- Email: [email protected]
Mutant prevention concentration of ciprofloxacin, enrofloxacin and nalidixic acid against Campylobacter jejuni
500
Letters to the Editor / International Journal of Antimicrobial Agents 31 (2008) 484–504
[7] Baker LV, Brown TJ, Maxwell O, Gibson AL, Fang Z, Yates MD, et al. Molecular analysis of isoniazid-resistant Mycobacterium tuberculosis isolates from England and Wales reveals the phylogenetic significance of the ahpC–46A polymorphism. Antimicrob Agents Chemother 2005;49:1455–64.
Kouassi Raymond N’Guessan ∗ Mireille Dosso Institut Pasteur de Cˆote d’Ivoire, Unit´e des Mycobact´eries Tuberculeuses et Atypiques, D´epartement de Bact´eriologie–Virologie, Abidjan, Cote d’Ivoire Euloge Ekaza Institut Pasteur de Cˆote d’Ivoire, Unit´e de Biologie Mol´eculaire, D´epartement de Bact´eriologie–Virologie, Abidjan, Cote d’Ivoire Jacquemin Kouakou Programme National de Lutte contre la Tuberculose, Abidjan, Cote d’Ivoire Vincent Jarlier National Reference Center for Mycobacteria, Department of Bacteriology–Hygiene, Facult´e de M´edecine Pierre & Marie Curie (Paris VI) site Piti´e-Salpˆetri`ere, Paris, France ∗ Corresponding
author. Tel.: +225 22 47 53 63. E-mail address: [email protected] (K.R. N’Guessan)
doi: 10.1016/j.ijantimicag.2008.01.008
Mutant prevention concentration of ciprofloxacin, enrofloxacin and nalidixic acid against Campylobacter jejuni Sir, Campylobacter jejuni is a leading cause of gastroenteritis in humans, the most common source of which is poultry [1]. Use of fluoroquinolones as one of the treatments of choice for campylobacteriosis in humans as well as of other bacterial infections both in human and veterinary medicine raises concern regarding the emergence of fluoroquinolone-resistant Campylobacter strains, with a potential increase of treatment failure both in veterinary and human therapy. The mutant prevention concentration (MPC), defined as the minimum inhibitory concentration (MIC) of the least drug-susceptible single-step mutant subpopulation, is a useful parameter to predict the efficacy of different antimicrobial agents in preventing the emergence of resistant bacteria during therapy [2]. No data are currently available on the MPCs of quinolones and fluoroquinolones against C. jejuni. The aim of this study was to evaluate the MPC of ciprofloxacin, enrofloxacin and nalidixic acid against fully susceptible strains of C. jejuni. Additionally, single-step mutants arising after exposure to sub-MPC antibiotic concentrations were investigated for the molecular basis of their fluoroquinoloneresistant phenotype.
Campylobacter jejuni ATCC 33560 (DSMZ, Braunschweig, Germany) was tested along with a C. jejuni poultry strain (named Cj 153/05). Both strains were susceptible to nalidixic acid, ciprofloxacin, enrofloxacin and erythromycin and carried wild-type quinolone resistancedetermining regions (QRDRs) of the gyrA gene. The MIC was determined by the agar dilution method according to the Clinical and Laboratory Standards Institute using C. jejuni ATCC 33560 as the quality control strain [3]. The MPC was determined using a previously reported protocol [4] with slight modifications: (i) the concentrated cellular suspension contained >109 colony-forming units/mL; and (ii) Mueller–Hinton agar (Difco, Milan, Italy) plates were supplemented with 5% defibrinated sheep blood and either enrofloxacin (Bayer Italia, Milan, Italy), ciprofloxacin (Bayer) or nalidixic acid (Sigma, Milan, Italy). After inoculation, all plates were incubated for 5 days at 42 ◦ C in microaerophilic conditions. From plates supplemented with antibiotic concentrations one or two dilutions lower than the MPC (sub-MPC), single-step mutants were randomly selected and tested for their MIC. Among all single-step mutants of each original strain, one representative mutant per each MIC value was chosen and tested for mutations in the QRDR of the gyrA gene using a polymerase chain reaction assay as described previously [5], with subsequent sequencing. Results of MICs and MPCs of all three antibiotics tested against both C. jejuni strains are summarised in Table 1. Ciprofloxacin MPCs