Thursday, October 27, 2016

ETX2514: A new Beta Lactamase Inhibitor


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.
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.
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.
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.
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.


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.

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.

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.
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?


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.
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.


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.

Friday, October 14, 2016

Understanding Autophagy: Nobel winning concept


The recent announcement of Nobel prize 2016 in Physiology or Medicine to Yoshinori Ohsumi is perhaps a topic trending online these days. Autophagy is a physiological process, innate to a cell and has multiple implications in immunology and infectious diseases biology. I decided to step in and write some details of interest. Autophagy (or sometimes also referred to as autophagocytosis) is defined as a physiological tightly regulated, destructive mechanism of the cell that disassembles unnecessary or dysfunctional components. It is important to note that unlike other cellular degradation machinery, autophagy removes large macromolecular complexes and organelles that have become obsolete or damaged. It also has a primary role in clearing invading microorganisms and toxic protein aggregates.

Photo 1: Dr Christian de Duve.
The roots of understanding autophagy come from an interesting observation by Christian de Duve studying the distribution of acid phosphatase but failed to detect any enzymatic activity in freshly isolated liver fractions. But the enzymatic activity reappeared upon storage for 5 days in a refrigerator. It was later understood that the proteolytic enzymes were sequestered in a membrane structure called the lysosome. Subsequent studies showed that in some cases they could also co-localise other cellular organelles with the lysosome. It became evident that the structures had the capacity to digest parts of the intracellular content and coined the term autophagy in 1963. There was some evidence that the process might have a role in human diseases, but nothing about the mechanism was understood.

Photo 2: Yoshinori Ohsumi.
Ohsumi carried the idea forward and test if the unicellular eukaryotic system also had autophagy. He theorised that if autophagy mechanism was present in yeast, inhibition of vacuolar enzymes would result in the accumulation of engulfed cytoplasmic components in the vacuole. By developing Saccharomyces cerevisiae strains that lacked the vacuolar proteases proteinase A, proteinase B and carboxypeptidase he showed abnormal vacuole formation testifying his ideas. By systematically studying genes in yeast and phenotype Ohsumi identified 15 different key genes that regulate the process, which forms the major basis for understanding autophagy and his Nobel Award.

Autophagy is a complex pathway mediated by multiple different proteins. Though the pathway operates on a general principle, they are divided into 3 major pathways based on the specifics of the mechanism

1. Macroautophagy
2. Microautophagy
3. Chaperone mediated autophagy

Macroautophagy is the process by which the substrates are sequestered within cytosolic double membrane vesicles termed autophagosomes. In Microautophagy, a cytoplasmic material is trapped in the lysosome/vacuole by membrane invagination. It is especially important for the survival of cells under starvation.

Fig 1: Types of Autophagy. Source
Chaperone-mediated autophagy (CMA) refers to targeting of proteins from the cytosol to the lysosomal membrane and then gaining access to the lumen by directly crossing its membrane. Proteins that undergo degradation by this pathway are identified through a recognition motif (such as amino acid sequence KFERQ motif). This allows for the removal of specific proteins thus becoming an efficient system for degradation of damaged or abnormal proteins. CMA is a multi-step process that involves following steps
  • Substrate recognition and lysosomal targeting
  • Substrate binding and unfolding
  • Substrate translocation
  • Substrate degradation in the lysosomal lumen

In general, Autophagy can be divided into multiple steps. Though most of these details have been worked on yeast models, mammalian genes have analogue proteins serving similar functions. Considering that the process is evolutionarily conserved the details are not expected to deviate much. The whole process in its simplest form can be described as forming a vesicle containing contents to be degraded and fusing a lysosome into it. The enzymes then degrade everything inside the pouch and contents are available for recycling. It is not clear, what are the exact signals that activate the process, but stress is known general factor.

