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Molecular insight into anti-infective effects of selected fruit phytochemicals against Streptococcus pyogenes

  

Admission to Candidacy Examination M.Sc. Program Requirement

 

Research Proposal 

Submitted by: Soheila Abachi

Department of Environmental Sciences Faculty of Agriculture Dalhousie University

June 16 2014

  

 

 

 

 

 

 

Supervisor:Supervisory committee members:

Dr. Vasantha Rupasinghe Dr. Song Lee (Faculty of Medicine) Dr. Bruce Rathgeber

 

   

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Abstract Group A streptococci (GAS) not only is important species of gram-positive extracellular bacterial pathogens but also is the most common cause of bacterial pharyngitis in children and adults. This bacterium has developed complex virulence mechanisms to compete with original flora and avoid its elimination. There are several essential steps for initiation of bacterial infectious diseases, adherence being the most important one followed by its stable association with the mucosal surface and formation of biofilm. Surface streptococcal ligands bind to specific receptors on host’s pharyngeal cells and start colonizing. Without strong host-pathogen interactions GAS cannot successfully adhere and would be eliminated by flow mechanisms of the hosts’ environment. Therefore, one of the strategies to control GAS is to inhibit or disrupt its adherence, biofilm formation or its metabolic activity. Various phytochemical components of medicinal plants have shown promising inhibitory effects against pathogenic Streptococcus pyogenes. Specific polyphenols and isoprenoids, among their broad health effects, have shown antibacterial activity against gram positive and specifically streptococci species. The mode of inhibition of phytochemicals against S. pyogenes is yet undefined. The proposed research aims to advance the understanding of the antimicrobial activity of selected fruit phytochemicals against GAS at molecular level by modern methods such as bacterial adherence assay. The results of this research may further be investigated for incorporating the effective extracts or compounds into antibacterial products such as HonibeTM dehydrated honey lozenges.

List of abbreviations and symbols ANOVA analysis of variance LC-MS liquid chromatography–mass spectrometry ATCC American type culture collection LTA lipoteichoic acid ATP adenosine triphosphate MBC minimum bactericidal concentration BHI brain heart infusion MgCl2 magnesium chloride C2H3N acetonitrile MgSO4 magnesium sulfate CDC center for disease control &prevention MH Mueller-Hinton CFU colony forming unit MIC minimum inhibitory concentration DMSO dimethyl sulfoxide MS mass spectrometry EDTA ethylenediaminetetraacetic acid NCCLS national committee of clinical laboratory

standards EPS exopolysaccharide NH4HCO3 ammonium bicarbonate F-ATPase F type ATP synthase OD optical density FBP fibronectin-binding proteins PBS phosphate-buffered saline GABHS group A β-hemolytic streptococcus Pi inorganic phosphate GAS group A streptococci RPMI Roswell Park Memorial Institute medium GC-MS gas chromatography–mass

