ANTIBACTERIAL ACTIVITY OF Argemone mexicana L.

AGAINST MULTI-DRUG RESISTANT Pseudomonas aeruginosa,

ISOLATED FROM CLINICAL SAMPLES

Mahesh C. Sahua, b, Nagen K. Debata b, Rabindra N. Padhya*

a
Department of Botany, B. J. B. Autonomous College, Bhubaneswar 751014, Orissa, India
b
Department of Microbiology, IMS & Sum Hospital Medical College, S O A University,

Kalinga Nagar, Bubaneswar-751003, Orissa, India

*
Communicating author e-mail: rnpadhy54@yahoo.com

ABSTRACT
_____________________________________________________________________
Twenty seven isolates of Pseudomonas aeruginosa were identified from clinical samples

from a hospital, using appropriate growth media. Among them, 22 strains were resistant to

cefotoxime-30µg/disc, 16 strains to amoxyclave-30µg, 15 strains to ofloxacin-5µg, 13 strains to

gentamicin-10 µg, 10 strains to piperacillin-100µg/tazobactam-10µg, 8 strains to amikacin-30µg,

7 strains to gatifloxacin-30µg, 6 strains to netilmicin-30µg, 4 strains to piperacillin100µg and 3

strains were resistant to imipenem-10µg and nitrofurantoin-300µg/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,

1
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 of P. aeruginosa. The

methanol-extract had the highest level of antipseudomonal activity both in cold and hot

extractions, confirmed by separate Kruskal–Wallis H tests. The Student’s 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 of A.

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
-----------------------------------------------------------------------------------------------------------
-
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

2
pathogens. For example, resistant strains of Staphylococcus intermedius, Campylobacter sp.,

Salmonella sp. and Escherichia 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

3
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 [15–21] or elsewhere [22–24] 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 or Argemone 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 of A. 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 of A. 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, Sigella sp. 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 of A. mexicana using against pathogenic and non-pathogenic bacteria

4
are described [29]. None of these reports record any antibiotic-susceptibility test of used

bacteria. Further, the literature on plant extracts including A. 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 MDR P. 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 of Argemone 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

5
prevent any fungal infection) at 37±1°C 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 of A. mexicana. A lot of

20g of powder-mass in an aliquot of 200 ml of a solvent was dissolved and stored at 4°C 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 -4°C 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

6
at 40°C 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-sensitive Pseudomonas 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 of P.

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 of P. aeruginosa in clinical

samples only and growing colonies were not from any nosocomial infection, repeated clinical

samples were plated. Isolated cells of P. aeruginosa were slender, non-capsulated with a polar

flagellum with dimensions, (1.5 to 3) x 0.5µm.

Biochemical identification test

Confirmatory biochemical tests done were the catalase test, the oxidase test and the

growth of colonies in King’s media. A prompt reaction of effervescence with a colony of P.

aeruginosa on exposure to a drop of hydrogen peroxide (H2O2) indicated the activity of the

7
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 25-30°C confirmed the presence of the oxidase

enzyme. Further, the bacterium was grown in King’s 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 King’s A medium were soluble in traces of chloroform and were found to

diffuse into the surrounding medium. When the colonies were grown King’s 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 King’s medium. These tests confirmed isolation of strains of P. aeruginosa. Pyocyanin

pigment production in King’s A medium conforms P. aeruginosa and the pigment soluble in

chloroform. Growth of P. aeruginosa in King’s B medium via production of fluorescein

(pyoverdin) insoluble in chloroform and soluble in water confirms presence of P. aeruginosa

[33].

Antibiotic sensitivity test

All the used 16 bacterial strains of P. aeroginosa were subjected to antibiotic sensitivity

test by the disc diffusion method, using a 4 mm thick Mueller–Hinton 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 37°C 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 for P. aeroginosa were placed at

8
equal distances from one another. Plates were incubated overnight for 18h at 37°C. 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 µl molten Mueller–Hinton agar.

Further, wells were filled with 100 µl aliquots of 30 mg/ml solvent-extracts of A. 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 37°C

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 µg/ml) for P. 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 of 10µl plant extract (50mg/ml) were soaked by sterile filter paper discs (5

mm diameter). Sterile filter paper discs were impregnated with 10µl of plant extract placed on

the surface of the medium and incubated at 37ºC for 24 h. The assessment of antibacterial

activity was based on the value of diameter of the inhibition zone formed around the disc [36].