were 16 mg/L and 8 mg/L for C. jejuni ATCC 33560 and Cj 153/05, respectively, whereas enrofloxacin MPCs were 4 mg/L and 2 mg/L and nalidixic acid MPC values were >128 mg/L and >64 g/L, respectively. According to resistance breakpoints [3,6], all the 38 single-step selected mutants were resistant to the respective antibiotic selective agent (Table 1). This observation confirms the single-step mechanism of acquired fluoroquinolone resistance in C. jejuni [7], in contrast to the well-known mechanism of multistep mutations required for fluoroquinolone resistance in other Gram-negative bacteria [2]. One mutation at codon 70, 86 or 90 (Thr86Ile, Ala70Ser, Asp90Tyr, Asp90Asn) was detected in the QRDR of the gyrA gene of each of the 14 C. jejuni representative resistant mutants selected (Table 1). Not surprisingly, Thr86Ile was the most frequent identified mutation, although previously unidentified (Ala70Ser) or rare (Ala90Tyr) mutations were also detected. Ciprofloxacin and enrofloxacin MPC values were higher than those detected for Escherichia coli and Salmonella Typhimurium in a previous study (2–16 mg/L vs. 0.06–2 mg/L) [4]. Recently, Drlica and Zhao [2] suggested that the MPC may overestimate the threshold needed to restrict mutant amplification for compounds showing a concentration-dependent killing activity such as fluoroquinolones and therefore sug-
Letters to the Editor / International Journal of Antimicrobial Agents 31 (2008) 484–504
501
Table 1 Mutant prevention concentrations (MPCs), minimum inhibitory concentrations (MICs) and quinolone resistance-determining region mutations of the gyrA gene of original fully susceptible strains and representative single-step MPC mutants of Campylobacter jejuni Antimicrobial/strain Ciprofloxacin Original strains C. jejuni ATCC 33560 C. jejuni 153/05 Single-step mutants Cj ATCC CIP 1 Cj 153/05 CIP 1 Enrofloxacin Original strains C. jejuni ATCC 33560 C. jejuni 153/05 Single-step mutants Cj ATCC ENR 1 Cj 153/05 ENR 1 Cj 153/05 ENR 2 Cj 153/05 ENR 7 Nalidixic acid Original strains C. jejuni ATCC 33560 C. jejuni 153/05 Single-step mutants Cj ATCC NA 3 Cj ATCC NA 4 Cj ATCC NA 5 Cj 153 NA 1 Cj 153 NA 4 Cj 153 NA 20 Cj 153 NA 21 Cj 153 NA 30
No. of selected mutants
4 6 – –
MIC (mg/L)
0.125 0.06 16 8
MPC (mg/L)
16 8
gyrA mutations
wt wt
– –
Thr86Ile Thr86Ile
3 7
0.125 0.06
4 2
wt wt
– – – –
2 2 4 8
– – – –
Thr86Ile Thr86Ile Thr86Ile Thr86Ile
4 14
8 4
>128 >64
– – – – – – – –
32 32 128 32 16 128 64 128
– – – – – – – –
wt wt Ala70Ser Ala70Ser Thr86Ile Thr86Ile Asp90Tyr Thr86Ile Asp90Asn Thr86Ile
wt, wild-type.
gest substituting the bacteriostatic MPC parameter with the area under the 24-h time–concentration curve (AUC24 ) value divided by the MPC as a better predictive index. Since no AUC24 /MPC threshold is currently available for the fluoroquinolone/C. jejuni combination, we decided to compare our MPC data with published maximum serum drug concentrations (Cmax ) in order to have a preliminary idea on whether fluoroquinolone monotherapy against C. jejuni may be suitable for restriction of mutant amplification or whether combination therapy is probably required. MPC values of ciprofloxacin against both C. jejuni strains tested were higher than the Cmax (4.4 mg/L) achievable in humans after a dose of 750 mg/day [8]. Regarding enrofloxacin, MPC values were equal to or higher than the Cmax (2.44 mg/L) achievable in chickens after a dose of 10 mg/kg [9]. Regarding nalidixic acid, both strains showed MPC values significantly higher than the Cmax of 20–40 mg/L achievable in patients with normal renal function [10]. In conclusion, this is the first report on MPCs of quinolones and fluoroquinolones against C. jejuni. Although more strains should be tested and a AUC24 /MPC threshold is still to be defined for the fluoroquinolone/C. jejuni
combination, our preliminary MPC data compared with published Cmax , and the selection of single-step mutants showing resistance to the selective agent, suggest that combination therapy may be required in order to restrict the selection of ciprofloxacin, enrofloxacin and nalidixic acid single-step mutants of C. jejuni.
