JAC Advance Access originally published online on September 7, 2007
Journal of Antimicrobial Chemotherapy 2007 60(5):1030-1037; doi:10.1093/jac/dkm344
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Effect of GrlA mutation on the development of quinolone resistance in Staphylococcus aureus in an in vitro pharmacokinetic model
1 Research Laboratories, Toyama Chemical Co., Ltd, Toyama, Japan 2 Department of Microbiology, Toho University School of Medicine, Tokyo, Japan
* Corresponding author. Tel: +81-76-431-8268; Fax: +81-76-431-8208; E-mail: yoshimi_oonishi{at}toyama-chemical.co.jp
Received 5 March 2007; returned 11 May 2007; revised 20 July 2007; accepted 13 August 2007
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Abstract |
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Objectives: To investigate the effect of GrlA mutation on the development of quinolone resistance in Staphylococcus aureus in an in vitro pharmacokinetic (PK) model and to examine the relationship between the emergence of resistance and PK/pharmacodynamic parameters.
Methods: A wild-type and a GrlA mutant of S. aureus were exposed to the Japanese clinical dose of ciprofloxacin in an in vitro PK model, and the development of resistance was measured by population analysis. In addition, several doses of four quinolones (pazufloxacin, ciprofloxacin, levofloxacin and tosufloxacin) were tested against the GrlA mutant. All model simulations were single-dose design and were conducted over 24 h.
Results: Four quinolones were tested against the GrlA mutant, and a resistant population emerged after treatment with 250 mg pazufloxacin intravenous drip infusion, 600 mg ciprofloxacin intravenous drip infusion, 200 mg levofloxacin oral dosing and 150 mg tosufloxacin oral dosing. The emergence of a resistant population was not induced by treatment with 500 mg pazufloxacin intravenous drip infusion, 2400 mg ciprofloxacin intravenous drip infusion, 400 mg levofloxacin oral dosing and 600 mg tosufloxacin oral dosing. Treatment with the clinical dose of ciprofloxacin induced the development of resistance in the GrlA mutant, but not in the wild-type strain.
Conclusions: These data suggest that the frequency of acquisition of additional mutations differs between the wild-type and the GrlA mutant of S. aureus. Also, GrlA mutation predisposes S. aureus to develop high-level quinolone resistance.
Keywords: S. aureus , resistant mutants , mutations
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Staphylococcus aureus causes a variety of local and systemic infections and is one of the most important community- and nosocomially acquired pathogenic organisms. This organism can easily become resistant to multiple antimicrobial agents including quinolones. At present, up to 89% of S. aureus isolates are resistant to antimicrobial agents in some areas of the world.1 Quinolone resistance develops mainly as a result of chromosomal mutations in the target of quinolones, topoisomerase IV or DNA gyrase. The GrlA subunit of topoisomerase IV and the GyrA subunit of gyrase are the most common sites of resistance mutations; topoisomerase IV mutations are the most critical, since they are the primary target for many fluoroquinolones in S. aureus.2,3
It has previously been reported that the frequency of emergence of resistant mutants after selection with various quinolones at constant concentration from wild-type, first-step and second-step mutants of S. aureus ranged from 10–8 to 10–10, and there was no difference in the frequency of the emergence of resistance in each strain.4 If the frequency of acquisition of mutations in wild-type and GrlA mutants was the same, there would be little difference in isolation frequency between GrlA mutants and double (GrlA and GyrA) mutants. The observation that clinical isolates of S. aureus with a single mutation within GrlA have been reported to comprise 
4–9% of isolates, whereas the majority of resistant clones have double or triple mutations in GrlA and GyrA5–7 suggests that mutation frequency between the wild-type to the GrlA mutant and the GrlA mutant to the double (GrlA and GyrA) mutant is different. These data also suggest that GrlA mutants are predisposed to selection of secondary mutations and development of highly resistant strains.
With regard to isolation frequency between in vitro studies and the real world, we speculated that the major difference in the emergence of quinolone-resistant mutants between in vitro and in vivo is the change of the time–drug concentration; that is, the drug concentration in an in vitro study is constant, but is variable in in vivo conditions in which it corresponds to the pharmacokinetic (PK) profiles of each drug.
