Antibacterial Effects of Iranian Mentha pulegium Essential Oil on Isolates of Klebsiella sp.
N. H. Jazani,
The aim of the present study was the evaluation of the antibacterial activity of Mentha pulegium essential oil on isolates of Klebsiella. Thirty nine isolates were collected from urine specimens submitted to two educational hospitals in Urmia, Iran. The susceptibility of isolates was determined using a broth microdilution method. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of isolates to Mentha pulegium essential oil were determined. The susceptibilities of isolates to different antibiotics were tested using agar disk diffusion method. The rates of resistance were determined to antibiotics as follows: gentamicin 46.1%, tobramycin 48.7%, ceftizoxime 41%, co-trimoxazole 46.1%, amikacin 33.3%, cephtazidime 51.3%, ciprofloxacin 30.8%, kanamycin 53.8%, nalidixic acid 30.8% ampicillin 79.5% and nitrofurantoin 41%. Mentha pulegium essential oil possessed antibacterial effect against all isolates of Klebsiella sp. with MIC and MBC values in the range of 1.9x10-3 to 4.9x10-4 mm3 mm-3. In this study clinical isolates of Klebsiella sp. showed very high resistance to tested antibiotics. These results suggest the potential use of the Mentha pulegium essential oil for the control of multi-drug resistant Klebsiella sp. infections. However, more adequate toxicological study must be carried out to verify the possibility of using it for fighting microorganisms in human.
The use of plants for healing purposes forms the origin of modern medicine
(Wargovich et al., 2001). Many drugs originate
from herbal sources: a century ago, most of the effective drugs were plant based.
The development of drugs from plants continues, with drug companies engaged
in pharmacological screening of herbs (Cetin et al.,
2006; Jarema, 2008; Vickers and
Mentha pulegium is one of the Mentha species known as pennyroyal. It
is native species of Asia and near East (Chalchat et al.,
2000). Mentha pulegium L. has been traditionally used as antiseptic
for treatment of cold, sinusitis, cholera, food poisoning, bronchitis and tuberculosis
(Zargari, 1990) and also as antiflatulent, carminative,
expectorant, diuretic, antitussive, menstruate (Newall et
al., 1996). Some pharmacological effect of Mentha pulegium L.
essential oil such as abortifacient effect in rat myometrium (Soares
et al., 2005), cytotoxic activity against different human cell lines
and its antioxidant effect (Mahboubi and Haghi, 2008)
were confirmed previously. Analysis of the Iranian essential oil of Mentha
pulegium revealed the presence of piperitone (38.0%), piperitenone (33.0%),
α-terpineol (4.7%) and pulegone (2.3%) as the major components. The antibacterial
effects of Mentha pulegium essential oil on limited strains of bacteria
have been investigated in earlier studies (Mahboubi and Haghi,
2008). However there is no studies in literature about the antibacterial
effects of this herb or its essential oil on Klebsiella sp.
Klebsiella sp. is a group of gram negative rods and they can cause different
kinds of infections especially in immunocompromised hosts. Multi-drug resistant
Klebsiella has been recognized as a cause of hospital acquired infections
worldwide. They are resistant to numerous antibiotics. Their resistance to antibiotics
restricts the choice of antibiotics for therapy (Gonzalez
and Schaeffer, 1999; Keynan and Rubinstein, 2007).
So, introducing of the new antimicrobial agents against this bacterium is one
of the most important goals in treatment of such infections. However, there
is no study on investigation the antibacterial effects of Mentha pulegium
on Klebsiella sp. In this study we evaluated the antibacterial activity
of Mentha pulegium essential oil on isolates of Klebsiellae sp.
MATERIALS AND METHODS
Essential oil: Mentha pulegium essential oil from Barij Essence
Pharmaceutical Company, Iran (commercial producers of plant essential oils and
aromatic substances) were used in this study. This oil was selected based on
literature survey and its use in traditional medicine. Analysis of the essential
oil by GC/MS revealed that the prominent components were piperitone (38.0%),
piperitenone (33.0%), α-terpineol (4.7%), 1,8-cineole (4.0%), piperitone
oxide (3.4%), menthone (3.1%), borneol (2.9%) and pulegone (2.3%) (Mahboubi
and Haghi, 2008).
Bacterial isolates: A total of 39 isolates were collected from urine
specimens submitted to two educational hospital clinical laboratories in Urmia,
Iran during a three months period from December 2006 until March 2007. The isolates
were further processed by the standard methods to identify as the Klebsiella
sp. (Baron and Finegold, 1990). Isolated bacteria were
maintained for long storage on skimmed milk medium (BBL) by adding 10% glycerol
in -60°C, cultures were maintained for daily use on Nutrient agar (BBL)
slants on 4°C. Klebsiella pneumoniae ATCC 10031 has been used as
Susceptibility testing: The Muller Hinton Agar (MHA) and Muller Hinton
Broth (MHB) media (Merck) were used for detection of antibiotic resistance of
isolates. The susceptibility of isolates to different antibiotics was tested
using agar disk diffusion method (Akram et al., 2007).