Acknowledgments The authors wish to thank Simonetta Braggio for excellent technical assistance. This work was partly presented at the 20th International ICFMH Symposium ‘Food Safety and Food Biotechnology: diversity and global impact’, 2 August–2 September 2006, Bologna, Italy. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required. References [1] Tauxe RJ. Epidemiology of Campylobacter jejuni infections in the United States and other industrialised nations. In: Nachamkin I, Blase
502
[2] [3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
Letters to the Editor / International Journal of Antimicrobial Agents 31 (2008) 484–504 M, Tompkins LS, editors. Campylobacter jejuni: current status and future trends. Washington, DC: American Society for Microbiology; 1992. Drlica K, Zhao X. Mutant selection window hypothesis updated. Clin Infect Dis 2007;44:681–8. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. 2nd ed. Approved standard M31-A2. Wayne, PA: NCCLS; 2002. Pasquali F, Manfreda G. Mutant prevention concentration of ciprofloxacin and enrofloxacin against Escherichia coli, Salmonella Typhimurium and Pseudomonas aeruginosa. Vet Microbiol 2007;119:304–10. Zirnstein G, Li Y, Swaminathan B, Angulo F. Ciprofloxacin resistance in Campylobacter jejuni isolates: detection of gyrA resistance mutations by mismatch amplification mutation assay PCR and DNA sequence analysis. J Clin Microbiol 1999;37:3276–80. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. Fourteenth informational supplement. M100-S14. Wayne, PA: NCCLS; 2004. Payot S, Cloeckaert A, Chaslus-Dancla E. Selection and characterisation of fluoroquinolone-resistant mutants of Campylobacter jejuni using enrofloxacin. Microb Drug Resist 2002;8: 335–43. Israel D, Gillum JG, Turik M, Harvey K, Ford J, Dalton H, et al. Pharmacokinetics and serum bactericidal titers of ciprofloxacin and ofloxacin following multiple oral doses in healthy volunteers. Antimicrob Agents Chemother 1993;37:2193–9. Anad´on A, Mart´ınez-Larra˜naga MR, Iturbe J, Mart´ınez MA, D´ıaz MJ, Frejo MT, et al. Pharmacokinetics and residues of ciprofloxacin and its metabolites in broiler chickens. Res Vet Sci 2001;71:101–9. Cuisinaud G, Ferry N, Pozet N, Zech PY, Sassard J. Nalidixic acid kinetics in renal insufficiency. Br J Clin Pharmacol 1982;14:489–93.
Fr´ed´erique Pasquali ∗ Alex Lucchi Gerardo Manfreda Department of Food Science, Alma Mater Studiorum, University of Bologna, del Florio 2, 40064 Ozzano dell’ Emilia (BO), Italy ∗ Corresponding author. Tel.: +39 051 209 7862; fax: +39 051 209 7852. E-mail address: [email protected] (F. Pasquali) doi: 10.1016/j.ijantimicag.2008.01.012
New acridinone derivative with trypanocidal activity Sir, Diseases caused by protozoal parasites remain an important human health problem, despite much progress in understanding the biology of these organisms. Present chemotherapies are inadequate, toxic or are becoming ineffective due to an increase in resistance. Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and affects around 15 million people [1]. Efforts have been addressed to replace the only available drug used to treat this disease, benznidazole, which presents severe side effects and variable efficacy [2]. It has been established for many years that the planar structure of tricyclic rings confers to acridinone derivatives the
ability to intercalate with DNA and interfere with various metabolic processes both in prokaryotic and eukaryotic cells [3]. During the past decade, natural and synthetic compounds of the acridinone family have been investigated for their bactericidal and antitumoral activities, and a few molecules have been selected for antibacterial or anticancer chemotherapy. Recently, various derivatives of the acridinone series also demonstrated powerful inhibitory activities towards Plasmodium, Leishmania and Trypanosoma parasites [4]. Our group has developed new synthetic alternatives based on the Ullman reaction that have allowed new acridinone derivatives to be obtained [5]. In the present study, we obtained for the first time 10-allyl-6-chloro-2-fluoro-9(10H)-acridinone (AD-1) and evaluated its in vitro trypanocidal activity. AD-1 was obtained from 4-chloro-2-(4-fluroanilino) benzoic acid, using Ullman modifications in the presence of water as solvent, according to the literature [5]. The acridone derivative was synthesised using a phase-transfer agent as reported previously [6]. The structure of AD-1 was deduced on the