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Molecules 2018, 23(5), 1158; https://doi.org/10.3390/molecules23051158

Article
Novel Guanidine Compound against Multidrug-Resistant Cystic Fibrosis-Associated Bacterial Species
1
Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan
2
CINDEFI (UNLP, CONICET-CCT La Plata), Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata 1900, Argentina
3
Sala de Microbiología, Hospital de Niños Sor María Ludovica, La Plata 1900, Argentina
4
CEQUINOR (UNLP, CONICET-CCT La Plata), Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata 1900, Argentina
*
Authors to whom correspondence should be addressed.
Received: 21 April 2018 / Accepted: 8 May 2018 / Published: 11 May 2018

Abstract

:
Chronic pulmonary infection is a hallmark of lung disease in cystic fibrosis (CF). Infections dominated by non-fermentative Gram-negative bacilli are particularly difficult to treat and highlight an urgent need for the development of new class of agents to combat these infections. In this work, a small library comprising thiourea and guanidine derivatives with low molecular weight was designed; these derivatives were studied as antimicrobial agents against Gram-positive, Gram-negative, and a panel of drug-resistant clinical isolates recovered from patients with CF. One novel compound, a guanidine derivative bearing adamantane-1-carbonyl and 2-bromo-4,6-difluouro-phenyl substituents (H-BDF), showed potent bactericidal activity against the strains tested, at levels generally higher than those exhibited by tobramycin, ceftazimide and meropenem. The role that different substituents exert in the antimicrobial activity has been determined, highlighting the importance of the halo-phenyl group in the guanidine moiety. The new compound displays low levels of cytotoxicity against THP-1 and A549 cells with a selective index (SI) > 8 (patent application PCT/IB2017/054870, August 2017). Taken together, our results indicate that H-BDF can be considered as a promising antimicrobial agent.
Keywords:
antimicrobials; thioureas; guanidines; drug-resistant; cystic fibrosis

1. Introduction

In recent years, increasing infections due to antibiotic-resistant pathogens have made the formerly routine therapy of many infectious diseases challenging, and in many instances, extremely difficult or impossible to be eradicated [1,2,3]. Multidrug resistance is specially associated with respiratory tract infection in cystic fibrosis (CF) [4] where opportunistic pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, Stenotrophomonas maltophilia and species of the Burkholderia cepacia complex (Bcc) infect patient’s lung and airways. Although for some patients the infection may occur only transiently, their acquisition most typically results in a chronic infection with acute debilitating exacerbations, causing a severe decline in respiratory function which contributes to disease progression and premature mortality [5,6]. In addition, they are important nosocomial pathogens affecting both immunocompetent and immunocompromised patients, and are responsible for a considerable proportion of infections in patients in Intensive Care Units (ICUs) worldwide [7]. Despite the emergence and dissemination of resistant bacteria and the need of more effective therapies, the development of new antimicrobial agents against these life-threatening infections is declining [8]. The impermeable nature of Gram-negative bacteria envelope, and the presence of multiple efflux pumps, in combination with other resistance mechanisms, has made the discovery of new effective antibacterial drugs very difficult [9].
Thioureas as well as guanidines represent two important groups of compounds due to their wide range of application as pharmaceutical agents. They possess a broad biological activity range including anti-inflammatory, anticancer, antiviral, antiparasitic, antifungal and antimicrobial properties [10,11]. Such a diverse range of biochemical behavior can be attributed to their flexible structure and the presence of nitrogen atoms in these molecules that make it possible to bear various substituents. For instance, it is well known that the 1-aroyl-3-(substituted-2-benzothiazolyl) thioureas exhibit potent antibacterial activity [12]. In addition, 1-(benzoyl)-3-(substituted) thioureas are antimicrobial agents [13] and the fluorinated analogues exhibit good antifungal activity [14]. Furthermore, due to efficient resonance stabilization of the charged protonated state, the guanidine groups have a relatively high acid dissociation constant which makes them stronger bases better suited for stable electrostatic interaction with the negative charged membranes of bacteria. This property improves the penetration of guanidine-bearing compounds through membranes and thus their biological activity [15,16]. On the other hand, the introduction of fluorine or appropriate fluorinated groups into organic compounds has advanced over recent decades in medicinal chemistry. The incorporation of fluorine atoms may contribute to increase metabolic stability, binding affinity and lipid solubility, thereby enhancing rates of absorption and transport of drugs in vivo [17,18]. Several studies further indicated that the incorporation of fluor and/or different electron withdrawing groups, such as bromo, chloro, acetyl, and nitro groups, on aromatic rings results in an improvement in antibacterial activity [16,19,20,21].
Taking into account the aforesaid biological and synthetic significance of thioureas and guanidines on one hand, and the multifunctional value of the electron withdrawing groups in drug design on the other, the endeavor of the current work was to investigate the activity of newly synthesized halophenyl substituted thioureas and guanidines against drug-resistant clinical isolates recovered from patients with CF.

2. Materials and Methods

2.1. Reagents and Equipment

1-adamantane carboxylic acid, thionyl chloride, triethylamine, potassium thiocyanate, mercury(II) chloride and substituted anilines were commercial products (Sigma-Aldrich, St. Louis, MO, USA) and were used as received. Analytical grade (Merck, Kenilworth, NJ, USA) acetone and dimethyl formamide, DMF, were dried and freshly distilled prior to use.
Melting points were recorded using a digital Gallenkamp (SANYO, Moriguchi, Japan) model MPD.BM 3.5 apparatus and are uncorrected. 1H and 13C NMR spectra were determined in CDCl3 at 300 MHz and 75.4 MHz, respectively, using a Bruker spectrophotometer (Billerica, Middlesex, MA, USA). FTIR spectra were acquired by a FTS 3000 MX spectrometer. Elemental analyses were conducted using a LECO-183 CHNS analyzer (LECO Corporation, MI, USA). Thin layer chromatography (TLC) was carried out on 0.25 mm silica gel plates (60 F254, Merck, Darmstadt, Germany). Visualization was achieved by ultraviolet light.

