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Journal of Clinical Microbiology, July 2006, p. 2672, Vol. 44, No. 7
0095-1137/06/$08.00+0 doi:10.1128/JCM.00958-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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Detection of Hypermutable Escherichia coli Strains in a Collection of Clinical Isolates by the Fosfomycin-Rifampin Disk Method
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Bacteria exhibiting elevated mutation frequencies may present an enhanced risk for the emergence of antibiotic resistance during chemotherapy (1). Galán et al. (2) described a simple method for detecting hypermutable strains in which commercial disks of fosfomycin (50 µg) and rifampin (30 µg) were placed on the surface of blood agar plates seeded with 100 µl of an overnight culture in Luria-Bertani broth and then the number of squatter colonies around the inhibition zones of the disks were counted after 24 h of incubation at 37°C.
We evaluated the ability of this method to detect hypermutable strains in a collection of 103 clinical isolates of Escherichia coli from the University Hospital Virgen Macarena of Seville, Spain, and compared the results with those obtained after determining the mutation frequency to rifampin by the conventional method (2). The collection included 64 isolates with an AmpC hyperproduction phenotype (defined as resistance to both cefoxitin and amoxicillin-clavulanate in the absence of extended-spectrum ß-lactamases) and quinolone resistance, 11 with an AmpC hyperproduction phenotype and quinolone susceptibility, and 28 susceptible to both cefoxitin and amoxicillin-clavulanate and susceptible or resistant to quinolones (13 and 15 strains, respectively). E. coli AB1157 (mutS::Tn10) (3), with a defective mutS gene, was used as a strong mutator control, and E. coli ATCC 25922 as a nonmutator control. All strains were kept frozen at –80°C until tested. Three independent colonies of each strain were evaluated and experiments repeated when discrepancies occurred. The results of the conventional method indicated that our collection included three strong mutators (mutation frequency of 
5 x 10–7), 26 weak mutators (mutation frequency of <5 x 10–7 to 5 x 10–8), and 74 nonmutators (mutation frequency of <5 x 10–8). The number of squatter colonies in the inhibition zones of fosfomycin and rifampin for E. coli ATCC 25922 were always in the range of nonmutators. E. coli AB1157 (mutS::Tn10) always yielded more than 70 squatter colonies in the inhibition zones of fosfomycin but less than 10 around the rifampin disk in 12 out of 15 determinations, although the mutation frequency to rifampin was always >5 x 10–7. According to the cutoff points established by Galán et al., this strain should be classified as a weak mutator. Figure 1 shows the results obtained after testing the 103 E. coli clinical isolates by the disk detection method compared to those obtained by the conventional method. Considering the cutoff values proposed by Galán et al., we observed discrepancies with the conventional method in 30 strains. The positive predictive value and negative predictive value of the disk detection method for any type of hypermutable strain were 53 and 81%, respectively. The low positive predictive value obtained discouraged us from considering the disk diffusion method for detecting hypermutable strains.
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FIG. 1. Mutation frequency categorization of 103 E. coli clinical isolates by the fosfomycin-rifampin diffusion test compared with that obtained by the conventional method. White bars, strains with <30 colonies around the fosfomycin disk and <10 around the rifampin disk; gray bars, strains with >30 but <70 colonies around the fosfomycin disk or >70 around the fosfomycin but <10 around the rifampin disk; black bars, strains with >70 colonies around the fosfomycin disk and >10 colonies around the rifampin disk; striped bars, strains with <30 colonies around the fosfomycin disk and >10 around the rifampin disk.
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- Chopra, I., A. J. O'Neill, and K. Miller. 2003. The role of mutators in the emergence of antibiotic-resistant bacteria. Drug Resist. Updates 6:137-145.[CrossRef][Medline]
- Galán, J. C., M. Tato, M. R. Baquero, C. Turrientes, F. Baquero, and J. L. Martínez. 2004. Fosfomycin and rifampin disk diffusion test for detection of Escherichia coli mutator strains. J. Clin. Microbiol. 42:4310-4312.
[Abstract/Free Full Text] - Pang, P. P., A. S. Lundberg, and G. C. Walker. 1985. Identification and characterization of the mutL and mutS gene products of Salmonella typhimurium LT2. J. Bacteriol. 163:1007-1015.
[Abstract/Free Full Text]
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M. Carmen Conejo* Gema Amblar Álvaro Pascual Evelio J. Perea Luis Martínez-Martínez Department of Microbiology School of Medicine University of Seville Av. Sánchez Pizjuán S/N 41009 Seville, Spain
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| * Phone: 34-955008287, Fax: 34-954377413 E-mail: cconejo{at}us.es |
Journal of Clinical Microbiology, July 2006, p. 2672, Vol. 44, No. 7
0095-1137/06/$08.00+0 doi:10.1128/JCM.00958-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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