basis of analysis of chemical shifts and coupling constants in the proton nuclear magnetic resonance (1 H NMR) spectra. Carbon (13 C) NMR assignation was performed using bidimensional techniques. A stock solution of 50 mM AD-1 in dimethyl sulfoxide (DMSO) was prepared and used with 1/1000 dilution (0.05 mM) to minimise the solvent’s influence on the experiments. T. cruzi epimastigotes (Tulahuen strain) were grown at 28 ◦ C in axenic medium containing brain–heart infusion, Na2 HPO4 , supplemented with 10% foetal calf serum, streptomycin and penicillin. At early stationary phase (≈7 days), cells were harvested by centrifugation and washed twice in medium containing NaCl, KCl, Na2 HPO4 and KH2 PO4 (pH 7.0). Cell numbers were ascertained using a Neubauer chamber. T. cruzi (108 cells) was incubated in the presence of 0.05 mM AD-1 for 30 min in phosphate-buffered saline medium in a humidified atmosphere at 37 ◦ C with 5% CO2 . Cells were labelled with Annexin-V and propidium iodide (PI) following the manufacturer instructions and were analysed in a FACSCalibur flow cytometer (Becton Dickinson). Apoptosis was quantified as the percentage of Annexin-V–fluorescein isothiocyanate (FITC)-positive and PI-positive cells over the total number of cells. Generation of intracellular reactive oxygen species (ROS) and the mitochondrial electrical membrane potential were measured by flow cytometry using the probes DHE and DioC6 (3), respectively. Results from experiments in triplicate were normalised using the F/FCCCP ratio, where F is the mean fluorescence intensity of DioC6 (3) (maximum fluorescence) and FCCCP is the mean fluorescence intensity in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) (minimum fluorescence). The protonophore CCCP dissipates the mitochondrial electrical membrane potential. Fig. 1 shows that 0.05 mM AD-1, when incubated for 30 min with the parasites, induces 52.4% death; 40.8% of Annexin-V-positive cells (apoptotic death; Fig. 1A) and 11.4% PI-positive cells (necrotic death; Fig. 1B).
Letters to the Editor / International Journal of Antimicrobial Agents 31 (2008) 484–504
[7] Baker LV, Brown TJ, Maxwell O, Gibson AL, Fang Z, Yates MD, et al. Molecular analysis of isoniazid-resistant Mycobacterium tuberculosis isolates from England and Wales reveals the phylogenetic significance of the ahpC–46A polymorphism. Antimicrob Agents Chemother 2005;49:1455–64.
Kouassi Raymond N’Guessan ∗ Mireille Dosso Institut Pasteur de Cˆote d’Ivoire, Unit´e des Mycobact´eries Tuberculeuses et Atypiques, D´epartement de Bact´eriologie–Virologie, Abidjan, Cote d’Ivoire Euloge Ekaza Institut Pasteur de Cˆote d’Ivoire, Unit´e de Biologie Mol´eculaire, D´epartement de Bact´eriologie–Virologie, Abidjan, Cote d’Ivoire Jacquemin Kouakou Programme National de Lutte contre la Tuberculose, Abidjan, Cote d’Ivoire Vincent Jarlier National Reference Center for Mycobacteria, Department of Bacteriology–Hygiene, Facult´e de M´edecine Pierre & Marie Curie (Paris VI) site Piti´e-Salpˆetri`ere, Paris, France ∗ Corresponding
author. Tel.: +225 22 47 53 63. E-mail address: [email protected] (K.R. N’Guessan)
doi: 10.1016/j.ijantimicag.2008.01.008
Mutant prevention concentration of ciprofloxacin, enrofloxacin and nalidixic acid against Campylobacter jejuni Sir, Campylobacter jejuni is a leading cause of gastroenteritis in humans, the most common source of which is poultry [1]. Use of fluoroquinolones as one of the treatments of choice for campylobacteriosis in humans as well as of other bacterial infections both in human and veterinary medicine raises concern regarding the emergence of fluoroquinolone-resistant Campylobacter strains, with a potential increase of treatment failure both in veterinary and human therapy. The mutant prevention concentration (MPC), defined as the minimum inhibitory concentration (MIC) of the least drug-susceptible single-step mutant subpopulation, is a useful parameter to predict the efficacy of different antimicrobial agents in preventing the emergence of resistant bacteria during therapy [2]. No data are currently available on the MPCs of quinolones and fluoroquinolones against C. jejuni. The aim of this study was to evaluate the MPC of ciprofloxacin, enrofloxacin and nalidixic acid against fully susceptible strains of C. jejuni. Additionally, single-step mutants arising after exposure to sub-MPC antibiotic concentrations were investigated for the molecular basis of their fluoroquinoloneresistant phenotype.
