TABLE OF CONTENTS

1. SUMMARY--------------------------------------------------------------------------- 2
2. INTODUCTION & LITERATURE REVIEW----------------------------------- 3
3. HYPOTHESIS------------------------------------------------------------------------ 5
4. STUDY OBJECTIVES-------------------------------------------------------------- 5
5. MATERIALS & METHODS------------------------------------------------------- 6
6. STATISTICAL ANALYSIS-------------------------------------------------------- 8
7. OUTCOME & UTILIZATION----------------------------------------------------- 8
8. PLAN OF WORK-------------------------------------------------------------------- 9
9. BUDGET FOR PROJECT---------------------------------------------------------- 10
10. ETHICAL DECLARATION------------------------------------------------------- 11
11. REFERENCES----------------------------------------------------------------------- 12

1
 SUMMARY

A biofilm is an aggregate of microorganisms in which cells adhere to each other on biotic

or abiotic surfaces. Biofilms appear early in the fossils record in 3.25 billion years ago.

Biofilm formation is controlled by special communication mechanism i.e. quorum

sensing. Biofilms have been found to be involved in a wide variety of microbial

infections in the body. Salmonella enterica, serovar Typhi also form biofilms in the

gallbladder and kidney. These biofilms form the chronic carrier state which is a risk

factor for developing cancer of gallbladder.

Honey has been extensively used as healing agent throughout human history besides its

widespread use as a popular food. The antibacterial properties of honey are mainly

attributed to the acidic pH , high osmolarity, release of hydrogen peroxide and plant

derived non-peroxide factors. Recently it has been known that antibacterial properties are

also due to MGO (methylglyoxal) and bee defensin 1 . Honey appears to be effective in

killing drug resistance biofilms. Biofilms are at least 500 times more resistant to

antibacterial agents than the organism. Hence it is not possible to treat biofilm with

conventional antibiotics.

This study has main aims to evaluate the efficacy of indigenous Beri honey in disruption

of S. typhi induced biofilm. MDR S. typhi will be used for this study. This will be

obtained from the Department of Microbiology, University of Health Sciences Lahore,

Pakistan. Manuka honey (standardized honey) will be used for comparison. Microtiter

plate method for Biofilm formation and detection will be used. The data will be analyzed

using the software statistical package for social sciences (SPSS version 17.0).

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INTRODUCTION / LITERATURE REVIEW

A biofilm is an aggregate of microorganisms in which cells adhere to each other on biotic

or abiotic surfaces. Biofilms are held together and protected by extracellular

1
polymericsubstance (EPS) composed of DNA, proteins, polysaccharides and lipids .
Biofilm production proceeds in two steps. First an attachment to a surface and second a

2
cell-to-cell adhesion by pilli . Biofilm formation is controlled by special interbacterial
communication mechanism i.e. quorum sensing which is taken place by AHL(N-Acyl

3
homoserine Lactose) in gram negative and oligopeptide in gram positive bactria .
Biofilms are ubiquitous. Every species of microorganism, not only bacteria and archaea,
have mechanisms by which they can adhere to surfaces and to each other. Biofilms will

4
form on virtually every non-shedding surface in a non-sterile aqueous environment .

The principal implants on which the biofilm infections are: central venous catheters, heart
valves, ventricular assist devices, coronary stents, neurosurgical ventricular shunts,
implantable neurological stimulators, arthro-prostheses, fracture-fixation devices, breast

2
implants, cochlear implants, intraocular lenses, dental implants etc . Biofilms have been
found to be involved in a wide variety of microbial infections in the body; by one

5
estimate 80% of all infections . Recently it has been noted that biofilm formation is well
established in coagulase negative staphylococcal infections, formation of dental plaque,

6
gingivitis, urinary tract infections, catheter infections, middle-ear infections, coating
contact lenses , endocarditis, infections in cystic fibrosis and infections of permanent

7
indwelling devices such as joint prostheses and heart valves . Bacterial biofilms may
impair cutaneous wound healing and reduce topical antibacterial efficiency in healing or

8
treating infected skin wounds .

