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ANTIBACTERIAL ACTIVITY OFArgemone mexicana L.
AGAINST MULTI-DRUG RESISTANTPseudomonas aeruginosa,
ISOLATED FROM CLINICAL SAMPLES
Mahesh C. Sahua,b, Nagen K. Debata b, Rabindra N. Padhya*
aDepartment of Botany, B. J. B. Autonomous College, Bhubaneswar 751014, Orissa, India
bDepartment of Microbiology, IMS & Sum Hospital Medical College, S O A University,
Kalinga Nagar, Bubaneswar-751003, Orissa, India
*Communicating author e-mail: [email protected]
ABSTRACT
_____________________________________________________________________
Twenty seven isolates ofPseudomonas aeruginosa were identified from clinical samples
from a hospital, using appropriate growth media. Among them, 22 strains were resistant to
cefotoxime-30g/disc, 16 strains to amoxyclave-30g, 15 strains to ofloxacin-5g, 13 strains to
gentamicin-10 g, 10 strains to piperacillin-100g/tazobactam-10g, 8 strains to amikacin-30g,
7 strains to gatifloxacin-30g, 6 strains to netilmicin-30g, 4 strains to piperacillin100g and 3
strains were resistant to imipenem-10g and nitrofurantoin-300g/disc, individually. Each strain
was resistant to several antibiotics at specified levels. Of these 27 clinical strains, 15 antibiotic-
resistant isolates and a totally antibiotic-sensitive standard strain (NTCC strain no. 10662) were
used in monitoring antimicrobial activity of leaf-extracts using 3 organic solvents (acetone,
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methanol and ethanol) and water of prickly poppy (Argemone mexicana L.). Cold and hot
extracts obtained using acetone, methanol and ethanol exhibited high levels of inhibition zones
monitored by agar-cup and disc-diffusion methods, against 16 strains ofP. aeruginosa. The
methanol-extract had the highest level of antipseudomonal activity both in cold and hot
extractions, confirmed by separate KruskalWallisHtests. The Students t-test was used and it
was ascertained that the hot extraction yielded promising antipseudomonal activity than cold
extraction, with methanol. Values of minimum inhibitory concentration (MIC) of extracts ofA.
mexicana using acetone, methanol and ethanol as solvents were 10, 8 and 8 mg/ml, respectively.
Similarly, corresponding values of minimum bactericidal concentration (MBC) were 32, 28 and
24 mg/ml for these solvents, respectively.
KEYWORDS:
Argemone mexicana, antimicrobial activity of leaf-extracts, multi-drug resistant bacterium,
Pseudomonas aeruginosa, MIC, MBC.
INTRODUCTION
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Millions of people belonging to all age groups die each year from infectious diseases and
additional millions suffer from several long-standing infections [1]. Often, it is found that some
drug-resistant pathogenic bacterium is the causative organism of any premature death or a long-
standing infection [2]. As it is known, the emergence of multi-drug resistant (MDR) strains of
pathogens is a natural process and indiscriminate uses of antibiotics (the broad-spectrum ones,
particularly), induce the development of MDR bacterial strains [3, 4].Furthermore, apart from
the use in human medicinal system, antibiotics are equally used in agriculture and the
management of livestock or poultry, which have accelerated the emergence of antibiotic resistant
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pathogens. For example, resistant strains ofStaphylococcus intermedius, Campylobacter sp.,
Salmonella sp. andEscherichia coli have been found in pet- and food- animals, as well as their
owners [5]. Eventually, older/earlier susceptibility ranges of pathogens to antibiotics get
changed/evolved, due to continual updating of genomes of pathogens due to natural process of
genetic recombination [6],probably for the fact that microbial evolution is a fast process and the
presence of a new antibiotic in an microbial mini-cosmos acts as the major factor for positive
selection pressure. Moreover, microbes do not get eliminated completely from any ecological
niche [7],as a fraction of population always survives with some specific survival mechanism [8].
