For Sigma Xi 2016

1. Development of a Novel Antiseptic to
Target MRSA and Pseudomonas
Janie Kim
Scripps Ranch High School, San Diego

2. Background Information
 Keratitis is the leading cause of corneal blindness.
 P. aeruginosa and S. aureus are responsible for most
keratitis cases.
 Multidrug-resistant P. aeruginosa and Methicillin-
resistant S. aureus are responsible for more than
131,000 infections per year.

3. Background Information
 Contact lens solutions need improvement.
 Pathogens can survive in current solutions for several
hours.
 Many contact lens users are non-compliant with
recommended disinfection methods, often reusing
solution or topping off with water.
 Several ophthalmological societies testified that contact
lens solutions need to be more antimicrobial.

4. The Goal
 To develop a novel combination of the antiseptic
compounds contained within contact lens solutions
that is better able to kill P. aeruginosa and S. aureus.
With a more potent antiseptic combination, improvements
could be made not only in contact lens solutions, but also in
many other commercial disinfection products that utilize the
same antiseptic components.

5. Hypothesis
 Testing antimicrobial compounds from
commercial contact lens solutions, both
individually and in various combinations, would
lead to the development of the ideal antiseptic
combination.

6. Procedures – General
Overview
Contact Lens Solution Microdilutions (MICs)
Antiseptic Compound Microdilutions (MICs)
Traditional Checkerboard Assay
Time-Kill Assay
ISO 14729 Stand-Alone Test Assay
Microdilutions (MICs), against clinical panel of bacteria

7. #1 – Contact Lens Solution
Microdilutions (MICs)
MRSA TCH 1516
Boston
Simplus
Boston
Advanced
Menicare Lobob Opti-Free
MinimalInhibitory
Concentration(%)
Boston
Simplus
Boston
Advanced
Menicare
GP
Lobob Opti-Free
MinimalInhibitory
Concentration(%)
P. aeruginosa PA01
This was done to identify which commercial contact lens solutions were the most
effective against MRSA and Pseudomonas. With these results, the component
compounds within those top-performing solutions could also be identified to be
tested.

8. #2 – Antiseptic Compound MICs
Minimum Inhibitory
Concentration
MRSA TCH
1516
P. aeruginosa
PA01
Chlorhexidine
Gluconate
2.5 PPM 15 PPM
Polyaminopropyl
Biguanide
2.5 PPM 20 PPM
EDTA 300 PPM 2,500 PPM
Benzyl
Alcohol
10,000 PPM 5,000 PPM
Contained
within
Boston
Simplus
Contained
within
MeniCare

9. Key Compounds Identified from Step
#2
 Chlorhexidine gluconate (Boston Simplus)
 Effective against MRSA
 Used in mouthwashes, surgical scrubs, and in treating umbilical
cords of newborn babies
 Polyaminopropyl biguanide (Boston Simplus)
 Effective against MRSA
 Used in wound dressings and many cosmetic/personal care
products
 EDTA (MeniCare)
 Effective against Pseudomonas
 Common food preservative, and is used in chelation therapy

10. #3 – Checkerboard Assay
 Blue wells bounded by
the green bars all have
FIC index values <0.5
(synergy).
 Green box:
approximate
concentrations used
for the next step:
 3.8 PPM CHD, 313 PPM
EDTA
This was done to test the combination of two promising compounds from two
different commercial contact lens solutions, chlorhexidine gluconate and EDTA.

11. #4 – Time-Kill Assay
 4 PPM CHD+300 PPM
EDTA: > 4 log10 reduction
in PA in 2 hours.
 FIC index of 0.39
 Individual 4 PPM CHD,
300 PPM EDTA, and
positive control: > 2 log10
increases in PA

12. The next step was to test 30 PPM chlorhexidine gluconate
and 5 PPM polyaminopropyl biguanide (the concentrations
from Boston Simplus) combined with 5000 PPM EDTA (the
concentration within MeniCare).
This combination was dubbed C30/P5/E5000.

13. #5 & #6 – ISO Assay & MICs
 In just one hour,
C30/P5/E5000 reduced the
concentration of MRSA by 5
log10 and Pseudomonas by
almost 6 log10.
 Compare this to the
recommended disinfection times
for Boston Simplus (4 hours) and
MeniCare (6 hours).
 C30/P5/E5000 was able to kill
several clinical strains of S.
aureus and P. aeruginosa at
concentrations as low as 3%
to 6%.
S. aureus MIC P.
aeruginosa
MIC
TCH1516 -
MRSA
3% PA01 6%
Sanger252 -
MRSA
6% PA103 -
Hemolytic
6%
UAMS1 -
MSSA
6% P4 - MDR 6%
These steps were done for the novel combination of 30 PPM CHD, 5 PPM PAPB,
and 5,000 PPM EDTA (C30/P5/E5000).

14. Discussions & Conclusions
 Novel combination
 Chlorhexidine & EDTA synergistic against P. aeruginosa.
 Equal anti-staphylococcal , 3-7 times more anti-
pseudomonal
 Very effective at low concentrations against drug-resistant
P. aeruginosa and S. aureus.
 Previous research has also shown that chlorhexidine,
polyaminopropyl biguanide, and EDTA are effective against
S. aureus and P. aeruginosa biofilms, acanthamoeba, and
fungal eye pathogens.

15. Advantages of C30/P5/E5000
 Effective at low concentrations against both Gram-negative
and Gram-positive drug-resistant bacterial pathogens.
 Bacteria do not easily develop resistance to C30/P5/E5000.
The three component preservatives strip away crucial ions such as Ca2+ and
Mg2+ from all over the cell membrane, rather than targeting one specific site
like most antibiotics.

16. Advantages of C30/P5/E5000
Continued
 More cost-efficient than any of its individual components:
Polyaminopropyl biguanide costs ~$3 per oz
EDTA costs ~$10 per oz
Pure chlorhexidine gluconate costs ~$200 per oz
 C30/P5/E5000 costs ~$10 per oz.
 C30/P5/E5000 can improve not only contact lens solutions,
but also many other disinfectant products.
E.g., Hand sanitizers, disinfecting wipes, mouthwashes, and surgical scrubs.

17. Summary
 Developed a novel antiseptic combination that is
effective against multidrug-resistant Pseudomonas
aeruginosa and MRSA at low concentrations, is
affordable, and can be integrated into a wide array of
commercial disinfecting products.

18. Acknowledgments
Mr. Leo Lin
Dr. Victor Nizet
Ms. Mitra Moshiri

19. References
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