spectrometry TE Tris-EDTA

HA hyaluronic acid THY Todd-Hewitt broth supplement with yeast extract

HCl hydrogen chloride HTEpiC human tonsil epithelial cells HY hyaluronatlyase

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Contents 1.  Introduction ................................................................................................................................. 4 2.  Literature review......................................................................................................................... 5 2.1.  Importance of Streptococcus pyogenes ...................................................................................... 5 2.2.  Pathogenicity of Streptococcus pyogenes ................................................................................... 5 2.3.  Pathophysiology of pharyngitis .................................................................................................. 6 2.4.  Modern medicine and development of drug-resistant strains ................................................ 7 2.5.  Emerging threat of antibiotics ................................................................................................... 7 2.6.  Folklore medicine ........................................................................................................................ 8 2.7.  Phytochemicals of berries ......................................................................................................... 10 3.  Research hypothesis and objectives ......................................................................................... 11 3.1.  General objective....................................................................................................................... 11 3.2.  Specific objectives ..................................................................................................................... 11 4.  Research methodology .............................................................................................................. 11 4.1.  Extract preparation .................................................................................................................. 13 4.1.1.  Polyphenol-rich extract and subsequent fractions preparation ........................................... 13 4.1.1.1.  Water-based extraction ............................................................................................................ 13 4.1.1.1.1. Purification of bioactive compounds from aqueous extracts ................................................ 13 4.1.1.2.  Ultrasonication-assisted ethanol extraction ............................................................................ 13 4.1.2.  Isoprenoid-rich extract and subsequent fractions preparation ............................................ 14 4.1.2.1.  Hydro-distillation technique .................................................................................................... 14 4.1.2.2.  Solvent based reflux system ..................................................................................................... 14 4.1.2.3.  Purification of bioactives of extracts obtained by hydro-distillation & reflux techniques . 14 4.1.3.  Determination of total isoperenoid and polyphenol content ................................................. 14 4.2.  Initial screening phase .............................................................................................................. 15 4.2.1.  Susceptibility testing- micro-dilution assay ............................................................................ 15 4.2.3.  Disk diffusion antibiotic sensitivity testing ............................................................................. 16 4.2.4.  Preliminary phytochemical analysis ........................................................................................ 16 4.3.  Understanding the mode of action phase ................................................................................ 16 4.3.1.  Adherence reduction or inhibition .......................................................................................... 16 4.3.1.1.  Preliminary bacterial adherence assay ................................................................................... 16 4.3.1.2.  Bacterial adherence Human Tonsil Epithelial Cells and human laryngeal HEp-2 cells .... 17 4.3.1.3.  Fibronectin cell adhesion assays .............................................................................................. 17 4.3.2.  ATPase assay ............................................................................................................................. 18 4.3.3.  Biofilm reduction or inhibition ................................................................................................ 18 4.3.3.1.  Biofilm inhibition assay ............................................................................................................ 18 4.3.3.2.  Total viable counts of biofilm ................................................................................................... 19 5.  Statistical analysis ..................................................................................................................... 19 6.  References .................................................................................................................................. 19  

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1. Introduction

Group A streptococcal infections range from none life threatening conditions such as mild skin

infection or pharyngitis to life-threatening and severe conditions such as necrotizing fasciitis, or

rheumatic fever including highly lethal Streptococcal toxic shock syndrome (1). Group A β-

hemolytic streptococcus (GAS) or S. pyogenes is the common cause of acute bacterial

pharyngitis, and is often called strep throat or sore throat (2). Pharyngitis is the most common

form of GABHS infections. Recorded cases of GAS pharyngitis are 15-36 percent in children (2)

and 5-15 percent in adults (3). On global scale, over 616 million new cases of GABHS

pharyngitis occur every year (4). Economical burden of the disease is estimated to be $224 to

$539 million among U.S. school-aged children every year and on average 7.3 million outpatient

physician visits take place by children aged 3-17 years each year in U.S. (2). Penicillin,

amoxicillin, erythromycin, and first-generation cephalosporins are the recommended antibiotics

for treatment of sore throat due to GABHS (5-7). Acute infections can lead to rheumatic fever

and post-streptococcal glomerulonephritis (kidney inflammation), which distress children

worldwide with disability and death, if antibiotic treatments fail or if the disease is left

unattended (8, 9). Rheumatic fever and rheumatic heart disease are known to be the leading

causes of cardiovascular death during the first five decades of life in underdeveloped countries

mainly concerning children (10). Streptococci have very specific virulence factors, which enable

them to cause such diverse infections (11-13).

Medicinal plants have long been used for the treatment of GAS infections including pharyngitis

in the form of tea, gargle, and drop. For example, gargle of infusions of bark and or leaves of

cashew plant (Anacardium occidentale), stickwort (Agrimony), mountain daisy (Arnica),

bayberry (Myrica cerifera), baobab (Adansonia digitata) (14-18) or tea of flowers of soft leafed

honeysuckle (Lonicera confuse) and leaves of cuajilote (Parmentiera aculeate) (19, 20). Natural

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products extracted from mainly medicinal plants as well as fruits of wild type, are the subject of

many studies as alternative substitutes to currently used antibiotics for the treatment of infectious

diseases and antibiotic-resistant human pathogens. With more in depth studies of the potent

components of these plant extracts and their mode of action, new drugs and antibiotics can be

developed for the soon to be post-antibiotic era.