3
Salmonella enterica, serovar Typhi also forms biofilms in the gallbladder and kidney.
These biofilms form in the chronic carrier state. This carrier state is a potential for
spreading the infection to the community. Besides, the gallstones have an 8.47-fold-
higher risk for developing cancer of the gallbladder. It may be noted with concern that
antibiotic treatment can be ineffective in Salmonella carriers with gallstones. To treat

9
these individuals usually requires surgery and gallstone removal .

Honey has been extensively used as healing agent throughout human history in addition

10
to its widespread use as a popular food . Miraculous healing properties of honey are
mentioned in almost all the Holy Scriptures viz The Holy Quran, The Holy Bible and The

11
Holy Torah . Allah Subhanhu Taala says in The Holy Quran,

“And Thy Lord taught the Bees: To build its cells in the mountains and the trees and in
(men’s) habitations. Then eat from every kind of fruit and travel the paths of your Lord,
which have been made easy for you to follow. From inside them comes a drink of varying
colours, containing healing for mankind. There is certainly a Sign in that for people who
reflect." (The Holy Qur'an. Al- Nahl 68-69).
In addition, the Prophet Muhammad (Peace Be Upon Him) said:
‘'Honey is a remedy for every illness while The Qur'an is a remedy for all illness of the
mind, therefore I recommend to you both remedies, the Qur'an and honey.’’

12
(Bukhari) .

The antibacterial properties of honey are mainly attributed to the acidic p H , high
osmolarity, release of hydrogen peroxide and plant derived non-peroxide factors

10
.Recently it has been known that antibacterial properties are also due to MGO

13
(methylglyoxal) and bee defensin 1 . A recent study revealed that quorum sensing can

14
be inhibited by honey . Honey appears to be effective in killing drug resistance
biofilms. In another study honey is also used in treating bacterial biofilms embedded in

15
chronic wound bacteria . Honey is also capable to disrupt the biofilm produced by

16
strains of P. aeruginosa and S. aureus .

4
 17
Biofilms are at least 500 times more resistant to antibacterial agents . Hence it is not
possible to treat biofilm with conventional antibiotics. Therefore there is passionate need
to explore such antibacterial agents which could disrupt bacterial biofilm. Therefore this
study has main aim to evaluate the efficacy of indigenous Beri honey against S. typhi
induced biofilm. It also overcomes the emerging issue of antibacterial resistance S. typhi
induced biofilm.

HYPOTHESIS

Beri honey has potential to disrupt the Salmonella typhi biofilm in vitro.

AIMS & OBJECTIVES

1. Formation and detection of the Salmonella enterica serovar Typhi biofilm in

microtiter plate.

2. Role of honey in prevention and disruption of the Salmonella enterica

serovar Typhi biofilm formed in microtiter plate.

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 MATERIALS & METHODS
Study Design

Experimental Study
Setting

The study will be conducted in the Department of Microbiology, University of Health

Sciences Lahore, Pakistan.

Duration

Six months after approval of synopsis.

MATERIALS

Honey
1. Pakistani Beri honey will be used in this study.
2. Beri honey has been selected because of its dark colour as the dark colour honeys
are considered to have high level of antioxidant as well as antibacterial property .
3. A recently study conducted at the Department of Microbiology, University of
Health Sciences Lahore, Pakistan revealed that out of one hundred sample of honey, Beri
honey, collected from Karak district has more antibacterial activity against MDR S. typhi.
4. Manuka honey (standardized honey / FDA approved) will be used for
comparison.

Bacterial Isolates
MDR S. typhi will be used for this study. This will be obtained from the Department of

Microbiology, University of Health Sciences Lahore, Pakistan.

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METHODS
Identification of Salmonella typhi
Before experiment the Salmonella typhi will be confirmed by Gram staining, colonial
morphology, biochemical and serological culture.

