High-level fluoroquinolone resistance in Streptococcus pneumoniae requires mutations in parC and gyrA

The mechanism of high-level fluoroquinolone resistance was studied in strains of Streptococcus pneumoniae, either selected in vitro or isolated from clinical samples. By using DNA from these high-level-resistant strains, low-level-resistant transformants (MIC of pefloxacin, > or = 32 micrograms/ml; MIC of ciprofloxacin, 4 micrograms/ml; MIC of sparfloxacin, 0.50 micrograms/ml) were obtained at high frequencies (ca.10(-2)), while high-level-resistant transformants (MIC of pefloxacin, > or = 64 micrograms/ml; MIC of ciprofloxacin, 16 to 64 micrograms/ml; MIC of sparfloxacin, > or = 8 micrograms/ml) were obtained only at low frequencies (ca.10(-4)). This suggested that mutations in at least two unlinked genes were necessary to obtain high-level resistance. Low-level resistance was associated with ParC mutations (change from Ser to Tyr at position 79 [Ser79Tyr], Ser79Phe, or Asp83Gly). ParC mutations were associated, in high-level-resistant strains and transformants, with alterations in the quinolone resistance-determining region of GyrA (Ser84Tyr, Ser84Phe, and/or Glu88Lys). Low-level resistance was shown to be necessary for expression of the gyrA mutations. No mutation in the region corresponding to the quinolone resistance-determining region of GyrB and no alteration of drug accumulation were found.

[1]  M. Cooper,et al.  In-vitro activity of sparfloxacin, a new quinolone antimicrobial agent. , 1990, The Journal of antimicrobial chemotherapy.

[2]  L. Fisher,et al.  Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance , 1996, Journal of bacteriology.

[3]  T. Ezaki,et al.  DNA gyrase mutations in quinolone-resistant clinical isolates of Neisseria gonorrhoeae , 1995, Antimicrobial agents and chemotherapy.

[4]  P. Heisig,et al.  Genetic evidence for a role of parC mutations in development of high-level fluoroquinolone resistance in Escherichia coli , 1996, Antimicrobial agents and chemotherapy.

[5]  D. Hooper,et al.  Genetic and biochemical characterization of norfloxacin resistance in Escherichia coli , 1986, Antimicrobial Agents and Chemotherapy.

[6]  L. Peterson,et al.  Detection of gyrA gene mutations associated with ciprofloxacin resistance in methicillin-resistant Staphylococcus aureus: analysis by polymerase chain reaction and automated direct DNA sequencing , 1992, Antimicrobial Agents and Chemotherapy.

[7]  W. M. Huang,et al.  Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone‐resistant isolates , 1994, Molecular microbiology.

[8]  G. Kaatz,et al.  Mechanisms of fluoroquinolone resistance in Staphylococcus aureus. , 1991, The Journal of infectious diseases.

[9]  S. Nakamura,et al.  Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli , 1990, Antimicrobial Agents and Chemotherapy.

[10]  J. Pemán,et al.  In vitro activity of sparfloxacin compared with those of five other quinolones , 1992, Antimicrobial Agents and Chemotherapy.

[11]  P. Appelbaum,et al.  Antimicrobial resistance in Streptococcus pneumoniae: an overview. , 1992, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[12]  K. Thomson,et al.  Dissociated resistance among fluoroquinolones , 1994, Antimicrobial Agents and Chemotherapy.

[13]  V. Ackerman,et al.  In vitro studies of ciprofloxacin and survey of resistance patterns in current isolates. , 1990, Diagnostic microbiology and infectious disease.

[14]  B. Murray,et al.  Analysis by PCR and direct DNA sequencing of gyrA mutations associated with fluoroquinolone resistance in Enterococcus faecalis , 1994, Antimicrobial Agents and Chemotherapy.

[15]  A. Khodursky,et al.  Topoisomerase IV is a target of quinolones in Escherichia coli. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[16]  R. Muñoz,et al.  Ser-127-to-Leu substitution in the DNA gyrase B subunit of Streptococcus pneumoniae is implicated in novobiocin resistance , 1995, Journal of bacteriology.

[17]  S. Brockbank,et al.  Cloning, sequencing, and expression of the DNA gyrase genes from Staphylococcus aureus , 1993, Journal of bacteriology.

[18]  J. Ghuysen,et al.  Nucleotide sequences of the pbpX genes encoding the penicillin‐binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime‐resistant mutant, C506 , 1989, Molecular microbiology.

[19]  S. Lacks,et al.  A study of the genetic material determining an enzyme activity in Pneumococcus , 1960 .

[20]  L. Peterson,et al.  Ciprofloxacin resistance in coagulase-positive and -negative staphylococci: role of mutations at serine 84 in the DNA gyrase A protein of Staphylococcus aureus and Staphylococcus epidermidis , 1991, Antimicrobial Agents and Chemotherapy.

[21]  S. Nakamura,et al.  Quinolone resistance-determining region in the DNA gyrase gyrB gene of Escherichia coli , 1990, Antimicrobial Agents and Chemotherapy.

[22]  L. Piddock,et al.  The selection and frequency of streptococci with decreased susceptibility to ofloxacin compared with other quinolones. , 1988, The Journal of antimicrobial chemotherapy.

[23]  J. Mainardi,et al.  In Vivo Selection of Streptococcus pneumoniae, Resistant to Quinolones, Including Sparfloxin. , 1995, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[24]  N. Moreau,et al.  Mechanisms of quinolone resistance in clinical isolates: accumulation of sparfloxacin and of fluoroquinolones of various hydrophobicity, and analysis of membrane composition. , 1993, Journal of Antimicrobial Chemotherapy.

[25]  B. Foleno,et al.  Induction of resistance of Streptococcus pneumoniae to quinolones in vitro. , 1993, Chemotherapy.

[26]  J. Crouzet,et al.  Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones , 1994, Molecular microbiology.

[27]  A. M. Sicard,et al.  Donor deoxyribonucleic acid length and marker effect in pneumococcal transformation , 1979, Journal of bacteriology.

[28]  J. Crouzet,et al.  Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus , 1995, Antimicrobial agents and chemotherapy.

[29]  M. Fox,et al.  Marker discrimination in transformation and mutation of pneumococcus. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[30]  H. Ito,et al.  Quinolone resistance mutations in the DNA gyrase gyrA and gyrB genes of Staphylococcus aureus , 1994, Antimicrobial Agents and Chemotherapy.

[31]  Y. Tokue,et al.  Incidence of various gyrA mutants in 451 Staphylococcus aureus strains isolated in Japan and their susceptibilities to 10 fluoroquinolones , 1995, Antimicrobial agents and chemotherapy.

[32]  L. Fisher,et al.  Nucleotide sequence of the Staphylococcus aureus gyrB-gyrA locus encoding the DNA gyrase A and B proteins , 1992, Journal of bacteriology.