may contain inhibitors which may hamper growth. Some groups have questioned this thinking. In reality, human blood for the preparation of blood agar is usually obtained from blood bank stored bags which have reached expiry date or has expired. The blood bank usually tests such blood samples for common TTIs (Transfusion transmittable infections) and hence the risk of using such samples is minimal. Since the application is to grow human pathogens they should, in theory, be able to overcome inhibitors if any and hemolysis obtained is significant since it closely mimics human scenario. A set of counter argument is that using blood samples from humans that have expired is likely to have degraded and hence, false results would be common.

Overall, it is recommended that sheep blood is used for best results and the sheep which is used for obtaining blood be occasionally tested for potential infection. Currently, most laboratories around the world rely on commercial vendors to supply the lab with readymade disposable sheep blood agar plates that are quality controlled, thus avoiding the hassles.

2. Catalase test:

Catalase is an enzyme produced by a few group of bacteria and their primary function is to neutralise hydrogen peroxide activity abundantly expressed by attacking immune cells.

Fig 1: SOD and Catalase activity.
From Prescott Textbook 5th Ed
I want to digress a little bit since it would be useful. In general, obligate aerobes and facultative anaerobes usually contain the enzymes superoxide dismutase (SOD) and catalase, which catalyse the destruction of superoxide radicals and hydrogen peroxide, respectively. Most strict anaerobes lack both enzymes or have them in very low concentrations and therefore cannot tolerate O2  There are multiple exceptions to these rules. Peroxidases also can be used to destroy hydrogen peroxide. Fig 2 is an illustration of the growth of bacteria with varying responses to oxygen and their catalase and SOD properties.

Catalase test is of 3 types-
  • Qualitative catalase
  • SQ (Semi Quantitative) catalase test
  • 68 C (Heat stable) catalase test
In routine microbiology, qualitative catalase is the most commonly performed test and hence conventionally called as “catalase test”. The other two variants of the test are performed for selective mycobacterium isolates.

Catalase test is a test for demonstrating the presence of catalase enzyme by decomposition of hydrogen peroxide to oxygen and water.

Photo 2: Catalase test. Source
The reagent used for qualitative catalase testing is 3% Hydrogen peroxide, though up to 6% is acceptable. The test can be done by mixing a colony with a few drops of H2O2 in a slide (Slide method), adding a small colony to test tube containing H2O2 (Tube method) or adding H2O2 to the culture plate/ slant directly (Direct method) and looking for the formation of bubbles within 10sec. Care should be taken that the culture medium from which colony is used is devoid of RBCs and inert materials (such as plastic applicator stick, nichrome wire etc) should be used for mixing the colonies with the reagent.

Pseudo-catalase reactions are false positive reactions. They can be identified by their weak and late reaction and seen in some cases of Aerococcus species.

Semi-Quantitative catalase and 68C catalase test are specific tests used for differentiating mycobacterium species.

Most Mycobacterium species possess catalase which differs by quantity and heat lability at 68 C. SQ catalase reagent contains 10% tween 80 and 30% H2O2  LJ medium is inoculated with test organism and incubated for 2 weeks at 37 C and then tween-hydrogen peroxide reagent is added and allowed to stay for 10 min. The height of bubble formation is measured and reported as <45mm or >45mm. M kansasii, M simiae, and most scotochromogens give >45mm. M avium complex, M xenopi, M gastri etc give <45mm.

Certain Mycobacterium loses its catalase activity when suspended in a pH of 7 at 65 C for 20 min. M tuberculosis, M bovis, M hemophilum etc possess heat labile catalase. The test is most useful in differentiating member of Nonchromogenic mycobacterium.

3. Oxidase test:

Photo 3: Oxidase Test. Source
The test identifies the presence of cytochrome c oxidase or indophenol oxidase. The test is based on the principle of Redox (Reduction-oxidation) reaction. Redox reactions are in simple terms, a set of reactions that involve the transfer of electrons. It is a bi-component reaction, involving oxidation which is a loss of electrons and, a reduction which is a gain of electrons. The oxidase test often uses a reagent, tetra-methyl-p-phenylenediamine dihydrochloride, as an artificial electron donor. When the reagent is oxidised it changes from colourless to a dark blue or purple compound, indophenol blue.

