j o u r n a l o f h e r b a l m e d i c i n e 3 ( 2 0 1 3 ) 65–75

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Antibacterial activity of Lantana camara L. against multidrug

resistant pathogens from ICU patients of a teaching hospital

Debasmita Dubey a , Rabindra N. Padhy b,∗

a Department of Microbiology, IMS & Sum Hospital Medical College, Siksha O Anusandhan University, Kalinga Nagar, Bhubaneswar
751 003, Odisha, India
b Central Research Laboratory, IMS & Sum Hospital Medical College, Siksha O Anusandhan University, Kalinga Nagar, Bhubaneswar

751 003, Odisha, India

a r t i c l e i n f o a b s t r a c t

Article history: The scientific basis for the use of the common shrub-weed plant Lantana camara L. was inves-
Received 12 July 2012 tigated by testing leaf extracts for antibacterial activity. Dried leaf powders were extracted
Received in revised form using a hot-solvent extraction method with eight polar to non-polar solvents in succession.
23 September 2012 Crude extracts were tested for antibacterial activity against three multidrug-resistant (MDR)
Accepted 17 December 2012 Gram-positive bacteria: methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyo-
Available online 4 February 2013 genes, and vancomycin-resistant Enterococcus faecalis (VRE); and five MDR extended-spectrum
␤-lactamase-producing Gram-negative bacteria: Acinetobacter baumannii, Citrobacter freundii,
Keywords: Proteus mirabilis, Proteus vulgaris and Pseudomonas aeruginosa. The MRSA strain was resis-
Antibacterial activity tant to 16 of 18 antibiotics, while Streptococcus pyogenes and VRE were resistant to 15 of 18
Lantana camara antibiotics. Similarly, A. baumannii and P. aeruginosa were resistant to 14 of 16 antibiotics. It
Minimum bactericidal was found that plant extracts with petroleum ether and water had the least antibacterial
concentration activity. Leaf extracts with dichloromethane and methanol registered the highest antibac-
Minimum inhibitory concentration terial activity on all bacterial strains. The minimum inhibitory concentration and minimum
Multidrug-resistant bacteria bactericidal concentration of two active leaf extracts, obtained with dichloromethane and
Phytochemical analysis methanol were determined. Phytochemical analysis of dichloromethane leaf extracts con-
firmed the presence of alkaloids, glycosides, terpenoids, saponins, flavonoids, and steroids,
but reducing sugars were also absent; and, in the methanolic leaf extract, alkaloids, ter-
penoids, saponins, flavonoids and steroids were present, but glycosides, reducing sugars
and tannins were absent. These findings point to the potential of the plant as a probable
source of bioactive compounds and provide a scientific basis for its folklore/ethnomedicinal
uses for infectious diseases.
© 2012 Elsevier GmbH. All rights reserved.

incineration is traditionally used for repelling mosquitoes

1. Introduction
Lantana camara L. (red sage, Verbenaceae family) is a shrub-
weed commonly found in South America, Africa, Asia and
Australia, where there are tropical and subtropical climates,
and is a prolific weed in Kenya. In Nigeria, L. camara leaf
(Egunyomi et al., 2010). In Indian Ayurveda, a decoction of fresh
root of the plant is used as a beneficial gargle for odontal-
gia. For all types of dysentery, cuts, wounds, and swellings,
infusion of powdered leaves is used by hill tribes (Ghisalberti,
2000), as the leaves have antimicrobial and termiticidal

