Antibacterial effects of oregano (Origanum vulgare) against gram negative bacilli

Abstract

The oil, aqueous infusion and decoction of oregano (Origanum vulgare), of the family Limiaceae, were asessed for antibacterial activity against 11 different genera of Gram–ve bacilli viz., Aeromonas hydrophila, Citrobacter sp., Enterobacter aerogenese, Escherichia coli, Flavobacterium sp., Klebsiella ozaenae, K. pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, S. paratyphi B, Serratia marcescens and Shigella dysenteriae, by disc diffusion method. Oregano oil exhibited the highest activity against Citrobacter species with mean zone of inhibition of 24.0 mm ± 0.5. The aqueous infusion also showed significant inhibitory activity against Klebsiella pneumoniae (20.1 mm ± 6.1 SD), Klebsiella ozaenae (19.5 mm ± 0.5 SD) and Enterobacter aerogenes (18.0 mm). Besides, all isolates were found resistant to the aqueous decoction of oregano seeds.

Full text

Pak. J. Bot., 39((2): 609-613, 2007.
ANTIBACTERIAL EFFECTS OF OREGANO (ORIGANUM
VULGARE) AGAINST GRAM NEGATIVE BACILLI
NAZIA MASOOD AHMED CHAUDHRY, SABAHAT SAEED AND PERWEEN TARIQ
Department of Microbiology,
University of Karachi, Karachi-75270, Pakistan
Abstract
The oil, aqueous infusion and decoction of oregano (Origanum vulgare), of the family
Limiaceae, were asessed for antibacterial activity against 11 different genera of Gram–ve bacilli
viz., Aeromonas hydrophila, Citrobacter sp., Enterobacter aerogenese, Escherichia coli,
Flavobacterium sp., Klebsiella ozaenae, K. pneumoniae, Proteus mirabilis, Pseudomonas
aeruginosa, Salmonella typhi, S. paratyphi B, Serratia marcescens and Shigella dysenteriae, by
disc diffusion method. Oregano oil exhibited the highest activity against Citrobacter species with
mean zone of inhibition of 24.0 mm ± 0.5. The aqueous infusion also showed significant inhibitory
activity against Klebsiella pneumoniae (20.1 mm ± 6.1 SD), Klebsiella ozaenae (19.5 mm ± 0.5
SD) and Enterobacter aerogenes (18.0 mm). Besides, all isolates were found resistant to the
aqueous decoction of oregano seeds.
Introduction
Microorganisms occur nearly everywhere in nature and affect the well being of
people in a great many ways. Many different microbial species normally inhabit various
parts of our bodies, such as the oral cavity, skin and intestinal tract. A variety of Gram
negative bacilli (GNB) are members of the intestinal microbiota of most animals and
humans as normal commensals or pathogens. They are mostly members of the family
Enterobacteriaceae but members of other taxonomical groups of Vibrionaceae are also
considered in this category. The GNB emerged rapidly, caused a large outbreak of
serious infections, and contributed to mortality (Herbert et al., 2007). The GNB vary in
the frequencies that they cause either intestinal and extra-intestinal infections. The
example of intestinal disorders are enteric fever, inflammatory and non-Inflammatory
enteritis. Among the extra-intestinal diseases, such as nosocomial urinary tract infections
(Yu et al., 2006), chronic obstructive pulmonary disease (Lin et al., 2007), neonatal
septicaemia (Iregbu et al., 2006), wound infections, meningitis, pneumonia, bloodstream
infection and bacteremia with septic shock are the most prevalent diseases (Donskey,
2006). During the past 20 years, changes in health care, infection-control practices, and
antimicrobial use and resistance may have influenced the frequency that these GNB are
associated with hospital-acquired infection (Gaynes & Edwards, 2005).
Previous research studies have documented increasing rates of antimicrobial
resistance of GNB (Gupta et al., 2006). Misuse of antibiotics and ineffective infection
control have been implicated in the development and spread of resistant GNB pathogens
which are associated with increased mortality and morbidity, prolonged hospitalization
and increased costs. It is evident from literature that new multi-drug-resistant GNB
strains have emerged and proliferated in the world. For example, the occurrence of
fluoroquinolone-resistant GNB colonizing community-dwelling people with spinal cord
dysfunction is common (Roghmann et al., 2006). It has also been reported that
ciprofloxacin-resistant GNB are becoming increasingly important and that they may
cause serious infections in children (Qin et al., 2006).

