The virulence factor genotypes of a large cohort of clinical, nosocomial environment and community environment isolates (184 in total) of Pseudomonas aeruginosa from Tasmania, Australia, were determined by PCR. The virulence factor genotype of the majority of isolates was highly conserved, with the exception of the virulence gene exoU, which demonstrated low prevalence (33 isolates; 18 %) in the population tested. Isolates collected from the environment of intensive therapy wards (intensive care unit and neurosurgical units) of the major tertiary referral hospital in Tasmania were found to be more likely (P,0.001 and P,0.05, respectively) to possess the virulence factor gene exoU than all other isolates. Adult cystic fibrosis isolates showed a decreased prevalence of the exoU gene (P,0.01) when compared to other clinical isolates (P,0.01), which may indicate decreased virulence. No specific virulence factor genotype was associated with the cystic fibrosis epidemic strains tested.

Limited experience and a lack of validated diagnostic reagents make Burkholderia pseudomallei, the cause of melioidosis, difficult to recognize in the diagnostic microbiology laboratory. We compared three methods of confirming the identity of presumptive B. pseudomallei strains using a collection of Burkholderia species drawn from diverse geographic, clinical, and environmental sources. The 95 isolates studied included 71 B. pseudomallei and 3 B. thailandensis isolates. The API 20NE method identified only 37% of the B. pseudomallei isolates. The agglutinating antibody test identified 82% at first the attempt and 90% including results of a repeat test with previously negative isolates. Gas-liquid chromatography analysis of bacterial fatty acid methyl esters (GLC-FAME) identified 98% of the B. pseudomallei isolates. The agglutination test produced four false positive results, one B. cepacia, one B. multivorans, and two B. thailandensis. API produced three false positive results, one positive B. cepacia and two positive B. thailandensis. GLC-FAME analysis was positive for one B. cepacia isolate. On the basis of these results, the most robust B. pseudomallei discovery pathway combines the previously recommended isolate screening tests (Gram stain, oxidase test, gentamicin and polymyxin susceptibility) with monoclonal antibody agglutination on primary culture, followed by a repeat after 24 h incubation on agglutination-negative isolates and GLC-FAME analysis. Incorporation of PCR-based identification within this schema may improve percentages of recognition further but requires more detailed evaluation.Burkholderia pseudomallei, the cause of melioidosis, can be difficult to reliably identify in the clinical microbiology laboratory. Many diagnostic laboratories have no experience of this species. Even in locations such as Southeast Asia and northern Australia where melioidosis is endemic, a preponderance of septicemic cases during the wet season results in a low expectation of B. pseudomallei in clinical specimens at other times of year (1). Practical difficulties for the diagnostic laboratory include the presence of closely related Burkholderia species in specimens from nonsterile sites and atypical colony morphology of some B. pseudomallei strains (4). Despite clear recommendations for screening suspect B. pseudomallei colonies (3), there is wide variation in the approaches used by diagnostic laboratories. Moreover, a lack of properly validated diagnostic test reagents means that laboratories have to rely on biochemical tests for definitive identification. The substrate utilization test panels in current diagnostic use can generate misleading identification profiles (6).These problems and the rarity of melioidosis outside of northern Australia and Southeast Asia highlight the need for a more standardized culture-based diagnostic pathway. In the present study we evaluated phenotypic identification methods used in Australia to develop a laboratory case definition of melioidosis. This approach aims to integrate previou...

SUMMARYDNA amplification techniques are being used increasingly in clinical laboratories to confirm the identity of medically important bacteria. A PCR-based identification method has been in use in our centre for 10 years for Burkholderia pseudomallei and was used to confirm the identity of bacteria isolated from cases of melioidosis in Ceará since 2003. This particular method has been used as a reference standard for less discriminatory methods. In this study we evaluated three PCR-based methods of B. pseudomallei identification and used DNA sequencing to resolve discrepancies between PCR-based results and phenotypic identification methods. The established semi-nested PCR protocol for B. pseudomallei 16-23s spacer region produced a consistent negative result for one of our 100 test isolates (BCC #99), but correctly identified all 71 other B. pseudomallei isolates tested. Anomalous sequence variation was detected at the inner, reverse primer binding site for this method. PCR methods were developed for detection of two other B. pseudomallei bacterial metabolic genes. The conventional lpxO PCR protocol had a sensitivity of 0.89 and a specificity of 1.00, while a real-time lpxO protocol performed even better with sensitivity and specificity of 1.00, and 1.00. This method identified all B. pseudomallei isolates including the PCR-negative discrepant isolate. The phaC PCR protocol detected the gene in all B. pseudomallei and all but three B. cepacia isolates, making this method unsuitable for PCR-based identification of B. pseudomallei. This experience with PCR-based B. pseudomallei identification methods indicates that single PCR targets should be used with caution for identification of these bacteria, and need to be interpreted alongside phenotypic and alternative molecular methods such as gene sequencing.

Abstract. The epidemiologic status of melioidosis in Sri Lanka was unclear from the few previous case reports. We established laboratory support for a case definition and started a nationwide case-finding study. Suspected Burkholderia pseudomallei isolates were collated, identified by polymerase chain reaction assay, referred for Matrix Assisted Laser Desorption Ionization-Time of Flight analysis and multilocus sequence typing (MLST), and named according to the international MLST database. Between 2006 and early 2014, there were 32 patients with culture-confirmed melioidosis with an increasing annual total and a falling fatality rate. Patients were predominantly from rural communities, diabetic, and male. The major clinical presentations were sepsis, pneumonia, soft tissue and joint infections, and other focal infection. Burkholderia pseudomallei isolates came from all parts of Sri Lanka except the Sabaragamuwa Province, the south central hill country, and parts of northern Sri Lanka. Bacterial isolates belonged to 18 multilocus sequence types, one of which (ST 1137) was associated with septicemia and a single-organ focus (Fisher's exact, P = 0.004). Melioidosis is an established endemic infection throughout Sri Lanka, and is caused by multiple genotypes of B. pseudomallei, which form a distinct geographic group based upon related sequence types (BURST) cluster at the junction of the southeast Asian and Australasian clades.

This study describes the isolation and characterization of several Burkholderia species from soil in northern Australia. Phenotypic and molecular tests indicate that these isolates belong to the species Burkholderia thailandensis and Burkholderia ubonensis. These observations significantly extend our knowledge of the geographic distribution of these 2 species. Evidence of these species in Australia has implications for bacterial identification in clinical laboratories, diagnostic serology tests, and environmental biodiversity studies.