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BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2004 61 (2) BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2004 61 (2)
Allicin and methicillin-resistant Staphylococcus aureus 2
15
40
CONTROL
Number of strains
Allicin
10 Mupirocin SENSITIVE
INTERMEDIATE
30
20
0
0 10 20 30 40 50
Zone size (mm)
10
Fig. 1. Number of strains and the size of zones of inhibition with
allicin (500 g/mL) and mupirocin against Staphylococcus aureus.
0
1000 500 250 125 62.5
Materials and methods Concentration of allicon (mg/ml)
Bacterial strains Fig. 2. Average diameter of zones of inhibition for allicin liquid
Thirty clinical isolates and one control strain (Oxford Strain, against Staphylococcus aureus strains that showed resistance,
NCTC 6571) of S. aureus were tested. All 30 clinical isolates intermediate resistance or sensitivity to mupirocin.
were obtained from the Royal London and St. Bartholomews
Hospitals and had been identified as showing multiple
antibiotic resistance. All strains were resistant to methicillin The bacterial inoculum used was approximately Log 6
and had no apparent epidemiological connection colony-forming units (cfu)/mL. Cultures were incubated
overnight at 37C and examined for growth the following
Allicin day. Subcultures of these were used to determine MBC.12
A 5000 g/mL solution of allicin in water was provided by
Allicin International. As it was an extracted product, the
purity of the allicin solution was tested and the Results
concentration confirmed by Allicin International using the
high-performance liquid chromatography (HPLC) method Mupirocin activity
of Lawson, Wang and Hughes.10 The control strain produced a 35-mm diameter zone of
inhibition to mupirocin. Of the clinical isolates, five strains
Antimicrobial activity were identified as fully susceptible (zone sizes 3345 mm), 12
The allicin liquid and allicin made up in aqueous cream were showed intermediate susceptibility (zone sizes 1223 mm)
tested for antimicrobial activity against MRSA using an agar and 13 strains were resistant (no zone of inhibition).
well diffusion method. Muller-Hinton agar plates were Variability of zone size was considerable, ranging from 045
inoculated using the methods recommended by the British mm. The most frequent result (13 strains) showed no zone of
Society for Antimicrobial Chemotherapy (BSAC).11 inhibition. The second most common group (11 strains)
Circular wells (6 mm) were cut in the agar culture media produced intermediate zone sizes (819 mm). Two strains
and filled with 100 L cream or liquid. The accuracy of the were highly susceptible, producing zone sizes of 45 mm.
method used to deliver these volumes was confirmed by (Fig. 1).
weighing the agar plates before and after the fluid and
cream were added. Allicin liquid activity
Mupirocin activity against the same MRSA strains was Allicin liquid was active against S. aureus strains down to 62.5
determined using a 5 g paper disk (Oxoid, UK) on Muller- g/mL (equal to 6.25 g of allicin in 100 L [the volume
Hinton agar plates added to each well]). No activity was detected below 62.5
g/mL, and concentrations of 250 g/mL and above were the
Minimum inhibitory and bactericidal concentrations of allicin most active.
liquid The average zone sizes produced against these strains
Standard methods based on those of Lorian12 were used to were 25 mm at 250 g/mL, 32 mm at 500 g/mL and 38 mm
determine minimum inhibitory concentration (MIC) and at 1000 g/mL. The spread of zone size to allicin was smaller
minimum bactericidal concentration (MBC). Strains were than that found with mupirocin (Fig. 1). Zone sizes ranged
cultured overnight at 37C in Muller-Hinton broth (Oxoid, from 2338 mm at 500 g/mL, and from 3144 mm at 1000
CM 405). Concentrations of allicin between 11000 g/mL g/mL (Figs. 2 and 3).
and broth containing no antimicrobial agent (negative
control) were assessed. Creams were not tested by this Allicin cream activity
method because the opacity they produced in the liquid The control strain produced a 20 (1.5) mm zone of inhibition
made determination of MIC impossible. compared to 23 ( 1.5) mm for mupirocin-susceptible MRSAs
BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2004 61 (2) BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2004 61 (2)
Allicin and methicillin-resistant Staphylococcus aureus 3
40
CONTROL
30 SENSITIVE
20
10
0
1000 500 250 125 62.5
Concentration of allicon (mg/ml)
Fig. 3. A typical zone of inhibition produced by 500 g/mL allicin Fig. 4. Average diameter of zones of inhibition for allicin cream
against MRSA. against Staphylococcus aureus strains that showed resistance,
intermediate resistance or sensitivity to mupirocin.
and 21 (1.5) mm for mupirocin-resistant MRSAs. When Table 1. Susceptibility of MRSA clinical isolates to a stabilised allicin
prepared in Boots aqueous cream, allicin was found to be extract, shown as the percentage of strains with different minimal
highly active against all strains at a concentration of 500 inhibitory concentrations (MIC) and minimum bactericidal
g/mL. No activity was detected below 125g/mL (Fig. 4). concentrations (MBC).
BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2004 61 (2) BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2004 61 (2)
Allicin and methicillin-resistant Staphylococcus aureus 4
been reported to have a range of potential targets. It is 4 Schentag JJ, Hyatt M, Carr J et al. Genesis of methicillin-resistant
reported to inhibit the acetyl CoA forming system, to inhibit Staphylococcus aureus (MRSA), how treatment of MRSA
DNA and protein synthesis, and to target RNA infections has selected for vancomycin-resistent Enterococcus
polymerase;1820 and these are responsible for the agents faecium and the importance of antibiotic management and
antibacterial effect. infection control. Clin Infect Dis 1998; 26: 120414.
More general proposals about the broad-spectrum activity 5 Walker ES, Vasquez JE, Dula R, Bullock H, Sarubbi FA.
of allicin were provided by Rabinkov et al. in 1998. This Mupirocin-resistant, methicillin-resistant Staphylococcus aureus:
group compared the importance of its antioxidant properties does mupirocin remain effective? Infect Control Hosp Epidemiol
with its thiol disulphide exchange activity and suggested 2003; 24: 3426.
that activity is related to allicins rapid reaction with thiol- 6 Hahn G. In: Koch HP, Lawson LD, eds. Garlic: the science and
containing proteins. In contrast, mupirocin (pseudomonic therapeutic application of Allium sativum L and related species (2nd
acid) inhibits protein synthesis by slowing the activity of edn). Baltimore Williams and Wilkins, 1996: 124.
RNA synthetase.13 7 Cavallito C, Bailey JH. Allicin, the antibacterial principal of
For a topical agent to produce maximum benefit, its Allium sativum. Isolation, physical properties and antibacterial
strength in any formulation should not only be sufficient to action. J Am Chem Soc 1944; 66: 194452.
inhibit growth but also to be bactericidal. The present study 8 Ankri S, Mirelman D. Antimicrobial properties of allicin from
demonstrated that the majority (88%) of strains had MBCs garlic. Microbes Infect 1999; 2: 1259.
for allicin of 128 g/mL and all the strains were killed by 9 Block E. The chemistry of garlic and onion. Sci Am 1985; 252:
allicin at 256 g/mL. 949.
Allicin liquid extracts were highly active against clinical 10 Lawson LD, Wang ZJ, Hughes BG. Identification and HPLC of
isolates of multiple antibiotic resistant S. aureus, including the sulphides and dialk(en)yl thiosulphinates in commercial
those strains that were identified as mupirocin-resistant. garlic products. Planta Med 1991; 57: 36370.
Although agar diffusion tests showed that the activity of 11 Andrews JM. BSAC standardised disc susceptibility testing
allicin was reduced in the cream formulation, comparison of method. J Antimicrob Chemother 2001; 48 (Suppl S1): 516.
zone sizes achieved with 250 g/mL in liquid showed 12 Lorian V. Antibiotics in laboratory medicine. Baltimore: Williams
similarity to those achieved with 500 g/mL in cream and Wilkins, 1980.
(average zone sizes of 25 mm and 23.7 mm, respectively). 13 Schmitz FJ, Jones ME. Antibiotics for treatment of infections
When made up in an aqueous cream base, 500 g/mL caused by MRSA and elimination of MRSA carriage. What are
allicin produced bactericidal levels high enough to support the choices? Int J Antimicrob Agents 1997; 9: 119.
the testing of allicin cream as a topical agent against S. 14 Lawson LD. The composition and chemistry of garlic cloves and
aureus, including MRSA. processed garlic. In: Koch HP, Lawson LD, eds. Garlic: the science
In conclusion, the present study demonstrated that liquid and therapeutic application of Allium sativum L and related species
and cream formulations containing allicin are active against (2nd edn). Baltimore: Williams and Wilkins, 1996: 37107.
S. aureus, including MRSA strains, showing both high and 15 Block E. The organosulphur chemistry of the genus Allium:
low levels of resistance to mupirocin. The aqueous cream implications for the organic chemistry of sulphur. Angew Chem
formulation showed reduced activity compared with allicin Int Ed Engl 1992; 31: 113578.
in water. At 500 g/mL, however, the cream was active 16 Lawson LD, Wang ZYJ. Changes in the organosulphur
against all the organisms tested, suggesting that this compounds released from garlic during aging in water, dilute
therapeutic concentration compares well with the 20,000 ethanol or dilute acetic acid. J Toxicol 1995; 14: 214.
g/mL mupirocin currently used for topical application.21 17 Hughes BG, Lawson LD. Antimicrobial effects of Allium sativum
L (garlic), Allium ampeloprasum (elephant garlic) and Allium cepa
This work was supported in part by a grant from Allicin L (onion), garlic compounds and commercial garlic supplement
International, Half House, Military Road, Rye, East Sussex. products. Phytotherapy Research 1991; 5: 1548.
18 Feldberg RS, Chang SC, Kotic AN et al. In vitro mechanism of
inhibition of bacterial cell growth by allicin. Antimicrob Agents
References Chemother 1988; 32: 17638.
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Alvarez S. Mupirocin resistance in methicillin-resistant RNAS polymerase by monomercuric derivative of flourescein
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BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2004 61 (2) BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2004 61 (2)