Advance Microbiology Presentation

Presented By: Balbeer SinghDated: 25th Oct 2013SZABIST, Karachi

Anti-Bacterial Activities of Serum from

the Komodo Dragon

Research Published on 29th August 2012

Different Biologists Contributions:

• Mark Merchant• Danyell Henry• Rodolfo Falconi• Becky Musher• Judith Bryja

Taxonomy • Phylum: Chordata• Class: Reptilia• Order : Squamata • Family: Varanidae • Species: Varanus Komodoensis

Features• Largest lizard on Earth• May live up to 100 years• Can run 20 kilometers per hour• Dive upto 4.5 meters ( 15 feets) deep in water• Kills its prey with a lethal bite• Believed to inject toxic substance into the

blood stream of the animals• Saliva contains potent and complex venom

Primary goal of the research was to characterize the antibacterial properties of Serum from the

Komodo Dragon

Nutrient BrothLow EEO AggroseBacterial Cultures

Gram Negative Bacteria

• Esherichia Coliform• Shigella Flexnerri• Klebsiella Oxytoca• Salmonella Tryphimurium• Providenica Stuartii.

Gram Positive Bacteria

• Staphylococcus Aureus• Streptococcus Epidermitis• Streptomyces Griseus

Collection of Blood Sample of Komodo Dragon

• Collected from komodo Dragons at the Houstan and San Antonio Zoo.

• Collected from 3 adults of weight 20 – 81.5 kg• Collected from 5 juveniles of weight 1.5-6.2 kg• Drawn from the tail vein• Transferred to vaccutainer tube• Allowed to clot for 5 hours before collecting the

serum.• Serum was pooled so that the average

antibacterial values can be generated• Stored at -20’C until used for bacterial assay.

Serum Volume Dependent Antibacterial Assays

• Took 200 samples of serum containing volumes of 20-200 ul in micro-titer plates.

• The plates were then inoculated with 5ul of bacterial culture.

• Then the samples were incubated for 24 hours at 37’C.

• Optical density of each sample was measured at 0,3,6,12 and 24 hour using Benchmark Plus micro-titer plate reader at 430 nm.

Kinetic Antibacterial Assay

• 500 ul of Komodo Dragon serum was inoculated with 20 ul of an E.Coli culture in log phase growth.

• 50 dilution of each sample were spread onto the surface of Nutrient Broth agar plates to determine the CFU’s for each sample.

• To determine the possible antibacterial mechanism, pooled serum was incubated with pronase ( isolated from Streptomyces Griseus), 5 mm EDTA or 5 mm phosphate for 30 minutes at ambient temperature.

Results of Serum Volume Dependent Antibacterial Assays

Results for Gram Negative Bacteria

• 10% of Komodoensis serum produced 44% decrease in E.Coli growth.

• 25% of serum produced 58% decrease in E.Coli growth.• 50% of serum poduced 75% decrease in E.Coli growth.• 75% of serum produced 93% decrease in E.Coli growth• 100% of serum produced 98% decrease in E.Coli

growth• 1 ul of serum has the capacity to kill approximately

2.24 million of E.Coli bacteria.

• 25% of serum produced 65% decrease in Klebsiella Oxytoca.

• 58% of serum completely inhibited the growth of Klebsiella Oxytoca in 24 hours.

• 10% of Serum inhibited Shigella Flexneri and Salmonella Typhimurium by 95% at 24 hours

• Providencia Staurti’s growth is completely inhibited by 10% Komodoensis serum.

Results for Gram Positive Bacteria

• 10% of komodoensis serum inhibited the growth of Staphylococcus Aureus by 64 % at 24 hours.

• 25% of serum inhibited 68% growth of Staphylococcus.

• 50% of serum inhibited 84% growth of Staphylococcus.

• 75% of serum inhibited 94% growth of Staphylococcus.

• 10% of serum inhibited 49% growth of Streptococcus Epidermitis

Results for Kinetic Antibacterial Assay

• Antibacterial activity occurred quickly as 18% of the bacteria (2.95 million) were killed in 5 mins.

• Further incubation resulted in 43% (7.05 million) of the bacteria killed in 10 mins after the exposure to serum

• In 20 mins 68% (11.2 million) of E.coli bacteria were killed.

• Treatment of serum with 5 Mm EDTA or 5 Mm of Phosphate only resulted in 6.3% or 8.7% of the antibacterial activity.

• This activity of Serum suggests that Komodo Dragon are resistant to Microbial Infections.

• It would be reasonable to expect the Komodo Dragons would have developed Innate Immune Mechanisms to combat possible infections by the bacteria in their own Saliva.

• Before this study ,crocodilians were considered to have the most potent serum complement defense system.

• The result in this study show that the potency of Komodo Dragon serum is much higher than the crocodilians