For a long period of time, plants have been a valuable source of natural products for maintaining human health. The use of plant extracts and phytochemicals, both with known antimicrobial properties, can be of great significance in therapeutic treatments [1]. Lately, there has been growing interest in the development of newer, natural and broader spectrum of antimicrobial agents due to the likelihood that these phytochemicals will find their way into antimicrobial drugs, the public’s increasing awareness of problems associated with the abuse of traditional antibiotics leading to rapid development of resistance against chemical antibiotics by bacterial strains [2].

The Mespilus germanica was already being cultivated about three thousand years ago in the Caspian Sea region of northern Iran and Azerbaijan. M. germanica L. belongs to Rosaceae family and it grows mainly in frost-free areas and on rocks and poor soils. M. germanica, known as the medlar or common medlar, is a large shrub or small tree and the name of the fruit of this tree. In Iran, they are abundant particularly in Guilan and Mazandaran province. The fruits are used as a nutrition component by the local population and are prepared by the local people as marmalade or pickle. The fruit is consumed as a medicinal remedy for example treatment of constipation, diuretic and to rid the kidney and bladder of stones in Iran [3].

Klebsiella pneumoniae organisms are often resistant to multiple antibiotics. Current evidence implicates plasmids as the primary source of the resistance genes. Klebsiella with the ability to produce extended-spectrum beta-lactamases ESBL is resistant to many classes of antibiotics. The most frequent resistances include resistance to aminoglycosides, fluoroquinolones, tetracyclines, chloramphenicol and trimethoprim/sulfamethoxazole [4]. Enterobacter aerogenes is a nosocomial and pathogenic bacterium that causes opportunistic infections including most types of infections. The majority is sensitive to most antibiotics designed for this bacteria class, but this is complicated by their inducible resistance mechanisms, particularly lactamase which means that they quickly become resistant to standard antibiotics during treatment, requiring change in antibiotic to avoid worsening of the sepsis [5]. Streptococcus pyogene can also cause disease in the form of post infectious “nonpyogenic” (not associated with local bacterial multiplication and pus formation) syndromes. These autoimmune-mediated complications follow a small percentage of infections and include rheumatic fever and acute post-infectious glomerulo-nephritis [6]. Listeria innocua is important because it is very similar to the food-borne pathogen Listeria monocytogenes but non-pathogenic in character [7].

Antibiotic resistance is a serious and growing phenomenon in contemporary medicine and has emerged as one of the pre-eminent public health concerns of the 21^st century, in particular as it pertains to pathogenic organisms. A world health organization (WHO) report released 2014 states, this serious threat is no longer a prediction for the future and it is happening right now in every region of the world and has the potential to affect anyone, of any age, in any country. Antibiotic resistance when bacteria change so antibiotics no longer work in people who need them to treat infections is now a major threat to public health [2].

The aim of this study is to investigate the antimicrobial effect of M. germanica on Streptococcus pyogenes PTCC 1447, L. innocua ATTC 33090, E. aerogenes ATTC 13048 and K. pneumoniae PTCC 1053 and its comparison with common therapeutic antibiotics.

This experimental study was conducted at industrial microbiology laboratory, department of food science and technology, Ferdowsi University of Mashhad in 2014.

Microorganisms and culture media: Four bacteria were tested in this study and include S. pyogenes PTCC 1447, L. innocua ATTC 33090, E. aerogenes ATTC 13048 and K. pneumoniae PTCC 1053. All culture media were purchased from Merck (Germany). Microorganisms were grown overnight on trypticase soy broth (TSB), under optimal conditions. Overnight cultures were adjusted to match a 0.5 McFarland standard and further diluted 1:100 with Mueller-Hinton broth (MHB). The dilution obtained served as the inoculums for antibacterial activity assay [8].

