Staphylococcus aureus is one of the most significant human pathogens responsible for nosocomial and community acquired infections. It can cause a range of infectious disease from mild conditions, such as soft tissue infections, to severe life-threatening debiliation, such as endocarditis 1. Despite the recent staphylococci infections, they are persisting as an important hospital and community pathogen 2. Methicillin resistance has become a major concern to the medical community due to the fact that they have an extraordinary ability to adapt rapidly to antibiotic stress 3. Among hospital isolates the frequency of methicillin resistant S. aureus (MRSA) is very high 4. There is need to have new chemicals for treatment of staphylococci infections.

The sulfonamides have, for many years, being widely studied for their chemotherapeutic activity. Their important role as antibacterial, antimalarial and antileprotic agents is well recognized 56. Recently, certain sulfonamides have been reported as showing interesting the antibacterial properties of sulfonamides have been extensively studied by Quantitive Structure-activity Relationship&Molecular Modeling (QSAR) method. 7. Antimicrobial therapy for infections with S. aureus often includes sulfonamides which are use to cure nosocomial infections 1. Sulfonamides are still an alternative option in order to cure methicillin resistant S. aureus (MRSA) staphylococci infections. Although the sulfonamide therapy has been reduced, owing to development of more effective antimicrobial agents and to the gradual increase in the resistance of bacterial species, clinical treatment with sulfonamides has undergone a revival by the combination sulfomethoxazole and trimethoprim.

Considering this background, the objective of this study some sulfonamides derivatives were tested in terms of antimicrobial activity with the purpose of revealing possible leading compounds for development of new antimicrobial agents against methicillin resistant S. aureus (MRSA) and methicillin sensitive S. aureus MSSA.

Methicillin-resistant staphylococci are resistant to all other penicillin, carbapenems, cephems and beta-lactam, beta-lactam inhibitor combinations 11. Consequently these antibiotics should not be used for treating of methicillin-resistant staphylococci infections. Moreover, recently several studies have shown that the methicillin-resistant staphylococci have started to gain resistance to some other widely used antibiotics (quinolone, macrolide group antibiotics, amino glycosides, tetracycline, trimetoprim sulphamethoxazole (SXT), clindamicin, chloramphenicol as well 12131415. The resistance increase trimetoprim-sulphametoxazole, which is an alternative medicine in the treatment of methicillin-resistant staphylococci infections, is recently received attention. Previously, trimetoprim-sulphametoxazole resistance has been shown to be 10–53% in Turkey while it was reportedly higher (47–79%) in European countries 16. Trimetoprim-sulphamethoxazole is used for the treatment of staphylococci infections. SXT has still maintain its' an alternative antibiotic potential for the treatment of MRSA and MSSA infections 1. In addition, sulphametizol, sulphamethaxozole or sulphisoxazole have been using for the treatment of E. coli urinary infection as a single antibiotics 1.

In this study, the in vitro antibacterial activity properties of the compounds tested on clinical isolates of 30 MRSA and 20 MSSA S. aureus by using new sulfonamide derivative compounds namely N-(2-Hydroxy-4-nitro-phenyl)-4-methyl-benzenesulfonamide(I), N-(2-Hydroxy-5-nitro-phenyl)-4-methyl-benzenesulfonamide, N-(5-Chloro-2-hydroxy-phenyl)-4-methyl-benzenesulfonamide(III>), N-(2-Hydroxy-5-methyl-phenyl)-4-methyl-benzenesulfonamide(IV). Some of the sulfonamides were found to be effective on S. aureus among the others. The strongest inhibition was detected by the effect of (N-(2-Hydroxy-4-nitro-phenyl)-4-metil-benzensulfonamide) (I). The similar results were obtained in previous studies against Nocardia species by the treatment of the same sulfonamide compound (Isik and Özdemir-Koçak, article in press in Microb. Res.)

First of all, we tried to find out (Minimal Inhibitory Concentrations) MIC values of sulfonamides derivatives against S. aureus isolates. The rate of MIC values showed alterations from 32 to 512 μg for 50 isolates. All data are given in Table 1 indicated that MIC values was not the same for all isolates i.e. showed variations in terms of resistance. It was given in the literature that treatment of some infections 300 μg sulfafurazol and sulfisoxazole ST are applied to patients 10. After determining the minimum and maximum MIC values, suitable concentration was selected for all isolates in order to proper comparison. It means, MIC values given in table 1 showed that sulfonamide derivatives I and II given here area potential antibacterial substances. It was also reported that I, and II numbered substances showed strong antibacterial activity against Staphylococcus aureus ATTCC 43300 and some other gram positive bacteria 17. Here tested compounds I, II, and III showed antibacterial activity against reference strain S. aureus ATTCC 29213 and their concentration were 32, 64 and 128 μg respectively. Compound I was found as a strongest antibacterial agent against S. aureus according to MIC values. The MIC values showed some alterations according to S. aureus strains given in Table 1.