Recently, many studies have been performed to examine the relationship between pharmacodynamic (PD) parameters and the evolution of resistance to quinolones using an in vitro PK model. To better understand the relationship between bacterial resistance and antibiotic concentrations, Drlica et al. proposed a concept in the context of quinolones and S. aureus by defining a mutant selection window (MSW) as the concentration range between MIC and mutant prevention concentration (MPC). They reported that regimens providing quinolone concentrations within MSW easily select resistant mutants, but those with levels above MPC prevent the emergence of resistant strains.8–10 Firsov et al.11 examined the changes in the susceptibility of S. aureus exposed to four fluoroquinolones in an in vitro system and reported that the greatest increases in MIC occurred at AUC0–24/MIC ratios that corresponded to antimicrobial concentrations that fell within the MSW over more than 20% of the dosing interval. They also reported bell-shaped relationships between simulated AUC/MIC and the emergence of resistance. Campion et al.12 examined the evolution of resistance in bacterial populations by exposing S. aureus to an in vitro-simulated clinical and experimental ciprofloxacin PK profile and reported that there was no relationship between the time that ciprofloxacin concentrations remained between MIC and MPC, and the degree of resistance or the presence or type of ciprofloxacin resistance mutations that appeared in grlA or gyrA.
Numerous reports have noted a relationship between the emergence of quinolone resistance and PD parameters such as AUC/MIC and time inside MSW (TMSW) relationships to resistance of S. aureus,11,13,14 whereas one study has found that AUC/MPC was the only parameter to correlate with development of resistance.15 The majority of these studies have only used quinolone-susceptible strains of S. aureus.
In the work reported here, we investigated the emergence of a resistant population in wild-type and GrlA mutants of S. aureus using ciprofloxacin in an in vitro PK model in order to verify our hypothesis that a GrlA mutant of S. aureus would be predisposed to selection of high-level quinolone-resistant mutants.
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Antimicrobial agents
Pazufloxacin and tosufloxacin were synthesized at the Research Laboratories, Toyama Chemical Co., Ltd (Toyama, Japan). Ciprofloxacin and levofloxacin were commercially purchased from LKT Laboratories, Inc. (St Paul, MN, USA).
Bacterial strains and susceptibility testing
S. aureus strain SA113, which is quinolone-susceptible, and S. aureus strain CR-3, which is a ciprofloxacin-selected first-step mutant of wild-type S. aureus SA113 and has a mutation in GrlA (Ser-80
Phe), were used in this study.16 Susceptibility testing was performed by the agar dilution method at an inoculum size of 106 cfu/mL at 24 h exposure with the organism grown on Mueller–Hinton agar (MHA). Agar plates were prepared to contain drugs at concentrations of 0.01–10 mg/L. The intervals of the drug concentration in agar plates were set at 0.01 over the range of 0.01–0.1 mg/L. Similarly, the intervals of drug concentration in agar plates were set at 0.1 and 1 over the range of 0.1 to 1 mg/L and 1 to 10 mg/L, respectively. MICs of pazufloxacin, ciprofloxacin, levofloxacin and tosufloxacin were 0.2, 0.2, 0.1 and 0.02 mg/L for strain SA113 and 0.3, 1.0, 0.4 and 0.2 mg/L for strain CR-3, respectively.
Determination of the frequency of spontaneous mutation at constant quinolone concentration
The frequency of spontaneous mutation at constant quinolone concentration was determined according to the method of Jones et al.17 with slight modification. Strains SA113 and CR-3 were cultured overnight in Mueller–Hinton broth (MHB) and concentrated by centrifugation to yield 109–1010 cfu/mL as the final inoculum. These suspensions were spread onto MHA containing eight times the MICs of pazufloxacin, ciprofloxacin, levofloxacin and tosufloxacin. After 48 h of incubation at 37°C, the number of viable cells was counted. The frequency of the occurrence of resistant mutants was calculated by dividing the number of resistant cells by the number of viable cells in the sample. For plates that generated no mutants, frequency was assumed to be less than 1 divided by the number of viable cells in the sample.
