Plant extracts display synergism with different classes of antibiotics

One manner in which plant-derived compounds exert their antibiotic potential is the synergism, a positive interaction between two compounds. Studies indicate that the use of plant extracts combined with antimicrobials may promote a significant reduction of the minimum inhibitory concentrations of antibiotics for bacterial strains. This study aimed to evaluate the activity of plant extracts and antibiotics as well as their combination on Staphylococcus aureus. The activity of 15 plant extracts was evaluated using diffusion assay. The minimum inhibitory concentrations (MICs) and the interactions between the extracts and antibiotics as well as compound emodin were evaluated with the checkerboard method. The active extracts were a hexane extract of the leaves of Baccharis dracunculifolia and the ethanol extracts of the leaves of Plectranthus ornatus, Inga edulis, Salvia officinalis and Senna macranthera. The Plectranthus ornatus extract displayed synergism with ampicillin (a β-lactam), kanamycin and gentamicin (aminoglycosides), with 8-fold reductions in the MIC. The same reduction was observed for the extracts of Salvia officinalis and Senna macranthera, which displayed the lowest MIC. Using these combinations resulted in a reduction in the minimum dose required for effective antimicrobial effects, which is interesting because it may decrease both the risk of side effects and the costs of treatment.


INTRODUCTION
The increase in bacterial resistance to currently known drugs is an issue of global concern and reinforces the need for new classes of antibacterial substances. Compounds

OBJECTIVE
Evaluate the activity of plant extracts and antibiotics as well as their combination on S. aureus isolates from animals affected with bovine mastitis.

Extract preparation
The leaves (500 g) of P. ornatus, Solanum cernuum, C. officinalis, C. citrates, S. officinale, I. edulis, J. cuspidifolia, A. indica, S. officinalis, B. orellana and O. basilicum (500 g) were extracted with ethanol (2 L) and the roots of S. macranthera (500 g) with dichloromethane for 120 h at room temperature. The solvent was removed under vacuum at 40 °C to obtain an ethanolic extract of each plant. The leaves of B. dracunculifolia, A. aculeate and seeds of H. anus were extracted with hexane by the same procedure. All extracts were stored at 4 °C.

Bacterial strains
S. aureus 3993 and 4125 strains, (identified by the Embrapa Dairy Cattle from the Milk Microbiology Laboratory) as an infectious reference microorganism which were isolated from animals with mastitis infections, were kindly provided by Embrapa/CNPGL, Juiz de Fora, MG, Brazil. The bacterial cultures were streaked on plates containing brain heart infusion agar (BHA; Himedia ®) and incubated for 16h at 37 °C. Müeller Hinton broth (Himedia ®) was used for testing antibacterial activities. The cell concentration was adjusted to 10 6 CFU mL −1 with an optical density set at 600 nm. Stock cultures were maintained in BHI agar containing 25% glycerol at -80°C.

Antimicrobial activity
Antimicrobial activity was assessed using the agar diffusion assay. For this purpose, 100 µL of a suspension containing 10 6 CFU.mL -1 was spread in Petri dishes containing Müeller Hinton agar (Himedia®). Holes of approximately 5 mm in diameter and 3 mm in height were created in the agar and were filled with 30 µL of the extracts at a concentration of 50 mg.mL -1 .The controls were prepared with 30 µL of DMSO and 5 mg.mL -1 of ampicillin (Sigma®, A9518). The plates were incubated for 24h at 37 ºC, and the subsequent inhibition zones were measured in mm. Inhibition zones greater than 7 mm were considered positive (NASCIMENTO et al., 2000). Student's t-test (p<0.05) was performed to compare the results of the inhibition zones obtained from the extracts with the positive control. Tests were performed twice in triplicate.

Minimal Inhibitory Concentration (MIC) assay
The activity of the extracts on bacterial growth was determined using the microdilution method described in CLSI (2003). The microorganisms were initially grown on BHI agar plates (Himedia®) and pre-incubated for 24 h at 37 ºC. Isolated colonies were then inoculated into Müeller-Hinton broth (Himedia®) and incubated at 37 ºC with shaking at 180 rpm until the exponential phase was reached. The culture was subsequently diluted to an optical density corresponding to the standard 0.5 on the McFarland scale (OD 620 =0.10). Microplate wells were filled with 100 µL of Müeller-Hinton broth that had extracts concentrations ranging from 0.1mg. ml -1 to 10 mg.mL -1 and 10 6 CFU.mL -1 of bacterial suspensions. Whereas the DMSO could be bactericidal a control of microbial growth in this solvent was done with 100 µL of bacterial suspension and 100 µL of Müeller-Hinton broth with DMSO at the highest concentration used in the preparation of the extract. After 24 h at 37 ºC, 4µL of p-iodonitrotetrazolium (INT, I8377, Sigma®) was added to each well, and the plate was incubated for an additional 2 h at 37 ºC. A change in the color of the medium from yellow to pink-violet was used as an indication of bacterial growth. The minimal inhibitory concentration of the antibiotics was determined by the same procedure, with concentrations ranging from 0.1 µg.mL -1 a 500 µg.mL -1 .

