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Denaturing HPLC (DHPLC) is used in a wide variety of genetic applications. Here we introduce a new application for this technique, the identification of bacteria. We combined the capability of DHPLC to detect sequence variation with the principles of rRNA genotyping analysis to develop a high-throughput method of identifying microorganisms. Thirty-nine bacterial species from a broad spectrum of genera were tested to determine if DHPLC could be usedfor identification. Most (36 of 39) species of bacteria had a unique peak profile that could be used as a molecular fingerprint. Furthermore, a blind panel of 65 different bacterial isolates was analyzed to demonstrate the diagnostic capability of this method to specifically identify Yersinia pestis and Bacillus anthracis. All the Y. pestis samples (10 of 10) and the majority of B. anthracis samples (12 of 14) were correctly identified. The procedure had an overall specificity of 100%, overall sensitivity of 91.7%, and a predictive value of 96.9%. The data suggest that DHPLC of products spanning regions of genetic variability will be a useful application for bacterial identification.

Colonization of the human nasopharyngeal region by Neisseria meningitidis is believed to lead to natural immunity. Although the presence of bactericidal antibody in serum has been correlated with immunity to meningococcal disease, mucosal immunity at the portal of entry may also play an important role. This study was undertaken to examine in mice the possibility of safely using native outer membrane vesicles (NOMV) not exposed to detergent as an intranasal (i.n.) vaccine. The mucosal and systemic responses of mice to intranasal and intraperitoneal (i.p.) vaccination with NOMV were compared over a range of doses from 0.1 to 20 μg. Intranasal vaccination of mice with NOMV induced a strong systemic bactericidal antibody response, as well as a strong local immunoglobulin A immune response in the lung as determined by assay of lung lavage fluid by enzyme-linked immunosorbent assay and lung antibody secreting cells by enzyme-linked immunospot assay. However, 8- to 10-fold-higher doses of NOMV were required i.n. compared to i.p. to elicit an equivalent bactericidal antibody response in serum. Some NOMV vaccine was aspirated into the lungs of mice during i.n. immunization and resulted in an acute inflammatory response that peaked at 1 to 2 days postimmunization and was cleared by day 7. These results indicate that i.n. delivery of meningococcal NOMV in mice is highly effective in eliciting the production of both a mucosal immune response and a systemic bactericidal antibody response.

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...