MPCs were determined according to the method of Blondeau et al.8 with slight modification. Briefly, cells were cultured in MHB and incubated for 24 h. The suspension was centrifuged and resuspended in MHB to yield a concentration of 1011 cfu/mL. A series of agar plates containing each quinolone was then inoculated with 1010 cfu of the tested strain. The quinolone concentrations of the plates were set in the same manner as susceptibility testing. The inoculated plates were incubated for 72 h at 37°C, at which time the MPC was recorded as the lowest quinolone concentration that completely inhibited bacterial growth.
In vitro PK model and bacterial killing curves
A computer-associated auto-simulation system (PASS-400, Dainippon Seiki, Co., Ltd, Kyoto, Japan) was used to simulate the serum concentration of the quinolone based on Cmax, AUCs and t1/2. The serum concentration after a single dosing of Japanese clinical doses of each quinolone was simulated. Japanese clinical dosing of each quinolone was as follows: 500 mg pazufloxacin intravenous drip infusion for 30 min (AUC 22 mg·h/L, Cmax 11 mg/L, t1/2
0.50 h, t1/2ß 1.9 h); 300 mg ciprofloxacin intravenous drip infusion for 60 min (AUC 7.5 mg·h/L, Cmax 3.3 mg/L, t1/2 0.12 h, t1/2ß 2.6 h); 200 mg levofloxacin oral dosing (AUC 20 mg·h/L, Cmax 2.0 mg/L, t1/2 6.0 h) and 150 mg tosufloxacin oral dosing (AUC 3.8 mg·h/L, Cmax 0.60 mg/L, t1/2 3.6 h), respectively.18–21
In addition, modified models that altered the dosage (i.e. both Cmax and AUC) 1/64–8-fold of the clinical values were established in order to study the effect of the dose of each quinolone on the emergence of a resistant population.
The initial inoculum size of 107 cfu/mL was prepared from overnight culture for all experiments. This initial inoculum was introduced into the central chamber (volume 100 mL) of the in vitro PK model and the model was run. All model simulations were conducted over 24 h. Single samples were taken from the central chamber at 0, 0.5, 1, 2, 4, 8 and 24 h for the assessment of viable bacteria. Viable bacterial counts were performed by spreading serial dilutions on MHA plates. After overnight incubation at 37°C, the number of viable cells was counted.
Emergence of resistance was assessed at 24 h by population analysis and susceptibility testing of regrown cells.
Population analysis and determination of the regrown cell inhibitory concentration.
The changes of populations after treatment were detected by population analysis. Population analysis detects the development of resistance more sensitively than measurement of MIC. At 24 h, culture broth containing regrown cells was drawn from the central chamber. Samples of 0.1 mL (109 cfu/mL) were spread on MHA plates containing each quinolone at 0.01–100 mg/L in order to quantify the quinolone-resistant population. If the bacterial concentration of the sample did not reach 109 cfu/mL, the sample was additionally incubated overnight at 37°C before being spread. Quinolone concentrations contained in the plates were set in the same manner as for susceptibility testing. The plates were incubated for 72 h at 37°C and the colonies were counted visually. The fraction of colonies recovered at each quinolone concentration was calculated by dividing the number of inocula applied on each plate into the number of colonies appearing on the drug-containing plate. Emergence of a resistant population was assessed by comparing the population of regrown cells with that of the study strain.
The lowest concentration that allows no colony growth on drug-containing agar plates in population analysis was defined as regrown cell inhibitory concentration (RCIC) in order to quantify the results of population analysis.
Susceptibility testing of regrown cells.
Susceptibility testing of regrown cells was performed by the broth microdilution method22 with the organism grown in Ca2+- and Mg2+-supplemented MHB at an inoculum size of 5 x 104 cfu/well. The culture broth used for population analysis was also used for susceptibility testing.
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Difference of acquired resistance to quinolones between the wild-type and its GrlA mutant
Frequency of spontaneous mutation at constant quinolone concentration. The frequencies of the occurrence of spontaneous mutants in strain SA113 and its GrlA mutant CR-3 at constant quinolone concentration were measured. In strain SA113, the frequency of the occurrence of spontaneous mutants resistant to quinolones was <3.2 x 10–10 to pazufloxacin, <3.2 x 10–10 to ciprofloxacin, 3.2 x 10–10 to levofloxacin and 6.4 x 10–10 to tosufloxacin, respectively. In GrlA mutant CR-3, the frequency of the occurrence of spontaneous mutants resistant to quinolones was 1.9 x 10–9 to pazufloxacin, <2.8 x 10–10 to ciprofloxacin, <2.8 x 10–10 to levofloxacin and 2.8 x 10–9 to tosufloxacin, respectively.
