Analysis of IR, NMR and invitro antibacterial Potency of Pistacia integerrima against 6 Clinically

17 Pages·2015·0.34 MB·English

Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 1174-1190
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 4 Number 6 (2015) pp. 1174-1 190
http://www.ijcmas.com

Original Research Article

Analysis of IR, NMR and invitro antibacterial Potency of Pistacia integerrima
against 6 Clinically Isolated Multidrug Resistant Bacteria

R.Bharathirajan1 and M.Prakash2*

1Resarch and Development Centre, Bharathiar University, Coimbatore, Tamil Nadu, India
2Department of Microbiology, Kanchi Shri Krishna College of Arts and Science, Kilambi,
Kancheepuram, Tamilnadu, India
*Corresponding author

A B S T R A C T

To investigate the antibacterial activity, isolation and identification of pure
compound usin g extraction procedure with five solvents, hexane, chloroform, ethyl

acetate, methanol and water to validate medicinal uses of Pistacia integerrima

(P.integerrima) in controlling infections; and to qualitatively estimate

phytochemical constituents of galls-extracts of the plant.The antibacterial activity
Keywords
of galls-extract s was evaluated by the agar-well diffusion method against clinically

isolated 6 Gram-positive and -negative multidrug resistant (MDR) pathogenic
Pista cia
bacteria in vitro. Values of minimum inhibitory concentration (MIC) and minimum
integ errima,
bactericidal co ncentration (MBC) of galls-extracts against each bacterium were
Gram-positive
obtained in a 9 6-well micro-titre plate, by broth dilution micro-titre plate technique.
bacteria,
To isolate pure compounds with a hope that they might be active against MDR
Gram -negative
bacteria cell, the chloroform extract of Pistacia integerrima showing maximum
bacte ria,
activity were subjected to TLC and finally pure compounds were subjected to IR

Multidrug
and NMR anal ysis for structural elucidation.The presence of tannins, flavonoids,
resistant
sterols, alkaloi ds, anthraquinone and coumarinsin different galls extracts was
bacte ria,
established. Pathogenic bacteria used were, Escherichia coli, Klebsiella
Mini mum
pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus (S. aureus),
inhib itory
methicillin res istant S. aureus and vancomycin resistant S. aureus along with
concentration,
standard bacte rial strains. These MDR bacteria had been recorded to have
Antibacterial
significant inhi bitions by galls extracts, obtained by extraction procedure with five
activ ity,
solvents. The chloroform extract against Staphylococcus aureus had the highest
Phyt ochemical
inhibition zone-size (14 mm). Cefotaxime 30 µg/disc was the positive/reference

constituents,
control and the diluting solvent, 10% dimethyl sulphoxide was the negative control.
GC IR.
Recorded MIC values of different extracts ranged between 0.48 and 13.20 mg/mL,
NMR . and MBC valu es were 2.58 to 30.00 mg/mL, for these bacteria. Galls-extracts with

water and chlo roform had shown significant antibacterial activity against bacteria.

On the basis o f spectroscopic data found compound 1 was proposed to be a β-
sitosterol. This study suggests that Pistacia integerrima galls-extract can be could
be used in trea ting infectious diseases, caused by the range of tested bacteria, as
complementary and alternate medicine.

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Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 1174-1190
Introduction Possess antiviral property (Mutail C, et al.,
DOI;4172).Stem resin is used in wounds
Presently in developing countries, synthetic
healing (Hussain F, et al., 2007).Leaves are
drugs are not only expensive and inadequate
used as fodder for cattle (Jan S, et al., 2008).
for the treatment of disease but are often