∗
Corresponding author. Formerly at: BJB Autonomous College, Bhubaneswar, India. Tel.: +91 674 6511205.
E-mail address: rnpadhy54@yahoo.com (R.N. Padhy).
2210-8033/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.hermed.2012.12.002
66 j o u r n a l o f h e r b a l m e d i c i n e 3 ( 2 0 1 3 ) 65–75
activities (Verma and Verma, 2006). The anti-tubercular activ-
ity of this plant against multidrug-resistant (MDR) tubercle
bacilli has been reported from Mexico (Jimenez-Arellanes
et al., 2003). In vitro antibacterial activity of a methanolic leaf
extract of L. camara against the non-resistant Gram-positive
(GP) and Gram-negative (GN) bacterial strains Bacillus sub-
tilis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas
aeruginosa and Escherichia coli, has been reported from India
(Udayprakash et al., 2011). Among the phytochemicals of L
camara, lantadines, anthraquinones and triterpenes have been
reported to also have antifungal properties (Kumar et al., 2006;
Juliani et al., 2002). However, teratological effects of the leaves
on rats have been recorded (Mello et al., 2005).
L. camara has been used in many parts of the world to
treat a wide variety of disorders (Ross, 1999). It has been used
in folk remedies for cancers and tumours. Leaves and flow-
ers of the plant are used for preparing a decoction that is
used against fever, influenza and stomach-ache. In Central
and South America, leaves are made into a poultice to treat
sores, chicken pox and measles. Fevers, colds, rheumatism,
asthma and high blood pressure have been treated with prepa-
rations of the plant (Irvine, 1961). In Ghana, an infusion of
the whole plant was used for bronchitis and the root pow-
dered in milk was given to children for stomach-ache (Irvine,
1961). In Asian countries, leaves have been used to treat cuts,
rheumatism, ulcers and intestinal worms. Decoctions were
applied externally for leprosy and scabies. It has been claimed
that a steroid, lancamarone, from the leaves has exhibited
cardiotonic properties (Sharma et al., 1992), and lantamine,
an alkaloid from the stem, bark and root showed antipyretic
and antispasmodic properties comparable to those of quinine
(Ghisalberti, 2000), but the validity of these claims has not
been confirmed. An alkaloid fraction from the leaves has been
reported to lower blood pressure and to accelerate deep res-
piration, and it was suggested that those might be useful in
reducing fevers and asthma attacks (Ghisalberti, 2000). More-
over, six phenolic compounds in an L. camara extract were
identified by high-pressure liquid chromatography (HPLC): sal-
icylic acid, gentisic acid, ␤-resorcylic acid, coumarin, ferulic
acid and 6-Me coumarin (Yi et al., 2006). These and several
other phytocompounds of the plant could be contributing, a
priori, to the recorded anecdotal health benefits.
Infectious diseases caused by drug/antibiotic-resistant
bacterial strains have always been a matter of clinical con-
cern. Mortality due to urinary tract infections (UTIs) has
been of utmost clinical concern in the last 4 decades, with
81% in 1973 and 50% in 2006 due to MDR P. aeruginosa,
for example (Giamarellos-Bourboulis et al., 2006; Pennington
et al., 1973). Authors, from the Institute of Medical Sci-
ences (IMS) and Sum Hospital, have previously reported MDR
P. aeruginosa as the primary candidate for community and
hospital-acquired UTIs with extended-spectrum ␤-lactamase
(ESBL) production (Sahu et al., 2012). Indeed, short courses
of empiric therapy (1–3 days) are usually completed for
UTIs before test results become available, promoting mis-
matches due to drug resistance. Here, the usual treatment
would be the use of cefepime, a cephalosporin that is tra-
ditionally used to treat clinically important strains of P.
aeruginosa, as empiric therapy for a UTI in an intensive
care unit (ICU), but when the infection is confirmed, the
antimicrobial stewardship programme subsequently uses
a carbapenem. Not surprisingly, 80–90% of uncomplicated
UTI cases worldwide in otherwise healthy adults occur
with the second-most important causative microbe, Acine-
tobacter species. Even community-acquired infections by
ESBL-producing Acinetobacter baumannii were reported in 2007
in the U.S. (DeBusscher et al., 2009). Furthermore, A. bauman-
nii has been a pandrug-resistant (resistant to all antibiotics
in use at present) bacterium, precipitating numerous global
catastrophic episodes with an ever-increasing resistance
pattern to cefepime, ceftazidime, ciprofloxacin, gentami-
cin, levofloxacin, piperacillin/tazobactam, and trimetho-
prim/sulfamethoxazole, as reported from North America,
Europe, Asia Pacific and Latin America (Perez et al., 2007).
Moreover, in the past decade, reports of occurrences of
community-acquired ESBL-producing E. coli isolates, the third
most common UTI causative have increased worldwide; but its
prevalence is still uncommon in the U.S., except from noso-
comial sources (Naas et al., 2007). Strains of E. coli, causing
enteropathogenic episodes were found to be resistant to 16
antibiotics from five groups in the author’s laboratory (Rath
et al., in press). Furthermore, Citrobacter freundii, which is a
widely prevalent nosocomial pathogen associated with many
infections, diarrhoea, septicaemia, meningitis and ailments in
urinary and respiratory tracts, was reported to be resistant to
carbapenem in China (Shen et al., 2009).
K. pneumoniae carbapenemase (KPC) enzyme and metallo-
␤-lactamase are the common armamentaria of carbapenem
resistance in Enterobacteriaceae (Kitchel et al., 2009; Cendejas
et al., 2010). In Thailand, Proteus mirabilis had been demon-
strated to have multidrug resistance, for example, to
broad-spectrum ␤-lactam derivatives, carbapenems, strep-
tomycin, tetracycline, sulphonamides, trimethoprim and
fluoroquinolones (Girlich et al., 2001). More recently, P. mirabilis
NKU (Northern Kentucky University) strain has been recorded
as showing resistance to a large number of antibiotics, there-
fore, their control is very difficult (Doublet et al., 2010).
Gram-negative bacteria are naturally transformable and
participate in DNA exchanges in natural unclean waters where
other Gram-negative bacteria are also present, sometimes
with drug resistance markers (Lorenz and Wackernagel, 1994;
Metzgar et al., 2004). The A. baumannii strain ADP 1 has a
remarkable natural competency 100-fold higher than CaCl2 -
induced E. coli (Metzgar et al., 2004).
Pathogenic bacterial strains gain multidrug resistance
due to their simple/plastic genomes and associated DNA
exchanges with other strains. Eventually, the MDR strains
are spread worldwide, causing disproportionate morbidity
and mortality. Many clinical and social factors also enhance
the problem of emergence of MDR pathogens, as discussed
elsewhere (Sahu et al., 2012). Obviously, this problem largely
affects economic and social/public health factors, leading to
the necessity of an urgent search for antimicrobials from alter-
nate sources. Plants provide a palpable source of drugs. The
efficacy of crude extracts of phytochemicals in controlling
MDR bacteria has been demonstrated by in vitro studies (Dubey
et al., 2012a; Dubey and Padhy, 2012; Arokiyaraj et al., 2012).
Phytocompounds in crude extracts have diverse structural
 j o u r n a l o f h e r b a l m e d i c i n e 3 ( 2 0 1 3 ) 65–75
complexity and as they are of non-microbial origin, no
microbe, no matter how well genetically equipped and devel-
oped can ever over-ride these coalesced chemicals in vitro. The
same is true in vivo when the phytocompounds are acces-
sorized with regular chemotherapy, since phytochemicals in
crude extracts remain as unbreakable barriers to microbial
pathogens. It would seem that accumulated ethnomedicinal
reports of different countries are idealistic without any sci-
entific verification, but should form the basis of further work
on drug targeting against MDR pathogens, as has been stated
in detail elsewhere (Dubey et al., 2012b). Taking recourse to
plants for new chemicals, from well-known and lesser-known
weeds as antimicrobials would be a prudent alternative,
not least because the Streptomyces source of antibiotics is
exhausted, but also because a large amount of pure phyto-
chemicals has been serving the health domain holistically.
Indeed, the many natural phytochemicals in crude extracts
encourages the preparation of complementary drugs for tar-
geting MDR pathogens. Several common MDR pathogens,
methicillin-resistant S. aureus (MRSA), MDR P. aeruginosa, MDR
A. baumannii and MDR tubercle bacillus, need to be controlled
carefully. A non-committal attitude towards these natural
chemicals would be a mistake. Obviously, host toxicity testing
remains an essential corollary in designing phytochemicals as
complementary medicines. There is also a widely held con-
sensus that phytocompounds can be used as drugs and it is
likely to be held in the future (see, MacLennan and Pendry,
2011), even by the World Health Organisation. L. camara, with
its unusually strong unpleasant aroma, was anticipated to
have control over MDR bacterial strains, emulating any dove-
tailed synthetic drug. As a report of the antibacterial activity
of L. camara upon resistant pathogenic strains was unavail-
able in the literature, this research on three GP and five GN
MDR bacteria, isolated from clinical samples was initiated to
quantify the in vitro control capacity of crude leaf extracts of
L. camara using several solvents.
2. Materials and methods
2.1. Preparations of plant extracts
Dried leaves of L. camara (Fig. 1) were powdered and the pow-
der was stored at room temperature in air-tight polythene
packs until use. Leaf extracts were obtained by the hot extrac-
tion method using eight non-polar to polar solvents. For each
of the solvents (petroleum ether, chloroform, ethyl acetate,
dichloromethane, acetone, methanol, ethanol and water), 40 g
of the leaf powder was extracted in a soxhlet extractor for 40
siphons or cycles, with a volume of 400 mL of solvent. Indi-
vidual solvent extracts were filtered and concentrated with
a rotary evaporator, run at 40 ◦ C. The sticky masses obtained
were 5.67, 4.12, 3.33, 3.56, 3.6, 3.77, 2.32 and 2.8 g of extract/40 g
of dry leaf powder for petroleum ether, chloroform, ethyl
acetate, dichloromethane, acetone, methanol, ethanol and
water, respectively. Each concentrated sticky mass was diluted
with an appropriate volume of 10% (v/v) dimethyl sulfoxide
(DMSO) to produce a 100 mg/mL stock solution that was stored
at −4 ◦ C until use.
67
Fig. 1 – Lantana camara.
2.2. Isolation and identification of pathogenic bacteria
Strains of eight bacteria were isolated from clinical samples
from patients of the ICU, IMS and Sum Hospital. Samples
were cultured on suitable media and the bacterial isolates
were identified by using standard biochemical procedures
(Table 1). Corresponding standard Microbial Type Culture Col-
lection (MTCC) strains of each bacterium (Table 1) were used
as reference controls. Three GP [MRSA, Streptococcus pyogenes,
vancomycin-resistant Enterococcus faecalis (VRE)] and five GN
(A. baumannii, C. freundii, P. mirabilis, Proteus vulgaris and P.
aeruginosa) bacteria were isolated and used in the study. For
pure-cultures of GP cocci, catalase and coagulase tests were
performed. The catalase test was carried out with a drop
of 3% H2 O2 that caused effervescence, indicating the pres-
ence of catalase enzyme. For the coagulase test, a lump of
test organism was emulsified with a drop of normal saline
water (0.89%) and a drop of human blood serum was added
to the suspension; clumping of cells was observed within
10 s in the presence of bound coagulase enzyme. When a
sample of GP cocci responded positively to both catalase
and coagulase tests, it was confirmed as S. aureus. Catalase-
negative colonies were also cultured on blood agar to check for
their haemolytic patterns, and a bacitracin test (Forbes et al.,
2007), was conducted. Catalase-negative GP colonies with ␤-
haemolysis (complete haemolysis of erythrocytes) on blood
agar and simultaneously sensitive to the bacitracin were iden-
tified as Group A streptococci or S. pyogenes. Catalase-negative,
␣-haemolytic (partial or green haemolysis of erythrocytes)
colonies were subjected to a bile-esculin test. The bile-esculin
medium contains esculin and peptone for nutrition and bile to
inhibit growth of GP bacteria, other than Group D streptococci
or enterococci. Ferric citrate was added as a colour indicator.
Organisms which split esculin molecules and use the liber-
ated glucose to supply energy release esculin into the medium.
The free esculin reacts with ferric citrate in the medium to
form a phenolic iron complex which turns the agar-slant
from dark brown to black. An agar-slant that was more than
half darkened within 48 h of incubation was bile-esculin pos-
itive, for the confirmation of E. faecalis; but the alternative
68 j o u r n a l o f h e r b a l m e d i c i n e 3 ( 2 0 1 3 ) 65–75

