Pharmaceutical Biology

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Antimicrobial Potential of Callistemon rigidus.

Sanjai Saxena & Charu Gomber

To cite this article: Sanjai Saxena & Charu Gomber (2006) Antimicrobial Potential of
Callistemon rigidus., Pharmaceutical Biology, 44:3, 194-201, DOI: 10.1080/13880200600685899
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Pharmaceutical Biology
2006, Vol. 44, No. 3, pp. 194–201

Antimicrobial Potential of Callistemon rigidus

Sanjai Saxena and Charu Gomber

Natural Product & Drug Discovery Research Group, Department of Biotechnology & Environmental Sciences,
Thapar Institute of Engineering & Technology, Patiala, Punjab, India

Abstract
In vitro investigation of the antimicrobial activity of a
crude methanol extract of leaves of Callistemon rigidus
R.Br. (Myrtaceae) revealed potential antibacterial activity
against a broad spectrum of multidrug-resistant human
pathogens. Agar well diffusion assays of the test extract
established significant concentration-dependent antibac-
terial activity against methicillin-resistant Staphylococcus
aureus, vancomycin-intermediate Staphylococcus aureus,
vancomycin-resistant Escherichia coli, extended spectrum
b-lactamase-producing E. coli, and multidrug-resistant
Pseudomonas spp.
in Cameroon, Australia, China, and Asia (Jirovetz
et al., 1997). Phytochemical analysis of the oil indicates
the presence of mono- and sesquiterpenes, which could
be responsible for their possible effects in folklore medi-
cine (Jirovetz et al., 1997).
However, there are no experimental studies carried
out on this plant to substantiate the folklore claims. This
study was therefore designed to evaluate broad-spectrum
antibacterial and anticandidal activity of the methanol
extract of the leaves of Callistemon rigidus.

Keywords: Agar well diffusion assay, antibacterial activity,
multidrug resistant, Myrtaceae.
Introduction
Plant-derived medicines have been a part of traditional
health care across the world for thousands of years
(Chariandy et al., 1999). With the emergence and spread
of antibiotic resistance in human pathogenic micro-
organisms, there is an increased need of screening natural
products for newer and effective chemotherapeutic
agents. Plants serve as a reservoir to numerous chemical
entities, which are a part of the defense mechanism in
response to microbial attack and adverse environmental
conditions. These biologically active compounds exhibit
antimicrobial properties (Cowan, 1999) and are largely
unexplored (Hamburger & Hostettmann, 1991).
Callistemon rigidus R.Br. (Myrtaceae) is a stiff upright
shrub indigenous to tropical countries and Australia.
Traditional medicinal systems advocate different med-
icinal properties of the plant. Leaves are used to cure
cough, bronchitis, and other respiratory tract infections
Materials and Methods
Plant collection
Fresh and healthy leaves (free from any visible contami-
nation) were collected in the month of October at the
Thapar Institute of Engineering and Technology cam-
pus by Ms. Charu Gomber and identified by Dr. Sanjai
Saxena with confirmation from Punjab Horticulture
Department. A specimen has been deposited at the
herbarium of the Department of Biotechnology &
Environmental Sciences and numbered as #7San03.
Samples were thoroughly washed under running water
and air-dried. The fresh weight was noted, and the
samples were subjected to drying at 37C and ground
into coarse powder.
Extraction
Cold percolation using methanol as the solvent was used
for extraction of the plant tissue. Pulverized plant
material (70 g) was soaked in 150 ml of methanol in clean
air-tight bottles. Extraction was done at 28C, 120 rpm
for 2 days. The solvent was filtered, and the extracts

Accepted: January 17, 2006

Address correspondence to: Dr. Sanjai Saxena, Natural Product & Drug Discovery Group, Department of Biotechnology &
Environmental Sciences, Thapar Institute of Engineering & Technology (Deemed University), Patiala, Punjab 147004, India.
Fax: 91-175-2393738; E-mail: sanjaibiotech@yahoo.com, ssaxena@tiet.ac.in

