MATERIALS AND METHODS - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/2113/13/13_chapter...

Chapter III

MATERIALS AND METHODS

3.1. PLACES OF RESEARCH WORK

The isolation, characterization and identification of bacterial and fungal

pathogens were done in the Clinical Microbiology Research Laboratory of PRIST

University, East Campus, Vallam, Thanjavur district, Tamil Nadu. The isolation,

identification and characterization of mycobacteria were done at K.A.P.V.

Medical Hospital, Tiruchirapalli, Molecular methods such as PCR, AGE and

PAGE were done at Government Hospital for Chest Diseases (STDC),

Puducherry, RT-PCR was done at Center for Molecular Biology Methods,

Kovalam, Chennai, CD4 count was done at Medical College, Thanjavur, DNA

sequencing for Mycobacteria was done at Bioserve, Bangalore. The protein

sequencing was done at Molecular Medicine, New Delhi. Protein sequencing for

Candida was done at Xceleris Laboratory, Ahmadabad. Phytochemical studies

were done at Armats Biotek Private Ltd., Chennai. Antimycobacterial activity of

herbal extracts was done at Tuberculosis Research Centre, Chetpet, Chennai.

3.2. STUDY GROUP

250 biosamples were collected from 200 HIV positive patients who were

attending the HIV counselling and testing centers of both the two medical colleges

and hospitals of Tiruchirapalli and Thanjavur districts of Tamil Nadu, India

(Lat.10° N and Long.79° E). All the HIV positive patients in the age group

between 1 and 80 with symptoms state as inclusion criteria and also in the age

group of above 80 as exclusion criteria with or without symptoms.

3.2.1. Research design

The present investigation gives a view, which is ideally suited to study the

prevalence of opportunistic bacterial and fungal pathogens in the HIV+ patients.

A broad outline of the research design is presented (Fig.1).

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3.2.2. Instrumentation

In this work, isolation and identification of bacterial and fungal pathogens

were done in UV safety cabinet. PCR amplification of mycobacterial DNA was

done by using Automated Thermal Cycler. The stained gels of PCR amplified

DNA was done in UV Transilluminator and photographed using

Geldocumentation Unit. The amplified gene products were sequenced by DNA

sequencer. Real time PCR was done in RT-PCR System. The proteomics of

serum albumin protein was done by Nano LC-MS. The medicinal herbal plant

extracts were obtained by using Soxhelet Apparatus (Plate I).

3.2.3. Collection of biosamples

250 biosamples were collected from clinically symptomatic HIV+ patients

of various hospitals, medical colleges and social welfare organizations of

Thanjavur and Tiruchirapalli districts during the period from August 2008 to July

2009 (in a sterile container after aseptic precaution). All the patients were

thoroughly evaluated by detailed history, clinical examination and biochemical

parameters including CD4 count.

3.3. ASPECTS OF STUDY

To realize the objectives of the present study outlined in the introductory

chapter the following experiments or investigations were carried out.

? Isolation, identification and characterization of bacterial pathogens in HIV+

patients.

? Isolation and identification of fungal pathogens in HIV+ patients.

? Antibiotic sensitivity of bacterial pathogens.

? Proportional antibiotic sensitivity of mycobacteria.

? Antibiotic sensitivity of fungal pathogens.



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? Molecular characterization of isoniazid and pyrazinamide drug resistant

mycobacteria.

? Molecular characterization of Candida albicans.

? Phytochemical analyses of ten medicinal herbal plant extracts.

? Antibacterial activity of ten medicinal herbal plant extracts.

? Antifungal activity of five medicinal herbal plant extracts.

? Identification of effective compounds by thin layer chromatography and

bioautography.

? Characterization of purified compound by GC-MS and FT-IR analyses.

Standard procedures and protocols were followed in the microbial,

biochemical, phytochemical and molecular studies which are listed in Table 1.

TABLE 1 The list of standard procedures and protocols

S.No. Test Aspect Reference

I. CD4 Count To count the CD4 cells inblood, thereby detect theimmune status of the HIV+

patients

Kahan and Jani,2001

II. EXAMINATION OF BIOSAMPLES1. Microscopic examination

i. Staining techniquesa. Gram staining Identification of bacteria Aneja, 2003b. Acid fast staining

Ziehl Neelson staining Identification ofM. tuberculosis

Venkataraman andAlexander, 1987

Flourescent staining Identification ofM. tuberculosis

Blair et al., 1970

c. Lactophenol cotton bluestaining

Identification of fungi James Cappucinoand NatalieSherman, 2009

ii. Hanging drop method Motility of bacteria Aneja, 2003



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2. Macroscopic examinationi. Modified Petroff’s

