Determination of anti-staphylococcal activity of thymoquinone in combinations with antibiotics by checkerboard method using EVA capmat™ as a vapor barrier

1 Introduction

Since their discovery, antimicrobial drugs have proved to be remarkably effective for the control of bacterial infections. However, it was soon evident that these pathogens are able to become resistant to many of the first effective drugs (Gold and Moellering, 1996). Staphylococcus aureus strains cause a broad spectrum of diseases, ranging from minor skin diseases to serious bloodstream infections (Daum, 2008). In the past, this bacterium has been successfully treated with penicillins, but many strains build up the resistance to ß-lactam antibiotics by the production of ß-lactamase (Andremont et al., 2011). Initially, the problem of bacterial resistance to antimicrobial drugs was solved by the discovery of new classes of antibiotics, such as the aminoglycosides, macrolides, and glycopeptides, as well as by the chemical modification of previously existing drugs (Gold and Moellering, 1996). Nevertheless, S. aureus has the ability to acquire resistance to practically all useful antibiotics and become multidrug-resistant (Gibbons, 2004).

One of the potential strategies how to overcome this problem in the treatment of staphylococcal infections is the use of antibiotics in combinations with other compounds (Drago et al., 2007). The best-known example of such a combination is the comedication of the ß-lactam antibiotics with clavulanic acid, which successfully aborts gained resistance (Wagner, 2011). In a number of previous studies, for example as reviewed by Hemaiswarya et al. (2008), plant substances were also observed to inhibit bacterial resistance and to potentiate synergistically the effect of conventional antimicrobial agents.

Thymoquinone (2-isopropyl-5-methyl-1,4-benzoquinone) (Tq) (Fig. 1) is one of the main bioactive constituent of Nigella sativa L. seeds (black seed or black cumin), which have been used traditionally for thousands years in the Middle East, Northern Africa and India as a natural remedy for various infectious diseases such as cough, bronchitis, pulmonary infection, fever, and influenza (Kokoska, 2011). Tq is also found in several medicinal plant species belonging to the other genera such as Monarda and Thymus (Taborsky et al., 2012). Besides its antimycotic, anti-oxidant, anti-inflammatory and anticancer effect, Tq exhibits significant antimicrobial activity against both Gram-negative and Gram-positive bacteria (Kokoska, 2011), including methicillin resistant S. aureus (MRSA) (Liu et al., 1996). Furthermore, it has recently been reported to potentiate the activity of some antibiotics. Halawani (2009) demonstrated synergism between Tq and common antibiotics (ampicillin, cephalexin, chloramphenicol, tetracycline, gentamicin, and ciprofloxacin) against S. aureus strains used disk diffusion technique. However, the general procedure for detecting synergy is the standard checkerboard method, in which two compounds are tested in serial dilutions and in all combinations of these dilutions together to find the concentrations of each compound, both alone and in combination, that produce some specified, easily determined effect. The interaction is then determined algebraically (according to its fractional inhibitory concentrations, FICs) or geometrically (Berenbaum, 1978; EUCAST, 2000). In another study, Kouidhi et al. (2011) reported increasing activity of tetracycline and benzalkonium chloride when combined with Tq at ½ of its minimum inhibitory concentration (MIC) using the microtiter plates assay. Nevertheless, our previously reported experiments suggest that the results of standard microdilution test can be influenced by Tq vapors that are able to strongly affect concentrations in adjoining wells on microtiter plate, which was confirmed by GC/MS analysis (Novy et al., 2014). With the aim of preventing this phenomenon, in this study we examined in vitro anti-staphylococcal effect of Tq in combination with oxacillin, penicillin, or tetracycline against nine strains of S. aureus including resistant strains by the broth microdilution method, where the microtiter plates were especially covered by ethylene vinyl acetate (EVA) cap mats.

Close

Figure 1

Chemical structure of thymoquinone.

Export to PPT

2 Materials and methods

3 Results and discussion

A comparison of results from EVA capmat™ covered and non-covered plates (Fig. 2) showed significant differences in the MIC values of tested compounds. In the case of the non-covered plates, it was impossible to calculate the MIC for Tq, because even the wells with the lowest concentration tested were without any bacterial growth. Moreover, the MIC of positive control oxacillin (0.03125 μg/ml) did not correspond with the CLSI (2009) limit (0.12–0.5 μg/ml), as it was 3.84 fold lower than the minimum of the recommended concentration range. On the other hand, when tested covered by EVA capmat™, Tq exhibited MIC value of 64 μg/ml and oxacillin MIC (0.25 μg/ml) lay in the reference interval. In accordance with our previous findings that Tq vapors are able to affect the S. aureus susceptibility testing on microtiter plates (Novy et al., 2014), these experiments showed that the results of checkerboard assay can also be affected by this phenomenon.

Close

Figure 2

The design of experiments demonstrating the influence of thymoquinone (Tq) vapors on bacterial growth (Staphylococcus aureus, ATCC 29213) after 24 h incubation using checkerboard method with (A) and without (B) use of EVA capmat™. C: non-infected medium control (sterility control); G: infected medium control (growth control); Ox: oxacillin; Tq: thymoquinone; X: corners of plate not used; grey colored wells signify staphylococcal growth after 24 h incubation.