DOI: 10.1080/13880200600685899 # 2006 Taylor & Francis Group, LLC

 Antimicrobial potential of C. rigidus
obtained were pooled to obtain the 7.5 g of residue. The
residue was stored at 20C until use.
Test microorganisms used
The test microorganisms used included Staphylococcus
aureus (31 isolates), Escherichia coli (18 isolates), Pseudo-
monas spp. (17 isolates), and Candida albicans (6 isolates)
(Table 1). The bacteria were maintained on trypticase soy
agar slants and yeasts on Saboraud dextrose agar slants.
Activation of cultures was carried out by streaking on a
Mueller-Hinton (MH) agar plate followed by incubation
overnight at 37C. A single colony was picked from this
plate and transferred to MH broth and incubated at 37C
prior to the test. In case of fungi, yeast extract peptone
dextrose (YEPD) agar was used for activation. Suscep-
tibility testing of all bacterial and fungal isolates was
done (NCCLS, 2002; EARSS, 2005) based on cultures
grouped as methicillin-resistant Staphylococcus aureus,
vancomycin-resistant Staphylococcus aureus, vancomycin-
intermediate Staphylococcus aureus, vancomycin-resistant
E. coli, extended spectrum b-lactamase-producing E. coli,
and multidrug-resistant S. aureus and Pseudomonas spp.
(Table 2). All isolates were resistant to one or more antibio-
tics. S. aureus NCTC 6571 and E. coli NCTC 10418 were
included as reference strains.
In vitro antibacterial assays
Antibacterial activity of the test extract was determined
by the agar well diffusion method (Reddish, 1929; Lehrer
et al., 1991). The plant extract was dissolved in DMSO
and tested at six different concentrations (16.65, 33.3,
66.6, 99.9, 199.8, and 399.9 mg). The assay was done by
preparing 4 wells of 5 mm diameter per 90 mm MH agar
plate (mean depth  4.00 mm) aseptically. Thirty micro-
liters of the test extract at 6 different concentrations
was dispensed in the test wells. Solvent blank was
included as control. The plates were left for 15 min for
diffusion after which the wells were sealed with molten
MH agar. These were then swabbed with 18 h old,
0.5 McFarland adjusted culture of test isolates and
incubated overnight at 37C. Antibacterial activity was
determined by measuring the zone of inhibition around
the test wells. The growth inhibition diameter was an
average of three different measurements.
Determination of anticandidal activity
This was performed using the agar well diffusion assay
(as described for the determination of anticandidal
activity) on YEPD agar.
Statistical analysis
Descriptive statistics using GraphPad Prism ver. 4.03
software was carried out for the assessment of effect of
195
different parameters, that is, concentration of the extract
and type of culture=isolate on the inhibitory activity
(diameter of inhibition zone) was studied by two-way
analysis of variance, that is, two-way ANOVA. Pearson
correlation was calculated to assess the concentration-
dependent antibacterial activity of the test extract against
the bacterial isolates.
Results and Discussion
The extract was active against both Gram-positive and
Gram-negative bacteria. There was no inhibitory
activity in the control wells. Among Staphylococcus
aureus isolates, the extract exhibited strong activity
against VISA, MRSA, and MARSA isolates, the
majority of which gave an appreciable zone size
(20.0 mm) at 199.8 mg. The exceptions to these were
Sau (G) 8 and Sau (G) 21 that resisted the extract at
all concentrations. The extract did not exhibit any
activity against vancomycin-resistant strains [Sau (G)
3 and Sau (G) 22] (Fig. 1).
Among Gram-negative isolates, barring Eco (G) 2, the
extract exhibited modest activity against all vancomycin-
resistant E. coli (VRE). Similarly, excluding Eco (G) 1
and Eco (G) 3, the extract displayed potential activity
against extended spectrum b-lactamase-producing
strains (Fig. 2). Tests against Pseudomonas species depict
the killing effect of the extract against all isolates exclud-
ing Pae (G) 1, Pae (G) 3, and Pae (G) 4 (Figure 3). The
extract was not active against any of the test isolates of
Candida albicans.
Agar well diffusion is a popular prescreen employed
by clinical microbiologists, pharmacognosists, and phy-
tochemists working on antimicrobial drug development
from plants (Nakamura et al., 1999; Alves et al., 2000).
Antimicrobial activity of numerous plants has been eval-
uated using agar well diffusion assay (Hoffmann et al.,
2004; Guven et al., 2005; Okoli & Iroegbu, 2005). The
current work is the first scientific documentation of the
antimicrobial potential of Callistemon rigidus against
multidrug-resistant bacteria.
Statistical analysis using ANOVA revealed a signi-
ficant effect (p < 0.0001) of the test parameters, viz.,
isolate type and concentration of extract on the inhibition
zone (Rambali et al., 2001; Zelenitsky et al., 2003; Larabi
et al., 2004). Pearson correlation is a very important
parameter in understanding the dose-drug behavior as
well as drug efficacy in terms of post antibiotic effect
(Stubbings et al., 2004). There was a highly significant
correlation between the dose of the extract and diameter
of inhibition zone against all the three groups of test
isolates. The R2 value of drug concentration to inhibi-
tion zone diameter was 0.9745 in Pseudomonas species,
followed by 0.92727 in Staphylococcal isolates and
0.9544 in Escherichia species (Figs. 4–6, respectively).
196 S. Saxena and C. Gomber