NAZIA MASOOD AHMED CHAUDHRY ET AL.,
610
Problematic organisms including non-fermentative Pseudomonas aeruginosa
(Wroblewska, 2006), multi-drug resistant Salmonella typhi (Akinyemi et al., 2000) and
other GNB producing extended-spectrum beta-lactamases (GNB-ESBL), pose a
particular difficulty for the healthcare community because they represent the problem of
multi-drug resistance to the maximum (Wroblewska et al., 2006). These organisms are
niche pathogens that primarily cause opportunistic healthcare-associated infections in
patients who are critically ill or immunocompromised. Multi-drug resistance is common
and increasing among GNB, and a number of strains have now been identified that
exhibit resistance to essentially all commonly used antibiotics, including
antipseudomonal penicillins, cephalosporins, aminoglycosides, tetracyclines,
fluoroquinolones, trimethoprim-sulfamethoxazole and carbapenems (McGawan, 2006).
The impact of antibiotic resistance on the outcome of infections due to GNB remains
highly controversial. Therapeutic options for multi-drug resistant GNB strains are
limited; for this reason, there are a continuous need for alternative new chemical entities
with such activities may be identified through a variety of approaches (Combes et al.,
2007).
Screening of natural medicinal plants is common because many infectious diseases
are known to have been treated with herbal remedies throughout the history of mankind.
Even today, plant materials continue to play a major role in primary health care as
therapeutic remedies in many developing countries (Naim & Tariq, 2006). In search for
alternative ways of infectious disease control; essential oil, aqueous infusion and aqueous
decoction from oregano were used in the present study to check their antibacterial
properties against GNB using standard disc diffusion method In vitro.
Materials and Method
Maintainance of isolates: A total of 100 bacterial isolates belonging to 11 different
genera of GNB viz., Aeromonas hydrophila (2), Citrobacter sp. (3), Enterobacter
aerogenes (2), Escherichia coli (22), Flavobacterium sp. (4), Klebsiella ozaenae (12),
Klebsiella pneumoniae (20), Proteus mirabilis (7), Pseudomonas aeruginosa (15),
Salmonella typhi (5), S. paratyphi B (2), Serratia marcescens (1) and Shigella
dysenteriae (5); were maintained on Nutrient Agar medium (Oxoid).
Preparation of aqueous infusion: Aqueous infusion of oregano seeds was prepared by
soaking 20g in 100ml sterile distilled water in sterile flask. The flask was kept for two
days with occasional shaking. The contents of flasks were filtered.
Preparation of aqueous decoction: Aqueous decoction of oregano seeds was prepared
by boiling 20g in 100ml sterile distilled water for 15minutes. The flask was then plugged
and removed from heat and allowed to cool. After cooling the contents of flask were
filtered.
Essential oil of oregano: Essential oil of oregano (Planter) was purchased from the local
market of Saddar, Karachi, Pakistan.
Screening of antibacterial activity: Screening of antibacterial activity was performed by
standard disc diffusion method (Saeed et al., 2006). Hundred sterilized discs of filter
paper (6 mm diameter) were soaked in 1 ml of infusion, decoction and oil, seperately, for
1-2 minutes and then used for screening. Thus potency of each disc was 10 μl. Mueller-

