Drugs 49 (Suppl. 2): 190-191, 1995 0012-6667/95/0002-0190/$01.00/0 -© Adis International Limited. All rights reserved.

Quinolone Resistance Mechanisms in Klebsiella pneumoniae Yong-hong Xiao} Akihiro Kitamura,2 Kazuki Hoshino2 and Kenichi Sato2

1 Department of Infectious Diseases, First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing, China

2 Exploratory Research Laboratories 1, Daiichi Pharmaceutical Co., Ltd, Tokyo, Japan

Along with the increasingly frequent use of quinolones in the clinical setting, Klebsiella pneu­moniae, one of the most common infectious patho­gens, is becoming resistant to quinolones. The pos­sible quinolone resistance mechanisms existing in K. pneumoniae were examined in this study.

1. Methods and Results

K. pneumoniae 900129 was a quinolone­susceptible clinical isolate; spontaneous quinolone­resistant mutants of this strain were selected by plating. K. pneumoniae G1, a first step mutant of 900129, was selected with nalidixic acid at a con­centration of 12.5 (2 x the MIC against 900129). K. pneumoniae G2, a second-step mutant of strain G1, was selected with ciprofloxacin at a concentra­tion of 1.56 (2 x the MIC against G1). MICs of nalidixic acid, ciprofloxacin, ofloxacin, tetracy­cline, chloramphenicol, imipenem and cephalori­dine against the 3 strains mentioned above were determined by the agar dilution method. The MICs of quinolones against G1 were 16- to 32-fold higher than those against 900129. Compared with G1, the MICs of quinolones to G2 were further increased by 2- to 4-fold. Both Gl and G2 had 4- to 32-fold lower susceptibilities to tetracycline and chloram-

phenicol than the parent strain. The susceptibility of G2 to ~-lactams was 2- to 4-fold lower than both G1 and the parent strain.

DNA gyrases from the 3 strains were purified. DNA supercoiling activity and doses required to inhibit supercoiling by 50% (IDsos) of quinolones were determined by methods described by Sato et al.[l] Gyrases from G1 and G2 were equally resistant to quinolones; IDsos were 3- to II-fold higher than those for gyrase from 900129. The IDsos of quinolones for the cross-reconstituted gyrase con­taining subunit A from G1 or G2 were the same as those for gyrases from G1 and G2. Exchange of subunit B of the gyrases did not affect the 1Dsos (table I). This indicates that modification of subunit A is one of the resistance mechanisms of K. pneumoniae.

Outer membrane proteins were prepared by the method of Sawai et alP] and analysed with sodium dodecyl sulfate-polyacrylamide gel electrophore­sis. Comparison of outer membrane protein pro­files with those of strain 900129 showed that G1

and G2 lacked a 42kD protein. Additionally, G2 lacked a protein of 34kD and 2 lower molecular weight proteins of 24.7 and 21kD.

Accumulation of ciprofloxacin by the bacterial cell was determined by the fluorometric method

Table I. Inhibitory concentrations of quinolones on supercoiling activity of reconstituted DNA gyrase from Klebsiella pneumoniae 900129

Antimicrobial agent 50% inhibitory dose (mg/L) for DNA gyrase ABa A1B1 A1B AB1 A2B2 A2B AB2

Nalidixic acid 10.46 98.65 118.3 10.03 115 107.5 13.64 Ciprofloxacin 0.12 0.97 1.15 0.13 0.91 0.95 0.15 Ofloxacin 0.35 0.91 1.18 0.34 0.95 1.01 0.33 a A and B, subunits from 900129; A 1 and B 1, subunits from G1; A2 and B2, subunits from G2.

Extended Abstract 191

O~--------.----------.---------.--------~--------~ o 10 20 30 40 50

Time (min)

Fig. 1. Uptake of ciprofloxacin by Klebsiella pneumoniae 900129, G, and G2 . Carbonyl-cyanide m-chloro-phenylhydrazone (CCCP) was added at time indicated by the arrow. Each point represents the mean of 3 independent assays.

described by Chapman and GeorgopapadakouPl The intracellular maximum concentration in 900129 was around 10 ng/mg of bacteria. In contrast, G I and G2 took up a 4 -and ll-fold lower amount of cipro­ftoxacin than 900129, respectively. In the presence of carbonyl-cyanide m-chloro-phenylhydrazone (CCCP), ciproftoxacin uptake by GI and G2 in­creased to the same as, or one-third of, that by strain 900129. The concentration of ciproftoxacin in 900129 was not affected by CCCP (fig. 1).

2. Conclusions

From these results, the following conclusions were reached: 1) mutation of subunit A of DNA gyrase was the major reason for quinolone resistance in K. pneumoniae G1 and G2; 2) drug active efflux of bacterial cells was also a quinolone resistance mechanism in K. pneumoniae G1 and G2; and 3)

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outer membrane protein alterations, especially in K. pneumoniae G2, may be involved in resistance by decreasing outer membrane permeability to anti­microbial agents.

References 1. Sato K, Inoue Y, Fujii T, et al. Purification and properties of

DNA gyrase from a fluoroquinolone-resistant strain Escher­ichia coli. Antimicrob Agents Chemother 1986; 30; 777-8

2. Sawai T, Himura R, Kawana N, et al. Outer membrane perme­ation of beta-Iactam antibiotics in Escherichia coli and Enterobacter cloacae. Antimicrob Agents Chemother 1981; 22; 585-92

3. Chapman JS, Georgopapadakou NH. Fluorometric assay for fleroxacin uptake by bacterial cells . Antimicrob Agents Chemother 1989; 33; 27-9

Correspondence and reprints; Dr Yang-hong Xiao, Depart­ment of Infectious Diseases, the First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 630042, China.

Drugs 49 (Suppl. 2) 1995