9
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 µl 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 µl

nutrient broth (Himedia, Mumbai), an aliquot of 20 µl bacterial inocula (109 CFU/ml) and a

5µl-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 37°C 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 of A. 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

Fehling’s 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,

10
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 Mayer’s reagent was added, and to another aliquot of

1ml of the filtrate, a few drops of Dragendorff’s 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 of 5 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].

11
 RESULTS
_______________________________________________________________________
Antibiotic sensitivity of clinical isolates of P. aeruginosa

In repeated attempts over a period of 3 months (November 2009 to February 2010), 27

strains of P. 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-30µg/disc and 3 strains were resistant to

imipenem-10µg/disc and nitrofurantoin-300µg/disc, individually (Table II). In summary, the order

of numbers of isolated strains is: cefotaxime-30µg> amoxyclave-30µg> ofloxacin-5µg>

gentamicin-10> piperacillin-100µg/tazobactam-10µg> amikacin-30µg> gatifloxacin-30µg>

netilmicin-30µg> piperacillin100µg> imipenem-10 or nitrofurantoin-300µg /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-

30µg> ofloxacin-5µg> gentamicin-10> piperacillin-100µg/tazobactam-10µg> amikacin-30µg>

12
gatifloxacin-30µg> netilmicin-30µg/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 H value was computed as 46.62, when tabulated H

values are 5.99 at p = 0.05 and 9.21 at p = 0.01, for degree of freedom (df) 4 extracts minus 1 =

3. Since tabulated values are for less than the computed H value, 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 µg/ml was 800. These clearly indicate that

methanol is the most suitable solvent for A. 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

13
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 µg/ml. As with cold extracts in hot

extraction also methanol-extract was the most effective against P. aeruginosa strains. Student’s

t test 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), at p = 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

of P. 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 of A.

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.

14
 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 MDR P.

aeruginosa have been isolated from random clinical samples (Table II). Moreover, antibiotics

used so for against any bacterial pathogen including P. 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 of A. 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 of P.

15
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 β-lactum and

aminoglycoside groups. In fact, P. aeruginosa is found to be resistant to antibiotics of β-lactum

derivatives used for control, due to low outer membrane permeability affording intrinsic

resistance; β-lactum molecule penetrates outer membrane of the bacterium at rates lower than

that of E. coli [42]. However, several third generation cephalosporin and fluroquinolones

derivatives such as, the ciprofloxacin have been proved to be useful [43]. Among the β-lactum

derivatives with antipseudomonal activity, piperacillin (acylureido penicillin), imipenem (a semi-

synthetic derivative of thienamycin, carbapenems), cefotaxime (cephems, first generation

cephalosporin) were found ineffective for P. aeruginosa. Further, the transfer of resistances to

both carbenicillin and gentamicin to a sensitive strain of P. 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 on P. 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 of A. 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

16
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 of P. 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

17
for facilities and encouragements. We are grateful to Dr. Ranjit Ghose, Principal, B. J. B.

Autonomous College for encouragements.

REFERENCES
_______________________________________________________________________
[1] W. H. O. (2004) The world health report: Changing history, Statistical annex, death by

cause, sex and mortality stratum in WHO regions, estimates for 2002, pp. 120–121.

Switzerland.

[2] Norrby, S. R. Nord, C.E. and Finch, R. (2005) Lack of development of new antimicrobial

drugs: A potential serious threat to public health. Lancet Infect. Dis. 5, 115–119.

[3] Walsh, C. (2003) Antibiotics: actions, origins, resistance. American Society for

Microbiology Press, Washington, DC.

[4] Levy, S. B. and Marshall, B. (2004) Antibacterial resistance worldwide: Causes,

challenges and responses. Nature Med. 10, S122–S129.

[5] Guardabassi, L., Schwarz, S. and Lloyd, D. H. (2004) Pet animals as reservoirs of

antimicrobial-resistant bacteria. J. Antimicrob. Chemother. 54, 321–32.

[6] Brown, S. A. (1987) Minimum inhibitory concentrations and post-antimicrobial effects as

factors in dosage of antimicrobial drugs. J. Am. Vet. Med. Assoc. 19, 871–872.

[7] Alexander, M. (1981) Why microbial predators and parasites do not eliminate their prey

and hosts? Ann. Rev. Microbiol. 35, 113–133.

[8] Potts, M. (1994) Desiccation tolerance of prokaryotes. Microbiol. Rev. 58, 755–805.

[9] Botzenhart, K. and Doring, G. (1993) Ecology and epidemiology of Pseudomonas

aeruginosa, In: Pseudomonas aeruginosa as an opportunistic pathogen. (Eds. Mario, C.