Export to PPT

For the detailed evaluation of combinatory anti-staphylococcal effect of Tq with antibiotics performed using EVA capmat™, the individual MICs of tested compounds as well as the MICs of their combinations with corresponding ΣFICs are summarized in Tables 1–3. The antimicrobial synergistic effect appeared in all combinations against at least one S. aureus strain. For Tq-oxacillin combination, synergy occurs against 3 out of 7 tested strains (ΣFIC 0.263–0.450). The resistance to oxacillin (MIC ⩾ 4 mg/L) (CLSI, 2009) was overcome in all these three strains. For Tq-penicillin and -tetracycline combinations synergy was acquired against 1 (ΣFIC 0.466), and 2 (ΣFIC 0.400–0.475) strains tested, respectively. Moreover, our results proved additive effect of Tq with all ATBs tested against majority of strains. According to our best knowledge, this is the first report of synergism between Tq and penicillin and oxacillin. Our findings support earlier reports on the enhancement of anti-staphylococcal activity of various classes of antibiotics by Tq and confirm its previously described synergistic effect with tetracycline (Halawani, 2009; Kouidhi et al., 2011). However, in view of the present study, we suggest that some specific results of the combinatory effect shown in previous reports can be influenced by Tq vapors, especially when tested at higher concentrations.

This study proved Tq as a compound able to potentiate anti-staphylococcal activity of oxacillin, penicillin and tetracycline and in some cases to overcome bacterial resistance. It is well known that the multidrug-resistance efflux pumps are one of the mechanisms responsible for a bacterial resistance to antibiotics, mostly to tetracyclines (Piddock, 2006). Kouidhi et al. (2011) discovered that Tq is able to cause efflux inhibition and therefore increases the intracellular concentration of 4,6-diamidino-2-phenylindole. Therefore we suggest that this mechanism could also contribute to the combinatory anti-staphylococcal effect of Tq-tetracycline, demonstrated in our experiments. On the other hand, staphylococcal resistance to β-lactams is caused mostly by the production of modified penicillin binding proteins (PBPs) with reduced affinity to antibiotics (Macheboeuf et al., 2006). Quinones are highly bioactive and make easily a complex with nucleophilic amino acids of proteins, often leading to inactivation of the protein and loss of function (Cowan, 1999). In view of those facts, we suggest that Tq may acts as an inhibitor of PBPs responsible for staphylococcal resistance.

From above mentioned, it can be assumed that Tq has different mode of action than tested antibiotics. In this case, some authors recommend to use a non-exclusive model for the ΣFIC calculation (ΣFIC = FICA + FICB + FICA ∗ FICB) (Miladinovic et al., 2013). Nevertheless when the data obtained in the present study were recalculated according to this suggested formula (data not shown), the results still showed many synergistic or additive effects even though the particular values slightly differ from the data calculated using exclusive model.

Although the application of quinones in drug development is subject of criticism because of their reactive unspecificity due to the redox potential (Baell and Walters, 2014), various quinone-based drugs are already used in pharmacological practices e.g. as laxative (Bruneton, 1999) and anti-inflammatory agents (Nguon et al., 2014). In addition, our previous study demonstrated that redox potential alone could not fully explain all aspects of pharmacological action of quinones (Landa et al., 2012). In preliminary clinical trials, Tq has been reported to possess no side effects when administered orally at doses up to 2600 mg/day to adult patients with solid tumors or hematological malignancies (Al-Amri and Bamosa, 2009). However, in case of in vitro studies there are controversial results showing relatively low toxicity of Tq (Gali-Muhtasiba et al., 2006; Banerjee et al., 2009) as well as certain cyto- and genotoxic effects (Khader et al., 2009). In view of the previous findings, Tq can be indicated as a promising compound applicable for further experiments focused on combinatory anti-staphylococcal effect. However, detailed examination of the toxicological profile of Tq-antibiotic combinations should be done to check the safety prior to its potential practical use.

The essential oils and their volatile constituents are frequently tested for their antibacterial combinatory effects using standard checkerboard assay (Langeveld et al., 2014). However, as it has been shown in our study, the evaluation of these interactions can be affected by the vapors when tested on microtiter plate by this method. According to our results, EVA capmat™ significantly avoids spreading of Tq vapors into the wells on microtiter plate and thus seems to be efficient protection against such affection. Moreover, our findings suggest that the results of assays on Tq cytotoxicity or anti-oxidative activity (Woo et al., 2013), which were previously performed on microtiter plates without any vapor protection, can also be affected by its vapors. Therefore, this capmat might be useful also when performing other biological assays with Tq or another volatile compound. However, detailed research aimed at the development of effective method able to avoid such errors on microtiter plates when testing of volatile compounds is needed.

In conclusion, this study indicated Tq as a compound able to enhance anti-staphylococcal effect of some antibiotics. Moreover, according to our best knowledge, this is the first report on Tq synergy with oxacillin and penicillin against S. aureus, including methicillin-resistant strains. The results of combinatory antimicrobial effect of Tq can be helpful in the research targeted on the new preparations to overcome multidrug resistance in the antimicrobial therapy. In addition, our results indicated that EVA capmat™ is effective vapor barrier when the volatile compounds are tested. Generally, these findings can influence further use and the development of improved methods for testing of volatile constituents performed on microtiter plates, such as combinatory antimicrobial assay.

Author contributions

The authors Johana Rondevaldova, Pavel Novy, Jan Urban, and Ladislav Kokoska approved the final version of the manuscript and listed below are the individual contributions of each author to the paper. Johana Rondevaldova participated on project design, testing of anti-staphylococcal combinatory activity, data evaluation, and manuscript preparation. Pavel Novy participated on comparative experiment with and without EVA capmat™. Jan Urban was responsible for preparation and maintenance of S. aureus cultures. Ladislav Kokoska designed the research protocol, and managed and coordinated laboratory experiments and manuscript preparation.