Saturday, 25 January 2014

Vancomycin Dependant Enterococcus (VDE)

Vancomycin Dependant Enterococcus (VDE)

Explanation of Enterococcus faecium’s curious response to the antibiotic Vancomycin

So you try to determine the Minimum Inhibitory Concentration (MIC) of Vancomycin against an Enterococcus isolate by E-test (epsilometer test) methodology and after appropriate incubation you obtain this curious result:

Enterococcus faecium's response to a Vancomycin E-test
(Mueller-Hinton Agar - 24+hours, 37˚C)

What the *$%#&!;.....??  The greatest growth is where the antibiotic concentration is the greatest and tapers off where the antibiotic concentration is the lowest.  It is kind of like shooting at a flock of ducks flying overhead, and only the ones that don’t get hit drop!!!

So what is happening here?  Let’s back up a bit and get some history:

Vancomycin is an important antibiotic as it is the last ‘common’ antibiotic active against most gram positive organisms.  Once an organism acquires resistance to vancomycin, the antimicrobial arsenal is greatly limited in what can be used to fight an infection.

Vancomycin resistance is plasmid mediated, meaning that vancomycin sensitive enterococci and acquire resistance from other organisms already vancomycin resistant.  In turn, these Vancomycin Resistant Enterococci (VRE) can pass the plasmid on to other organisms.  This may result in an outbreak of resistant organisms which are challenging to treat and may be particularly devastating in severely debilitated patients.

There are eight known vancomycin resistance genotypes in enterococci with those known as Van-A being most prevalent, followed by Van-B.  Van-C offers low level intrinsic resistance to E.gallinarum & E.casseliflavus.  The remaining genotypes have not proven to be significant in the clinical setting.

Enterococci expressing the Van-A genotype are resistant to both vancomycin & teicoplanin.  Expression of the Van-B genotype conveys resistance to vancomycin but the enterococcus remains susceptible to teicoplanin.  Van-A resistance is generally higher (16 – 516 µg/ml) than that provided by Van-B (4 – 64 µg/ml).

In order to prevent nosocomial (hospital acquired) infections, many facilities require a rectal swab be taken from newly admitted patients in order to screen for VRE.
Various methods & media can be employed for this screening.  Our facility utilizes Oxoid® Brilliance Chromogenic VRE media.  On this media, E.faecalis appears as light blue colonies while E.faecium appears purple.  Other organisms are repressed or appear uncoloured.

Enterococcus faecium on Oxoid ® Brilliance Chromogenic Media (24hrs at 37˚C)

Suspicious colonies are investigated further by determining the actual MIC of vancomycin using the E-test as mentioned above.  Enterococci with MIC’s greater than 8 µg/ml are considered to be VRE.  (Identifications can be confirmed using common microbial identification platforms or traditional tests).
Patients known to harbour VRE’s can be isolated and contact precautions implemented to reduce the likelihood of dissemination.

The antibiotic sensitivity plate above shows three different organisms subjected to an e-test in order to determine their susceptibility to vancomycin.  Organism (1) is an enterococcus susceptible to vancomycin (VSE), (2) shows an enterococcus resistant to vancomycin (VRE), and (3) shows a curious response to vancomycin I had never before encountered.  This organism exhibits vancomycin dependence! (VDE).

Note: the E-test is a strip impregnated with a continuously varying concentration of antibiotic along its length.   On the Vancomycin E-test strip the concentration varies from 0.016 µg/ml to 256 µg/ml.  The MIC value is where the growth/no-growth intersects the strip.  The zone of inhibition is narrowest as it approaches the point of intersection and widest at the top of the strip where the concentration is the greatest.

