Letters to the Editor / International Journal of Antimicrobial Agents 43 (2014) 383–393
Ethical approval: Not required.
References [1] Gelfand MS, Mazumder SA, Cleveland K. Minocycline for the treatment of community-acquired Staphylococcus aureus infections. Int J Antimicrob Agents 2014:43. [2] Cunha BA. Minocycline, often forgotten but preferred to trimethoprim– sulfamethoxazole or doxycycline for the treatment of community-acquired meticillin-resistant Staphylococcus aureus skin and soft-tissue infections. Int J Antimicrob Agents 2013;42:497–9. [3] Schwartz BS, Graber CJ, Diep BA, Basuino L, Perdreau-Remington F, Chambers HF. Doxycycline, not minocycline, induces its own resistance in multidrugresistant, community-associated methicillin-resistant Staphylococcus aureus clone USA300. Clin Infect Dis 2009;48:1483–4. [4] Cunha BA. Minocycline versus doxycycline for meticillin-resistant Staphylococcus aureus (MRSA): in vitro susceptibility versus in vivo effectiveness. Int J Antimicrob Agents 2010;35:517–8. [5] Cunha BA. Methicillin-resistant Staphylococcus aureus: clinical manifestations and antimicrobial therapy. Clin Microbiol Infect 2005;11(Suppl. 4):33–42. [6] Ruhe JJ, Monson T, Bradsher RW, Menon A. Use of long-acting tetracyclines for methicillin-resistant Staphylococcus aureus infections: case series and review of the literature. Clin Infect Dis 2005;40:1429–34. [7] Ruhe JJ, Smith N, Bradsher RW, Menon A. Community-onset methicillinresistant Staphylococcus aureus skin and soft-tissue infections: impact of antimicrobial therapy on outcome. Clin Infect Dis 2007;44: 777–84. [8] Cunha BA. Oral antibiotic therapy of serious systemic infections. Med Clin North Am 2006;90:1197–222. [9] Kucers A, (Editor). Kucers’ the Use of Antibiotics. etc. 5th ed. Oxford, UK: Butterworth Heinemann; 1997. pp. 719–63. [10] Cunha BA, editor. Antibiotic essentials. 12th ed. Sudbury, MA: Jones & Bartlett; 2013. pp. 8–16, 132–5, 595–7, 647–8, 680–2.
Burke A. Cunha Infectious Disease Division, Winthrop-University Hospital, Mineola, NY, USA State University of New York, School of Medicine, Stony Brook, NY, USA E-mail address: [email protected] 6 January 2014 http://dx.doi.org/10.1016/j.ijantimicag.2014.01.003
Importance of chemical modification at C-7 position of quinolones for glutathione-mediated reversal of antibacterial activity Sir We have previously shown that glutathione (GSH) modulates the antibacterial activity of fluoroquinolones, aminoglycosides and -lactams [1]. We further established that GSH-mediated decreased fluoroquinolone activity can be attributed to neutralisation of antibiotic-induced oxidative stress in bacteria [1]. Different chemical groups at the C-6 and C-7 positions in quinolones affect their activity and spectrum [2]. Accordingly, they can also influence the GSH-mediated protection phenotype against fluoroquinolones. In the present study, we investigated whether chemical modification at positions C-6 and C-7 of the quinolone play a role in GSH-mediated inhibition of fluoroquinolone activity. The effect of GSH on bacterial susceptibility to quinolone derivatives was measured using Escherichia coli K-12 strain MG1655. Different quinolone derivatives represented the selective presence or absence of C-6 fluoro and C-7 diamine groups (Table 1). Minimum inhibitory concentrations (MICs) of the derivatives were determined by the agar dilution method in the presence and absence of 10 mM GSH as outlined previously [1]. Briefly, an inoculum containing 104 –105 CFU was applied to Lysogeny broth agar plates with increasing antibiotic concentrations. The MIC was
387
defined as the lowest concentration of antibiotic that prevented visible growth after 20 h of incubation at 37 ◦ C. We first examined whether GSH decreased the antibacterial activity of a classical quinolone, namely nalidixic acid, on the same lines as the fluoroquinolone ciprofloxacin reported previously [1]. However, nalidixic acid susceptibility of E. coli was unaltered in presence of GSH (Table 1). The disparity in response of GSH towards these quinolones could be attributed to their structure, as both of them have the same site of action in bacteria. Apart from nalidixic acid, the activities of oxolinic acid and cinoxacin were also not affected by GSH. Importantly, all of these derivatives lacked 6-fluoro and 7-diamine groups, showing that one or both of these