Classically, 1% tetra-methyl-p-phenylenediamine dihydrochloride is freshly prepared daily and impregnated into a filter paper and dried. The colonies are smeared on the paper and look for colour change within 10 sec. Commercially available discs are now available which contains N, N-dimethyl-p-phenylenediamine oxalate, ascorbic acid and α-naphthol, a combination which is more stable thus avoiding the requirement of daily preparation.

Modified oxidase test is a special test used only for Gram positive, catalase positive cocci. It is commonly referred as Microdase test. Micrococcus oxidase enzyme is not readily accessible for reaction. This problem is overcome by the use of DMSO which permeabilizes the cell and permits access of reagents to oxidase enzyme. 

4. Oxidative fermentation test:

The test was invented by Hugh and Leifson and thus sometimes also known as Hugh- Leifson Test or OF- glucose test. Bacteria can degrade glucose in a fermentative or oxidative manner. In either of the case, the end products are a mixture of acids which is indicated by an indicator. Bromothymol blue or Bromocresol purple are commonly used indicators. Bromothymol blue has a pH range of 6.0 - 7.6 and Bromocresol purple has a pH range of 5.2-6.8, both of which gives yellow colour in the acidic range. The medium contains a high concentration of carbohydrate and low concentration of peptic digest which reduces the possibility of utilising peptic digest to produce an alkaline condition which masks the acidity produced. The agar concentration is also kept low, which enables the determination of motility. Careful observation of the medium for breaks or rise in the medium can also be used to indicate gas production.

Photo 4: Oxidative-fermentative (OF) test. Source
The test uses 2 tubes both containing OF medium and inoculated with bacteria. One is covered with a sterile mineral oil. This keeps the tube in an anaerobic condition. The tubes are then incubated for 24–48 hours. If the medium in the anaerobic tube turns yellow, then the bacteria are fermenting glucose. If the tube with oil doesn't turn yellow, but the open tube does turn yellow, then the bacterium is oxidising glucose. If the tube with mineral oil doesn't change, and the open tube turns blue, then the organism neither ferments nor oxidises glucose. Instead, it is oxidising peptones which liberate ammonia, turning the indicator blue. Motility can be observed in the medium by looing for growth trail. It should be noted that there are certain bacteria that take their own time to work the process and hence the test is not ideally read negative before 5 days of incubation.

Modified OF tests are used in special circumstances. For example, for testing halophiles, the OF medium is integrated with high salt concentration. There are certain bacteria that prefer other sugars, instead of glucose in which case OF medium containing other sugars can be prepared. For testing staphylococcus and micrococcus, Baird-Parker modification of the medium is recommended.

5. IMViC test:
Photo 5: IMViC test for E Coli.
Source

IMViC reaction is a set of four reactions helpful in identification of Enterobacteriaceae and related members. The tests include
  1. Indole test
  2. Methyl Red test
  3. Voges Proskauer test
  4. Citrate utilisation test
Indole Test:

Indole is an aromatic heterocyclic organic compound with a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring, which is derived from tryptophan using the enzyme tryptophanase. Most people recommend peptone water for testing indole production. Peptone water basically consists of Peptic digest of animal tissue and sodium chloride. A peptic digest is obtained by acid hydrolysis or enzymatic digestion. Acid hydrolysis method is harder and tryptophan is usually lost or reduced to very low levels by this method. The enzymatic method is much milder and uses trypsin and chymotrypsin combination. In each case, there is some loss of tryptophan and reduced availability of free tryptophan. Since most of the commercially available peptone is an enzymatic digest, peptone water should still work. However, if the nature of peptone is not known or results are not good it is recommended to add 1% tryptophan to peptone water and used for detecting indole.

Indole is a gas and can be detected easily with many different reagents. The most commonly used include Ehrlich's or Kovacs. Kovac’s reagent consists of para-dimethyl amino benzaldehyde in isoamyl alcohol and concentrated HCl. Ehrlich’s reagent uses Ethanol instead of Isoamyl alcohol and is more sensitive in detecting indole production especially in anaerobes and non-fermenters. Another sensitive alternative is p-Dimethylaminocinnamaldehyde (DMACA) in acidic solution impregnated into a filter paper, used as a spot test. 

Methyl Red (MR) test:

Fig 2: The Embden-Meyerhof pathway for glucose dissimilation.
Bacteria can utilise glucose through Embden-Meyerhof fermentation pathway and get into one of the end products, depending on the species. It can either produce homolactic acid or  mixed acids containing a combination of lactic acid, acetic acid, formic acid, succinate and ethanol and gas formation if the bacterium possesses the enzyme formate dehydrogenase, which cleaves formate to the gases. Certain species can further get down the pathway and form 2, 3 butanediol from the condensation of 2- pyruvate. See Fig 2 for details.

