ANTIBACTERIAL ACTIVITY EVALUATION OF 15 EUCALYPTUS SPECIES ESSENTIAL OILS AGAINST CLINICALLY RELEVANT PATHOGENIC BACTERIA

This study aims to evaluate the potential of 15 Eucalyptus species essential oils as alternatives to clinical surface disinfectants with known association to ant ibiotic resistance. Four reference pathogenic bacte ri were tested: Pseudomonas aeruginosa (ATCC10145), Escherichia coli (CECT434), Staphylococcus aureus (CECT976) and Listeria monocytogenes (ATCC15313). Gram-positive bacteria revealed higher sensitivity than Gram-negative. Essential oils from E. bosistoana, E. botryoides, E. camaldulensis, E. cinerea and E. citriodora showed bacterial inhibition for Gram-positive, eve n higher than with gentamicin and ciprofloxacin (positive controls). L monocytogenes was the most sensitive and P. aeruginosa demonstrated resistance to all essential oils. The antimicrobial potential values were 6.25, 6.25 and 12.5% for E. coli, S. aureus and L. monocytogenes, respectively. This study reveals that Eucalyptus essential oils may be useful in order to control pathogenic bacteria as potential complementary treatment or as disinfectants in clin ical/hospital environments.


INTRODUCTION
The introduction of antibiotics after World War I resulted in a dramatic decrease in death numbers due to bacterial infections. However, the increase of antibiotic resistance is the reason for this medical emergency (Schjørring and Krogfelt, 2010), which has lead inevitably to the emergence and dissemination of resistant bacteria and resistance genes. The inefficacy of the conventional antibiotics is due in part to their often excessive and inappropriate use (Saavedra et al., 2010).
Reduced susceptibility of microorganisms to antimicrobial products may be acquired through mutation, by plasmid or transposon acquisition, or by the microorganisms' intrinsic properties conferring reduced susceptibility to antimicrobial agents (Alekshun and Levy, 2007;Simões et al., 2009). Therefore it becomes of high importance to take a closer look at the traditional and complementary medicine.
The Eucalyptus genus belongs to Myrtaceae family and includes more than 700 species (Francisco et al., 2001). These species are distributed all over the world and Portugal is not an exception. There are many evidences that Eucalyptus species EOs have a strong antibacterial (Cimanga et al., 2002;Chung et al., 2007), analgesic, anti-inflammatory and antioxidant effects (Cruz et al., 2001;Silva et al., 2003).
Nowadays, hospital infections are a serious concern for health authorities. In order to contain and prevent drug multi-resistant bacteria there have been intensive studies with plant extracts and EOs that show the ability to eliminate or decrease infections (Cimanga et al., 2002;Chung et al., 2007;Takahashi et al., 2004).
Few studies have been published about the potential effect of Eucalyptus EOs as disinfectants of surfaces in clinical and hospital environments associated to the antibiotic resistance phenomena. Thus the aim of the present work is to evaluate the potential of these compounds as alternatives to the traditional clinical disinfectants like bleach that are known to be toxic for human health. In the present study the in vitro antibacterial activity of the selected Eucalyptus EOs were tested against four clinically significant bacteria largely associated with antibiotic resistance: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Listeria monocytogenes. The essential oils were isolated by hydrodistillation for 3 h using a Clevenger-type apparatus according to the European Pharmacopoeia (CE, 2002). The Eucalyptus species composition in volatiles was reported previously (Faria et al., 2011).

Bacterial Strains
In the present study four bacterial strains were tested: Two Gram-negative (Pseudomonas aeruginosa ATCC10145 and Escherichia coli CECT434) and two Gram-positive (Staphylococcus aureus and Listeria monocytogenes). These strains were obtained from American Type Culture Collection (ATCC) and from the Spanish Type Culture Collection (CECT). There are several studies on the effects of essential oils with the same bacteria but with different strains (Cimanga et al., 2002;Chung et al., 2007).

Antibacterial Assay
Antibacterial activity was tested using the disc diffusion method describe by Bauer et al. (1966) with some adjustments. Colonies of bacteria were picked from 24 h cultures in BHI solid medium, inoculated into 4 mL of 0.9% NaCl solution. The cultures were adjusted to 0.5 McFarland standards. A loop of bacteria from the agar-slant stock was cultured in nutrient broth overnight and spread with a sterile cotton swab into Petri dishes (90 mm of diameter) containing 20 mL of Mueller-Hinton Agar (Oxoid). Sterile filter paper discs (6 mm in diameter) (Oxoid) impregnated with 10 µL of the EO were placed on the agar plate seeded with respective bacteria and the plates were incubated in an inverted position 24 h at 37°C. The equivalent volume of solvent, dimethyl sulfoxide (DMSO), served as negative control. Gentamicin (10 µg/disc) and ciprofloxacin (5 µg/disc) (Oxoid) were used as positive control. The results were obtained by measuring the diameter in mm of the inhibitory or clear zones around the disc (Saavedra et al., 2010). All tests were performed in triplicate and the antibacterial activity was expressed as the mean of inhibition zone diameters (mm).
The data were analyzed using the statistical program SPSS version 14.0 (Statistical Package for the Social Sciences). The mean and standard deviation within samples were calculated for all cases. Because low sample numbers contributed to uneven variation, nonparametric Wilcoxon test was used. Statistical calculations were based on confidence level equal or higher than 95% (p<0.05 was considered statistically significant).