Table 1. Culture collection.

Species Culture ID Source Repository

PUS cultures
Staphylococcus aureus Sau (A) 1 Pus discharge AIIMS
Staphylococcus aureus Sau (A) 2 Pus culture AIIMS
Staphylococcus aureus Sau (G) 1 Pus for C=S GMCP
Staphylococcus aureus Sau (G) 2 Pus for C=S GMCP
Staphylococcus aureus Sau (G) 3 Pus for C=S GMCP
Staphylococcus aureus Sau (G) 10 Pus for C=S GMCP
Escherichia coli Eco (A) 1 Pus Culture AIIMS
Escherichia coli Eco (G) 1 Pus culture (C=S) GMCP
Escherichia coli Eco (G) 2 Pus for C=S GMCP
Escherichia coli Eco (G) 3 Pus for C=S, ileostomy GMCP
Pseudomonas aeruginosa Pae (G) 1 Pus culture GMCP
Pseudomonas aeruginosa Pae (G) 2 Pus for C=S GMCP
Pseudomonas aeruginosa Pae (G) 3 Pus for C=S GMCP
Pseudomonas aeruginosa Pae (G) 4 Pus for C=S GMCP
Pseudomonas spp. P (G) 1 Pus for C=S GMCP
Urine cultures
Staphylococcus aureus Sau (A) 3 Urine culture AIIMS
Staphylococcus aureus Sau (G) 4 Urinary pathogen GMCP
Staphylococcus aureus Sau (G) 5 Urinary pathogen GMCP
Staphylococcus aureus Sau (G) 11 Urine for C=S GMCP
Escherichia coli Eco (A) 2 Urine culture AIIMS
Escherichia coli Eco (A) 3 Urine culture AIIMS
Escherichia coli Eco (G) 4 Urine C=S GMCP
Escherichia coli Eco (G) 6 Urine C=S, prostatectomy GMCP
Escherichia coli Eco (G) 7 Urine C=S GMCP
Pseudomonas aeruginosa Pae (G) 5 Urine C=S GMCP
Pseudomonas aeruginosa Pae (G) 6 Urine for C=S GMCP
Pseudomonas spp. P (A) 3 Urine culture AIIMS
Blood cultures
Pseudomonas spp. P (A) 1 Drain culture AIIMS
Pseudomonas aerogenes Par (A) 1 (Endocarditis) AIIMS
Pseudomonas aerogenes Par (A) 2 (Endocarditis) AIIMS
Pseudomonas spp. P (G) 2 Blood culture GMCP
Escherichia coli Eco (A) 4 Blood culture AIIMS
Staphylococcus aureus Sau (G) 6 Blood culture GMCP
Staphylococcus aureus Sau (G) 12 Blood culture GMCP
Vaginal swabs
Escherichia coli Eco (G) 9 Vaginal swab for C=S GMCP
Escherichia coli Eco (G) 10 Vaginal swab for C=S GMCP
Staphylococcus aureus Sau (G) 7 Vaginal swab for C=S GMCP
Staphylococcus aureus Sau (G) 13 Vaginal swab for C=S GMCP
Gastrointestinal pathogens
Escherichia coli Eco (NDRI) NDRI, Karnal
E. coli O157:H7 Eco (G) 14
E. coli O157:H7 Eco (G) 15
Tracheal cultures
Pseudomonas spp. P (A) 2 Tracheal culture AIIMS
Wound subcultures
Staphylococcus aureus Sau (A) 4 Wound subculture AIIMS
Staphylococcus aureus Sau (G) 14 Wound swab GMCP
Burn swabs
Staphylococcus aureus Sau (G) 15 Face abcess (Swab) GMCP
Staphylococcus aureus Sau (G) 9 Burn swab for C=S GMCP
Staphylococcus aureus Sau (G) 16 Pus for C=S GMCP