adulterated and show side effects (Shariff
Infections with both Gram-positive (GP) and
ZU. 2001). So there is a need to search for
Gram-negative (GN) bacteria have clinically
plants of medicinal value. Throughout
become intractable, slowly, due to the
history, plants have provided human with an
emergence of multidrug resistant (MDR)
essential source of medicine (Foster.
strains. Among GP pathogens, strains of
1999).The World Health Organization
Staphylococcus aureus (S. aureus),
estimated that about an 80% population of
methicillin resistant S. aureus (MRSA) and
developing countries relies on traditional
vancomycin resistant S. aureus (VRSA),
medicines, mostly plant drugs, for their
strains of Enterococcus sp. are noteworthy
primary health care needs(WHO 2008, and
(Dubey D,et al., 2012).Moreover, GN
Loeraa JA, et al., 2007). Particularly in rural
bacteria, Acinetobacter sp., Klebsiella
India, uses of raw plant products as well as
pneumoniae (K. pneumoniae), Citrobacter
some concoction of plant products in
freundii (C. freundii), Escherichia coli (E.
Ayurvedic medicines are sought after to a
coli) and Pseudomonas aeruginosa (P.
great proportion, because of cheap
aeruginosa) are commonly found as
availability, and in urban areas too those are
pathogens of urinary tract; while E. coli, K.
increasingly popular for cultural nuances
pneumoniae, are pathogens of
that exist(De Silva T, et al., 2009).In ethno-
gastrointestinal tract. Presently, these
botanical literature of India, several
pathogens are too MDR, recorded in several
hundreds of plants are known to have the
reports (Dubey, et al., 2012).
potential to treat many diseases and one of

those popular ones Pistacia integerrima
The resistance of pathogenic bacteria to
(P.integerrima).
antibiotics is of high clinical concern. Rather

the concept of the control of drug resistance
Pistacia integerrima belonging to family
is a matter of clairvoyance for dovetailed
Anacardiaceaeis a native to Asia widely
antimicrobials today. A suitable epitome is
distributed in south Alpine Himalayas,
the superbug, multidrug resistant (MDR) S.
Pakistan, Afghanistan. Growing at an
aureus in the human health domain
altitude of 800-1900 m (Pant S, et al., 2010).
worldwide, as its different strains or rather
It is an important medicinal plant and is used
incarnations have generated β-lactamase
as anti-inflammatory, antidiabetic agent, a
activities in degrading all sorts of penicillin
blood purifier, a remedy for gastrointestinal
derived antibiotics, in addition to resistance
disorders and as a expectorant. In India it is
to other groups/generations of antibiotics
used as an herbal remedy for aliments such
(Clarke CR. 2006).Multidrug resistance of
as cough, asthma, fever, vomiting and
Staphylococcus, Pseudomonas, Escherichia
diarrhea (Pant S, et al., 2010 and Uddin G,
and a few more pathogenic bacteria to a
et al., 2012).it is hepatoprotective (Uddin G,
wide range of antibiotics has been reported
et al., 2011). Galls in combination of other
to have been due to non-prudent uses of
drugs are also used against snake bite and
same antibiotics against infections of food-
scorpion sting (Ahmad S, et al., 2010); and
and pet-animals worldwide(Middleton J,et
Galls possess antimicrobial property (Uddin
al., 2005).
G, et al., 2012).
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Including man(Maple PAC, et al., 1989). Materials and Method
MDR-MRSA strains carry resistance
markers for other antibiotics and instances Preparation of Plant Extract
of resistance up to 23 antibiotics in some
strains have been reported (Middleton J,et The air-dried powdered galls material (in 40
al., 2005). g lots) of Pistacia integerrima was extracted
with 400 mL volumes of solvents, hexane,
The emergence of VRSA is of further chloroform, ethyl acetate, methanol and
concern. Today, the management of the distilled water. By using soxhlet apparatus
consortium of MDR strains of both GN and for solvent extraction done in a lot of 40 g of
GP pathogens has become increasingly powder-mass was placed in the extractor and
difficult because of the β-lactamase a volume of 400 mL of a solvent was used
production by Staphylococcus, Bacillus, during 24 h of soxhletion, till colourless
Pseudomonas, Proteus, Klebsiella, extracts precipitated in the extractor. After
Neisseria, Salmonella, Haemophilus and a filtration, each extract was concentrated by
few more pathogens(Rath S,et al., 2012);and the rotary evaporator.
pandrug resistance (PDR, resistance of
bacteria to all antibiotics in present use) to The resultant sticky-mass was dried in a
different classes of antibiotics in GN ones desiccator; the solid mass was stored in a
(Falagas ME, et al., 1989). suitable volume of 10% dimethyl sulphoxide
(DMSO) with a drop of Tween-80. The solid
Meek appreciation of failures in the control extract was dissolved in a required volume
of MDR strains would be inhuman, which of 10% DMSO and a drop of Tween-80 for
generates the impetus on a systematic global a final concentration of 30 mg/mL. The
search for new drugs from natural resources stock concentration of each extract was
like plants, worldwide (Davidovich C, et al., maintained at 30 mg/mL, for further use.
2008 and Dubey D, et al., 2012); chemicals
from plants could be chosen for the control Phytochemical Screening
in a future crusade against MDR pathogens.
Moreover, accumulated ethnomedicinal Extracts of galls of Pistacia integerrima
reports of different countries lend using hexane, chloroform, ethyl acetate,
themselves well to the basic information methanol and distilled water were subjected
needed for further work on drug-targeting to various chemical tests in order to
against MDR pathogens (Dubey D, et al., determine the secondary plant constituents:
2012).
Test for the Presence of Anthraquinones
In the present study, crude galls extracts of
Pistacia integerrima with 5 solvents, An aliquot of 0.5 mL of the extract was
hexane, chloroform, ethyl acetate, methanol shaken with 10 mLof benzene, filtered and
and distilled water were used to monitor an aliquot 5 mL of 10% ammonia solution
antibacterial property against 6 clinically was added to the filtrate and the mixture was
isolated MDR bacterial strains and isolated shaken, the presence of a pink, red or violet
the bioactive compound-1 from Pistacia colour in the ammoniac (lower) phase
integerrima characterize by TLC, IR and indicated the presence of anthraquinones
NMR spectral analysis. (Sofowara A. 1993).

