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Senior Research

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Charlene Caoili

on 27 March 2017

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Transcript of Senior Research

Retrieved from:http://www.richardsmedical.com/images/microcuff_endotracheal.jpg
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Antibiofilm activity of pomegranate extract against
Staphylococcus epidermidis
Charlene Mae Caoili
December 3, 2014
BIO3099: Research Seminar
Community of microorganisms attached to a substrate producing extracellular polymeric substance (EPS)
Able to form on almost any surface
Formed substrate reduces the ability of antibiotic treatment
(Otter et al., 2014)
Staphylococcus epidermidis
Normally present human skin and mucous membranes
Mostly moist/wet
Staphylococcus genus; "gram-positive" bacteria
icaA, icaD, icaB, and icaC genes
(Cho, Naber, Hacker, & Ziebuhr, 2002)
encodes the production of
-acetylglucosamine polysaccharide intercellular adhesion
ica operon
S. epidermidis
positive reference biofilm forming
S. epidermidis
RP62A (ATCC 35984)
ATCC 12228
non-biofilm forming
(Zhang et al., 2003; Cramton et al., 1999)
Li et al. (2005) conversion of
S. epidermidis
strains from commensal to invasive with the expression of
Retrieved from: http://cdn.c.photoshelter.com/img-get/I0000A2Auhlc04ms/s/600/600/3009061.jpg
Retrieved from: http://iai.asm.org/content/67/10/5427/F2.large.jpg
S. epidermidis
has become important associated pathogen in nosocomial infections due to indwelling medical devices
Adequate disinfection on hospital surfaces
Biofilms not restricted to moist/wet hard surfaces
Catheters, endotracheal tube, or any implanted device for a prolonged period of time
(Zhang et al., 2003)
Dry and fabric surfaces: Neely & Maley (2000) examined five common hospital materials
smooth 100% cotton, 100% cotton-terry, 60% cotton – 40% polyester, 100% polyester, and 100% polypropylene plastic
The ellagic acid in pomegranate will be an effective antibiofilm agent against pre-formed
S. epidermidis
biofilms and inhibit formation.
Bakkiyaraj, D., Nandhini, J. R., Malathy, B., & Pandian, S. K. (2013) The anti-biofilm potential of pomegranate (Punica granatum L.) extract against human bacterial and fungal pathogens. Biofouling: The Journal of Bioadhesion and Biofilm Research, 29(8), 929-937. doi:10.1080/08927014.2013.820825

Chen, M., Yu, Q., & Sun, H. (2013). Novel Strategies for the Prevention and Treatment of Biofilm Related Infections. International Journal of Molecular Sciences, 14, 18488-18501. doi:10.3390/ijms140918488

Cho, S., Naber, K., Hacker, J., & Ziebuhr, W. (2002). Detection of the icaADBC gene cluster and biofilm formation in Staphylococcus epidermidis isolates from catheter-related urinary tract infections. International Journal of Antimicrobial Agents, 19(6), 570-575. doi:10.1016/S0924-8579(02)00101-2

Cramton, S. E., Gerke, C., Schnell, N. F., Nichols, W. W., & Gotz, F. (1999). The Intercellular Adhesion (ica) Locus Is Present in Staphylococcus epidermidis and Is Required fro Biofilm Formation. Infection and Immunity, 67(10), 5427-5433. Retrived from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC96900/pdf/ii005427.pdf

Dahham, S. S., Ali, M. N., Tabassum, H., & Khan, M. (2010). Studies on Antibacterial and Antifungal Activity of Pomegranate (Punica granatum L.). American-Eurasian J. Agric. & Environ. Sci., 9(3), 273-281. Retrieved from http://idosi.org/aejaes/jaes9%283%29/8.pdf

Das, J.R., Bhakoo, M., Jones, M. V., & Gilbert, P. (1998). Changes in the biocide susceptibility of Staphylococcus epidermis and Escherichia coli cells associated with rapid attachment to plastic surfaces. Journal of Applied Microbiology, 84, 852-858. Retrieved from http://web.a.ebscohost.com/ehost/pdfviewer/pdfviewer?sid=af025d79-6443-48e8-900a-2caeb3277586%40sessionmgr4001&vid=7&hid=4114

