Modulation of the norfloxacin resistance in Staphylococcus aureus by Cordia verbenaceae DC

Some species of Staphylococcus are frequently recognized as agents of opportunistic infections12. S. aureus, S. epidermidis, S. saprophyticus and S. haemolyticus are the most important species responsible for human infections. S. aureus represents the main agent of purulent infections (e.g. osteomyelitis, furuncle, carbuncle, abscess, myocarditis, endocarditis, pneumonia, shunt-associated meningitis, bacterial arthritis)34.

There has been scientific interest in chemical and pharmacological investigations of the biological properties of medicinal plants5–8. Medicinal plants have been the source of many medications that are now applied in clinical practice. The use of extracts as antimicrobial agents demonstrated a low risk to the resistance development because these are complex mixtures9. Many plants have been evaluated not only for direct antimicrobial activity but also as resistance-modifying agents. Various chemical compounds, synthetic or from natural sources, have direct activity against many species of bacteria, enhancing the activity of a specific antibiotic, reversing the natural resistance of bacteria to specific antibiotics, causing the elimination of plasmids and inhibiting the active efflux of antibiotics through the plasma membrane15.

The genus Cordia is present in all tropical and subtropical zones worldwide10. Cordia verbenaceae DC is distributed in all regions of Brazil11. The bulk extract of aerial parts is used in folk medicine as a topical anti-inflammatory product and several studies demonstrated a phototoxic, antibacterial and modulatory effect of antibiotic activity12–14. The anti-inflammatory activity observed is thought to be due the flavonol artemetine15.

The aim of this study was to verify the phytochemical composition of the methanol and hexane extracts of C. verbenacea and determine the modulatory antibiotic activity of these extracts against the norfloxacin in S. aureus.

Material & Methods

Bacterial strains: The bacterial strains utilized were the clinical isolate S. aureus 358 (SA358) and the strains S. aureus ATCC25923 (SA-ATCC25923) and S. aureus 1199B (SA1199B). The strain 1199B overexpress the norA gene, encoding the NorA efflux protein, the main protein of the efflux pump responsible by the extrusion of norfloxacin and other biocides16. All isolates were maintained on slants with heart infusion agar (HIA, Difco Laboratories Ltd., USA). Before the assay, the cells were grown overnight at 37°C in brain heart infusion broth (BHI, Difco, USA).

Plant material: Leaves of C. verbenacea DC. were collected in the county of Crato, Ceará, Brazil. The plant material was identified and voucher specimens were deposited in the Herbario Prisco Bezerra of Universidade Federal do Cearα - UFC, as N°. 044171.

Preparation of methanol and hexane extracts of C. verbenaceae DC.: For the preparation of the extracts, leaves were collected and submersed in two reservoirs with methanol or hexane for 72 h. The extract was filtered and concentrated under vacuum using a rotary evaporator (model Q-344B - Quimis, Brazil) and an ultrathermal bath (model Q-214M2 - Quimis, Brazil)1. Overall, 31.4 g of leaves yielded 1.74 g of methanol extract (MECV) and 107.47 g leaves yielded 1.72 g of hexane extract (HECV). The extracts solutions utilized in the tests were dissolved using DMSO (Dimethyl Sulphoxide) to a concentration of 10 mg/ml and diluted with distilled water to obtain a final concentration of 1024 μg/ml.

Phytochemical tests: The phytochemical tests to detect the presence of heterosides, saponins, tannins, flavonoids, steroids, triterpenes, cumarins, quinones, organic acids and alkaloids were performed according to the methods described by Matos17. The tests were based on the visual observation of colour changes and observations of precipitates after the addition of specific reagents (Table I).

Table I Phytochemical prospection of methanol and hexane extracts of C. verbenaceae DC

Drugs: Norfloxacin was purchased from Bayer S.A., Brazil, and all the other drugs were purchased from Sigma Chemical Co., USA.

