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Bhoj R Singh et al., Asian Journal of Pharmaceutical Technology & Innovation, 03 (13); 2015; 01 - 05
www.asianpharmtech.com
1
Asian Journal of Pharmaceutical Technology & Innovation
ISSN: 2347-8810
Research Article
Received on: 11-05-2015
Accepted on: 17-05-2015
Published on: 15-08-2015
Corresponding Author
Antimicrobial Activity of Rose Geranium
(Pelargonium roseum) Essential Oil on
Bacteria of Veterinary Clinical Origin
*Dr. Bhoj R Singh
Act. Head of Division of Epidemiology,
Indian Veterinary Research Institute,
Izatnagar-243122, India, Ph. No. +91-
8449033222
Bhoj R Singh*1, Ravi Kant Agrawal2, Sakshi Dubey1, Monika
Bhardwaj1, Prasanna Vadhana1
ABSTRACT
In the study on antimicrobial activity of rose geranium oil (GEO), 167
bacterial strains belonging to 22 genera and 49 species were tested. In
total only seven strains were sensitive to GEO. Out of 15 reference
strains included in the study only one (Streptococcus equi ssp. equi
MTCC-3522) and six of the 120 isolates from clinically sick animals
were found sensitive. None of the 26 isolates from environment (soil,
water and air) and six from healthy animals was sensitive to GEO. All
resistant strains (160) had minimum inhibitory concentration (MIC)
more than 2µL of GEO / mL of medium. Sensitive strains’ GEO MIC
ranged between 0.2 µL/ mL to 2µL/ mL. The minimum MIC was for
Pasteurella langaensis (0.2 µL/ mL) strain followed by strain of
Streptococcus equi ssp. equi MTCC-3522 (0.4µL/ mL), Strept. intestnalis
and Strept. pyogenes (0. 4µL/ mL), Strept. equi ssp. equi (0.8µL/ mL),
Bacillus polymyxa (1.5 µL/ mL) and Pseudomonas aeruginosa (2.0µL/
mL). The study emphasizes need for evolution of some short of central
universally accepted guidelines to perform and report antimicrobial
activity of herbal antimicrobials so that the comparable data can be
analyzed for future meta-analytical and clinical purposes.
Email: brs1762@ivri.res.in;
brs1762@gmail.com
Key-words: Pasteurella, Brucella, Klebsiella, Escherichia,
Streptococcus, Staphylococcus, Geranium oil
Cite this article as:
Bhoj R Singh, Ravi Kant Agrawal, Sakshi Dubey, Monika Bhardwaj, PrasannaVadhana, Antimicrobial Activity of Rose
Geranium (Pelargonium roseum) Essential Oil on Bacteria of Veterinary Clinical Origin, Asian Journal of Pharmaceutical
Technology & Innovation, 03 (13); 2015. www.asianpharmtech.com
1Division of Epidemiology, Indian Veterinary Research Institute, Izatnagar-243122, India.
2Division of Livestock Products Technology, Indian Veterinary Research Institute, Izatnagar-243122, India.
Bhoj R Singh et al., Asian Journal of Pharmaceutical Technology & Innovation, 03 (13); 2015; 01 - 05
www.asianpharmtech.com
2
Introduction:
Geranium oil is extracted from fragrant plants of Pelargonium species especially P. graveolens, however,
much appreciated rose fragrance of rose geranium may also be obtained from P. roseum. Main constituents of
geranium essential oil1 include citronellol (~26.7%) and geraniol (~13.1%), nerol (~8.7%), citronellyl formate
(~7.1%), isomenthone (~6.3%) and linalool (~5.2%). Composition does not vary significantly among geranium
essential oil (GEO) of different origin2.
Due to its antidepressant, anti-inflammatory, antiseptic, astringent, cicatrisant, cytophylactic, diuretic,
deodorant, haemostatic, insect repellent, styptic, tonic, vermifuge and vulnerary properties GEO has widely
been used for therapeutic purposes3. The GEO is oil of choice in aromatherapy to treat acne, sore throat,
anxiety, depression, and insomnia. Due to its anti-inflammatory property GEO is used to reduce pain and
inflammation. For its antiseptic properties it has been used on wounds, burns, frostbites, fungal infections,
athlete’s foot, eczema and hemorrhoides. It not only protects as a natural insect repellent but its topical
application helps to heal insect bites and stop itching4.