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Test for Saponins drops of Dragendorff’s reagent were added.
Turbidity or precipitation in tubes due to
An aliquot of 0.5 mL of an extract was either of these reagents indicated the
dissolved in an aliquot of 10 mL of distilled presence of alkaloids in the extract (Brain
water in a test-tube was shaken vigorously KR, etal., 1975).
for 30 s and then allowed to stand for 45
min. The appearance of a frothing, which Test for Coumarins
persists on warming indicated the presence
of saponins(Brain KR, etal., 1975). Moistened plant extract (0.5g) was taken in
a small test tube and covered it with filter
Test for Flavonoids paper moistened with 1N NaOH. The test
tube was placed for few minutes in boiling
To a portion of the dissolved extract, a few water. Then filter paper was removed and
drops of 10% ferric chloride solution were examined in UV light for yellow florescence
added. A green or blue colour indicated the to indicate the presence of coumarins(Brain
presence of flavonoids (Brain KR, etal., KR, etal., 1975).
1975).
Isolation and Identification of Pathogenic
Test for Steroids/Terpenes Bacteria

A lot of 500 mg of the extract from the From (Kanchipuram Dist.) hospitalized
rotary evaporator was dissolved in an aliquot patients of wards and cabins of Hospital, 6
of 2 mL of acetic anhydride and cooled at 0 bacterial strains (Three GP species, S.
to 4°C, to which a few drops of 12 N aureus, MRSA, VRSA.; and Three GN
sulphuricacid were carefully added. A bacteria, K. pneumoniae, E. coli and P.
colour change from violet to blue-green aeruginosa,) were isolated. All these 6
indicated the presence of a steroidal nucleus strains were identified by standard
(Sofowara A. 1993). biochemical tests and were maintained as
axenic cultures in suitable media, as
Test for Tannins described previously (Dubey D,et al., 2012
and Rath S, et al., 2012).
A fraction of 0.5 g of the extract was
dissolved in 5 mL of water followed by a Different clinical samples (Pus, Swabs,
few drops of 10% ferric chloride. A blue Urine, and Body Fluids) were collected from
black, green, or blue-green precipitate would patients of wards, cabins, intensive care unit,
indicate the presence of tannins (Brain KR, neonatal care unit in the hospital, and were
etal., 1975). used for the growth of bacteria in nutrient
agar,
Test for Alkaloids
MacConkey agar, blood agar, eosin
A lot of 0.5 g of ethanol extract (from rotary methylene blue (EMB) agar, and xylose
evaporator) was stirred with an aliquot of 5 lysine deoxycholate (XLD) agar. Microbial
mL of 1% HCl on a steam bath and filtrated; type culture collection (MTCC) strain of
to an aliquot of 1 mL of the filtrate, a few each bacterium was used as the reference
drops of Mayer’s reagent was added, and to control during identification (see Table 1).
another aliquot of 1 mL of the filtrate, a few
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Antibiotic Sensitivity Pattern details of methods of determinations of
minimum inhibitory concentration (MIC)
All bacterial strains were subjected to and minimum bactericidal concentration
antibiotic sensitivity test by Kirby-Bauer's (MBC) were described previously (Sahu
method, using a 6 mm thick Mueller-Hinton MC, et al., 2012).
agar medium, as described previously(Perez
C, et al., 1990);and results were determined
Isolation of Phytochemical Compound
basing upon the standard guidelines(Clinical

Laboratory Standard Institute Performance Chloroform fraction were selected on the
standard for antimicrobial susceptibility
basis of its antibacterial activity. Silica gel
testing 2011). For the control, ciprofloxacin was used for column chromatography.
30 µg/disc was used and it was sensitive to
Samples were loaded after adsorption on
all test bacteria and its inhibition zone range silica gel by making a uniform and even
was 16-19 mm.
layer. Mobile phases starting from hexane to
methanol in different ratios step by step
Antibacterial Activity Tests were used. Different fractions obtained. All
collected fractions were subjected to Thin
Antibacterial activity tests were performed
Layer Chromatography (TLC). Solvent
both by agar well diffusion and disc system used for TLC was chloroform and
diffusion methods. For the agar well
ethylacetate in the ratio of 50:50, 75:25,
diffusion method (Rath S, et al., 40:60 but still minor impurities were
2012).bacterial lawn was prepared as
observed. Further purification was carried
described previously (Perez C, et al., 1990). out by preparative TLC. For mobile phase,
but the agar was 6 mm thick. Wells (6 mm
chloroform and ethyl acetate were used in
depth) were punched in 30 min old agar the ratio of 8:2. TLC plates were visualised
lawn and each well was based by 50 µL
under UV light at 254nm. A very fine single
molten MHA medium. Further, wells were line was observed. Boundary lines were
filled with 100 µL aliquots of 30 mg/mL
drawn and plates were cut from those
solvent-extracts of Pistacia integerrima boundaries to separate that part of TLC
(diluted from the original stock by 10%
containing compound. Strips of TLC plates
DMSO to 30 mg plant extract/mL). Plates were cut in small parts, added in 250ml
were incubated at 37°C for 24 h.
conical flask and first washed and ethanol
Antibacterial activities were evaluated by successively 3-4 times to completely remove
measuring the diameter of zone of
compound from the plate. Resultant sample
inhibition. It was confirmed that 10% were filtered and solvent was allowed to
DMSO had no inhibitory effect on the
evaporate on rotary evaporator. Finally
bacterium. Gentamicin 40 µg/mL was taken compound was confirmed for its purity and
as the reference-control.
spectroscopic data were taken (Ahmad NS,
et al., 2010).
Antibacterial Activity and Determination
of MIC and MBC The melting points were determined on a
Perfit apparatus and are uncorrected. The IR
Antibacterial activities of plant-extracts spectra were recorded in KBr pellet on Win
were recorded by the agar-well diffusion FT-IR S 135 Instrument (Bio-rad.USA).1H
method, as described previously (Rath S, et (300MHz) and 13C (75MHz) spectra were
al., 2012 and Perez C, et al., 1990). The recorded by Brukerspectro- spin NMR
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Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 1174-1190
instrument in CDCL using TMS as internal were noted, and extracts with the rest other
3
standard. EIMS were scanned at 70 eV on solvents had medium levels of phyto-
jeol D-300 instrument (Jeol USA) (Ahmad constituents (Table3)
S, et al., 2010).
Antibiotic sensitivity pattern