Secondly, antimicrobial activity of compounds was tested according to disc diffusion method on the base of MIC values. 500 μg concentrations were chosen as a suitable concentration which showed an effect to all S. aureus isolates. All data related to inhibition zones against S. aureus were given in Table 2. As regard to results the strongest inhibition was observed in case of compound I. In general, the ratio of inhibition caused by Compound I, II and III were 84%, 50% and 36% respectively (Table 3).

Early and recent researchers have suggested that sulfonamides are useful for the treatment of some staphylococci infections, especially against urinary infections 1. In this study, some sulfonamides derivatives were tested in terms of antimicrobial activity with the purpose of revealing possible leading compounds for the development of new antimicrobial agents. Outcome of the study showed that sulfonamide derivatives I and II have proved to be effective enough, which is comparable with previous studies 17. It was reported that, sulfonamide I and II showed the highest inhibitory effect on gram positive bacteria i.e. S. aureus, N. asteroides, N. farcinia and B. subtilis. On the contrary, they did not lead to significant inhibitory effect on gram negative bacteria and also yeast and mould namely E. coli, P. aeruginosa, E. cloaceae, a yeast C. albicans and a mould A. niger 17.

Many attempts have been made to relate the antibacterial behavior of sulfonamides with molecular structure 6. It was shown here that antimicrobial activities of the sulfonamides were increased when introducing electron withdrawing groups into the benzene ring of the compounds (Table 2). The compound I has NO₂ group in para position according to the sulfonamide group which showed the strongest effect on the S. aureus. Ionization is important factor on antimicrobial effect of sulfonamides due to increasing solubility. The behaviors of o-substituted acids are often anomalous 18. Their strength is sometimes found to be much greater than expected due to direct interaction between the adjacent groups. For example, o-hydroxybenzoic acid is 10 times stronger than p-hydroxybenzoic acid. In the case of the sulfonamides, possibly the OH group in the ortho position stabilize the developing negative charge on the nitrogen through intramolecular hydrogen bonding, and in this way the ionization increases. As can be seen in table 2 the compound I is more effective against the bacteria possibly due to increasing ionization of N-H group which is enhanced by p-NO₂ group. OH group in ortho position is also supports ionization by involving intramolecular hydrogen bonding.

Introducing NO₂ group to meta position (compound II) reduces antimicrobial activity in general this is because of lesser effect of this position on N-H group. Chlorine substituted sulfonamide III has a weaker antimicrobial activity than NO₂ substituted ones due to less electron withdrawing ability of the chlorine. This is also supported by the results obtained by compound IV which has CH₃ group in meta position. Methyl group donates electron to the benzene ring therefore reduces the ionization dramatically of the compound. The correlation of antimicrobial activity with chemical facts of the current study are also in line with biological activity results 19.

Antimicrobial activities of the sulfonamides depend on substituent and their position in the benzene ring. While electron releasing group decreases, electron withdrawing groups enhanced the activity of the sulfonamides against S. aureus isolates. Although sulfonamide-based therapy is generally effective, optimal treatment could be guided by antimicrobial susceptibility testing of isolates. Moreover, experimental data show that compound I may also be considered as a broad spectral effective sulfonamide at 128 μg (78% in total isolates) against MRSA and MSSA S. aureus isolates. Although 30 out of 50 S. aureus isolates showed resistance to oxacillin antibiotic, 21 of them were susceptible mainly to compound I and II.

Sulfonamides are still an alternative option in order to cure MRSA staphylococci infections. However, increasing resistance against sulfonamides is a serious problem recently that has been taken attention. Therefore, there is need to have new chemicals for treatment of staphylococci and other bacterial infections. Outcome of the study has two noteworthy features. This study may help to suggest an alternative possible leading compound for development of new antimicrobial agents against MRSA and MSSA resistant S. aureus. It was also shown here that that clinical isolates of 50 S. aureus have various resistance patterns against to four sulfonamide derivatives. It may also be emphasized here that in vitro antimicrobial susceptibility testing results for S. aureus species need standardization with further studies and it should also have a correlation with in vivo therapeutic response experiments

MRSA: methicillin resistant S. aureus; MSSA: methicillin susceptible S. aureus; SXT: trimetoprim sulphamethoxazole; DMSO: dimethylsulfoxid

The authors declare that they have no competing interests.

All authors have read and approved the final manuscript. RÖ participated in the analysis of the data and coordinated and drafted the manuscript. YG carried out the antimicrobial activity tests and the data analysis. YB participate the production of sulfonamide derivatives and approving the final manuscript.