Mutant Prevention Concentration of Tigecycline for Klebsiella Pneumoniae Isolates with Four Different Resistance Statuses

Key words: tigecycline; Klebsiella pneumoniae; mutant prevention concentration. Dear Editor, Klebsiella pneumoniae (K. pneumoniae) is one of the most important causes of healthcare-associated infections, especially among hospitalized patients [1]. Since most clinical isolates of K. pneumoniae are resistant to fluoroquinolones and carbapenems, clinicians have to use tigecycline as the last resort. Unfortunately, the prevalence of tigecycline-resistant K. pneumoniae seems to be increasing [2,3]. The potential development of resistance to tigecycline during treatment is of concern. In this study, in order to determine whether tigecycline restricts the selective enrichment of resistant mutant subpopulations of K. pneumoniae, we measured the mutant prevention concentrations (MPCs) of tigecycline for 91 K. pneumoniae clinical isolates with four different resistance statuses, and assessed their propensity of developing resistance to tigecycline. The study Ninety one K. pneumoniae isolates were collected from Beijing Hospital, China. The isolates were divided into four groups according to their resistance statuses: (1) thirty-two isolates were carbapenem-resistant (resistant to either meropenem, impenem or ertapenem) and fluoroquinolone-resistant (resistant to either ciprofloxacin or levofloxacin); (2) twenty-nine isolates were carbapenem-and fluoroquinolone-susceptible; (3) twenty-two isolates were carbapenem-susceptible and fluoroquinolone-resistant; (4) eight isolates were carbapenem-resistant and fluoroquinolone-susceptible. Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922 and K. pneumoniae ATCC BAA-2146 were used as the quality control strain and were included in each susceptibility test. The minimum inhibition concentrations (MICs) were determined by the method of agar plate assays according to the Clinical and Laboratory Standards Institute guidelines [4]. The MICs for the strains were interpreted in accordance with FDA guidelines for tigecycline, MIC ≤2 µg/mL and ≥8 µg/mL were classified as susceptible and resistant, respectively [5,6]. MPCs for K. pneumoniae of the four groups were performed with a previously described procedure with modifications [7]. Briefly, each isolate was cultured on the Mueller-Hinton (MH) agar and incubated at 37°C for 24 hours, then transferred to 500 mL of MH and incubated for another 24 hours. The suspension was centrifuged at 5000 g for 10 minutes. Bacteria were re-suspended with 3 mL fresh MH broth to a concentration about 10 10 c.f.u. ml –1. Aliquots of 0.1 mL of the suspension were plated respectively onto a series of agar plates containing various concentrations of tigecycline. After incubation at 37°C for 72 hours, the bacterial colonies were counted. The MPC was defined as the lowest drug concentration at which the growth of K. pneumoniae on agar plate was completely inhibited. Correlations …