Okay, what gives? First, let's explore Vancomycin Resistance a bit further:

Vancomycin binds to the terminal D-Ala:D-Ala structure in the peptidoglycan layer of the enterococcal cell wall. This prevents the crosslinks from forming and the pentapeptide structures from extending during synthesis. Cell wall formation is terminated, or rather the cell wall is weakened without the cross-links and therefore the integrity is compromised and the bacterial cell is subject to variations in osmotic pressure.  Eventually the cell will burst if not strengthened with the cross-links.  Think of each peptidoglycan molecule as a brick and the pentapeptide bridge as the mortar holding the bricks together. (see diagram below)

In both Van-A & Van-B genotypes, the gene cluster acts to a) detect the presence of vancomycin and start transcription of specific resistance genes, b) form and incorporate D-Ala:D-Lac into the growing peptidoglycan wall, and c) eliminate any D-Ala:D-Ala precursors, thereby eliminating the vancomycin sensitive pathway of peptidoglycan formation.

In other words, vancomycin binds to D-Ala:D-Ala, however by the enterococcus substituting D-Ala:D-Lac into the structure, vancomycin will no longer "functionally" bind rendering the organism vancomycin resistant.

Exactly why does the substitution of D-Ala:D-Lac make the enterococcus resistant to vancomycin?  My university biochemistry was rusty and I wondered if the Lactate molecule in D-Lac was larger or more complex than the Alanine molecule in D-Ala and the vancomycin was sterically inhibited or prevented from binding by shear size.  Did this substituted molecule block the vancomycin? In the sentence previous to the above diagram, I said that the vancomycin does not "functionally" bind to the D-Ala:D-Lac side chain of the peptidoglycan cell wall component.  Looking at the structure of D-Ala:D-Alanine compared to D-Ala:D-Lactate you can see in the diagram below that they are almost identicle.  The difference lies in the substitution of an oxygen molecule in the D-Ala:D-Lac for the amine group (NH) in D-Ala:D-Ala.

Vancomycin is the large molecule at the top third of this graphic.  By substituting D-Ala:D-Lac for D-Ala:D-Ala, the amine (NH) is replaced by and oxygen (O) and the hydrogen bond shown in red reduces the total number of hydrogen bonds from 5 to 4.

In more detail:

Now, vancomycin binds to the D-Ala:D-Ala in the peptidoglycan side chain via 5 hydrogen bonds (kind of an electrostatic bonding which can only occur between Hydrogen and Nitrogen, Oxygen or Fluorine)  However, with the substitution of D-Ala:D-Lac into the side chain, and the removal of the amine group, there is one less hydrogen bond capable of forming.  You say "big deal"?  Well, actually it is a big deal as this loss of one hydrogen bond weakens the bond between the vancomycin molecule and the enterococcal peptidoglycan side chain by three fold, or 1000X.  While the vancomycin molecule still attaches to some degree, it is not in a configuration that can effectively block or prevent the Trans-glycosylase enzyme from forming the pentapeptide cross-link between the neigbouring peptidoglycan wall components

Vancomycin Dependence (VDE):
It has been proposed that vancomycin dependence may develop from the loss of a functional D-Ala:D-Ala ligase in the VRE strain, which is then unable to survive unless vancomycin induces the production of D-Ala: D-Lac ligase. This dependence involves mutations to the dll gene which encodes the enterococcal D-Ala:D-Ala ligase protein.

In other words, Vancomycin induction of the Van A or Van B ligase would compensate for the absence of the native ligase by producing D-Ala:D-Lac allowing for cell wall precursor synthesis. Since these ligases are only induced in the presence of vancomycin, the organisms cannot grow in the absence of this antibiotic unless it reverts to the vancomycin resistant form.

Revertant Mutant Enterococci:
If a particular strain of enterococcus becomes dependent on vancomycin for its growth and survival, it would seem logical that removing vancomycin would cause the organism to die. Surprisingly, this is not always the case as the organism may undergo a ‘revertant’ mutation. The enterococcus may undergo another genetic change that restores the D-Ala:D-Ala ligase function. The organism may enter a cyclical mutational change allowing it to shift between resistant and dependant phenotypes.
Withdrawal of vancomycin may not be adequate to eliminate vancomycin dependent strains.

On the first photograph of this post, the colonies randomly scattered throughout the agar surface, away from the E-test strip may be revertant colonies.  These colonies were not apparent after 24 hours however these colonies appeared after sitting on the bench for approximately another 16 hours.