Li, H., Xu, L., Wang, J., Wen, Y., Voung, C., Otto, M., & Gao, Q. (2005). Conversion of Staphylococcus epidermidis Strains from Commensal to Invasive by Expression of the ica Locus Encoding Production of Biofilm Exopolysaccharide. Infection and Immunity, 73(5), 3188-3191. doi:10.1128/IAI.73.5.3188-3191.2005

Neely, A. N., & Maley, M. P. (2000). Survivial of Enterococci and Staphylococci on Hospital Fabrics and Plastic. Journal of Clinical Microbiology, 38(2), 724-726. Retrived from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC86187/pdf/jm000724.pdf

O'Toole G.A. (2011). Microtiter Dish Biofilm Formation Assay. Journal of Visualized Experiments, 47, doi:10.3791/2437

Otter, J.A., Vickery, K., Walker, J., Pulcini, E. d., Stoodley, P., Goldenberg, S.D., … Edgeworth, J. D. (2014). Surface-attached cells, biofilms and biocide susceptibility: implications for hospital cleaning and disinfection. Journal of Hospital Infection, 1-46. doi:10.1016/j.jhin.2014.09.008

Otto, Michael. (2009). Staphylococcus epidermidis – the “accidental” pathogen. Nature Reviews Microbiology, 7(8), 555-567. doi:10.1038/nrmicro2182

Pettit, R.K., Weber, C. A., Kean, M. J., Hoffmann, H., Pettit, G. R., Tan, R., … Horton, M. L. (2005). Microplate Alamar Blue Assay for Staphylococcus epidermidis Biofilm Susceptibility Testing. Antimicrobial Agents and Chemotherapy, 49(7), 2612-2617. doi:10.1128/AAC.49.7.2612-2617.2005

Sadovskaya, I., Vinogradov, E., Flahaut, S., Kogan, G., & Jabbouri, S. (2005). Extracellular Carbohydrate-Containing Polymers of a Model Biofilm-Producing Strain, Staphylococcus epidermidis RP62A. Infection and Immunity, 73(5), 3007-3017. doi:10.1128/IAI.73.5.3007-30017.2005

Wei, W., Cao, Z., Zhu, Y., Wang, X., Ding, G., Xu, H., … Li, Y. (2006). Conserved genes in a path from commensalism to pathogenicity: comparative phylogenetic profiles of Staphylococcus epidermidis RP62A and ATCC12228. BMC Genomics, 7(112). doi:10.1186/1471-2164-7-112

Wiegand, I., Hilpert, K., & Hancock, R. E. W. (2008). Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nature Protocols, 3(2), 163-17. doi: 10.1038/nprot.2007.521

Zhang, Y., Ren, S., Li, H., Wang, Y., Fu, F., Yang, J., … Wen, Y. (2003). Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228). Molecular Biology, 49(6), 1577-1593. doi:10.1046/j.1365-2958.2003.03671.x

I would like to thank Dr. Cheung for mentoring me, Dr. Pappas, and the Manhattanville Biology Department.
et al.
, 2005)
(Li et al., 2005)
More resistant than planktonic batcterium
(Pettit et al., 2005)
(Wei et al., 2006)
Pomegranate Extract
Punica granatum
Homeopathic anti-biofilm agent
Antibiotic treatment could develop in resistance, e.g. MRSA (methicillan-resistant
Staphylococcus aureus
Bakkiyaraj et al. (2014):
Observed pomegranate extract inhibited formation in
S. aureus
Disrupted pre-formed biofilms in
C. albicans
(Bakkiyaraj et al., 2014)
Ellagic acid identified as major component in pomegranate
Bioactive tannin with various properties (e.g. antioxidant, anticancer, anti-inflammatory)
ATCC 12228-
expressed biofilm formation
Bacterial Strains