2. Literature review

2.1. Importance of Streptococcus pyogenes

Particular virulence factors enable S. pyogenes to attach to host tissues, elude the immune

response, and spread by penetrating the host tissue layers and then colonizing (21). Hyaluronic

acid bacterial capsule, surrounds the bacterium and protects it from phagocytosis by neutrophils

(22). In addition to the capsule, several factors embedded in the cell wall facilitate attachment of

the bacteria to various host cells, including M protein, lipoteichoic acid, and fibronectin-binding

protein (23). S. pyogenes pilli promotes pharyngeal cell adhesion, aggregation to human cells and

biofilm formation (24).

2.2. Pathogenicity of Streptococcus pyogenes

There are several important steps for initiation of GAS infectious diseases. First is the bacteria’s

capacity to adhere to host tissues and its competition with the normal flora present on

nasopharynx surface. After successful attachment, they establish interaction with salivary

glycoproteins, extracellular matrix, serum components, host cells and other microbes and

assemble in cell aggregates (25). Then bacteria begin colonizing the host tissue, producing exo-

polysaccharide (EPS), differentiating into EPS-encased micro-colonies, and developingcomplex

communities called mature biofilm (25, 26). Not only biofilm offers GAS a protected

environment but also plays an important role in its pathogenicity which has been proposed and

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experimentally supported in recent studies (25, 27, 28). Proliferation of GAS in the pharynx

leads to the invasion of the host tissues. It has been reported that streptococci adhere in two

steps. First weak reversible adhesion probably mediated by hydrophobic interactions for example

between lipoteichoic acid (LTA) and the binding domains on the host cell. Second firm

irreversible adhesion mediated by composite multivalent interactions for example adherence of

protein F1 (a fibronectin binding protein) to fibronectin (a glycoprotein of the extracellular

matrix) (29-34). GAS has several surface proteins and produces numerous extracellular products

that facilitate permeation and successive evasion of the host’s immune system. Center for disease

control and prevention (CDC) report says 65% of human bacterial infections involve biofilms

and treatment of these biofilm-associated nosocomial infections cost more than $1 billion

annually (35). Antibiotic treatment has been indicated for streptococcal pharyngitis. Even though

GABHS is the common cause of bacterial pharyngitis, but other bacteria also could cause acute

pharyngitis including Actinomyces spp., Arcanobacterium haemolyticum, Bacteroides spp.,

Borrelia spp., Bordetella pertussis, Chlamydophila pneumoniae, Chlamydia trachomatis,

Corynebacterium diphtheria, Corynebacterium pyogenes and others (36-52). Streptococcal

pharyngitis results from the proliferation of GAS in the pharynx (1, 10).

2.3. Pathophysiology of pharyngitis

Signs of GAS pharyngitis overlap the symptoms of non-streptococcal pharyngitis meaning that

proper diagnosis of the disease on the basis of clinical presentation is often impossible (3).

Clinical presentations suggestive of GAS pharyngitis in children aged 5–15 years are: sudden

onset of sore throat fever, headache, nausea, vomiting, abdominal pain, tonsillopharyngeal

inflammation, patchy tonsillopharyngeal exudates, palatal petechiae, anterior cervical adenitis

(tender nodes), winter and early spring presentation, and scarlatiniform rashes (44, 53).

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2.4. Modern medicine and development of drug-resistant strains

Antimicrobials’ mechanism of action can be briefly described based on disruption of several

activities and processes including cell wall synthesis, plasma membrane integrity, nucleic acid

synthesis, ribosomal function, and folate synthesis (54). For patients with penicillin allergy, U.S.

treatment guidelines recommend erythromycin. In instances where gastrointestinal side effects of

erythromycin is observed, physicians prescribe the FDA-approved second-generation macrolides

azithromycin and clarithromycin (54). Penicillin derivatives (ampicillin or amoxicillin),

cephalosporins, and macrolides are all effective against GABHS. According to a survey, from

1995 to 2003 in U.S. physicians prescribed antibiotics to 53% of children with sore throat (2).