If the surviving fraction of cells escapes to the environment, re-infection of a pathogen is usual,
and favourable situations for nosocomial infection for a pathogenic bacterium are myriad [9].
The mechanism of arrival of resistant pathogens is as follows: once a population of a bacterium
reaches 107 cfu, chance mutation yields at least a single cfu that is resistant to any antimicrobial
drug (6 Brown 1987), resulting in a proportion of the population that has probably undergone a
first-step mutation, indicating a low-level resistance. The infection might be controlled;
nevertheless surviving (resistant) isolates face less competition and can have unimpeded
proliferation [10, 11]. In healthy patients in whom infection has been controlled, the emergence
of these first-step mutants may not be an issue. In patients with immune-suppression and
unhealed injuries, however, mutants emerge as a new population. In fact, the two main goals of
drug treatment are stopping bacterial growth and preventing the evolution of drug resistance, as
discussed [3, 12]. A mutant preventive concentration (MPC) or the highest point in MIC-range
of the isolates, of the drug necessary to prevent (or inhibit) the emergence of the first-step
mutants is not prescribed, normally [13]. However, synergistic treatment of two drugs helps
prevent emergence of resistant mutants, notwithstanding the antagonistic situation wherein one
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of drugs would partially inhibit the other, resulting in the rapid emergence of resistant mutants
[14].
As new MDR strains of pathogens arriving at the community cause hazards in
managements of infectious diseases, there are searches for alternate antimicrobial substances to
control MDR pathogens from several popular sources including medicinal plants. From
ethnomedicinal information, it is evident that plants with little edible plant-part, most often are
reported from Orissa [1521]or elsewhere [2224] to have healing effects on infectious diseases.
An overview on work published on antimicrobial activity of plants Dubey et al. [25] indicated
that a limited work is reported against MDR pathogenic bacteria, especially isolated from clinical
samples.
Particularly, the prickly poppy orArgemone mexicana L. (Papaveraceae), an analgesic,
antispasmodic, possibly hallucinogenic and sedative weed-plant is known to lend itself as a
traditional healing agent in treatments of malaria, warts, cold sores, skin diseases, itches and a
few more [26]. Scientifically, leaf-extracts ofA. mexicana were used to examine antibacterial
potentiality against several pathogenic bacteria: NTCC strains (wild or drug-sensitive strains) of
Staphylococcus aureus, Escherichia coli, Proteus sp., Klebsiella pneumonia and Pseudomonas
aeruginosa [27]. Antimicrobial activities of extracts ofA. mexicana with solvents, petroleum
ether, benzene, chloroform, methanol and ethanol on NTCC cultures (wild strains) of E. coli,K.
pneumoniae, Proteus mirabilis,P. aeruginosa, Salmonella typhi, S. paratyphi, Sigellasp. and
S. aureus were reported [28]. Till date, pathogenic bacteria on which antimicrobial activity of
plants have been described are not used for any antibiotic profiling. Therefore, it could not be
confirmed whether those studies were done on MDR strains of pathogens. Several other reports
on the antibacterial activity ofA. mexicana using against pathogenic and non-pathogenic bacteria
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are described [29]. None of these reports record any antibiotic-susceptibility test of used
bacteria. Further, the literature on plant extracts includingA. mexicana against P. aeruginosa
does not record any work on multidrug resistant strain. Obviously, the accumulated literature on
plants as sources of antimicrobial agents record a lot of work on wild or drug-sensitive strains of
pathogens [30].