2.2. Synthesis of Compounds

Thirteen compounds were synthesized and their structures were confirmed by a combination of elemental analysis, infrared and nuclear magnetic resonance spectroscopy. 1-(Adamantane-1-carbonyl)-3-substituted thiourea compounds were prepared by the addition reaction between adamantyl isothiocyanate with a variety of suitably substituted anilines [22,23,24,25]. The starting material 1-adamantane carbonyl chloride was obtained via the reaction of 1-adamantane carboxylic acid with thionyl chloride. A solution of adamantane-1-carbonyl chloride in dry acetone was treated with an equimolar quantity of potassium thiocyanate in dry acetone to yield the adamantane-1-carbonyl-isothiocyanate as intermediate (Figure 1). A treatment of the latter with an equimolar quantity of cyclohexylamine (for compound 1, Table 1) and a variety of substituted anilines (compounds 27, Table 1) in acetone produced the thiourea derivatives. In a typical procedure, a freshly distilled solution of adamantane-1-carbonyl chloride (10 mmol) in dry acetone (50 mL) was added dropwise to a suspension of potassium thiocyanate (10 mmol) in acetone (30 mL) and the reaction mixture was refluxed for 30 min under nitrogen. After cooling to room temperature, a solution of the substituted aniline (10 mmol) in acetone (10 mL) was added and the resulting mixture refluxed for 2–4 h. The reaction mixture was poured into cold water and the precipitated thioureas were recrystallized from suitable solvents.
Three 1-acyl-3-(2-bromo-4,6-difluoro-phenyl)thioureas (compounds 810, Table 1) were synthesized in a similar way by treating the corresponding acyl chloride derivatives (1-naphthoyl chloride, 2,4-dichloro-benzoyl chloride and 4-methyl-benzoyl chloride, respectively) with potassium thiocyanate in dry acetone followed by the addition of 2-bromo-4,6-difluoro-aniline.
For the synthesis of guanidine derivatives (compounds 1113, Figure 2), the general method proposed by Vencato and coworkers [26] was applied (Figure 1). In a typical procedure triethylamine (2.8 mL, 20 mmol) and selected anilines (10 mmol) were added successively to a stirred solution of the corresponding 1-(adamantane-1-carbonyl)thiourea (10 mmol) in DMF (20 mL) at 10 °C followed by the addition of mercury(II) chloride (2.72 g, 10 mmol). The reaction mixture was stirred at room temperature for 12 h and then filtered to remove the HgS. The filtrate was extracted with EtOAc/H2O (1:1) (3 × 5 mL), the organic phase dried over anhydrous Na2SO4, and concentrated in vacuum to leave an oily residue which recrystallized on standing.
1-(Adamantane-1-carbonyl)-3-cyclohexylthiourea (1). Yield 68%, semisolid; FT-IR (KBr, ν, cm−1): 3336 (NH), 3034 (Ar-CH), 2926 (CH2), 2909, 2849 (CH2, CH), 1675 (C=O), 1575, 1457, 1370 (C=S). 1H NMR (300 MHz, CDCl3): δ 13.08 (br s, 1H, NH, D2O exchangeable); 6.25 (1H, s, broad, NH); 4.09 (br s, 1H, NH, D2O exchangeable); 3.94 (1H, m, CH), 2.1 (br s, 3H, adamantane-CH), 1.95 (s, 6H, adamantane-CH2), 1.94–2.02 (2H, dd, CH2), 1.60–1.76 (4H, m, CH2 × 2), 1.79 (m, 6H, adamantane-CH2) 1.18–1.45 (4H, m, CH2 × 2); 13C NMR (75 MHz, CDCl3): 179.1 (C=S); 178.46 (C=O), 54.37 (CH), 41.98, 41.90, 39.2, 38.5, 36.4, 36.0, 33.03 (CH2-4), 32.81 (CH2-2), 31.6, 28.0, 24.75 (CH2-3), 27.7, (adamantane-C) 25.41 (CH2-3), 24.75 (CH2-3); Anal. Calcd for C18H28N2OS (320.19): C, 67.46; H, 8.81; N, 8.74; S, 10.00%; Found: C, 67.46; H, 8.81; N, 8.74; S, 10.00%.
1-(Adamantane-1-carbonyl)-3-phenylthiourea (2). Yield 72%, mp 108–110 °C. FT-IR (KBr, ν, cm−1): 3336 (NH), 3034 (Ar-CH), 2909, 2849 (CH2, CH), 1679 (C=O), 1575, 1457, 1375 (C=S); 1H NMR (300 MHz, CDCl3): δ 12.71 (br s, 1H, NH, D2O exchangeable); 7.63 (br s, 1H, NH, D2O exchangeable); 7.23–7.33 (m, 2H, Ar); 7.38–7.43 (m, 2H, Ar), 8.40–8.48 (m, 1H, Ar); 2.08 (s, 3H, adamantane-CH), 1.69 (s, 6H, adamantane-CH2), 1.58 (q, 6H, adamantane-CH2, J = 8.6 Hz); 13C NMR (75 MHz, CDCl3): 179.6 (C=S); 170.12 (C=O); 143.05 (C-9); 41.51, 39.25, 38.69, 38.49, 36.44, 36.14, 28.05, 27.86, 27.78, (adamantane-C); Anal. Calcd for C18H22N2OS (314.45): C, 68.75; H, 7.05; N, 8.91; S, 10.20%; Found: C, 68.83; H, 7.10; N, 8.98; S, 10.14%.