methodCulture processing of sputum Allen and Baker,

1968ii. Culture techniques To isolate bacteria and fungi Aneja, 2003

III. Biochemical characterization1. Bacteria

1. Catalase test To detect the presence ofcatalase enzyme .

Kubica and Pool.,1960

2. Oxidase test To detect the presence ofoxidase enzyme

Cappucino andSherman, 2007

3. Indole test To detect the production ofindole.

Aneja, 2003

4. Methyl red test To detect the ability ofmicroorganisms to oxidizeglucose

Aneja, 2003

5. Voges proskauer To differentiate entericpathogens

6. Citrate utilization test To detect the utilization ofcitrate substrate.

Aneja, 2003

7. Urease test To detect the presence ofurease enzyme.

Aneja, 2003

8. Hydrogen sulphideproduction test

To detect fermentative natureof bacteria by H2S production

Cappucino andSherman, 2007

9. Sugar fermentation test Detect the fermentation ofsugars by pathogens.

Aneja, 2003

2. Mycobacteria1. Catalase test To detect the presence of

catalase enzymeKubica and Pool.,1960

2. Niacin test To detect the production ofniacin

Venkataraman andPrabakar, 1970

3. PNB test To detect the conversion of p-nitrobenzoic acid

Tsukamura andTsukamura, 1964

4. Nitrate reduction test To detect the utilization ofnitrate in the medium.

Angeby et al., 2002

IV. Antibiotic sensitivity testing of bacteria1. Disc diffusion method To detect the sensitivity of

isolated bacteriaBauer et al., 1996



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2. Proportional sensitivitymethod

To detecting the sensitivity ofisolated mycrobacteria.

Tripathy et al.,1970

V. Molecular Studies1. Isoniazid and Pylozinamide drug resistant Mycobacterium

tuberculosisa. Preliminary work Isolation of DNA Vijdea et al., 2008b. PCR Amplification of inhA, katG

and pncA genesCousins et al., 1992

c. Agarose gelelectrophoresis

To determination the size ofDNA of the clinical isolate

Helen et al., 2003

d. DNA sequencing To detection the nucleotidesequences of DNA of clinicalisolate

Altschul et al.,1997

e. RT-PCR To detect the molecular sizeof the DNA of the M.tuberculosis.

Heid et al., 1996

f. SDS-PAGE To determine the molecularsize of DNA of the resistantbacterial strain by band size

Zhang et al., 1992

2. Molecular characterization of Candida albicans1. Preliminary work Isolation of DNA Christine et al.,

19992. Agarose gel

electrophoresis [AGE]To purify of DNA Sambrook and

Russel, 20073. PCR To amplify DNA Christine et al.,

19994. DNA sequencing To determine the sequences

of DNA moleculeChristine et al.,1999

5. Phytogenetic analysis1. MEGA BLAST2. MEGA4

Analysis the similaritybetween the isolated strainand closely related strains

Christine et al.,1999

VI. Effect of medicinal herbal plants on the microbialpathogens1. Phytochemical analyses To detect the

phytocompounds ofmedicinal herbal plants

Trease and Evans,1996

2. Antimicrobial studiesa. Well diffusion for

bacteriaTo detect the antibacterialactivity of medicinal plants

Fazeli et al., 2007,



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b. Luciferase reporterphage assay forMycobacteria

To screen theantimycobacterial activity ofherbal extracts.

Sivakumar et al.,2007

c. Well diffusion assay forfungi

To detect the antifungalactivity of medicinal plantsagainst fungi

Zgoda and Porter,2001

d. TLC and bioautography To separate the antimicrobialcompounds

Wagner and Bladt,1996

3. Characterization analysesa. GC-MS Separation and

characterization of volatilecomponents in thephytochemical compounds.

Parasuraman et al.,2009

b. FT-IR Identification of functionalgroups of phytochemicalcompounds.

Sivaraju andKannan, 2010

3.4. CD4 COUNT PROCEDURE (Kahan and Jani, 2001)

20 µl of whole blood was added to a Partec test tube containing EDTA as

an anticoagulant. 20 µl of CD4 mAB PE was added and mixed gently and

incubated for 15 minutes at room temperature to protected from light, 800 µl of no

lyse buffer was then added and shaked or vortexed gently.

3.5. EXAMINATION OF BIOSAMPLES

3.5.1. Microscopic examination (Aneja, 2003)

Microbiological examination of pathogens was done using binocular

microscope (40X) following standard procedures and protocols (Table 1).

Staining techniques were performed to identify the bacterial, mycobacterial

and fungal pathogens namely gram’s staining, acid fast staining and lactophenol

cotton blue (LCB) staining respectively. Motility test was also examined for the

preliminary identification of the bacterial pathogens.



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3.5.2. Macroscopic examination on isolation of biosamples

3.5.2.1. Processing of samples (Allen and Baker, 1968)

Biosamples normally does not require processing for culturing bacteria and

fungi. Exceptionally they must be processed before handling them for HIV+

patients particularly sputum and urine was done by Modified Petroff’s Method.

3.5.2.2. Culture technique (Aneja, 2003)

The specific media used for the isolation of different bacteria were Nutrient

Agar. Lowenstein Jensen agar, Cetrimide agar, MacConkey agar, Mannitol salt

agar, blood agar, Chocolate agar, XLD agar and Eosine Methylene blue agar.

Specific media used for the isolation of different fungi were Rose Bengal agar,

Potato dextrose agar, Saborauds dextrose agar, Czapek dox agar, Chrom agar and

Brain heart infusion agar, which is prepared by the following standard procedures.

3.6. CHARACTERIZATION OF BACTERIA BY BIOCHEMICAL TESTS

(Aneja, 2003)

The following were biochemical tests performed for characterization of

identified bacterial pathogens namely Catalase, Oxidase, Indole, Methyl Red,

Voges Proskauer, Citrate Utilization, Urease, Hydrogen sulphide reduction and

Sugar fermentation tests (Aneja, 2003). The mycobacterial characterization

methods namely niacin test (Venkataraman and Prabakar, 1970) nitrate reduction

(Angeby et al., 2002) and PNB tests (Tsukamura and Tsukamura, 1964).

