Lantana montevidensis Briq improves the aminoglycoside activity against multiresistant Escherichia coli and Staphylococcus aureus.

OBJECTIVE: In this work, we report the antibacterial and modulatory activity of Lantana montevidensis Briq.
MATERIALS AND METHODS: The antibacterial activities of leaf (LELm) and root (RELm) extracts alone or in association with aminoglycosides were determined by a microdilution test. Multiresistant strains of Escherichia coli (Ec 27) and Staphylococcus aureus (Sa 358) were used.
RESULTS: The results show the inhibitory activity of LELm against E. coli (minimal inhibitory concentration [MIC] 16 μg/mL) and S. aureus (MIC 128 μg/mL). The synergistic effect of the extracts and aminoglycosides was verified too. The maximum effects were obtained with RELm with gentamicin against E. coli with MIC reduction (312 to 2 μL).
CONCLUSION: The data from this study are indicative of the activity antibacterial of extracts of L. montevidensis and its potential in modifying the resistance of aminoglycosides.

Introduction

The search for new antibacterial agents is important due to the progressively increasing resistance of clinically important pathogens to known classes of antibiotics.[1] With the increased incidence of resistance to antibiotics, natural products from plants could be an interesting alternative.[23] In the past years, many plants have been evaluated not only for antimicrobial activity, but also for resistance-modifying agents.[45]

Resistance occurs due to random genetic mutations in the bacterial cell that alter its sensitivity to a single drug or to chemically similar drugs through a variety of mechanisms.[6] Many bacteria are able to develop changes in their sensitivity, but Staphylococcus aureus and Escherichia coli have been recognized for the increasing resistance to conventional antibiotics.

S. aureus has persisted as one of the most important hospital and community pathogens; apart from causing different kinds of intoxication, it is usually involved in diverse tissue and/or organ infections.[7] E. coli is one of the microorganisms that has been associated with intestinal and urinary tract infections. Pathogenic and commensal strains of E. coli have different rates of resistance and can carry different genes.[8]

Lantana is a genus of about 150 species of perennial flowering plants popularly used as antirheumatics, stimulants, antibacterials, biologic controls, and as ornamental plants.[9-12] Phytochemical studies of the Lantana species led to the isolation of triterpenes, steroids, and flavonoids.[13] Leaf extracts of Lantana have shown a broad spectrum of biological activities.[14-17] Lantana montevidensis Briq (Verbenaceae), a shrub native to Brazil and Uruguay, is popularly known as “chumbinho.” It was introduced in many countries as an ornamental plant and considered as an invasive species in many parts of the world. The leaf infusions are used in folk medicine to treat fever, influenza, asthma, bronchitis, and many other diseases.[1018]

The methanolic leaf extract has shown an antiproliferative activity against tumor cells and the flavonoid rich fraction was effective against human gastric adenocarcinoma, human uterine carcinoma, and melanoma cell lines.[14] However, the antimicrobial modulatory activity of L. montevidensis extracts was not previously demonstrated and this justifies this work.

In the present study, the ethanolic leaf and root extracts of L. montevidensis from Cariri Cearense, Brazilian Northeast, were evaluated as modifiers of aminoglycoside resistance.

Materials and Methods

Plant Material

Leaves and roots of Lantana montevidensis Briq were collected in March, 2009, from the Small Aromatic and Medicinal Plants Garden of the Natural Products Research Laboratory (LPPN) at University Regional do Cariri (URCA), Crato of county, Ceará state, Brazil. A voucher specimen was sent to the Herbarium Caririense Dárdano de Andrade Lima (HCDAL), Department of Biological Sciences (URCA), which is deposited on the registration no. 1619.

Preparation of Extracts

The LELm and RELm were prepared using the cold extraction method.[19] A total of 400 g of the fresh leaves and 714 g of the roots was placed in a flask containing cold ethanol and left for 72 h at an ambient temperature. A rotary vacuum pump extractor was used to remove the ethanol from the extracts (under reduced pressure, 80ºC). The extracts were weighed and stored.

Antibacterial Activity and Minimal Inhibitory Concentration

The antibacterial activity of the LELm and RELm was investigated employing a microdilution method, recommended by National Committee for Clinical and Laboratory Standards M7-A6.[20] In the assay were used two multiresistant strains obtained from a clinical material: E. coli (Ec 27) from sputum and S. aureus (Sa 358) from a surgical wound.

