Anti-Staphylococcal Activity of Eugenia Jambolana L. Against Methicillin–Resistant Staphylococcus Aureus

Abstract

The antibacterial activity of ethanol extracts of Eugenia jambolana L. and E. uniflora L. (Myrtaceae) were evaluated by agar-well diffusion method and MICs by microdillution method. E. jambolana showed antibacterial activity against MRSA strains (MIC < 1024 μg/mL). E. uniflora did not show significant antibacterial activity (MIC > 1024 μg/mL). It is therefore suggested that extracts from E. jambolana could be used as an anti– Staphylococcus agent. When compared with Methicillin and Gentamicin, the extract was more effective against, being a promising antibacterial agent. Based on the author's knowledge, this is the first report about the antibacterial activity of leave ethanol extract from Eugenia jambolana.

Keywords:

• Eugenia jambolana
• Eugenia uniflora
• Agar-well diffusion
• Antibacterial
• Antimicrobial
• Staphylococcus aureus
• MRSA

INTRODUCTION

With resistance to antibiotics on the rise, natural products could be an interesting alternative.[1,2] Many plants have been evaluated not only for direct antimicrobial activity, but also as possible sources of resistance modifying agents.[3,4] There is a growing interest in the use of biologically active compounds isolated from plants in the treatment of infections caused by antibiotic-resistant microorganisms.[5,6]

Eugenia jambolana L. and E. uniflora L. (Myrtaceae) fruits and leaves are used as foods and in folk medicine, respectively, because their traditionally purported antimicrobial activity,[7,8] and have been extensively studied because of their biological activities.[9] Several phytochemicals have been isolated from E. jambolana, such as triterpenoids from the leaves,[10] essential oil from the leaves, stem and fruits,[11] and oleanolic acid from the flowers.[12] E. uniflora yielded compounds such as the flavonoids myricitrin, quercetin and its 3-L-ramnoside quercitrin, steroids and/or triterpenoids, mono and triterpenic compounds, tannins, anthraquinones and phenols, cineol and essential oils.[13,14] Aminoglycosides are potent bactericidal antibiotics targeting the bacterial ribosome, and the increase of bacterial resistance to aminoglycosides is widely recognized as a serious health threat.[15] In Escherichia coli, the main mechanisms of resistance to aminoglycosides are active drug efflux and enzymatic inactivation.[16] Today, resistance to this and other classes of antibiotics represents a serious social and health problem, with an economic impact of millions of dollars on the public health systems of the whole world, mainly in developing countries.[17] The aim of this study was to determine the antibacterial effect of ethanol extracts of Eugenia jambolana and E. uniflora by testing them on the growth and survival of S. aureus strains isolated from clinical samples.

MATERIALS AND METHODS

Plant Material

Leaves of E. uniflora and E. jambolana were collected in the rainy season (April 2008) in the county of Crato, Ceará State, Brazil. The plant material was identified by Dra. Arlene Pessoa, and voucher specimens have been deposited with the identification numbers 3106 and 3107 at the Herbarium “Dárdano de Andrade Lima” of Universidade Regional do Cariri – URCA, respectively.

Preparations of Extracts

200 g of leaves were dried with no sunlight exposition and powdered at room temperature. The powdered material was extracted by maceration using 1 L of 95% ethanol as solvent at room temperature. The mixture was allowed to stand for 72 h at room temperature. The extracts were then filtered and concentrated under vacuum in a rotary evaporator under 60°C and 760 mm/Hg of temperature and pressure, respectively.[18] Each 200 g of aerial parts yield 5,6 g of extract. For the tests, the extract was diluted in DMSO, sterilized by filtration, and the highest concentration of DMSO remaining after dilution in broth caused no inhibition of bacterial growth.

Strains

Escherichia coli (ATCC 8539 and ATCC10536), Pseudomonas aeruginosa (ATCC 25619 and ATCC9027), Staphylococcus aureus (ATCC 6538 and ATCC25923) were used as positive controls. The clinical strains and methicillin-resistant Staphylococcus aureus (MRSA) were obtained from the Laboratório de Genética de Microrganismos - UFPB. All strains were stocked at room temperature on heart infusion agar slants (HIA, Difco) and, prior to assay, the cells were grown overnight at 37°C in brain heart infusion medium (BHI, Difco).