3.7. ANTIBIOTIC SENSITIVITY OF ISOLATED PATHOGENS

3.7.1. Antibiotic sensitivity test for bacterial pathogens (Bauer et al., 1966)

Krby-Bauer disc diffusion method is commonly employed for antibiotic

sensitivity tests for bacterial. The antibiotic discs were for used Amikacin,

Ampicillin, Carbennicillin, Cefixime, Ciprofloxacin, Norfloxacin, Rifampin,



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Streptomycin, Tetracycline and Vancomycin. The diameters of antibiotics

compared with standard charts.

3.7.1.1. Proportional sensitivity test method for mycobacteria (Tripathy et al.,

1970)

The antibiotic sensitivity of mycobacterial strains was done by proportional

sensitivity method. For the purpose of calculation, the number of colonies growing

on the drug-contianing medium was expressed as proportion of the estimated

number of colonies on the acidified drug-free medium.

Calculation

The CFU was calculated by using the following formula (Roberts et al.,

1991)

CFU =

No. of CFU (colony forming units) on drug slopes ×dilution factor

× 100No. of CFU (colony forming units) on drug free slopes ×dilution factor

1 (or) more than 1 per cent is resistant and less than 1 per cent is sensitive

(S) to concern drug (Always count the colonies from the higher dilution to lower

dilution (that is, S4, S3, S2, S1). If the colony count is more than 10 CFU only

consider for the calculation.

3.7.2. Antibiotic sensitivity tests for fungal pathogens (Bauer et al., 1966)

Antifungal activity test carried out by using the disc diffusion method. Ten

antibiotic discs namely amphotericin B, fluconazole, flucytosine, griseofulvin,

itraconazole, ketoconazole, miconazole, nystatin, trimethoprim and voriconazole

were placed upon the media isolated with inocula to detect the antibiotic

sensitivity of the isolated fungal organisms.



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3.8. MOLECULAR CHARACTERIZATION OF ISONIAZID RESISTANT

STRAIN OF M. tuberculosis

3.8.1. PCR amplification of isolated DNA (Cousins et al., 1992 and Helen

et al., 2003)

The isolated DNA was amplified using IS6110 primer in an automated

thermal cycler (Eppendorf). This confirms the template DNA of isolated DNA as

Mycobacterium tuberculosis.

3.8.1.1. Mycobacterial DNA extraction (Mani et al., 2003)

Mycobacterial DNA was extracted with the slight modifications of usual

DNA extraction method.

3.8.1.2. Isolation of inhA and katG gene from clinical isolate strain (Vijdea

et al., 2008)

The Mycobacterial DNA was extracted as stated above and the Taq

polymerase, dNTPs, MgCl2, Milli Q water, 10x Buffer, DMSO, Template forward

and reverse primer were used for amplification of each respective gene. The PCR

cycling parameters were 94°C for 5 minutes; followed by 40 cycles of 94°C for 1

minute, 57°C for 1 minute and 74°C for 1 minute and a final extension of 74°C for

5 minutes. The PCR was then kept at hold at 4°C for 15 minutes.

3.8.1.3 Agarose gel electrophoresis (Helen et al., 2003)

The amplified PCR product inhA and katG gene from clinical isolate strain

were run on 2 per cent agarose gel and purify the PCR product using PCR

purification kit (Invitrogen).



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3.8.1.4. PCR cleanup procedure

The amplified PCR products was purified using charges witch PCR clean

up kit (Invitrogen catalog No.CS12000) prior to given for DNA sequencing.

3.8.2. DNA sequencing analysis (isoniazid inhA and katG genes) (Williams

et al., 1995; Altschul et al., 1997)

The purified electrophoresed PCR product of inhA and katG genes were

and subjected to DNA sequencing analysis. The purified PCR product was directly

sequenced in an automated DNA Sequencer at Bioserve in Bangalore. The

nucleotide sequence obtained was analyzed using BLASTn Bioinformatics tool

available at National Center for Biotechnology Information to know the specificity

of PCR amplification and to identify the nucleotide variation. The sequence was

further subject for BLASTx to know the amino acid changes in comparison with

the wild type M. tuberculosis (H37Rv).

3.8.3. RT-PCR analysis (Marin et al., 2004)

It is necessary to add internal control (IC) in the reaction mix. IC allows the

user to determined and control the possibility of PCR inhibition. The internal

control (IC) (1 µl/reaction) was added and the result will be shown in the VIC/JOE

channel. The kit can be used for quantitative (or) qualitative Real-time PCR.

A positive control defined as 1 × 10-1 IU/ml. The positive control (1 × 10-1 IU/ml)

was taken as the starting high standard in the first tube. Respectively 36 µl of

molecular grade water was pipetted out into next three tubes. Three dilutions were

done. To generate a standard curve on the Real-time PCR system, all four dilution

standard should be used and defined as standard with specification of the

corresponding concentrations.



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RT-PCR protocol (Torres et al., 2003)

RT-PCR analysis was done by the method.3 µl Master Mix + 5 µl Sample / Positive control / Negative control

Reaction Plate / Tube

RT-PCR instrument

45°C for 10 minutes, 1 cycle

95°C for 15 minutes, 1 cycle

60°C for 60 seconds, 40 cycles

Fluorescence was measured at was measured at 60°C.

