Pseudomallei Clinical Isolates Research Articles

Use of multiplex PCR patterns as genetic markers for Burkholderiapseudomallei

A simple PCR-based typing method was developed to differentiate between strains of Burkholderia pseudomallei. Two pairs of primers, based on sequences from two specific DNA probes, were used to amplify the bacterial DNA by multiplex PCR. We evaluated the PCR method for epidemiological typing of B. pseudomallei and compared this with restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) methods. In RFLP, the DNA of B. pseudomallei was digested with HindIII and the pKKU-S23L was used as a probe while 5′ GTTTCGCTCC 3′ primer was used in RAPD. DNA was obtained from 37 B. pseudomallei environmental and clinical isolates from humans or animals. These isolates were also classified by their ability to assimilate l-arabinose. A total of 21 type patterns were identified by multiplex PCR. Among human and animal isolates, multiplex PCR revealed ten types, all of which were arabinose negative (Ara −), whereas six of the 11 types of environmental isolates were Ara −. There are two environmental patterns that also were found in clinical isolates. The RFLP technique showed 12 different types in the 37 isolates, and three of these contained both Ara + and Ara − isolates. The RAPD technique revealed 33 different types in the 37 isolates. Multiplex PCR, therefore, is the genetic marker that best correlates with the ability of the organism to assimilate l-arabinose. Moreover, two types (M4, M15) correlated with disseminated septicemic melioidosis in the northeast Thailand. If a greater number of isolates are tested, the multiplex PCR technique may prove to be useful for rapid epidemiological typing of B. pseudomallei.

Rapid identification of Burkholderia pseudomallei in blood cultures by latex agglutination using lipopolysaccharide-specific monoclonal antibody.

Melioidosis, an infection caused by Burkholderia pseudomallei, is endemic in Southeast Asia. The septicemic form of melioidosis is the leading cause of death due to community-acquired bacteremia in the northeastern part of Thailand. The delay in isolation and identification of the causative organism is a major contributing factor to the high mortality. The present study describes the evaluation of a latex agglutination test for rapid identification of the bacteria directly from blood cultures. The Bps-L1 monoclonal antibody recognized the lipopolysaccharide antigen of 96.8% of B. pseudomallei clinical isolates and was highly specific for B. pseudomallei. The diagnostic value of the latex agglutination test based on Bps-L1 monoclonal antibody was prospectively evaluated in an area endemic for melioidosis. The agglutination test kit was evaluated in 88 blood cultures with gram-negative bacteria identified with Gram staining. The sensitivity and specificity of the test kit were both 100%. These results indicated that the detection of B. pseudomallei lipopolysaccharide by specific monoclonal antibody in a latex agglutination format is clinically useful for the rapid identification of the bacteria in blood cultures in areas endemic for melioidosis.

Construction of a specific DNA probe for diagnosis of melioidosis and use as an epidemiological marker of Pseudomonas pseudomallei

Pseudomonas pseudomallei is a causative agent of melioidosis. The disease manifestations range from fulminant sepsis to asymptomatic seroconversion. In septicemic cases, a mortality rate of 80-90% is reported. Rapid and specific diagnosis has become important to the clinical microbiology laboratory. We have developed a P. pseudomallei-specific DNA probe. The cloned fragment, herein designated pKKU-S23L, contained 1·5 kb of P. pseudomallei chromosomal DNA. A radioactively labelled pKKU-S23L insert could detect 1·5 ng of its genomic DNA or 40 000 P. pseudomallei cells. The probe was highly specific for P. pseudomallei DNA and did not cross-hybridize with DNAs prepared from other related bacteria. Using pKKU-S23L as a probe in total cellular DNA digestions and Southern blot hybridization, we were able to classify 60 P. pseudomallei clinical isolates obtained from individual melioidosis patients into eight categories. Therefore, this probe has a potential not only for use in development of specific detection of bacterial DNA in clinical specimens but also for application in epidemiological studies of P. pseudomallei.