1-(Adamantane-1-carbonyl)-3-(4-methyl-3-fluorophenyl)thiourea (3). Yield 69%, mp 174–176 °C. FT-IR (ν, cm−1): 3436, 3034, 2909, 1675, 1585, 1457, 1368. 1H NMR (300 MHz, CDCl3): δ 12.47 (br s, 1H, NH, D2O exchangeable); 8.53 (br s, 1H, NH, D2O exchangeable); 7.19 (s, 1H, Ar), 7.59 (s, 1H, Ar), 7.81(d, 2H, J = 8.6 Hz, Ar), 2.37 (s, 3H, Ar-CH3) 2.14 (brs, 3H, adamantane-CH), 1.95 (s, 6H, adamantane-CH2), 1.79 (q, 6H, adamantane-CH2, J = 8.6 Hz); 13C NMR (75 MHz, CDCl3): 178.9 (C=S), 177.1 (C=O), 161.7 (Ar), 136.7 (Ar), 135.1 (Ar), 136.7 (Ar), 129.7, 141.4, 124.2 (ArCs), 21.2 (Ar-CH3) 38.44, 36.14, 27.86, 21.78, (adamantane-C); Anal. Calcd for C19H23FN2OS (346.15): C, 65.87; H, 6.69; N, 8.09; S, 9.25%; Found: C, 65.739; H, 6.72; N, 7.97; S, 9.23%.
1-(Adamantane-1-carbonyl)-3-(2-nitrophenyl)thiourea (4). Yield 73%, mp 160–162 °C. FT-IR (KBr, ν, cm−1): 3336 (NH), 3034 (Ar-CH), 2909, 2849 (CH2, CH), 1682 (C=O), 1586, 1543 (NO2 asymmetric) 1457, 1368 (C=S), 1340 cm-1 (NO2 symmetric); 1H NMR (300 MHz, CDCl3): δ 12.71 (br s, 1H, NH, D2O exchangeable); 7.63 (br s, 1H, NH, D2O exchangeable); 7.23–7.33 (m, 2H, Ar); 7.38–7.43 (m, 2H, Ar); 2.08 (s, 3H, adamantane-CH), 1.69 (s, 6H, adamantane-CH2), 1.58 (q, 6H, adamantane-CH2, J = 8.6 Hz); 13C NMR (75 MHz, CDCl3): 179.6 (C=S); 170.12 (C=O); 143.05 (C-9); 41.51, 39.25, 38.69, 38.49, 36.44, 36.14, 28.05, 27.86, 27.78, (adamantane-C); Anal. Calcd for C18H21N3O3S (359.44): C, 60.15; H, 5.89; N, 11.69; O, 13.35; S, 8.92%; Found: C, 60.21; H, 5.93; N, 11.71; S, 8.89%.
1-(Adamantane-1-carbonyl)-3-(4-acetyl-phenyl)thiourea (5). Yield 160–161 °C. FT-IR (KBr, ν, cm−1): 3336 (NH), 3034 (Ar-CH), 2909, 2849 (CH2, CH), 1679 (C=O), 1575, 1457, 1375 (C=S). 1H NMR (300 MHz, CDCl3): δ 12.74 (br s, 1H, NH, D2O exchangeable), 9.83 (br s, 1H, NH, D2O exchangeable), 7.91 (d, 2H, J= 8.6 Hz, Ar); 7.73 (d, 2H, J = 8.6 Hz, Ar), 2.3 (s, 3H, CH3CO), 2.08 (s, 3H, adamantane-CH), 1.69 (s, 6H, adamantane-CH2), 1.58 (q, 6H, adamantane-CH2, J = 8.6 Hz); 13C NMR (75 MHz, CDCl3): δ 193.6 (CO), 179.6 (C=S), 174.5 (C=O), 143.0, 138.0, 132.6, 127.8, 28.1 (CH3), 41.51, 39.25, 38.69, 38.49, 36.44, 36.14, 28.05, 27.86, 27.78, (adamantane-C); Anal. Calcd for C20H24N2O2S (356.47): C, 67.39; H, 6.79; N, 7.86; S, 8.99%; Found: C, 67.42; H, 6.83; N, 7.81; S, 8.91%.
1-(Adamantane-1-carbonyl)-3-(2,3-dichlorophenyl)thiourea (6). Yield 79%, mp 196–198 °C. FT-IR (KBr, ν, cm−1): 3336 (NH), 3034 (Ar-CH), 2909, 2849 (CH2, CH), 1675 (C=O), 1575, 1457, 1370 (C=S). 1H NMR (300 MHz, CDCl3): δ 12.74 (br s, 1H, NH, D2O exchangeable); 8.70 (br s, 1H, NH, D2O exchangeable); 8.03 (d, 1H, J = 8.6 Hz Ar), 7.96 (d, 1H, J = 8.6 Hz Ar), 7.90 (d, 1H, J = 8.6 Hz Ar), 7.83 (d, 1H, J = 8.6 Hz Ar), 7.57 (m, 3H, Ar), 2.1 (br s, 3H, adamantane-CH), 2.03 (s, 6H, adamantane-CH2), 1.81 (q, 6H, adamantane-CH2, J = 8.6 Hz); 13C NMR (75 MHz, CDCl3): 178.9 (C=S); 134.10 (Ar), 128.6, 126.9 125.3,123.64, 121.67 (ArCs), 41.94, 41.90, 39.2, 38.6, 36.1, 36.0, 31.6, 28.0, 27.8, (adamantane-C); Anal. Calcd for C18H2o Cl2N2OS (383.34): C, 56.40; H, 5.26; N, 7.31; S, 8.36%; Found: C, 56.40; H, 5.26; N, 7.31; S, 8.36%.
1-(Adamantane-1-carbonyl)-3-(2-bromo-4,6-difluorophenyl)thiourea (7). Yield 70%, mp 194–196 °C. FT-IR (KBr, ν, cm−1): 3336 (NH), 3034 (Ar-CH), 2909, 2849 (CH2, CH), 1675 (C=O), 1575, 1457, 1370 (C=S). 1H NMR (300 MHz, CDCl3): δ 11.93 (br s, 1H, NH, D2O exchangeable), 9.61 (br s, 1H, NH, D2O exchangeable), 7.48–7.44 (m, 1H, Ar), 7.29-7.22 (m, 1H, Ar), 2.08 (t, 10H, adamantane-H, J = 6.0 Hz), 1.80 (t, 6H, adamantane-H, J = 4.8 Hz); 13C NMR (75 MHz, CDCl3): 182.3 (C=S), 179.2 (C=O), 163.3, 160.6, 159.8, 157.2, 123.9, 115.8, 104.5, 103.8 (ArCs), 41.9, 37.6, 35.8, (adamantane-C); Anal. Calcd for C18H19F2BrN2OS (429.32): C, 50.36; H, 4.46; N, 6.53; S, 7.47; Found: C, 50.24; H, 4.51; N, 6.57; S, 7.36%.