3.8.4. SDS-PAGE electrophoresis and nano LC/MS analysis

SDS-PAGE was used to separate the proteins from the clinically isolated

M. tuberculosis, the suspected protein spot of the gel was cut by a sterile surgical

blade and it was transferred to the 2 ml eppendorff tube containing 1.5 ml of milli

Q water. The protein sample was subjected to NANO LC/MS in the Center for

Molecular Medicine at New Delhi.

3.8.5. Phylogenetic analysis

Mycobacterial DNA was extracted from TB coinfected HIV patient. The

respective 16s RNA gene was isolated and amplified by using Taq polymerase,

dNTPs, MgCl2, Milli Q water, 10x Buffer, DMSO, template, forward and reverse

primer in the Thermal cycles. The PCR cycling parameters were 94°C for 5

minutes: followed by 40 cycles of 94°C, 57°C and 74°C for 1 minute and a final

extension of 74°C for 5 minutes. The PCR was then kept at hold at 4°C for 15

minutes. The amplified PCR product was withdrawn from thermal cycler and run



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on a 2 per agarose gel in TAE buffer. The ethidium bromide stained gels were

observed in a UV transilluminator and photographed using a Geldoc. The

amplified PCR product were purified using charge switch PCR cleanup kit

(Invitrogen catalog No.CSI2000). The purified PCR products was directly

sequenced in an automated DNA sequencer at Bioserve in Bangalore. The

nucleotide sequences were analyzed using CLUSTAL W algorithm.

3.9. MOLECULAR CHARACTERIZATION OF PYRAZINAMIDE

RESISTANCE STRAIN OF M. tuberculosis

PCR amplification of isolated DNA was done by using the method of

Cousins et al., 1992. Mycobacterial DNA extraction was done by using the

method of Mani et al., 2003. Isolation of pncA gene from TB confected HIV

patient was done by using the method of Vijdea et al., 2008. Agarose gel

electrophoresis was done by using the method of Helen et al., 2003. The amplified

PCR products were purified using charge switch PCR clean up kit (Invitrogen

catalog No.CS12000) prior to given for DNA sequencing. DNA sequencing

analysis (Pyrazinamide-pncA) was done by using the method of Williams et al.

(1995). RT-PCR analysis was done by using the method of Marin et al. (2004).

SDS-PAGE was done by using the method of Zhang et al. (1992).

Nano LC/MS process was done following the similar method for isoniazid

resistant strains described above. The protein sample was subjected to NANO

LC/MS in Center for Molecular Medicine at New Delhi. The amplified PCR

products were purified using charge switch PCR clean up kit (Invitrogen catalog

No.CS12000). The purified PCR product was directly sequenced in an automated

DNA Sequencer at Bioserve in Bangalore. The nucleotide sequence obtained was

analyzed using CLUSTAL W algorithm.



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3.10. MOLECULAR CHARACTERIZATION OF Candida albicans

3.10.1. PCR amplification of isolated fungi (Christine et al., 1999)

3.10.1.1. DNA isolation from cultured cells using master pureTM Yeast DNA

purification kit

The culture was harvested from 25 ml culture media after the 5 days of

incubation. The culture was rinsed with several volumes (original culture

volume)of 0.1 M MgCl2. The culture pellet was transferred to a chilled mortar and

grind the mycelium to a powder in the presence of liquid nitrogen. The powder

was transferred to microcentrifuge tube and continue with cell lysis and

precipitation of DNA.

3.10.1.2. Cell lysis and precipitation of DNA

The yeast cell lysis solution was thoroughly mixed to ensure uniform

composition before dispensing. 300 ml of Yeast Cell Lysis solution was added to a

microcentrifuge tube. The cells were suspended by either vortex mixing of

pipetting the cells repeatedly using a 1 ml capacity pipette tip. The suspended cells

were incubated at 65°C for 15 minutes. The samples were placed on ice for 5

minutes 150 ml of MPC protein precipitation reagent was added and vortex mixed

for 10 seconds. Pellet cellular debris was pelleted by centrifugation in a

microcentrifuge for 10 minutes at 10,000 rpm. The supernatant was transferred to

a clean microcentrifuge tube and 500 ml of isopropanol was added and mixed

thoroughly by inversion. The DNA was pelleted out by centrifugation in a

microcentrifuge for 10 minutes at 10,000 rpm. The supernatant was removed by

pipetting and discard. The pellet containing the DNA was washed with 0.5 ml of

70 per cent ethanol. The ethanol was carefully removed by pipetting and

discarded. The DNA pellet was briefly centrifuged and remaining ethanol was

removed. The DNA was suspended in 35 ml of TE Buffer. The DNA was stored at

4°C.



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3.10.1.3. RNase A treatment

The presence of RNase A may interfere with subsequent PCR amplification

of fungal genomic DNA. 1 ml of 5 mg/ml RNase A was added to the purified

DNA and it was incubated at 37°C for 30 minutes.

3.10.1.4. Quantitation and quality assessment of DNA

The DNA stock samples were quantified using Nanodrop

spectrophotometer at 260 and 280 nm using the convention that one absorbance

unit at 260 nm wavelength equals 50 µg DNA per ml. The UV absorbance was

checked at 260 and 280 nm for determination of DNA concentration and purity.

Purity of DNA was judged on the basis of optical density ratio at 260:280 nm. The

DNA ratio between 1.8 and 2.0 was considered to be of good purity.

Concentration of DNA was estimated using the formula.