Killing curve and emergence of a resistant population using an in vitro PK model. The killing curves of strain SA113 and strain CR-3 treated with the clinical dose of ciprofloxacin are shown in Figure 1. For both strains, the bacterial count of drug-free control rapidly increased to 2 log cfu/mL within 8 h after inoculation.
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In the case of wild-type strain SA113, the dosing of ciprofloxacin reduced the bacterial count more than 3 log cfu/mL within 8 h, but regrowth was observed, and the final bacterial count became comparable to the first inoculum size at 24 h. In the case of the GrlA mutant strain CR-3, although only 2 log cfu/mL of bacterial reduction was observed within 4 h after inoculation, drastic regrowth was observed and the bacterial count became comparable to that of the control at 24 h. The population analyses of culture broth after treatment were performed. Little difference in population was observed before and after treatment in strain SA113; however, a resistant population was drastically amplified after treatment with ciprofloxacin in strain CR-3 (Figure 2).
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These observations suggest that the frequency of acquisition of additional mutations differs between the wild-type and the GrlA mutant in in vitro PK model-simulated ciprofloxacin treatment in contrast to mutation frequency at constant ciprofloxacin concentration. Therefore, in our opinion the prevention of mutation in the GrlA mutant rather than the wild-type would lead directly to the prevention of high-level quinolone resistance in the clinical field. Thus, we examined the relationship between the emergence of a resistant population and PK/PD parameters using the GrlA mutant.
Relationship between the emergence of a resistant population and the PK/PD parameters
Next, we examined the relationship between the emergence of a resistant population and PK/PD parameters using four quinolones against GrlA mutant strain CR-3.
MPC determination. MICs and MPCs for strain CR-3 are shown in Table 1.
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Killing curves. The killing curves of strain CR-3 treated with pazufloxacin, ciprofloxacin, levofloxacin and tosufloxacin are shown in Figure 3. The bacterial count of drug-free control rapidly increased 2 log cfu/mL within 8 h after inoculation. A 3 log cfu/mL decrease (99.9% killing) within 8 h was observed at 1/4- to 1-fold the clinical dose of pazufloxacin, 2–8-fold for ciprofloxacin, 1/2–2-fold for levofloxacin and 1/2–4-fold for tosufloxacin; however, at almost all doses, regrowth was observed and the bacterial concentration was close to the plateau level of the control at 24 h.
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Emergence of resistant populations. The population analysis of CR-3 and culture broth after simulation was performed to assess the emergence of resistant populations (Figure 4). The emergence of resistant populations was not observed after treatment at 1/64- and 1-fold the clinical dose of pazufloxacin, 1/2- and 8-fold for ciprofloxacin, 1/8- and 2-fold for levofloxacin and 1/4- and 4-fold for tosufloxacin. At the clinical dose of pazufloxacin, 8-fold for ciprofloxacin, 2-fold for levofloxacin and 4-fold for tosufloxacin, a 3 log cfu/mL decrease in the bacterial count within 8 h was observed (Figure 3). At 1/64-fold the clinical dose of pazufloxacin, 1/2-fold for ciprofloxacin, 1/8-fold for levofloxacin and 1/4-fold for tosufloxacin, a slight decrease in the bacterial count was observed (Figure 3). However, resistant populations increased after treatment at 1/16-, 1/4- and 1/2-fold the clinical dose for pazufloxacin, 1-, 2- and 4-fold for ciprofloxacin, 1/4-, 1/2- and 1-fold for levofloxacin and 1/2-, 1- and 2-fold for tosufloxacin, respectively. Moreover, the MICs of each quinolone increased after treatment with these doses (Figure 4).