This work describes antimicrobial activity of leaves of a nearly-poisonous weed, A.
mexicana on several clinically isolated MDRP. aeruginosa and a numbered NTCC wild strain,
which is sensitive to antibiotics originally prescribed against this bacterium. This communication
embodies an antibiotic profile of 27 clinically isolated strains of P. aeruginosa,the urinary tract
infection causing opportunistic pathogen. Further, it is recognized as a dangerous pathogen
owing to its resistance to many antibiotics and its capacity to acquire further resistance against
progressively newly introduced antimicrobial agents [31]. This organism is reported to be
responsible for nosocomial infection in patients compromised for trauma, burns, respiratory
illness, cancer, surgical wound and HIV [32]. It is anticipated that, this work would be an
example of the use of too many numbers of MDR strains isolated from clinical samples of a
common pathogen in a study of antimicrobial activity of a nearly-poisonous weed plant.
MATERIALS AND METHODS
_______________________________________________________________________
Plant materials used in the study were of leaves ofArgemone mexicana L. (Papaveraceae)
collected during December 2009 to January 2010, locally. Voucher specimens of the plant are
present in the Parija Herbarium of Post-Graduate Botany Department, Utkal University, Vani
Vihar, Bhubaneswar. Generally, A. mexicana grows as a weed in non-arable lands. Collected
leaves were washed, rinsed with distilled water and dried (after soaking with paper towel to
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prevent any fungal infection) at 371C for 24 h in an incubator and those were further shade-
dried for 7 days without exposing to sunlight, in laboratory. Dried leaves were powdered with a
blender and the powder-mass was stored in air-tight polythene packs till use.
Preparations of plant extracts
For cold extracts, 3 solvents (non-polar to polar solvent, in the order) namely, acetone,
methanol and ethanol were used individually for 3 lots of powder-mass ofA. mexicana. A lot of
20g of powder-mass in an aliquot of 200 ml of a solvent was dissolved and stored at 4C for 48
h. Further, the extract was filtered with Whatman No. 1 filter paper and was concentrated by a
rotary evaporator till a sticky-mass was obtained for each extraction. The sticky-mass was
weighed and values of individual sticky masses from rotary evaporator were 80, 100, 80, 60,
110, 160, 200 and 180 mg, using individually 8 solvents, chloroform, ethyl acetate, acetone,
dichloromethane, petroleum ether, methanol, ethanol and water, respectively. Each concentrated
sticky masses was stored in an appropriate volume of 10% DMSO-solution (v/v dimethyl
sulfoxide or DMSO/distilled water); stocks were stored at -4C till use against the bacterium,
Pseudomonas aeruginosa.
Plant extracts with cold solvents using acetone, methanol and ethanol exhibited distinct
zones of inhibition on Mueller-Hinton agar against the bacterium (P. aeruginosa). Further, these
3 solvents only were used for hot extraction. A sample lot of 40g of the shade-dried powder of
A. mexicana was extracted in a soxhlet extractor for 40 siphons or cycles, with an aliquot 400 ml
acetone, until colorless extract was obtained on the top of the extractor. Repetitions of extraction
were done successively with methanol (40 siphons) and ethanol (40 siphons) with the same
sample lot. Individual extracts were filtered and were concentrated with a rotary evaporator, run
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at 40C to get sticky-masses that were weighed and found 0.2, 0.6 and 0.8 mg from extracts
using acetone, methanol and ethanol, respectively. Those were stored with 10% DMSO solution
and final concentrations 150 mg sticky-masses/ml for acetone and 200 mg sticky-masses/ml for
individually both methanol and ethanol were obtained.
Isolation and identification of the bacterium
Totally antibiotic-sensitivePseudomonas aeruginosa NTCC strain no. 10662 was used in
the study. For obtaining antibiotic-resistant isolates of the bacterium, clinical samples, urine,
sputum, throat-swab, pus, tracheal aspirate, blood and a few more were collected from various
kinds of patients. Little lots of samples were cultured in suitable nutrient agar (Table I), for 18
24h for isolation and concomitant identification of the bacterium, P. aeruginosa. Non-lactose-
fermenting-colorless colonies were formed on MacConkey-agar, which too formed green-
pigmented colonies on further streaking on nutrient agar (Hi-media) indicated presence ofP.
aeruginosa. Green-pigmented colonies had inhibitory activity on growth of any other bacteria
around them. These were confirmed as Gram-negative bacilli and were identified as P.
aeruginosa with specific biochemical tests. To confirm the presence ofP. aeruginosa in clinical
samples only and growing colonies were not from any nosocomial infection, repeated clinical
samples were plated. Isolated cells ofP. aeruginosa were slender, non-capsulated with a polar
flagellum with dimensions, (1.5 to 3) x 0.5m.