Concentration of DNA (mg/ml) = OD 260 × 50 × dilution factor

3.10.1.5. Agarose gel electrophoresis (Sambrook and Russel, 2001)

The quality and purity of DNA were checked by agarose gel

electrophoresis. Agarose 0.8 per cent (w/v) in 0.5X TAE (pY 8.0) buffer was used

for submarine gel electrophoresis. Ethidium bromide (1%) was added @ 10 µl/100

ml. The wells were charged with 5 µl of DNA preparations mixed with 1 µl gel

loading dye. Electrophoresis was carried out at 80 V for 30 min at room

temperature. DNA was visualized under UV using UV transilluminator. The DNA

was used further for PCR.

3.10.1.6. Polymerase chain reaction (PCR)

Internal transcribed spacer (ITS) gene fragment was amplified by PCR

genomic DNA using ITS gene universal primers: IF and IR.



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Details of primers used for PCR

IF: 5′-TCC GTA GGT GAA CCT GCC G-3′

IR: 5′-TCC TCC GCT TAT TGA TAT GC-3′

PCR was carried out in a final reaction volume of 25 µl in 200 µl capacity thin

wall PCR tube. PCR tubes containing the mixture were tapped gently and spin

briefly at 10,000 rpm. The PCR tubes with all the components were transferred to

thermal cycler. The PCR protocol designed for 30 cycles for the primers.

3.10.1.7. Visualization of PCR product

To confirm the targeted PCR amplification, 5 µl of PCR product from each

tube was mixed with 1 µl of 6X gel loading dye and electrophoresed on 1.2 per

cent agarose gel containing ethidium bromide (1 per cent solution @ 10 µl/100

ml) at constant 5V/cm for 30 min in 0.5 X TAE buffer. The amplified product was

visualized as a single compact band of expected size under UV light and

documented by gel documentation system (Gene Genius, SynGene Bio Imaging

System, UK).

3.10.1.8. Purification of PCR product

Amplified PCR product was purified using Exosap enzyme as per the

protocol given in table 3 and further used for sequencing reaction.

3.10.2. Sequencing of purified DNA

Sequencing of Purified Internal Transcribed Spacer (ITS) Gene Segment

The concentration of the purified DNA was determined and was subjected

to automated DNA sequencing on ABI 3730×1 genetic analyzer (Applied

Biosystems, USA). Sequencing was carried out using BigDye® Terminator v3.1

Cycle sequencing kit following manufacturers instructions.



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Cycle sequencing

Cycle sequencing was performed following the instructions supplied along

with BigDye® Terminator v3.1 Cycle Sequencing Kit. The reaction was carried

out in a final reaction volume of 20 µl using 200 µl capacity thin wall PCR tube.

The cycling protocol was designed for 25 cycles with the thermal ramp rate of 1°C

per second. After cycling, the extension products were purified and mixed well in

10 µl of Hi-Di formamide. The contents were mixed on shaker for 30 minutes at

300 xg. Eluted PCR products were placed in a sample plate and covered with the

septa. Sample plate was heated at 95°C for 5 min, snap chilled and loaded into

autosampler of the instrument.

Electrophoresis and data analysis was carried out on the ABI 3730xl

Genetic analyzer using appropriate Module, Basecaller, Dyeset/Primer and Matrix

filers.

3.10.3. Sequence analysis by bioinformatics tools

The obtained sequenced data of forward and reverse reactions were used to

great a consensus sequence of large subunit DNA (LSU DNA). Both end of the

sequence was verified with the chromatogram file and edited if required. The

sequence was converted into FASTA format and saved in notepad. Internal

transcribed spacer (ITS) gene sequence were used to carry out BLAST (Basic

local alignment search tool) with nr database of NCBI Genbank using

MEGABLAST algorithm. The BLAST data was arranged in maximum percentage

identify and first ten sequence was selected and exported in FASTA format. Based

on maximum identity score and query coverage the best highly identical 10

sequences were selected and aligned using multiple alignment software program

ClustaW (MEGA4 tool). The evolutionary history was inferred using the

Neighbour-joining method. The bootstrap consensus tree inferred from 500

replicates is taken to represent evolutionary history of the taxa analyzed. The



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evolutionary distances were computed using the Kimura 2-parameters method.

Phylogenetic analysis was conducted in MEGA4.

Software tools

3.10.3.1. BLAST

BLAST® (Basic Local Alignment Search Tool) is a set of similarity search

programs designed to explore all of the available sequence databases regardless of

whether the query is protein or DNA. The BLAST programs have been designed

for speed, with a minimal sacrifice of sensitivity to distant sequence relationships.

The scores assigned in a BLAST search have well-defined statistical

interpretation, making real matches easier to distinguish from random background

hits. BLAST uses a heuristic algorithm that seeks local as opposed to global

alignments and is therefore able to detect relationships among sequences that share

only isolated regions of similarity (Altschul et al., 1990).

Algorithm: MEGABLAST

The best way to identify an unknown sequence is to see if that sequence

already exists in a public database. If the database sequence is a well-characterized

sequence, then one will have access to a wealth of biological information

MEGABLAST, discontiguous-megablast and blast all an beused to accomplish

this goal. However, MEGABLAST is specifically designed to efficiently find log

alignment between very similar sequences and thus is the best tool to use to find

the identical match to your query sequence. In addition to the expect value

significance cut-off, MEGABLAST also provides an adjustable percent identity

cut-off for the alignment, which provides cut-off in addition to the significance

cut-off threshold set by expect value.

