ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1996, p. 35 39 Vol. 40, No. 1 0066-4804/96/$04.00 0 Copyright 1996, American Society for Microbiology Influence of ph on Adaptive Resistance of Pseudomonas aeruginosa to Aminoglycosides and Their Postantibiotic Effects YAN-QIONG XIONG, JOCELYNE CAILLON, HENRI DRUGEON, GILLES POTEL,* AND DENIS BARON Laboratoire d Antibiologie Clinique et Expérimentale, Faculté demédecine, Centre Hospitalier Universitaire, 44035 Nantes, France Received 12 June 1995/Returned for modification 30 August 1995/Accepted 27 October 1995 Adaptive resistance to aminoglycosides in Pseudomonas aeruginosa and other gram-negative bacilli is usually induced by the initial exposure to the drug. We investigated the influence of ph on the adaptive resistance of a clinical P. aeruginosa strain to aminoglycosides in vitro and on their postantibiotic effects. For adaptive resistance, the first-exposure concentrations of both amikacin and netilmicin were one, two, four, and eight times the MIC of each drug and the second-exposure concentrations were two times the MIC of each drug. Adaptive resistance was greater and more prolonged with higher initial aminoglycoside concentrations, and the bactericidal effects of the aminoglycosides were concentration dependent at ph 7.4. At ph 6.5, the killing rates of amikacin and netilmicin were far lower than those observed at ph 7.4. At ph 5.5, amikacin and netilmicin exerted practically no bactericidal effect on the P. aeruginosa strain used. However, with media at ph 5.5 and 6.5, adaptive resistance of P. aeruginosa preexposed to amikacin and netilmicin was also clearly exhibited, with the degree of adaptive resistance depending on the bactericidal effects of both drugs on nonpreexposed controls. Maximal adaptive resistance occurred between 0 and 4 h after preexposure. The postantibiotic effects of amikacin and netilmicin against the P. aeruginosa strain were shown to be concentration dependent and were reduced at acidic phs. No changes in outer and inner membrane proteins occurred during the adaptiveresistance interval. Adaptive resistance for Pseudomonas aeruginosa and other gram-negative bacilli after exposure to aminoglycosides has been recently described. This reversible form of resistance develops within 2 to 3 h of the initial exposure to an aminoglycoside and then disappears during growth for several hours in a drug-free medium (2, 5, 6). The mechanism of adaptive resistance is not completely understood but is probably related to the reversible downregulation of aminoglycoside uptake during the period of accelerated energy-dependent drug transport (5) and to the decreased proton motive force during the adaptive-resistance interval (reduced killing) (14). Aminoglycosides need to cross the bacterial cytoplasmic membrane before initiating lethal effects. It is well known that increased aminoglycoside uptake is associated with an enhanced bactericidal effect. Previous data have demonstrated that aminoglycoside transport is regulated by, the electrical component of the proton motive force; more specifically, uptake is dependent on (3, 9, 10, 16). Eisenberg et al. indicated that there is generally an increase in the magnitude of at the expense of ph (the transmembrane difference in the H concentration) (9). Moreover, acidic phs seem to have a deleterious effect on aminoglycoside bactericidal activity, although only a few studies have investigated this aspect (12). The purpose of this study was to determine the influence of ph (7.4, 6.5, and 5.5) on the adaptive resistance of P. aeruginosa to amikacin and netilmicin and on the postantibiotic effects (PAEs) of amikacin and netilmicin on P. aeruginosa in vitro as well as to monitor changes in outer and inner membrane proteins during the adaptive-resistance interval. * Corresponding author. Mailing address: Laboratoire d Antibiologie, Faculté de Médecine, 44035 Nantes, France. Phone: (33) 40-08-38-63. Fax: (33) 