1. Background
Staphylococcus aureus (S. aureus) is one of the most common nosocomial bacteria of infectious diseases such as endocarditis, osteomyelitis and food poisoning (1). With the spread of β-lactam resistant S. aureus, methicillin was synthesized from penicillin. Very soon methicillin-resistant S. aureus strains were identified. Methicillin-resistant S. aureus (MRSA) is one of the major nosocomial bacteria that leads to epidemiologic and clinical disorders. This organism can be transmitted among hospital personnel and hospitalized patients. In addition, MRSA has many virulence factors that create serious risks in sick or healthy individuals (2). In New York City hospitals, MRSA accounts for 29% of nosocomial infections and 50% of associated deaths (3). The replacement of antibacterial agents with herbal medicines may overcome the above-mentioned resistant bacteria.
One of these treatment modalities is Zataria multiflora, referred as “Avishen-e- Shirazi” in Persian, which is a very famous Iranian folk medicine. Z. multiflora is a member of the Lamiacea family and the most effective compounds in this medication are Thymol and Caracrol which have antibacterial effects (4). This plant grows in the south of Iran, Pakistan, India, and Afghanistan. The dried leaves of the plant were used in the food industry as a preservative and also for its flavor (5). The extracts stimulate innate immunity (6) and inhibit the growth organisms such as fungi and bacteria (4).
2. Objectives
In this study, we investigated the effects of alcoholic Z. multiflora extracts on 75 clinical MRSA isolates from patients in Faghihi Hospital (Shiraz University of Medical Sciences, Iran) during 2010-2012.
3. Materials and Methods
3.1. MRSA Isolation
The cultured samples were from wounds, abscesses, ears, nose, blood cultures and groins. 232 S. aureus strains were isolated from patients in Faghihi Hospital (Shiraz University Of Medical Sciences, Iran). We diagnosed S. aureus by the tube coagulase test, DNase test and growth in mannitol salt agar from mentioned specimens (2). For isolating MRSA, we used “a plate containing 6 µg/mL of oxacillin in Mueller Hinton Agar supplemented with NaCl (4% w/v; 0.68 mol/L)” (7). After culturing the specimens, 75 strains were diagnosed as MRSA from the mentioned bacteria.
3.2. Extract Preparation
Maceration method was used to prepare Z. extracts (8). First, some dried leaves of Z. multiflora were ground. Next, this was mixed with 80% ethanol and kept in a dark bottle. After 48 h of incubation in a dark room, it was filtered. It was then totally concentrated by the Rotary evaporator (Heidolph, Germany) and the alcohol-free extract was obtained. The extract was then frozen at 25◦C and finally the frozen extract was powdered by the freeze dryer (Zerbus, Germany).
3.3. Determination of Antimicrobial Activities of the Extract Against MRSA
The antibacterial activities of the extract against 75 clinical isolates of bacteria were determined by standard methods (2). Disk diffusion agar method was used to examine the susceptibility of all clinical isolates of the bacteria against selected antibiotics (9). Some of them were resistant and the others were sensitive to the current antibiotics.
3.4. Determination of Minimum Inhibitory Concentration (MIC) of the Extract Against MRSA
MICs were determined by using the broth micro dilution method recommended by the CLSI protocol with some modifications (9). To determine the antimicrobial activities of Zataria extracts against the bacteria, the initial concentration of the extract was prepared with 200 µg/mL in Dimethyl Sulfoxide (DMSO) as the solvent (Merck, Germany). Next, serial dilutions of the extract from the 2-512 µg/mL suspension were prepared in 96-well micro titer plates (Sigma, USA), using Muller Hinton broth (Merck, Germany).
The bacterial suspension was adjusted to 0.5 McFarland standards and then diluted to 1:1000 (10). 100 micro liters of the bacterial suspension in 1.5×105 cfu/mL concentration was added to each well except the negative control and the initial concentration of the extract. The cultured micro plate was incubated at 35◦C for 18 hours. After 24 hours of incubation, the tests were studied. The components of the positive control well included media, bacterial suspension and maximum concentration of the solvent. There were media and solvent in the negative control well. After incubation, the micro titer plates were studied. Wells without sediments indicated no growth of bacteria. The first well, which showed no growth, was considered as the MIC.
