Inhibitory effect of N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide on Haemophilus spp. planktonic or biofilm-forming cells

During this study, we have investigated in vitro activity of N-substituted-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide derivatives with N-ethyl, N-(4-metoxyphenyl) and N-cyclohexyl substituents against Gram-negative Haemophilus influenzae and H. parainfluenzae bacteria. A spectrophotometric assay was used in order to determine the bacterial growth and biofilm formation using a microtiter plate to estimate minimal inhibitory concentration (MIC) and minimal biofilm inhibitory concentration (MBIC). Among the tested N-substituted pyrazole derivatives, only N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide showed a significant in vitro activity against both planktonic cells of H. parainfluenzae (MIC = 0.49–31.25 μg ml−1) and H. influenzae (MIC = 0.24–31.25 μg ml−1) as well as biofilm-forming cells of H. parainfluenzae (MBIC = 0.24–31.25 μg ml−1) and H. influenzae (MBIC = 0.49 to ≥31.25 μg ml−1). The pyrazole compound exerted higher inhibitory effect both on the growth of planktonic cells and biofilm formation by penicillinase-positive and penicillinase-negative isolates of H. parainfluenzae than the activity of commonly used antibiotics such as ampicillin. No cytotoxicity of the tested compound in vitro at concentrations used was found. The tested pyrazole N-ethyl derivative could be considered as a compound for the design of agents active against both pathogenic H. influenzae and opportunistic H. parainfluenzae, showing also anti-biofilm activity. This appears important because biofilms are determinants of bacterial persistence in long-term and recurrent infections recalcitrant to standard therapy.

Haemophilus spp. rods, generally known as Gram-negative microbiota of the upper respiratory tract, are able to live as planktonic cells or colonize natural and artificial surfaces as biofilm-forming cells Chin et al., 2005;Musk and Hergenrother, 2006;Galli et al., 2007;Moxon et al., 2008;Kosikowska and Malm, 2009;Murphy et al., 2007;Drago et al., 2012;Ü nal et al., 2012). Both pathogenic Haemophilus influenzae and opportunistic H. parainfluenzae can cause acute, chronic, invasive or non-invasive infections. These microorganisms may form a biofilm which is a virulence determinant which contributes to recurrent or chronic infections. H. influenzae is the most pathogenic bacteria colonizing the mucous membranes of the respiratory tract of young children or sporadically elderly people. H. influenzae, mainly serotype b (Hib), is frequently associated with different diseases, e.g., otitis media, chronic bronchitis, and pneumonia (Agrawal and Murphy, 2011). Other illnesses caused by this species include osteomyelitis, arthritis, sepsis, phlegmon cellulitis, or abscesses. Non-typeable H. influenzae (NTHi) is one of the main causes of airway infection in chronic obstructive pulmonary disease, of recurrent otitis media in infants and children, sinusitis in children and adults, pneumonia in adults, lower respiratory tract infection in adults, and recurrent respiratory tract infections in patients with chronic bronchitis (Murphy, 2003;Erwin and Smith, 2007).
To determine the power of the tested compound as an anti-biofilm agent, the MBIC/MIC ratio was assessed. The most frequently MBIC/MIC ratio ranged from 0.5 to 2 lg ml -1 , indicating comparable activity of the compound either against planktonic or biofilm-forming cells of H. parainfluenzae and H. influenzae (Fig. 1). Only in some cases, MBIC/MIC ratio was lower for H. parainfluenzae and was higher for H. influenzae, indicating strain-and species-dependent activity of the tested compound. Figure 2 shows the activity of N-ethyl-3-amino-5-oxo-4phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide on the growth or biofilm formation by penicillinase-negative (S85Pen-) and penicillinase-positive (S86Pen?) H. parainfluenzae. In the case of penicillinase-positive isolate, the activity of the compound was significantly higher both on the growth and on the biofilm formation.
The in vitro cytotoxicity of the tested N-ethyl-3-amino-5oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide compound was presented as percentage viability of vero cells used as an experimental model. According to the results shown in Table 2, after 48 h of incubation, no cytotoxic effect was observed up to 200 lg ml -1 concentration of the tested compound. The most widely used as a measurement of compound's toxicity is the half maximal effective concentration (EC 50 ), as the concentration of the compound where 50 % of its maximal effect is observed; in case of the tested compound EC 50 = 278.8 lg ml -1 . This means that this compound was not toxic to eukaryotic cells at concentrations exerting inhibitory effect against Haemophilus spp., including anti-biofilm activity.