To represents different classes of antibacterial agents commonly used for treatment
of Klebsiella infections, gentamicin, tobramycin, ceftizoxime, co-trimoxazole,
amikacin, cephtazidime, ciprofloxacin, kanamycin, nalidixic acid, ampicillin
and nitrofurantoin were used in present study (Hi-media, Mombay, India).
Determination of antimicrobial activity of Mentha pulegium essential oil: The susceptibility of Klebsiella isolates to Mentha pulegium essential oil was determined using a broth microdilution method based on CLSI guidelines. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of isolates to Mentha pulegium essential oil were determined.
Mueller-Hinton Broth (MHB; Oxoid) was supplemented with 0.002% (v/v) tween
80 (Sigma) (MHB-T) to enhance dispersion of the Mentha pulegium essential
oil (Papadopoulos et al., 2006). The initial concentration
of Mentha pulegium essential oil in the first tube contains MHB-T was
1/2. This was used to prepare serial doubling dilutions over the range 0.03-25%
(v/v). 1.5x106 inoculums of the isolates were added to each concentration
in MHB-T. A tube containing growth medium without Mentha pulegium essential
oil and an un-inoculated tube were used as a positive and negative growth control,
respectively. Antibacterial activity was measured by determining MICs and MBCs.
The MIC was the lowest concentration of Mentha pulegium essential oil
that resulted in a clear tube. Ten microlitres from each tube was spot-inoculated
onto Nutrient Agar (NA) and incubated overnight at 37°C to determine the
MBC. The highest dilution that inhibits bacterial growth on nutrient agar after
overnight incubation was taken as MBC (Baron and Finegold,
1990; Papadopoulos et al., 2006). Experiments
were performed at least three times and the modal value selected.
The rates of resistance to different antibiotics for 39 isolates of Klebsiella have been showed in Table 1. Ampicillin (79.5%) and kanamycin (53.8%) showed the highest rate of resistance and ciprofloxacin and nalidixic acid (30.8%) demonstrated the lowest. Twenty percent of isolates showed resistance to the 11 tested antimicrobials.
Results showed that Mentha pulegium essential oil possessed antibacterial effect against all isolates of Klebsiella sp. with MIC and MBC values in the range of 1.9x10-3 to 4.9x10-4 mm3 mm-3 (Table 2).
Also MIC and MBC amounts for Klebsiella pneumoniae ATCC 10031 was 4.9x10-4 mm3 mm-3.
Klebsiella is an opportunistic pathogen and is a causative agent of
several kinds of infections in humans. It is one of the major pathogens in nurseries,
intensive care units and in hospital wards in spite of many effective antibiotics
||The rates of resistance to different antibiotics for 39 isolates of Klebsiella
from two educational hospitals in a three months period
|Gm: Gentamicin, Tob: Tobramycin, Ct: Ceftizoxime, SXT: Co-trimoxazole,
AN: Amikacin, CAZ: Cephtazidime, Cp: Ciprofloxacin, K: Kanamycin, NA: Nalidixic
Acid, Am: Ampicillin and FN: Nitrofurantoin
||Antibacterial activity of Mentha pulegium essential oil against
39 isolates of Klebsiella
*MIC amount for each isolate was equivalent to MBC
The use of broad spectrum antibiotics in hospital environments exerts selective
pressure on bacteria, results in promoting infections by multi-antibiotic resistant
isolates. Present finding showed that the most useful antibiotics for infections
caused by Klebsiella sp. were amikacin, nalidixic acid and ciprofloxacin.
Resistance to some antibiotics such as ampicillin, gentamicin, kanamycin and
nitrofurantoin showed increases in comparison with previous studies in different
countries (Randrianirina et al., 2007;
Akram et al., 2007).
Iranian people used the Mentha pulegium plant against infectious diseases
and find it to be efficacious against these problems without any scientific
base to explain of this action. The antibacterial effects of Mentha pulegium
essential oil has been investigated previously and significant antimicrobial
activity against Gram-positive bacteria especially Staphylococcus aureus
has been shown (Mahboubi and Haghi, 2008). Other
study showed piperitone, one of the main components of Mentha pulegium
essential oil completely inhibited Aspergillus flavus at low concentrations
(Cardenas-Ortega et al., 2005), however the antibacterial
effects of Mentha pulegium essential oil is much lower on E. coli
and Salmonella typhimurium (Mahboubi and Haghi, 2008).
These results suggest the potential use of Mentha pulegium essential oil for the control of Klebsiella infections. However, more adequate toxicological study must be carried out to verify the possibility of using it for fighting microorganisms in human body.
This study has been supported by a research grant (Grant No. 550/87) from student research committee of Urmia University of Medical Sciences.
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