Fig 2: Generalised autophagic pathway. Source

Fig 2, is a simplification of Major events in Autophagy pathway. The process of autophagy involves the following steps

Autophagosome nucleation

Fig 3: Proposed process of Vesicle formation. Source
This step is also known as Initiation. The initiation of autophagy can be triggered by a variety of extracellular signals which includes nutrient starvation, stress, microbial infection, toxins etc. An important target of these signals is TOR (Target of Rapamycin), a kinase that inhibits the autophagic pathway until this protein is inactivated by dephosphorylation. Class III PI3Ks are also good inducers of autophagy activation. The first step is to form a phagophore or isolation membrane. There are multiple proteins that are involved in this process, and they are called as ATGs (Autophagy-related proteins). Atg5–Atg12–Atg16 complex is recruited to the sequestration crescent, a double-membrane-bound structure that engulfs cytosolic constituents to become the closed, double-membrane-bound autophagosome. The process of vesicle formation is shown in Fig 3.

Growth and completion

The first step is to add phosphatidylethanolamine to Atg8. The carboxy-terminal amino acids of Atg8 are cleaved by cysteine protease Atg4 thereby leaving a conserved glycine residue. Cleaved Atg8 is then transiently linked to the Atg7 protein, then to Atg3, and finally to phosphatidylethanolamine. Modified Atg8 remains associated with autophagosomes until destruction at the autolysosomal stage. Cleaved Atg-8 enables fusion of autophagosome with a lysosome.

Autophagosome target and fusion

Autophagosomes fuse with endosomal vesicles mediated by small GTPases, such as the RAB proteins and acquire LAMP1 and LAMP2. These structures fuse with lysosomes and acquire cathepsins and acid phosphatases to become mature autolysosomes.

Table 1: ATG functional groups.
I have avoided very intricate details of the pathway to avoid too much complexity. However, it would be useful remember that there are 36 ATG proteins known and most of them are retained in mammalian cells. Autophagy set of proteins is classified into 5 functional groups. See Table 1 for details.

Since one of the functions of autophagy pathway is to clear intracellular pathogens, inhibition of the pathway becomes important for the survival of the pathogen. Many different microbial proteins can achieve this goal. For example, Herpesvirus US11, Vaccinia E3L, Influenza virus NS1 inhibit PKR activation by dsRNA; HIV TAT forms complex with PKR, inhibiting its kinase activity; Legionella pneumonia delays acquisition of LAMP1; M tuberculosis blocks phagosome maturation. Intracellular microbial pathogenesis is full of such examples. Degradation of microbial proteins by autophagy are the source of peptides for presentation to the immune system.

Jeremy Berg captures the importance of Nobel award to workings of Ohsumi, "The process of autophagy that he discovered is now part of the fabric of modern cell biology and medicine. Researchers now consider defects in this pathway when trying to understand diseases." Ohsumi comments, "The human body is always repeating the auto-decomposition process or cannibalism, and there is a fine balance between formation and decomposition. That's what life is about.” He also noted that even though he has been studying the mechanism of autophagy for more than 27 years, he still doesn't think he fully understands it and hopes he could learn more.


1. Glick D, Barth S, Macleod K. Autophagy: cellular and molecular mechanisms. The Journal of Pathology. 2010;221(1):3-12. 

2. Kaur JDebnath J. Autophagy at the crossroads of catabolism and anabolism. Nature Reviews Molecular Cell Biology. 2015;16(8):461-472.

3. Kirkegaard K, Taylor M, Jackson W. Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nature Reviews Microbiology. 2004;2(4):301-314.

Wednesday, October 12, 2016

Rhinovirus C


Let me first throw a question. What would be the most common set of infection that a good majority of the world population would have had at least once in a lifetime? Well, the answer is probably a big list which includes Herpes viruses, Adenovirus, Enteroviruses, Rhinovirus etc. Of these, Rhinovirus are perhaps the most ignored.

Rhinoviruses belong to Enterovirus group (Picornaviridae Family). Structurally they are Non-enveloped, roughly spherical with single-stranded positive sense RNA. The viral genome encodes a single polyprotein which is cleaved by virally encoded proteases to produce 11 proteins. VP1, VP2, VP3, and VP4, make up the viral capsid and remaining nonstructural proteins are involved in viral genome replication and assembly. Rhinovirus is different from other rhinoviruses in that they don't grow at 37 C. Instead, they prefer a temperature of about 35 C which is normally seen in upper respiratory tract.