ANTIBACTERIAL EFFECTS OF OREGANO AGAINST GRAM-VE BACILLI 611
Hinton agar (MHA) (Merck) was used as base medium and Mueller-Hinton broth (MHB)
was used for the preparation of inoculum. Four to five isolated colonies of tested
organisms were picked by sterile inoculating loop and inoculated in tubes of MHB (5 ml
in each). The inoculated tubes were incubated at 35-37oC for 24 hours and matched with
0.5 McFarland nephelometer turbidity standard (Saeed & Tariq, 2005). A sterile cotton
swab was dipped into the standardized bacterial test suspension to inoculate entire surface
of a MHA plate. Discs of infusion, decoction and oil were placed on the surface of
inoculated plates with the help of sterile forcep. The inoculated plates were incubated at
35-37oC for 24 hours. After incubation inhibition zone diameters were measured to the
nearest millimeter (mm).
Statistical analysis: Mean zone of inhibition and standard deviations were calculated.
Results and Discussion
Oregano has been a valuable source of natural products for maintaining human
health for a long period of time, especially in last decade, with more intensive studies for
natural therapies (Force et al., 2000). The volatile oil of oregano has been used
traditionally for respiratory disorders, indigestion, dental caries, rheumatoid arthritis, and
urinary tract disorders. As a medicinal plant, oregano has been used as antifungal,
anticoccidial, antispazmolytic, antibacterial (Ertas et al., 2005), antioxidant (Lamaison et
al., 1991), antiaggregant (Okazaki et al., 2002) and antiinflammatory (Kelm et al., 2000)
agent. In addition, it has stimulating effect of digestion and antiseptic (Cabuk et al.,
2003). The most important components of oregano are the limonene, gamma-cariofilene,
rho-cymenene, canfor, linalol. Alpha-pinene, carvacol and thymol (Arcila-Lozano et al.,
2004). Among them thymol and carvacrol are the main components of the essential oil of
oregano (Tian & Lai, 2006), which are responsible for its antioxidative, antimicrobial and
antifungal effects (Proestos et al., 2005).
Antibacterial activity of aqueous infusion, aqueous decoction and oil of oregano
seeds was evaluated against 11 different GNB that are known to cause infections in
human. In the present study, the oregano oil revealed the highest antibacterial activity. It
was followed by aqueous infusion while aqueous decoction of oregano did not show
inhibitory potential against tested bacterial isolates (Table 1).
The oregano oil exhibited significant inhibitory activity against Citrobacter spp.,
(24.0 mm ± 0.5 SD), Salmonella typhi (22.4 mm ± 1.5 SD) and Escherichia coli (19.0
mm ± 2.2 SD) (Table 1). It has long been acknowledged that oregano oil is among the
most active against strains of E. coli and also presents antimicrobial activity against
pathogenic microorganisms like Salmonella. choleraesuis, S. typhi, S. typhimurium and
many others related GNB strains of Enterobacteriaceae family (Penalver et al., 2005)
and H. pylori (Stamatis et al., 2003). Similarly, the results of present study are in
accordance with the reports on oregano oil against GNB viz., Proteus vulgaris,
Aeromonas hydrophila, Klebsiella pneumoniae and Escherichia coli (Baydar et al.,
2004).
In the present study, the antibacterial effect of aqueous infusion of oregano was next
to oil and exhibited significant inhibitory activity against Klebsiella pneumoniae (20.1 ±
6.1SD), Klebsiella ozaenae (19.5 ± 0.5SD) and Enterobacter aerogenes (18.0 ± 00SD).
It is interesting to note that aqueous infusion of oregano inhibited all type of tested
bacterial strains. The present study also described the antibacterial activity of aqueous
decoction of oregano seeds. It was found that the aqueous decoction of oregano seeds did
not posses any antibacterial effect against tested GNB.

NAZIA MASOOD AHMED CHAUDHRY ET AL.,
612
Table 1. Antibacterial activities of oil, aqueous infusion and aqueous decoction of
oregano seeds against Gram negative bacteria
Mean zone of inhibition in mm ±
standard deviation
Organisms No. of
isolates Oil Infusion Decoction
Aeromonas hydrophila 2 12.8 ± 3.3 10.5 ± 1.5 ---
Citrobacter sp. 3 24.0 ± 0.5 10.0 ---
Enterobacter aerogenes 2 --- 18.0 ± 0.0 ---
Escherichia coli 22 19.0 ± 2.2 15.8 ± 1.5 ---
Flavobacterium sp. 4 11.6 ± 0.9 8.8 ± 1.5 ---
Klebsiella ozaenae 12 --- 19.5 ± 0.5 ---
Klebsiella pneumoniae 20 11.7 ± 1.6 20.1 ± 6.1 ---
Proteus mirabilis 7 --- 10.2 ± 1.3 ---
Pseudomonas aeruginosa 15 --- 10.8 ± 1.0 ---
Salmonella typhi 5 22.4 ± 1.5 16.1 ± 3.8 ---
Salmonella para typhi B 2 12.0 ± 1.0 13.0 ± 1.0 ---
Serratia marcescens 1 12.0 13.0 ---
Shigella dysenteriae 5 9.5 ± 0.5 11.0 ± 0.7 ---
The study demonstrated that oregano represents an economic source of natural
mixtures of antibacterial compounds that can be as effective as modern medicine to
combat pathogenic microorganisms and safe alternative to treat infectious diseases.
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(Received for publication 19 February 2007)