Antibacterial, antifungal, and antioxidant properties of GEO have been explored all over the globe. It has
been shown to inhibit many food-borne microbes and reference strains of Aspergillusniger, Candida albicans,
Basillus subtilis, Brevibacterium linens, Enterobacter aerogenes, Enterococcus faecalis, Escherichia coli, Klebsiella
pneumoniae, Listeria monocytogenes, Mycobacterium smegmatis, Proteus mirabilis, Proteus vulgaris,
Pseudomonas aeruginosa, Salmonella Enteritidis, Salmonella Typhi, Salmonella Typhimurium, Staphylococcus
aureus, Staph. epidermidis, Streptococcus mutans and Yersinia enterocolitica5-11. However, contrasting reports of
its non activity on gram positive bacteria12 are also cited in literature. The wide range of variation in its
affectivity as antimicrobial leads to confusion due to use of only limited number of bacterial strains to test and
also variation in amount of GEO used (1.5 µL to 15µL) in disc diffusion assay1, 8, 9, 11. Therefore, this study was
planned to do an elaborate testing using 15 reference, 26 environmental, 120 clinical and six commensal (from
healthy animals) bacteria isolates and testing them all at the same level of GEO concentration (2 µL) of discs.
Material and Methods:
Bacterial isolates tested: Bacterial strains either reference (15) or isolated from different sources (from
apparently healthy animals 6, sick animals 120, air 10, water 7, soil 9) and available in different laboratories at
Indian Veterinary Research Institute, Izatnagar (Table. 1) were revived and checked for purity and identity
using standard bacteriological techniques13, 14. Pure cultures were stored on nutrient blood agar (BBL, Difco)
slants for the period of testing for sensitivity assay for GEO.
Sensitivity assay for GEO: All the bacterial strains were tested in duplicate using disc diffusion method15, 16 on
Mueller Hinton (MH) agar (BBL, Difco). However, for Streptococcus, Brucella and Pasteurella strains brain heart
infusion (BHI) agar (BBL, Difco) was used for sensitivity assays. The GEO discs were prepared to contain 2µL of
GEO in each disc as described earlier16. The rose geranium oil (GEO) used in the study was purchased from
Shubh Flavours and Fragrances Pvt. Ltd. New Delhi.
Minimum inhibitory concentration (MIC) determination: It was determined using agar well diffusion assay16
as it is done for antibiotics. Geranium oil (GEO) dilutions (8) were made in sterile dimethyl sulphoxide (DMSO)
to contain 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0 µL of GEO in 50 µL of DMSO. The eight dilutions of GEO in DMSO
were aseptically transferred to 8 peripheral wells of pre-inoculated (with 1:1000 diluted overnight growth of
test strain) while central well was filled with 50 µL of sterile DMSO. Plates were incubated under desired
atmosphere (5% CO2 enriched for Brucella and aerobic for other bacteria) for 24 h. Plates were read for growth
inhibition zone and results were interpreted16.
To find out the statistical significance in association of sensitivity of bacteria and source of bacteria χ2
test was performed using MS Office Excel-2007.
Results
Of the 167 bacterial strains tested with disc diffusion assay for GEO sensitivity inhibition zone of 6 mm
to 20 mm was evident for only seven strains. Only one of the 15 reference strains of 11 species belonging to
nine genera tested, a strain of Streptococcus equi ssp. equi (MTCC-3522) was sensitive to GEO (Table. 1). Out of
120 isolates of 32 species belonging to 16 genera of potentially pathogenic bacteria from clinically sick animals
only six isolates were sensitive to GEO. None of 26 isolates from environment (soil 9, water 7, and air 10)
belonging to 8 genera and 9 species was sensitive to GEO in the study. Similarly of the six isolates from
apparently healthy animals (belonging to 6 species of four genera) none was sensitive to GEO. Of the 101 Gram
Bhoj R Singh et al., Asian Journal of Pharmaceutical Technology & Innovation, 03 (13); 2015; 01 - 05
www.asianpharmtech.com
3
negative bacterial isolates and 66 Gram positive isolates only two and five strains were sensitive to GEO.
Maximum zone of inhibition (20 mm) was recorded for Pasteurella langaensis and minimum (6-7 mm) for
Bacillus polymyxa and Pseudomonas aeruginosa while for all the four streptococci, sensitive to GEO, inhibition
zone was 7mm to 10 mm.