The test is done on glucose phosphate peptone water. The MR test looks for the ability of bacteria to produce large amounts of acid resulting in significant decrease in the pH of the medium below 4.4. This acidic nature is indicated by methyl red (p-dimethylaminoaeobenzene-O-carboxylic acid) indicator which is yellow above pH 5.1 and red at pH 4.4. The test should be ideally read at 48 hrs since the test looks for sustained pH.

Voges-Proskauer (VP) test:

VP test is actually an extension of MR test and looks for the ability to produce butylene products. Acetoin (3-hydroxybutanone) is an intermediate in the reaction which is looked for using 40% KOH and alpha-naphthol. If acetoin is present, it is oxidised in the presence of air and KOH to diacetyl which reacts with guanidine components of peptone, in the presence of alpha- naphthol to produce a red colour. The test is read along with MR test.

Citrate test:
Fig 3: Citrate utilisation pathway.
Source

The test looks for the ability of a bacteria to utilise citrate as a sole source of carbon. For the bacteria to be able to do so, it requires 2 components- Citrate permease and citrate lyase. Citrate permease is a group of uptake proteins that allows the cell to uptake citrate and then lyase which converts citrate to oxaloacetate and acetate. The oxaloacetate is then metabolised to pyruvate and CO2.

The organism is inoculated into Simmon's or Koser's citrate medium. Simmons citrate agar contains sodium citrate as the sole source of carbon, ammonium dihydrogen phosphate as the sole source of nitrogen, other nutrients, and the pH indicator bromothymol blue. The bacteria converts the ammonium dihydrogen phosphate to ammonia and ammonium hydroxide, which creates an alkaline environment in the medium. At pH 7.5 or above, bromthymol blue turns royal blue which is otherwise green. Most people make the mistake of reading the reaction by colour. In some cases, the alkalinization doesn't occur (Or takes longer time) but colonies can be seen. Colony formation should be taken as an evidence of growth which is a reflection of the ability of the bacteria to utilise sole carbon source.

6. Triple Sugar Iron Test:

Triple sugar Iron is a complex test with multiple readouts. The test was first designed proposed by Sulkin and Willett which was later modified by Hajna for identification of Enterobacteriaceae members. The test medium is TSI (Triple sugar Iron agar). The test medium contains 3 sugars- Glucose (0.1%), lactose and sucrose (1% each). Phenol red serves as the indicator. The medium contains a butt and a slant. Ferrous sulphate serves as an indicator for H2S production. The medium is inoculated with a stab method on the butt and stroke method on the slant. The lower portion of the butt acts as an anaerobic condition since it is nearly inaccessible.

The first thing that happens when bacteria is inoculated, is to utilise the glucose. The amount of glucose is purposefully kept low (Nearly 10 times less in comparison to other sugars). If the organism can metabolise glucose in anaerobic conditions and aerobic conditions both the butt and slant becomes acidic turning the colour of indicator to yellow. This happens within 6-8 hours of inoculation. If the bacteria can utilise lactose or sucrose (or both), the acidification of medium continues and the medium remains yellow. If it cannot, the bacteria starts utilising amino acids by decarboxylation of peptone making the medium alkaline thus reversing the first acidic step. This gives a more reddish appearance. Phenol red has a pH range from 6.8 (yellow) - 8.2 (red). If the bacteria is a strict aerobe (ex Pseudomonas aeruginosa) the reactions occur only in the slant and the butt remains no change or non-reactive. If the bacteria is a facultative anaerobe, the reaction will be seen in both butt and slant. In general, more amounts of acids are liberated in butt region (fermentation) than in the slant (respiration).

Production of gas is evidenced by breaks or rising of the agar medium. Thiosulphate is reduced to H2S by several species of bacteria which combines with ferric ions of ferric salts to produce the insoluble black precipitate of ferrous sulphide. Reduction of thiosulphate proceeds only in an acid environment. There are several combinations of reactions possible that can be read. Following are the most common.
Fig 4: TSI reaction readouts. Source
  • The  organism ferments glucose but does not ferment lactose or sucrose. The slant becomes red and butt remains yellow. It is reported as K/A (Alkaline slant/Acid butt)- remember butt is more acidic.
  • The organism in addition to glucose ferments lactose and (or) sucrose. The slant and butt remain yellow. It is reported as A/A (Acid slant/Acid butt).
  • If the organism is non-fermenter, Instead of sugars, peptone is utilised as an alternate source of energy under the aerobic condition on the slant which makes it alkaline indicated by the red colour while there is no change in the colour of the butt. It is reported as K/NC (Alkaline slant/No change)
In addition to the above gas and H2S is reported. Reactions in TSI should not be read after 24 hours of incubation because eventually sugars will be exhausted and decarboxylation reactions will take over making the medium alkaline.