Results and Discussion
Antibiotic profile of each bacterial strain
was determined using specified antibiotic
Isolation and Biochemical Identification
discs (Table 4).
of Bacteria

Specific colony morphology of each It was recorded that antibiotics (µg/disc),
gentamicin 30 was resistant to six bacteria;
pathogen was noted, for which a
corresponding MTCC strain was used, norfloxacin 300 was resistant to three and
sensitive to Three strains; nitrofurantoin 300
parallely (Table 1). For example, After
growth, a single colony was subjected to was resistant to one and sensitive to five
strains; amikacin 30 was resistant to one and
Gram-staining and basing upon it, other
biochemical tests were performed for sensitive to five bacteria; cefotaxime 30 was
resistant to six; imipenem 10 was resistant to
identification E. coli was negative for
oxidase, Voges-Proskauer, citrate and urease five and sensitive to one;
piperacillin/tazobactam 100/10 was resistant
tests; it was positive for catalase, indole, and
methyl red, triple sugar iron and nitrate to five and sensitive to one bacterium;
gatifloxacin 30 was resistant to Three and
reduction tests. (Table 2).Similarly, the rest
bacteria were typified. Three GPs, S. aureus, sensitive to Three isolates; ofloxacin 5 was
resistant to Three and sensitive to Three
MRSA, VRSA. And three GNs, E. coli, K.
pneumonia., and P. aeruginosa, were strains; netilmicin 30 was resistant to four
and sensitive to two isolates; amoxyclav 30
isolated.
was recorded as resistant to four and
sensitive to two bacteria.
Phytochemical Analyses

Antibacterial Activities
From phytochemical analyses, it was

ascertained that alkaloids were present in
Five pairs P. integerrima galls extracted
galls-extracts, obtained with ethyl acetate,
with hexane, chloroform, ethyl acetate,
methanol and water. flavonoids were present
methanol and water. Here screened for anti-
in extracts, obtained with hexane,
bacterial activity against cited GP bacteria
chloroform, ethyl acetate and water.
and three GN bacteria. Water extract only
Tannins obtained with chloroform, ethyl
had the highest antibacterial activity against
acetate and water extracts. Sterols were
E.coli. (Table 5). Galls-extracts with water
present in extracts, obtained with hexane
have shown significant antibacterial activity
and methanol. Coumarins were present in
against all bacteria.
water extract only.