Campylobacter jejuni ATCC 33560 (DSMZ, Braunschweig, Germany) was tested along with a C. jejuni poultry strain (named Cj 153/05). Both strains were susceptible to nalidixic acid, ciprofloxacin, enrofloxacin and erythromycin and carried wild-type quinolone resistancedetermining regions (QRDRs) of the gyrA gene. The MIC was determined by the agar dilution method according to the Clinical and Laboratory Standards Institute using C. jejuni ATCC 33560 as the quality control strain [3]. The MPC was determined using a previously reported protocol [4] with slight modifications: (i) the concentrated cellular suspension contained >109 colony-forming units/mL; and (ii) Mueller–Hinton agar (Difco, Milan, Italy) plates were supplemented with 5% defibrinated sheep blood and either enrofloxacin (Bayer Italia, Milan, Italy), ciprofloxacin (Bayer) or nalidixic acid (Sigma, Milan, Italy). After inoculation, all plates were incubated for 5 days at 42 ◦ C in microaerophilic conditions. From plates supplemented with antibiotic concentrations one or two dilutions lower than the MPC (sub-MPC), single-step mutants were randomly selected and tested for their MIC. Among all single-step mutants of each original strain, one representative mutant per each MIC value was chosen and tested for mutations in the QRDR of the gyrA gene using a polymerase chain reaction assay as described previously [5], with subsequent sequencing. Results of MICs and MPCs of all three antibiotics tested against both C. jejuni strains are summarised in Table 1. Ciprofloxacin MPCs were 16 mg/L and 8 mg/L for C. jejuni ATCC 33560 and Cj 153/05, respectively, whereas enrofloxacin MPCs were 4 mg/L and 2 mg/L and nalidixic acid MPC values were >128 mg/L and >64 g/L, respectively. According to resistance breakpoints [3,6], all the 38 single-step selected mutants were resistant to the respective antibiotic selective agent (Table 1). This observation confirms the single-step mechanism of acquired fluoroquinolone resistance in C. jejuni [7], in contrast to the well-known mechanism of multistep mutations required for fluoroquinolone resistance in other Gram-negative bacteria [2]. One mutation at codon 70, 86 or 90 (Thr86Ile, Ala70Ser, Asp90Tyr, Asp90Asn) was detected in the QRDR of the gyrA gene of each of the 14 C. jejuni representative resistant mutants selected (Table 1). Not surprisingly, Thr86Ile was the most frequent identified mutation, although previously unidentified (Ala70Ser) or rare (Ala90Tyr) mutations were also detected. Ciprofloxacin and enrofloxacin MPC values were higher than those detected for Escherichia coli and Salmonella Typhimurium in a previous study (2–16 mg/L vs. 0.06–2 mg/L) [4]. Recently, Drlica and Zhao [2] suggested that the MPC may overestimate the threshold needed to restrict mutant amplification for compounds showing a concentration-dependent killing activity such as fluoroquinolones and therefore sug-
Letters to the Editor / International Journal of Antimicrobial Agents 31 (2008) 484–504
501
Table 1 Mutant prevention concentrations (MPCs), minimum inhibitory concentrations (MICs) and quinolone resistance-determining region mutations of the gyrA gene of original fully susceptible strains and representative single-step MPC mutants of Campylobacter jejuni Antimicrobial/strain Ciprofloxacin Original strains C. jejuni ATCC 33560 C. jejuni 153/05 Single-step mutants Cj ATCC CIP 1 Cj 153/05 CIP 1 Enrofloxacin Original strains C. jejuni ATCC 33560 C. jejuni 153/05 Single-step mutants Cj ATCC ENR 1 Cj 153/05 ENR 1 Cj 153/05 ENR 2 Cj 153/05 ENR 7 Nalidixic acid Original strains C. jejuni ATCC 33560 C. jejuni 153/05 Single-step mutants Cj ATCC NA 3 Cj ATCC NA 4 Cj ATCC NA 5 Cj 153 NA 1 Cj 153 NA 4 Cj 153 NA 20 Cj 153 NA 21 Cj 153 NA 30
No. of selected mutants
4 6 – –
MIC (mg/L)
0.125 0.06 16 8
MPC (mg/L)
16 8
gyrA mutations
wt wt
– –
Thr86Ile Thr86Ile
3 7
0.125 0.06
4 2
wt wt
– – – –
2 2 4 8
– – – –
Thr86Ile Thr86Ile Thr86Ile Thr86Ile
4 14
8 4
>128 >64
– – – – – – – –
32 32 128 32 16 128 64 128
– – – – – – – –
wt wt Ala70Ser Ala70Ser Thr86Ile Thr86Ile Asp90Tyr Thr86Ile Asp90Asn Thr86Ile
wt, wild-type.