Amoxicillin was mostly prescribed (26% of visits), penicillin (7%), first-generation

cephalosporins (3%), and erythromycin (2%) (2). β-Lactam and macrolide class of antibiotics are

recommended and prescribed for GAS pharyngitis. The mechanisms of action of these

antibiotics differ from one another. Peptidoglycan polymerization which leads to cell wall

synthesis is inhibited by ß-lactams such as penicillins and cephalosporins (1). In other hand,

macrolides are capable of inhibiting a number of protein synthesis stages occurring on the

ribosome but do not usually interfere with amino acid activation or attachment to a particular

tRNA. Most of the macrolides have an affinity for 70S ribosomes (prokaryotic) versus 80S

(eukaryotic), resulting in macrolides selective toxicity (1). Erythromycin and clindamycin all

interfere with ribosome function (1, 10, 55). According to the researchers of internal medicine at

the University of Missouri, current GAS antibiotic treatments “interfere with critical biological

processes in the pathogen to kill or stop its growth leading to endurance of stronger strains of

harmful bacteria and prosper of resistant bacteria” (56).

2.5. Emerging threat of antibiotics

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Bacteria employ some basic mechanisms to resist an antimicrobial agent such as altering the

drug receptor and making the target insensitive to inhibitor (antibiotic), or decreasing the

physiological importance of target molecule to the bacteria’s pathogenicity, producing new

enzyme molecule, and replacing the inhibited target. Some penicillin-resistant streptococci,

group D streptococci and S. pneumonia, have developed resistance by altering the penicillin-

binding proteins. Streptococci species have developed resistance to macrolides such as

clindamycin and erythromycin through altering 23S RNA (57) and as emphasized earlier in the

introduction, CDC has prioritized erythromycin-resistant GAS as concerning threat in their 2013

report. Macrolides resistance among GABHS isolates in the United States is on rise, possibly

because of azithromycin overuse (58). This climb in certain areas of the United States and

Canada reaches 8-9 percent (59). GAS is not yet resistant to penicillin but some of the

streptococci such as S. pneumonia have developed resistance to penicillin through β-lactam

hydrolysis (60). All these mechanisms put forth strong selective pressure that favors emergence

of antibiotic-resistant strains.

2.6. Folklore medicine

Plants produce diverse secondary metabolites, most of which are phenols or their oxygen-

substituted derivatives such as tannins that could be the raw materials for future drugs. Herbs and

spices used by humans contain useful medicinal compounds including antibacterial chemicals

and researches have recorded many of these compounds shown to inhibit growth of pathogenic

bacteria (61). These agents appear to have structures and modes of action that are distinct from

those of the antibiotics in current use, suggesting that cross-resistance with drugs already in use

may be minimal. Observations have shown that herbal preparations work very well, but

extensive research and purification of such antibacterial agents are required to make an

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encouraging statement for the use of phytochemicals. In the period of 1981–2006, 109 new

antibacterial drugs were approved of which 69% originated from natural products, and 21% of

antifungal drugs were natural derivatives or compounds mimicking natural products (62).

Various medicinal plants have recently been tested for antimicrobial activity and all have proven

that phytochemicals particularly polyphenols exhibit significant antibacterial activity against

different strains of GAS such as HITM 100, ATCC 19615 and clinical isolates. Few examples of

these plants are: Wild maracuja (Passiflora foetida), white weed (Ageratum conyzoides),

Calabash tree (Crescentia cujete), bush-banana (Uvaria chamae), ginger (Zingiber Officinale),

bitter kola (Garcinia Kola), and little gourd (Coccinia grandis) (63-74). Adhesion reduction of

S. pyogenes DSM2071 to HEp-2 cells have been tested with (-)-epigallocatechin and (-)-

epigallocatechin-3-O-gallate, flavan-3-ols, at concentration of 30 μg/ml and the reported results

are 15% and 40% respectively (75). Also Morin, a flavonol, at concentration of 225μM reduced

the biofilm biomass of S. pyogenes MGAS6180 by 60 % (76). The anti-adhesive properties of

root extract of Pelargonium sidoides have been studied against GAS attachment to human

epithelial type 2 (HEp-2) cells. Results show that after pre-treatment of GAS with 30µg/ml of

methanol insoluble and methanol soluble fractions, adhesion of GAS to HEp-2 cells was

inhibited by 30-35 percent. Further analysis has revealed that the proanthocyanidins content of

the fraction is of prodelphinidin nature and inhibition of the adhesion is in a specific rather than

non-specific fashion. Successful inhibition of adhesion and hydrophobic interactions could

reduce and or prevent the S. pyogenes caused occurrence of sore throat (75).