1-(1-naphtyl)-3-(2-bromo-4,6-difluoro-phenyl)thiourea (8). Yield 81%, mp 174–176 °C. FT-IR (KBr, ν, cm−1): 3336 (NH), 3034 (Ar-CH), 1671 (C=O), 1585, 1451, 1372 (C=S). 1H NMR (300 MHz, CDCl3): δ 11.98 (br s, 1H, NH, D2O exchangeable); 11.29 (br s, 1H, NH, D2O exchangeable); 8.89–6.71 (m, 9H, Ar); 13C NMR (75 MHz, CDCl3): 168.9, 164.3, 145.1, 132.0, 134, 120.4, 125.1, 129.6, 116, 103.1 (ArCs); Anal. Calcd for C18H11 F2BrN2OS (421.97): C, 51.32; H, 2.63; N, 6.65 S, 7.61%; Found: C, 51.24; H, 2.60; N, S, 6.61, 7.57%.
1-(2,4-dichloro-phenyl)-3-(2-bromo-4,6-difluoro-phenyl)thiourea (9). Yield 81%, mp 174–176 °C. FT-IR (KBr, ν, cm−1): 3336 (NH), 3034 (Ar-CH), 2909, 2849 (CH2, CH), 1675 (C=O), 1575, 1457, 1370 (C=S). 1H NMR (300 MHz, CDCl3): δ 12.07 (br s, 1H, NH, D2O exchangeable); 11.35 (br s, 1H, NH, D2O exchangeable); 7.61 (s, 1H, Ar), 7.58 (d, 1H, J = 8.3 Hz, Ar), 7.58 (d, 1H, J = 8.3 Hz, Ar), 7.49 (s, 1H, Ar), 7.19 (s, 1H, Ar); 13C NMR (75 MHz, CDCl3): 181.9 (C=S); 170.3 (C=O), 168.2, 159.5, 134.10 (Ar), 141.4, 134.1, 130.1, 129.4, 127.3, 128.6, 126.9, 124.2, 119.7, 114.9 (ArCs); Anal. Calcd for C14H17 Cl2 F2BrN2OS (439.88): C, 38.21; H, 1.60; N, 6.37; S, 7.28%; Found: C, 37.28; H, 1.62; N, 6.33; S, 8.30%.
1-(4-methylphenyl)-3-(2-bromo-4,6-difluoro-phenyl)thiourea (10). Yield 69%, mp 174–176 °C. FT-IR (KBr, ν, cm−1): 3436 (NH), 3034 (Ar-CH), 2909, 1675 (C=O), 1585, 1457, 1368 (C=S). 1H NMR (300 MHz, CDCl3): δ 12.74 (br s, 1H, NH, D2O exchangeable); 11.31 (br s, 1H, NH, D2O exchangeable); 7.19 (s, 1H, Ar), 7.59 (s, 1H, Ar), 7.81 (d, 2H, J = 8.6 Hz, Ar), 2.51 (s, 3H, Ar-CH3); 13C NMR (75 MHz, CDCl3): 178.9 (C=S), 173.1 (C=O), 134.10 (Ar), 181.7, 141.4, 130.1, 128.6, 126.9, 124.2, 119.7, 114.9 (ArCs), 19.4 (Ar-CH3); Anal. Calcd for C15H11 F2BrN2OS (385.97): C, 46.77; H, 2.88; N, 7.27; S, 8.32%; Found: C, 46.81; H, 2.92; N, 7.23; S, 8.28%.
1-(Adamantane-1-carbonyl)-2,3-bis(2-bromo-4,6-difluoro-phenyl)guanidine (11). Yield 70%, mp 148–149 °C. FT-IR (KBr, ν, cm−1): 3336, 3413, 3245, 3128, 3043, 3034, 2909, 2849, 1675, 1575, 1457, 1370. 1H NMR (300 MHz, CDCl3): δ 9.79 (br s, 1H, NH, D2O exchangeable); 8.04 (br s, 1H, NH, D2O exchangeable); 7.17–7.13 (m, 2H, Ar), 7.06–6.98 (m, 2H, Ar), 2.0 (br s, 3H, adamantane-H), 1.94–1.89 (br m, 3H, adamantane-H), 1.78–1.60 (br m, 10H, adamantane-H); 13C NMR (75 MHz, CDCl3): 178.2 (C=O), 174.2 (C=N), 154.9, 151.8, 148.6, 131.9, 114.6, 107.8, 103.2 (ArCs), 40.9, 37.9, 35.8, (adamantane-C); Anal. Calcd for C24H21F4Br2N3O (603.2): C, 47.78; H, 3.51; N, 6.97%; Found: C, 48.1; H, 3.49; N, 7.01%.
1-(Adamantane-1-carbonyl)-2-(2-bromo-4,6-difluoro-phenyl)-3(2,6-di-bromo-4-fluoro-phenyl)guanidine (12). Yield 70%, mp 144–145 °C. FT-IR (KBr, ν, cm−1): 3413, 3245, 3128, 3043, 3034, 2909, 2849, 1675, 1575, 1457, 1370. 1H NMR (300 MHz, CDCl3): δ 11.94 (br s, 1H, NH, D2O exchangeable); 9.66 (br s, 1H, NH, D2O exchangeable); 7.48 (m, 1H, Ar), 7.23 (m, 2H, Ar), 7.01 (m, 1H, Ar), 1.99–1.84 (m, 10H, adamantane-H), 1.79–1.59 (m, 6H, adamantane-H); 13C NMR (75 MHz, CDCl3): 179.2 (C=O), 174.2 (C=N), 160.4, 159.8, 157.2, 151.9, 147.2, 140.2, 123.9, 115.8, 114.3, 104.5 (ArCs), 41.9, 37.6, 35.8 (adamantane-C); Anal. Calcd for C24H21F3Br3N3O (664.2): C, 43.40; H, 3.19; N, 6.33%; Found: C, 43.21.1; H, 3.52; N, 6.97%.
1-(Adamantane-1-carbonyl)-2,3-bis-(2-nitro-phenyl)guanidine (13). Yield 70%, mp 156 °C. FT-IR (KBr, ν, cm−1): 3336, 3413, 3245, 3128, 3043, 3034, 2909, 2849, 1675, 1575, 1457, 1370. 1H NMR (300 MHz, CDCl3): δ 11.94 (br s, 1H, NH, D2O exchangeable); 9.66 (br s, 1H, NH, D2O exchangeable); 7.48 (m, 1H, Ar), 7.23 (m, 2H, Ar), 7.01 (m, 1H, Ar), 1.99–1.84 (m, 10H, adamantane-H), 1.79–1.59 (m, 6H, adamantane-H); 13C NMR (75 MHz, CDCl3): 179.2 (C=O), 174.2 (C=N), 160.4, 159.8, 157.2, 151.9, 147.2, 140.2, 123.9, 115.8, 114.3, 104.5 (ArCs), 41.9, 37.6, 35.8 (adamantane-C); Anal. Calcd for C24H25N5O5 (463.5): C, 62.19; H, 5.44; N, 15.11%; Found: C, 61.97.1; H, 5.42; N, 6.93%.