Table 1 – Isolation and maintenance of clinically isolated bacteria with colony characteristics and biochemical test results.
Bacterium MTCC no. of Media used Colony Biochemical
standard strain characteristics test reactions
Methicillin resistant 7443 Blood agar Medium to large, smooth, Positive: catalase, coagulase
Staphylococcus aureus entire, slightly raised, creamy
yellow, with green/␤-hemolytic
colonies
Nutrient agar As above without hemolytic
activity
Chromogenic Blue coloured smooth, entire,
MRSA agar slightly raised

Streptococcus 1928 Blood agar Grey, round, large and Positive: bacitracin
pyogenes ␤-hemolytic colonies Negative: catalase

Vancomycin 439 Blood agar Grey, round, small. Positive: bile esculin test
resistant ␣-Hemolytic zones Negative: catalase,
Enterococcus faecalis bacitracin

Acinetobacter baumannii 1425 Nutrient agar Colourless smooth, opaque, Positive: catalase, VP, citrate
raised and pinpoint Negative: oxidase, indole, MR,
MacConkey agar Colourless smooth, opaque, nitrate
raised, NLF TSI: ND
CLED agar Blue coloured opaque raised Urease test: V
NLF

Citrobacter freundii 1658 MacConkey agar Late LF light pink after 48 h Positive: catalase, MR,
citrate, nitrate
Negative: oxidase, indole,
VP, urease
TSI: A/A H2 S