The maximum size of zone of inhibition had
Anthraquinone were present in methanol
been recorded due to the water extract, as 15
extract, Saponins not present in any extracts;
mm against K. pneumoniae and MRSA
in water extract the maximum number of
which was 18 mm in the case of
phyto-constituents and the hexane and
ciprofloxacin 30 µg/disc. Detailed
chloroform extract had the least number
information of antibacterial activities of
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Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 1174-1190
extracts and inhibition zone sizes were 1.16 mg/mL, the maximum MIC value was
recorded (Table 5) as 5.91 mg/mL, due to methanol and water
extracts. With VRSA, the minimum MIC
MIC and MBC Values value was with the chloroform extracts as
1.16 mg/mL, and the maximum MIC value
Particular galls-extracts obtained with was with the hexane extract as 13.20
different solvents that have shown mg/mL.
significant antibacterial activity in the agar-
well diffusion method were further used for With E. coli, hexane, chloroform, ethyl
the determination of MIC and MBC values acetate, methanol not detected MBC value
with bacteria, in a 96-well micro-titre plate water extract as 13.30 mg/mL.With K.
(Table 6). MIC values of all extracts ranged pneumoniae, the minimum MBC value was
from 0.48 to 13.20 mg/mL, and MBC values 2.62 mg/mL in methanolic extract and the
ranged from 2.58to 30.00 mg/mL. The MIC maximum value with hexane, chloroform
value of the hexane extract ranged between and ethyl acetate extract was 13.20 mg/mL.
2.62 and 13.20 mg/mL, the MBC value With P. aeruginosa, the minimum MBC
ranged between 5.91 and 30.00 mg/mL; with value with methanol extract was 2.62
chloroform extract, the MIC value ranged mg/mL, the maximum MBC value with
from 0.48 to 5.91 mg/mL, the MBC value hexane, chloroform and water extract was
ranged between 1.16 and 13.20 mg/mL; for 13.20 mg/mL. With S. aureus, the minimum
ethyl acetate extract the MIC values ranged MBC value with chloroform extract was
between 1.16 and 5.91 mg/mL, the MBC 1.16 mg/mL, while the maximum value with
value was 2.58 to 13.20 mg/mL; the MIC hexane and water extract was 13.20 mg/mL.
value with the Methanol extract was 1.16- With MRSA, the minimum MBC value with
5.91 mg/mL and the MBC value ranged ethyl acetate extract was 2.58 mg/mL, the
between 2.62and 13.20 mg/mL. The MIC maximum value with methanol and water
value with the water extract was 2.62-5.91 extract was 13.20 mg/mL. With VRSA, the
mg/mL and the MBC value ranged between minimum MBC value with chloroform
5.91 and 13.30 mg/mL. extract was 2.62 mg/mL, the maximum
value with hexane extract was 30.00 mg/mL.
With E. coli, hexane, chloroform, ethyl
acetate, methanol not detected MIC value Identification and Characterization of
water extract as5.91 mg/mL. With K. Compound Isolated From Chloroform
pneumoniae, the lowest MIC was with the Fraction
methanolic extract as 1.16 mg/mL, and the
highest MIC value was with the hexane, Compound was obtained a white amorphous
chloroform, ethyl acetate extract as 5.91 material, with a melting point range of 136-
mg/mL. With P. aeruginosa, the lowest 139°C.Dried crystals were reconstituted in
MIC was with Methanol extract at 1.16 chloroform and spotted on silica TLC plates
mg/mL, the highest MIC value was with and TLC was carried out using mobile phase
hexane, chloroform, water extract as 5.91 of chloroform ethyl acetate (8:2 ratio) was
mg/mL. With S. aureus, the minimum MIC detected on plates as the pink spot at the
value was as 0.48 mg/mL and with the Rfvalue of 0.3 on staining with 50% H SO .
2 4
chloroform extract, and the maximum MIC The compound was found to be UV active at
value was with the hexane and water extract 254nm. IR and NMR spectral analysis were