2.3. Bacterial Strains

The antibacterial activity of the compounds was tested against the reference strains Escherichia coli ATCC25922, Bordetella bronchiseptica 9.73H+ [27], Pseudomonas aeruginosa ATCC15692, Burkholderia cenocepacia J2315, Pandorea apista DSM16535, Staphyloccocus aureus ATCC6538, Bacillus cereus ATCC10876. A total of forty non-fermenting Gram-negative bacilli and two Methicillin-Resistant Staphylococcus aureus (MRSA) clinical isolates collected from sputum samples of patients with CF attended at different hospitals and CF Centers in the period 2004 to 2017 were used in this study. They were selected from the collection of microorganisms CAMPA (Colección Argentina de Microorganismos Patógenos y Ambientales) of CINDEFI, at the Faculty of Exact Sciences in La Plata University [28]. All Bcc isolates were identified by PCR-recA technology (amplification, PCR-recA RFLP HaeIII, and sequencing). Additionally hisA, gyrB, or other gene from the current multilocus sequence typing (MLST) scheme were sequenced when the identification remained ambiguous [29,30]. The isolates were maintained both as lyophilized and frozen at −80 °C in Trypticase-soy broth with 10% (v/v) glycerol until further analysis.

2.4. Antimicrobial Activity Assays

The in vitro susceptibility tests (Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) tests) were determined using the micro-dilution method according to guidelines of the Clinical and Laboratory Standards Institute (CLSI) [31]. Briefly, serial two-fold dilutions of each compound were prepared (final volume of 50 μL) in 96-well polypropylene microtiter plates (Sarstedt, Nümbrecht, Germany) with Mueller Hinton (MH) broth. Each dilution series included control wells without any compound and control wells without bacteria. Then, a total of 50 μL of the adjusted inoculum (approximately 5 × 105 cells/mL) in MH broth was added to each well. The MIC was taken as the lowest concentration of antimicrobial compound resulting in the complete inhibition of visible growth after 18 h of incubation at 37 °C. Minimal bactericidal concentration (MBC) assay was performed following MIC assay. After reporting the MIC assay value, 10 μL aliquots of the medium were taken from wells with no visible bacterial growth. These were plated on LB agar and incubated for 24 h to allow colony growth. The lowest concentration of the compound at which no growth occurred on LB plates was denoted as the MBC. Results are mean values of at least two independent determinations.

2.5. Checkerboard Assay

The activity of compound 11 in combination with meropenem, tobramycin and ciprofloxcin was analyzed using the checkerboard broth dilution method [32] to determine the fractional inhibitory concentration indices (FICIs), calculated as: FICI = (MICH-BDFcomb/MICH-BDFalone) + (MICantibioticcomb/MICantibioticalone) (comb, combination). The calculated FICI was interpreted as synergistic (FICI ≤ 0.5), additive (0.5 < FICI < 1), indifferent (1 ≤ FICI < 4.0), or antagonistic (FICI ≥ 4.0), according to the previously published methods [33].

2.6. Cytotoxicity Assays

A trypan blue exclusion assay [34] was performed to check the cytotoxicity of compound 11 against THP-1 human monocytic leukemia cells (ATCC, TIB-202, Manassas, VA, USA) and A549 alveolar epithelial cells (ATCC, CCL185, Rockville, MD, USA). Cells were routinely maintained in Complete Medium RPMI-1640 and Dulbecco’s Modified Eagle’s medium (DMEM), respectively, supplemented with 10% heat-inactivated fetal bovine serum (FBS). For the cytotoxicity assay, cells were seeded at a density of 5 × 104 per well in a 96 well plate and were incubated with serial dilutions of compound 11 to a total of 200 μL, at 37 °C in a humidified atmosphere of 5% CO2 for 24 h. Two negative controls were included: cells in drug-free culture media and cells treated for 24 h with the maximum concentration of the drug solvent used in the experiment (4% dimethyl sulfoxide). Cells were subsequently stained with 0.2% trypan blue and incubated for 3 min at room temperature. The number of dye-excluding cells was counted by microscopy. A minimum of 200 cells were counted and the percent viability was calculated in comparison to the control. The IC50 value was defined as the highest drug concentration at which 50% of the cells are viable relative to the control. Results are mean values of at least five independent determinations. The selectivity index (SI) was calculated as the ratio of IC50 and the MIC [35].

3. Results and Discussion

3.1. Chemistry

A series of 11 novel closely related compounds belonging to the thiourea family (compounds 110) and a guanidine derivative (11) was prepared (Figure 1 and Table 1). Primary amines substituted with different electron withdrawing groups were subjected to the addition reaction with isothiocyanates in order to be transformed into the corresponding thioureas by using the general method originally proposed by Douglas and Dains [22] (Figure 1). The substitution on both nitrogen positions (1 and 3) of the thiourea group was varied in order to better understand the role of different substituents in the biological activity. To rationalize this aspect, a series of closely related 1-(adamantane-1-carbonyl)-3-mono substituted thioureas was firstly prepared by taking into account the well-known capacity of the adamantyl group to enhance antibacterial activity [36,37,38,39,40]. Thus, several thioureas were prepared bearing the adamantyl group in R1 (compounds 17, Table 1).
Moreover, taking into account the improvement in antibacterial activity exerted by the presence of phenyl groups substituted with electron withdrawing groups [16,19,20,21], a second group of thioureas (compounds 710, Table 1) was substituted in R2 with the 2-bromo-4,6-difluoro-phenyl group. Finally, the effect of replacing the thiocarbonyl (C=S) with aryl-guanidino functionality (Ar-N=C) was evaluated in compound 7, in which N-3 of the guanidine was substituted with the 2-bromo-4,6-difluoro-phenyl group. To this end, the procedure proposed by Vencato et al. [26] was applied and the acyl thiourea derivatives were treated with mercury(II) chloride under basic conditions in the presence of 2-bromo-4,6-difluoroaniline to produce the corresponding guanidine derivative (compound 11, Figure 1 and Table 1) [26,41].
Obtained compounds were purified by flash chromatography. FTIR, 1H-NMR and 13C-NMR spectra and elemental analysis confirmed the identity of the products (see Materials and Methods). In the 1H-NMR of most of the compounds, the characteristic signals of adamantyl moiety: a 6H quartet at δ = 1.75–1.79 ppm (adamantane-CH2), a 6H, singlet at 1.95–1.98 (adamantane-CH2) and a 3H, singlet around 2.08 ppm (adamantane-CH), besides N-H amide and thioamide singlets at δ = 8.5–8.7 and 12.7–13.0 ppm were clearly observed. In the 13C-NMR, characteristic signals for adamantyl moiety at δ = 27.7, 36.1–36.4, 38.6–38.5 and 41.5 ppm, as well those at δ = 170–179 for carbonyl and δ = 178–182 ppm for thiocarbonyl carbons, were observed. The acyl thioureas were also characterized by their IR spectra, with intense absorptions around 3300–3400 (νNH), 1670 (νC=O), 1580 (δNH), and 1380 (νC=S) cm−1 [24,42,43].
The guanidine derivative 11 was characterized by two typical NH absorptions at ca. 3400 and 3240 cm−1, the C=O stretching at around 1670 cm−1 and the absence of thiocarbonyl stretching when the FTIR spectra are compared with the corresponding thiourea reagent. The characteristic C=N stretching modes of the guanidine group are observed as an intense absorption at ca. 1575 cm−1. In 1H-NMR, two broad NH singlets appeared besides the aromatic protons. The carbonyl carbons are observed at 178–179 ppm in the 13C-NMR spectrum, while the (C=N-Ar) appeared upfield at 174 ppm compared to the thiocarbonyl carbon.