Proteus mirabilis NA MacConkey agar LLF light pink after 48 h Positive: catalase, oxidase, VP,
Blood agar Swarms on blood agar with citrate, urease, nitrate
␤-hemolysis Negative: indole, MR
CLED agar Translucent blue TSI: K/A H2 S

Proteus vulgaris 1771 Blood agar Swarms on blood agar with Positive: catalase, oxidase,
␤-hemolysis citrate, urease, nitrate
Negative: indole, MR, VP
CLED agar Translucent blue TSI: K/A H2 S

Pseudomonas 1688 Nutrient agar Large, irregular opaque with Positive: catalase, oxidase,
aeruginosa bluish green pigment citrate, urease, nitrate
Negative: indole, MR, VP
TSI: ND
MTCC: Microbial Type Culture Collection; LF: lactose fermenting; NLF: non-lactose fermenting; LLF: late lactose fermenting; CLED: cysteine
lactose electrolyte deficient; NA: not available. MR: methyl red; VP: Voges-Proskauer; TSI: triple sugar iron; A/A H2 S: acid in slant and butt with
hydrogen sulfide gas production; K/A H2 S: alkali in slant and acid in butt with hydrogen sulfide gas production; A/A gas: acid in slant and butt
with gas production; ND: not done; V: variable.