Fig 1: Genetic Phylogeny of Human Rhinovirus Virus.
Historically, rhinoviruses were classified into serotypes and genotypes. The viruses were grown in cell cultures and then subsequently typed. Currently, Human rhinovirus (HRV) is currently classified to 3 species HRV-A, HRV-B and HRV-C with a total of 160 identifiable subtypes.

The 3rd group of HRV doesn't grow in cell culture systems and thus was missed in earlier times. With the ability to do the whole genome sequences directly from the sample, HRV-C has been identified. The first detection was in 2006, in respiratory samples collected from patients in Queensland and New York City. An approximate estimate of the genetic phylogeny of HRV is shown in Fig 1. It is expected that this phylogeny will change with an increase in the number of sequences. In fact, there is some data to hint that HRV-Cγ is very diverse.

Interestingly, HRV C is associated with more fulminant respiratory infections and some studies have shown it to be associated with Asthma. For example, Casas et al showed that of nasopharyngeal aspirates tested from 16 infant patients infants hospitalized after an apparently life-threatening event, 6 aspirates had HRV-C. In another study by Bizzintino et alHRV- C was  detected in 59.4% of children with acute asthma and was associated with more severe asthma.

Fig 2: Structural modelling of RV-C15 binding to
CDHR3 receptor. Source
HeLa Cells are used for culture and study of HRV biology. The special problem of inability of HRV-C to infect and grow in HeLa cell line led to the idea that the receptor is different. This problem was studied by Bochkov et al. The virus grows considerably well in a primary organ or cell cultures derived from sinus tissue. Studying the genetic expression profile in susceptible cells and narrowing the list 12 membrane proteins were identified. Expression of the gene in cells was used to test the receptor and found that the protein Cadherin-related family member 3 (CDHR3) when expressed in HeLa cells lead to replication.

The exact role of CDHR3 is not well understood. What is known is it is a member of the cadherin family of transmembrane proteins and functionally, are involved in cell adhesion, epithelial polarity, cell-cell interaction, and differentiation. Te gene is strongly linked with early childhood asthma with severe exacerbations. Even more interesting is the finding that a mutation in the gene causing a change from cysteine to tyrosine at amino acid 529, increases virus binding and virus replication in HeLa cells that are artificially induced to synthesise CDHR3.

Rhinovirus vaccine hasn't been an issue of great interest earlier since HRV-A and HRV-B causes a very low level of clinical impact. However, there is a renewed interest to make a vaccine and study a detailed biology of HRV-C especially in context with Asthma. Rhinovirus C is resistant to current known antiviral drugs. In a recently published study in PNAS, researchers have analysed the cryo-EM atomic structure of RV-C15a. The structure showed why the virus is different from other HRV. Palmenberg comments, "We found some interesting things. Unlike normal rhinoviruses, this one has spikes on the surface of the particles. We had not anticipated that."

Interesting enough, the structure has been sufficiently enlightening. The structre shows possible virus receptor contact regions, which is important in drug and vaccine design. The study also found that about 30% of the virus particles were empty and didn't contain any genetic material. These structures called as native empty particle (NEP) are also proposed as a possible vaccine candidate. Earl etal has a commentary piece running suggesting that Cryo-EM could serve as a possible tool to find vaccine targets of interest, especially in those cases where classic technologies maynot be applicable.


1. Lauber CGorbalenya A. Toward Genetics-Based Virus Taxonomy: Comparative Analysis of Genetics-Based Classification and the Taxonomy of Picornaviruses. Journal of Virology. 2012;86(7):3905-3915. 

2. Lau S, Yip C, Woo P, Yuen K. Human rhinovirus C: a newly discovered human rhinovirus species. Emerging Health Threats Journal. 2010;3(0). 