as 5.91 mg/mL. With MRSA, the minimum done. The IR spectral analysis revealed a
MIC value was with ethyl acetate extract broad peak at 3426.3 cm-1 for OH group
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Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 1174-1190
The peaks at 2936, 2832, 2366 cm-1 al., 1994). Pistacia integerrima galls
indicate C=C group. The peaks at 1596.4 extracts presence alkaloids, terpenoids,
and 1032 cm-1 indicating the presence of flavonoids and tannins. Terpenoids and
C=O and C-O stretches, respectively flavonoids in bark and leaves while roots of
(Yamaguchi, 1970). Elemental analysis and Pistacia integerrima were found to contain
molecular weight determination indicated terpenoids and tannins. Fruits of Pistacia
C -H O as its molecular formula (Fig-2). integerrima reported to possess tannins,
29 50
In which IR peaks were obtained at 3426.89, essential oil, resin, pistacienoic acid,
2924.52, 2855.1, 1738.51, 1057.31 cm-1. triterpene alcohol and triterpenoic acid
Further, evidence for its characterization (Prajapati ND, et al., 2006). Methanol
came from its 1H NMR spectrum which extract and its ethyl acetate fraction of
exhibited a broad triplet at δ 5.36 and a Pistacia integerrima were found to contain
multiple at 3.50 corresponding to H-6 phenolics and flavonoids (Joshi UP, et al.,
olefinic proton and H-3α proton 2009).
respectively. Rest of protons appeared in the
high field region in between 0.7-2.0 ppm. Pistacia integerrima represents one of those
plants having broad-spectrum activities
The presence of flavonoids, tannins, (Yamin B, et al., 2015). Antibacterial and
alkaloids, sterols, coumarins and antifungal activity of Pistacia integrrima
anthraquinonein galls-extracts was stem were determined however, less
established; flavonoids were present in four- antibacterial activity and more antifungal
extract, whereas saponins absent in all activity was observed (Aqil F, et al., 2003).
extracts; alkaloids were present in three
extracts, obtained with ethyl acetate, Crude and fractionated extracts of Pistacia
methanol and water, in this study. As integerrima stem were tested against
reported, alkaloids, terpenoids, flavonoids, different pathogenic bacteria. Crude extract
reducing sugar, soluble starch, and tannins showed maximum activity against
were found in P. integerrima galls-extracts Pseudomonas picketti. All fractions showed
obtained with various fraction(Uddin G, et pronounced activity against Salmonella
al., 2012);and alkaloids, tannins, flavonoids setubaland Staphylococcus aureus, however,
and sterols were recorded with P. maximum inhibition (19.66mm) were
integerrima stem bark chloroform and ethyl shown by aqueous fraction against Bacillus
acetate fractions (Muhammad I, et al., subtilis (Bibi Y, et al., 2011).Methanolic
2011). Monoterpenes triterpenoids sterols, bark extract of Pistacia integerrima and
dihydromalvalic acid and flavonoids have solvent based fractions were subjected to
been isolated from the different parts of anti-microbial activity and outstanding
Pistacia species (Ahmad NS, et al., 2010 antibacterial activity was shown by ethyl
and Ahmad NS, et al., 2008). acetate fraction against Staphylococcus
aureus, however, extracts proved inactive
Pistaciai ntegerrima contain different for anti-fungal activity (Shafiqur Rahman,
phytochemicals including alkaloids, Ismail M, et al., 2011).
flavonoids, tannins, saponins, sterols and
essential oils. Phytochemical analysis of MRSA strains reported from Nepal were at
Pistacia integerrima leaf was carried out 40.1% of the total bacterial isolates, and
and was found to contain carotenoids, those strains were multiple resistant to