gest substituting the bacteriostatic MPC parameter with the area under the 24-h time–concentration curve (AUC24 ) value divided by the MPC as a better predictive index. Since no AUC24 /MPC threshold is currently available for the fluoroquinolone/C. jejuni combination, we decided to compare our MPC data with published maximum serum drug concentrations (Cmax ) in order to have a preliminary idea on whether fluoroquinolone monotherapy against C. jejuni may be suitable for restriction of mutant amplification or whether combination therapy is probably required. MPC values of ciprofloxacin against both C. jejuni strains tested were higher than the Cmax (4.4 mg/L) achievable in humans after a dose of 750 mg/day [8]. Regarding enrofloxacin, MPC values were equal to or higher than the Cmax (2.44 mg/L) achievable in chickens after a dose of 10 mg/kg [9]. Regarding nalidixic acid, both strains showed MPC values significantly higher than the Cmax of 20–40 mg/L achievable in patients with normal renal function [10]. In conclusion, this is the first report on MPCs of quinolones and fluoroquinolones against C. jejuni. Although more strains should be tested and a AUC24 /MPC threshold is still to be defined for the fluoroquinolone/C. jejuni
combination, our preliminary MPC data compared with published Cmax , and the selection of single-step mutants showing resistance to the selective agent, suggest that combination therapy may be required in order to restrict the selection of ciprofloxacin, enrofloxacin and nalidixic acid single-step mutants of C. jejuni.
Acknowledgments The authors wish to thank Simonetta Braggio for excellent technical assistance. This work was partly presented at the 20th International ICFMH Symposium ‘Food Safety and Food Biotechnology: diversity and global impact’, 2 August–2 September 2006, Bologna, Italy. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required. References [1] Tauxe RJ. Epidemiology of Campylobacter jejuni infections in the United States and other industrialised nations. In: Nachamkin I, Blase
502
[2] [3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
Letters to the Editor / International Journal of Antimicrobial Agents 31 (2008) 484–504 M, Tompkins LS, editors. Campylobacter jejuni: current status and future trends. Washington, DC: American Society for Microbiology; 1992. Drlica K, Zhao X. Mutant selection window hypothesis updated. Clin Infect Dis 2007;44:681–8. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. 2nd ed. Approved standard M31-A2. Wayne, PA: NCCLS; 2002. Pasquali F, Manfreda G. Mutant prevention concentration of ciprofloxacin and enrofloxacin against Escherichia coli, Salmonella Typhimurium and Pseudomonas aeruginosa. Vet Microbiol 2007;119:304–10. Zirnstein G, Li Y, Swaminathan B, Angulo F. Ciprofloxacin resistance in Campylobacter jejuni isolates: detection of gyrA resistance mutations by mismatch amplification mutation assay PCR and DNA sequence analysis. J Clin Microbiol 1999;37:3276–80. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. Fourteenth informational supplement. M100-S14. Wayne, PA: NCCLS; 2004. Payot S, Cloeckaert A, Chaslus-Dancla E. Selection and characterisation of fluoroquinolone-resistant mutants of Campylobacter jejuni using enrofloxacin. Microb Drug Resist 2002;8: 335–43. Israel D, Gillum JG, Turik M, Harvey K, Ford J, Dalton H, et al. Pharmacokinetics and serum bactericidal titers of ciprofloxacin and ofloxacin following multiple oral doses in healthy volunteers. Antimicrob Agents Chemother 1993;37:2193–9. Anad´on A, Mart´ınez-Larra˜naga MR, Iturbe J, Mart´ınez MA, D´ıaz MJ, Frejo MT, et al. Pharmacokinetics and residues of ciprofloxacin and its metabolites in broiler chickens. Res Vet Sci 2001;71:101–9. Cuisinaud G, Ferry N, Pozet N, Zech PY, Sassard J. Nalidixic acid kinetics in renal insufficiency. Br J Clin Pharmacol 1982;14:489–93.