Database “nr”



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All GenBank + EMBL + DDBJ + PDB sequences (but no EST, STS, GSS,

or phase 0, 1 or 2 HTGS sequences). No longer “non-redundant” due to

computational cost.

3.10.3.2. MEGA4

MEGA is an integrated tool for conducting automatic and manual sequence

alignment, inferring phylogenetic trees, mining web-based databases, estimating

rates of molecular evolution and testing evolutionary hypotheses.

3.11. EFFECT OF MEDICINAL HERBAL PLANTS ON THE MICROBIAL

PATHOGENS

3.11.1. Collection of samples

Fresh herbal leaves of Acalypha indica L., Adhatoda vasica L., Calotropis

procera L., Datura metel L., Ocimum basilicum L., Solanum trilobatum L.,

Tylophora indica M., Vitex negundo L. and Withania somnifera Dunal and root

parts of Acorus calamus L. were collected from the herbal garden of PRIST

University, Thanjavur and were identified with the help of a botanist (Plaet II(A)).

The leaves and root parts of respective medicinal plants were washed with distilled

water separately and used for this study.

3.11.1.1. Medicinal herbal plants

Acalypha indica L.

Kingdom : Plantae

Order : Malpighiales

Family : Euphorbiaceae

Genus : Acalypha

Species : indica

Common Name : Kuppai meni.



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Acorus calamus L.

Kingdom : Plantae

Order : Acorales

Family : Acoraceae

Genus : Acorus

Species : calamus

Common Name : Sweet flag, sweet sledge.

Adhatoda vasica L.

Kingdom : Plantae

Order : Lamiales

Family : Acanthaceae

Genus : Adhathoda

Species : vasica

Common Name : Adulsa(vasaka)

Calotropis procera L.

Kingdom : Plantae

Order : Gentianales

Family : Apocynaceae

Genus : Calotropis

Species : procera

Common Name : Sodom apple

Datura metel L.

Kingdom : Plantae

Phylum : Tracheophyta

Class : Magnoliopsida



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Order : Solanales

Family : Solanaceae

Genus : Datura

Species : metel

Ocimum basilicum L.

Kingdom : Plantae

Order : Lamiales

Family : Lamiaceae

Genus : Ocimum

Species : basilicum

Common Name : Sweet basil

Solanum trilobatum L.

Taxonomic hierarchy of Solanum trilobatum L.

Kingdom : Plantae

Phylum : Tracheophyta

Subphylum : Euphyllophytina

Class : Magnoliopsida

Order : Solanales

Family : Solanaceae

Genus : Solanum

Species : trilobatum

Tylophora indica M.

Taxonomic hierarchy of Tylophora indica L.

Kingdom : Plantae

Order : Gentianales



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Family : Apocyanaceae

Genus : Tylophora

Species : indica

Common name : Indian ipecac

Vitex negundo L.

Kingdom : Plantae

Order : Lamiales

Family : Verbenaceae

Genus : Vitex

Species : negundo

Common Name : Nirgund

Withania somnifera D.

Taxonomic hierarchy of Withania somnifera L.

Kingdom : Plantae

Division : Angiosperma

Class : Dicotyledoneae

Order : Tubiflorae

Family : Solanaceae

Genus : Withania

Species : somnifera Dunal

3.11.2. Preparation of medicinal herbal plant extracts

The leaves of nine medicinal plants and root materials of one plant were

carefully washed with tap water, rinsed with distilled water and air dried for one

hour. After washing the roots were cut into small pieces. Then the leaves and

small root segments were dried at room temperature. After drying process, they

were ground into powder and stored at room temperature in sterile pockets.



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3.11.3. Extraction method (Plate IIB-D)

The medicinal herbal plant leaves and root extracts were obtained by

soxhlet apparatus. For this extraction, four different solvents such as hexane,

ethylacetate, diethyl ether and methanol were used. After extraction, the solvent

was removed by condensation of the extracts.

3.11.4. Phytochemical analysis (Trease and Evans, 1996)

The herbal extracts were subjected to various phytochemical tests

separately. Different qualitative chemical tests were performed for determining the

phytoconstituents present in the plant extracts. Phytochemical analysis were done

according to the procedure of phytochemical methods. Methanolic extract of

plants were used for qualitative phytochemical analysis. The results of the

analyses of ten herbal extracts were tabulated.

Detection of tannins: 0.5 g of the dried powdered sample of each plant was

separately added with 20 ml of water and boiled in a test tube and filtered. A few

drops of 0.1 per cent of ferric chloride solution was added and observed for

brownish green or blue black colouration which indicated the presence presence of

tannin.

Detection of saponins

The 50 mg of each plant extract was diluted with distilled water and made

upto 20 ml. The suspension was shaken in a graduated cylinder for 15 mins. A 2

cm layer of foam indicated the presence of saponins.

Detection of flavonoids

Shinoda’s test: In a test tube containing 0.5 ml of extract 5-10 drops of diluted

HCl and small piece of ZnCl or magnesium were added and the solution was

boiled for few minutes. Appearance of reddish brown colour indicated the

presence of flavonoids.



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Alkaline reagent test: A few drops of diluted NaOH was added to the extract. An

intense yellow colour was produced and become colourless after of few drops of

diluted acid indicated the presence of flavonoids.

Detection of Terpenoids: 0.5 g of crude powder was dissolved in 5 ml of

methanol. 2 ml of the extract was treated with 1 ml of 2, 4-dinitrophenyl hydrazine

dissolved in 100 ml of 2 M HCl. A yellow-orange colouration was observed as an

indication of terpenoids.