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The RCIC of each quinolone for CR-3 was 2 mg/L for pazufloxacin, 6 mg/L for ciprofloxacin, 4 mg/L for levofloxacin and 2 mg/L for tosufloxacin. The RCICs of each quinolone after treatment at the dose that prevented the emergence of resistant populations were comparable to the RCICs before treatment. On the other hand, increases in RCICs were observed after treatment at the dose that increased the resistant population.
The ratio to the clinical dose of quinolone versus the RCICs was profiled, and these graphs revealed bell-shaped patterns (Figure 5). In addition, the RCICs of four quinolones after treatment at the dose of quinolones that most increased the resistant population were different. Pazufloxacin prevented the emergence of a resistant population at the clinical dose, whereas ciprofloxacin, levofloxacin and tosufloxacin increased the resistant population at the clinical dose.
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The PK/PD parameters of the dose that prevented the emergence of a resistant population or most increased the resistant population are shown in Tables 2 and 3. In relation to the parameters that prevented the emergence of a resistant population at higher doses, that is, 500 mg intravenous drip infusion over 30 min for pazufloxacin, 2400 mg intravenous drip infusion over 60 min for ciprofloxacin, 400 mg oral dosing for levofloxacin and 600 mg oral dosing for tosufloxacin, the ranges of Cmax/MIC, AUC0–24/MIC and time above MIC (h) were 10–37, 60–98 and 9–22 for the MIC-related parameters, respectively. For MPC-related parameters, the ranges of Cmax/MPC, AUC0–24/MPC and time above MPC (h) were 0.30–1.4, 1.2–5.6 and 0–0.35, respectively. TMSW (h) ranged from 8.8 to 22. On the other hand, in relation to the parameters that increased the resistant population, that is, 250 mg intravenous drip infusion over 30 min for pazufloxacin, 600 mg intravenous drip infusion over 60 min for ciprofloxacin, 200 mg oral dosing for levofloxacin and 150 mg oral dosing for tosufloxacin, the ranges of Cmax/MIC, AUC0–24/MIC and time above MIC (h) were 3.0–18, 15–49 and 4.5–16 for the MIC-related parameters, respectively. For MPC-related parameters, the ranges of Cmax/MPC, AUC0–24/MPC and time above MPC (h) were 0.075–0.69, 0.30–2.8 and 0, respectively. TMSW (h) ranged from 4.5 to 16.
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Discussion |
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In the present study, we investigated the emergence of quinolone resistance using a wild-type and its GrlA mutant in an in vitro PK model in order to verify our hypothesis that a GrlA mutant of S. aureus would be predisposed to development of high-level quinolone-resistant mutants.
In vitro PK models are widely used to study relationships between PK and emergence of resistance. Experiments should be designed to mimic dosing regimens and be of sufficient duration to reflect a clinical situation and allow for the emergence of resistant variants. We found that a resistant population emerged after a single dosing of ciprofloxacin (300 mg) in the GrlA mutant of S. aureus. Therefore, we considered that the effect of GrlA mutation on development of resistance cannot be assessed accurately by multiple-dose experiments and designed single-dose experiments in this study.
Using other quinolones (pazufloxacin, levofloxacin and tosufloxacin), we found that resistant populations emerged with a single dosing of the GrlA mutant. Moreover, additional single GyrA mutations were found in strains isolated from the resistant population after the dosing of each quinolone. A single dosing at the clinical dose 1- and 2-fold for ciprofloxacin selected strains that had additional mutations of Ser-85Pro and Ser-84Leu in GyrA, respectively (data not shown). On the other hand, the emergence of a resistant population was not observed in S. aureus SA113, which is a quinolone-susceptible strain, after the clinical dosing of ciprofloxacin. In another study, it has been reported that the MIC of the tested strains increased by multiple dosing of an in vitro PK model in quinolone-susceptible S. aureus.11 This led to the assumption that mutation from the GrlA mutant to a high-level resistant mutant would progress quickly by exposure to quinolones, whereas the mutation from wild-type to the GrlA mutant would progress gradually. This was also consistent with the fact that the isolation frequency of the GrlA mutant was quite low compared with that of the double (GrlA and GyrA) mutant in clinical isolates5–7 and suggested that the GrlA mutant would act as a trigger leading to high-level quinolone-resistant mutants.