non-darkening of the agar was taken as the negative result
(Forbes et al., 2007).
For pure-cultures of GN bacilli, the following tests were
carried out in succession along with the catalase test.
1. Oxidase test: A sample of bacterial colony was rubbed
onto a filter paper impregnated with tetramethyl-p-
phenylenediamine dihydrochloride and the dye indophe-
nols; the zone of the filter paper turns blue/purple in the
positive result, while the negative result is shown by no
change of colour.
2. Indole test: An aliquot of 0.5 mL of Kovac’s reagent (p-
dimethylaminobenzaldehyde, isoamyl alcohol and HCl)
was added to an aliquot of 5 mL of 48-h old grown culture
(test culture). A formation of a cherry red or purple red ring
at the interface of the broth culture and the reagent indi-
cated the indole production from tryptophan in the sample.
3. Methyl red test (MR test): The test culture was added to an
aliquot of 5 mL of sterile MRVP broth (7 g of peptone, 5 g of
glucose, 5 g of potassium phosphate, at pH 6.9), and incu-
bated for 48 h at 37 ◦ C. To this culture, five drops of methyl
red were added as an indicator. If the total solution turns
red, the test is positive for the formation of organic acids
as end products.
4. Voges-Proskauer test (VP test): The test culture was added to
an aliquot of 5 mL of sterile MRVP broth and incubated for
48 h at 37 ◦ C. To this culture tube, ten drops of VP I reagent
(5% ␣-napthol, in absolute alcohol) and two to three drops
of VP II reagent (40% KOH solution) were added and the
mixture was allowed to stand for 15–20 min for the reac-
tion to complete. A positive result was indicated by the
 j o u r n a l o f h e r b a l m e d i c i n e 3 ( 2 0 1 3 ) 65–75
mixture turning red i.e., production of a neutral product,
acetoin, from the fermentation of glucose by the organism,
and alternatively yellow colour production indicated the
negative result.
5. Citrate test: The test culture was inoculated onto a slant of
Simon citrate agar that was incubated for 48 h at 37 ◦ C. The
agar changing colour from green to blue indicated that the
organism used citrate as the sole source of carbon.
6. Urease test: The test organism was inoculated with a slant
of Christensen’s urea agar (peptone, glucose, sodium chlo-
ride, mono-potassium phosphate, urea, phenol red, and
distilled water at pH 6.8). The hydrolysis of urea yielding
ammonia gas increases the pH, which changes the colour
of the medium from off-white to pink/orange, the positive
result.
7. Triple-sugar-iron test (TSI test): Two or three drops of test
broth culture were inoculated on triple-sugar-iron agar
slant and subsequently, a stab was made to the butt of the
slant. The tube was incubated for 37 ◦ C for 48 h; the black
colour appearance indicated H2 S production.
8. Nitrate test: An aliquot of 5 mL of nitrite broth (5 g of
peptone, 3 g of beef extract, 1 g of KNO3 and 1000 mL
of distilled water) was inoculated with one drop of 24-
h-old broth test culture and was incubated for 48 h at
37 ◦ C. From the development of red colour within 30 s of
adding a few drops of the reagent A (5 g of ␣-napthol in
1000 mL of 30% acetic acid) and reagent B (5 g of sulphanilic
acid in 1000 mL of acetic acid) the positive result was
inferred. No colour change suggested the negative result
(Forbes et al., 2007).
2.3. Antibiotic susceptibility test
All bacterial strains including the standard MTCC strains of
each bacterium (Table 1) were subjected to antibiotic sensitiv-
ity tests by the Kirby-Bauer’s method/disc diffusion method,
using a 4-mm thick Mueller-Hinton (MH) agar medium (HiMe-
dia, Mumbai) (Bauer et al., 1966). An aliquot of 0.1 mL of
0.5 McFarland equivalents, approximately from an exponen-
tially growing culture was spread on agar for the development
of lawn of a strain of a bacterium at 37 ◦ C in a BOD incu-
bator (Remi CIM-12S). On the lawn agar of each plate,
eight high-potency antibiotic discs (HiMedia) of 18 prescribed
antibiotics were placed at equal distances from one another.
Plates were incubated for 18 h at 37 ◦ C and were examined
for the size of zones of inhibition around each disc, fol-
lowing the standard antibiotic susceptibility test chart of
Clinical Laboratory Standard Institute (CLSI) guidelines (CLSI,
2011).
2.4. Detection of MRSA, VRE and ESBL strains
2.4.1. Detection of MRSA by chromogenic agar media test
Pure clinical isolates of S. aureus were streaked onto MRSA
agar media (Hichrome-MeReSa agar media, HiMedia) and were
incubated for 24 h at 37 ◦ C. Colonies appearing blue after the
incubation period were detected as MRSA strains, and non-
MRSA strains appeared white, according to guidelines of the
media supplier.
69
2.4.2. Detection of VRE by the vancomycin screen agar
plate method
Screening for vancomycin resistance was carried out by the
screen agar method on both MH agar and brain heart infu-
sion (BHI) agar (HiMedia). The vancomycin screen agar plate
was prepared by an addition of 6 mg/L of vancomycin to BHI
agar and MH agar. The inoculum suspension was prepared by
transferring colonies of E. faecalis from an overnight-growth
culture on nutrient agar plates to sterile saline to produce a
suspension that matched the turbidity of the 0.5 McFarland
standard. An aliquot of 0.1 mL of the suspension was spread
on to vancomycin screen agar plates and incubated for 24 h at
37 ◦ C. Any visible bacterial growth indicated vancomycin resis-
tance. In addition, S. aureus MTCC 7443 and E. faecalis MTCC 439
were used as methicillin- and vancomycin-susceptible control
strains, respectively (Tenover et al., 2001).
2.4.3. Detection of ESBL producers
A double disc diffusion synergy test was used for the deter-
mination of ESBL producers in five GN bacteria. In this test,
synergy was determined between a disc of augmentin (a com-
bination of 20 ␮g of amoxicillin and 10 ␮g of clavulanic acid)
and two discs of third-generation cephalosporin antibiotics
(30 ␮g of ceftazidime and 30 ␮g of cefotaxime) all placed at a
distance of 30 mm apart on a lawn culture of a test bacterium,
on an MH agar plate. The test organism was considered to
produce ESBL if the zone size around both test antibiotic discs
increased towards the augmentin disc. This increase occurred
because the clavulanic acid present in the augmentin disc was
inactivated by the ESBL enzyme produced by the test organism
(Vercauteren et al., 1997).
2.5. Antibacterial activity test by the agar well
diffusion method
The antibacterial activities of the eight different solvent
extracts of the plant were tested using the agar well diffu-
sion method (Perez et al., 1990). One strain from each bacterial
species showing resistance to the maximum number of antibi-
otics was further used for monitoring antibacterial activities
of plant extracts. A bacterial lawn was prepared as described
above, with the agar being 6 mm thick. Wells (6 mm deep)
were punched in 30-min old agar lawn and each well was
lined by 50 ␮L of molten MH agar. Wells were then filled with
100 ␮L aliquots of 30 mg/mL of solvent extracts of L. camara