Detection of Glycosides: Each plant extract of about 50 mg was hydrolysed with

concentrated HCl for 2 hours on a water bath, filtred and the hydrolysate was

subject to the test.

Borntrager’s test: To 2 ml of filtrate hydrolysate, 3 ml of chloroform was added

and shaken. Chloroform layer was separated and 10 per cent ammonia solution

was added to it. Appearance of pink colouration suggested the positive response

for anthraquinones indicated the presence of phenolic compounds.

Detection of Phenolic compounds

Ferric chloride test: 50 mg of each plant extract dissolved in 5 ml of distilled

water. To this, few drops of neutral 5 per cent ferric chloride solution was added.

A dark green colour indicated the presence of phenolic compounds.

Gelatin test: 50 mg of the extract dissolved in 5 ml of distilled water, 2 ml of 1

per cent solution of gelatin containing 10 per cent sodium chloride was added to it.

White precipitate indicated the presence of phenolic compounds.

Lead acetate test: 50 mg of the extract was dissolved in 5 ml of distilled water.

To this 3 ml of 10 per cent lead acetate solution was added. A bulky white

precipitate formed as an end point.

3.11.5. Detection of fixed oils and fats

Saponification: A few drops of 0.5 N alcoholic potassium hydroxide solution was

added to a small quantity of extract along with a drop of phenolphthalein. The



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mixture was heated on a water bath for 2 hours. The formation of foam as a partial

neutralization of alkali indicated the presence of fixed oils and fats.

3.11.6. Detection of Alkaloids

Mayers Test: 50 mg of solvent free extract was stirred with few ml of diluted HCl

and filtered. To 1.2 ml of filtrate, 0.1 ml of Mayer’s reagent was added. A white

creamy precipitate indicated the presence of alkaloids.

Wagners Test: 5 g of the powdered sample was extracted by boiling in 50 ml of

distilled water in a water bath for 30 min. It was then filtered into a test tube and

the filtrate collected. The filtrate was tested with Wagner’s reagent and results

compared to blanks.

Hagers Test: 5 g of the powdered sample was extracted by boiling in 50 ml of

distilled water in a water bath for 30 min. It was then filtered into a test tube and

the filtrate collected. The filtrate was tested with Hager’s reagents and results

compared to blanks.

3.11.7. Detection of Carbohydrates

The extract (100 mg) was dissolved in 5 ml of water and filtered. The

filtrate was subjected to the following tests.

a) Molish’s test: To 2 ml of filtrate of each plant extract, 2 drops of alcoholic

solution of α-napthol was added. The mixture was shaken well and 1 ml of

concentrated sulphuric acid was added slowly along the sides of the test tube

and allowed to stand. The formation of violet ring indicated the presence of

carbohydrates.

b) Fehling’s test: One ml of each Fehling’s solution A and B was mixed with the

2 ml of plant extract and boiled on water bath. An yellow red precipitate was

formed indicated the positive reaction of Fehlings test.



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c) Benedict’s test: One ml of benedicts reagents was added with 2 ml of plant

extract and boiled in a water bath for 2 minutes. The formation of brick red

precipitate indicated the positive reaction of benedicts test.

3.11.8. Detection of proteins and aminoacids

The 100 mg each plant extract was dissolved in 10 ml of distilled water and

filtered through Whatmann no:1 filter paper. The filtrate was subjected to proteins

and amino acids determination.

Millon’s test: To 2 ml of filtrate, few drops of Millon’s reagent was added. A

white precipitate indicated the presence of proteins.

Biuret test: An aliquot of 2 ml of filtrate was mixed with one drop of 2 per cent

copper sulfate. To this 1 ml of ethanol (95%) was added followed by excess of

potassium hydroxide pellets. The pink colour in the ethanolic layer indicated the

presence of proteins.

3.11.9. Detection of Phytosterols

Libermann-Burchard test: 50 mg of plant extract was mixed with drops of acetic

anhydride and sulfuric acid. The appearance of a blue-green colour is a positive

test for cholesterol.

3.11.10. Antimicrobial activity of herbal extracts

3.11.10.1. Well diffusion assay for antibacterial activity (Fazeli et al., 2007)

Agar well diffusion assay is used widely to determine the antibacterial

activity of crude extract containing unknown components (Perez et al., 1990). 24

hours growing culture of bacterial pathogens namely E. coli, S. typhi,

P. aeruginosa, E. aerogenes, S. aureus, K. pneumoniae, P. vulgaris, C. botulinum,

S. pneumoniae and S. dysentriae were swabbed separately on the nutrient agar

media containing plates, different extracts namely hexane, ethyl acetate and



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methanol with three types of concentrations used viz., 100 µg (10 µl), 250 µg (25

µl) and 300 µg (30 µl).

3.11.10.2. Well diffusion assay for antifungal activity (Zgoda and Porter,

2001)

Agar well diffusion assay is also used to determine the antifungal activity

of medicinal herbal plants. Three types of solvents have been used namely

ethylacetate, diethyl ether and methanol. Various fungal pathogens namely

C. albicans, A. niger, A. flavus, A. terreus, H. capsulatum, P. marneffei,

B. dermatidis, C. neoformans, F. moniliforme and F. solani were swabbed

separately on the SDA media containing plates. The wells about 10 mm in

diameter were made by using cork borer. Different concentrations of plant extracts

namely 100 µg (10 µl), 500 µg (50 µl) and 1000 µg (100 µl).

