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.Yersinia pestis is a gram-negative bacillus belonging to the Enterobacteriaceae family. This organism can cause several different forms of disease, including bubonic, pneumonic, and septicemic plague, depending on the route of exposure (26). Bubonic plague usually infects one group of lymph nodes, and infection can travel to the lungs. Pneumonic plague is transmitted by airborne droplets. Infection takes place within hours and causes bronchopneumonia. Plague pneumonia can be treated if it is recognized early. Delay of therapy results in high mortality rates (30), making the organism an effective biological warfare agent.Ciprofloxacin is an antibiotic that has been used to treat many bacterial diseases, including Enterobacteriaceae infections (6, 7, 14, 19-21, 27, 32). Ciprofloxacin belongs to the fluoroquinolone class of antibiotics that inhibit bacterial DNA replication by inhibiting the activity of DNA gyrase. The major weakness of this class of compounds is that a single mutation in the DNA gyrase gene may make the bacteria resistant to antibiotic activity. In the event of a biological attack involving this agent, rapid detection of antibiotic-resistant bacteria would have critical importance.In bacteria, DNA contains a little less than one helical turn for each 10.4 bp. This leaves the bacterial genome slightly unwound, and negative supertwists are unfavorable energetically. Topoisomerases are enzymes that alter the number of times a DNA strand wraps around itself. In prokaryotes, type II topoisomerase is a DNA gyrase which cleaves the double strand and introduces negative supercoils (9). DNA gyrase consists of two 100-kDa A subunits encoded by the gyrA gene and two 90-kDa B subunits encoded by the gyrB gene (35). Mutations in either or both of these genes may make an organism resistant to ciprofloxacin.Denaturing high-performance liquid chromatography (DHPLC) is a quick and sensitive method for detecting genetic mutatio...

Effective Plague Vaccination via Oral Delivery of Plant Cells Expressing F1-V Antigens in Chloroplasts

“…However, a "natural" strain resistant to multiple antibiotics was isolated in 1995 in the Ambalavao district of Madagascar (27). The possibility of the occurrence of such multidrug-resistant strains in the natural environment, the ease of generating such strains under laboratory conditions (39,41), and the potential use of such strains for bioterrorist attack, together with the rapidity and high lethality of the disease (for a review, see reference 5), indicate that it is necessary to search for alternatives to antibiotics.…”

Fluoroquinolone and multidrug resistance phenotypes associated with the overexpression of AcrAB and an orthologue of MarA in Yersinia enterocolitica

“…However, few studies analysing the mutations acquired within the gyrA gene among a set of ciprofloxacin-resistant Y. pestis mutants (derivatives of the avirulent strain KIM5) obtained in vitro have been performed (Lindler et al, 2001;Hurtle et al, 2003). On the other hand, more recent studies performed with Y. enterocolitica clinical isolates have also been carried out (Sanchez-Cespedes et al, 2003;Capilla et al, 2004).…”

Use of PCR-DHPLC with fluorescence detection for the characterization of the bacterial diversity during cassava (Manihot esculenta crantz) fermentation

“…Denaturing high-performance liquid chromatography (DHPLC) has been pointed out as a rapid, reliable, and highly sensitive method for screening microbial rDNA amplicons for clinical (Hurtle et al, 2003) and industrial (Hutzler et al, 2010) applications. DHPLC has also been used as an accessory method in the clinical diagnosis of human diseases such as Von Willebrand syndrome (Kakela et al, 2006) and cancer (Li et al, 2008).…”