Mauro, B. and Herman, F.). Plenum, New York. pp. 1–18.

18
[10] Drlica, K. (2003) The mutant selection window and antimicrobial resistance. J.

Antimicrob. Chem. 52, 11–17.

[11] Gould, I. M. and Mackenzie, F. M. (2002) Antibiotic exposure as a risk factor for

emergence of resistance: the influence of concentration. Soc. Appl. Microbiol. 31, 78S–

84S.

[12] Bliziotis, I.A., Samonis, G., Vardakas, K. Z., Chrysanthopoulous, S. and Falagas, M. E.

(2005) Effect of aminoglycoside and β -lactam combination therapy versus β-lactam

monotherapy on the emergence of antimicrobial resistance: A meta-analysis of

randomized, controlled trials. Clin. Infect. Dis. 41, 149–158.

[13] Boothe, D. M. (2006) Principles of antimicrobial therapy. Vet. Clin. Small Anim. 36,

1003–1047.

[14] Hegreness, M., Shoresh, N., Damian, D., Hartl, D. and Kishony, R. (2008) Accelerated

evolution of resistance in multidrug environments. PNAS, USA 105, 13977–13981.

[15] Behera, K. and Misra, M. K. (2005) Indigenous phytotherapy for genito-urinary diseases

used by the kandha tribe of Orissa, India. J. Ethnopharmacol. 102, 319–325.

[16] Panda, T. and Padhy, R. N. (2008) Ethnomedicinal plants used by tribes of Kalahandi

district, Orissa. Indian J. Tradit. Know. 7, 242–249.

[17] Pattanaik, C., Reddy, C. S. and Murthy, M. S. R. (2008) An ethnobotanical survey of

medicinal plants used by the Didayi tribe of Malkangiri district of Orissa, India.

Fitoterapia 79, 67–71.

[18] Ahmad, I. and Beg, A. Z. (2001) Antimicrobial and photochemical studies on 45 Indian

medicinal plants against multi-drug resistant human pathogens. J. Ethnopharmacol. 74,

113–123.

19
[19] Jagtap, S. D., Deokule, S. S. and Bhosle, S. V. (2006) Some unique ethnomedicinal uses

of plants used by the korku tribe of Amravati district of Maharashtra, India. J.

Ethnopharmacol. 107, 463– 469.

[20] Samy, R. P., Thwina, M. M., Gopalakrishnakone, P. and Ignacimuthu, S. (2008)

Ethnobotanical survey of folk plants for the treatment of snakebites in southern part of

Tamilnadu, India. J. Ethnopharmacol. 115, 302–312.

[21] Singh, A. and Singh, P. K. (2009) An ethnobotanical study of medicinal plants in

Chandauli district of Uttar Pradesh, India. J. Ethnopharmacol. 121, 324–329.

[22] Macia, M. J., Garcia, E. and Vidaurre, P. J. (2005) An ethnobotanical survey of

medicinal plants commercialized in the markets of La Paz and El Alto, Bolivia.

J. Ethnopharmacol. 97, 337–350.

[23] Al-Qura’n, S. (2007) Ethnobotany of folk medicinal aquatic plants in Jordan. The

Botanical Rev. 73, 51–65.

[24] Vitalini, I. S., Tomè, F., Fico, G. (2009) Traditional uses of medicinal plants in

Valvestino (Italy). J. Ethnopharmacol. 121, 106–116.

[25] Dubey, D., Rath, S., Sahu, M. C., Debata, N. K. and Padhy, R. N. (2010)

Antimicrobials of plant origin against multi-drug resistant bacteria including the TB

bacterium and economics of plant-drugs – Introspection. Indian J. Tradit. Know. (in

press).

[26] Wilcox, M. L., Graz, B. and Falquet, J. (2007) Argemone mexicana decoction for the

treatment of uncomplicated falciparum malaria. Trans. R. Soc. Trop. Med. 101, 1190–

1198.

20
[27] Bhattacharjee, I., Chatterjee, S. K., Chatterjee, S. and Chandra, G. (2006) Antibacterial

potentiality of Argemone mexicana solvent extracts against some pathogenic bacteria.

Mem. Inst. Oswaldo Cruz. 101, 645–648.

[28] Mohana, D. C., Satish, S. and Raveesha, K. A. (2008) Antibacterial evaluation of some

plant extracts against some human pathogenic bacteria. Adv. Biol. Res. 2, 49–55.