Revertant strains have not been observed in clinical situations and the presence of VDE does not appear to affect the patient’s clinical outcome.

These are the kind of microbiological oddities that give this blog its title “Fun With Microbiology”!


1. Bacterial resistance to vancomycin: five genes and one missing hydrogen bond tell the story.
C.T. Walsh et al: Chemistry & Biology: January 1996, 3:21-28

2. Vancomycin resistance in enterococci: reprogramming of the d-Ala–d-Ala ligases in bacterial peptidoglycan biosynthesis.
V.L. Healy et al: Chemistry & Biology: Volume 7, Issue 5, 1 May 2000, Pages R109–R119

3. Vancomycin-Resistant Enterococci: Mechanisms and Clinical Observations.
H.S.Gold; Clinical Infectious Diseases (CID)  33: 210-218, July 2001

4. Crystal Structure of Vancomycin
Martina Schäfer, Thomas R Schneider & George M Sheldrick.
Structure: 15 December, 1996, 4: 1509-1515

5. The cytoplasmic peptidoglycan precursor of vancomycin-resistant Enterococcus faecalis terminates in lactate.
S. Handwerger et al: J Bacteriol. Sep 1992; 174(18): 5982–5984.

6. Final Diagnosis – Vancomycin-Dependent Enterococcus VDE:  (no source address/author provided. )

7. Vancomycin Dependent Enterococcus faecium Isolated from Stool following Oral Vancomycin Therapy:  LIsa L. Dever, et al.) J Clin Micro. Vol. 33, No. 10  Pg. 2770 – 2773, 1995

8. An Outbreak of Vancomycin-Dependent Enterococcus faecium in a Bone Marrow Transplant Unit.  B.D. Kirkpatrick et al: CID Vol 29: pg. 1268 – 1273, 1999

9.  Noscomial Infection with Vancomycin Dependent Enterococci:  Paul A. Tambyah et al, Emerg. Infect. Dis. Vol. 10, No.7 July 2004

10. Vancomycin analogues active against vanA-resistant strains inhibit bacterial transglycosylase without binding substrate.
L.Chen et al: Proc Natl Acad Sci U S A. May 13, 2003; 100(10): 5658–5663.

Thursday, 9 January 2014

Fasciola hepatica

Fasciola hepatica (Trematode – Parasite)

Geographic Distribution & Pathogenicity

Fasciola hepatica is commonly known as the sheep liver fluke and is a common parasite in herbivores.  With cosmopolitan distribution, human infections have been reported in many parts of the world.  Fasciola hepatica is most frequently found in countries where sheep raising is common, such as China, Taiwan, India, Indonesia and other parts of Asia. 

Fasciola hepatica is responsible for the disease fascioliasis, also known as fasciolopsiasis or simply, sheep liver fluke infection.  The infection may have first been recognized as early as 1379 when the effects were noticed between certain water plants and the sheep that had eaten them.  Fascioliasis is considered a zoonotic disease (passed from animals to man).

Symptoms:  The infection may produce symptoms of biliary obstruction and cholangitis.  Symptoms may include upper right quadrant pain, fever, chills and jaundice.  Symptoms may depend on the worm burden and light infections may be asymptomatic.

Life Cycle & Morphology:

Worms: The Fasciola fluke is quite large and may measure as large as 3 cm by 1.5 cm in size.  The anterior end of the worm (fluke) has a distinctive cone shaped projection. The interior organs of the worm appear extensively branched.  The adult worms live in the bile ducts of the liver and the gallbladder.

