METHODOLOGY by KMs2B3

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									                                       METHODOLOGY



       The flowchart diagram of the research is shown in Fig.2, Appendix.



Moringa oleifera Leaves

       The M. oleifera leaf samples were purchased from a local market in Guadalupe, Makati.

Mature, healthy-looking leaves with no blemishes were chosen. The sample was submitted to the

Bureau of Plant Industry Malate, Manila for the verification of its identity. Bureau of Plant

Industry confirmed the sample to be Moringa oleifera after 2 days upon being handed over.


Moringa oleifera Extract Preparation

   Leaves were rinsed through several passages in sterile distilled water. Leaves were dried in a

plant drier for 8 hours and then grinded. Grinded, dried Moringa oleifera leaves were mixed with

distilled water at 100 g dry wt/L, and boiled for 5 minutes. The extract was filter-sterilized using

0.45 um membrane filter. An aliquot was removed for testing and the remainder of the decoction

were maintained at−80 ⁰C. Method is adapted from Adonizio et al. (2006).



Bacterial Cultures

       Chromobacterium violaceum ATCC 12472 was generously provided by Dr. Robert J.C.

McLean of the Department of Biology, Texas State University-San Marcos, USA. The bacterium

is a type strain and is used to detect potential quorum signal inhibitors evidenced by loss of

pigment production. It was isolated from the freshwater of Malaya.

       Pseudomonas aeruginosa PAO1 is known for its C4 and 3-oxo-C12 HSL production.

Strain PAO1, a derivative of the original Australian PAO isolate, is used as positive control for
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QSI. Several of the virulence factors produced by this strain are QS-mediated (Latifi et al.,

1995). The bacterium is an opportunistic pathogen especially to immuno-compromised

individuals. Dr. Robert J.C. McLean of the Department of Biology, Texas State University-San

Marcos, USA munificently provided the PAO1.

       Staphylococcus aureus was obtained from the stock culture of the microbiology

laboratory of the De La Salle University. This bacterium is frequently part of the flora found in

the nose and on skin of humans. It can cause a wide variety of illnesses from simple skin

infections to life-threatening diseases.


Disk-diffusion Assay for Quorum Sensing Inhibitory Activity of Moringa oleifera Extract

on Chromobacterium violaceum


       Chromobacterium violaceum ATCC 12472 was grown overnight in Luria-Bertani broth

at ambient temperature with shaking. One hundred microliter of the culture was inoculated to

5ml LB broth and grown at 30 ⁰C for 4 hours. Turbidity was adjusted to MacFarland 0.5 before

being inoculated into the LB plates.


       Twenty microliters of the undiluted extract was loaded onto sterile 6mm disks prepared

from Whatman filter paper (no.3) with the final concentration of 2mg/ml. Disks were loaded

with bromo-furanone (SIGMA), which has been tested to contain QSI compounds, and distilled

water were used for positive and negative controls, respectively. The prepared disks were dried

at 50 ⁰C. Disks were placed onto prepared LB plates inoculated with 100 ul of the bacterium and

spread plate using sterile bent glass rod. Plates were incubated at 30⁰C overnight. QS inhibition

was evaluated by the presence of colorless zone having viable cells around the disks. The
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production of violacein is induced in the presence of AHL’s (McLean, 2004). Upon inhibition of
the AHL production, the violacein production is also inhibited. The diameter of the ring of

colorless colonies around the disks was measured using the Zoomstereo microscope. Method is

adapted from Adonizio et al. (2006).




Disk-diffusion Assay for Antibacterial Activity of Moringa oleifera Extract on Pseudomonas

aeruginosa


       Pseudomonas aeruginosa PAO1 were grown overnight in Luria-Bertani broth at 37 ⁰C

with shaking. One hundred ul of the cultures was subcultured for 4 hours, and the turbidity

adjusted to MacFarland 0.5 before being inoculated into the LB plates.


       Six disks were used. Three disks were impregnated with 20ul of the extract and the other

three disks with distilled water for negative controls. Disks were placed onto prepared LB plates

spread with 100ul of the four- hour P. aeruginosa culture. Plates were incubated at 37 ⁰C

overnight.


       The presence of growth around the disks showed that M. oleifera does not inhibit the

growth of P. aeruginosa, and that the decrease in virulence factor production in the presence of

the extract is not due to its antibacterial activity on PAO1.


Establishing Pseudomonas aeruginosa Growth Curve


       In several studies, it was noted that the quorum sensing happens in the late-log phase of

the P. aeruginosa PAO1. Autoinducers that are produced constitutively generally reach a

threshold during the late-log to the stationary 19
                                                phase (Rumbaugh et al., 2000). Pearson et al.
(1995) reported that lasR and rhlR are expressed in a growth-dependent manner, with activation

of each gene occurring during the last half of the log-phase growth of P. aeruginosa.


       The growth curve of the P. aeruginosa PAO1 was established and the mid-log phase was

determined to be at 6 hour of incubation and was able to approximate the time for the

introduction of the extract before quorum sensing is activated at its peak at the late log phase.


   A. Establishing the Growth Curve


       PAO1 was grown for 16-18 hours in LB at 37 ⁰C. An aliquot of the culture was adjusted

to MacFarland 0.5 for standardization of inoculum concentration (1-2 x 108 cells/ml).