 

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3. Research hypothesis and objectives

Phytochemicals of the Nova Scotia’s cultivated and wild fruits can suppress or inhibit the growth

of S. pyogenes in a specific approach of adherence and biofilm formation inhibition under

experimental conditions.

3.1. General objective

Identifying and understanding the mode of action of phytochemicals of the ten selected

cultivated edible berries and wild fruits (Table 1) with inhibition activity against S. pyogenes.

3.2. Specific objectives

1) Polyphenol-rich and isoprenoid-rich extract preparation with different techniques.

2) Assessment of inhibition activity of the selected fruit crops extracts against S. pyogenes.

3) Chemical characterization of extracts with inhibition activity against S. pyogenes.

4) Investigation of mechanism of inhibition and disruption of ATPase activity, adherence and

biofilm formation of selected extracts/phytochemicals.

4. Research methodology

S. pyogenes ATCC® 19615™ (isolated from pharynx of child following episode of sore throat)

and ATCC 49399™ will be purchased from American Type Culture Collection (ATCC) and

inoculum will be prepared according to the manufacturer’s instructions in bio-safety laboratory

level 2 (culture: ATCC® Medium 44: Brain Heart Infusion (BHI) agar/broth). I will freeze

portions of 18 -24 h culture in BHI broth in small culture tubes and will store at -80°C for use as

needed. To prepare working inoculums,0.8 ml of the thawed frozen culture will be transferred to

8 ml of BHI broth and incubated at 37°C (78).

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Table 1: Selected fruit crops Common name Botanical

name Family Major phytochemicals Ref.

Cultivated berry Blackcurrant Ribesnigrum

Grossulariaceae

PA; caffeic acid, m-coumaric acid, p-coumaric acid, ferulic acid, sinapic acid AN; cyanidin, delphinidin

(79-81)

Blueberry

Vacciniumcorymbosum

Ericaceae PA; gallic acid, syringic acid, vanillic acid and chlorogenic acid AN; cyanidin, delphinidin, petunidin, Malvidin, peonidin FL; quercetin, myricetin, syringetin

(82)

Cranberry

Vacciniummacrocarpon

Ericaceae PA; benzoic acid, protocatechuic acid, vanillic acid, p-coumaric acid FL; quercetin, myricetin FL3; monomer, dimer and trimer of proanthocyanidins

(83-85)

Honeysuckle

Loniceracaerulea

Caprifoliaceae

PA; chlorogenic acid, AN; cyanidin, peonidin, pelargonidin, FL; quercetin, FL3; proanthocyanidins, catechin

(86-88)

Partridgeberry

Vacciniumvitis-idaea

Ericaceae AN; cyaniding, FL;quercetin, kaemferol FL3; proanthocyanidins

(89)

Wild berry Bearberry

Arctostaphylosalpina

Ericaceae AN;cyanidin, peonidin, delphinidin, petunidin, malvidin, pelargonidin FL;quercetin

(90)

Chokeberry Aroniaarbutifolia

Rosaceae PA;neochlorogenic acid, chlorogenic acid AN;cyaniding, FL;quercetin

(91)

Juniper berry Juniperuscommunis

Cupressaceae

TR; myrcene, α-pinene, β-pinene, sabinene, α-cadinol

(92)

Rosehip

Rosa rugosa Rosaceae PA;gallicacid, protocatechuicacid, gentisicacid, p-hydroxybenzoicacid, vanillicacid, caffeicacid, syringicacid, p-coumaricacid, ferulicacid, salicylicacid FL;quercetin, kaemferol, FL3; catechin, procyanidin-B2, HT; ellagitannins, gallotannins, TA; betulinic acid, oleanolic acid, ursolic acid

(93-95)

Staghorn

Rhushirta Anacardiaceae

PA;ellagic acid,caffeic acid AN;peonidin, FL;quercetin

(96)

Abbreviations:AN; Anthocyanins, FL; Flavonols, FL3; Flavan-3-ols, HT; Hydrolysable tannins, PA; Phenolic acid, TA; Triterpene acids, TR; Terpenes

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4.1. Extract preparation

4.1.1. Polyphenol-rich extract and subsequent fractions preparation

4.1.1.1. Water-based extraction

Aqueous extracts will be prepared as described by Gunathilake et al. (97) with modifications.