Eggs:  The eggs (ova) are large (80-150 µm by about 60 -90 µm) and broadly elliptical in appearance.  They are operuclated but the operculum is rather small in relation to the egg and rather inconspicuous.  The eggs are unembryonated when passed in the feces.  When passed into water, they undergo embryonation and subsequently miracidia are hatched (usually in 1 – 2 weeks).  Fasciola hepatica requires an intermediate host for development, which in this case is a freshwater snail (Lymnaea sp).  The miracidia within the snail mature and emerge as cercariae which then attach to aquatic vegetation (eg. watercress) where they undergo encystation.  Humans are infected by the ingestion of uncooked aquatic vegetation on which the metacercariae are encysted.  The metacercariae excyst (hatch) in the duodenum and migrate through the intestinal wall into the peritoneal cavity.  The larvae penetrate the liver and wander through the parenchyma for up to 9 weeks.  The larvae finally enter the bile ducts where they mature and in about three to four months and begin to produce eggs, which are ultimately passed out in the feces.  The adult worms may live for up to a year.


Diagnosis is made by the detection of the characteristic eggs in the patient’s faeces.  One problem in identification is that the species Fasciolopsis buski produces eggs which are almost indistinguishable from those produced by Fasciola hepatica.  Life cycles of these two trematodes are very similar.  In some areas of the orient where these two species overlap, the clinical evaluation of symptoms aids the diagnosis of these faciolid eggs.  The size of the operculum opening may also assist in diagnosis where the Fasciola hepatica’s operculum is larger than that of Fasciolopsis buski (measurements to follow).  Putting pressure on the coverslip of a concentrated faecal specimen with the eraser end of a pencil may be sufficient to cause the operculum to pop open and better reveal itself.  Molecular methods may provide a definitive identification.

 Fasciola hepatica egg in faecal concentrate.  Bile-stained shell and inconspicuous operculum.
  Egg measures 151 µm  by 75 µm.  (DMD-108)

 Fasciola hepatica egg with Iron Heamatoxylin stain. (DMD-108)

Fasciola hepatica egg showing the operculum (OP). Measurement reads 28.82 µm.  (DMD-108)

*   *   *

Saturday, 4 January 2014

Okay, Why so much Fungus???

This blog is called ‘Fun With Microbiology’, not’ Fun With Mycology’ – so what gives?  Why the disproportionate number of Fungal posts?

Well, this blog came about primarily because, after my injury, I needed some way to fill my time after regular work hours while waiting for my ride home.  With my return to work, I discovered that the laboratory had acquired some new “Toys” for photographing interesting microbiological specimens.  Surprisingly, few were interested in utilizing them, whereas with my previous interest in photography, I was only too happy to take command of these tools.

This blog is primarily about photography - about describing microbiology through pictures.  It is also about “interesting” cases – some of the less commonly encountered observations usually not described in text books.   (eg Mycobacteria in a gram stain, Strongyloides  tracks on agar plates, VDE –bacteria that need  antibiotics to survive.) Finally, it is about sharing my photographs with anyone who may find them of interest.

So, why so much fungus?  Well, bacteria exhibit a limited number morphological variations, as I’ve attempted to illustrate (right).  Generally speaking, an E.coli cell looks pretty much like a Salmonella cell, looks like a Citrobacter cell.  While important, yet often subtle, differences in morphology do occur in bacteria, I have chosen not to pursue them on this site.  Bacteria will be documented when the topic best lends itself to photographic interpretation.  

 Generally speaking, there are a limited number of bacterial morphotypes to explore with photography.

While I wish to pursue Parasitology a bit more extensively, I find myself limited to what cases present themselves in our acute care community hospital.

Numerous fine textbooks on Clinical Mycology are in print (see sidebar); however I have frequently found that the description ‘in text’ relates poorly to the one or two small supporting photographs offered.  Fungi are three dimensional organisms whose structure varies as they mature.  I’ve attempted to document these fascinating organisms from all angles and in all pertinent stages of development.

In summary, whether saprophytic contaminants or clinically significant isolates. fungi are the most photogenic microbiological organisms and present themselves in sufficient numbers to keep this blog going.

Finally, I offer my apologies for the rather lame name of this Blog.  My wife suggested I explore ‘blogging’ as a way to pass the time while bed bound, recovering from a catastrophic injury.  I jokingly chose this name never thinking I’d develop it past the few print photos I had taken years earlier.  Too late to change it now…