3.2. Biological Activity

3.2.1. Antimicrobial Evaluation of Newly Synthesized Compounds

All obtained compounds were tested in vitro for their MIC and MBC against two reference Gram-negative non-fermentative bacilli strains, Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia J2315. These species play a critical role in morbidity and mortality associated with CF and they were selected on the basis of their high level of resistance to a variety of antimicrobial substances [44,45,46,47]. The results of antimicrobial activity are summarized in Table 1. The MIC and MBC values of meropenem, tobramycin and ceftazidime, three commonly used antibiotics for the treatment of chronic pulmonary bacterial infections [48], were analyzed in parallel. It is apparent from the results that only the guanidine derivative 11, namely H-BDF, showed a MIC value less than 2 µg/mL, and comparable or superior activity than standard drugs. Interestingly, this compound has the lowest MIC and MBC against B. cenocepacia J2315, a strain particularly resistant to meropenem [44].
A first look into structural activity relationship (SAR) indicates that, independent of the halogens introduced in the phenyl group, thiourea derivatives have poor or no antimicrobial activity. However, the replacement of thiourea in compound 7 for the guanidine group (compound 11) greatly improves antimicrobial activity. We next evaluated the impact of introducing changes in the phenyl ring of compound 11 in the biological activity. To this end, the guanidine derivatives 12 and 13 (Figure 2) were synthesized and characterized. Compound 13 was designed to evaluate the effect of changing the substitution of the halophenyl groups by the incorporation of another electron withdrawing group (nitro) in N-2 and N-3, whereas compound 12 evaluates the effect of introducing a small change in N-3 by the substitution of bromine by fluorine in position 6.
The antimicrobial activity of the new compounds was tested against P. aeruginosa PAO1 and B. cenocepacia J2315 as well as other Gram-negative and Gram-positive reference strains. As shown in Table 2, when the phenyl group substituent of compound 11 was altered by the introduction of a nitro group at the meta position (compound 13), the guanidine derivate completely lost its inhibition potency, suggesting that not only the guanidine group but also the identity and/or position of the phenyl substitutions are decisive for the antibacterial activity. Moreover, whereas compound 11 exhibited very good inhibitory and bactericidal activity against all tested strains, compound 12, in which the 2-bromo-4,6-difluoro-phenyl group in N-2 was substituted by 2,6-dibromo-4-fluoro-phenyl ring, showed only moderate microbicidal activity, suggesting that the presence of fluorine atom in position 6 of the phenyl group in N-2 is critical to ensure high inhibition and bactericidal potency.

3.2.2. Cytotoxic Evaluation of H-BDF

As limited human cellular toxicity is an important feature for an antibiotic compound, the toxicity of H-BDF was evaluated using the human monocytic leukemia cell line THP-1 and the human lung epithelial cell line A549, commonly employed in toxicity evaluation of new compounds for pulmonary application [49,50]. The IC50 for compound 11 was 38.4 ± 5.4 µg/mL for A549 and 15.5 ± 3.1 µg/mL for THP-1 cells. On the basis of the MIC and IC50 values, the selectivity indices were calculated for standard strains (Table 3). It is generally considered that the ratio for a good therapeutic index for a drug should be >10, which is a cut-off point ensuring that overdose does not put the life of the patient in danger [35]. Good SI values were obtained with compound 11 suggesting that H-BDF can be considered as a promising antibacterial agent.

3.2.3. Synergistic Effects between H-BDF and Conventional Antibiotics

Developments of alternate antibacterial strategies to potentiate the antimicrobial activity of conventional antibiotics have become increasingly important due to the emerging threat of multi-drug resistant infection [51]. As many clinical isolates exhibit resistance to meropenem, ciprofloxacin and tobramycin, three of the different classes of antibiotics commonly used to treat CF pulmonary exacerbations [52], we next studied the ability of H-BDF to potentiate the antimicrobial activity of these antibiotics toward the multidrug-resistant strain B. cenocepacia J2315. To this end, the relationship between H-BDF and meropenem, tobramycin, and ciprofloxacin was assessed via a standard checkerboard assay [29]. Treatment with H-BDF reduced the minimum inhibitory concentration of ciprofloxacin and meropenem below their clinical sensitivity breakpoints (≤4 µg/mL and ≤1 µg/mL, respectively). Fractional inhibitory concentration calculations revealed that H-BDF exhibited a synergistic interaction with meropenem and ciprofloxacin with FICIs values of 0.3 and 0.4, respectively, and an additive interaction with tobramycin with a FICI value of 0.75. This preliminary study suggests that in addition to being used as antimicrobial agent alone, H-BDF has the potential to be used in combination with other antibiotics.

3.2.4. Activity of Compound H-BDF against Multidrug-Resistant Clinical Isolates Recovered from Respiratory Samples of CF Patients

Respiratory infections with opportunistic pathogens with intrinsic antibiotic resistance to most clinically available antimicrobials are life-threatening in patients with CF [53,54,55]. Although P. aeruginosa and S. aureus remain the most common pathogens in CF lung infections, other bacteria such as species within the Bcc, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans, have emerged as significant opportunistic human pathogens in the last decades [56,57,58,59]. To investigate whether the guanidine derivative H-BDF would have clinical utility against current multidrug resistant bacteria, we determined the MIC and MBC of compound 11 against thirty eight Bcc clinical isolates, one Achromobacter xylosoxidans, one Stenotrophomonas maltophilia and two MRSA recovered from sputum samples of CF patients and selected on the basis of their high level of resistance to a variety of antimicrobial substances [55] (Table 4). MIC values of compound H-BDF were generally lower than those of meropenem, ceftazimide and tobramycin. In total, 69% of Bcc clinical isolates had H-BDF MIC values less than or equal to 4 µg/mL whereas only 41% of isolates were classified as susceptible to meropenem (MIC values ≤4 µg/mL), 49% were classified as susceptible to ceftazimide (MIC values ≤8 µg/mL), and 2.6% of isolates were classified as susceptible to tobramycin (MIC values ≤4 µg/mL). The activity of compound H-BDF against B. cenocepacia strains was impressive, with 92% susceptible at 4 µg/mL compared with only 31% susceptible to meropenem at 4 µg/mL, and 69% susceptible to ceftazimide at 8 µg/mL (Table 4). Interestingly, some clinical isolates were resistant to more than 16 antibiotics, such as B. seminalis CBC040 [55] had H-BDF MIC values ≤ 4 µg/mL. Indeed, H-BDF was active against two methicillin-resistant S. aureus clinical isolates with MIC values varying from 1 to 2 µg/mL. In conclusion, compound H-BDF was active in vitro against a significant number of multi-resistant clinical isolates recovered from CF patients.