(diluted from the original stock of plant extracts of individ-
ual organic solvents and aqueous extract with DMSO 10%
to 30 mg of plant extract/mL). Plates were incubated at 37 ◦ C
for 18–24 h. Antibacterial activities were evaluated by mea-
suring the diameter of inhibition zones. Each solvent extract
was tested three times and data from the third repeated
experiment are presented. Aliquots of 100 ␮L of linezolid
(30 ␮g/mL) for GP bacteria and 100 ␮L of imipenem (10 ␮g/mL)
for GN bacteria were used as reference controls for all solvent
extracts. MH agar was used for MRSA, while blood agar was
used for S. pyogenes and VRE. Extracts causing a zone of inhibi-
tion of 20 mm or more were considered highly active and those
having a zone of inhibition of <20 mm were considered mod-
erately active. Linezolid or imipenem with an average zone of
70 j o u r n a l o f h e r b a l m e d i c i n e 3 ( 2 0 1 3 ) 65–75
inhibition of 20 mm and no zone of inhibition by 10% solution
of DMSO were taken as reference controls.
2.6. Determinations of minimum inhibitory and
minimum bactericidal concentrations
The minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) of the active plant extracts were
determined. Original stock solutions of plant extracts were
prepared with both dichloromethane and methanol at 100 mg
of plant extract/mL in 10% DMSO solution with distilled water.
Each stock solution was diluted to obtain final concentrations
of 0, 1.562, 3.125, 6.25, 12.5, 25, 50 and 100 mg/mL with 10%
DMSO solution. A separate experiment was conducted for
each solvent extract. An aliquot of 80 ␮L of each dilution of
a solvent extract was delivered into a well on a 96-well (12 × 8)
micro-titre plate, along with an aliquot of 100 ␮L of MH broth
(HiMedia), an aliquot of 20 ␮L of bacterial inocula (109 CFU/mL)
and a 5 ␮L-aliquot of 0.5% 2,3,5-triphenyl tetrazolium chloride
(TTC). After pouring all the above into a well, the microplate
was incubated inside the BOD incubator at 37 ◦ C for 18 h. The
development of pink colouration in a well indicated bacterial
growth due to TTC and the absence of the colour was taken as
inhibition of bacterial growth. The 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. Bacteria
from each well of the microplate were sub-cultured on a nutri-
ent agar plate; the level of dilution, where no bacterial growth
on the nutrient agar plate was observed, was noted as the MBC
value.
2.7. Preliminary phytochemical analyses
1. Test for reducing sugars. The presence of free reducing sug-
ars was ascertained by Fehling’s test (Anonymous, 2004).
2. Test for anthraquinones. The extract (0.5 g) was shaken
with an aliquot of 10 mL of benzene. The mixture was fil-
tered and an aliquot 5 mL of 10% ammonia solution was
added to the filtrate and the final mixture was shaken; the
appearance of pink, red or violet colour in the ammoniac
(lower) phase indicated the presence of anthraquinones
(Brain and Turner, 1975).
3. Test for saponins. The extract (0.5 g) was dissolved in an
aliquot of 10 mL of distilled water in a test tube, which was
shaken vigorously for 30 s and was subsequently allowed to
stand for 45 min. The appearance of frothing on warming
the mixture indicated the presence of saponins (Harborne,
1998).
4. Test for flavonoids. A few drops of 10% ferric chloride solu-
tion were added to a portion of the dissolved extract. A
green or blue colour indicated the presence of flavonoids
(Sofowara, 1993).
5. Test for steroids/terpenes. Concentrated mass extract
(500 mg) from the rotary evaporator was dissolved in an
aliquot of 2 mL of acetic anhydride and the mixture was
cooled to 0–4 ◦ C, when a few drops of 12N sulphuric acid
were carefully added. A colour change from violet to
blue-green indicated the presence of a steroidal nucleus
(Sofowara, 1993).
6. Test for tannins. The extract (0.5 g) was dissolved in 5 mL
water followed by addition of a few drops of 10% ferric chlo-
ride. A blue-black, green or blue-green precipitate indicated
the presence of tannins (Harborne, 1998).
7. Test for alkaloids. Plant extract (0.5 g) was stirred with an
aliquot of 5 mL of 1% HCl in a steam bath and the mix-
ture was filtrated. A few drops of Mayer’s reagent (1.36 g of
HgCl2 and 5 g of KI in 100 mL distilled water) were added to
an aliquot of 1 mL of the filtrate, and a few drops of Dra-
gendorff’s reagent (two solutions in 1:1 ratio: ‘solution A’
with 0.85 g of bismuth nitrate, 10 mL of glacial acetic acid
and 40 mL of distilled water and ‘solution B’ with 8 g of KI in
30 mL of distilled water) were added to another aliquot of
1 mL of the filtrate. Turbidity or precipitation in tubes due to
either of these reagents indicated the presence of alkaloids
in the extract (Harborne, 1998).
8. Test for resins. An aliquot of 10 mL of 1% copper acetate
solution was added to 10 mL of diluted extract and the
mixture was shaken vigorously, a separate green colour
indicated the presence of resin (Sofowara, 1993).
9. Test for glycosides. An aliquot of 5 mL of each extract was
mixed with 2 mL of glacial acetic acid (1.048 g/mL), one drop
of 1% FeCl3 solution, and mixed thoroughly. Then 1 mL of
12 N H2 SO4 was added. A brown ring at the interface indi-
cated the presence of glycosides (Sofowara, 1993).
2.8. Thin layer chromatography
Both extracts (with dichloromethane and methanol) were sub-
jected to thin layer chromatography (TLC) for the separation of
polar and non-polar compounds (Venkatachalam et al., 2011).
Two solvent systems were used (n-hexane:dichloromethane,
1:1, v/v and chloroform:methanol:water, 7.5:1.5:0.5, v/v) for
separation. The plates were sprayed with the Godin reagent
(1% vanillin + 3% perchloric acid + 10% sulphuric acid, w/v)
solution, which produced a violet colour.
3. Results
The colony characteristics and biochemical tests that enabled
identification of the bacteria are detailed in Table 1. MRSA
were differentiated from S. aureus as those that produced blue-
pigmented, round and opaque colonies on chromogenic agar.
Further confirmation of colonies as MRSA was carried out by
observing growth in the presence of oxacillin 1 ␮g/disc on an
MH agar plate.
Antibiotic susceptibility tests for three GP and five GN
bacteria were carried out; 18 antibiotics from six different
groups were used against GP bacteria and 16 antibiotics
from five different groups were used against GN bacteria.
The results of these tests are recorded in the antibiogram
presented in Table 2. All isolated GN bacteria were ESBL pro-
ducers.
The antibacterial activity of eight solvent extracts was
monitored by the agar well diffusion method (Perez et al.,
1990), on separate lawn-cultures of eight bacterial isolates
(three GPs and five GNs). Dichloromethane leaf extracts regis-
tered the maximum sizes of zones of inhibition against MRSA
(29 mm) and P. mirabilis (29 mm). Similarly, the methanolic leaf
Table 2 – Antibiogram of the selected clinically isolated pathogenic organisms.
Bacterium Susceptibility to prescribed antibiotics

