Journal of Antimicrobial Chemotherapy (2002) 50, 421-424
© 2002 The British Society for Antimicrobial Chemotherapy
Significance of low-level resistance to ciprofloxacin in Klebsiella pneumoniae and the effect of increased dosage of ciprofloxacin in vivo using the rat granuloma pouch model
1 Department of Clinical Microbiology, Aarhus University Hospital, Aarhus Kommunehospital, DK-8000 Aarhus C; 2 Department of Clinical Microbiology, Viborg Sygehus, DK-8800, Viborg, Denmark
Received 21 November 2001; returned 3 April 2002; revised 24 May 2002; accepted 17 June 2002
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Abstract |
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This study was designed to compare the killing effect of ciprofloxacin on strains of Klebsiella pneumoniae with different MICs of ciprofloxacin in vivo using the rat granuloma pouch infection model. Five different strains were used: one ciprofloxacin-susceptible strain (MIC 0.06 mg/L); one strain highly resistant to ciprofloxacin (MIC 8 mg/L); and three nalidixic acid-resistant strains with low-level resistance to ciprofloxacin (MIC 0.25–0.5 mg/L). The efficacy of ciprofloxacin was evaluated 3 h after bacterial challenge (treating an acute infection) or after 3 days (treating a late infection) with a single intraperitoneal injection of ciprofloxacin (40 and 200 mg/kg). Ciprofloxacin was bactericidal against both growing K. pneumoniae (acute infection model) and non-growing K. pneumoniae (late infection model), but the extent of killing was significantly higher on growing bacteria and against ciprofloxacin-susceptible K. pneumoniae. A peak concentration of ciprofloxacin, at the infection site, <3 x MIC was not sufficient for optimal bacterial elimination. However, it was possible to compensate for the lower killing in low-level ciprofloxacin-resistant K. pneumoniae by increasing the dosage of ciprofloxacin from 40 to 200 mg/kg, consistent with the higher MIC.
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Introduction |
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Fluoroquinolones have excellent clinical activity against Enterobacteriaceae including Klebsiella, but the frequency of ciprofloxacin-resistant Klebsiella pneumoniae has increased worldwide in recent years, including in Denmark.1,2 Moreover, an increase in nalidixic acid resistance with a concomitant low-level resistance to ciprofloxacin has been seen.3,4 The development of resistance to fluoroquinolones is generally a stepwise process, with successive genetic mutations resulting in gradual increasing resistance.2 The MIC breakpoint of 1 mg/L recommended by BSAC (www.bsac.org.uk) or NCCLS5 testing for resistance to ciprofloxacin has recently been challenged,4,6 because of the increasing incidence of treatment failures in cases of nalidixic acid-resistant and low-level ciprofloxacin-resistant salmonellae, with MICs between 0.25 and 1 mg/L,3,7 which have 10–100 times higher MICs than isolates that lack resistance mechanisms (www.srga.org). It is possible that this decreased susceptibility or low-level resistance can be overcome merely by increasing the dosage of ciprofloxacin, analogous to the treatment of intermediate-level penicillin-resistant pneumococci with high-dose penicillin.8 However, there are no clinical or experimental animal data to support such a high dosage regimen for the treatment of low-level ciprofloxacin-resistant K. pneumoniae or other Enterobacteriaceae. Consequently, this study using an animal model was designed to compare and evaluate the in vivo antimicrobial efficacy of ciprofloxacin against low-level resistant K. pneumoniae.
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Materials and methods |
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Strains, antibiotics and media
Five clinical isolates of K. pneumoniae were included in the study: one strain was fully susceptible to ciprofloxacin (MIC 0.06 mg/L); three strains had low-level resistance to ciprofloxacin (MIC 0.25–0.5 mg/L); and one strain was highly resistant to ciprofloxacin (MIC 8 mg/L). A molecular characterization of the low-level ciprofloxacin-resistant strains was not carried out. Ciprofloxacin was provided by Bayer Danmark A/S (Copenhagen, Denmark). Gastric mucin, croton oil and olive oil were obtained from Sigma Chemical Company (St Louis, MO, USA).
MICs were determined with the Etest (AB Biodisk, Solna, Sweden) methodology.
Rat granuloma pouch model
All animal experiments were approved by the Danish animal ethics committee. Animals were housed two per cage and had free access to a pelleted diet and drinking water.
The granuloma-pouch technique as described by Selye9 was used with a few modifications: male Wistar rats weighing 210–235 g were obtained from M&B A/S (Ry, Denmark). Air (10–20 mL) was injected subcutaneously at the back of the rat through a 25-gauge ‘butterfly’ (Valu-Set; Becton Dickinson, Sandy, UT, USA), which generated a pouch. Then, 1 mL of 1% croton oil in olive oil was injected into the pouch. Experiments were started 7 days after induction of the granuloma pouch, at which time the animals weighed 227–300 g. K. pneumoniae was suspended in 5% gastric mucin in saline and 1 mL of the suspension (107–108 cfu) was injected into the granuloma pouch of each rat. Therapy of infected rats was commenced after 3 h (treating an acute infection) or 3 days (treating a late infection) after challenge with a single intraperitoneal injection of ciprofloxacin (40 or 200 mg/kg).
