Cheon-Kwon Yoo

Sensitive and Rapid Quantitative Detection of Anthrax Spores Isolated from Soil Samples by Real-Time PCR

Quantitative analysis of anthrax spores from environmental samples is essential for accurate detection and risk assessment since Bacillus anthracis spores have been shown to be one of the most effective biological weapons. Using TaqMan real‐time PCR, specific primers and probes were designed for the identification of pathogenic B. anthracis strains from pag gene and cap gene on two plasmids, pXO1 and pXO2, as well as a sap gene encoded on the S‐layer. To select the appropriate lysis method of anthrax spore from environmental samples, several heat treatments and germination methods were evaluated with multiplex‐PCR. Among them, heat treatment of samples suspended with sucrose plus non‐ionic detergent was considered an effective spore disruption method because it detected up to 105 spores/g soil by multiplex‐PCR. Serial dilutions of B. anthracis DNA and spore were detected up to a level of 0.1 ng/μl and 10 spores/ml, respectively, at the correlation coefficient of 0.99 by real‐time PCR. Quantitative analysis of anthrax spore could be obtained from the comparison between CT value and serial dilutions of soil sample at the correlation coefficient of 0.99. Additionally, spores added to soil samples were detected up to 104 spores/g soil within 3 hr by real‐time PCR. As a consequence, we established a rapid and accurate detection system for environmental anthrax spores using real‐time PCR, avoiding time and labor‐consuming preparation steps such as enrichment culturing and DNA preparation.

Molecular Characterization of Korean Bacillus anthracis Isolates by Amplified Fragment Length Polymorphism Analysis and Multilocus Variable-Number Tandem Repeat Analysis

ABSTRACT We analyzed the genetic relationships and molecular characteristics of 34 Bacillus anthracis isolates from soil and clinical samples in various regions of Korea and 17 related Bacillus species, using the amplified fragment length polymorphism (AFLP) and multilocus variable-number tandem repeat (MLVA) approaches. Triplicate AFLP profiles of these strains showed high reproducibility and identified 376 polymorphisms. AFLP phylogenetic analysis of B. anthracis isolates showed a high level of similarity, 0.93, and this monomorphic fragment profile proved to be useful to differentiate B. anthracis strains from other Bacillus species. The B. cereus group was separated from other Bacillus species at a level of similarity of 0.68. Among them, some B. cereus strains showed genetic interspersion with B. thuringiensis strains. The evolutionary pattern of nucleotide differences among B. anthracis strains with the eight MLVA markers showed nine MLVA types. Three MLVA types, M1 to M3, were pathogenic B. anthracis isolates and were assigned as new genotypes belonging to the A4 and B3 clusters, compared with 89 genotypes deduced from previous data. This indicates that differences in cluster prevalence and distribution may be influenced more by MLVA markers on two plasmids loci and human activity. Consequently, we suggest that the novel MLVA type may represent significant evidence for historic adaptation to environmental conditions of the Asian continent, particularly Korea. Therefore, MLVA techniques may be available for molecular monitoring on anthrax-release-related bioterrorism and further study is required for the continuous epidemiological study of variable anthrax collections.

Application of real-time PCR for quantitative detection of Clostridium botulinum type A toxin gene in food.

The TaqMan real‐time PCR method for the quantitative detection of C. botulinum type A was developed based on sequence‐specific hybridization probes. The validity of this assay was verified by using 10 genera of 20 strains, including reference strains of C. botulinum types A, B, C, D, E and F. The detection limit of this assay was evaluated on C. botulinum type A, using a 10‐fold dilution series of DNA and spores. The DNA and spores were detected up to level of 0.1 ng/ml and 102 spores/ml, respectively. Spore spiked food sample preparation prior to the real‐time PCR was performed by two methods, heat treatment and GuSCN. The detection limits after heat treatment showed 102 spores/ml for spiked sausage slurry, and 103 spores/ml for spiked canned corn slurry, while detection limits after GuSCN precipitation showed 102 spores/ml in both sausage and canned corn. Therefore the real‐time PCR assay after GuSCN precipitation is useful for the quantification of C. botulinum type A because it showed identical CT values in both pure spore solutions and food slurries. We suggest that quantitative analysis of C. botulinum type A by TaqMan real‐time PCR can be a rapid and accurate assessment method for botulinal risk in food samples.

The Poly-γ-d-Glutamic Acid Capsule of Bacillus anthracis Enhances Lethal Toxin Activity

ABSTRACT The poly-γ-d-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infectious disease. The PGA capsule disguises B. anthracis from immune surveillance and allows its unimpeded growth in the host. The PGA capsule recently was reported to be associated with lethal toxin (LT) in the blood of experimentally infected animals (M. H. Cho, et al., Infect. Immun. 78:387-392, 2010). The effect of PGA, either alone or in combination with LT, on macrophages, which play an important role in the progression of anthrax disease, has not been thoroughly investigated. In this study, we investigated the effect of PGA on LT cytotoxicity using the mouse macrophage cell line J774A.1. PGA produced a concentration-dependent enhancement of the cytotoxicity of LT on J774A.1 cells through an enhancement in the binding and accumulation of protective antigen to its receptors. The increase of LT activity was confirmed using Western blot analysis, which showed that the combination of PGA and LT produced a greater degree of degradation of mitogen-activated protein kinase kinases and an increased level of the activation of the proform of caspase-1 to its processed form compared to the effects of LT alone. In addition, mice that received a tail vein injection of both PGA and LT had a significantly increased rate of death compared to that of mice injected with LT alone. PGA had no effect when added to cultures or administered to mice in the absence of LT. These results emphasize the importance of PGA in the pathogenesis of anthrax infection.