3.1. Preparation of the Plant

The M. germanica were collected from countryside of Aliabad-e-Katul (Golestan province, Iran) and dried at room temperature. Samples were crashed and transferred into glass container and preserved until extraction procedure was performed in the laboratory and taxonomic identification was performed by the faculty of science herbarium, Ferdowsi university of Mashhad, Mashhad, Iran [9].

3.2. Suspension Preparation

Fresh cultivated S. pyogenes PTCC 1447, L. innocua ATTC 33090, E. aerogenes ATTC 13048 and K. pneumoniae PTCC 1053 colonies were suspended in 5 mL of 0.85% normal saline. Suspension was mixed for 15 seconds with a vortex (Germany). Then its concentration was adjusted to 1.5 × 10⁸ CFU/mL based on a standard 0.5 McFarland [10].

3.3. Extract Preparation

The M. germanica were pulverized into a coarse powder and 25 g grinded powder was soaked in 125 mL ethanol 96° (Merck-Germany) or water, for 24 hours (shaking occasionally with a shaker). After one day of dissolving process, materials were filtered (Whatman no. 1 filter paper) and centrifuged in 9000 g for 15 minutes. Then extracts were evaporated using rotary evaporator and dried extracts were obtained and stored at 4°C in air tight screw-cap tube [11].

3.4. Antibiotics Susceptibility Test

The test was done by using the agar disk diffusion method or the Kirby-Bauer method as recommended by the national committee for clinical laboratory standard (NCCLS) on bacterial strains of S. pyogenes PTCC 1447, L. innocua ATTC 33090, E. aerogenes ATTC 13048 and K. pneumoniae PTCC 1053. The antibiotic discs used in this test are erythromycin, ampicillin, gentamicin, ciprofloxacin, clindamycin and vancomycin. To perform this test, the accurate identification of species of bacteria, S. pyogenes, L. innocua, E. aerogenes and K. pneumoniae colonies of strains tested in Mueller Hinton broth (MHB) and then suspended in standard 0.5 McFarland was conducted to compare. If turbidity ensures it matches perfectly sterile cotton swab using four different bacteria in the culture medium Mueller Hinton agar (MHA) has cultivated and antibiotic discs used in sterile conditions with respect to the distance of two centimeters away from each other placed on medium Mueller Hinton agar plates. After incubation for 18 to 24 hours at 37°C, the results obtained with the standard provided by the national committee for clinical laboratory standards were compared to susceptible and resistant [12, 13].

3.5. Determining Antimicrobial Activities Using the Disc Diffusion Method

The agar disc diffusion method was employed to determine antimicrobial activity in this study. In the disk agar diffusion method 1.5×10⁸ CFU/mL (equivalent to 0.5 McFarland standards) of standard culture of each strain was cultured on agar surface at the first step, and then it was spread on the surface of agar by sterile glass spreader. After the inoculated plates had dried sufficiently the discs were kept over the agar plates using sterile forceps at various concentrations (10, 20, 30, and 40 mg/mL) [14].

3.6. Determining Antimicrobial Activities Using the Pour Plate Method

The pour plate technique can be used to determine the number of microbes/mL in a specimen. It has the advantage of not requiring previously prepared plates and is often used to assay bacterial contamination of foodstuffs. Ethanol extract (0.2 g) were added to 5 mL of sterile distilled water. After that, it was stirred with vortex system to assist being steady, subsequently 1 mL of this solution was added to sterile plates. The final concentration of the extract was 2 mg/mL. At the next step, the sterilized Mueller Hinton agar (Merck-Germany) medium were added to the plates and placed at room temperature. One loop of each standard strain culture media was cultured inoculums on these medium. The plates were incubated for 24 hours at 37°C [15].