triterpenoids, catechins and flavonoids trimethoprim/sulfamethoxazole, cephalexin,
(Ansari SH, et al., 1993 and Ansari SH,et amikacin, ciprofloxacin and norfloxacin, in
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Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 1174-1190
addition to the usual penicillin derivatives, Most often than not, an infection from a
but all those were vancomycin sensitive MDR bacterial strain leads to a disease,
(Tiwari HK,et al., 2009). particularly when an emulating control-
agent/antimicrobial is absent, i.e., the
But the most effective way to prevent employed antibiotic has been won over by it.
clinical crisis due to MRSA has been with Indeed, in the presence of a stress factor-an
daptomycin, nowadays (Holloway K. 2000 antibiotic, the bacterial cell undergoes
and Sorlozano A, et al., 2009). In Brazil, intrinsic or acquired genetic changes via,
about 40% to 60% nosocomial infections in conjugation/transformation, involving
urinary and respiratory tracts, boils and exchanges of resistance markers,
surgical wound infections were by MRSA exemplified with the mar-locus of E. coli
alone, and the presence of mecA gene with (George AM, et al., 1983).if at least, the
those was proved, probably because of such natural selection for the emergence of
greater infection prevalence (Raja NS, mutants is slow. Spontaneous mutation in
2007).In a study from Malaysia, it was bacteria occurs at the rate, 1 in 107 cells
reported that among 287 pathogens, 52% usually. Eventually, some drug-resistant
were GNs with Proteus sp. 25%, P. mutant predominates with the replacement
aeruginosa 25%, K. pneumoniae 15%, E. of all sensitive strains by the resistant strain,
coli 9%, and the rest 45% were GP bacteria the later serving as if a doppelgänger. Since,
with S. aureus 40%, Group B Streptococci the emergence of resistant mutants is a self-
25% and Enterococcus sp. 9%; antibiograms repetitive process in conditions ideal for
indicated the susceptibility to imipenem and pathogens, serial/continual resistant events
amikacin in GN and vancomycin in GP to a gamut of diverse antibiotics land at the
bacteria(Kato T, et al., 1998).Among emergence of multidrug resistance in a
intracellular pathogens isolated, both S. bacterium, at least in an aged/immune-
aureus and Staphylococcus epidermidis compromised body. Indeed, the horizontal
were frequently present, the latter species transfer of genetic materials from one
being coagulase-negative Staphylococcus; organism to another appears faster than
and S. aureus strains were mostly MRSA. mutational changes, a phenomenon
Indeed, S. aureus was not invasive popularly called as, evolution of quantum
intrinsically, but MRSA was reported as leaps, operates naturally(Groisman EA,
invasive through eye. Further, in a classical 1996). It is because, genes for the drug-
study from New York, it was reported that resistance mechanism are operative in
the colonization rate of MRSA was more in antibiotic-producing cells, and those are
intravenous drug abusers (Berman DS, et transferred naturally to sensitive
al., 1987). strains(Martinez JL, et al ., 2002), as an
event of natural selection. Ultimately,
While analyzing the infection dynamics of antibiotic resistance remains as the clinical
pathogens, it was obvious that antibiotic determinant of the pathogenesis. Slowly, the
sensitive pathogens have a limited capacity use of numbers of antibiotics for the control
of virulence as the employed antibiotic of infectious diseases in last decades have
controls them. At several levels, the host led to multiple resistances in one cell, the
defence system also helps the control of MDR strain of a species, paradigmatically
pathogens when the later are in a smattering with any of notorious pathogens.
number.