Regulation of hemagglutinin/protease expression by the VarS/VarA–CsrA/B/C/D system in Vibrio cholerae

In this study, through the analysis of Vibrio cholerae 2740-80 mutant strains produced by the cholera toxin subunit B gene containing Mariner-based transposon, we found that disruption of the varS gene, a member of the recently reported sensory system VarS/VarA-CsrA/B/C/D, resulted in altered expression of hemagglutinin/protease A. To further investigate the connection between VarS and HapA, we generated an additional varS mutant, V. cholerae 2740-80-VS, and examined the effect of this mutation on expression of HapA and of genes in the VarS/VarA-CsrA/B/C/D system. 2740-80-VS showed decreased expression of varS, csrB/C, hapR, and hapA along with increased biofilm production. Interestingly, expression of the alternative sigma factor sigma(s), which is important for adaptation to environmental stress, was also decreased in this mutant. These results indicate that the VarS/VarA-CsrA/B/C/D system is involved in the control of HapA expression and biofilm production in V. cholerae 2740-80 through HapR regulation, and also that VarS/VarA controls expression of sigma(s) for HapA regulation.

Seroepidemiology of spotted fever group and typhus group rickettsioses in humans, South Korea.

The prevalence of spotted fever group (SFG) and typhus group (TG) rickettsioses was investigated in 3,362 sera by immunofluorescence assay. The serum samples were obtained from patients with acute febrile episodes in South Korea from December 1992 to November 1993. The number of polyvalent positive sera against SFG rickettsial agents at the level of 1:40 dilution was 269 (8%) in Rickettsia sibirica, 482 (14.34%) in R. conorii, and 546 (16.24%) in R. akari. Many of the positive sera contained immunoglobulin (Ig) M antibodies rather than IgG antibodies. These results strongly suggest that SFG rickettsioses are prevalent in Korea. For TG rickettsial agents, the number of positive sera was 1,096 (32.60%) in R. typhi and 951 (28.29%) in R. prowazekii. Only a few epidemic typhus positive sera contained IgM antibodies. The result suggests that recent and/or primary infections of epidemic typhus were very rare in Korea during the said period. Among seven patients who had high titers (1:5,120) of IgG antibody to R. prowazekii, six were over 50 years old. The result suggests that Brill‐Zinsser disease was prevalent in Korea.

Complete Genome Sequence of Bacillus anthracis H9401, an Isolate from a Korean Patient with Anthrax

Bacillus anthracis H9401 (NCCP 12889) is an isolate from a Korean patient with gastrointestinal anthrax. The whole genome of H9401 was sequenced. It is a circular chromosome containing 5,480 open reading frames (ORFs) and two plasmids, pXO1 containing 202 ORFs and pXO2 containing 110 ORFs. H9401 shows high pathogenicity and genome sequence similarity to Ames Ancestor.

The Vibrio cholerae VarS/VarA two-component system controls the expression of virulence proteins through ToxT regulation.

Although the conditions for inducing virulence protein expression in vitro are different, both classical and El Tor biotypes of Vibrio cholerae have been reported to regulate the expression of virulence proteins such as cholera toxin (CT) and toxin-coregulated pili (Tcp) through the ToxR/S/T system. The transcription activator ToxR responds to environmental stimuli such as pH and temperature and activates the second transcriptional regulator ToxT, which upregulates expression of virulence proteins. In addition to the ToxR/S/T signalling system, V. cholerae has been proposed to utilize another two-component system VarS/VarA to modulate expression of virulence genes. Previous study has shown that VarA of the VarS/VarA system is involved in the regulation of virulence proteins in the classical V. cholerae O395 strain; however, no further analysis was performed concerning VarS. In this study, we constructed varS mutants derived from the classical O395 and El Tor C6706 strains and demonstrated that VarS is also involved in the expression of the virulence proteins CT and Tcp from the V. cholerae classical and El Tor strains. This expression is through regulation of ToxT expression in response to environmental changes due to different toxin-inducing conditions.

Outbreak of Ciprofloxacin-Resistant Shigella sonnei Associated with Travel to Vietnam, Republic of Korea

We investigated an October 2014 outbreak of illness caused by Shigella sonnei in a daycare center in the Republic of Korea (South Korea). The outbreak strain was resistant to extended-spectrum cephalosporins and fluoroquinolones and was traced to a child who had traveled to Vietnam. Improved hygiene and infection control practices are needed for prevention of shigellosis.

Antimicrobial resistance patterns (1999-2002) and characterization of ciprofloxacin-resistant Neisseria gonorrhoeae in Korea.

Background: Antimicrobial susceptibilities of Neisseria gonorrhoeae were monitored during 4 years. In Korea, ciprofloxacin-resistant N. gonorrhoeae has dramatically increased after recommendation as a therapeutic drug. Goal: The goal of this study was to determine the resistance patterns and characterize Korean ciprofloxacin-resistant N. gonorrhoeae. Study Design: Antimicrobial susceptibilities were performed. PFGE profile and DNA sequencing of gyrA and parC genes were used to characterize the ciprofloxacin-resistant isolates in Korea. Results: Tetracycline, ofloxacin, ciprofloxacin-resistant isolates were increased and among them, the proportion of isolates resistant to ciprofloxacin increased remarkably from 1% in 1999 to 48.8% in 2002. Fifteen different types by PFGE profile were identified. Major alteration type was M12 (67%), which have amino acid substitution in gyrA (S-91→F, D-95→G) and parC (S-87→A). Conclusion: We could conclude that resistance for ciprofloxacin was remarkably increased during 4 years. Ciprofloxacin-resistant N. gonorrhoeae was supposed by the spread of several strains that had a small number of origins.