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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Introduction |
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Materials and methods |
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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 0.250.5 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 210235 g were obtained from M&B A/S (Ry, Denmark). Air (1020 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 227300 g. K. pneumoniae was suspended in 5% gastric mucin in saline and 1 mL of the suspension (107108 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 1824 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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Discussion |
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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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Footnotes |
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References |
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