[29] Singh, S. K., Pandey, V. D., Singh, A. and Singh, C. (2009) Antibacterial activity of seed

extracts of Argemone mexicana L. on some pathogenic bacterial strains. Afr. J.

Biotechnol. 8, 7077–7081.

[30] Mahady, G. B., Huang, Y., Doyle, B. J. and Locklear, T. (2008) Natural products as

antibacterial agents. In: (Ed. Atta-ur-Rahman). Studies in natural products chemistry.

Elsevier B.V., Amsterdam. pp. 423–444.

[31] Livermore, D. M. (2002) Multiple mechanisms of antimicrobials resistance in

Pseudomonas aeruginosa. Our worst nightmare? Clin. Infect. Dis. 34, 634–640.

[32] Shepp, D. H., Tang, I.T-L., Romundo, M. B. and Kaplan, M. H. (1994) Serious

Pseudomonas aeruginosa infection in AIDS. J. AIDS 7, 823–831.

[33] Pitt, T. L. (1990) Pseudomonas. In: Principle of bacteriology, virology and immunology.

(Eds. Parker, T., Leslie, M. and Collier, H.). Vol 2, 8th Edn. Edward Arnold, New York.

pp. 255–273.

[34] Bauer, A. M., Kirby, W. M. M., Sherris, J. C. and Turk, M. (1966) Antibiotic

susceptibility testing using standard single disc method. Amer. J. Clin. Pathol. 45, 493–

496.

21
[35] Perez, C., Paul, M. and Bazerque, P. (1990) Antibiotic assay by agar-well diffusion

method. Acta Biol. Medicin. Experim. 15, 113–115.

[36] Rios , J. L., Recio, M. C. and Vilar, A. (1988) Screening methods for natural products

with antimicrobial activity, a review of the literature. J. Ethnopharmacol. 23, 127–149.

[37] Brain K. R., Turner T. D. (1975) The practical evaluation of phytopharmaceuticals.

Wright-Scientica, Bristol. pp. 57–58.

[38] Sofowara, A. (1993) Medicinal plants and traditional medicine in Africa. Spectrum

Books, Ibadan. pp. 151-153.

[39] Harborne, J. B. (1998) Phytochemical methods –– a guide to modern techniques of plant

analysis. Chapman and Hall, London. pp. 1–203.

[40] Trease, G. and Evans, W. C. (1983). Textbook of pharmacognosy. 12th ed, Tindall,

London. pp. 343–383.

[41] Konrad, B. and Gerd, D. (1993) Ecology and epidemiology of Pseudomonas

aeruginosa. In: Pseudomonas aeruginosa as an opportunistic pathogen. (Eds. Mario, C.,

Mauro, B. and Herman, F.). Plenum, New York. pp. 1–18.

[42] Arenstein, A. W. and Cross, S. A. (1993) Local and disseminated disease caused by

Pseudomonas aeruginosa. In: Pseudomonas aeruginosa as an opportunistic pathogen.

(Eds. Mario, C., Mauro, B. and Herman, F.). Plenum, New York. pp. 223–244.

[43] Bellido, F. and Hancock, R. E.W. (1993) Susceptibility and resistance of Pseudomonas

aeruginosa to antimicrobial agents. In: Pseudomonas aeruginosa as an opportunistic

pathogen. (Eds. Mario, C., Mauro, B. and Herman, F.). Plenum, New York. pp.321–348.

22
[44] Milatovic, D. and Brawny, A. (1987) Development of resistance during antibiotic

therapy. Eur. J. Clin. Microbiol. 6, 234–244.

[45] Philips, I. and Shannon, K. I. (1997) Aminoglycosides and aminocyclitois. In:

Antibiotics and chemotherapy: anti- infective agents and their use in therapy. (Eds.

Francis, O.G., Lambert, P., Harold-Finch, R.G. and David, G.). 7th ed, Churchill,

Livingstone. pp. 78–102.

[46] Ibrahim, H. A. and Ibrahim, H. (2009) Phytochemical screening and toxicity

evaluation on the leaves of Argemone mexicana L. (Papaveraceae). Int. J. P. App.
Sci. 3, 39–43.
[47] Kreger, A. S. (1983) Pathogenesis of Pseudomonas aeruginosa in ocular diseases. Rev.
Infect. Dis. 5, 931–935.
[48] Pennington, J. E., Reynolds, H. Y. and Carbone, P. P. (1973) Pseudomonas pneumonia:

a retrospective study of 36 cases. Am. J. Med. 55, 155–160.

23