Fig. 1 The effect of N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide compound on biofilms formation by Haemophilus spp. on the basis of MBIC/MIC ratio Figure 2 shows the activity of N-ethyl-3-amino-5-oxo-4-phenyl-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. Fig. 2 The effect of N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide compound

and ampicillin on the penicillinase-negative (filled diamond S85Pen−) and penicillinase-positive Stattic order (filled square S86Pen+) Haemophilus parainfluenzae planktonic or biofilm-forming cells (broth without bacteria: OD570 = 0.09–0.11) The in vitro cytotoxicity of the tested N-ethyl-3-amino-5-oxo-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 μg ml−1 concentration of the tested compound. The most widely used as a measurement of compound’s toxicity is the half maximal effective concentration (EC50), as the concentration of the selleck kinase inhibitor compound where

Y27632 50 % of its maximal effect is observed; in case of the tested compound EC50 = 278.8 μg 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. 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 Compound concentration (μg ml−1)

Cell viability (in %) ± SD x 500 37.95 ± 7.7 200 82.15 ± 5.7 100 89 ± 6.6 50 93.55 ± 4.2 25 97.4 ± 1.7 12.5 98.05 ± 1.8 6.25 98.75 ± 2.0 3.15 100 ± 0.0 0 (control) 100 ± 0.0 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 ZD1839 clinical trial 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 (Kilian, 2007; Murphy et al., 2007).