(Continued)
 Antimicrobial potential of C. rigidus 197

Table 1. Continued.

Species Culture ID Source Repository

Staphylococcus aureus Sau (G) 17 Pus for C=S GMCP

Staphylococcus aureus Sau (G) 18 Pus for C=S GMCP
Staphylococcus aureus Sau (G) 19 Pus for C=S GMCP
Pseudomonas aeruginosa Pae (G) 7 Pus for C=S GMCP
Ear discharge swabs
Staphylococcus aureus Sau (G) 8 Ear discharge swab GMCP
Pseudomonas aeruginosa Pae (G) 8 Ear discharge C=S GMCP
Pseudomonas aeruginosa Pae (G) 11 Ear discharge swab GMCP
Catheter tip isolates
Staphylococcus aureus Sau (G) 20 Tip for C=S GMCP
Staphylococcus aureus, coagulase negative Sau (G) 21 Tip for C=S GMCP
Eye isolates
Staphylococcus aureus Sau (G) 22 Swab for C=S GMCP
Other cultures
Staphylococcus aureus Sau (A) 5 AIIMS
Staphylococcus aureus Sau (MTB) Pathogenic isolate—protease Karolinska Institute, Sweden
Staphylococcus aureus Sau (FI) 1 Food isolate Abhijit Ganguli
Staphylococcus aureus Sau (FI) 2 Food isolate Abhijit Ganguli
Staphylococcus aureus Sau (LHMC) Antibiotic sensitivy testing (AST) LHMC
Escherichia coli Eco (LHMC) Antibiotic sensitivy testing LHMC
Escherichia coli Eco (G) 16 Urinary pathogen GMCP
Escherichia coli Eco (A) 5 Resistant to all drugs AIIMS
Pseudomonas aeruginosa Pae (G) 12 GMCP
Pseudomonas aeruginosa Ps (VP) Multidrug resistant GMCP
Pathogenic yeast
Candida albicans G1 Cal (JNU) 1 Drug-resistant pathogen Dr. R. Prasad, JNU
Candida albicans G2 Cal (JNU) 2 Drug-resistant pathogen Dr. R. Prasad, JNU
Candida albicans G3 Cal (JNU) 3 Drug-resistant pathogen Dr. R. Prasad, JNU
Candida albicans G4 Cal (JNU) 4 Drug-resistant Pathogen Dr. R. Prasad, JNU
Candida albicans Cal (G) 1 Oral candidiasis GMCP
Candida albicans Cal (G) 2 Vaginal swab GMCP

AIIMS, All India Institute of Medical Sciences, New Delhi, India; GMCP, Government Medical College, Patiala, India; NDRI,
National Dairy Research Institute, Karnal, India; LHMC, Lady Harding Medical College, New Delhi, India.