3.1.10.3. Screening of antimycobacterial activity of Withania somnifera

extracts (Sivakumar et al., 2007)

Antimycobacterial activity of W. somnifera was done by Luciferase

reporter phage assay. The procedure (Riska et al., 1999) was optimized and

modified by Dr. Vanaja Kumar Tuberculosis Research Centre. Chetpet. Chennai,

India. Fifty-microliter bacterial suspension of M. tuberculosis equivalent to

MacFarlands No.2 standard was added to 400 µl of G7H9 with and without the

test compound. Hexane and methanolic extracts of W. somnifera separately added

with 1 per cent DMSO (Vehicle solvent). Two different concentrations of hexane

and methanol extracts were used viz., 100 and 500 µg. For each sample, two drug-

free controls and two drug concentrations were prepared and this set up was

incubated for 72 h at 37°C. After incubation 50 µl of the high titer Luciferase

reporter phage (phAE 129) and 40 µl of 0.1 M CaCl2 were added to all the vials

and this setup was incubated at 37°C for 4 h. After incubation 100 µl of the



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mixture was taken from each tube into a luminometer cuvette and equal amount of

working D-luciferin (0.3 mM in 0.05 M sodium citrate buffer. pH 4.5) solution

was added. The RLU was measured after 10 s of integration in the Luminometer

(Monolight 2010). Duplicate readings were recorded for each sample and the

mean and standard deviation values were calculated. The percentage reduction in

the RLU was calculated for each test sample and compared with control.

3.11.10.4 Thin layer chromatography (TLC) (Wagner and Bladt, 1996)

In order to separate antimicrobial principles, the crude leaf extracts showed

high antibacterial and antifungal activity was subjected to thin layer

chromatography. The separation of the compound depends on the usage of the

solvent. Here the solvent used were 5 and 10 per cent methanol in chloroform.

1 mg/ml concentration of the effective plant extract was spotted on the TLC plates

and dried. It was then run with both ultraviolet and iodine chamber. Thin layer

chromatography was performed on Merck TLC F254 plates, with chloroform:

Methanol (95:5) as mobile phase. The separated components were visualized

under visible and ultraviolet light (254 and 360 nm). The Retention Factor (RF)

value was calculated by using the following formula.

Distance traveled by soluteRF = _________________________

Distance traveled by solvent.

The compounds from the spots were scrabbed and used for further screening.

3.11.10.5. Bioautography (Beague and Kline, 1972)

Bioautography is a rapid aid in the bioassay guided isolation and

fractionation of antibacterial compounds and its fractions. In this approach, the

activity of plant extracts against the pathogens are determined on chromatograms

in accordance with the bioautography procedure. Developed chromatography

plates of crude extract was dried overnight sprayed with a suspension of actively



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growing cells of bacteria and fungi and incubated at 37 and 24°C respectively in a

chamber at 100°C relative humidity for 18 hrs. Plants were sprayed with MTT-3

(4,5-Dimethyl thiazol-2.5- Diphenyl tetrazolium Bromide) (5 mg/ml). Clear zones

on the chromatogram indicate inhibition of growth after incubating for hours at

37°C. The method was chosen for its simplicity, low cost, accuracy and rapid

result that make it ideal for bioassay guided isolation (Eloff, 1998).

3.11.11. Characterization studied on herbal plants

3.11.11.1. Gas chromatography and mass spectrometry of purified TLC

compound of effective antibacterial and antifungal herbal plants

(Parasuraman et al., 2009)

GC-MS techniques involves the separation and characterization of volatile

components in a test sample of phytochemical compound. Instrument control

parameter were shown in Fig. Here the compounds from both the herbal extracts

of W. somnifera and A. India L. acquired through TLC were tested in this GC-MS

analysis. In this analysis, a capillary column was used. An inert gas helium was

used as a carrier gas. The components of a compound was evaporated in the

injector of GC equipment and segregated in the column by adsorption and

absorption technique with suitable temperature of oven about 325°C controlled by

software. The GC column was heated in the oven, each and every component of

the test compound was eluted from the column, the time termed as Retention Time

(RT) was noted. The eluted components were tested in the mass detector. The

spectrum of the unknown components were compared with the spectrum of known

components stored in the NIST library, thus the name, molecular weight and

structure of the components of the test compound were ascertained. It is not

possible to make an accurate identification of a molecules by gas chromatography

or mass spectrometry alone. The mass spectrometry normally requires a very pure

sample while gas chromatography using a traditional detector detects multiple



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molecules that happen to take the same amount of tie to travel through the column

have the same retention time which results in two or more molecules to co elute.

Therefore when an identifying mass spectrum appears at a characteristic retention

time in a GC-MS analysis, it typically tends to increased certainty that analyte of

interest was in the sample.

3.11.10.2. FT-IR analysis of effective compound (Sivaraju and Kanna, 2010)

FT-IR analyses were done by the procedure. ATR model FT-IR

Spectrophotometer (Bruker Co., Germany) was used for the analysis of the

methanolic crude extracts of A. indica and W. somnifera. The compound acquired

through TLC was subjected to FT-IR. The samples was individually milled with

potassium bromide (KBr) to form a very fine powder. The powder was then

compressed into a thin pellet which can be analyzed. The spectrum was recorded

using Attenuated Total Reflectance (ATR) technique beach measurement.



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