Won Keun Seong

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.

Allergic rhinitis in laboratory animal workers and its risk factors

BACKGROUND The workers in an animal laboratory are exposed to laboratory animal allergens (LAAs). OBJECTIVES To evaluate the difference of sensitization to LAAs and the symptoms according to the exposure levels and to investigate the risk factors for sensitization to LAAs. METHODS The subjects were divided into 3 groups according to the presence or absence of exposure: 74 subjects were in the direct exposure group, 33 subjects were in the indirect exposure group, and 30 subjects were in the control group. Each group answered the questionnaire and underwent skin prick tests that included 10 common allergens and 10 LAAs. The levels of total IgE and specific IgE to mouse and rat urine allergen were measured by enzyme-linked immunosorbent assay in 2 exposure groups. Allergic symptoms, skin sensitization, and serum IgE level were compared between the study groups. RESULTS Twenty-five (34%) of the 74 subjects in the direct exposure group experienced allergic symptoms since their exposure to laboratory animals. The subjects in the direct and indirect exposure groups had more sensitization to LAAs than did the control subjects. The direct exposure group had more positive results for total IgE than did the indirect exposure group. The subjects in the direct exposure group with atopy had more severe allergic symptoms than the subjects in the indirect exposure group with atopy. Atopy and total IgE level were risk factors for the sensitization to LAAs for the direct exposure group (odds ratios, 7.47 and 7.33, respectively). CONCLUSIONS Indirect exposure may be as risky for sensitization to LAAs as direct exposure. More careful protection is needed for laboratory animal workers with atopy.

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.

Comparative Proteome Analysis of the Outer Membrane Proteins of in Vitro-Induced Multi-Drug Resistant Neisseria gonorrhoeae

Antimicrobial‐resistant gonococcus has been a major problem in sexually transmitted disease control. Outer membrane proteins (OMPs) of Neisseria gonorrhoeae were suggested to have influence on its resistance to antibiotics. So, in this work, we provide a proteomic analysis tool for examining the OMPs of N. gonorrhoeae and also provide a comparative analysis of the OMPs between the susceptible parent strain (92WT) and the resistance‐induced isogenic mutant (92mu13) to determine the OMPs responsible for resistance. The 2‐D gel spots of 92mu13 differed from 92WT particularly in porin, pilus secretion protein (PilQ) and enzymes. PilQ expression in 92mu13 was considerably reduced by abrupt termination at nucleotide 2,112. This made it difficult to form a high molecular mass (HMM) pore at the outer membrane; it is suspected that reduction of PilQ serves a role in antibiotic resistance in N. gonorrhoeae. The amount of porin was not changed but its isoelectric point (pI) shifted to a basic region, which is caused by the alteration of an amino acid of porin and it is suggested to relate to the development of antimicrobial resistance. Differential regulation of the enzymes involved in metabolism was found in 92mu13, believed to represent an adaptation of N. gonorrhoeae to the antibiotic environment.

Enteric Bacteria Isolated from Diarrheal Patients in Korea in 2014

Objectives The aim of this study was to characterize the pathogens responsible for causing diarrhea according to season, region of isolation, patient age, and sex as well as to provide useful data for the prevention of diarrheal disease. Methods Stool specimens from 14,886 patients with diarrhea were collected to identify pathogenic bacteria from January 2014 to December 2014 in Korea. A total of 3,526 pathogenic bacteria were isolated and analyzed according to season, region of isolation, and the age and sex of the patient. Results The breakdown of the isolated pathogenic bacteria were as follows: Salmonella spp. 476 (13.5%), pathogenic Escherichia coli 777 (22.0%), Vibrio parahaemolyticus 26 (0.74%), Shigella spp. 13 (0.37%), Campylobacter spp. 215 (6.10%), Clostridium perfringens 508 (14.4%), Staphylococcus aureus 1,144 (32.4%), Bacillus cereus 356 (10.1%), Listeria monocytogenes 1 (0.03%), and Yersinia enterocolitica 10 (0.3%). The isolation rate trend showed the highest ratio in the summer season from June to September for most of the pathogenic bacteria except the Gram-positive bacteria. The isolation rate of most of the pathogenic bacteria by patient age showed highest ratio in the 0–19 year age range. For isolation rate by region, 56.2% were isolated from cities and 43.8% were isolated from provinces. Conclusion Hygiene education should be addressed for diarrheal disease-susceptible groups, such as those younger than 10 years, aged 10–19 years, and older than 70 years, and monitoring for the pathogens is still required. In addition, an efficient laboratory surveillance system for infection control should be continued.

Plasmid-mediated transfer of CTX-M-55 extended-spectrum beta-lactamase among different strains of Salmonella and Shigella spp. in the Republic of Korea

We screened 10 CTX-M-55-producing Shigella and Salmonella isolates from a national surveillance in Korea. The blaCTX-M-55 was located on the IncI1 (n=5), IncA/C (n=4) and IncZ (n=1) plasmids, downstream of ISEcp1, IS26-ISEcp1 and ISEcp1-IS5 sequences, respectively. These results indicate that CTX-M-55 has disseminated to other bacteria by lateral plasmid transfer.