Whole frozen fruits will be juiced. The pomace will be macerated with deionized water, 25°C for

5 min and filtered through sieves and a 1.5-μm glass microfiber filter paper. The filtrate and juice

will be combined and then concentrated.

4.1.1.1.1. Purification of bioactive compounds from aqueous extracts

Polyphenols of aqueous extracts will be fractioned as described by Sekhon-Loodu et al. (98).

Polyphenols will be eluted with step gradient of ethanol. Fractions will be concentrated by using

a rotary evaporator at 40 °C to viscous liquid followed by freeze-drying to obtain extract powder.

4.1.1.2. Ultrasonication-assisted ethanol extraction

The fruits will be processed into a dry powder using a freeze drier. Polyphenols will be extracted

using ethanol and ultrasonic bath as described by Rupasinghe et al. (99). Briefly, dehydrated fruit

will be mixed with ethanol in glass stoppered Erlenmeyer flasks. After vortexing the mixture, it

will be exposed to 60 min (10 min interval between each 15 min exposure), 20-28 °C,

ultrasonication in ultrasonic bath of 20 kHz/1000 Watts. After filtration of the mixture, the

solvent will be evaporated and freeze-dried. To eliminate the interferences of sugar in the

analysis, solid phase extraction using column chromatography will be performed. Aliquots of

sample will be loaded onto C18 Sep-Pak cartridge, which has been previously conditioned with

ethanol and will be subsequently washed with ethanol to remove the sugars. Column will be

dried with nitrogen gas. Fractionation of polyphenols will be performed as described above

(4.1.1.1.1).

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4.1.2. Isoprenoid-rich extract and subsequent fractions preparation

4.1.2.1. Hydro-distillation technique

Hydro-distillation technique will be performed as described by Orio et al. (100) with

modifications. Briefly, aliquot of fresh or dried fruits will be placed in the steam distillation

system (glass balloon filled with 1.5 L deionized water with glass balloon filled with the plant

material). Each distillation timing and plant steaming would be about 1.5 h.

4.1.2.2. Solvent based reflux system

Fruit powder will be prepared as previously described in this proposal. Isoprenoid-rich extract

will be prepared by reflux as described by Thilakarathna et al. (101). Under heated reflux system

with ethyl-acetate as solvent, fruit powder will be refluxed for 2 h. The extracting solvent will be

removed under pressure followed by n-hexane wash and centrifugation. This will be repeated

until off-white solid extract is obtained. Extract will be dried under N2 and the remainder solvent

will be removed by vacuum oven, 33°C.

4.1.2.3. Purification of bioactives of extracts obtained by hydro-distillation &

reflux techniques

Fractionation of isoprenoid-rich fraction with macroporous styrenic based polymeric bead type

resin will be performed as described by Puttarak et al. (102). Briefly, beads will be conditioned

with methanol, loaded into a column, washed twice with methanol and H2O. I will dissolve crude

extract in ethanol and water, filter through cotton wool, load solution into column, elute with

25%, 75%, 100% ethanol and ethyl acetate. Under reduced pressure at 45°C elutes will be dried.

4.1.3. Determination of total isoperenoid and polyphenol content

The total phenolic, flavonoid, and anthocyanin contents will be determined by Folin-Ciocalteu

assay, aluminum chloride colorimetric method, and pH differential method, respectively as

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described by Rupasinghe et al. (103) and Ratnasooriya et al. (104). Total triterpenoid content

assay will be determined by a colorimetric method as described by Chang et al. (105).