3. Bochkov Y, Watters K, Ashraf S, Griggs T, Devries M, Jackson D et al. Cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates rhinovirus C binding and replication. PNAS. 2015;112(17):5485-5490. 

4. Liu Y, Hill M, Klose T, Chen Z, Watters K, Bochkov Y et al. Atomic structure of a rhinovirus C, a virus species linked to severe childhood asthma. PNAS. 2016;113(32):8997-9002.

5. Earl L and  Subramaniam S. Cryo-EM of viruses and vaccine design. PNAS. 2016;113(32):8903-8905. 

Nobel Awards- 2016


I have been out for a few days and hence haven't been online for a short while. The whole time was also mixed with some intense personal research and hence I didn't get the time to write a post. For the time being, I want to drop a quick a post on Nobel Awards 2016. Nobel Prize is considered as the most prestigious academic award. The recipients of the award are chosen by the Nobel foundation constituted by Nobel committee of Royal Swedish Academy of Sciences, Nobel committee of Karolinska Institutet and Norwegian Nobel Committee. The award consists of a citation, gold medal and money. However, the fame is considered far superior for the award.

1. Physiology / Medicine:

Photo 1: Yoshinori Ohsumi. Source
The award goes to Yoshinori Ohsumi (Professor; Tokyo Institute of Technology) for his pioneering work on autophagy. He had previously been recognised for his work on Autophagy by Kyoto Prize; 2012. Autophagy is a normal physiological phenomenon where the cells can recycle self-components. It is an important part of the cellular machinery since it allows for clearing up clogs, remove damaged organelles, proteins etc. As evidenced by studies, many different disease conditions (such as Alzheimer's) is a possibility due to dysregulation of autophagy process. Autophagy is a topic relevant to immunology and maybe in future I will write in detail about the process.

2. Chemistry

Photo 2: Fraser Stoddart, Bernard Feringa
and Jean-Pierre Sauvage. Source
The award is shared between Jean-Pierre Sauvage (University of Strasbourg), Fraser Stoddart (Northwestern University) and Bernard Feringa (University of Groningen) who have been involved in creating nanoscale machines. Ability to make machines at atomic scales using simple carbon atoms is achieved in less than a few handful of specialised labs. The field is still in its infancy but has tremendous technical applications. For example, it has been proposed that nano-machines could be useful in attacking cancerous cells, by specifically engineering them to do so. I specifically like a quote by Ferringa on how nano-machines are going to be useful in future, "People were saying, why do we need a flying machine? Now we have a Boeing 747 and an Airbus. That’s a little bit how I feel. The opportunities are great.”

3. Physics

Photo 3: Michael Kosterlitz, David
Thouless and Duncan Haldane.
The award goes to Michael Kosterlitz, David Thouless and Duncan Haldane for theoretical discoveries of topological phase transitions and topological phases of matter. The theory explained behaviours that experimentalists discovered at the surfaces of materials, and inside extremely thin layers such as superconductivity and magnetism. Thouless and Kosterlitz showed that topological phase transitions (Came to be known as Kosterlitz–Thouless transition) in which material switches between states with different topologies were possible in thin layers of materials. Haldane was involved in working with the concept of topology and how it applies to chains of magnetic atoms. Understanding of this mechanism is currently explored in the creation of quantum computing technologies.

Apart from the above, Nobel Prize 2016, is also announced for Peace to Juan Manuel Santos "for his resolute efforts to bring the country's more than 50-year-long civil war to an end" and Economics to Oliver Hart and Bengt Holmström "for their contributions to contract theory". 


1. Richard Van Noorden & Heidi Ledford. Medicine Nobel for research on how cells 'eat themselves'. doi:10.1038/nature.2016.20721

2. Richard Van Noorden & Davide Castelvecchi. World’s tiniest machines win chemistry Nobel. doi:10.1038/nature.2016.20734

3. Elizabeth Gibney& Davide Castelvecchi. Physics of 2D exotic matter wins Nobel. doi:10.1038/nature.2016.20722