Serological Correlate of Protection in Guinea Pigs for a Recombinant Protective Antigen Anthrax Vaccine Produced from Bacillus brevis

Objective Recombinant protective antigen (rPA) is the active pharmaceutical ingredient of a second generation anthrax vaccine undergoing clinical trials both in Korea and the USA. By using the rPA produced from Bacillus brevis pNU212 expression system, correlations of serological immune response to anthrax protection efficacy were analyzed in a guinea pig model. Methods Serological responses of rPA anthrax vaccine were investigated in guinea pigs that were given single or two injections (interval of 4 weeks) of various amounts of rPA combined with aluminumhydroxide adjuvant. Guinea pigs were subsequently challenged by the intramuscular injection with 30 half-lethal doses (30LD50) of virulent Bacillus anthracis spores. Serumantibody titerswere determined by anti-PA IgGELISA and the ability of antibodies to neutralize the cytotoxicity of lethal toxin on J774A.1 cell was measured through the toxin neutralizing antibody (TNA) assay. Results To examine correlations between survival rate and antibody titers, correlation between neutralizing antibody titers and the extent of protection was determined. Toxin neutralization titers of at least 1176 were sufficient to confer protection against a dose of 30LD50 of virulent anthrax spores of the H9401 strain. Such consistency in the correlation was not observed from those antibody titers determined by ELISA. Conclusion Neutralizing-antibody titers can be used as a surrogate marker.

A waterborne outbreak of multiple diarrhoeagenic Escherichia coli infections associated with drinking water at a school camp

BACKGROUND In June 2015, a local public health laboratory was notified that students had developed gastroenteritis symptoms after attending a camp. An outbreak investigation was conducted to determine the extent and cause of the outbreak. METHOD A retrospective cohort study was conducted to determine the correlations between the illness and specific exposures at the school camp. All attendees were interviewed with a standard questionnaire that addressed clinical symptoms, food consumption, and environmental exposures. Clinical specimens were cultured using standard microbiological methods for bacterial and viral pathogens. The genetic relationships of all isolates were determined using pulsed-field gel electrophoresis (PFGE). RESULTS A total 188 patients with symptoms of diarrhoea, abdominal pain, and nausea were identified. The completed questionnaires suggested that the consumption of drinking water was likely to be linked to this outbreak. Using microbiological methods, enterohaemorrhagic Escherichia coli, enteropathogenic E. coli, and enteroaggregative E. coli were isolated, and the isolates from both patient stool and environmental water samples displayed indistinguishable XbaI-PFGE patterns. The water system in the camp used groundwater drawn from a private underground reservoir for cooking and drinking. The environmental investigation revealed some problems with the water supply system, such as the use of inappropriate filters in the water purifier and a defect in the pipeline between the reservoir and the chlorination device. CONCLUSIONS This outbreak points to the importance of drinking water quality management in group facilities where underground water is used and emphasizes the need for periodic sanitation and inspection to prevent possible waterborne outbreaks.

Dermabacter jinjuensis sp. nov., a novel species of the genus Dermabacter isolated from a clinical specimen.

A Gram-stain-positive, catalase-positive, facultatively anaerobic, non-motile, coryneform bacterium, designated strain 32T, was isolated from a closed pus sample from a patient having finger necrosis in Korea. Strain 32T was considered as representing a novel species according to its initial identification by matrix-assisted laser desorption/ionization-time-of-flight MS. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 32T belonged to the genus Dermabacter and was closely related to Dermabacter hominis DSM 7083T (=ATCC 49369T) (98.34 % similarity). Optimal growth was observed at 30-40 °C and pH 7. Growth occurred in the presence of 0-6 % (w/v) NaCl. Menaquinones MK-8, MK-7 and MK-9 were the major respiratory quinones. The major polar lipids were phosphatidylethanolamine, phosphatidylcholine, glycolipid and two unknown lipids. The major cellular fatty acids were anteiso-C17 : 0, anteiso-C15 : 0, iso-C16 : 0 and iso-C15 : 0. The DNA G+C content of strain 32T was 62.58 mol%, and the mean level of DNA-DNA relatedness between strain 32T and D. hominis ATCC 49369T was 49±1.6 %. Based on the phenotypic and genotypic characteristics, strain 32T is confirmed to represent a novel species of the genus Dermabacter, for which the name Dermabacter jinjuensis sp. nov. is proposed. The type strain is 32T (=NCCP 16133T=DSM 101003T).

Outbreak of CTX-M-15-Producing Enterotoxigenic Escherichia coli O159:H20 in the Republic of Korea in 2016

ABSTRACT We investigated an outbreak of enterotoxigenic Escherichia coli (ETEC) O159:H20 associated with the consumption of a tossed-noodle dish in a high school in 2016. Thirty-three ETEC strains isolated from clinical and food samples were genetically indistinguishable. The outbreak strains were resistant to third-generation cephalosporins and harbored a blaCTX-M-15 gene on a 97-kb self-transferable IncK plasmid. This is the first outbreak caused by CTX-M-15-producing ETEC strains.