Table 2. Culture classification based on susceptibility testing.

Resistance No. of isolates Culture ID

Vancomycin-resistant S. aureus 2 Sau (G)22, Sau (G)3

Vancomycin-intermediate S. aureus
Methicillin-resistant S. aureus
Multi-antibiotic-resistant S. aureus
Vancomycin-resistant E. coli
Extended spectrum beta-lactamase-producing E. coli
Multi-antibiotic-resistant Pseudomonas
3 Sau (G)1, Sau (G)7, Sau (G)17
13 Sau MTB, Sau (G)2, Sau (G)3, Sau (G)4, Sau (G)9,
Sau (G) 10, Sau (G)11, Sau (G)12, Sau (G)16, Sau (G)18,
Sau (G)19, Sau (G)20, and Sau (FI) 1.
22 Sau (A) 3, Sau MTB, Sau (G)2, Sau (G)3, Sau (G)4 , Sau (G)5,
Sau (G)7, Sau (G)8, Sau (G)10, Sau (G)11, Sau (G)12,
Sau (G)13, Sau (G)14, Sau (G)15, Sau (G)16, Sau (G)18,
Sau (G)19, Sau (G)20, Sau (G)21, Sau (G)22, Sau (FI)1,
and Sau (FI)2
8 Eco (A) 3, Eco (A) 4, Eco (A) 5, Eco(G)2, Eco (G)3, Eco (G)4,
Eco (G)6, Eco (G)7
13 Eco (A) 1, Eco (A) 2, Eco (A) 3, Eco (A) 4, Eco (A) 5,
Eco (G)1, Eco(G)2, Eco (G)3, Eco (G)4, Eco (G)7,
Eco (G)15, Eco (G)16, and EcNd.
3 Pae (G)4, Pae (G)8, and Pae (G)12
198 S. Saxena and C. Gomber

Figure 1. Maximum inhibition zone against S. aureus isolates.

Figure 2. Maximum inhibition zone against E. coli isolates.

 Antimicrobial potential of C. rigidus 199

Figure 3. Maximum inhibition zone against Pseudomonas isolates.

Figure 4. Agar well diffusion assay of test extract against S. aureus.

200 S. Saxena and C. Gomber

Figure 5. Agar well diffusion assay of test extract against E. coli.

Figure 6. Agar well diffusion assay of test extract against Pseudomonas spp.
 Antimicrobial potential of C. rigidus
Conclusion
The current investigation is the first experimental docu-
mentation of the antimicrobial efficacy of leaf extract of
Callistemon rigidus. The in vitro antimicrobial activity of
the methanol extract of leaves of Callistemon rigidus
against all isolates (predominantly toward MRSA,
MARSA, ESBL, and VISA strains) signifies that bioactive
compounds may be present. Preliminary isolation with
column chromatography and further antibacterial testing
has confirmed the presence of single active compounds
in the methanol extract of leaves of Callistemon rigidus.
Currently, work is being conducted to further isolate and
identify these active constituents.
Acknowledgments
We are grateful to Dr. Rajendra Prasad, Department of
Life Sciences, Jawaharlal Nehru University, New Delhi,
Dr. Aarti Kapil, Department of Microbiology, All India
Institute of Medical Sciences, Prof. S. Arvidson, Micro-
biology & Tumor Biology Centre, Karolinska Institute,
Sweden, and Dr. Amarjit Kaur Gill, Department of
Microbiology, Government Medical College, Patiala
for providing us multidrug-resistant cultures of S. aureus,
E. coli, Pseudomonas, and C. albicans. We acknowledge
Dr. S. Natesh and Dr. M. Aslam for their continuous
inspiration for the current work, and Charu Gomber
acknowledges DBT, Government of India, for providing
a fellowship under the project number BT=PR=3982=
PBD=17=257=2003.
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