Detection and Identification of Ciprofloxacin-Resistant Yersinia pestis by Denaturing High-Performance Liquid Chromatography

ABSTRACT Denaturing high-performance liquid chromatography (DHPLC) has been used extensively to detect genetic variation. We used this method to detect and identify Yersinia pestis KIM5 ciprofloxacin-resistant isolates by analyzing the quinolone resistance-determining region (QRDR) of the gyrase A gene. Sequencing of the Y. pestis KIM5 strain gyrA QRDR from 55 ciprofloxacin-resistant isolates revealed five mutation types. We analyzed the gyrA QRDR by DHPLC to assess its ability to detect point mutations and to determine whether DHPLC peak profile analysis could be used as a molecular fingerprint. In addition to the five mutation types found in our ciprofloxacin-resistant isolates, several mutations in the QRDR were generated by site-directed mutagenesis and analyzed to further evaluate this method for the ability to detect QRDR mutations. Furthermore, a blind panel of 42 samples was analyzed by screening for two mutant types to evaluate the potential diagnostic value of this method. Our results showed that DHPLC is an efficient method for detecting mutations in genes that confer antibiotic resistance.

[1]  V. McGovern.  Taking a world view of asthma. , 2002, Environmental health perspectives.

[2]  F. Tenover,et al.  DNA Gyrase and Topoisomerase IV Mutations Associated with Fluoroquinolone Resistance in Proteus mirabilis , 2002, Antimicrobial Agents and Chemotherapy.

[3]  W. Hurtle,et al.  Denaturing HPLC for identifying bacteria. , 2002, BioTechniques.

[4]  A. Salyers.  AN OVERVIEW OF THE GENETIC BASIS OF ANTIBIOTIC RESISTANCE IN BACTERIA AND ITS IMPLICATIONS FOR AGRICULTURE , 2002, Animal biotechnology.

[5]  P. Caumette,et al.  Type II Topoisomerase Quinolone Resistance-Determining Regions of Aeromonas caviae, A. hydrophila, and A. sobria Complexes and Mutations Associated with Quinolone Resistance , 2002, Antimicrobial Agents and Chemotherapy.

[6]  F. Tenover,et al.  Genetic Analyses of Mutations Contributing to Fluoroquinolone Resistance in Clinical Isolates ofStreptococcus pneumoniae , 2001, Antimicrobial Agents and Chemotherapy.

[7]  Luther E. Lindler,et al.  Detection of Ciprofloxacin-ResistantYersinia pestis by Fluorogenic PCR Using the LightCycler , 2001, Journal of Clinical Microbiology.

[8]  S. M. Friedman,et al.  Mutation in the DNA Gyrase A Gene ofEscherichia coli That Expands the Quinolone Resistance-Determining Region , 2001, Antimicrobial Agents and Chemotherapy.

[9]  L. Lagae,et al.  De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy. , 2001, American journal of human genetics.

[10]  P. Oefner,et al.  Denaturing high‐performance liquid chromatography: A review , 2001, Human mutation.

[11]  F. Collins,et al.  Germline and somatic mutation analyses in the DNA mismatch repair gene MLH3: Evidence for somatic mutation in colorectal cancers , 2001, Human mutation.

[12]  H. Brauch,et al.  DHPLC-based germline mutation screening in the analysis of the VHL tumor suppressor gene: usefulness and limitations , 2001, Human Genetics.

[13]  K. Jinneman,et al.  Listeria monocytogenes Lineage Group Classification by MAMA-PCR of the Listeriolysin Gene , 2001, Current Microbiology.

[14]  E. L. Pettit,et al.  Genotyping of two mutations in the HFE gene using single-base extension and high-performance liquid chromatography. , 2001, Analytical chemistry.

[15]  A. Hall,et al.  The use of denaturing high-pressure liquid chromatography for the detection of mutations in thiopurine methyltransferase. , 2001, Journal of biochemical and biophysical methods.

[16]  C. Walker,et al.  Determination of loss of heterozygosity in frozen and paraffin embedded tumors by denaturating high-performance liquid chromatography (DHPLC). , 2001, Journal of biochemical and biophysical methods.

[17]  L. Valera,et al.  Susceptibility of bacterial isolates to gatifloxacin and ciprofloxacin from clinical trials 1997-1998. , 2000, International journal of antimicrobial agents.

[18]  L. Schriml,et al.  Use of denaturing HPLC to map human and murine genes and to validate single-nucleotide polymorphisms. , 2000, BioTechniques.

[19]  E. Gross,et al.  A highly sensitive, fast, and economical technique for mutation analysis in hereditary breast and ovarian cancers , 1999, Human mutation.

[20]  G. Zirnstein,et al.  Ciprofloxacin Resistance in Campylobacter jejuniIsolates: Detection of gyrA Resistance Mutations by Mismatch Amplification Mutation Assay PCR and DNA Sequence Analysis , 1999, Journal of Clinical Microbiology.

[21]  P. Oefner,et al.  Rapid quantification of gene expression by competitive RT-PCR and ion-pair reversed-phase HPLC. , 1996, BioTechniques.

[22]  J. Ruiz,et al.  Mutation in the gyrA gene of quinolone-resistant clinical isolates of Acinetobacter baumannii , 1995, Antimicrobial agents and chemotherapy.

[23]  J Ruiz,et al.  Association between double mutation in gyrA gene of ciprofloxacin-resistant clinical isolates of Escherichia coli and MICs , 1994, Antimicrobial Agents and Chemotherapy.

[24]  H. Dupont.  Use of quinolones in the treatment of gastrointestinal infections , 1991, European Journal of Clinical Microbiology and Infectious Diseases.

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

[26]  T. Sekiya,et al.  Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. , 1989, Genomics.

[27]  S. Ho,et al.  Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. , 1989, Gene.

[28]  J. Hoogkamp-Korstanje,et al.  Treatment of chronic bacterial prostatitis with ciprofloxacin , 1987, Pharmaceutisch Weekblad.

[29]  Scully Be,et al.  Treatment of serious infections with intravenous ciprofloxacin. , 1987 .

[30]  M. Gellert,et al.  DNA gyrase: an enzyme that introduces superhelical turns into DNA. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[31]  A. Bauer,et al.  Antibiotic susceptibility testing by a standardized single disk method. , 1966, American journal of clinical pathology.

[32]  H. Poppel,et al.  Clinical experience with ciprofloxacin in the treatment of urinary tract infections: A review , 2005, Infection.

[33]  J. Yamagishi,et al.  Quinolone-resistant mutations of the gyrA gene of Escherichia coli , 2004, Molecular and General Genetics MGG.

[34]  T Foitzi,et al.  Allelic discrimination using fluorogenic probes and the 5' nuclease assay , 1999 .

[35]  E. Lander,et al.  Characterization of single-nucleotide polymorphisms in coding regions of human genes , 1999 .

[36]  P. Oefner,et al.  Denaturing HPLC-identified novel FBN1 mutations, polymorphisms, and sequence variants in Marfan syndrome and related connective tissue disorders. , 1997, Genetic testing.

[37]  Robert C. Wolpert,et al.  A Review of the , 1985 .