The Laboratory Diagnosis of Melioidosis in a Korean Patient

Burkholderia pseudomallei is a gram-negative opportunistic intracellular pathogen that causes an acute and fatal septicemic melioidosis in humans. The organism is mainly found in Southeastern Asia and Northern Australia. Recently, we encountered a case of melioidosis in a Korean patient and performed the laboratory diagnosis of melioidosis. As a result, a gram negative bacterium was isolated from a melioidosis patient, and it was identified as B. pseudomallei on DNA sequencing of 16S ribosomal RNA with 99.9% homology and biochemical examination of VITEK gram-negative identification card. Also, DNA from cultured bacteria was tested in multiplex PCR, a 245 bp fragment amplified from the metalloprotease gene and a fragment of variable size ranging from 400~700 bp resulting from amplification of the 10 bp repetitive element for B. pseudomallei were confirmed after electrophoresis. The bacterium was sensitive to ceftazidime, imipenem and meropenem but resistant to ticarcillin. So far, there are no domestic cases of melioidosis in Korea, however, due to the increase in international travelers, the incidence of melioidosis is likely to increase. We report a recent case of melioidosis in a Korean patient.

Genetic Diversity among Virulent Mega Plasmids pXO1 and pXO2 of Bacillus anthracis Isolated in Korea

We compared genetic variations in virulence mega plasmids pXO1 and pXO2 of twenty-seven Bacillus anthracis strains from Korean patients and environmental samples together with those of Bacillus anthracis Sterne, Pasteur and A2012 standard strains. Genetic variations were analyzed in twenty-three variable regions (ten and thirteen variablenumber tandem repeats and insertion/deletions in pXO1 and pXO2, respectively). The pXO1 plasmids were classified into 7 groups and pXO2 plasmids to 12 groups. Discrete phylogenic lineages could be differentiated between environmental and clinical strains by UPGMA (unweighted pair group method with average) method. In addition, clinical strains showed more variations than environmental isolates. The pXO2 plasmid appeared genetically more unstable than pXO1. A general plasmid genotype could be suggested for Korean soil isolates since they mostly clustered into a representative group.

Epidemiological Characterization of Shigella flexneri Isolates in Korea and the Analysis of Pulsed - Field Gel Electrophoresis Patterns

We investigated the molecular epidemiological characteristics of the Shigella flexneri strains primarily isolated in the provincial health center from 1998 to 1999. Among 289 isolates of S. flexneri, 270 isolates (93A%) were confirmed as S. flexneri serotype 2a. The monthly isolation rate of S. flexneri strains was different from that of S, sonnei. S. flexneri strains were not isolated from July to August in 1998 but were isolated rarely during the same period in 1999. Shigella strains were isolated at higher rates in the areas of Chungbuk (64.4%), Busan (8.2%), Jeonnam (6.8%) in 1998 and in the areas of Busan (10.6%), Gangwon (9.3%), Gyeongnam (29.2%), Jeonbuk (4.2%) and Jeonnam (20.8%) in 1999. In these areas, the large outbreaks occurred with relatively high isolation rates of Shigella strain. Among 289 strains, 172 (59.5%) S. flexneri strains were isolated from female patients. Eighty-eight (30.4%) Shigella strains were isolated among the high risk age group of over 61 years. With the antimicrobial susceptibility tests, 284 isolates (98.3%) showed multiple resistance to more than four antibiotics, but all isolates were sensitive to ciprofloxacin, ceftriaxone and cefoxitin. We could divide on isolates into 5 groups (A, B, C, D and E) by analyzing PFGE patterns. Group A subdivided as 16 subgroups and 270 (93.4%) strains belong to the group A. The PFGE patterns of strains isolated from outbreaks revealed that the was only little difference corresponding one to three bands among strains. This result indicates that our isolates are genetically related.

Development of a Rapid Detection Method for Yersinia pestis by Polymerase Chain Reaction

A polymerase chain reaction (PCR) method for detection of the pathogenic Yersinia pestis from other Yersinia spp. was developed. Five Y. pestis strains, ninety-two other Yersinia species and twenty-four Enterobacteriaceae strains were collected in Korea and from other countries. Oligonucleotide primers were designed from pathogenic gene of antiphagocytic protein capsule gene (fra 1) and plasminogen activator gene (pla). The 428 bp DNA fragment was amplified from five Y. pestis which contained the fra I gene. No product was amplified from other Yersinia species and other strains of the Enterobacteriaceae. The 439 bp DNA fragment was amplified from three K pestis which contained the pla gene. No product was amplified from two Y. pestis, other Yersinia species and other strains of the Enterobacteriaceae. These showed that the designed primers were specific for detection of Y. pestis among other Yersinia species and Enterobacteriaceae strains. Amplification was successful whether the template was derived from purified DNA or from aliquots of boiled bacterial suspension. The detection limits were 100 pg of DNA and 100 colony forming units (CFU) for fra I and 100 pg DNA and 10 CFU for pla, respectively. Our results prove that the PCR method using specific primers for Y. pestis is a rapid and convenient procedure for routine clinical detection and identification of Y. pestis.