Antimicrobial Assay

The solid medium diffusion technique using agar wells was used for screening the extracts for antibacterial activity. 100 μL of the bacterial suspension (approximately 10⁵ CFU/mL) was uniformly spread on sterile heart infusion agar Petri dishes, and 50 μL of EEEJ and EEEU, both at 10 mg/mL, were added inside agar wells of 6 mm in diameter. The plates were incubated at 37°C for 24 h. The data of antibacterial activity were used only when the growth inhibition zone had a diameter ≥10 mm. MICs were determined bu microdillution method[19] by adding 100 μL of each strain suspended in BHI (final concentration 10⁵ colony-forming units/mL) to a 96-well microtiter plate with wells containing 100 μL of twofold serial dilutions of extracts. The final concentrations of EEEJ and EEEU were 512, 256, 128, 64, 32, 16, and 8 μg/mL. MIC was defined as the lowest concentration required for growth inhibition. The minimal bactericidal concentration (MBC) was determined by inoculating BHI agar plates with samples from non-growth wells. The MRSA strains 007 and 441 were assayed with methicillin and gentamicin (SIGMA Co.) at final concentrations of 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, and 1 μg/mL. All plates were incubated aerobically for 24 h at 37°C. MBC was defined as the lowest concentration showing no growth. All antimicrobial assays were performed twice and the results were expressed as the average of two repetitions. The solutions of the antibiotics were prepared using the recommendations of Clinical and Laboratory Standards Institute—CLSI.[20]

RESULTS AND DISCUSSION

In the last years, there has been great scientific interest in chemical and pharmacological investigations regarding the biological properties of medicinal plants. It is known that medicinal plants have been the source of many drugs now in use in clinical procedures (e.g., morphine, emetine, and rutin, to name just a few). The use of extracts as antimicrobial agents is associated with a low risk of the development of microbial resistance to their action because they are complex mixtures, making it more difficult for microbial agents to adapt.[21]

Table 1 shows the inhibition zones generated by EEEJ and EEEU assayed against clinical isolates of S. aureus. EEEJ inhibited the growth of all strains with zones of 14–18 mm (17.5 mm ± 3.5). Six strains showed inhibition zones with a diameter ≥17 mm. The smallest inhibition zones (14 mm) were found with the MRSA strains 296I, 02H and 05H, while the largest one (18 mm) was found with the MRSA strains 192C, 10C and 19L. EEEU inhibits only 5 MRSA strains with inhibition zones between 12–14 mm (13 mm ± 1).

Table 1 Origin, resistance profile of Staphylococcus aureus strains and inhibitory activity of E. jambolana and E. uniflora.