40-08-46-54. MATERIALS AND METHODS Organism. The P. aeruginosa strain studied was isolated from respiratory secretions and proved susceptible to -lactams, quinolones, and aminoglycosides. Antibiotics. Amikacin and netilmicin, supplied by Bristol Laboratories (Paris, France) and Schering-Plough Laboratories (Levallois-Perret, France), respectively, were dissolved in sterile water. Susceptibility testing. MICs were determined by macrodilution in Mueller- Hinton broth supplemented with Ca 2 and Mg 2 (1), with a final bacterial inoculum of 10 5 to 10 6 CFU/ml. MICs and minimum bactericidal concentrations were defined as the lowest concentrations completely inhibiting growth and killing 99.9% of the inoculum, respectively. PAE. The PAE was assessed by the repeated-washing method, as previously described (4). Cultures were incubated at 37 C in various concentrations of amikacin or netilmicin at different phs (ph 7.4, 6.5, and 5.5) for 1 h prior to removal of the antibiotic by two washes (8,000 g for 10 min) and resuspension of the bacterial pellet in fresh drug-free medium. The PAE was then assessed, and the following equation was formulated for quantitation of the PAE: PAE T C, where T is the time required for the CFU count in the test culture to increase 1 log 10 (10-fold) above that observed immediately after drug removal and C is the time required for the CFU count in an untreated control culture to increase by 1 log 10 above that observed immediately after completion of the same procedure used on the test culture after drug removal. Adaptive resistance in vitro. Adaptive resistance was induced as previously described (5). An overnight culture was diluted 10-fold in Mueller-Hinton broth and incubated with different concentrations of amikacin or netilmicin for 1 h at 37 C. An identical sample of the same culture was simultaneously incubated in drug-free broth and used as a control. After 1 h, bacteria were harvested by centrifugation at 8,000 g for 10 min and washed twice with drug-free medium. The pellet was resuspended and diluted in drug-free broth to yield a suspension of 5 10 4 viable postexposure CFU/ml and was incubated at 37 C. Aliquots of the cultures were removed hourly for 7 h and reexposed to two times the MIC (2MIC) of amikacin or netilmicin. Colony counts were determined before and after exposure, and the difference was expressed as bacterial killing after 90 min. The bacterial killing values for the culture with prior exposure to amikacin or netilmicin were compared with those for the control culture with no prior exposure to antibiotics. Adaptive resistance was observed when the bactericidal effect was less than that noted at the same time point in the control experiment. Outer and inner membrane proteins. Whole-cell preparations and cellular subfractions were isolated as previously described (18). An overnight culture was diluted 10-fold in Mueller-Hinton broth (T 1 ) and incubated with 8MIC of amikacin or netilmicin for 1 h (until T 0 )at37 C. An identical sample of the same culture was simultaneously incubated in drug-free broth and used as a control. After 1 h, bacteria were harvested by centrifugation at 8,000 g for 10 min and 35
36 XIONG ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 1. Antibacterial effects of first and second exposures to amikacin (AMK) against P. aeruginosa in medium at ph 7.4. (A) Bactericidal effect of first amikacin exposure on control culture. (B to E) Bactericidal effects of second amikacin exposure on test cultures. For these panels, the first exposures were MIC, 2MIC, 4MIC, and 8MIC of amikacin, respectively. FIG. 2. Antibacterial effects of first and second exposures to netilmicin (NET) against P. aeruginosa in medium at ph 7.4. (A) Bactericidal effect of first netilmicin exposure on control culture. (B to E) Bactericidal effects of second netilmicin exposures on test cultures. For these pan first exposure is MIC, 2MIC, 4MIC, and 8MIC of netilmicin, respectively.
