In vitro antibacterial activities of p-toluenesulfonyl-hydrazinothiazoles and hydrazinoselenazoles against multi-drug resistant Gram-negative phenotypes

Background Bacterial multidrug resistance (MDR) constitutes a major hurdle in the treatment of infectious diseases worldwide. The present study was designed to evaluate the antibacterial activities of synthetic p-toluenesulfonyl-hydrazinothiazoles against multidrug resistant Gram-negative bacteria. Methods The broth microdilution method was used to determine the minimal inhibitory concentrations (MIC). Results The results demonstrated that the best activities were obtained with hydrazinoselenazoles. p-Chloro-benzyliden-selenosemicarbazide, 4-methyl-2-[(4-chloro-benzyliden)-hydrazinyl]-1,3-selenazole, p-chloro-benzoyl-selenosemicarbazide and 4-chloromethyl-2-[(4-chlorobenziliden)-N-acetyl-hydrazinyl]-1,3-selenazole were more active than the choramphenicol on Klebsiella pneumoniae KP63. Tested alone, the lowest MIC value of 16 mg/L was obtained with p-methoxy-benzyliden-selenosemicarbazide against Enterobacter aerogenes ATCC13048, K. pneumoniae ATCC112296 and KP55. Tested in the presence of an efflux pump inhibitor, phenylalanine arginine β-naphthylamide (PAβN), the activity of p-chloro-benzyliden-selenosemicarbazide, 4-methyl-2-[(4-chloro-benzyliden)-hydrazinyl]-1,3-selenazole, p-chloro-benzoyl-selenosemicarbazide and p-methoxy-benzyliden-selenosemicarbazide significantly increased with MIC values below 10 mg/L obtained respectively on 43.8 %, 31.3 %, 62.5 % and 100 % of the 16 tested bacterial strains. The lowest MIC value of 0.5 mg/L in the presence of PAβN was recorded with p-chloro-benzoyl-selenosemicarbazide against Escherichia coli ATCC8739 and KP55 as well as p-methoxy-benzyliden-selenosemicarbazide against E. aerogenes KP55. p-Chloro-benzyliden-selenosemicarbazide and p-chloro-benzoyl-selenosemicarbazide contained the same pharmacophore as p-methoxy-benzyliden-selenosemicarbazide. Conclusion This study indicates that p-chloro-benzyliden-selenosemicarbazide, p-chloro-benzoyl-selenosemicarbazide and p-methoxy-benzyliden-selenosemicarbazide could be explored more to develop novel antimicrobial drugs to fight MDR bacterial infections.


Background
Bacterial multidrug resistance (MDR) constitutes a major hurdle in the treatment of infectious diseases worldwide. Bacterial efflux systems, especially AcrAB-TolC pumps in Enterobacteriaceae or MexAB-OprM in Pseudomonas aeruginosa, are involved in multidrug resistance of pathogenic Gram-negative bacteria [1][2][3][4][5]. Clinically, the continuous emergence of Gram-negative MDR bacteria drastically reduces the efficacy of the antibiotic arsenal leading globally to an increase of the frequency of therapeutic failure [6]. Consequently, new antibacterials are needed to fight these bacterial pathogens, but progress in developing them has been slow [7]. p-Toluenesulfonyl-hydrazinothiazoles and hydrazinoselenazoles are synthetic compounds with established anticancer, analgesic and anti-inflammatory activities [8][9][10][11]. The aim of the present work was to determine the antibacterial activities of a panel of p-toluenesulfonyl-hydrazinothiazoles and hydrazinoselenazoles against different bacterial strains expressing MDR phenotype. Furthermore, we highlighted possible pharmacophoric cores amongst the active compounds.

Microbial strains and culture media
The studied microorganisms included sensitive and resistant strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes, Escherichia coli obtained from the American Type Culture Collection (ATCC) as well as clinical strains. Their bacterial feature were previously reported [5,[14][15][16]. Nutrient agar were used for the activation of the tested Gram-negative bacteria [17].

INT colorimetric assay for MIC and MBC determinations
The Minimum Inhibitory Concentration (MIC) determinations on the tested bacteria were conducted using rapid p-iodonitrotetrazolium chloride (INT) colorimetric assay according to described methods [12] with some modifications [18,19]. The tested samples and RA were first of all dissolved in DMSO/Mueller Hinton Broth (MHB). The final concentration of DMSO was lower than 2.5 % and does not affect the microbial growth [20,21]. The solution obtained was then added to Mueller Hinton Broth, and serially diluted two fold (in a 96-wells microplate). One hundred microlitre (100 μL) of inoculum 1.5 x 10 6 CFU/mL prepared in appropriate broth was then added [18,19]. The plates were covered with a sterile plate sealer, then agitated to mix the contents of the wells using a plate shaker and incubated at 37°C for 18 h. The assay was repeated thrice. Wells containing adequate broth, 100 μL of inoculum and DMSO to a final concentration of 2.5 % served as negative control. The MIC of samples was detected after 18 h incubation at 37°C, following addition (40 μL) of 0.2 mg/mL of INT and incubation at 37°C for 30 min. Viable bacteria reduced the yellow dye to a pink. MIC was defined as the sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth [12]. To evaluate the role of efflux pumps in the susceptibility of Gram-negative bacteria, compounds were tested in the presence of PAßN (at 30 μg/mL) and MIC was determined as above.