Biochemical identification test
Confirmatory biochemical tests done were the catalase test, the oxidase test and the
growth of colonies in Kings media. A prompt reaction of effervescence with a colony ofP.
aeruginosa on exposure to a drop of hydrogen peroxide (H2O2) indicated the activity of the
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catalase enzyme. The oxidase reaction was carried out by touching and spreading a well isolated
colony on an oxidase disc (Himedia Laboratories, disc number, DD a 018). The production of
deep purple blue on the disc within 5-10s at 25-30C confirmed the presence of the oxidase
enzyme. Further, the bacterium was grown in Kings A medium[33], for pyocyanin production,
which was observed after 24 h of growth. It was conformed that blue-green pyocyanin pigment
of colonies grown in Kings A medium were soluble in traces of chloroform and were found to
diffuse into the surrounding medium. When the colonies were grown Kings B medium,
fluorescein (pyoverdin) imparting yellow tinge to culture were observed and the colour material
was insoluble in chloroform and soluble in water; some colonies developed bright red pyorubin
which was found to be water soluble. No pyomelanin, a brown to black pigment was seen in any
of the Kings medium. These tests confirmed isolation of strains ofP. aeruginosa. Pyocyanin
pigment production in Kings A medium conforms P. aeruginosa and the pigment soluble in
chloroform. Growth of P. aeruginosa in Kings B medium via production of fluorescein
(pyoverdin) insoluble in chloroform and soluble in water confirms presence ofP. aeruginosa
[33].
Antibiotic sensitivity test
All the used 16 bacterial strains ofP. aeroginosa were subjected to antibiotic sensitivity
test by the disc diffusion method, using a 4 mm thick MuellerHinton agar medium [34]. An
aliquot of 0.1 ml of 38.9 41.2 x 1010 CFU/ml, approximately from an exponentially growing
culture was spread on agar for development of lawn of any strain of the bacterium; freshly
inoculated plates were incubated for 30 min at 37C in a BOD incubator (Remi CIM -12s), for
drying the water of inocula. Further on the lawn-agar of each plate, 5 to 8 high potency
antibiotic-discs (Himedia, Mumbai) of 8 prescribed antibiotics forP. aeroginosa were placed at
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equal distances from one another. Plates were incubated overnight for 18h at 37C. Those were
examined and diameter of zone of inhibition was measured to the nearest millimeter value.
Antibacterial activity test by agar-well diffusion method
For monitoring antibacterial activity by the agar-well diffusion method [35], bacterial
lawn was prepared as described above, but the agar was 6 mm thick. Wells (6 mm depth) were
punched in 30 min old agar lawn and each well was based by 50 l molten MuellerHinton agar.
Further, wells were filled with 100 l aliquots of 30 mg/ml solvent-extracts ofA. mexicana
(diluted from the original stock of plant extract of individual organic solvent, by 10% v/v,
DMSO to 30 mg plant extract/ml, and that of the aqueous extract). Plates were incubated at 37C
for 18-24 h. Antibacterial activities were evaluated by measuring the diameter of zone of
inhibition. Experiment of each solvent extract was conducted thrice and data of the third repeated
experiment are presented. It was confirmed that 10% DMSO had no inhibitory effect on the
bacterium. Gentamicin, the prescribed dose of antibiotic (at 40 g/ml) forP. aeroginosa was
taken as the reference-control for all the solvent extracts. Sterile water was taken as the control
for experiments with both cold and hot aqueous extracts.