The brain heart infusion (BHI 3.8%) broth was used for the bacterial growth (24 h, 35 ± 2ºC). The inoculum was an overnight culture of each bacterial species in the BHI broth diluted in the same medium to a final concentration of approximately 1 × 108 CFU/mL (0.5 NTU - McFarland scale). After this, the suspension was dilluted to 1 × 106 CFU/mL in 10% BHI. A total of 100 μL of each dilution was distributed in 96-well plates plus essential oils, achieving 5 × 105 CFU/mL as the final concentration of the inoculum.

The initial solution of the LELm and RELm was performed using 10 mg of each extract dissolved in 1 mL of dimethyl sulfoxide (DMSO) to obtain an initial concentration of 10 mg/mL. From this concentration, several dilutions were made in distilled water in order to obtain a stock solution of 1024 μg/mL. Further serial dilutions were performed by the addition of the BHI broth to reach a final concentration in the range of 8-512 μg/mL).

All experiments were performed in triplicate and the microdilution trays were incubated at 35 ± 2ºC for 24 h. The antibacterial activity was detected using a colorimetric method by adding 25 μL of the resauzurin staining (0.01%) aqueous solution in each well at the end of the incubation period. The minimal inhibitory concentration (MIC) was defined as the lowest extract concentration able to inhibit the bacteria growth, as indicated by resauzurin staining (dead bacterial cells are not able to change the staining color by visual observation – blue to red).

Antibiotic Modifying Activity

In order to evaluate of the LELm and RELm as modulators of antibiotic resistance, the MICs of aminoglycosides (neomycin, kanamycin, amikacin, and gentamicin) against the analyzed strains were determined in the presence or absence of the extracts using the microdutiltion test. Subinhibitory concentrations (MIC 1/8) in 10% BHI were used.

The antibiotic solutions (5000 μg/mL) were prepared in distillated water for use on the same day. A total of 100 μL of the antibiotic solution, using serial dilutions (1:2), was added to the wells containing 10% BHI and the diluted bacterial suspension (1:10). Microplates were incubated for 24 h at room temperature and the antibacterial activity was determined as described before.

Results

The antimicrobial properties of the extracts against two bacterial strains by using a microdilution assay for in vitro susceptibility testing was investigated. It was verified for the inhibitory activity, clinically relevant, against E. coli (MIC 16 μg/mL) and S. aureus (MIC 128 μg/mL), as shown in Table 1.

Table 1

Values of the minimal inhibitory concentration (MIC) for the leaf and root extracts of L. montevidensis

Table 2 shows effects of extracts on aminoglycoside activities (MIC ×1/8). Reduction in MICs for all analyzed antibiotics was observed, when the extracts were added to the culture medium. The maximum effect was seen for the activity of gentamicin on E. coli by RELm, with sevenfold reduction in the MIC (312 to 2 μg/mL).

Table 2

Minimal inhibitory concentration (MIC) values for the aminoglycoside in the absence and presence of the leaf and root extracts of L. montevidensis

Discussion

The antibacterial assay using chloroformic and methanolic extracts of L. camara leaves and three pentacyclic triterpenes isolated from hexanic extracts of L. hispida leaves showed an effective antibacterial activity against Mycobacterium tuberculosis.[1517] The ethanol extract of the leaves of L. camara was reported to exhibit an activity against S. aureus, Klebsiella pneumoniae, and E. coli.[21] Many triterpenes, naftoquinones, flavonoids, alkaloids, and glycosides isolated from the Lantana species are known to posses different biological activities, including antibacterial properties.[13]

Aminoglycosides are potent bactericidal antibiotics targeting the bacterial ribosome. Several mechanisms have evolved in bacteria which confer them with antibiotic resistance. These mechanisms can chemically modify the antibiotic, render it inactive through physical removal from the cell, or modify the target site so that it is not recognized by the antibiotic. In E. coli, the main mechanisms of resistance to aminoglycosides are active drug efflux and enzymatic inactivation.[2223]

Many substances were claimed to be modulators of the antibiotic activity, such as phenothiazines,[24] diterpenes,[25] flavones, and phenolic derivatives.[26] However, no single drug is reported to revert the aminoglycoside resistance.[27]

Several reports indicate different antibiotic combinations assayed in vitro and used in the clinics. However, the combinations of natural products and clinically used antibiotics are less reported. The data obtained in this study are indicative of the potential antibacterial and modulatory activity of the LELm and RELm.