All GEO resistant strains (160) had GEO MIC more than 2µL/ mL while for sensitive strains it ranged
between 0.2 µL/ mL to 2µL/ mL (Table. 1). Minimum MIC was for P. langaensis followed by that for Strept. equi
ssp. equi MTCC-3522 (0.4µL/ mL), Strept. intestnalis and Strept. pyogenes (0. 4µL/ mL), Strept. equi ssp. equi
(0.8µL/ mL), B. polymyxa (1.5 µL/ mL) and Pseudomonas aeruginosa (2.0µL/ mL).
Table 1. Sensitivity of some reference and bacterial isolates for rose geranium (Pelargonium roseum) essential oil
(GEO)
Source of
Strains (Nos.)
No. of
strains
sensitive
to GEO
GEO MIC
for test
strains
Bacterial strains resistant to GEO(Nos.)
Bacterial strains
sensitive to GEO
4Apparently
healthy
animals (6)
0
>2µL/mL
One strains each of Staphylococcus capitis
ssp. urealyticus, Staph. caseolyticus, Staph.
chromogenes, Agrobacterium tumefaciens,
Klebsiella pneumoniae, Pseudomonas
aeruginosa
Nil
4Clinically
sick animals
(120)
6
0.2µL/mL
to 2µL/mL
for
sensitive
strains and
>2µL/mL
for
resistant
Aeromonas caviae (1), A. sobria (1), Bacillus
marcerans (1), B. polymyxa (1), Bacillus spp.
(5), Brucella abortus (3), Burkholderia spp.
(5), Dermatophilus congolensis (2),
Enterobacter agglomerans (2), Erwinia
ananas (1), Escherichia coli (37), E.
fergusonii(3), Hafnia alvei (1), Klebsiella
oxytoca (1), Pasteurella canis (1), Proteus
mirabilis (1), Proteus vulgaris (3),
Pseudomonas aeruginosa (2), Staphylococcus
aureus (3), Staph. capitis ssp. urealyticus (2),
Staph. haemolyticus (1), Staph. hyicus (1),
Staph. intermedius (2), Staph. sciuri (6),
Streptobacillus moniliformis(1),
Streptococcus equi ssp. zooepidemicus (16),
Strept. Intestinalis (4), Strept. Milleri (1),
Strept. pneumoniae(1), Strept. Pyogenes (5)
One strains each of
Streptococcus
intestinalis,
Streptococcus
pyogenes,
Streptococcus equi ssp.
equi, Pseudomonas
aeruginosa, Bacillus
polymyxa and
Pasteurella langaensis
1-4Reference
strains (15)
1
0.4 µL/mL
for
sensitive
and for
others
>2µL/mL
Bordetella bronchiseptica (MTCC3838),
Brucella abortus strain 19 (S-19, and BS-
19)1, Burkholderia cepacia (MTCC438), B.
gladioli (MTCC1888), B. pseudomallei
(MTCC7183 and MTCC7259), Erwinia
herbicola (MTCC7100 and MTCC7100/1),
Pasteurella multocida (P-52 and SORON)2,
Salmonella Gallinarum (E-79)3, Staph.
aureus (BM-100)4, Yersinia enterocolitica
(MTCC3100)
Streptococcus equi ssp.
equi (MTCC3522)
4Air (10)
0
>2µL/mL
E. coli (1), Pseudomonas aeruginosa (1),
Staph. epidermidis (8)
Nil
4Soil (9)
0
>2µL/mL
E. coli (3), Enterobacter agglomerans (2),
Proteus mirabilis (1),
Pseudomonasaeruginosa (3)
Nil
4Water (7)
0
>2µL/mL
Citrobacter freundii (1), E. coli (1), Erwinia
chrysanthemii (1), Klebsiella pneumoniae
(3), Proteus vulgaris (1)
Nil
Strains numbers prefixed with MTCC were procured from The Microbial Type Culture Collection and Gene Bank (MTCC),
Institute of Microbial Technology (IMTECH), Chandigarh, 1National Brucella Centre, 2Pasteurella Laboratory, 3National
Salmonella Centre (Vet.), 4Division of Epidemiology, Indian Veterinary Research Institute, Izatnagar, Bareilly.