Activity of hydrazinoselenazoles
The best activities were obtained with p-toluenesulfonylhydrazinothiazoles; When they were tested at up to 256 mg/L, hydrazinoselenazole displayed selective antibacterial activities. Compound    The lowest MIC value of 0.5 mg/L was recorded with compounds 14 against E. coli ATCC8739 and KP55 as well as 16 against E. aerogenes KP55 (Table 1). When they were combined with EPI, hydrazinoselenazoles 12, 13, 14 and 16 were more active than choramphenicol on at least two of the tested bacterial species.

Structure activity relationship and identification of pharmacophoric moiety
When regarding the structure-activity relationship, it appears that amongst the tested compounds, p-toluenesulfonyl-hydrazinothiazoles were less active than hydrazinoselenazoles. Within p-toluenesulfonyl-hydrazinothiazoles, it was also found that compound 8 scaffold is the most active and that any modification of its structure resulted to a decrease of antibacterial activity. Within hydrazinoselenazoles, it was also found that compound 16 [active on 14/16 (87.5 %) and 16/16 (100 %) tested bacteria in the absence or presence of EPI respectively; MIC values below 10 mg/L on all tested bacteria in the presence of EPI and lowest MIC value of 0.5 mg/L] can be considered as the phamacophoric moiety. Within this moiety, the presence of a free selenoamide is primordial for the antibacterial activity, as compounds 12 and 14 (bearing a free selenoamide) were also very active, especially when they were combined with EPI (Fig. 2). The presence of chloride substituent seems to increase the affinity of hydrazinoselenazole 12 and 14 to bacteria efflux pumps. In fact in the absence of EPI, the activity of  Fig. 2 Representation of pharmacophoric groups detected in the tested compounds compounds 12 and 14 (with chloride substituent) were much lower than that of 16 (with -OCH 3 substituent).

Discussion
Bacterial multidrug resistance represents a major problem in the treatment of infectious diseases. In the present study, we tested a panel of bacterial strains including both reference ATCC strains and MDR phenotypes expressing active efflux pumps [4,5,22]. In fact, tripartite drug efflux pumps, mainly those clinically reported as AcrAB-TolC in Enterobacteriaceae or as MexAB-OprM in P. aeruginosa tested in the present study, play a key role in multidrug resistance of pathogenic Gram-negative bacteria [23,24]. p-Toluenesulfonyl-hydrazinothiazoles and hydrazinoselenazoles are synthetic compounds that previously displayed good anticancer, analgesic and antiinflammatory activities [8][9][10][11]. Herein, we investigated the ability of such compounds to combat MDR Gramnegative bacteria expressing active efflux pumps. As results, it was found that hydrazinoselenazoles were much more active than p-toluenesulfonyl-hydrazinothiazoles. However, all the tested chemicals were substrates of bacterial efflux pumps. Interestingly, when efflux pumps were blocked by EPI, significant increase of the activity of three compounds 12, 14 and 16 was observed. More importantly, the obtained activity was better than that of the reference drug chloramphenicol in many cases ( Table 1), suggesting that the three compounds can be used in combination with PAβN to fight bacterial infections involving MDR phenotypes. The antimicrobial activity of a compound has been defined as significant when MIC is below 10 mg/L, moderate when 10 mg/L < MIC < 100 mg/L or low when MIC > 100 mg/L [25,26]. In this study, the MIC values below 10 mg/L was noted with hydrazinoselenazoles 12, 14 and 16 on 43.8 %, 62.5 % and 100 % tested bacterial strains when they were tested in the presence of EPI, highlighting their possible use in combination with PAβN in the control of MDR bacterial infections.
Regarding the involvement of MDR bacteria in treatment failures and the re-emergence of infectious diseases [1,27,28], these findings can be considered very promising. In fact, Pseudomonas aeruginosa is an important nosocomial pathogen highly resistant to clinically used antibiotics, causing a wide spectrum of infections and leading to substantial morbidity and mortality [29] and was found sensitive to the three compounds. MDR Enterobacteriaceae, including K. pneumoniae, E. aerogenes, P. stuartii and E. coli, have also been classified as antimicrobial-resistant organisms of concern in healthcare facilities [28,30].

Conclusions
Regarding the medical importance of the studied microorganisms, the results obtained in this study demonstrated that hydrazinoselenazoles are strong source of inspiration in antibacterial drug discovery. Thus, the present data showed that hydrazinoselenazoles 12, 14 and 16 are promising antibacterial agents to combat MDR phenotypes, but are also substrates of efflux pumps. They should therefore be combined with an EPI to combat MDR phenotypes. We also found that compound 16 skeleton was the pharmacophoric moiety of the tested hydrazinoselenazoles and that the presence of a free selenoamide scaffold is necessary for their antibacterial activity. However, more compounds should be further synthesized to fully investigate the structure-activity relationship for this series of hydrazinoselenazoles. Toxicological studies will also be performed to ensure the safety of the active hydrazinoselenazoles, especially compound 16.