Aminogycosides ␤-lactams Cephalosporins Fluoroquinolones Glyco-peptide Sulfonamide Stand-alones

Ac Ge Am Ak Ox Pt Ce Cf Ci Gf Na No Of Va Cot Ch Nf Te
MRSA R R R R R R R R S R R R R R R S R I
S. pyogenes R R R R R R R R S R R R R R S S R R
VRE R R R R R R R R S R R R R R S S R R
A .baumannii R S R R Nd R R R R R R R R Nd R S R R
C. freundii S S R R Nd S R R S R S S S Nd R S I S
P. mirabilis R R R R Nd R R R I R R R R Nd R S R R

j o u r n a l o f h e r b a l m e d i c i n e 3 ( 2 0 1 3 ) 65–75
P. vulgaris R R R R Nd R R R I R R R R Nd R I I R
P. aeruginosa R I R R Nd R R R S R R R R Nd R S R R

MRSA: methicillin resistant S. aureus; VRE: vancomycin resistant Enterococcus; R: resistant; S: sensitive; I: moderately sensitive; Antibiotics (␮g/disc): Ac: amikacin 30; Ak: amoxyclav 30; Am: ampicillin
10; Ce: ceftriaxone 30; Cf: cefpodoxime 10; Ch: chloramphenicol 30; Ci: ciprofloxacin 5; Co-t: co-trimoxazole 25; Ge: gentamicin 10; Gf: gatifloxacin 5; Na: nalidixic acid 30; Nf: nitrofurantoin 300; No:
norfloxacin 10; Of: ofloxacin 5; Ox: oxacillin 30; Pt: piperacillin/tazobactam 100/10; Te: tetracycline 30; Va: vancomycin 30; Nd: not done.