			Inhibition (mm)
Strain	Origin	PRP ^a	E. jambolana	E. uniflora
E. coli ATCC10536	–	–	21	12
E. coli ATCC8539	–	–	15	–
P. aeruginosa ATCC25619	–	–	15	–
P. aeruginosa ATCC9027	–	–	–	–
S. aureus ATCC6538	–	–	15	12
S. aureus ATCC25923	–	–	14	12
MRSA 06C	Surgical wound	1	–	–
MRSA 365	Surgical wound	2	16	11
MRSA 358	Surgical wound	2	–	–
MRSA 296I	Abscess	3	14	–
MRSA 171C	Surgical wound	4	15	13
MRSA 015	Surgical wound	2	16	–
MRSA 02H	Surgical wound	5	14	12
MRSA 01	Windpipe secretion	6	17	–
MRSA 007	Surgical wound	2	15	12
MRSA 05H	Surgical wound	7	14	14
MRSA 441	Surgical wound	2	17	–
MRSA 10C	Surgical wound	8	18	–
MRSA 192C	Surgical wound	2	18	–
MRSA 19L	Surgical wound	9	18	–
^aPhenotipic Resistance Profile: PRP; 1: Oxacilin, Penicilin, inductive Eritromicin, Kanamicin, Streptomicin, Gentamicin, Amikacin, Tobramicin, Chloramphenicol, Rifampicin, Novobiocin; 2: Oxacilin, Gentamicin, Tobramicin, Amikacin, Kanamicin, Neomicin, Paramomicin, Butirosin, Sisomicin, Netilmicin; 3: Oxacilin, Penicilin, inductive Eritromicin, Kanamicin, Streptomicin, Gentamicin, Amikacin, Tobramicin, Rifampicin; 4: Oxacilin, Penicilin, inductive Eritromicin, Kanamicin, Streptomicin, Gentamicin, Amikacin, Tobramicin, Tetraciclin-minociclin; 5: Oxacilin, Penicilin, inductive Eritromicin, Kanamicin, Streptomicin, Gentamicin, Amikacin, Tobramicin, Chloramphenicol, Tetraciclin-minociclin; 6: Oxacilin, Ampicilin, Amoxixilin, Cefalotin, Cafadroxil, Cefalexin, Clindamicin, Ciprofloxacin, Gentamicin, Gatifloxacin, Penicilin, Ampicilin-Sulbactan; 7: Oxacilin, Penicilin, Constituve Eritromicin, Kanamicin, Streptomicin, Gentamicin, Amikacin, Tobramicin, Tetraciclin-minociclin; 8: Oxacilin, Penicilin, Constituve Eritromicin, Kanamicin, Streptomicin, Gentamicin, Amikacin, Chloramphenicol, Tetraciclin; and 9: Oxacilin, Penicilin, inductive Eritromicin, Kanamicin, Streptomicin, Gentamicin, Amikacin, Tobramicin, Chloramphenicol, Tetraciclin-minociclin.

Table 2 shows the anti-staphylococcal effect of EEEJ and EEEU compared to the aminoglycoside gentamicin and the β-lactam methicillin. MIC and MBC values for EEEJ were 512–1024 μg/ml and ≥1024 μg/mL for the S. aureus strains 007 and 441, respectively. The EEEJ was 1–2 times more effective in inhibiting S. aureus growth than these drugs against strain 007, but not against strain 441. MIC and MBC values for EEEU were ≥1024 μg/ml in both strains, being similar to the effects of the antibiotics assayed, indicating that EEEU and EEEJ did not show an important antibacterial activity against the strains used.

Table 2 Comparative MICs and MBCs of Ethanol Extracts of E. jambolan a and E. uniflor a and antibiotics against MRSA strains isolated from clinics (ྒྷg/mL)

Strain	Methicilin MIC/MBC	Gentamicin MIC/MBC	E. jambolana MIC/MBC	E. uniflora MIC/MBC
MRSA 441	≥1024/≥1024	≥1024/≥1024	≥1024/≥1024	≥1024/≥1024
MRSA 007	≥1024/≥1024	≥1024/≥1024	512/1024	1024/≥1024

In contrast to our findings, many researchers have claimed that E. uniflora and E. jambolana possess antibacterial activity. The antibacterial activity of Eugenia jambolana was activated using UV-A light.[22] The essential oil and the methanol extract of E. uniflora have been assayed against various bacteria including Escherichia coli, Staphylococcus aureus, Salmonella typhimirium and others, with positive results.[8,23–25] However, the methanol and aqueous extracts of E. jambolanum have been tested against several bacteria using the agar disk diffusion method, and the MIC values were similar to those obtained in this study.[26] In the Mirtaceae family, there is a large variety of active principles against microorganisms including essential oils, flavonoids, and tannins.[7] Other possibilities are the use of natural products obtained from plants of Eugenia genus as Modifying antibiotic activity, as observed with plants from other families as Mentha arvensis and Turnera ulmifolia.[6,27,28]

The ethanol extract of E. jambolana showed antibacterial activity, mainly against Staphylococcus aureus, including the MRSA strains. E. uniflora did not show antibacterial activity. Compared with the conventional antibiotics, methicillin and gentamicin, E jambolana showed a similar or better MIC. Based on this result, the extracts are not of clinical relevance for direct use as antibacterial drugs, but E. jambolana could be a source of secondary metabolites with antibacterial potential, being necessary complementary studies about human toxicity and bioavailability to this application, but the good knowledge about this natural products is important to help guide those populations who use these products with scientific information.