3.7. Minimum Inhibitory Concentration (MIC)

The lowest concentration (highest dilution) of test agent preventing appearance of turbidity (growth) is considered to be the minimal/minimum inhibitory concentration (MIC). At this dilution the test agent is bacteriostatic. Serial dilution method was determined MIC. Various concentrations (2, 4, 8, 16, 32, 64, 128, 256 mg/mL) of extracts were prepared in 10 cm experimental tubes. Each tube contained 9 mL of Muller Hilton was sterilize by autoclaving. After cooled, 1 mL of different concentrations (2, 4, 8, 16, 32, 64, 128, 256 mg/mL) of each extract was added to each tube. The tubes were incubated for 24 hours at 37°C. The lowest concentration that did not permit any visible growth when compared with the control was considered as the minimum inhibitory concentration [16].

3.8. Minimum Bactericidal Concentration (MBC)

The minimum bactericidal concentration (MBC) or the minimum lethal concentration (MLC) of an antibacterial which is defined as the maximum dilution of the product that will kill a test organism can be determined by sub-culturing last clear MIC tube onto growth medium and examining for bacterial growth. Serial dilutions are made of the products in bacterial growth media. MBC was determined according to agar dilution method with slight modifications. The MBC were determined by incorporating various concentrations of extracts (2, 4, 8, 16, 32, 64, 128, 256 mg/mL) in Muller Hilton Broth for bacteria [17].

3.9. Statistical Analysis

The experimental results were expressed by means ± standard deviation. The data were analyzed using one way analysis of variance (ANOVA) using SPSS-18. In the one-way ANOVA, we have tested the values of a quantitative variable in the different groups.

The results of the antimicrobial effects of ethanolic and aqueous extracts, by pour plate method were showed 2 mg/mL concentration of ethanolic extract, were quite effective on reduce of growth S. pyogenes, L. innocua, E. aerogenes and K. pneumonia; and were had prevent growth over the medium. The aqueous extract, only had antimicrobial effect in 2 mg/mL concentration on growth of, S. pyogenes and L. innocua.

The results the antimicrobial effects of ethanolic and aqueous M. germanica extracts, by the agar diffusion method were presented in Table 1. The results the antimicrobial effects of common therapeutic antibiotics, by Kirby-Bauer method were presented in Table 2. The results of MIC and MBC of ethanolic and aqueous M. germanica extracts were presented in Tables 3 and 4.

According to the result of this study, antimicrobial activity was assessed by inhibition diameters which were found to range from 8 - 21.5 mm for the two extracts against all the bacterial strains tested. The M. germanica extract showed the more effective impact on the growth S. pyogene and L. innocua than E. aerogenes and K. pneumoniae. The results showed 2 mg/mL concentration of ethanolic extract, were quite effective on reduce of growth S. pyogenes, L. innocua, E. aerogenes and K. pneumonia and were had prevent growth over the medium. The aqueous extract, only had antimicrobial effect in 2 mg/mL concentration on growth of, S. pyogenes and L. innocua.

Many plant extracts owe their potency to the presence of substances such as tannins, phenolics, micro elements, essential oils, peptides, unsaturated long chain aldehydes, alkaloids and so on. These substances are usually found in various parts of the plants like roots, leaves, shoots and bark [18]. Many plants have therefore become sources of important drugs and the pharmaceutical industries have come to consider them as a source of bioactive agents that can be used in the preparation of synthetic medicine [19].

The results of the present study clearly showed that M. germanica extracts showed antibacterial activity against tested pathogenic bacterial strains including antibiotic resistant strains. The effectiveness of the active compounds present in plant extracts cause the production of growth inhibition zones that appear as clear areas surrounding the disk. Antibacterial activity may be due to active components which are present in plant extracts. However, some plant extracts were unable to exhibit antibacterial activity against tested bacterial strains. These bacterial strains may have some kind of resistance mechanisms e.g. enzymatic inactivation, target sites modification and decrease intracellular drug accumulation [20] or the concentration of the compound used may not be sufficient. No inhibition was observed with controls, which proves that solvents could not act as antibacterial agents. Further investigations were done using extracts of M. germanica only, since they showed considerable antibacterial activity. In almost all tests, ethanol extract showed better inhibition against all tested bacterial strains, indicating that active ingredients in plant materials could be extracted into ethanol. However, highest antibacterial activity was observed against S. pyogenes.