Table 3 – Antimicrobial assay by agar-well diffusion method of eight hot solvent leaf-extracts of L. camara against MDR bacterial strains (zone of inhibition in mm).
Strain Petroleum ether Chloroform Ethyle acetate Dichloromethane Acetone Methanol Ethanol Water Linezolid/imipenem
(30/10 ␮g/ml)
MRSA 10 21 13 29 13 27 12 15 29
S. pyogenes 15 22 23 19 18 26 22 22 29
VRE 17 15 14 25 16 29 10 13 33
A .baumannii 08 12 12 19 13 19 14 13.5 31
C. freundii 11 18 14 23 19 21 16 13 26
P. mirabilis 10 19 14 29 14 23 14 14 29
P. vulgaris 18 18 15 19 15 22 19 17 26
P. aeruginosa 09 23 12 25 19 22 11 12 29

71
72 j o u r n a l o f h e r b a l m e d i c i n e 3 ( 2 0 1 3 ) 65–75

isolated MDR bacteria in this study. Therefore, this work has

Table 4 – MIC and MBC of two bioactive leaf-extracts of L.
camara against MDR bacterial strains (mg/ml). overriding importance over other similar research into non-
resistant bacteria of diverse classes and medicinal plants,
Strain Dichloromethane Methanol
which is widely available in the literature (Dubey et al., 2012b).
MIC MBC MIC MBC Long-term hospitalization leads to increased susceptibility
MRSA 6.25 25 3.125 25 of patients to both GP and GN bacteria, particularly, MRSA,
S. pyogenes 3.125 12.5 6.25 25 MDR P. aeruginosa and MDR A. baumannii. It has also been
VRE 6.25 25 6.25 25 reported that 51.5% of MRSA-infected patients had already
A .baumannii 3.125 25 1.562 12.5 been infected at the time of admission to hospital, which indi-
C. freundii 6.25 50 3.125 25 cates introduction of some new MRSA strains to hospital from
P. mirabilis 3.125 25 6.25 50
the community (Slonczewski and Foster, 2009). Moreover, in
P. vulgaris 3.125 25 3.125 25
England and Wales, less than 2% of S. aureus strains were
P. aeruginosa 12.5 50 6.25 50
methicillin resistant in 1990, but in 2002, about 42% of S. aureus
MIC: minimum inhibitory concentration; MBC: minimum bacteri-
strains were methicillin resistant. As of 2006, an estimated
cidal concentration.
300,000 MRSA infections have led to 5000 deaths (Carnicer-
Pont et al., 2006). Vancomycin has always been the choice of
extract registered the maximum size of zone of inhibition drug against MRSA infections, however, our results recorded
against VRE (29 mm). The petroleum-ether leaf extract and resistance to it. Not surprisingly, vancomycin-resistant S.
aqueous leaf extract registered very low antibacterial activity aureus (VRSA) has been an additional clinical concern, as
compared with the other six solvent extracts. The antibac- reported from IMS and Sum Hospital (Dubey et al., in press).
terial activities of all other solvent extracts are presented in Enterococci are primarily opportunistic pathogens. Exces-
Table 3. sive use of broad-spectrum antibiotics in hospitals and
The MIC and MBC values of dichloromethane and community centres could be the cause of rapid emergence of
methanolic leaf extracts were determined as they registered drug-resistant enterococci in social conditions, as is the case
the maximum antibacterial activities. The MIC and MBC val- in India. The first report of VRE appeared in 1988 and later
ues of these leaf extracts against the eight MDR bacterial those had spread into nosocomial settings (Uttley et al., 1988).
strains are presented in Table 4. In the U.S., S. pyogenes (Group A Streptococcus) had records of
From the qualitative phytochemical analysis of the nosocomial transmission, from patients to healthcare workers
dichloromethane leaf extract of L. camara, the presence of in causing fulminant invasive diseases, sore throat with hip
alkaloids, glycosides, terpenoids, saponins, flavonoids, and and joint pains (Lacy and Horn, 2009). Levofloxacin-resistant
steroids were confirmed, and reducing sugars were absent. S. pyogenes strains with an MIC of 16 ␮g/mL were prevalent in
Whereas, in the methanolic leaf extract, alkaloids, terpenoids, the U.S. (Richter et al., 2003). Most GP bacteria such as, Entero-
saponins, flavonoids and steroids were present, while glyco- coccus sp. (including VRE), S. aureus (including MRSA) and S.
sides, reducing sugars and tannins were absent (Table 5). pyogenes survive for months on dry surfaces. Many GN species
TLC of dichloromethane and methanolic extracts was such as, A. baumannii, E. coli, Klebsiella sp., P. aeruginosa and
performed with silica gel 60 F254 as the stationary phase. Shigella sp. also survived equally for months on dry surfaces.
For the dichloromethane extract, the mobile phase was Bacteria such as, Bordetella pertussis, Haemophilus influenzae, P.
the mixture n-hexane:dichloromethane (1:1); and for the mirabilis and Vibrio cholerae persisted for only a few days on
methanolic extract the mobile phase was the mixture, chloro- dry surfaces. This clearly suggested the potency of nosoco-
form:methanol:water (7.5:1.5:0.5). TLC plates were examined mial spreads of the above mentioned bacteria (Kramer et al.,
in an ultraviolet (UV) chamber at 254 nm; the Godin reagent 2006). The multidrug resistance of S. pyogenes was linked to
was used for detection of invisible components of chro- major virulence factor the M-protein, which is coded by the
matogram. UV active compounds, component-1 (retardation emm gene (Wahl et al., 2007).
factor or Rf value = 0.123 (colouration violet, the compound In a study from Chennai, India, it was reported that, among
would be glycoside), and component-2 (Rf = 0.343 (colouration common weeds, Antigonon leptopus, Croton sparsiflorus and L.
blue, the compound would be phenolic acid) were observed in camara had remarkable antibacterial activities against B. sub-
the dichloromethane extract. Three spots having Rf values of tilis, K. pneumoniae, S. aureus and E. coli (a MTCC drug-sensitive
0.569 (colouration yellow the compound would be flavonoid), strain); the methanolic extracts of L. camara particularly, had
0.868 (colouration light blue, the compound would be pheno- better antibacterial activities in comparison to two other
lic acid), and 0.96 (colouration yellow, the compound would be solvent extracts (acetone and chloroform), as well as other
flavonoid) in the methanolic extract at 254 nm under UV light plants used (Udayprakash et al., 2011). Lantadine, a triter-
were observed. Some of the components were visible after penoid was the first isolated toxic compound from L. camara.
spraying the Godin reagent under UV light (Sofowara, 1993; The Indian ecotype of L. camara had lantanoic and lantic
Harborne, 1998). acid, which were also toxic (Ghisalberti, 2000). Later, euphane
triterpene lactones were also isolated from the methanolic
extracts, which were potential inhibitors of human throm-
4. Discussion bin which helps in blood clotting. The review gave a vivid
description of the chemicals in L. camara (Ghisalberti, 2000);
Leaf extracts using methanol or dichloromethane had the a number of iridoid glycosides, furanonaphtoquinones and
best control activity over three GP and five GN clinically flavonoids (3-methoxyquercetin, 3,7 dimethoxyquercetin, and
 j o u r n a l o f h e r b a l m e d i c i n e 3 ( 2 0 1 3 ) 65–75
Table 5 – Preliminary phytochemicals analysis of L. camara.
Solvent Alkaloids Glycosides Terpenoids Reducing sugars
Dichloromethane + + + −
Methanol + − + −
+: present; −: absent.
3,7,49-trimethoxyquercetin), a flavone glycoside and cama-
raside and three new pentacyclic triterpenoids, camarin,
lantacin and camarinin were isolated from the methano-
lic extracts of L. camara. Its phytocompounds were active
against both S. aureus and Salmonella typhi (Barre et al., 1997).
Multi-solvent extracts of Oroxylum indicum had antibacterial
activities on GN bacteria. Hexane, chloroform and carbon
tetrachloride extracts showed significant activity on Bacillus
megaterium, Salmonella paratyphi, Vibrio mimicus, Vibrio para-
haemolyticus, P. aeruginosa, B. cereus, B. subtilis and E. coli (Dubey
et al., 2012a). Both aqueous and ethanolic extracts of the
plants Aegle marmelos, Azadirachta indica and Withania som-
nifera were highly effective against MDR isolates of several
MDR uropathogens: A. baumannii, C. freundii, Klebsiella oxytoca,
P. mirabilis, P. vulgaris and P. aeruginosa. Plants A. indica, Ter-
minalia arjuna and Terminalia alata contained the full range
of phytochemicals (alkaloids, glycosides, terpenoids, reduc-
ing sugars, saponins, tannins, flavonoids and steroids), which
could be attributed to the significant anti-uropathogenic activ-
ities (Rath et al., 2012).
L. camara is a weed. Any plant with a characteristic set of
unpalatable/aromatic phytochemicals would cause aversion
to grazing animals and eventually be established as a weed.
This plant has succeeded as a weed due to its aroma and
perennial nature. Broadly speaking a successful weed would
have the coveted phytochemicals suitable for the control of
pathogens; thus, such plants need a microbiological evalua-
tion, possibly with MDR bacteria, which has been carried out
here. The many chemicals found present in it make it more
successful than any pure compound against MDR bacteria.
The multiple antibiotic resistances in pathogenic bacteria
has become a serious matter of concern, as reasons such as
natural evolutionary modes of obtaining both intrinsic and
extrinsic resistance patterns (Perez et al., 1990) are blamed
for rapid and prolific occurrences of MDR bacteria. Pathogenic
bacteria evolve new strains, gaining resistance to recently
used antibiotics and drugs, an event which recurs indepen-
dently and in the last few decades there has been an increase
in the occurrence of MDR pathogens worldwide. Concomitant
to the search for a new generation of drugs for increased MDR
pathogenic bacteria, continual efforts to search for control
agents from plants and related sources has been undertaken
(Dubey et al., 2012b).
This study is unique compared to most other research in
the field of monitoring antimicrobial activities of medicinal
plants, as drug-sensitive bacteria obtained from type cul-
ture collection centres as well as MDR strains of pathogens
obtained directly from patients were used. It is purely aca-
demic research, as it lacks toxicity studies with animals;
nonetheless, MDR human pathogens were directly used for
monitoring antimicrobial activities of the plant, with ethnobo-
tanical information against infectious elements universally
73
Saponins Tannins Flavonoids Steroids
+ + + +
+ − + +
recorded. It has however been reported that a methanolic
extract of L. camara was non-toxic to mice (Kirimuhuzya et al.,
2009). Thus, this plant could be further pursued for use as a
complementary medicine against MDR pathogens.
5. Conclusion
Effective in vitro control of MDR strains of GP and GN bacteria,
MRSA, S. pyogenes, VRE, A. baumannii, C. freundii, P. mirabilis, P.
vulgaris and P. aeruginosa (the latter five being the most likely
UTI pathogens), by extracts of the plant L. camara frequently
used traditionally by several cultures was recorded. The large
selection of chemotherapeutic drugs and dovetailed antibi-
otics against MDR pathogens could plausibly help to overcome
the present resistant infection problem, if the principle of
synergism with suitable phytochemicals as complementary
medicine in a revised therapeutic module is adopted in the
crusade against MDR pathogens.
Conflicts of interest
We declare that we have no conflict of interest.
Acknowledgements
This work (a part of the PhD thesis of D. Dubey, an INSPIRE
Fellow of DST, Govt. of India, New Delhi) was supported by
the INSPIRE Fellowship grant (Code No. IF10503), awarded in
BJB Autonomous College, Bhubaneswar. We are grateful to Dr.
D.K. Roy, Dean, IMS and Sum Hospital for extended facilities.
We are grateful to Dr. S.C. Patnaik, Principal, BJB Autonomous
College, for encouragement.
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