Antibacterial test (MIC): MIC (minimal inhibitory concentration) of extracts was determined using the microdilution assay18. Bacterial inoculum (100 μl) was suspended in BHI 10 per cent to a final concentration of 10⁵ cfu/ml and distributed in 96-well microtiter plates. Each well received 100 μl of each extract solution. The final concentrations of the extracts varied between 512-8 μg/ml using two-fold serial dilutions18. MICs were recorded as the lowest concentrations of the extracts required to inhibit growth. The same method were used to evaluate the MIC of norfloxacin, with a drug concentration ranging between 2.5 to 0.0012 mg/ml18. The plates were incubated for 24 h at 37 °C.

Modulation assay: To evaluate the modulatory antibiotic activity of the extracts, the MIC of antibiotic was determined in the presence or absence of MECV and HECV using a sub-inhibitory concentration (MIC/8 = 32 μg/ml). The modulation assay was realized following the plate modulation method, as described by Stavri et al19. All experiments were performed in triplicate.

Results & Discussion

Methanol extract of C. verbenaceae DC showed MIC of ≥1024 μg/ml for both strains SA 358 and SA-ATCC 25923. Hexane extract gene ≥1024 μg/ml MIC for SA358 and 256 μg/ml for SA-ATCC 25923 strain. The results demonstrated that HECV was more effective against the assayed bacteria.

Table II shows the modulatory antibiotic activity of the extracts when associated with norfloxacin. When the extracts were incorporated into the growth medium with a sub-inhibitory concentration of 32 μg/ml, an enhancement of 50 per cent in the inhibition zone was observed as compared with the antibiotic alone.

Table II Enhancement of the inhibition zone by C. verbenaceae

With the increase of the bacterial resistance to antibiotics, the use of natural products from plants could represent an interesting alternative1. Some plant extracts and phytochemicals are known to have antimicrobial properties. Several studies have been conducted in different countries, demonstrating the efficacy of this approach20. Many plants have been evaluated not only for direct antimicrobial activity but also as resistance modifying agents21.

The antibacterial activity of C. verbenacea was demonstrated against Gram- positive and -negative bacteria14, as well as the phototoxic and modifying antibiotic activity against aminoglycoside, another class of antibiotics that inhibits the protein synthesis1213. These activities can be related with the presence of phytocompounds with low polarity as tannins, flavonoids and terpenes. These compounds are usually found in higher concentrations on apolar fractions or extracts, as the hexane extract of C. verbenacea. That could be the reason for a better antibacterial and modulatory activity of HECV, with lower MIC values and a better enhancement of norfloxacin activity.

About the tannins, the antimicrobial properties appear to be associated with the hydrolysis of an ester bond with gallic acid, thereby serving as a mechanism of natural defence against microbial infections. The tannins, epicatechin and catechin from Vaccinium vitisidaea L. demonstrated a strong antimicrobial activity against bacteria and fungi22. Flavonoids are synthesized by plants in response to microbial infection23 and are effective against a broad range of microorganisms. The activity is probably due to their capacity to form complexes with extracellular soluble proteins associated with the bacterial cell wall. Some lipophilic flavonoids can also cause rupture of the plasma membrane of microorganisms24. Terpenes occur in the form of diterpenes, triterpenes, tetraterpenes as well as hemi- and sesquiterpenes. Terpenenes or terpenoids are active against bacteria2526.

The mechanisms by which extracts can inhibit the growth of microorganisms are varied, and can be due to the hydrophobic nature of some components. Due to this fact, these can show a higher interaction with the lipid bilayer of the cell membrane, affecting the respiratory chain and production of energy27 or even enhancing the permeability of the cell membrane to antibiotics2829. The interference with bacterial enzyme systems can also be a potential mechanism of action30. These mechanisms of action may be due to combination of antibiotic with extract at a sub-inhibitory concentration applied directly to the culture medium15. This synergy approach may lead to the development of a new generation of phytopharmaceuticals31.

In conclusion, the results indicated that C. verbenacea DC. could serve as a source of plant-derived natural products that modify antibiotic resistance for use against multidrug-resistant bacteria.