At 0, 2, 6 and 24 h after initiation of drug therapy, samples (
0.5 mL) of abscess fluid were drawn from the infected pouch through a 23-gauge syringe and serially diluted in saline, and then 20 µL samples were plated in quadrants on 5% blood agar plates. To eliminate ciprofloxacin carry-over, only diluted samples were plated. Plates were incubated at 35°C for 18–24 h, and then colonies were counted (lower limit of detection 500 cfu/mL). Simultaneously, the kinetics of ciprofloxacin in the abscess fluid were determined. In addition, the susceptibility to ciprofloxacin of K. pneumoniae recovered from abscess fluid sampled at 24 h was determined.
Pharmacokinetic studies
The concentration of ciprofloxacin in serum and abscess fluid was determined by bioassay10 (agar diffusion technique with 6 mm disc using Escherichia coli ATCC 25922 as the test strain with a day-to-day coefficient of variance below 11%, and a lower limit of detection of 0.2 mg/L). Ciprofloxacin was determined in serum at 0, 0.25, 0.5, 1, 2, 4 and 6 h. The terminal elimination half-live (T) of ciprofloxacin in serum and abscess fluid was calculated using a one-compartment model with zero-order absorption and first-order elimination using the computer program Prism version 3.0 for Windows (GraphPad Software, San Diego, CA, USA).
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Results |
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No animals died or had systemic complications from the infection. Results from time–kill curves in vivo are shown in Figure 1 (acute infection model) and in Figure 2 (late infection model). The number of cfu, in abscess fluid, at the start of treatment varied between 5.8 log10 and 7.1 log10 cfu/mL. The bactericidal activity of ciprofloxacin was significantly higher when treating the infection caused by the susceptible K. pneumoniae strain as compared with treatment of infections caused by K. pneumoniae with low-level resistance to ciprofloxacin. The bactericidal effect of a 40 mg/kg dose of ciprofloxacin on the susceptible strain was comparable to a 200 mg/kg dose of ciprofloxacin on the low-level resistant strains with both the acute infection model (2.6 log10 versus 2.9 log10 decrease in cfu/mL) and with the late infection model (2.1 log10 versus 1.9 log10 decrease in cfu/mL). Ciprofloxacin had no detectable bactericidal activity against the highly ciprofloxacin-resistant K. pneumoniae strain. No bacterial regrowth was observed at 24 h in any experiments. K. pneumoniae recovered in abscess fluid at 24 h remained as susceptible as before the experiments (within a two-fold dilution step in MIC).
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Treatment of rats with a single dose of ciprofloxacin at 40 versus 200 mg/kg produced peak levels of 3.3 versus 13.7 mg/L, and 0.7 versus 3.2 mg/L in serum and abscess fluid, respectively. Ts in serum varied between 56 and 73 min, and in abscess fluid, between 7.1 and 7.8 h.
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Discussion |
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The efficacy of antibiotics in the treatment of abscesses is of great interest because abscesses usually require surgical drainage for cure, and, therefore, should represent the ultimate challenge for an antimicrobial agent. The rat granuloma pouch infection model represents a suitable model for studying and comparing the effectiveness of antibacterial agents for the treatment of infections caused by either growing or non-growing bacteria.11,12
We found, as expected, that ciprofloxacin was bactericidal against both growing K. pneumoniae (acute infection model) and non-growing K. pneumoniae (late infection model), but the extent of killing was significantly higher on growing bacteria and against ciprofloxacin-susceptible K. pneumoniae. Ciprofloxacin had no activity against the highly ciprofloxacin-resistant strain, which had an MIC above the peak concentration. A peak concentration of ciprofloxacin at the infection site <3 x MIC was not sufficient for optimal bacterial elimination. However, it was possible to compensate for the lower killing in low-level ciprofloxacin-resistant K. pneumoniae by increasing the dosage of ciprofloxacin from 40 to 200 mg/kg, consistent with the higher MIC. Clinical studies are needed to corroborate these animal observations, and we do not know to what extent these data can be extrapolated to Enterobacteriaceae other than K. pneumoniae.
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Acknowledgements |
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This study was funded by a research grant from SSAC Foundation for Research in Antimicrobial Chemotherapy.
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Footnotes |
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* Corresponding author. Tel: +45-89-49-35-25; Fax: +45-89-49-35-50; E-mail: fuursted{at}rocketmail.com
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References |
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