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Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 1174-1190
Table.1 Isolation and Characterization of Pathogenic Clinical Isolates with
Individual Colony Characteristics

Bacteria Standard strain Agar media Colony morphology
Nutrient agar Flat dry, irregular colonies
E. coli MTCC 443
MacConkey agar LF, flat dry pink, irregular colonies

Flat dry, irregular colonies, with
EMB agar
metallic green colour
K. pneumoniae MTCC 4031 MacConkey agar LF, pink, mucoid colonies
Large, irregular opaque colonies, with
P. aeruginosa MTCC 1688 Nutrient agar
bluish green pigment
Medium to large, smooth, entire,
S. aureus, MRSA, Blood agar slightly raised, creamy yellow, with
MTCC 7443
VRSA green/β hemolytic colonies

As in blood agar without hemolytic
Nutrient agar
activity

MRSA: methicillin resistant S. aureus; VRSA: vancomycin resistant S. aureus; LF: lactose fermenting colonies; EMB:
eosin methylene blue agar

Table.2 Summary of Results of Biochemical Tests of Six Pathogenic Bacteria

Bacterium Catalase Oxidase Indole MR VP Citrate Urease TSI NT
E. coli +ve -ve +ve +ve -ve -ve -ve A/G +ve
K. pneumoniae +ve -ve -ve -ve +ve +ve +ve A/GH S +ve
2
P. aeruginosa +ve +ve -ve -ve -ve +ve +ve -ve +ve
S. aureus,
+ve +ve nd nd nd nd +ve nd nd
MRSA, VRSA
MR: methyl red; VP: Voges-Prausker; TSI: triple sugar iron; NT: nitrate reduction; A/G: acid and gas production; A/GH2S:
acid-gas and hydrogen sulfide production; nd: not done; +ve: positive; -ve: negative

Table.3 Qualitative Phytochemical Analysis of Extracts of P. integerrima
galls with different Solvents

Constituents He Ch Et MeOH H O
2
Alkaloids - - + + +
Coumarins - - - - +
Flavonoids + + + - +
Anthraquinone - - - + -
Saponins - - - - -
Tannins - + + - +
Sterols + - - + -
He: Hexane;Ch: Chloroform; Et: Ethyl acetate; Me: Methanol; H O: Water +: presence; -: absence of phytoconstituent
2

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