groups could be important for the GSH-mediated protection phenotype. Likewise, the MIC of flumequine was not affected in the presence of GSH. Flumequine is a unique antibiotic without any diamine residue, having a C-6 fluoro group attached to a quinolone pharmacore. Therefore, it unequivocally demonstrated that a GSH-mediated decreased MIC is not mediated through the C-6 fluoro group of the quinolone. On the other hand, the MIC of pipemidic acid increased 10-fold in the presence of GSH, establishing that GSH can decrease the antibacterial activity of non-fluorinated quinolones as well. Moreover, the presence of a C-7 piperazinyl group in quinolones is both necessary and sufficient for GSH to counter its antibacterial action. Like ciprofloxacin, the MIC of norfloxacin increased 100fold in the presence of GSH, confirming the importance of a C-7 piperazinyl moiety in this phenotype. A more than 10-fold augmentation in the MIC of ofloxacin, pefloxacin, lomefloxacin and gatifloxacin by GSH established that secondary chemical modifications in the C-7 piperazinyl group do not affect the abovementioned phenotype. Decreased moxifloxacin and gemifloxacin susceptibility implied that the presence of any diamine group at the C-7 position is adequate for GSH-mediated reversal of quinolone activity, as these derivatives have chemically different diamine groups. Of the 14 different quinolones tested in the current study, GSH decreased the activity of all 10 derivatives having a diamine group at the C-7 position. Moreover, these quinolones are structurally different at C-1, C-5 and C-8 positions, implying that chemical modification at these positions does not play an apparently significant role in the GSH-mediated protection phenotype. Notably, C-2, C-3 and C-4 positions in quinolones are not amenable to modification owing to their imperative role in binding and inhibition of DNA gyrase and DNA topoisomerase IV [2]. Taken together, these data reveal that a diamine group at the C-7 position in quinolones determines its response towards GSH, which is a major cellular antioxidant. More than one factor could be playing a role here. First, the presence of diamine affects the transport of quinolones [2] by inhibiting their efflux. In addition, as per a previous report [3], GSH might influence the efflux of quinolones. The current findings of GSH-mediated increased MICs of fluoroquinolones (having C-6 fluoro and C-7 diamine groups) but not of quinolones (lacking a C-7 diamine group) indicate that fluoroquinolones could be the preferential substrates over quinolones for GSH-induced antibiotic efflux. Our proposition is substantiated by a previous report showing involvement of a similar efflux system in ciprofloxacin-resistant, nalidixic-acid-sensitive bacteria [4]. Second, the presence of a diamine group in quinolones can predispose these molecules to oxidative stress induction on interaction with their biological targets. Accordingly, GSH reduces the activity of fluoroquinolones [1] but not of quinolones. Lastly, GSH-mediated modification at the diamine binding pocket in DNA gyrase/topoisomerase IV can also contribute towards selective quinolone susceptibility. The proposed scheme is supported by a preceding independent report [5]. Although these propositions are yet to be experimentally validated in order to completely
388
Letters to the Editor / International Journal of Antimicrobial Agents 43 (2014) 383–393
Table 1 Effect of glutathione (GSH) on the minimum inhibitory concentration (MIC) of different quinolone derivatives in Escherichia coli K-12 strain MG165 Antibiotic
MIC (g/mL) Without GSH
With GSH
Inhibition of antibacterial action by GSH
Chemical groups at C-6 and C-7 position in quinolone molecule 6-H 7-H 6,7-Heterocyclic ring 6,7-Heterocyclic ring 6-F 7-H 6-H 7-Piperazinyl 6-F 7-Piperazinyl 6-F 7-Piperazinyl 6-F 7-3-Methylpiperazinyl 6-F 7-3-Methylpiperazinyl 6-F 7-2-Methylpiperazinyl 6-F 7-2-Methylpiperazinyl 6-F 7-[(3R,5S) 3,5-dimethylpiperazin-1-yl] 6-F 7-diazabicyclo 6-F 7-[(4Z)-3-(aminomethyl)-4-methoxyimino-pyrrolidin-1 yl]
Nalidixic acid
4
4
No
Oxolinic acid Cinoxacin Flumequine
1 4 0.8
1 4 0.8
No No No
Pipemidic acid
4
Norfloxacin
0.001
0.1
Yes
Ciprofloxacin
0.03
0.3
Yes
Ofloxacin
0.05
>1.0
Yes
Pefloxacin
0.01
1.0
Yes
Lomefloxacin
0.001
0.01
Yes
Gatifloxacin
0.001
0.01
Yes
Sparfloxacin
Ethical approval: Not required.