Although the control of bacterial infections has been effective since the discovery of antimicrobial drugs, widespread drug resistance among bacteria has led to a search for new antibacterial agents. However, the finding of biofilm phenotype bacteria, showing usually intrinsic insensitivity to available drugs at standard dosing effective against planktonic cells, has created a necessity to pay more attention to targeted anti-biofilm agents. In this work, we have found that the N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide possessed good in vitro activity either against free-floating (planktonic) or biofilm-forming cells of Haemophilus spp. Haemophili rods, e.g., pathogenic H. influenzae or opportunistic H. parainfluenzae are found to be a part of proper microflora of the upper respiratory tract Murphy et al., 2007). Under favorable conditions, these bacterial species may be the etiologic agents of various and unspecified infections, including those associated with biofilm formation (Trollfors et al., 1985;Black et al., 1988;Mitchell and Hill, 2000;Chin et al., 2005;Musk and Hergenrother, 2006;Rele et al., 2006;Galli et al., 2007;Moxon et al., 2008;Cardines et al., 2009;Drago et al., 2012;Bjarnsholt, 2013). It has been estimated that the biofilms protect microbes from the immune system, antimicrobials, predation or stresses, and are crucial for the development of recurrent and opportunistic diseases (Costerton et al., 1999, Donlan, 2002;Prakash et al., 2003;Jain et al., 2007;Wolcott and Ehrlich, 2008). The pyrazole derivatives are potent and selective inhibitors against DNA gyrase (Reece and Maxwell, 1991;Tanitame et al., 2004;Tse-Dinh, 2007;Farag et al. 2008;Liu et al., 2008;Shiroya et al., 2011). Considering a possible mechanism of anti-biofilm activity of N-ethyl-3-amino-5oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide, it should be noted that several classes of chemical compounds, e.g., pyrazole or thioamide derivatives, may act as quorumsensing inhibitors (Hentzer and Givskov, 2003;Schillaci et al. 2008;Brackman et al., 2009;Kociolek, 2009;Oancea, 2010). Quorum-sensing phenomenon, which is one of the ways to control biofilms, is a chemical form of bacterial communication via signaling molecules essential for bacterial communities to regulate the group and to synchronize the behavior (Hastings and Greenberg, 1999;Van Houdt et al., 2004;Raffa et al., 2005;Waters and Bassler, 2005;Musk and Hergenrother, 2006;Bjarnsholt and Givskov, 2007;Amer et al., 2008;Labandeira-Rey et al., 2009;Deep et al., 2011). In agreement with the data provided by the literature, pyrazole compounds may act as inhibitors that target this cell-cell signaling mechanism (Tanitame et al., 2004;Musk and Hergenrother, 2006;Tse-Dinh, 2007;Schillaci et al., 2008;Brackman et al., 2009;Oancea, 2010). The number of literature data dealing with regulatory mechanisms controlling the haemophili biofilm formation and a possible effect of different chemical compounds on this process is strongly limited. In our opinion, comparable activity of the tested compound having the ethyl substituent against planktonic or biofilm-forming cells of haemophili rods may be due to the dual activity of pyrazole--main inhibitory effect against DNA gyrase and additional activity  Fig. 2 The effect of N-ethyl-3amino-5-oxo-4-phenyl-2,5dihydro-1H-pyrazole-1carbothioamide compound and ampicillin on the penicillinasenegative (filled diamond S85Pen-) and penicillinasepositive (filled square S86Pen?) Haemophilus parainfluenzae planktonic or biofilm-forming cells (broth without bacteria: OD 570 = 0.09-0.11) Table 2 The effect of N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide on vero cells line viability It should be mentioned that Lux-S family of quorumsensing regulatory systems involved in production of autoinducer 2 (AI-2), occurring in many bacterial species and functioning as interspecies signaling system, have been identified in H. influenzae or H. ducrei (Bassler, 1999;Vendeville et al., 2005;Armbruster et al., 2009;Swords, 2012). Ü nal et al. (2012) have showed that in nontypeable H. influenzae, the two-component signaling system QseB/C was involved in biofilm formation. Daines et al. (2005) and Armbruster et al. (2009) have observed the role of LuxS and AI-2 luxS-dependent factors which control biofilm formation in non-typable H. influenzae, but they considered as controversial its importance as virulence factor in pathogenesis of the biofilm-associated infections.
