1. Background
The currently used feed additives such as antibiotics, probiotics and prebiotics in broiler diets to enhance nutrient utilization, play important roles in antibiotic resistance development among pathogens and saprophyte bacteria (1). The search for components with antimicrobial activities has recently gained increasing importance, due to the growing worldwide concern about the alarming increase in the rate of infections by antibiotic-resistant microorganisms. Due to their antibacterial activities against bacterial pathogens, medicinal plants are very important in human health. Cumin (Cuminum cyminum L.) originates from Egypt and Ethiopia and is much cultivated in Arabia, Malta, Sicily, India and China. Cumin seeds are used to flavor foods and liquors and its oil is utilized in perfumes and cosmetics. C. cyminum Linn. is an annual plant of the Umbelliferae family with antioxidant, anticholesterol and antimicrobial properties. Urinary tract infections (UTIs) are the second most common type of infections in the body and E. coli is the most common bacterial pathogen causing UTI (2). Different antimicrobial agents with high levels of activity against Gram-negative bacilli, including amikacin, ciprofloxacin, fosfomycin, gentamicin and nitrofurantoin, have shown acceptable levels of activities against this bacterium. Unfortunately, rapid appearance and development of drug resistance among these bacteria have caused a lot of difficulties in the modern world.
2. Objectives
In the present study, antibacterial activities of C. cyminum Linn. essential oil against multidrug resistant (MDR) E. coli strains isolated from UTIs were determined, using microdilution method.
3. Materials and Methods
3.1. Isolation of Bacteria
A total of 12 strains of E. coli were isolated from urine cultures of hospitalized patients (Zabol, southeastern Iran) suffering from UTIs, during 2011-2012. The isolated bacteria were identified and evaluated by Gram staining and standard biochemical tests (3).
3.2. Antibiotic Susceptibility Test
Susceptibility to all antibiotics was tested using standard disc diffusion method, as recommended by Clinical and Laboratory Standards Institute (CLSI) (4). Briefly, a colony suspension was prepared using sterile normal saline, equivalent to the 0.5 McFarland standard, and spread over the Mueller Hinton agar plate. Afterwards, the antibiotic discs were transferred aseptically to the surfaces of the inoculated media plates. Antibiotics and their concentrations were as follows: ceftazidim: 30 μg, tetracycline: 30 μg, erythromycin: 15 μg and ceftazidime: 30 μg. E. coli ATTCC 25922 was used as the control strain.
3.3. Plant Materials
Seeds of C. cyminum Linn. were collected from suburban areas of Kerman (southeastern Iran) and dried at room temperature in Kerman Azad University herbarium. Afterwards, the samples were crashed, transferred into a glass container, and preserved until the extraction process was performed in the laboratory.
3.4. Distillation of Essential Oil
The seeds were ground prior to the operation, and then, 300 g of the ground powder was submitted to water distillation for four hours, using a Clevenger apparatus. The distilled essential oil was dried over anhydrous sodium sulfate, filtered, and stored at 4ºC.
3.4. Minimum Inhibitory Concentration of Essential Oil
The broth microdilution method was used to determine the minimum inhibitory concentration (MIC). Briefly, serial double dilutions of the extract in Mueller Hinton broth, containing 0.5% (V/V) tween 80 over the ranges of 250, 100, 50 and 10 ppm, were prepared and added to a 96-well microtiter plate. To each well, 10 μL of indicator solution and 10 μL of Mueller Hinton broth were added. Finally, 10 μL of bacterial suspension (106 CFU/mL) was added to each well to achieve a concentration of 104 CFU/mL of the bacteria. The plates were wrapped loosely with cling film to prevent the bacteria dehydration. The plates were prepared in triplicates, and placed in an incubator at 37°C for 18–24 hours. MIC was defined as the lowest concentration of the essential oil, at which the microorganism did not demonstrate any visible growth. Average of the three values was calculated to provide the MIC values for the tested extract.
4. Results
4.1. Antibiotic Susceptibility
Antibiotic full resistance profile of the E. coli isolates was as follows: tetracycline (75%), erythromycin (58.4%), ceftazidime (50%) and cefixime (41.7%). Moderate resistance was observed in only 25% and 8.3% of the isolates against erythromycin and cefixime, respectively (Table 1). About 25% of the E. coli isolates showed resistance to all the antibiotics, whereas 8.3% and 25% showed resistance to three and two antibiotics, respectively (Table 2).