4. Conclusions

We have reported the synthesis and preliminary evaluation of the antimicrobial activity of 13 novel thiourea and guanidine derivatives. The results evidenced that H-BDF, a guanidine derivative bearing adamantane-1-carbonyl and two 2-bromo-4,6-di-fluoro-phenyl groups, can be considered as a promising antimicrobial agent, since it exhibited higher in vitro antibacterial potency against Gram-positive and Gram-negative reference strains than previously reported guanidine compounds [10,11,15]. Moreover, the novel compound was active in vitro against a panel of multidrug-resistant clinical isolates recovered from sputum samples of patients with CF. Preliminary studies further suggest that H-BDF was able to significantly potentiate antibacterial synergy with meropenem and ciprofloxacin. From the structure activity relationship, it can be concluded that the antimicrobial activity depends mainly on the presence of a guanidine group. It has been proposed that most of the biological properties of guanidine derivatives are related to their strong basicity due to efficient resonance stabilization of the charged protonated state. The pKa of H-BDF was not determined; however, it is expected that under physiological conditions, the guanidine group exists mainly in its protonated form [60]. We can hypothesize that under this state, the guanidine moiety may alter bacterial outer membrane permeability by binding to a negatively charged site in the lipopolysaccharide layer, causing cell death. This mechanism of action have been proposed for several guanidine derivatives with antibacterial activity [61]. Alternatively, the protonated forms may interact with the active site of proteins and enzymes altering its function [11]. By analyzing the role that different substituents exert in the antimicrobial activity, the importance of the halo-phenyl group in the guanidine moiety was also demonstrated. The substituted fluorine in position 6 of the phenyl group in N-2 may contribute to increase binding affinity and/or lipid solubility [18]. Also, the electron-withdrawing group may activate the guanidine binding moiety to enhance its interaction with amine groups present in the cell membrane. Future studies will be directed towards elucidating the targets of H-BDF and the mechanisms of action.
Importantly, this compound displays low levels of cytotoxicity against THP-1 and A549 cell lines. Future research will be performed to evaluate its efficacy and safety in animal models of infection in order to validate its development as a novel antimicrobial.

5. Patents

“Antimicrobials compounds”. Patent application PCT/IB2017/054870, August 2017.

Author Contributions

Aamer Saeed, Alejandra Bosch , Yanina Lamberti and Mauricio Federico Erben conceived and designed the experiments; Aamer Saeed, Diana L. Nossa González, Marisa Bettiol , and Yanina Lamberti performed the experiments; Aamer Saeed, Alejandra Bosch , Diana L. Nossa González, Yanina Lamberti and Mauricio Federico Erben analyzed the data; Alejandra Bosch and Marisa Bettiol contributed reagents and clinical isolates; Yanina Lamberti, Aamer Saeed, Alejandra Bosch, and Mauricio Federico Erben wrote the paper.