The change in the structure of the substituent has a significant impact on the physicochemical properties of the compound (Hulzebos et al., 2001;Martin et al., 2002). In our study, we synthesized and tested derivatives differing from each other by the type of substituents in the thioamide group. The best results were obtained for the compound having the ethyl substituent. From the microbiological point of view, the key factor is the presence of ethyl group which only slightly increases the mass and the volume of the compound compared to derivatives with cyclohexyl or 4-metoxyphenyl substituents. Additionally, lower molecular weight of ethyl derivative can have a significant effect on the antimicrobial properties of this compound. In our opinion, replacement of ethyl group on cyclohexyl or 4-metoxyphenyl in the tested pyrazole derivatives causes a significant decrease of their activity against Haemophilus spp. Besides, ethyl substituent has a limited conformational freedom which may affect selectivity (Graham, 2001). This is very important information from the point of view of the further modifications of these derivatives and their activity against either biofilm-forming cells or against mature biofilm of Haemophilus spp. In addition, further work is needed to evaluate the role of pyrazole derivatives during biofilm formation and their influence either on adhesive capabilities of haemophili rods or on quorum-sensing phenomenon.

Bacterial strains
The haemophili reference species from American Type Culture Collection (ATCC)--H. influenzae ATCC 10211, H. parainfluenzae ATCC 7901, and H. parainfluenzae ATCC 51505 were included. Besides, 20 clinical isolates of H. parainfluenzae and 11 clinical isolates of H. influenzae from the museum of Department of Pharmaceutical Microbiology of Medical University of Lublin were used.

Growth conditions
The Haemophilus chocolate agar (HAEM, bioMerieux) medium with PolyVitex and hemoglobin or tripticasein soy broth (TSB) ? Haemophilus test medium supplement (HTMS)--TSB (Biocorp) medium supplemented with HTMS (HTMS SRO158E, Oxoid) with growth factors for haemophili (25 lg ml -1 of NAD and 15 lg ml -1 of hematin) were used. Chocolate agar is blood agar medium that has been heated to open the pyrrole ring, forming haemin (a required growth factor for bacteria lacking hemolysins), providing optimal growth conditions for H. influenzae and other fastidious bacteria (Rennie et al., 1992;Han et al., 2006). In clinical microbiology, the TSB medium is used in a variety of procedures, e.g., for the microbiological test procedure of culture media according to the standards (NCLSI, 2000(NCLSI, , 2004. However, according to our results, TSB supplemented with HTMS is good as a primary enrichment medium directly inoculated with the various bacteria (Kosikowska and Malm, 2009). The standardized bacterial suspensions with an optical density of 0.5 McFarland standard--150 9 10 6 colony-forming units ml -1 in sterile 0.85 % NaCl were prepared. A stock solutions of N-substituted-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide derivatives at a concentration of 50 mg ml -1 in dimethyl sulfoxide (Sigma) were prepared. The medium with DMSO at a final concentration and without the tested compounds served as control--no microbial growth inhibition was observed.
The bacterial cultures in TSB?HTMS medium with addition of 30 % glycerol were stored at -70°C. Before each experiment, bacterial strains were subcultured on HAEM medium and incubated overnight at 35°C in about 5 % CO 2 atmosphere.
In order to assay the influence of the tested pyrazole derivatives on the growth of haemophili rods, 198 ll of TSB?HTMS medium without (control) and with a series of twofold dilution of the tested compounds in the range of final concentration from 1,000 to 62.5 lg ml -1 was inoculated with 2 ll of the standardized microbial suspension (total volume per each well --200 ll), and then incubated for 18 h at 35°C in the presence of about 5 % CO 2 . Then in order to assay the influence of the tested N-ethyl-3-amino-5oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide with highest inhibitory effect against the planktonic cells of Haemophilus spp., 198 ll of TSB?HTMS medium without (control) and with a series of twofold dilution of the tested compound in the range of final concentration from 0.12 to 31.25 lg ml -1 was inoculated with 2 ll of the standardized microbial suspension (total volume per each well--200 ll), and then incubated for 18 h at 35°C in the presence of about 5 % CO 2 . After incubation, spectrophotometric measurements of optical density at wavelength k = 570 Scheme 1 Synthesis and structure of N-substituted-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide (OD 570 ) of the bacterial cultures with or without the tested compound were done by using a microplate reader (ELx800 BioTek) in order to determine MIC. The MIC values were determined by comparison to the growth of a control (compound-free) medium. Ampicillin was used as a reference antimicrobial agent on selected H. parainfluenzae (penicillinase-positive or penicillinase-negative strains) isolates at the same conditions. The blank control wells without or with twofold dilution of the tested compounds added to TSB?HTMS broth without bacterial suspension were incubated under the same conditions. The experiments were performed in triplicate.