Fr´ed´erique Pasquali ∗ Alex Lucchi Gerardo Manfreda Department of Food Science, Alma Mater Studiorum, University of Bologna, del Florio 2, 40064 Ozzano dell’ Emilia (BO), Italy ∗ Corresponding author. Tel.: +39 051 209 7862; fax: +39 051 209 7852. E-mail address: [email protected] (F. Pasquali) doi: 10.1016/j.ijantimicag.2008.01.012
New acridinone derivative with trypanocidal activity Sir, Diseases caused by protozoal parasites remain an important human health problem, despite much progress in understanding the biology of these organisms. Present chemotherapies are inadequate, toxic or are becoming ineffective due to an increase in resistance. Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and affects around 15 million people [1]. Efforts have been addressed to replace the only available drug used to treat this disease, benznidazole, which presents severe side effects and variable efficacy [2]. It has been established for many years that the planar structure of tricyclic rings confers to acridinone derivatives the
ability to intercalate with DNA and interfere with various metabolic processes both in prokaryotic and eukaryotic cells [3]. During the past decade, natural and synthetic compounds of the acridinone family have been investigated for their bactericidal and antitumoral activities, and a few molecules have been selected for antibacterial or anticancer chemotherapy. Recently, various derivatives of the acridinone series also demonstrated powerful inhibitory activities towards Plasmodium, Leishmania and Trypanosoma parasites [4]. Our group has developed new synthetic alternatives based on the Ullman reaction that have allowed new acridinone derivatives to be obtained [5]. In the present study, we obtained for the first time 10-allyl-6-chloro-2-fluoro-9(10H)-acridinone (AD-1) and evaluated its in vitro trypanocidal activity. AD-1 was obtained from 4-chloro-2-(4-fluroanilino) benzoic acid, using Ullman modifications in the presence of water as solvent, according to the literature [5]. The acridone derivative was synthesised using a phase-transfer agent as reported previously [6]. The structure of AD-1 was deduced on the basis of analysis of chemical shifts and coupling constants in the proton nuclear magnetic resonance (1 H NMR) spectra. Carbon (13 C) NMR assignation was performed using bidimensional techniques. A stock solution of 50 mM AD-1 in dimethyl sulfoxide (DMSO) was prepared and used with 1/1000 dilution (0.05 mM) to minimise the solvent’s influence on the experiments. T. cruzi epimastigotes (Tulahuen strain) were grown at 28 ◦ C in axenic medium containing brain–heart infusion, Na2 HPO4 , supplemented with 10% foetal calf serum, streptomycin and penicillin. At early stationary phase (≈7 days), cells were harvested by centrifugation and washed twice in medium containing NaCl, KCl, Na2 HPO4 and KH2 PO4 (pH 7.0). Cell numbers were ascertained using a Neubauer chamber. T. cruzi (108 cells) was incubated in the presence of 0.05 mM AD-1 for 30 min in phosphate-buffered saline medium in a humidified atmosphere at 37 ◦ C with 5% CO2 . Cells were labelled with Annexin-V and propidium iodide (PI) following the manufacturer instructions and were analysed in a FACSCalibur flow cytometer (Becton Dickinson). Apoptosis was quantified as the percentage of Annexin-V–fluorescein isothiocyanate (FITC)-positive and PI-positive cells over the total number of cells. Generation of intracellular reactive oxygen species (ROS) and the mitochondrial electrical membrane potential were measured by flow cytometry using the probes DHE and DioC6 (3), respectively. Results from experiments in triplicate were normalised using the F/FCCCP ratio, where F is the mean fluorescence intensity of DioC6 (3) (maximum fluorescence) and FCCCP is the mean fluorescence intensity in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) (minimum fluorescence). The protonophore CCCP dissipates the mitochondrial electrical membrane potential. Fig. 1 shows that 0.05 mM AD-1, when incubated for 30 min with the parasites, induces 52.4% death; 40.8% of Annexin-V-positive cells (apoptotic death; Fig. 1A) and 11.4% PI-positive cells (necrotic death; Fig. 1B).