Disc diffusion method
Aliquots of10l plant extract (50mg/ml) were soaked by sterile filter paper discs (5
mm diameter). Sterile filter paper discs were impregnated with 10l of plant extract placed on
the surface of the medium and incubated at 37C for 24 h. The assessment of antibacterial
activity was based on the value of diameter of the inhibition zone formed around the disc [36].
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Determinations of MIC and MBC
Original stock solutions of plant extracts prepared with acetone, methanol and ethanol
(hot extracts) were 150, 200 and 200 mg plant extract/ml, respectively in 10% DMSO solution
with distilled water. Each stock solution was diluted to obtain final concentrations, 0, 10, 20, 25,
30, 40, 60, 70, 75, 80, 90 and 100 mg /ml with DMSO solution. Separate experiment was
conducted for each solvent-extract. An aliquot of 80 l of each dilution of a solvent-extract
was released to a well on a 96 welled (12 x 8) micro-titer plate along with an aliquot of 100 l
nutrient broth (Himedia, Mumbai), an aliquot of 20 l bacterial inocula (109 CFU/ml) and a
5l-aliquot of 0.5 % of 2,3,5-triphenyl tetrazolium chloride (TTC). After pouring all the above
to a well, the microplate was incubated inside at 37C for 18 h. A pink colouration in a well
indicated bacterial growth due to TTC and the absence of any colour was taken as inhibition of
bacterial growth. First well of the microplate was the control without any plant extract. The MIC
value was noted at the well, where no colour was manifested. Further, bacteria from each well of
the microplate were sub-cultured on a nutrient agar plate; the level of dilution, where no bacterial
growth on the nutrient agar plate was observed, was noted as the MBC value.
Phytochemical Screening
Extracts of leaves ofA. mexicana using ethanol, methanol and acetone were subjected
to various chemical tests in order to determine the secondary plant constituents: 1. Test for
reducing sugars,To an aliquot of 2 ml of any extract, an aliquot of 5ml of a mixture (1:1) of
Fehlings solution I and II was added and the mixture was boiled for five minutes; a brick-red
precipitate indicated the presence of free reducing sugars [37]. 2. Test for the presence of
anthraquinones.An aliquot of 0.5g of the extract was shaken with 10 ml of benzene, filtered and
an aliquot 5 ml of 10 % ammonia solution was added to the filtrate and the mixture was shaken,
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the presence of a pink, red or violet colour in the ammoniac (lower) phase indicated the presence
of anthraquinones [38, 39]. 3. Test for saponins. An aliquot of 0.5g of an extract was dissolved
in an aliquot of 10 ml of distilled water in a test-tube was shaken vigorously for 30 seconds and
then allowed to stand for 45 minutes. The appearance of a frothing, which persists on warming
indicated the presence of saponins [38]. 4. Test for flavonoides,To a portion of the dissolved
extract, a few drops of 10 % ferric chloride solution were added. A green or blue colour
indicated the presence of flavonoides [37]. 5. Test for steroids/terpenes. A lot of 500 mg of the
extract from the rotary evaporator was dissolved in an aliquot of 2 ml of acetic anhydride and
cooled at 0 to 4 C, to which a few drops of 12N sulphuric acid was carefully added. A colour
change from violet to blue-green indicated the presence of a steroidal nucleus (38 Sofowara
1993). 6. Test for tannins. 0.5 g of the extract was dissolved in 5 ml of water followed by a few
drops of 10 % ferric chloride. A blue-black, green, or blue-green precipitate would indicate the
presence of tannins [38]. 7. Test for alkaloids. A lot of 0.5g of ethanol extract (from rotary
evaporator) was stirred with an aliquot of 5 ml of 1% HCl on a steam bath and filtrated; to an
aliquot of 1ml of the filtrate, a few drops of Mayers reagent was added, and to another aliquot of
1ml of the filtrate, a few drops of Dragendorffs reagent were added. Turbidity or precipitation
in tubes due to either of these reagents indicated the presence of alkaloids in the extract [38]. 8.