3.5. Determination of Minimum Bactericidal Concentration (MBC) of the Extract Against MRSA
10 micro liters of MIC of the extract and the previous concentrations were sub cultured via the spotted form on Muller Hinton Agar (MHA) (Merck, Germany) for determination of MBC. All of the inoculated plates were incubated at 35◦C for 18 hours. The first spotted culture on MHA, which showed no growth, was considered as the MBC.
4. Results
As Table 1 shows, MIC and MBC were measured for MRSA and MSSA. The alcoholic extract of Avishen-e-Shirazi inhibited the growth of all of the MRSA strains in the range of 2 to 16 µg/mL. Also, it inhibited the growth of S. aureus , S. epidermidis , S. saprophyticus and ATCC 25923 strain of S. aureus by 8-16 µg/mL. The MBC of the extract that could destroy all of the examined bacteria was 512 µg/mL or more. 47 isolates (62.2%) of MRSA were killed with this extract.
Table 1.The Comparison of MIC and MBC (μg/ml) of Alcohol Extracts of Z . multiflora on MRSA
| Bacteria (No.) | MIC a, μg/mL (No.) | MBC a, μg/mL, No. |
|---|---|---|
| S.aureus (MRSA) (75)a (75) | 2 (5) | 2 (0) |
| 4 (12) | 4 (0) | |
| 8 (30) | 8 (0) | |
| 16 (28) | 16 (3) | |
| 32 (0) | 32 (13) | |
| 64(0) | 64 (6) | |
| 128 (0) | 128 (7) | |
| 256 (0) | 256 (10) | |
| 512 (0) | 512 (7) | |
| S. aureus (ATCC25923) | 16 (1) | 512 (1) |
| S. aureus (MSSA) a | 16 (1) | 256 (1) |
| S. epidermidis (1) | 16 (1) | 256 (1) |
| S. saprophyticus | 8 (1) | 512 (1) |
aAbbreviations: MIC, Minimum Inhibitory Concentration; MBC, Minimum Bactericidal Concentration; MRSA, Methicillin Resistant S. aureus; MSSA, Methicillin Sensitive Staphylococcus aureus
5. Discussion
Nowadays, the drug resistant bacteria can create the most important infectious diseases. One aspect of such problem is the incidence of multidrug resistant bacteria like MRSA in hospitals. For resolving this problem, many investigators noticed the effect of herbal extracts instead of antibiotics (4, 5). Based on the previous studies, the Avishen-e-Shirazi extract can inhibit the growth of enterohemorrhagic Escherichia coli (11), Salmonella sp and Shigella sp (12), S. aureus (13), Klebsiella (14), Enterococcus (15), Pseudomonas aeruginosa (16), and Acinetobacter baumannii, Alcaligenes, Chryseobacterium meningosepticum in the NFB (Non Fermentative Gram Negative Bacteria ) group.(13).
In this study the effect of alcoholic Z. multiflora extract on MRSA was studied. Based on the obtained results, we found that the micro broth dilution method is more sensitive than tube macro broth dilution and disk agar diffusion as used by previous investigators (14). Moreover, we noticed that the alcoholic Zataria extracts could inhibit the growth of MRSA strains and kill 62.2%. The extract could also inhibit the growth of MSSA, ATCC 25923 of S. aureus, S. epidermidis and S.saprophyticus as well as MRSA. There were no differences between the mentioned bacteria due to inhibitory and bactericidal effects of the extract. This research shows that low concentrations of Zataria extracts inhibit the growth of MRSA efficiently. The results have shown that the inhibitory effect of the extract is better than its bactericidal effect on the mentioned bacteria. Due to the oral intake of Z. multiflora, we suggest that the high concentration of the extract could be administered for prevention of bacterial growth. We also suggest some experiments to be performed using the extracts in the form of lotions or creams on skin, cutaneous and subcutaneous lesions on lab animals in order to cure or prevent staphylococcal lesions.