VOL. 40, 1996 INFLUENCE OF ph ON ADAPTIVE RESISTANCE 37 Downloaded from http://aac.asm.org/ FIG. 3. Antibacterial effects of first and second exposures to amikacin (AMK) against P. aeruginosa in media at different phs. (A to C) Bactericidal effects of first amikacin (2MIC) exposure on control cultures at ph 7.4, 6.5, and 5.5, respectively. (D to F) Bactericidal effects of a second amikacin (2MIC) exposure on test cultures at ph 7.4, 6.5, and 5.5, respectively. The first exposure was to 8MIC of amikacin. washed twice with drug-free medium. The pellet was resuspended and diluted in drug-free broth and incubated at 37 C for7h.att 1,T 0,T 2, and T 7, cells of the control, amikacin, and netilmicin groups were harvested by centrifugation at 8,000 g for 10 min at 4 C (A 600, 0.4 to 0.5). The cells were disrupted by sonication. The differentiation of outer and inner membrane proteins was based on the differential solubilities of these proteins in Sarkosyl. The reaction was stopped with 20% Sarkosyl, and the samples were prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (13). RESULTS PAE. The PAEs of amikacin and netilmicin against P. aeruginosa were shown to be concentration dependent and were reduced at acidic phs (Table 1). At ph 7.4, the PAE of amikacin and netilmicin after exposure to 8MIC of each drug was about 1 h. With lower concentrations, the duration of the PAE was reduced. At ph 6.5, the PAEs of amikacin and netilmicin were shorter than at ph 7.4. At ph 5.5, amikacin exhibited practically no PAE at 4MIC, and netilmicin had only a 0.6-h PAE at 4MIC. Adaptive resistance. The results of adaptive resistance to amikacin and netilmicin (with different concentrations of these compounds after 1 h of preexposure) in the P. aeruginosa strain in ph 7.4 medium are shown in Fig. 1 and 2, respectively. on January 14, 2019 by guest TABLE 1. PAEs for various concentrations of amikacin and netilmicin at different phs a ph MIC/MBC (mg/liter) AMK PAE (h) for drug at concn (mg/liter) NET AMK NET 2 4 8 16 4 8 16 32 7.4 2/4 4/8 0.10 0.35 0.90 1.10 0.13 0.25 0.80 1.00 6.5 2/4 4/8 ND ND 0.70 0.95 ND ND 0.65 0.75 5.5 4/8 8/16 ND ND 0.25 0.10 ND ND 0.00 0.60 a Abbreviations: MBC, minimum bactericidal concentration; AMK, amikacin; NET, netilmicin; ND, not determined.
38 XIONG ET AL. ANTIMICROB. AGENTS CHEMOTHER. Downloaded from http://aac.asm.org/ FIG. 4. Antibacterial effects of first and second exposures to netilmicin (NET) against P. aeruginosa in media at different phs. (A to C) Bactericidal effects of first netilmicin (2MIC) exposure on control cultures at ph 7.4, 6.5, and 5.5, respectively. (D to F) Bactericidal effects of a second netilmicin (2MIC) exposure on test cultures at ph 7.4, 6.5, and 5.5, respectively. The first exposure was to 8MIC of netilmicin. Figure 1 shows that the addition of 2MIC of amikacin (final concentration, 4 mg/liter) in the control culture (without 1-h preexposure) produced bacterial killing of 1.3 to 2.3 log CFU/ ml/90 min at each hourly interval for 7 h (Fig. 1A). The test culture (with 1-h preexposure to 4MIC or 8MIC of amikacin) showed the development and subsequent decline of unstable adaptive resistance when amikacin (4 mg/liter) was added during 7-h growth periods in drug-free medium (Fig. 1D and E). However, with 1-h preexposure to the MIC and 2MIC of amikacin, adaptive resistance was not clearly exhibited (Fig. 1B and C). Figure 2, representing the netilmicin control (Fig. 2A), shows that the addition of 2MIC of netilmicin (final concentration, 8 mg/liter) produced bacterial killing of 1.5 to 2.3 log CFU/ml/90 min at each hourly interval for 7 h. The test culture (Fig. 2B to 2E) clearly showed unstable adaptive resistance to the readdition of netilmicin (8 mg/liter), even with 1-h preexposure to the MIC. The effects of ph (7.4, 6.5, and 5.5) on the adaptive resistance of P. aeruginosa to amikacin and netilmicin are shown in Fig. 3 and 4, respectively. At ph 6.5, the bacterial killing by amikacin (Fig. 3B) was far lower than that at ph 7.4 (Fig. 3A) but was less so for netilmicin (Fig. 4B vs. 4A). At ph 5.5, amikacin and netilmicin had practically no bactericidal effect on the P. aeruginosa strain (Fig. 3C and 4C). For the test group (8MIC of amikacin or netilmicin for 1 h ofpreexposure), the degree and duration of adaptive resistance at ph 6.5 and 5.5 depended on the bactericidal effect of the control, although adaptive resistance was still clearly exhibited. The