The test cannot differentiate between lactose and sucrose fermenters. For this, a modification called as Kligler Iron Agar (KIA) which combines features of Kligler's Lead Acetate medium and Russell's Double Sugar Agar can be used. This medium doesn't have sucrose.
Fig 5: Fermentation test.
Source
7. Carbohydrate fermentation test:

The test is usually done on a Carbohydrate Fermentation Broth (Contains trypticase, Sodium chloride, and Phenol red) with 1% sugar which is to be tested. A durham's tube is kept in an inverted position which accumulates gas in case of gas production. The phenol red indicator turns yellowish if there is fermentation leading to acidic pH change. Alternatively, Andrade's indicator may be used.

Most often, a single sugar may not be sufficient enough to make a distinction and combination of multiple sugars are used. The most commonly used include- lactose, sucrose, xylose, mannose, arabinose, trehalose and maltose etc.

8. Urease test

Urease is an enzyme belonging to belong to the superfamily of amidohydrolases and phosphotriesterases. It catalyses the hydrolysis of urea into ammonia and Carbon dioxide. 
(NH2)2CO + H2O → CO2 + 2NH3
The formation of ammonia causes alkalinization of the medium, and the pH change is indicated by a change to pink at pH 8.1. Certain organisms can rapidly hydrolyze urea and their speed of hydrolysis can indicate the organism. A test called the CLO test (Campylobacter-like organism test), is a rapid urease test for diagnosis of Helicobacter pylori). A biopsy of mucosa is taken from the antrum of the stomach and is placed into Urea broth. A positive test in less than 30 min may be obtained indicating the pylori infection. Another method called Urea breath test is based on a similar idea but detection is based on isotope measurement.
As already mentioned there are so many Phenotypic biochemical tests that can be performed for identifying an organism. However, with some experience and training, most organisms can be identified with few tests mentioned above at least up to the genus level.

Thursday, October 20, 2016

Lyme disease: A possible link to Swiss agent?

Greetings

For most of the infections, the culprit is a single pathogen. That is what we are taught and what we believe. The exception are the cases of hospital acquired infection, where of course a lot of them have a polymicrobial cause. There are many different cases of infection, where the treatment response is straightforward and in some cases rather complicated. The argument that it is because of genetics and strain variation doesn't seem to hold true in many case scenarios.

Lyme disease also known as Lyme borreliosis is an infection caused by a bacteria called Borrelia burgdorferi. It is a thin, spiral, motile, extracellular bacterium belonging to the family Spirochaetaceae. The first isolate of this disease-causing spirochete was only obtained in 1981 when Burgdorfer demonstrated a spirochete in Ixodes ticks collected from Shelter Island. B burgdorferi is primarily seen in the United States. The related species Borrelia afzelii and Borrelia garinii are seen in Europe and Asia. All 3 species are collectively referred to as B. burgdorferi sensu lato. Rodents are the primary reservoir of Borrelia species.

Photo 1: Ixodes scapularis.
Source
B burgdorferi infects a wide range of vertebrate animals including small mammals, lizards, and birds. Ixodes species transmit B. burgdorferi between multiple hosts and are the only known natural transmission agents. Humans are actually an accidental host. Analysis of genetic sequence showed that it possess most genes similar to other bacteria but lack any specific identifiable pathogenesis associated genes. This is mostly because B burgdorferi is not designed to infect human and not a classic human pathogen.

Ixodes species  have a three-stage life cycle to be completed in a time period of 2 years- larva, nymph and adult. They need one blood meal per stage. Transovarial transmission does not occur commonly and thus each generation of tick acquires  B burgdorferi through fresh infection.