Test for resins. To an aliquot of 10 ml of the extract an aliquot of 10 ml of cupper acetate
solution 1% was added and shaken vigorously and, a separate green colour indicated the
presence of resin [40]. 9. Test for glycosides. An aliquot of5 ml of each extract was mixed with
an aliquot of 2 ml of glacial acetic acid (1.048 - 1.049g/ml), one drop of ferric chloride solution
(1%), and mixed thoroughly. To this mixture, an aliquot of 1 ml of 12N H2SO4 was added. A
brown ring at the interface indicated the presence of glycosides [38].
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RESULTS
_______________________________________________________________________
Antibiotic sensitivity of clinical isolates of P. aeruginosa
In repeated attempts over a period of 3 months (November 2009 to February 2010), 27strains ofP. aeruginosa were obtained from clinical samples and each strain was resistant to 11
antibiotics at levels, monitored by disc diffusion method, detailed in Table II. Among the
isolated 27 strains, cefotaxime-resistant isolates of the bacterium were the maximum (81.5%)
and imipenem-resistant and nitrofurantoin-resistant isolates were the minimum (11.1%); in other
words, 22 strains were resistant to cefotoxime-30g/disc and 3 strains were resistant to
imipenem-10g/disc and nitrofurantoin-300g/disc, individually (Table II). In summary, the order
of numbers of isolated strains is: cefotaxime-30g> amoxyclave-30g> ofloxacin-5g>
gentamicin-10> piperacillin-100g/tazobactam-10g> amikacin-30g> gatifloxacin-30g>
netilmicin-30g> piperacillin100g> imipenem-10 or nitrofurantoin-300g /disc (Table II).
Maximum number of clinical isolates was from urine and minimum number from pleural fluid
(Table II).
A strain sensitive to all antibiotics used obtained from NTCC, Pune, India with the number
10662 was used in parallel to 15 clinically isolated strains of originally obtained 27 strains, in the
study of monitoring antipseudomonal activities of plant extracts; 8 types of antibiotic discs were
tested against individual clinical isolates, i.e., nitrofurantoin, gentamicin and singular piperacillin
were not used (Tables III). Resistance of clinical isolates to other used antibiotics could be
arranged in the decreasing order of numbers of resistant strains among 15 isolates: amoxyclave-
30g> ofloxacin-5g> gentamicin-10> piperacillin-100g/tazobactam-10g> amikacin-30g>
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gatifloxacin-30g> netilmicin-30g/disc that are analyzed in Table 4. Strains moderately
sensitive to selected antibiotics were also observed (Table III).
Antibacterial activity test by agar-well diffusion method
Antipseudomonal activities of 8 solvent-extracts using chloroform, ethyl acetate,
acetone, and dichloromethane petroleum ether, methanol, ethanol and water (non-polar to polar
solvent in the order) were used by the agar-well diffusion method on lawns of 15 bacterial
isolates and the NTCC strain. It was found that cold solvent-extracts, when tested against 16
pseudomonad strains, the following solvent-extracts: chloroform, ethyl acetate, dichloromethane,
petroleum ether and water had no zone of inhibition. But, rest three cold-extracts using acetone,
methanol, and ethanol had prominent antibacterial activities (Table V). Kruskal-Wallis H test
was applied to the dataset of antibacterial activity cold-extracts with acetone, methanol and
ethanol. It was found that Kruskal-Wallis Hvalue was computed as 46.62, when tabulated H
values are 5.99 atp = 0.05 and 9.21 atp = 0.01, for degree of freedom (df) 4 extracts minus 1 =
3. Since tabulated values are for less than the computedHvalue, both at p = 0.05 and 0.01, the
null hypothesis that there is no difference between inhibitory zones due to 3 cold solvent-extracts
is outright rejected. Differences in values of zones of inhibition of individual 3 cold-solvent-
extracts are highly significant (Table IV). Secondly, the total rank signs recorded in Table 4
for the methanolic extract is 747, against similar values due to acetone and ethanol as 166.5 and
341.5, respectively, while that of gentamicin 40 g/ml was 800. These clearly indicate that
methanol is the most suitable solvent forA. mexicana in cold extraction (Table 4). Similar result
of highest antimicrobial activity of methanol was also obtained in the hot extraction (Table V).