duration of adaptive resistance was similar with media at different phs (about 3 to 4 h for amikacin and 5 h for netilmicin [Fig. 3D to F and 4D to F]), whereas the intensity was more marked at ph 6.5 than at ph 7.4. After initial drug exposure, the degree of adaptive resistance was very marked at 2 h for amikacin and from 2 to 4 h for netilmicin. Outer and inner membrane proteins. No changes in outer and inner membrane proteins occurred during the adaptiveresistance interval (data not shown). DISCUSSION Few studies have investigated the influence of ph on adaptive resistance of P. aeruginosa to aminoglycosides. As is well known, the bactericidal effect of the aminoglycosides is relative to the ph of the medium (7, 8). In our experiments at ph 5.5, amikacin and netilmicin had practically no bactericidal effect on P. aeruginosa. At ph 6.5, the killing by amikacin and netilmicin was far lower than that at ph 7.4. This resistance phe- on January 14, 2019 by guest
VOL. 40, 1996 INFLUENCE OF ph ON ADAPTIVE RESISTANCE 39 nomenon in media at acidic phs may have been due to a failure of drug uptake. In order to reach their ribosomal intracellular targets, aminoglycosides must traverse the cytoplasmic membranes of bacteria. This process of aminoglycoside uptake contains three energy-dependent phases (EDP). The first phase is binding to the cell surface by ionic interactions. The second phase (EDP-I) requires metabolic energy from the electron transport system that establishes a proton gradient between the inside and outside of the cytoplasmic membrane and is dependent on many factors (e.g., the ph of the medium). The final stage (EDP-II) involves the binding of the drug to ribosomes and necessitates active protein synthesis (8, 17). This final stage could be different from one aminoglycoside to another, possibly explaining some differences in killing rates. Some studies (9, 10, 15) demonstrated that increased aminoglycoside uptake is clearly associated with an enhanced bactericidal effect. Moreover, aminoglycoside uptake is dependent on, the electrical component of the proton motive force. Both aminoglycoside uptake and the subsequent lethal effect are proportional to the magnitude of. However, the magnitude of generally increases as the ph (transmembrane difference in the H concentration) decreases. Over the external ph range from 5.0 to 7.5, both and aminoglycoside uptake increased. Aminoglycoside uptake did not occur at ph 5.0 but was demonstrated at about ph 6.0. Thus, aminoglycoside uptake was clearly dependent on the magnitude of (9, 10, 15). The mechanism of adaptive resistance, although not clearly understood, could be related to reversible aminoglycoside-induced downregulation of drug uptake by bacteria, especially during the period of accelerated energy-dependent drug transport (4), and to a decrease in the magnitude of the electrical potential ( ) during the adaptive-resistance interval (14). We found no changes in outer and inner membrane proteins during the adaptive-resistance interval. The precise mechanisms of the PAE are unknown. Gottfredsson et al. (11) have demonstrated that DNA synthesis, as measured by thymidine uptake, decreases during the PAE and starts to recover about 20 to 40 min prior to growth by viable counts. We have shown that the PAEs of amikacin and netilmicin against the P. aeruginosa strain were reduced at acidic phs. Thus, the mechanism of PAE might be related to ph and. As demonstrated in other studies (2, 5, 6), adaptive resistance is an unstable, reversible form of resistance which develops within 1 to 3 h after initial exposure to an aminoglycoside and disappears several hours after its removal. Our experiments confirm the results of Barclay et al. (2), who used a dynamic model in vitro. They showed that higher initial aminoglycoside concentrations produce greater levels of bactericidal activity and more marked adaptive resistance at ph 7.4. Moreover, we found that adaptive resistance was obtained at lower preexposure concentrations of netilmicin than of amikacin. We evaluated the effects of ph (7.4, 6.5, and 5.5) on the adaptive resistance of P. aeruginosa to aminoglycosides. At ph 6.5 and 5.5, the degree and duration of adaptive resistance depended on the bactericidal effect of the control, although adaptive resistance was still clearly exhibited from 1 to 4 h after the first drug exposure. 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