References [1] Gelfand MS, Mazumder SA, Cleveland K. Minocycline for the treatment of community-acquired Staphylococcus aureus infections. Int J Antimicrob Agents 2014:43. [2] Cunha BA. Minocycline, often forgotten but preferred to trimethoprim– sulfamethoxazole or doxycycline for the treatment of community-acquired meticillin-resistant Staphylococcus aureus skin and soft-tissue infections. Int J Antimicrob Agents 2013;42:497–9. [3] Schwartz BS, Graber CJ, Diep BA, Basuino L, Perdreau-Remington F, Chambers HF. Doxycycline, not minocycline, induces its own resistance in multidrugresistant, community-associated methicillin-resistant Staphylococcus aureus clone USA300. Clin Infect Dis 2009;48:1483–4. [4] Cunha BA. Minocycline versus doxycycline for meticillin-resistant Staphylococcus aureus (MRSA): in vitro susceptibility versus in vivo effectiveness. Int J Antimicrob Agents 2010;35:517–8. [5] Cunha BA. Methicillin-resistant Staphylococcus aureus: clinical manifestations and antimicrobial therapy. Clin Microbiol Infect 2005;11(Suppl. 4):33–42. [6] Ruhe JJ, Monson T, Bradsher RW, Menon A. Use of long-acting tetracyclines for methicillin-resistant Staphylococcus aureus infections: case series and review of the literature. Clin Infect Dis 2005;40:1429–34. [7] Ruhe JJ, Smith N, Bradsher RW, Menon A. Community-onset methicillinresistant Staphylococcus aureus skin and soft-tissue infections: impact of antimicrobial therapy on outcome. Clin Infect Dis 2007;44: 777–84. [8] Cunha BA. Oral antibiotic therapy of serious systemic infections. Med Clin North Am 2006;90:1197–222. [9] Kucers A, (Editor). Kucers’ the Use of Antibiotics. etc. 5th ed. Oxford, UK: Butterworth Heinemann; 1997. pp. 719–63. [10] Cunha BA, editor. Antibiotic essentials. 12th ed. Sudbury, MA: Jones & Bartlett; 2013. pp. 8–16, 132–5, 595–7, 647–8, 680–2.
Burke A. Cunha Infectious Disease Division, Winthrop-University Hospital, Mineola, NY, USA State University of New York, School of Medicine, Stony Brook, NY, USA E-mail address: [email protected] 6 January 2014 http://dx.doi.org/10.1016/j.ijantimicag.2014.01.003
Importance of chemical modification at C-7 position of quinolones for glutathione-mediated reversal of antibacterial activity Sir We have previously shown that glutathione (GSH) modulates the antibacterial activity of fluoroquinolones, aminoglycosides and -lactams [1]. We further established that GSH-mediated decreased fluoroquinolone activity can be attributed to neutralisation of antibiotic-induced oxidative stress in bacteria [1]. Different chemical groups at the C-6 and C-7 positions in quinolones affect their activity and spectrum [2]. Accordingly, they can also influence the GSH-mediated protection phenotype against fluoroquinolones. In the present study, we investigated whether chemical modification at positions C-6 and C-7 of the quinolone play a role in GSH-mediated inhibition of fluoroquinolone activity. The effect of GSH on bacterial susceptibility to quinolone derivatives was measured using Escherichia coli K-12 strain MG1655. Different quinolone derivatives represented the selective presence or absence of C-6 fluoro and C-7 diamine groups (Table 1). Minimum inhibitory concentrations (MICs) of the derivatives were determined by the agar dilution method in the presence and absence of 10 mM GSH as outlined previously [1]. Briefly, an inoculum containing 104 –105 CFU was applied to Lysogeny broth agar plates with increasing antibiotic concentrations. The MIC was
387
defined as the lowest concentration of antibiotic that prevented visible growth after 20 h of incubation at 37 ◦ C. We first examined whether GSH decreased the antibacterial activity of a classical quinolone, namely nalidixic acid, on the same lines as the fluoroquinolone ciprofloxacin reported previously [1]. However, nalidixic acid susceptibility of E. coli was unaltered in presence of GSH (Table 1). The disparity in response of GSH towards these quinolones could be attributed to their structure, as both of them have the same site of action in bacteria. Apart from nalidixic acid, the activities of oxolinic acid and cinoxacin were also not affected by GSH. Importantly, all of these derivatives lacked 6-fluoro and 7-diamine groups, showing that one or both of these groups could be important for the GSH-mediated