Evaluation of the interactions between plant extracts and antibiotics
The checkerboard method, which is commonly used for measurement of interactive

RESULTS
This study evaluated the antimicrobial potential of plant extracts from Viçosa, Minas Gerais, Brazil. The activity of these extracts was tested on strains of S. aureus isolated from cows affected with mastitis. Five of the 15 plant extracts tested displayed antimicrobial activity, as indicated by inhibition zones greater than 7 mm (Table I)  The MICs for the active extracts were determined (Table II). Among the plant extracts tested, S. macranthera displayed the lowest MIC (0.2 mg.mL -1 ), followed by S. officinalis (0.3 mg.mL -1 ). The highest MICs were observed for the extracts of P. ornatus (2.0 mg.mL -1 ) B. dracunculifolia (5.0 mg.mL -1 ) and I. edulis (7.0 mg.mL -1 ). In addition to the antimicrobial potential of plant extracts, in this study, we investigated the combined effect of the active extracts with antibiotics traditionally used to treat bovine mastitis. First, the MICs of five antibiotics, namely ampicillin, kanamycin, chloramphenicol, gentamicin, and tetracycline were determined (Table III). The checkerboard method was used to determine the interactions between the antibiotics and the active extracts. The concentrations of the extracts and antibiotics varied between 1/8 MIC to 2MIC when combined.
Based on the lower combined concentrations where bacterial growth inhibition was observed, the fractional inhibitory concentration index (FIC) was calculated and the type of interaction was determined (Table IV).
In almost all combinations of extracts and antibiotics tested, additive or synergistic interactions were observed. Only the interaction between S. officinalis and chloramphenicol was indifferent. No antagonistic interactions were detected.  The P. ornatus extract displayed synergism with ampicillin (β-lactam), kanamycin and gentamicin (aminoglycosides), with 8-fold reductions in the MIC. The same reduction was observed for S. officinalis extract, which demonstrated synergism with ampicillin, kanamycin, gentamicin and tetracycline but displayed an indifferent interaction with chloramphenicol. The same results observed for the S. officinalis extract were observed for the S. macranthera extract, except for the interaction between chloramphenicol and S. macranthera extract, which was additive.

DISCUSSION
S. aureus is an important human pathogen and also a relevant pathogen that causes diseases in animals. In dairy cattle this bacteria causes mastitis, an inflammation of the udder that causes significant economic losses worldwide. Antibiotic therapy is commonly used by  Based on the checkerboard assay, synergism was observed between the P. ornatus extract and ampicillin, kanamycin or gentamycin, with an 8-foldreductionin the MIC. The extract of S. officinalis also displayed synergism with ampicillin, kanamycin, gentamicin, and tetracycline, though the extract displayed an indifferent interaction with chloramphenicol. An 8-fold decrease in the MIC value was also detected. The same reduction was observed for the extract of S. macranthera; however, its interaction with chloramphenicol was considered additive. No specificity to a group of antibiotics was observed which suggests that the crude extract of these plants might contain a mixture of compounds that potentiate the activities of different antibiotics. These compounds facilitate a reduction in the minimum dose required for effective antimicrobial activity. This is interesting because smaller doses can reduce the chance of side effects as well as reduce the costs of treatment. However, for the purposes of therapeutic use, the mode of action of plant extracts should be explored togain a comprehensive view of the molecular mechanisms involved in their interactions.

CONCLUSION
Synergism against the two strains of S. aureus was observed between the extracts of P. ornatus, S. officinalis and S. macranthera and different antibiotics used in the treatment of bovine mastitis. The interactions displayed between the extracts of S. officinalis and S. macranthera and chloramphenicol were additive and between the extract of P. ornatus and chloramphenicol was indifferent. The present results are promising and may enhance the use of natural products instead of antibiotics or these can be used in association with active extracts to reduce the use of existing antibacterial drugs. Using these combinations can result in a reduction in the minimum dose required for effective antimicrobial effects, which is interesting because it may decrease both the risk of side effects and the costs of treatment of infectious diseases.