4.2. Initial screening phase

4.2.1. Susceptibility testing- micro-dilution assay

Susceptibility testing will be performed as described by Xiao et al. (106) with modifications. The

minimum inhibitory concentration (MIC) of fruit extracts and chemical fractions against GAS

will be determined by micro-dilution assay according to procedures developed by the National

Committee of Clinical Laboratory Standards (NCCLS 2006). A series of test tubes containing

different concentrations of extract or chemical fractions will be prepared by serial two-fold

dilution of the previously prepared Mueller-Hinton (MH) broth. A 96-well plate will be prepared

as follows: 100 µl of microorganism suspension (108 CFU/ml) will be added to 100 µl extract

solution and vibrated gently for 1 min and then incubated in an anaerobic incubator (37°C) for 48

h. For the determination of minimum bactericidal concentration (MBC), aliquots of incubated

test tubes with concentrations higher than the MIC will be sub-cultured on MH agar and

incubated in an anaerobic incubator (37°C under a 5% CO2 atmosphere) for 48 h. All of the

assays will be performed in triplicate using two controls (M-H broth with 1.0%, w/v, glucose as

negative control, M-H broth with 1.0%, w/v, glucose and 0.05%, w/v, penicillin as positive

control (106).

4.2.2. Bacterial growth assay

Bacterial growth assay will be performed as described by Sun et al. (107). Briefly, S. pyogenes

cultures will be grown and maintained static for 15–20 h, diluted 1:100 into 50 ml aliquots MH

medium, and cultured with different concentrations of extracts, or DMSO vehicle alone. OD600

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will be measured at 2, 3, 4, 6, 8, 10, 11, 12, and 24 h to monitor growth. Bacterial growth will be

monitored by measuring OD600 and viable cell count by serial dilution method (106-108).

4.2.3. Disk diffusion antibiotic sensitivity testing 

Kirby Bauer paper method will be performed as described by Fabio et al. (65). Each extract (10

μl) will be applied to a sterile filter paper disc (6 mm) placed on the surface of inoculated plates

(duplicate plates for each extract will be used). After overnight incubation at 37 °C, the

inhibition zones will be measured. Control plates will be prepared by placing sterile water for

negative and antibiotics as positive controls (65).

4.2.4. Preliminary phytochemical analysis

The analysis by gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–

mass spectrometry (LC-MS) will be performed according to the methods of Erkan et al. (109)

and Rupasinghe et al. (110, 111). Briefly, after isolating the most effective extracts or fractions

based on MIC and MBC, preliminary phytochemical analysis will be carried out for determining

the active constituents. Preliminary phytochemical analysis could be as well carried out using

chemicals and reagents as described by Jigna and Sumitra et al. (112, 113). 

4.3. Understanding the mode of action phase

4.3.1. Adherence reduction or inhibition

4.3.1.1. Preliminary bacterial adherence assay

Adherence to glass surface assay will be performed as described by Somanah et al. (114) with

modifications. To 3 ml BHI broth, 50 µl standardized microbial suspension (2 108 CFU/ml),

300 µl of the extract or its fractions, and 100 µl tween 80 (0.001%) will be added. Penicillin will

be used as positive control. All tubes will be inclined at an angle of 30° and incubated at 37°C

for 24 h. After overnight incubation, the supernatant will be decanted into a clean tube and

adhered cells will be removed by the addition of 3 ml sodium hydroxide (0.5 M). Both test tubes

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will be centrifuged (1048 g, 15 min) and the aqueous supernatant discarded. Bacterial cells will

be re-suspended in 3 ml sodium hydroxide (0.5 M) and the percentage of adherence will be

quantified at Abs.600 nm by applying Islam et al. formula (115):

Adherence % Abs. 600nm adhered cells Abs. 600nm nonadhered cells

Abs. 600nm of adhered cells 100

4.3.1.2. Bacterial adherence Human Tonsil Epithelial Cells and human

laryngeal HEp-2 cells

Bacterial adherence to HTEpiC and HEp-2 cells (HEp2 (ATCC® CCL23 ™)) will be performed

as described by Edwards et al. (116) with modifications. HTEpiC (isolated from human normal

tonsil tissue) and HEp-2 purchased from ScienCell Research Laboratories and ATCC,

respectively, will be maintained in accordance to the manufacturer’s instructions.