S. epidermidis strains RP62A (ATCC 35984) and ATCC 12228 will be maintained at 35°C in Mueller Hinton agar (MHA). Confirmation of biofilm formation will be done similar to Pettit et al. (2005), with which the strains will be grown in cation-adjusted Mueller Hinton II broth (MHIIB) for 24 hours in a flat-bottom, polystyrene, non-tissue-culture-treated 94 microtiter plates. Ten μl of 1% solution of Congo red will added to each well with brief shaking applied before a 10 minute incubation at room temperature. Liquid will be removed from the wells and then gently washed with saline (Pettit et al., 2005). The stained biofilms will be then visualized by phase contrast microscopy where ATCC 12228 will be compared to RP62A.

Pomegranate Extraction

The pomegranate extract will be purchased from BulkSupplements.com that is listed to contain 40% ellagic acid. Most commercial pomegranate extract powder is subjected to aqueous extract. The pomegranate aqueous extract (PAE) will be dissolved in sterile Milli-Q water (Bakkiyaraj, Nandhini, Malathy, & Pandian, 2013).

S. epidermidis strains RP62A (ATCC 35984) and ATCC 12228
Pomegranate Aqueous Extract (PAE)
BulkSupplements.com pomegranate extract powder
(Bakkiyaraj, Nandhini, Malathy, & Pandian, 2013)
40% ellagic acid
Whole fruit with Pericarp
Country of origin: China
Powder Color: Grey
Pomegranate aqueous extract (PAE) will be dissolved in sterile Milli-Q water
Bacterial Strains
35°C in Mueller Hinton agar (MHA)
Pettit et al. (2005) biofilm formation conformation:
cation-adjusted Mueller Hinton II broth (MHIIB) for 24 hours
flat-bottom, polystyrene, microtiter plates
Stained with 10 μl of 1% Congo red
Stained biofilms will be visualized; ATCC 12228 will be compared to RP62A.
Clinical and Laboratory Standards Institute (2006) microdilution guidelines
Antibiofilm Activity of Pomegranate
Minimum Inhibitory Concentration (MIC)
Extract concentrations (100 – 1000 μg/ml inoculated with a 2% (v/v) inoculum (~〖10〗^5 bacterial cells)
Incubated 37°C for 24 hours and absorbance at 600 nm
Growth control culture plated to verify CFU/ml
Minimum concentration of extract to inhibit growth determined as MIC
(Bakkiyaraj et al., 2013)
(Wiegand, Hilpert, & Hancock, 2008)
PAE at various concentrations (μg/ml above,below, and at the MIC)
Absorbance measured at 600 nm after incubation
Wells washed 3X with distilled water (DW)
Stained with 0.4% crystal violet (CV) solution & washed again
Destained with 95% alcohol
Absorbance measured at 570 nm
Biofilm inhibition equation:
Biofilm inhibitory concentration (BIC): lowest concentration that produces inhibition of biofilm formation
Biofilm Disruption of PAE
Biofilms will be grown without PAE in microtiter plate
BIC of pomegranate aqueous extract added
Incubated at 37°C for 24 hours, washed 3X with DW
Stained with 0.4% crystal violet
Destained with 95% alcohol and absorbance at 570 nm measured
Biofilm disruption following equation:
Controls: without PAE, broth alone (negative control)
Control: Phosphate-buffered saline (PBS) and fresh media plated in a set of wells
(Bakkiyaraj et al., 2013)
(Bakkiyaraj et al., 2013)
Biofilm Formation on Various Surfaces
Microttiter Biofilm Assay - crystal violet stain
Various surfaces found in the hospital environment:
Moist plastic
Dry plastic
Moist polyester
Dry polyester
Effects of PAE under a nutrient limited and a nutrient rich condition
(Bakkiyaraj et al., 2013)
(Bakkiyaraj et al., 2013)
(Neely & Maley, 2000)
1. Transformed
S. epidermidis
ATCC 12228-
will be less susceptible to pomegranate extract.
Working Hypotheses
2. Disinfection and prevention of formation of
S. epidermidis
biofilms on dry surfaces will be less susceptible to pomegranate extract than wet surfaces.
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