In this study, we used the population analysis and the MICs for regrown culture to evaluate the emergence of resistance. The results of population analysis and MICs were compared, the changes of MICs tended to be smaller than those estimated from degree of increase of resistant populations in some doses (Figure 4). This observation suggests that the population analysis would detect the development of resistance more sensitively than measurement of MICs. Population analysis was performed by inoculation with 108 cfu of regrown cells on an agar plate containing quinolone, whereas measurement of MICs was performed at an inoculum size of 5 x 104 cfu/well. This difference in inoculum size would be a reason for difference in sensitivity to detection of development of resistance.
When the PK parameters (Cmax and AUC) of each quinolone were altered theoretically, there was a dose at which a high-level resistant mutant was selected, but the quinolone-resistant population rarely emerged at lower or higher doses. The ranges of the risky dose of each quinolone, based on the actual clinical dose that developed high-level quinolone resistance, were 1/16–1/2-fold (31–250 mg intravenous drip infusion over 30 min) for pazufloxacin, 1–4-fold (300–1200 mg intravenous drip infusion over 60 min) for ciprofloxacin, 1/4–1-fold (50–200 mg oral dosing) for levofloxacin and 1/2–2-fold (75–300 mg oral dosing) for tosufloxacin, respectively. In contrast, the ranges of the dose of each quinolone at the higher side of the clinical dose not developing high level-quinolone resistance were 1-fold (500 mg intravenous drip infusion over 30 min) for pazufloxacin, 8-fold (2400 mg intravenous drip infusion over 60 min) for ciprofloxacin, 2-fold (400 mg oral dosing) for levofloxacin and 4-fold (600 mg oral dosing) for tosufloxacin, respectively. From the quinolones tested, only pazufloxacin prevented the increase of the resistant population at the Japanese clinical dose. In Japan, only two products, ciprofloxacin and pazufloxacin, can be used as quinolones for intravenous infusion. The clinical dose of ciprofloxacin in Japan (300 mg) is lower than in the USA and European Union (400–500 mg). This Japanese dose probably increases the quinolone-resistant population of S. aureus; however, in this study, the dose of ciprofloxacin that prevented the emergence of a resistant population was 2400 mg, suggesting that the clinical dose in the USA and European Union would not be sufficient to prevent the emergence of resistance.
By analysing the population data in detail, additional information was obtained, that is: (i) the RCICs of four quinolones after treatment at the dose that most increased the resistant population were different; (ii) the relationship between the ratio to the clinical dose of each quinolone and the RCIC of each quinolone after simulation showed a bell-shaped pattern. This result was in agreement with a study that reported bell-shaped relationships between the simulated AUC/MIC and the emergence of resistance;11,14 and (iii) the clinical dose of ciprofloxacin, levofloxacin and tosufloxacin but not pazufloxacin obviously caused an increase in resistant populations.
The PK/PD parameters of the dose that prevented the emergence of resistant populations and the highest dose that increased the resistant population were compared (Table 4). There was an obvious difference in only the value of AUC0–24/MIC. These data suggest that AUC0–24/MIC may be related to the development of resistance. These results are consistent with previous studies,13,14 in spite of many experimental restrictions in the single-dose experiment. It has been reported that other parameters, i.e. AUC/MPC, TMSW, are related to the emergence of resistance in S. aureus;11,15 however, in this study, a clear relationship between these parameters and the emergence of resistance was not found. The reason for this is still unknown, but it may be related to methodological differences in the design or execution of the experiments. More detailed study is required in order to investigate the relationship between PK parameters and the emergence of resistance.
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In conclusion, the data obtained in this study suggest that GrlA mutation predisposes S. aureus to develop high-level quinolone resistance and are consistent with the finding that the isolation frequency of GrlA mutants is quite low in clinical isolates. Of the four quinolones tested, pazufloxacin displayed unique characteristics in both potent antibacterial activity and the prevention of the emergence of high-level quinolone-resistant populations from the GrlA mutant of S. aureus at the Japanese clinical dose. It is necessary to use an appropriate quinolone that does not select a high-level resistant mutant from GrlA mutants at clinical dosing.
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No funding was received for the study.
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None to declare.
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