For adherence assays, about 105 cells/ml will be seeded onto 12-mm-diameter glass coverslips in

the bottoms of 24-well tissue culture plates. After overnight growth at 37°C in 5% CO2

atmosphere, the cells will be washed with PBS (pH 7.4) and inoculated with 500 μl of the GAS

inoculum and the test compounds/extract. After incubation, the coverslips will be washed with

PBS, and removed by aspiration. Host cells and adherent bacteria will be fixed with 95%

methanol, air-dried, heat fixed, gram stained and viewed under microscope. The attachment will

be expressed as the average number of GAS chains per cell (116).

4.3.1.3. Fibronectin cell adhesion assays

Fibronectin binding inhibition assay will be done as described by Okada et al. (117) with

modifications. Fibronectin (extracellular matrix glycoprotein) purchased from Sigma, will be

coated onto 96-well ELISA plates. Plates will be incubated at 37 °C for 24 h. Streptococci from

overnight cultures re-suspended in PBS (pH 7.2) with a density of 1 × 108 cells/ml, and a 100 μl

aliquot of the bacterial suspension and 100 μl of extract/test compound will be added to each

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well. The amount of streptococci bound to each protein will be determined by ELISA using a

rabbit antiserum specific for the S. pyogenes cell wall carbohydrate and an alkaline phosphate-

conjugated anti-rabbit IgG antiserum.

4.3.2. ATPase assay

Preparation of permeabilized cells will be performed as described by Belli et al.(118).

ATPase assay will be performed as described by Belli et al.(118)and Gregoireet al. (119) with

modifications. Using permeabilized cells of S. pyogenes F-ATPase activity will be assayed in

terms of the release of inorganic phosphate. The amount of Pi liberated is directly proportional to

the activity of the transporter. ATPase activities will be expressed as micromoles of phosphate

released from ATP per milligram of cell protein per minute extrapolated from the linear portion

of the phosphate release curve.

4.3.3. Biofilm reduction or inhibition

4.3.3.1. Biofilm inhibition assay

Biofilm biomass assay will be performed as described by O'Toole et al.(120) and Pitts et al.

(121). Briefly, S. pyogenes will be cultured overnight in MH broth and its concentration will be

standardized at 109 CFU/ml. Diluted bacterial culture (1 × 106 CFU/ml) will be used to inoculate

the MTP containing a range of concentrations of extracts or fractions. On each well 100 μl of the

bacterial culture will be added to 100 μl of the tested antibacterial compounds resulting in total

volume of 200 μl/well. Biofilms will be grown for 24 h at 37°C. Wells will be washed twice with

saline water, and then will be air dried for 30-60 min. Plates will then be stained with 125 μl

crystal violet (1% w/v) and incubated at room temperature for 15-20 min. Following 3 times of

rinsing with saline water, plates will be re-solubilized using 95% (v/v) ethanol and incubated for

another 5-10 min. Absorbance readings will be taken using microtitre plate reader at A595 nm (80).

The effect of fractions on the biomass of S. pyogenes biofilms will be determined by comparing

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the biomass of treated biofilms with untreated biofilms (122). I will follow the same procedure

for 3 and 4 days old biofilms. I will add the extracts on 3-4 days old biofilms.

4.3.3.2. Total viable counts of biofilm

Biofilm viability measurement will be performed as described by Pettit et al. (123). The effect of

fractions on biofilms will be determined by comparing CFU/ml of the treated biofilms with

untreated biofilms.

5. Statistical analysis

The data will be analyzed using ANOVA (analysis of variance) with the level of significance at

1% and 5%, and difference among the groups will be tested by the F-test. If significant

difference is observed pair-wise comparisons will be used between all the groups using Tukey’s

method.

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Timeline

Year Semester Course work Research

1

Winter2014

AGRI5730 – directed studies in food & bio-product sciences AGRI5700 – communication skills SATI3000 – statistics

ATC preparation

Summer2014 - ATC completion Crude extraction & fractionation

Fall2014 AGRI5630 – intermediate statistical methods

Crude extraction & fractionation continued Initial screening phase

2

Winter2015 To be determined Mode of action (MOA) study

Summer2015 Teaching assistantship MOA study continued Data analysis, article writing

Fall2015 - Data analysis, Thesis writing and defense