The Kruskal-Wallis H value was computed as 57.18. The differences in values of zones of
inhibition among 3 hot solvent-extracts were statistically significant both at p = 0.05 and 0.01, as
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computed H value is far more than tabulated values, 5.99 or 9. 21. The total rank signs are
recorded in Table V of data of agar-well diffusion and disc diffusion methods for methanolic,
acetone or ethanol extracts along with gentamicin 40 g/ml. As with cold extracts in hot
extraction also methanol-extract was the most effective against P. aeruginosa strains. Students
ttest was performed between cold and hot extraction data of values of zones of inhibition. As
the calculated t= 2.192 is more than the tabulated t=2.05, at df = 28 (15+15-2), atp = 0.05, the
difference between values of zones of inhibition of cold extract and hot extract with methanol is
statistically significant, at p = 0.05. Thus, the hot methanol extraction caused higher values of
zones of inhibition of all strains than the cold methanol extraction with A. mexicana, monitored
in the agar-cup method.
Determinations of MIC and MBC
The hot acetone extract yielded the MIC value of 10 mg/ml on the NTCC strain and 15
clinical isolates. But MIC values of these 16 strains due to methanol and ethanol extracts were
found to be same, that is 8 mg/ml. MBC values (or lethal concentration 100) for all the 16 strains
ofP. aeruginosa were 32, 28 and 24 mg/ml in response to acetone, methanol and ethanol
extracts (hot extraction), respectively.
Phytochemical analysis
During Phytochemical analysis of acetone, methanol and ethanol extraction ofA.
mexicana was found that flavonoides, tannins, sterols/terpenes and alkaloids were present in all
the 3 types of extract. The results of other tests for colour, pH, reducing sugar, anthraquinone,
saponins, resins and glycosides are summarized in Table IV.
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DISCUSSION
_______________________________________________________________________
P. aeruginosa is a well-known opportunistic bacterial pathogen contaminating inanimate
articles of hospitals such as, sinks, drains and many medical equipments, thereby it is marked as
the notorious one in nosocomial spread, and even it is found from soil, but is rare in clinical
isolates of healthy individuals, as it is known [41]. In this study, 27 strains of MDRP.
aeruginosa have been isolated from random clinical samples (Table II). Moreover, antibiotics
used so for against any bacterial pathogen includingP. aeruginosa are from microbial sources.
Gradually, the target bacterium develops resistance to those antibiotics readily and survives.
Moreover, crude drugs from eukaryotic systems (as plants) have an array of compounds against
which resistance can never be developed in any pathogen. In fact, eukaryotic compounds in
crude extracts when employed against a pathogen, a synergistic effect is achieved with the
eventual control of the pathogen; that is how certain plants are reported to be highly successful
and popular in managing infections in traditional health care systems, worldwide. By the by,
the development of scientifically un-approved drugs has proliferated so much that the plant-
based crude-medicine trade in local and international markets is popular, worldwide. Studies on
crude phyto-extracts in monitoring of antimicrobial activities of plants are of practical
importance. Further, many pure phytochemicals have too crept in to the scientific medicinal
system [25]. Herein, the efficiency ofA. mexicana in the control of several types of infections in
the age-old low-cost health-care module of marginalized and poverty-stricken aborigine-
folklore of India in crude plant extract [15- 20]; A. mexicana belongs to the plant-family,
Papaveraceae to which another the most successful medicinal plant, Papaver somniferum,
(yielding morphine and many more) belongs, in controlling in vitro MDR strains ofP.