Biofilm assay
In order to assay the effect of N-ethyl-3-amino-5-oxo-4phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide on Haemophilus spp. biofilm formation, the method based on staining with 0.1 % crystal violet described previously by Kaplan and Mulks (2005) with some modifications (Kosikowska and Malm, 2009) was used. The activity of the tested compound against biofilm formation was determined on the basis of MBIC, defined as the lowest concentration of the compound at which the biofilm formation was inhibited (Č ernohorská and Votava, 2008). In order to assay the influence of the tested compound on the biofilm formation by haemophili rods, 198 ll of TSB?HTMS medium without (control) and with a series of twofold dilution of the tested compound in the range of final concentration from 0.12 to 31.25 lg ml -1 was inoculated with 2 ll of the standardized microbial suspension (total volume per each well--200 ll), and then incubated at 35°C in the presence of about 5 % CO 2 . After overnight incubation of bacterial culture, the medium above the culture was decanted and then the plates were washed extensively several times with distilled water to remove nonadherent or loosely adherent cells, dried in inverted position and stained with 200 ll of 0.1 % crystal violet. The plates were left for 15 min to stain the cells, then washed extensively under distilled water to remove unbound dye. Next, in order to elicit a response to each of the wells, 200 ll of isopropyl alcohol (Color Gram 2 R 3-F, bioMerieux) was added and the plates were left at room temperature for 15 min to solubilize the dye. The optical density of the alcohol-dye solution in each well was read at wave length k = 570 (OD 570 ) by using a microplate reader (BioTek ELx800). Ampicillin was used as a reference compound. The blank control wells without or with twofold dilution of the tested compound added to TSB?HTMS broth without bacterial suspension were incubated under the same conditions. The experiments were performed in triplicate.

Cytotoxicity assay
The vero cell culture from the American Type Culture Collection (ATCC--84113001) was used in the experiment. The minimum essential medium Eagle (MEM, Sigma) media were supplemented with 10 % fetal bovine serum (Sigma), 100 U ml -1 of penicillin, and 0.1 lg ml -1 of streptomycin (Polfa-Tarchomin, Poland). The cell culture was incubated at 37°C for 24 h in the 5 % CO 2 atmosphere. A stock solution of N-ethyl-3-amino-5-oxo-4phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide at a concentration of 50 mg ml -1 was dissolved in DMSO (Sigma). The initial concentration of the examined compound in the MEM medium was 500 lg ml -1 .
100 ll of the vero cell culture prepared was plated into 96-well polystyrene microplates (NUNC) at a cell density 2 9 10 4 cells per well. After 24 h incubation at 37°C, the media were removed and the cells were treated with a solution of the tested compound diluted in the MEM medium including 2 % of serum. The following final concentrations were applied: 3. 15, 6.25, 12.5, 25, 50, 100, 200, and 500 lg ml -1 . At the same time, the cytotoxicity of solvents was examined. The control cell culture was supplemented with media including 2 % of serum only. The cell cultures were incubated for 48 h at 37°C in the 5 % CO 2 atmosphere. Cytotoxicity of the tested compound was estimated using the MTT method, described by Takenouchi and Munekata (1998). The MTT method is a quantitative colorimetric toxicity test, based on the transformation of yellow, soluble tetrazolium salts (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to purple-blue insoluble formazane. This process occurs naturally in mitochondria of living cells. After 48 h incubation with compounds, cell cultures were supplemented with 10 ll of 5 mg ml -1 MTT solution per well, and further incubated for 4 h at 37°C. Afterwards, 100 ll of water solution, including 50 % dimethylformamide and 20 % SDS, per well was added and after the all-night incubation the absorbance was measured by the 96-well plastic plate reader (Organon Teknika) at wavelengths of k = 540 and 620 nm. The medium with DMSO at tested concentration range without the tested compound served as control--it was not toxic to vero cells line. The experiments were carried out in duplicates.
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