The study
Ninety one K. pneumoniae isolates were collected from Beijing Hospital, China.The isolates were divided into four groups according to their resistance statuses: (1) thirty-two isolates were carbapenemresistant (resistant to either meropenem, impenem or ertapenem) and fluoroquinolone-resistant (resistant to either ciprofloxacin or levofloxacin); (2) twenty-nine isolates were carbapenem-and fluoroquinolonesusceptible; (3) twenty-two isolates were carbapenemsusceptible and fluoroquinolone-resistant; (4)  The minimum inhibition concentrations (MICs) were determined by the method of agar plate assays according to the Clinical and Laboratory Standards Institute guidelines [4].The MICs for the strains were interpreted in accordance with FDA guidelines for tigecycline, MIC ≤2 µg/mL and ≥8 µg/mL were classified as susceptible and resistant, respectively [5,6].MPCs for K. pneumoniae of the four groups were performed with a previously described procedure with modifications [7].Briefly, each isolate was cultured on the Mueller-Hinton (MH) agar and incubated at 37°C for 24 hours, then transferred to 500 mL of MH and incubated for another 24 hours.The suspension was centrifuged at 5000 g for 10 minutes.Bacteria were re-suspended with 3 mL fresh MH broth to a concentration about 10 10 c.f.u.ml -1 .Aliquots of 0.1 mL of the suspension were plated respectively onto a series of agar plates containing various concentrations of tigecycline.After incubation at 37°C for 72 hours, the bacterial colonies were counted.The MPC was defined as the lowest drug concentration at which the growth of K. pneumoniae on agar plate was completely inhibited.Correlations between MICs and MPCs were analysed using SPSS software, version 18.0 (IBM Corp., Armonk,NY,USA).
Table 1 shows the distribution of MICs and MPCs of tigecycline for the 91 K. pneumoniae clinical isolates.Using the resistant breakpoint of tigecycline (8 µg/mL), 13 tigecycline-resistant K. pneumoniae isolates were obtained, and the tigecycline-resistance rate was 14.3% (13/91).MPC 90 and MPC range for the four groups' strains are shown in table 2. The MPCs of tigecycline with 91 K. pneumoniae isolates were 2-to 256-fold higher than the susceptibility breakpoint (2 µg/mL).Furthermore, the MPCs of tigecycline for the cabapenem-and fluoroquinolone-resistant isolates were found to be 8-fold higher than those for cabapenem and quinolones-susceptible isolates.However, there was no significant difference of the MPC values between cabapenem-resistant and quinolones-susceptible and cabapenem-susceptible and quinolones-resistant isolates.As shown in table 1, the MPCs for the isolates ranged over 6 2-fold dilutions even with the same MIC values.In addition, low correlations between MPCs and MICs were observed for all the91 K. pneumoniae isolates (r 2 = 0.53), or the 13 tigecycline-resistant isolates (r 2 = 0.32), as well as the 78 tigecycline-susceptible isolates (r 2 = 0.29), which is consistent with previous reports.[8] Conte JE et al. [9] reportedthat the maximum concentration of drug in serum (C max ) and in alveolar cells was 0.38±0.06µg ml-1 and 15.2±7.6µgml-1, respectively, after intravenous injection administration of the recommended dosage of 50 mg tigecycline every 12 hours.Our data showed that MPC 90 values of tigecycline were 128 mg ml -1 and 32mg ml -1 for cabapenem and quinolones-resistant and cabapenem and quinolones-susceptible K. pneumoniae clinical isolates,respectively, which were much higher than the tigecycline concentrations in serum and lung tissue.Therefore, at the recommended dosage, the tigecycline concentrations would fall into the mutant selective window (drug concentration range between MIC and MPC), and likely to lead to the enrichment of resistant mutant subpopulations.Our result are in agreement with a recent study from Korea, by Myung-Jin Choi et al. [10]; they reported that the MICs and MPCs of tigecycline for K. pneumoniae isolates ranged between 0.5-1 mg/L and 4-16 mg/L, respectively, suggesting that the current clinical dosage regimen may lead to the development of tigecycline-resistant mutants.
The following limitations of our study should be considered.First, all of the studied isolates were recovered from a single hospital, which might increase the biases of our results.Second, Myung-Jin Choi et al. [10] found that up-regulation of the efflux pumps was associated with tigecycline resistance.The efflux pump expression levels were not tested in the singlestep mutants in our study.Further studies are warranted to validate the relationship between efflux pump expression and tigecycline resistance.

Conclusions
Our data indicate that tigecycline therapy may be prone to the emergence of resistance with K. pneumoniae.Therefore, the continuous monitoring of K. pneumoniae susceptibility and patients responsiveness to tigecycline treatment is recommended.

Table 1 .
Distribution of MICs and MPCs of tigecycline for the K. pneumoniae clinical isolates.

Table 2 .
Comparison of the MICs before and after mutant prevention concentration and MPCs of K. pneumoniae isolates with four different resistance statuses.