protection phenotype. Likewise, the MIC of flumequine was not affected in the presence of GSH. Flumequine is a unique antibiotic without any diamine residue, having a C-6 fluoro group attached to a quinolone pharmacore. Therefore, it unequivocally demonstrated that a GSH-mediated decreased MIC is not mediated through the C-6 fluoro group of the quinolone. On the other hand, the MIC of pipemidic acid increased 10-fold in the presence of GSH, establishing that GSH can decrease the antibacterial activity of non-fluorinated quinolones as well. Moreover, the presence of a C-7 piperazinyl group in quinolones is both necessary and sufficient for GSH to counter its antibacterial action. Like ciprofloxacin, the MIC of norfloxacin increased 100fold in the presence of GSH, confirming the importance of a C-7 piperazinyl moiety in this phenotype. A more than 10-fold augmentation in the MIC of ofloxacin, pefloxacin, lomefloxacin and gatifloxacin by GSH established that secondary chemical modifications in the C-7 piperazinyl group do not affect the abovementioned phenotype. Decreased moxifloxacin and gemifloxacin susceptibility implied that the presence of any diamine group at the C-7 position is adequate for GSH-mediated reversal of quinolone activity, as these derivatives have chemically different diamine groups. Of the 14 different quinolones tested in the current study, GSH decreased the activity of all 10 derivatives having a diamine group at the C-7 position. Moreover, these quinolones are structurally different at C-1, C-5 and C-8 positions, implying that chemical modification at these positions does not play an apparently significant role in the GSH-mediated protection phenotype. Notably, C-2, C-3 and C-4 positions in quinolones are not amenable to modification owing to their imperative role in binding and inhibition of DNA gyrase and DNA topoisomerase IV [2]. Taken together, these data reveal that a diamine group at the C-7 position in quinolones determines its response towards GSH, which is a major cellular antioxidant. More than one factor could be playing a role here. First, the presence of diamine affects the transport of quinolones [2] by inhibiting their efflux. In addition, as per a previous report [3], GSH might influence the efflux of quinolones. The current findings of GSH-mediated increased MICs of fluoroquinolones (having C-6 fluoro and C-7 diamine groups) but not of quinolones (lacking a C-7 diamine group) indicate that fluoroquinolones could be the preferential substrates over quinolones for GSH-induced antibiotic efflux. Our proposition is substantiated by a previous report showing involvement of a similar efflux system in ciprofloxacin-resistant, nalidixic-acid-sensitive bacteria [4]. Second, the presence of a diamine group in quinolones can predispose these molecules to oxidative stress induction on interaction with their biological targets. Accordingly, GSH reduces the activity of fluoroquinolones [1] but not of quinolones. Lastly, GSH-mediated modification at the diamine binding pocket in DNA gyrase/topoisomerase IV can also contribute towards selective quinolone susceptibility. The proposed scheme is supported by a preceding independent report [5]. Although these propositions are yet to be experimentally validated in order to completely
388
Letters to the Editor / International Journal of Antimicrobial Agents 43 (2014) 383–393
Table 1 Effect of glutathione (GSH) on the minimum inhibitory concentration (MIC) of different quinolone derivatives in Escherichia coli K-12 strain MG165 Antibiotic
MIC (g/mL) Without GSH
With GSH
Inhibition of antibacterial action by GSH
Chemical groups at C-6 and C-7 position in quinolone molecule 6-H 7-H 6,7-Heterocyclic ring 6,7-Heterocyclic ring 6-F 7-H 6-H 7-Piperazinyl 6-F 7-Piperazinyl 6-F 7-Piperazinyl 6-F 7-3-Methylpiperazinyl 6-F 7-3-Methylpiperazinyl 6-F 7-2-Methylpiperazinyl 6-F 7-2-Methylpiperazinyl 6-F 7-[(3R,5S) 3,5-dimethylpiperazin-1-yl] 6-F 7-diazabicyclo 6-F 7-[(4Z)-3-(aminomethyl)-4-methoxyimino-pyrrolidin-1 yl]
Nalidixic acid
4
4
No
Oxolinic acid Cinoxacin Flumequine
1 4 0.8
1 4 0.8
No No No
Pipemidic acid
4
Norfloxacin
0.001
0.1
Yes
Ciprofloxacin
0.03
0.3
Yes
Ofloxacin
0.05
>1.0
Yes
Pefloxacin
0.01
1.0
Yes
Lomefloxacin
0.001
0.01
Yes
Gatifloxacin
0.001
0.01
Yes
Sparfloxacin