On the basis of the above results, it showed that ethanol extract of M. germanica exhibited a greater inhibition compared with aqueous extract. Parekh et al. reported that most of the antimicrobial active compounds were soluble in polar solvent such as alcohol instead of water [21]. This result is comparable to the study by de Souza et al. using alcohol extract of L. sibiricus that showed effective antibacterial activity on Bacillus subtilis [22]. Bajwa and Shafique showed that methanol fraction of A. rabiei exhibited more promising results in suppressing the fungal growth rather than aqueous extract [23]. This was also reported by Zafar et al. where chloroform extract of Melia azedarch leaves was active against Fusarium chamdosporum while water extract of the leaves did not show any positive results [24].

In this study, by increasing the amount of alcoholic extract on disc diffusion method, inhibition zone around the disc was increased. Various sizes of inhibition zones were measured by a ruler in millimeters. The maximum size of inhibition zone for M. germanica ethanol extract in the disc method was 21.5 mm (40 mg/mL) and M. germanica aqueous extract was 20 mm (40 mg/mL), respectively. The results show that ethanolic and aqueous extract of M. germanica in all concentrations (10, 20, 30 and 40 mg/mL) had the inhibitory effect on S. pyogenes, L. innocua, E. aerogenes and K. pneumonia.

The results of MIC and MBC assay revealed that M. germanica have more antibacterial effect on Gram-positive bacteria compared to Gram-negative bacteria. So that Gram-positive bacteria such as S. pyogenes and L. innocua and Gram-negative bacteria such as E. aerogenes and K. pneumonia showed lowest and highest MIC, respectively. The MIC of ethanolic extract of M. germanica for S. pyogenes, L. innocua, E. aerogenes and K. pneumonia were 2, 4, 32 and 32 mg/mL, respectively. But MIC of the aqueous extract of M. germanica for S. pyogenes, L. innocua, E. aerogenes and K. pneumonia were 4, 8, 64 and 32 mg/mL, respectively. The MBC of ethanolic extract of M. germanica for S. pyogenes, L. innocua, E. aerogenes and K. pneumonia were 4, 8, 64 and 64 mg/mL, respectively. But MBC of the aqueous extract of M. germanica for S. pyogenes, L. innocua, E. aerogenes and K. pneumonia were 8, 16, 128 and 64 mg/mL, respectively. This is probably due to presence of lipopolysaccharide in cell wall of Gram-negative bacteria. Lipopolysaccharides of cell wall can prevent influx of active compounds to cytoplasmic membrane of these bacteria [25]. In study of Khalil and Li reported the sage essential oil was effective against both Gram positive (Streptococcus) and Gram negative bacteria (E. coli, S. typhi) and the antibacterial effect against Gram positive bacteria was more than Gram negative bacteria which had similar result with this study [26]. Arias et al. investigated the antibacterial effects of aqueous and ethanolic extract of Acacia aroma They reported that Gram positive bacteria (Bacillus cereus and Bacillus Subtlis) are more sensitive than Gram negative bacteria (Escherichia coli) to plant extracts. This result is consistent with the findings of this study [27]. Also, they reported that ethanolic extract compared to the aqueous extract was more effective and has a greater inhibitory effect [27]. These results are consistent with reported by Karsha and Laskhmi, that Piper nigrum L. extract is more susceptible to Gram positive compared against Gram negative. It might because P. nigrum L. extract altered the membrane permeability results in the leakage of nucleic acid and protein into the extracellular medium [28]. The results of this study provide informative data for the use of the extracts from M. germanica against bacterial microbial infections. However, the clinical application of this plant are needed more and larger studies and if successful and standardization of results. These plants can use as an alternative instead of inert and ineffective anti microbial drugs currently used.