Table 1. Antimicrobial Susceptibility of Escherichiacoli isolates a
| Ceftazidime | Erythromycin | Cefixime | Tetracycline | |
|---|---|---|---|---|
| Sensitive | 6 (50) | 2 (16.6) | 6 (50) | 3 (25) |
| Intermediate | 0 | 3 (25) | 1 (8.3) | 0 |
| Resistant | 6 (50) | 7 (58.4) | 5 (41.7) | 9 (75) |
a Data are presented in No. (%).
| Bacterial Code | Antibiotic Susceptibility | MIC for Essential Oil, ppm | Resistance Pattern | |||
|---|---|---|---|---|---|---|
| A1 | A2 | A3 | A4 | |||
| 1 | R | R | R | R | 100 | A1, A2, A3, A4 |
| 2 | R | I | R | S | 250 | A1, A3 |
| 3 | S | R | R | R | NO | A2, A3, A4 |
| 4 | R | R | R | R | 100 | A1, A2, A3, A4 |
| 5 | R | R | R | R | 50 | A1, A2, A3, A4 |
| 6 | S | S | I | S | 50 | - |
| 7 | S | S | S | S | 10 | - |
| 8 | I | S | S | R | 100 | A4 |
| 9 | R | S | S | R | 250 | A1, A4 |
| 10 | R | S | I | R | 100 | A1, A4 |
| 11 | S | S | S | R | NO | A4 |
| 12 | R | R | R | R | 100 | A1, A2, A3, A4 |
a Abbreviations: I, intermediate; MIC, minimum inhibitory concentration; NO, Any inhibition; R, resistant; S, sensitive.
b A1, erythromycin; A2, cefixime; A3, ceftazidime; A4, tetracycline.
4.2. Minimum Inhibitory Concentration Assessment for Essential Oil
Different inhibitory effects of essential oil against most E. coli isolates were demonstrated in Table 2. The essential oil had inhibitory effects against most of the isolates. About 8.3% and 16.6% of the E. coli isolates showed the lowest MICs (10 and 50 ppm, respectively), while moderate (100 ppm) and highest (250 ppm) MIC values were seen in 41.6% and 16.6% of the isolates, respectively.
5. Discussion
In the present study, E. coli strains were resistant to four of the agents, including tetracycline (75%), erythromycin (58.3%), ceftazidime (50%), cefixime (41.6%). Different results were reported by other investigators in different geographical areas. For example, Shayan et al. reported antibiotic susceptibility of the AmpC-producing E. coli isolates as follows: erythromycin (92.3%), tetracycline (92.2%) nalidixic acid (84.6%), cefixime (84.6%), difloxacin (84.6%) azithromycin (76.9%), amoxicillin (76.9%), trimethoprim-sulfamethoxazole (76.9%) and gentamicin (76.9%) (5). Madani et al. reported antimicrobial resistance to ampicillin (91.4%), cotrimoxazole (61.1%), cefixime (46.8%), gentamicin (43.3%), ceftazidime (38.8%) and nalidixic acid (38.5%) (6). In the study of Heidari-Soureshjani et al. the highest resistance was reported to ampicillin (85.71%), nalidixic acid (78.78%), and ciprofloxacin (46.51%) (7). In the recent years, essential oils of plants have been in high demand from the manufacturers of foods flavoring, fragrance, cosmetics, and pharmaceutical industries, due to the growing interest of consumers to ingredients from natural sources. In our study, 8.3% and 16.6% of the E. coli isolates showed the lowest MICs (10 and 50 ppm respectively), while moderate (100 ppm) and highest (250 ppm) MIC values were seen in 41.6% and 16.6% of the isolates, respectively. Inhibitory effects of Cumin extract on E. coli 0:157 has been demonstrated previously (8). Other authors have also shown the antimicrobial activities of hexane extract and volatile components (9), water extracts or juices (10), and methanolic extracts of C. cyminum against different bacterial strains. For example, in the study of Vaishnavi et al. Cumin seeds were effective at lower concentration against Salmonella typhi and E. coli O:157 isolates (11). Soniya et al. reported the largest diameter of inhibition zone related to methanol extracts of C. cyminum against Bacillus subtilis, E. coli and Proteus sp. (12). As the study of Steffanini et al. reported, essential oil of C. cyminum was active against different Gram-negative bacteria, including E. coli, Pseudomonas. aeruginosaandSalmonella sp. with inhibitory zones of 18 mm, 10 mm and 23 mm, respectively (13). Con et al. reported that Cumin had inhibitory effect against Staphylococcus. aureus and Micrococcus luteus (14). Akgul and Kivanc reported that Cumin exhibited an inhibitory effect against S. aureus, Klebsiella pneumonia and P. aeruginosa (15). essential oil of C. cyminum can be used for protection against some bacteria.