Determination of Antimicrobial Potential
The bacteria to be tested (the three strains susceptible to the EOs tested) were picked from overnight cultures in Brain Heart agar (Oxoid). The method used is one previously reported with minor modifications (Sarker et al., 2007). Since we are using dilutions of essential oils we chose to use the term of Antimicrobial Potential (AP instead of Minimum Inhibitory Concentration (Table 3). A small portion of bacteria was transferred into a bottle with 50 mL of Mueller Hinton broth (Oxoid), capped and placed in an incubator overnight at a 37°C. After 12-18 hours of incubation, the bacteria suspension was adjusted, using aseptic preparation, in order to match the optical density in the range of 0.5-1.0 that was measured at 500 nm. The resazurin solution was prepared by dissolving a 270 mg tablet in 40 mL of sterile distilled water. A vortex mixer was used to ensure that it was a well-dissolved and homogenous solution, followed by dilution until 50% with sterile distilled water. The plates used were prepared under aseptic conditions (96 well plate, Orange Scientific). A volume of 100 µL of Mueller Hinton broth was used in each well together with 100 µL of essential oil in the first line. From the first well (belonging to the first horizontal line) were taken 100 µL, added to the next well and then this step is repeated to each of the following wells in the vertical line, allowing a serial dilution of decreasing concentration. For each essential oil were considered the percentages of the dilutions performed and therefore for the pure essential oil was considered 100% and for the other concentrations 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78%. In each of the wells were also added 20 µL of the bacteria suspension and 20 µL of resazurin solution (50%). The plates were then placed in an incubator set at 37°C for 18-24 h. All tests were performed in triplicate and the Antimicrobial Potencial (AP) was then assessed visually by the colour change of the resazurin in each well (blue to pink in the presence of bacteria growth).

Antibacterial Screening
All 15 EOs tested had antibacterial activity at least in one of the studied bacteria (  (Aires et al., 2009) (Table 1), it was found that some of the tested EOs were more efficient than the antibiotic in the inhibition of bacteria growth. The EOs tested were moderately effective for all bacteria except to P. aeruginosa. The first five species were visibly effective against L. monocytogenes and S. aureus showing halos (mm) with more than twice of the ones found for traditional antibiotics.

Antimicrobial Potencial (AP)
The bacteria strains E. coli, S. aureus and L. monocytogenes that revealed sensitivity to almost all of the pure EOs (Table 1 and 2) were then tested against dilutions (50, 25, 12.5, 6.25, 3,13, 1.56 and 0.78% v/v) of these pure EOs in order to determine the Antimicrobial Potencial (AP) (

