sample research proposal

1. 1
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

2. 2
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-ATPaseF 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

3. 3
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

4. 4
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

5. 5
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

6. 6
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).

7. 7
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

8. 8
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

9. 9
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).

10.
Figure 1
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11. 11
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).

12. 12
Table 1: Selected fruit crops
Common name Botanical
name
Family Major phytochemicals Ref.
Cultivated berry
Blackcurrant Ribesnigrum Grossulari
aceae
PA; caffeic acid, m-coumaric acid, p-
coumaric acid, ferulic acid, sinapic acid
AN; cyanidin, delphinidin
(79-
81)
Blueberry Vacciniumco
rymbosum
Ericaceae PA; gallic acid, syringic acid, vanillic acid
and chlorogenic acid
AN; cyanidin, delphinidin, petunidin,
Malvidin, peonidin
FL; quercetin, myricetin, syringetin
(82)
Cranberry Vacciniumma
crocarpon
Ericaceae PA; benzoic acid, protocatechuic acid,
vanillic acid, p-coumaric acid
FL; quercetin, myricetin
FL3; monomer, dimer and trimer of
proanthocyanidins
(83-
85)
Honeysuckle Loniceracaer
ulea
Caprifolia
ceae
PA; chlorogenic acid, AN; cyanidin,
peonidin, pelargonidin, FL; quercetin, FL3;
proanthocyanidins, catechin
(86-
88)
Partridgeberry Vacciniumviti
s-idaea
Ericaceae AN; cyaniding, FL;quercetin, kaemferol
FL3; proanthocyanidins
(89)
Wild berry
Bearberry Arctostaphyl
osalpina
Ericaceae AN;cyanidin, peonidin, delphinidin,
petunidin, malvidin, pelargonidin
FL;quercetin
(90)
Chokeberry Aroniaarbutif
olia
Rosaceae PA;neochlorogenic acid, chlorogenic acid
AN;cyaniding, FL;quercetin
(91)
Juniper berry Juniperusco
mmunis
Cupressac
eae
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 Anacardia
ceae
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

13. 13
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).

14. 14
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

15. 15
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

16. 16
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

17. 17
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

18. 18
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

19. 19
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