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Antimicrobial Agents and Chemotherapy, January 2006, p. 368-370, Vol. 50, No. 1
0066-4804/06/$08.00+0 doi:10.1128/AAC.50.1.368-370.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Departamento de Microbiología, Hospital Universitario Virgen Macarena, Facultad de Medicina, Universidad de Sevilla, Seville, Spain
Received 24 June 2005/ Returned for modification 21 August 2005/ Accepted 28 October 2005
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ABSTRACT |
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Isolation of extended-spectrum-ß-lactamase (ESBL)-producing E. coli and Klebsiella pneumoniae strains is increasing in Spain, and a high proportion of these isolates are recovered from outpatients with uncomplicated UTIs (4, 7, 11). It is common to find that the same plasmid coding for ESBL also contains genes conferring resistance to several groups of antimicrobial agents, such as aminoglycosides and cotrimoxazole (7, 10). The concurrence of quinolone resistance, particularly in ESBL-producing K. pneumoniae, is frequent (6, 10, 13), there being few alternatives for the appropriate oral treatment of uncomplicated UTIs caused by ESBL-producing microorganisms.
The present study was designed to assess the in vitro activity of fosfomycin by three different methods against 428 ESBL-producing strains, made up of 290 (68%) E. coli and 138 (32%) K. pneumoniae isolates.
The strains were collected in two Spanish studies: the first, developed in 2000, included 40 hospitals representing different regions of Spain, and the second was carried out in Seville, Spain, at the University Hospital of Virgen Macarena (UHVM), using strains recovered between 1995 and 2001 (7, 11). In both studies ESBL production was determined by broth microdilution according to CLSI guidelines (5).
The strains collected from the multicenter study included 170 E. coli and 70 K. pneumoniae strains, while those obtained from the UHVM included 120 E. coli and 68 K. pneumoniae strains. One-hundred thirty-two out of 290 (45.5%) E. coli and 16 out of 138 (11.6%) K. pneumoniae strains were isolated from outpatients with community-acquired infections, and among these isolates found in the community, 66 (22.7%) E. coli and 9 (6.5%) K. pneumoniae strains were isolated from urine samples of women with uncomplicated UTIs.
The MICs of fosfomycin (Zambon, Milan, Italy) were determined by the agar dilution and broth microdilution methods in cation adjusted Mueller-Hinton medium, supplemented with 25 mg/liter of G-6-P (glucose-6-phosphate; Sigma Chemical Co.). A twofold dilution across a range of 0.25 to 512 mg of fosfomycin per liter was used. The inocula were prepared to achieve 1 x 104 CFU per spot (agar dilution) or 5 x 105 CFU/ml (broth microdilution) according to CLSI recommendations (5). At the same time, disks containing 200 µg fosfomycin and 50 µg G-6-P (Oxoid) were tested following the method outlined by CLSI (9).
Fosfomycin breakpoints for the interpretative criteria for E. coli were used according to CLSI guidelines (5, 9). Because of the absence of accepted breakpoints for K. pneumoniae, those proposed by CLSI for E. coli have been used (5, 9).
Susceptibility results of the broth microdilution and disk diffusion methods were compared with those obtained from the agar dilution method used as the reference method. Agreement and discrepancies between the evaluated and reference method were classified as complete agreements, very major errors, major errors, and minor errors. Differences in proportions among paired categorical data were calculated by the McNemar test and for independent categorical data by the chi-square test. The Wilcoxon test was used for comparing MICs. Statistical significance was established as P < 0.05, and very significant was established as P < 0.01.
By the agar dilution method, 417 (97.4%) strains were susceptible to fosfomycin, with MICs of 
64 µg/ml. The fosfomycin 50% minimum inhibitory concentration (MIC50) was 2 µg/ml, and the MIC90 was 32 µg/ml (range, 0.5 to 512 µg/ml). Most isolates with intermediate susceptibility or resistance to fosfomycin (2.6%) were K. pneumoniae isolates: four resistant strains (MIC, 
256 µg/ml) and six intermediate strains (MIC, 128 µg/ml). Only one strain of E. coli of all 290 tested showed intermediate susceptibility to fosfomycin (MIC, 128 µg/ml). K. pneumoniae isolates have the highest MICs for fosfomycin (MIC50 and MIC90 of 16 to 64 µg/ml) but are still within the susceptible range, whereas more than 90% of E. coli isolates yielded very low MICs (4 µg/ml) (Fig. 1). These results are similar to those described in previous reports of non-ESBL-producing isolates (2, 8, 14), confirming that fosfomycin retains its activity against ESBL-producing isolates, and cross-resistance with other classes of antimicrobial agents is not a problem at present.
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However, in common with others (1, 3, 6) who have used Mueller-Hinton broth supplemented with G-6-P, we observed only minor differences (average, ±1.93 dilutions; range, 0 to 7). In 241 (56.4%) strains the MIC broth values were higher than those obtained with agar (P < 0.001). The complete percentage agreement between the broth microdilution method and agar dilution results was higher for E. coli (98.6%) than for K. pneumoniae strains (72.5%) (P < 0.001). Table 1 compares the categories obtained by the microdilution broth and disk diffusion methods with those obtained by the agar dilution method used as reference. For both evaluated methods, the error rate observed for K. pneumoniae was significantly higher than that for E. coli (P < 0.001).
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When broth microdilution or disk diffusion methods are used to determine the susceptibility to fosfomycin of K. pneumoniae, the results for resistance and intermediate susceptibility should be confirmed by the reference method. Because most automated systems for antimicrobial susceptibility testing are microdilution-based methods, resistance to fosfomycin may be overestimated in laboratories employing such systems, especially for isolates other than E. coli.
Our data suggest an excellent in vitro activity of fosfomycin against ESBL-producing E. coli and K. pneumoniae strains. Since fosfomycin is only approved in the United States for E. coli UTI treatment, further clinical studies are required to assess the clinical efficacy of fosfomycin for the treatment of UTIs caused by ESBL-producing K. pneumoniae.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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