DISCUSSION
Plant EOs and extracts have been used in traditional medicine since remote time. Those products have also a particular interest in food preservation (Lis-Balchin and Deans, 1997), alternative medicine (Ben-Arye et al., 2011) and parasites control (Yang et al., 2004). It becomes of great importance to do extensive studies in order to determine if some EOs could be used to treat some bacterial infections or used as disinfectants in clinical environments. Eucalyptus species are some of the plants most described as efficient in antibacterial treatments of some infections such as those of the upper respiratory tract infections (Ben-Arye et al., 2011). Another main concern in modern medicine is drugresistant bacteria and its exponential growth, so the main objective of this assay is to find some compounds that can be used in complementary medicine or as powerful disinfectants against pathogenic bacteria.
In the present in vitro study were tested all bacteria against antibiotics (gentamicin and ciprofloxacin) to determine their susceptibility ( Table 1). S. aureus is the most sensitive bacteria followed by E. coli, L. monocytogenes and P. aeruginosa in the case of gentamicin. Ciprofloxacin express more activity against E. coli, P. aeruginosa, S. aureus and L. monocytogenes, respectively.
There are hundreds of species of Eucalyptus, but in this study 15 were tested that are previously described in literature as the most economic and world distributed species (Cimanga et al., 2002;Chung et al., 2007;Francisco et al., 2001;Evtuguin et al., 2003;Freitas et al., 2008). Its importance at several industries, such as pharmaceutical, cosmetic and paper, justifies the necessity to develop more studies with Eucalyptus. There are several studies on the antimicrobial activity of Eucalyptus EOs (Cimanga et al., 2002;Chung et al., 2007;Lis-Balchin and Deans, 1997;Ghalem and Mohamed, 2008). The results of the present work show that Gram-positive bacteria are more sensitive to the EOs in general than Gram-negative bacteria. This fact can be related to the different cell wall structure of Grampositive and Gram-negative bacteria (Gootz, 2010). The present in vitro study also showed that the Eucalyptus EOs inhibited bacterial growth but their effectiveness varied. The most active EOs were E. bosistoana, E. botryoides, E. camaldulensis, E. cinerea and E. citriodora, which induced total inhibition of bacterial growth in L. monocytogenes and S. aureus. A previous study (Lis-Balchin and Deans, 1997) with L. monocytogenes revealed that E. citriodora and E. radiata caused a total inhibition of every strains tested. The EO of E. camaldulensis and E. globulus have a stronger activity in S. aureus than E. coli from clinical isolates (Ghalem and Mohamed, 2008). The chemical composition of this EO is very heterogeneous because E. camaldulensis is rich in 1,8-cineole and E. citriodora only has 1.2% of this molecule. On the other hand, E. citriodora is rich in citronellal (72.7%) and the EO of E. camaldulensis does not possess this compound (Cimanga et al., 2002). So it is probable that this bactericide effect is due to the presence of some minor compound of the Eucalyptus EO. Previously authors indicated that globulol (an EO minor component) extracted from E. globulus leaf has a strong activity against fungus such as Alternaria solani, Fusarium graminearum, Rhizoctonia solani, Venturia pirina and some bacteria like Xanthomonas vesicatoria and Bacillus subtilis (Manliang et al., 2008). Other authors also relate antimicrobial activity of globulol from E. globulus L. fruits (Tan et al., 2008). Other cause can be the fact that the composition of each essential oil depends on the age of the plant, climate region, season of the year, EOs extraction method, (Cimanga et al., 2002).
P. aeruginosa is one of the most difficult bacteria to treat in hospital infections. The results found in Table 1 demonstrate that this bacteria, in particular this strain have a strong resistance to all EOs tested. But there are some works in which P. aeruginosa is sensitive to peppermint, orange and Eucalyptus EO (Cimanga et al., 2002).
Previous studies reported an inhibition zone (7-20 mm) of ten Eucaliptus species EOs (Cimanga et al., 2002) against E. coli with higher values, while against S. aureus the values found (8-25 mm) were within the range of the values presented in our study. This previous report, when comparing the strains S. aureus and E. coli, revealed the same tendency shown in the present study with higher inhibition zone attributed to S. aureus. Other authors reported values of minimum inhibitory concentration for E. globulus EO of 125 µg mL −1 against S. aureus and 250 µg mL −1 for E. coli (Dessi et al., 2001). When comparing the values obtained for these two bacteria, this last study reveals the lowest MIC for S. aureus, while in the present study (Table 3) the lowest AP was found for E. coli (E. globulus EO). Lis-Balchin and Deans (1997) previously reported the effect of EOs from E. radiata and E. citriodora showing antimicrobial effect against several strains of L. monocytogenes.

CONCLUSION
The 15 Eucalyptus EOs revealed a strong activity against Gram-positive bacteria which may indicate that in their constituents there are some compounds that can be used as a pharmacological active ingredient or as a main component of a powerful antiseptic disinfectant for clinical surfaces. In general, our results reveal a high susceptibility of the pathogenic bacteria tested to the 15 Eucalyptus EOs, with exception of P. aeruginosa. The development of clinical surfaces disinfectants from the Eucalyptus EOs may be of great importance, as alternatives to other chemical products that are currently being used, that are known for their toxicity for the human health. This is a preliminary study and therefore it is necessary in future to perform more studies to evaluate the level of toxicity of these EOs regarding its antibacterial action since the current applications are more focused on topical use as an antiseptic, as well as the chemical composition of the EOs tested and also evaluate their potential application as disinfectants or even antibacterial agents.

ACKNOWLEDGEMENT
The reachers acknowledge the Centro de Biotecnologia Vegetal, Instituto de Biotecnologia e Bioengenharia (Universidade de Lisboa, Faculdade de Ciências de Lisboa, Departamento de Biologia Vegetal, C2, Campo Grande, 1749-016 Lisboa, Portugal) for the kind supply of Eucalyptus species essential oils. The authors also acknowledge the financial support provided by the Portuguese Foundation for Science and Technology (Carla Dias-SFRH/BGCT/33354/2008 and Maria do Carmo Vasconcelos-SFRH/BPD/70310/2010).

Author's Contributions
All authors equally contributed in this work.

Ethics
This article is original and contains unpublished material. The corresponding author confirms that all of the other authors have read and approved the manuscript and no ethical issues involved.