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Volumn 288, Issue 5469, 2000, Pages 1251-1253

High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection

Author keywords

[No Author keywords available]

Indexed keywords

AMIKACIN; CEFTAZIDIME; FOSFOMYCIN; GENTAMICIN; IMIPENEM; NORFLOXACIN; RIFAMPICIN; TICARCILLIN; TOBRAMYCIN;

EID: 0034685940     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.288.5469.1251     Document Type: Article
Times cited : (1219)

References (33)
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    • note
    • Mutation frequency measurement. One bacterial colony was resuspended in 20 ml of Mueller-Hinton Broth (MHB) and grown at 37°C overnight Bacterial cells were then collected at 3000 rpm for 5 min and resuspended in 1 ml of MHB. A 100-μl sample from this suspension as well as from successive dilutions was plated onto Mueller-Hinton Agar (MHA) plates, with and without rifampicin (300 μg/ml) or streptomycin (500 μg/ml). In all cases, the isolates were originally susceptible to such concentrations of rifampicin and streptomycin. Colony counting was performed in plates containing 100 to 1000 colonies after 36 hours of culture. Ten colonies growing under high antibiotic concentrations were streaked onto another plate with antibiotic in order to assess the stability of mutants. To avoid mutation jackpot (the recovery, by chance, of a vast number of mutants), all experiments were performed in triplicate and the mean value was recorded. In the rare cases (less than 2%) in which one discrepant result was found, a new triplicate experiment was performed. In this work, one strain was considered a mutator when the corresponding mutation frequencies for both rifampicin (300 μg/ml) and streptomycin (500 μg/ml) were at least 20-fold higher than those observed for the PAO1.
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    • note
    • Mutator gene analysis. BLASTing the E. coli mutator gene sequences (TIGR) against the database of PAO1 (Pseudomonas Genome Project) allowed us to obtain the corresponding putative sequences of P. aeruginosa mutator genes: mutS, mutL, mutT, mutY, mutM, mutD, and uvrD. Specific primers were designed to amplify these genes in the studied strains by PCR.
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    • note
    • DNA from these four isolates was checked by amplifying in the same PCR tube another gene with higher molecular weight (uvrD) that, in contrast to mutY, was correctly amplified.
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    • note
    • 2 decrease in mutation frequency) was found in mutator strains from 4 out of the 11 CF patients with mutators. No significant change in mutation frequency was detectable in non-mutator strains or with the plasmid lacking the mutS gene.
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    • note
    • We thank B. Levin, J. M. Gómez-Gómez, I. Matic, and J. L. Martinez for useful discussions and comments, and two anonymous referees for suggestions; H. Escobar (Unit of Cystic Fibrosis) for continuous support; and R. Garber (PathoGenesis) for BLASTing the E. coli mutS sequence against the database of PAO1. We also thank L. de Rafael for English correction, O. Santisteban for help with preparation of the figures, and A. Vindel (National Center for Microbiology, Carlos III Institute, Majadahonda, Madrid) for help with serotyping and phagetyping of Pseudomonas isolates. Sequence data for this work were obtained from The Institute for Genomic Research (TIGR) and the Pseudomonas Genome Project.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.