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aeruginosa. Reminded that it is the causative organism in bacteremic form of pneumonia of aged
and immune-compromised patients in bringing 80% mortality [42].
Most antibiotics showing antipseudomonal activity belong to the -lactum and
aminoglycoside groups. In fact, P. aeruginosa is found to be resistant to antibiotics of -lactum
derivatives used for control, due to low outer membrane permeability affording intrinsic
resistance; -lactum molecule penetrates outer membrane of the bacterium at rates lower than
that ofE. coli [42]. However, several third generation cephalosporin and fluroquinolones
derivatives such as, the ciprofloxacin have been proved to be useful [43]. Among the -lactum
derivatives with antipseudomonal activity, piperacillin (acylureido penicillin), imipenem (a semi-
synthetic derivative of thienamycin, carbapenems), cefotaxime (cephems, first generation
cephalosporin) were found ineffective forP. aeruginosa. Further, the transfer of resistances to
both carbenicillin and gentamicin to a sensitive strain ofP. aeruginosa was attributed to R-
plasmid mediated transfer [44]. Moreover, protein synthesis inhibiting aminoglycosides are
gentamicin, amikacin and tobramycin that are commonly used against P. aeruginosa [45]. In the
present study,P. aeruginosa is resistant to gentamicin and amikacin. Records onP. aeruginosa
offering resistances to many antibiotics, by production of antibiotic-inactivating enzymes and/or
alteration of target sites are available [45].
It has been reported that in the ethanolic extract ofA. mexicana flavonoides, tannins,
sterols, terpins, alkaloids and some reducing sugar were present, while anthraquinone, saponins
and resins were absent [46]. But, this study records presence of flavonoides, tannins,
sterols/terpenes and alkaloids in three types of plant extracts and these constituents are expected
to be effective in the present anti-pseudomonad activity. The effectivity of ethanolic extract on
antipseudomonal activity in the present study was found medium (compared to that of
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methanolic extract), probably due partial extraction of phytochemicals by ethanol in the
successive extraction procedure followed. It has been shown that in an acute toxicity test of
exposing the ethanolic extract to live mouse, the lethal dose50 (LD50) was 400mg/kg body weight,
typically in a study [46].
This study of antibiogram of clinical isolates ofP. aeruginosa should be helpful to
establish appropriate treatment regimen in a situation of changing epidemiology of the organism.
More particularly, corneal infections are rapidly progressive and require immediate appropriate
chemotherapy for control of the infection [47]. Furthermore, the basis of ethnotherapeutic use of
this nearly poisonous plant against infectious diseases as reported often from various countries is
found suitable for control of P. aeruginosa. This organism is found in infections of skin
sometimes with bacteria laden fluid lesions, subcutaneous nodules, metastatic abscess, bacillary
endocarditis, pneumoniae, cystic fibrosis, recurrent urinary tract infections, musculoskeletal
infections and meningitis. As discussed, of these the mortality rate from bacteremic form of
pneumonia is approximately 80% [42, 48]. This study would possibly help development of
antipseudomonal drugs, especially for MDR strains, from A. mexicana with methanolic extracts.
Further, quantification of quantification of chemicals in with methanolic extracts of the plant
HPLC is in progress.
ACKNOWLEDGEMENTS
______________________________________________________________________________
This work (part of PhD thesis of MCS) was supported by a research project Monitoring
antimicrobial activities of ethnomedicinal plants of Kalahandi district, from Department of
Science and Technology, Govt. of Orissa, Bhubaneswar awarded to RNP.We are grateful to Dr.
B. N. Panda, MD, Medical Superintendent, and Dr. D. K. Roy, MD, Dean, IMS & Sum Hospital
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for facilities and encouragements. We are grateful to Dr. Ranjit Ghose, Principal, B. J. B.
Autonomous College for encouragements.
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