       In a 500 ml Erlenmeyer flask with 198 ml nutrient broth, 2 ml of the standardized culture

was diluted 100-fold. One hundred ul (1-2 x 106 cells/ml) of this the starting culture was

subjected to 10-fold serial dilutions and each dilution was plated for counting viable colonies.

The viable counts were determined every hour for the duration of 12 hours of incubation of the

culture at 37⁰C with shaking at 100 rpm.


   B. Effect of M. oleifera Extract on the Log Phase Growth and on Virulence Factor

       Production of P. aeruginosa PAO1


   PAO1 was grown for 16-18 hours in LB at 37 ⁰C. An aliquot of the culture was adjusted to

MacFarland 0.5 for standardization of inoculum concentration (1-2 x 108 cells/ml).


       Concentrated extract of M. oleifera was used to determine its antibacterial effect and QS

inhibition on P. aeruginosa PAO1. One ml of the extract was added to 9 ml of mid log phase (6

hour) culture of PAO1 as determined in the growth curve. At late-log phase, its culture with the

extract was plated for counting viable colonies. Five ml of the mixture was centrifuged at 10,000
rpm for 5 mins. and supernatant was filter-sterilized. This was used for the virulence factor

production assays. Each assay were performed in triplicates.(Fig.___, Appendix)


       A negative control set-up was prepared parallel to that describe above, but with the

addition of sterile distilled water in place of the plant extract, which had served for comparison

of the viable cell counts and virulence factor production of P. aeruginosa PAO1treated with

plant extract (Fig. ____).


Dilution Assay for Determination of Effect of M. oleifera Extract on P. aeruginosa at

Different Concentrations


       Mid log phase culture was prepared. One ml of M. oleifera extract with 200mg/ml will be

added to 9ml of the prepared culture to achieve the final 20 mg/ml extract concentration. Two-

fold serial dilution of 200mg/ml was carried out to achieve the following concentrations: 100

mg/ml, 50mg/ml, 25 mg/ml, 12.5 mg/ml, and 6.25 mg/ml. Each concentrations was added with 9

ml of the mid log of the culture to obtain 10mg/ml, 5 mg/ml, 2.5 mg/ml, 1.25 mg/ml, and 0.625

mg/ml extract concentrations, respectively. One ml sterile distilled water and 9ml of culture

broth was mixed to obtain 0mg/ml concentration (Fig. ______). The media were incubated at 37

⁰C for 2 hours to allow interaction of the extracts and the culture. Method is adapted from Kudi

and Myint (1999).


       The effect of the extracts on QS inhibition of PAO1 as manifested by its virulence factor

production were determined using the following assays in triplicates:


Pseudomonas aeruginosa Virulence Assays



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          The supernatants of the different P. aeruginosa PAO1 late log phase cultures with and

without the extract were used in the virulence assays. Methods are adapted from Adonizio et al.

(2006).

LasA Staphylolytic Assay

          LasA protease activity was determined by measuring the ability of culture supernatants to

lyse boiled S. aureus cells. A 100 μl aliquot of P. aeruginosa LB late log phase culture

supernatant with or without plant extract was added to 900 μl of boiled S. aureus suspension.

OD600 will be recorded after 0, 5, 10, 20, 30, 45, and 60 minutes. This was used to determine the

ability of the produced protease enzymes to cleave the protein or lyse the cell of the S. aureus.

LasB Elastolytic Assay


          The LasB elastolytic assay was used to identify whether there has been a decrease in

elastolytic enzyme production, which is equivalent to the decrease in the synthesis of the

virulence protein enzyme LasB. The shades of red in the solution indicate the presence of the

produced elastase enzymes. The darker the red coloration the more elastase enzymes have been

produced, while the lighter the shade of red denotes the opposite. Elastase cleaves elastin Congo

red which becomes evident in the shades of the red coloration of the solution.


          The elastolytic activity of late log phase culture supernatants exposed and not exposed to

extract was determined using elastin Congo red (ECR). A 100 μl aliquot of the culture supernate

was added to 900 μl of ECR buffer (100 mM Tris, 1 mM CaCl2, pH 7.5) containing 20 mg ECR.

It was incubated with shaking for 3 hours at 37 °C. Insoluble ECR was removed by

centrifugation, and the absorption of the supernatant was measured at 495 nm. Reagent blank

without any cells alone and with extract was used as negative control.



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Swarming Motility

       Method is adapted from Vattem et al., 2006.

       One hundred microliters of sterile M. oleifera extract was spread-plated unto 0.3% LB

agar plates, and were allowed to dry for 3 hours at 30⁰C. Luria-Bertani plate without the extract

was used as the negative control. The plates were point-inoculated at the center with 2 ul

(approximately 3 x 106 cells/spot) of the PAO1 late log phase culture adjusted to MacFarland 0.5

exposed and not exposed to extract, and were incubated at 37 ⁰C for 24 hours. The extent of

swarming will be determined by measuring the diameter of the motility swarms.


       Swarming motility of the P. aeruginosa PAO1 is one of the virulence processes that is

controlled by quorum sensing. Determining the competence of the P. aeruginosa PAO1

swarming motility has provided information on inhibition in quorum sensing.

								
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