Serological identification of Salmonella typhi using Antisera BD DifcoTM

Serological identification of Salmonella typhi will be performed by anti sera (Salmonella

O antisera, Salmonella H antisera and Salmonella Antiserum Vi) manufactured by BD

DifcoTM

Biofilm formation and detection by microtiter plate method

Three wells of a sterile 96-well flat-bottomed plastic tissue culture plate (TPP
Switzerland) with a lid will be filled with 200 µl of bacterial suspension each. Negative
control wells will contain broth only. The plates will be covered and incubated
aerobically for 24 h at 37ºC. Then, the content of each well will be aspirated, and each
well will be washed three times with 250 µl of sterile physiological saline. The plates will
be vigorously shaken in order to remove all non-adherent bacteria. The remaining
attached bacteria will be fixed with 200 µl of 99 % methanol per well, and after 15 min
plates will be emptied and left to dry. Then, the plates will be stained for 5 min with 0.2
ml of 2% crystal violet used for Gram staining per well. Excess stain will be rinsed off
by placing the plate under running tap water. After the plates are air dried, the dye bound
to the adherent cells will be resolubilized with 160 µl of 33% (v/v) glacial acetic acid per
well. The optical density (OD) of each well will be measured at 570 nm by using an
automated ELISA reader. The reading will be performed two times: (i) before addition of
glacial acetic acid, as in standard microtiter-plate test and (ii) after glacial acetic acid was
added.

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For the purposes of comparative analysis of test results, we will be introduced
classification of adherence capabilities of tested strains into four categories. All strains
will be classified into the following categories: non-adherent (0), weakly (+), moderately
(+ +), or strongly (+ + +) adherent, based upon the ODs of bacterial films. We defined the

cut-off OD ( OD ) for the microtiter-plate test as three standard deviations above the
c
mean OD of the negative control. Strains were classified as follows:
OD ≤ OD non-adherent
c

ODc < OD ≤ 2 X OD weakly adherent

2 X OD < OD ≤ 4 X OD moderately adherent

c

4 X OD < OD strongly adherent

c

Then we will introduce the different concentration of honey in above mentioned steps of
biofilm formation to see whether honey will inhibit the formation of biofilm. After
formation of biofilm as mentioned above steps , we will again use different concentration
of honey to see whether it disrupt the biofilm. All tests will be carried out three times and

15,18
results were averaged .

STATISTICAL ANALYSIS

The data will be entered and analyzed using SPSS 17.0(Statistical Package for Social

Sciences). Mean ± SD will be given for quantitative variables.

One way ANOVA will be applied to observe group mean differences. Post Hoc Tukey

test will also be applied to observe which group means differ. A p-value of <0.05 will be

considered as statistically significant.

OUTCOME AND UTILIZATION

This study is likely to provide an effective treatment of infections caused by biofilms.

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 PLAN OF WORK

Months(2010- 1 2 3 4 5 6 7 8 9 1 1 1 1 1 1 1 1 1 1 2 2 2 2 24
2011) 0 1 2 3 4 5 6 7 8 9 0 1 2 3
Course work
and synopsis
writing
Advanced
Studies and
Research
Board
M.Phil Part 1
exam
Lab Work
Thesis
writing
M.Phil Part 2
exam

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BUDGET FOR PROJECT:

Sr # ITEM UNITS COST

1. Beri honey 1Kg RS.1500/
2. Manuka honey 500g RS.7200/

20+UMF
3. 99% Methanol 1 Unit RS.1500/
4. Muller Hinton 500g RS.10000/

Broth
5. MacConkey 500g RS.4400/

Agar
6. API 20E 25/box RS.18000/
7. Sterilized petri 150 RS.1470/

dishes
8. Microtitration 30 RS.4500/

plates 96 well

GRAND TOTAL : RS. 48,570/-

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 11) Namias N. Honey in the management of infections. Surg Infect 2003;2:219- 26.

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