Photo 2: Erythema migrans caused
by B burgdorferi. Source
B burgdorferi is inoculated into the skin by the bite of an infected Ixodes tick containing tick saliva and bacteria. Tick saliva contains immunosuppressive molecules which help bacteria multiply and migrate radially within the dermis layer. The host inflammatory response to the bacteria in the skin leads to clinical signs of this infection, a distinctive 'bullseye' rash (The classic sign- Erythema chronicum migrans), which occurs at the site of the tick bite three to 32 days after the tick bite. See Photo 2. The bacteria has the capacity for antigenic variation which helps in avoiding immune attack. Laboratory diagnosis of the bacteria is not attempted through culture, but rather by serology and PCR. Culture is difficult due to the requirement of specialised culture techniques and hence done only in specialised laboratories. Serology is not considered as a standard since ELISA's are positive only after the infection has advanced.

The treatment is a short course of antibiotics and most people recover without any sequelae. In a subset of the cases, the patients suffering extends for months or even longer than a year. This is called as Post-treatment Lyme disease syndrome (PTLDS) or chronic Lyme disease. There is no clear understanding of mechanics of this condition and research is focussed on this problem.

Photo 3: After initial tests, Burgdorfer suspected the Swiss Agent caused Lyme. He shared the strong evidence with a close colleague in Switzerland to see whether he could verify the findings in patients there. Source
STAT news has obtained lab notes documents from Burgdorfer’s personal papers found in his garage after his death in 2014. The notes indicate that in late 1970's Burgdorfer had results indicating that he suspected "Swiss agent" or Rickettsia helvetica. But later somehow he was convinced that it is B burgdorferi was the cause which was published in Science in 1982. But the notes indicated that he was still doubtful of the Swiss agent and was constantly communicating with his close colleagues about the possibility.

There are several speculations about this whole story. Certain Lyme experts theorise that Lyme patients who test negative for the infection might be suffering from an illness caused by R helvetica. Another group of experts think that Patients with PTLDS R helvetica occurs as a co-infection. Of course, there is no definitive proof for either. I read somewhere, (unable to recall the source) that following the above CDC has decided to conduct PCR on 30000 samples from patient samples to see of they can find the Swiss agent. Ian Lipkin a virus hunter (If I can call so) has collected 5,000 ticks from New York and Connecticut to look for viruses in them and identified 20 new viruses in these ticks so far. He explains, “Everyone wants to get to the bottom of this. All of this is critical to  finding out why some people respond to antibiotics and some people don’t, and whether or not the antibiotics being used are appropriate, and trying to find ways to link different bacteria and different viruses to different syndromes.

Rickettsia helvetica was first isolated from Ixodes ricinus ticks in Switzerland and is currently in the list of an unconfirmed pathogen. Except for some case reports nothing is clear about the pathogen. In a study by Nilson et al; 2013 20 of 206 patients (0.09%) had seroreactivity to Rickettsia species from patients seeking medical care for erythema migrans or flu-like symptoms after suspected or observed tick bite in the south-east of Sweden. The same also showed that less than 1% of healthy blood donor were also serologically positive. This situation is an example in a case of complexity in identifying if R helvetica is really a pathogen of interest.

That begs the question if similar cases exist anywhere else? As a matter of fact, there does. There is some research suggesting Trichomonas vaginalis a protozoal pathogen involved with Sexually transmitted infections is more aggressive when accompanied with its dsRNA virus (endosymbiotic Trichomonasvirus) mostly through modulating inflammatory cytokines. There is similar evidence in the latest publication by Fasel et al; 2016 suggesting that Leishmania-RNA-viruses has similar role in Leishmaniasis.

It should be noted that in the above cases, these are viruses that modulate the outcome, but R helvetica is no less than intracellular cytoplasmic pathogen in operational terms.


References

Radolf J, Caimano M, Stevenson B, Hu L. Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. Nature Reviews Microbiology. 2012; 9;10(2):87-99.

Tilly K, Rosa P, Stewart P. Biology of Infection with Borrelia burgdorferi. Infectious Disease Clinics of North America. 2008;22(2):217-234.

Fichorova R, Lee Y, Yamamoto H, Takagi Y, Hayes G, Goodman R et al. Endobiont Viruses Sensed by the Human Host – Beyond Conventional Antiparasitic Therapy. PLoS ONE. 2012;7(11):e48418.

Hartley M, Bourreau E, Rossi M, Castiglioni P, Eren R, Prevel F et al. Leishmaniavirus-Dependent Metastatic Leishmaniasis Is Prevented by Blocking IL-17A. PLOS Pathogens. 2016;12(9):e1005852.