Acknowledgments

This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica, ANPCyT (PICT-2013-2130) and Universidad Nacional de La Plata (11/X794), Argentina. Y.L. and M.F.E. are members of the Scientific Career of CONICET. D.L.N.G. is a doctoral fellow of CONICET. A.B. is member of CIC PBA.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Synthesis of acyl thiourea and guanidine derivatives. Reagents and conditions: (i) Acyl chlorides and KSCN in dry acetone, 2 h, reflux. (ii) Primary amines in dry acetone. (iii) HgCl2, substituted aniline and Et3N in dry DMF.
Figure 1. Synthesis of acyl thiourea and guanidine derivatives. Reagents and conditions: (i) Acyl chlorides and KSCN in dry acetone, 2 h, reflux. (ii) Primary amines in dry acetone. (iii) HgCl2, substituted aniline and Et3N in dry DMF.
Molecules 23 01158 g001
Figure 2. Chemical structure of guanidine derivative compounds 1113. Molecular weights (g/mol) are shown in parentheses.
Figure 2. Chemical structure of guanidine derivative compounds 1113. Molecular weights (g/mol) are shown in parentheses.
Molecules 23 01158 g002
Table 1. Activities of newly obtained compounds and common antibiotics used in clinical treatments against Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia J2315.
Table 1. Activities of newly obtained compounds and common antibiotics used in clinical treatments against Pseudomonas aeruginosa PAO1 and Burkholderia cenocepacia J2315.
EntryR1R2R3Molecular Weight (g/mol)Chemical StructureP. aeruginosa PAO1B. cenocepacia J2315
MIC (µg/mL)MBC (µg/mL)MIC (µg/mL)MBC (µg/mL)
1C10H15 aC6H11 320.19 Molecules 23 01158 i001>128nd>128nd
2C10H15 aC6H5-314.45 Molecules 23 01158 i002>128nd>128nd
3C10H15 a3-F-4-CH3-C6H3-385.97 Molecules 23 01158 i003>128nd>128nd
4C10H15 a2-NO2-C6H4-359.44 Molecules 23 01158 i004>128nd>128nd
5C10H15 a4-CH3CO-C6H4-356.47 Molecules 23 01158 i005>128nd>128nd
6C10H15 a2,3-di-Cl-C6H3-383.34 Molecules 23 01158 i006>128nd>128nd
7C10H15 a2-Br-4,6-di-F-C6H2-428.32 Molecules 23 01158 i007>128nd>128nd
8C10H7 b2-Br-4,6-di-F-C6H2-421.97 Molecules 23 01158 i008>128nd>128nd
92,4-di-Cl-C6H32-Br-4,6-di-F-C6H2-439.88 Molecules 23 01158 i009>128nd>128nd
104-CH3-C6H42-Br-4,6-di-F-C6H2-385.97 Molecules 23 01158 i010>128nd>128nd
11C10H15 a2-Br-4,6-di-F-C6H22-Br-4,6-di-F-C6H2603.2 Molecules 23 01158 i0110.5428
Tobramycin 467.51 22>128>128
Meropenem 383.46 14864
Ceftazimide 546.57 2216128
a 1-adamantyl, b-naphthyl
Table 2. Anitmicrobial activities of new compounds 11, 12 and 13 against Gram-negative and Gram-positive bacteria—minimal inhibitory concentrations (MIC, µg/mL) and minimal bactericidal concentration (MBC, µg/mL).
Table 2. Anitmicrobial activities of new compounds 11, 12 and 13 against Gram-negative and Gram-positive bacteria—minimal inhibitory concentrations (MIC, µg/mL) and minimal bactericidal concentration (MBC, µg/mL).
Compound11 (H-BDF)1213TobramycinMeropenemCeftazimide
OrganismMIC/MBCMIC/MBCMIC/MBCMIC/MBCMIC/MBCMIC/MBC
Gram-negative bacteria
Bordetella bronchiseptica 9.73H+0.5/216/64>128/>12864/640.125/0.258/64
Escherichia coli ATCC259221/264/64>128/>12816/160.03125/0.06251/1
Pseudomonas aeruginosa PAO10.5/432/>128>128/>1282/21/42/2
Burkholderia cenocepacia J23152/864/128>128/>128>128/>1288/6416/128
Pandorea apista DSM165351/264/128>128/>12832/128>128/nd128/nd
Gram-positive bacteria
Staphyloccocus aureus ATCC65380.25/18/64>128/>1282/2<0.125/<0.258/8
Bacillus cereus ATCC108762/264/64>128/>1288/32<0.125/<0.251/1
nd: no data.
Table 3. Selective Indices (SI) of compound 11 against different cell lines.
Table 3. Selective Indices (SI) of compound 11 against different cell lines.
Cells
OrganismsA549THP-1
Gram-negative bacteria
Bordetella bronchiseptica 9.73H+76.830.9
Escherichia coli ATCC2592238.415.45
Pseudomonas aeruginosa PAO176.830.9
Burkholderia cenocepacia J231519.27.7
Pandorea apista DSM1653538.415.45
Gram-positive bacteria
Staphyloccocus aureus ATCC6538153.661.8
Bacillus cereus ATCC1087619.27.7
Table 4. Microbial susceptibility of multi-resistant isolates recovered from patients with cystic fibrosis.
Table 4. Microbial susceptibility of multi-resistant isolates recovered from patients with cystic fibrosis.
H-BDFTobramycinMeropenemCeftazidime
Clinical Isolates aMIC (µg/mL)MBC (µg/mL)MIC (µg/mL)MBC (µg/mL)MIC (µg/mL)MBC (µg/mL)MIC (µg/mL)MBC (µg/mL)
Achromobacter xylosoxidans
A. xylosoxidans HNA 0010.1250.25RndS8Snd
Burkholderia cenocepacia
B. cenocepacia CAMPA 6690.252SndSndRnd
B. cenocepacia CAMPA 1533416RndR64S16
B. cenocepacia CAMPA 119424RndRndRnd
B. cenocepacia CAMPA 54428RndRndS8
B. cenocepacia CAMPA 1771816RndI32Rnd
B. cenocepacia CAMPA 81728RndRndS8
B. cenocepacia CAMPA 54824RndRndS8
B. cenocepacia CAMPA 825 (CBC 033) b416RndIndS32
B. cenocepacia CAMPA538 (CBC 035) b24RndI16S16
B. cenocepacia CAMPA 81728RndRndS16
B.cenocepacia CAMPA 53114RndSndSnd
B.cenocepacia CAMPA 993 (CBC 024) b14RndSndSnd
B.cenocepacia HE001464RndRndRnd
Burkholderia cepacia
B. cepacia CAMPA 545416RndRndS16
B. cepacia CAMPA 233 (CBC 012) b24RndS8S16
B. cepacia CAMPA 26032ndRndR32Rnd
B. cepacia CAMPA 91432ndRndR32R64
B. cepacia CAMPA 88632ndRndR32R128
B. cepacia CAMPA 99832ndRndR64S32
B. cepacia CAMPA 103964ndRndR32Rnd
B. cepacia CAMPA 853 (CBC 001) b32ndRndI64I64
B. cepacia CAMPA 860 (CBC 007) b64ndRndI64R64
B. cepacia CAMPA 66048RndS4Rnd
B. cepacia CAMPA 721 (CBC 011) b232RndS64Rnd
Burkholderia contaminans
B. contaminans HNBC0010.251RndRndSnd
Burkholderia multivorans
B. multivorans CAMPA 661(CBC 015) b24RndS4S8
B. multivorans CAMPA 153028RndRndS4
B. multivorans CAMPA 647 (CBC 017) b44RndS4S8
B. multivorans CAMPA 653 (CBC 018) b28RndS4S8
B. multivorans CAMPA 623(CBC 019) b28RndS8Rnd
B. multivorans CAMPA 832 (CBC 020) b416RndS32Rnd
B. multivorans CAMPA 987 (CBC 021) b24RndS8Rnd
B. multivorans CAMPA 997 (CBC 022) b48RndS8Rnd
Burkholderia seminalis
B. seminalis CAMPA 23132ndRndIndR32
B. seminalis CAMPA 261 (CBC 039) b32ndRndS16S16
B. seminalis CAMPA 475 (CBC 040) b48RndIndRnd
B. seminalis CAMPA 22718RndRndRnd
Burkholderia vietnamiensis
B. vietnamiensis CAMPA 992 (CBC 038) b32ndRndS8S16
Staphylococcus aureus
S. aureus CAMPA 1909216128nd>128nd>128nd
S. aureus CAMPA 19081432>128>128nd>128nd
Stenotrophomonas maltophilia
S. maltophilia CAMPA 1911216>128nd>128nd>128nd
nd = non-determined. R= resistant, I = intermediate, S = sensible (according to the criteria set up by the CLSI). Meropenem (≤4 µg/mL S, 8 µg/mL I, ≥16 µg/mL R). Ceftazidime (≤8 µg/mL S, 16 µg/mL I, ≥32 µg/mL R). Tobramycin (≤4 µg/mL S, 8 µg/mL I, ≥16 µg/mL R). a Isolates recovered from patients with chronic infections in the period 2004–2017. b Isolates recovered from patients with cystic fibrosis whose complete antibiotic susceptibilities to 17 antimicrobial agents were previously reported (reference [55]).

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