Discussion
Bhoj R Singh et al., Asian Journal of Pharmaceutical Technology & Innovation, 03 (13); 2015; 01 - 05
www.asianpharmtech.com
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It was apparent from the results that all six isolates sensitive to GEO were isolated from sick animals
only and none from the healthy animals or their environment. It might be due to the fact that none of the type
of bacteria found sensitive to GEO, except P. aeruginosa, was included in the study was from healthy animals
and environment. The P. aeruginosa strain sensitive to GEO had MIC just near the cut off limit to decide
sensitivity. Therefore, the results cannot be compared on the basis of source except for strains of those bacteria
which were detected in healthy as well as sick animals and their environment. Statistical analysis indicated that
source of bacteria could not be associated (p, >0.1) with their sensitivity to GEO.
Both of the Streptococcus equi ssp. equi (one reference and other from strangled horse), only one of the
five Streptococcus intestinalis, one of 6 Streptococcus pyogenes and a Streptococcus pneumoniae isolates, were
sensitive to GEO. However, none of 16 Streptococcus equi ssp. zooepidemicus, one Streptococcus milleri, all from
clinically sick animals, irrespective of the animal source was sensitive to GEO. The observation indicated that it
cannot be predicted even for the streptococci that which one of the strain may be sensitive to GEO. Probability
of being sensitive to GEO can also not be defined for strains of other bacteria. Even for the most sensitive P.
langaensis, it cannot be generalized as it was the lonely isolate in the study and testing of more number of
strains is required. However, from the earlier studies (mostly on reference strains) one may get a false
impression that GEO is an effective antimicrobial acting against wide range of microbes5- 11. Observations in this
study corroborate with only few studies12 indicating the fact that GEO possess very limited antimicrobial utility
similar to so many other herbal antimicrobials17-19.
Our study seems to contrast earlier observations5-11 but the difference in observations might be due to
the fact that most of the earlier studies were done on reference sensitive strains using discs impregnated in to
GEO or disc containing more amount of oil ranging between 3.5 µL to 15 µL1, 8, 9, 11. Besides, in earlier studies
different concentrations of GEO were used in discs for different bacteria in contrast to uniform concentration
used in the present study (2 µL/ disc). Therefore, it is now the time to decide over fixing the amount of herbal
product(s) to be used for determining herbal drug sensitivity similar to the standards available for
antimicrobial drugs15. In lack of standards, confusing literature will keep on emerging giving false impression of
affectivity of herbal drugs on microbes. Besides, the lot of data generated in different labs using different
standards poses difficulty in meta-analysis of the information to draw a useful conclusion for future clinical use
of herbal antimicrobials.
In the present study using cut off limit of 2 µL/ disc or 2 µL/ mL for deciding sensitivity by disc diffusion
assay or MIC through agar well method, respectively was used throughout the study irrespective of microbes
tested. The uniformity in testing provides more lucidity in understanding the comparative sensitivity of
different bacteria to GEO. In earlier studies GEO MIC has been reported to be the lowest for Streptococcus
mutans in range of 0.06µL/ mL to 1.25µL/ mL9, 11, the figures are quite close to our observations on GEO
sensitive Streptococcus strains with MIC ranging from 0.4-0.8 µL/ mL. However, none of the 27 staphylococci
and majority of the streptococci tested in our study was sensitive to GEO and had MIC ≥2 µL/ mL which is
contrast to earlier observation on MIC (0.25–2.50 μL/ mL) for Staph. aureus1. It might be due to several reasons
including the variation in the genetic background of the strains and origin of GEO used in the study.
Conclusion
The study conclude that GEO is effective as antimicrobial only on few strains of some bacteria. For
evolution of consistent and clinically useful literature on antimicrobial activity of herbal antimicrobials some
central agency should be constituted to guide the researchers to use the standard concentrations of different
preparations based on the scientific data on, method of using (topical/ systemic/ perentral), toxicity in target
tissues, organs and animals/ host, biological availability, serum levels and diffusion in agar or any other testing
media etc. Similarly for determining MIC and minimum bactericidal concentration (MBC) of herbal
antimicrobials, often insoluble in broth media and aqueous environment even after use of tween and other
solvents, suitable methodology needs to be standardized.
Acknowledgements
Authors are thankful to Joint Director (R) and Director of Indian Veterinary Research Institute, Izatnagar for
providing required funds and laboratory facilities to conduct the research. Authors also extend their
indebtedness to all the laboratory in-charges who provided the needed reference strains in the study. We thank
to Mr. HC Joshi and Mr. Laikur Rahman for laboratory assistance.
Source(s) of support: Indian Veterinary Research Institute, Izatnagar-243122, India
Bhoj R Singh et al., Asian Journal of Pharmaceutical Technology & Innovation, 03 (13); 2015; 01 - 05
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5
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