Surveillance of Bacillus cereus Isolates in Korea from 2012 to 2014

OBJECTIVES: To investigate the prevalence and toxin production characteristics of non-emetic and emetic Bacillus cereus strains isolated via the laboratory surveillance system in Korea. METHODS: A total of 667 B. cereus strains were collected by the Korea National Research Institute of Health laboratory surveillance system from 2012 to 2014. The collected strains were analyzed by geographical region, season, patient age, and patient sex. Additionally, the prevalence rates of enterotoxin and emetic toxin genes were evaluated. RESULTS: The isolation rate of B. cereus strains increased during the summer, but the isolation rate was evenly distributed among patient age groups. Emetic toxin was produced by 20.2% of the isolated strains. The prevalence rates of five enterotoxin genes (entFM, nheA, cytK2, hblC, and bceT) were 85.0, 78.6, 44.5, 36.6, and 29.7%, respectively, among non-emetic strains and 77.8, 59.3, 17.8, 11.9 and 12.6%, respectively, among emetic strains. Thus, the prevalence rates of all five enterotoxin genes were lower in emetic B. cereus. CONCLUSION: The prevalence of enterotoxin genes differed between non-emetic and emetic B. cereus strains. Among emetic B. cereus strains, the prevalence rates of two enterotoxin genes (cytK2 and hblC) were lower than those among the non-emetic strains. In both the emetic and non-emetic strains isolated in Korea, nheA and entFM were the most prevalent enterotoxin genes.

Genome Sequencing Analysis of Atypical Shigella flexneri Isolated in Korea

OBJECTIVES: An atypical Shigella flexneri strain with a plural agglutination pattern [i.e., reacting not only with serum samples containing type antigen II but also with serum samples containing group antigens (3)4 and 7(8)] was selected for genome sequencing, with the aim of obtaining additional comparative information about such strains. METHODS: The genomic DNA of atypical S. flexneri strain NCCP 15744 was sequenced using an Ion Torrent PGM sequencing machine (Life Technologies, USA). The raw sequence data were preprocessed and reference-assembled in the CLC Assembly Cell software (version 4.0.6; CLC bio, USA). RESULTS: Ion Torrent sequencing produced 1,450,025 single reads with an average length of 144 bp, totaling ~209 Mbp. The NCCP 15744 genome is composed of one chromosome and four plasmids and contains a gtrX gene. Among the published genome sequences of S. flexneri strains, including 2457T, Sf301, and 2002017, strain NCCP 15744 showed high similarity with strain 2002017. The differences between NCCP 15744 and 2002017 are as follows: i) NCCP 15744 carries four plasmids whereas 2002017 carries five; ii) 19 genes (including CI, CII, and cro) were lost in the SHI-O genomic island of NCCP 15744 and six genes were gained as compared with strain 2002017. CONCLUSION: Strain NCCP 15744 is genetically similar to 2002017, but these two strains have different multilocus sequence types and serotypes. The exact reason is unclear, but the 19 lost genes may be responsible for the atypical seroconversion of strain NCCP 15744.

First Report of Salmonella Serotype Tilene Infection in Korea / 대한임상미생물학회지

Salmonellosis is a common food- and water-borne disease and is also a major zoonosis. Currently, the isolation of rare Salmonella serotypes is increasing every year in Korea. Among them, the Salmonella serotype Tilene was first isolated from two people who visited a hospital located in Andong-si in 2013. Clinical symptoms were weak or non-existent. There was no clear epidemiological connection between the two cases. However, it was assumed that both were independently exposed to a single infectious agent. Perhaps due to their geographical proximity, molecular epidemiological analysis showed the same result between the isolated strains. This serotype has increasingly reported an association with hedgehogs. Recently, the importation of exotic animals, including hedgehogs, as pets has been gradually increasing. Thus, it is recommended that high-risk groups avoid contact with exotic pets.

Modified Method of Multilocus Sequence Typing (MLST) for Serotyping in Salmonella Species

Multilocus sequence typing (MLST) of Salmonella is useful method for replacing serotyping using antisera but is limited by difficulties associated with in polymerase chain reaction (PCR). We optimized the PCR reaction, especially annealing temperature and extension time (94degrees C for 2 min; 40 cycles at 94degrees C for 30 sec, 56.8degrees C for 1 min, 72degrees C for 2 min; and 72degrees C for 10 min). The degradation of PCR product by thermostable nucleases was inhibited by using template DNAs treated proteinase K or purified by a commercialized preparation kit. The resulting modified MLST was used as accurate and fast typing method.

The Prevalence and Characteristics of Bacteria Causing Acute Diarrhea in Korea, 2012 / 대한임상미생물학회지

BACKGROUND: Through change in the climate and living environment, bacterial pathogens that cause diarrhea also change. This study sought to determine the characteristics of pathogens according to species, isolated region, and patient age/sex using National Surveillance Data for diarrhea, and to provide basic data for the prevention of diarrheal disease. METHODS: From January to December 2012, stool specimens were collected from 21,180 diarrheal patients in Korea to identify the pathogenic bacteria involved. Pathogenic bacteria were analyzed according to isolated region and patient age/sex. Identification and analysis of the pathogens were conducted based on the Guidelines of the National Institute of Health Diagnostic Laboratory: Disease-specific protocol (2005). RESULTS: Among the 21,180 stool specimens, pathogenic bacteria known to cause diarrhea were isolated from 2,444 stool specimens (11.5%). The isolation rate was highest in the summer (from June to September) for most pathogenic bacteria, except Escherichia coli, Staphylococcus aureus, and Clostridium perfringens. The isolation rate of pathogenic bacteria based on patient age was highest in children under the age of 10. CONCLUSION: Hygiene education should be addressed in diarrheal disease-susceptible groups, such as children under 10, people in their 50s, and those greater than 70 years old, and ongoing monitoring for pathogens is needed. In addition, an efficient information system and surveillance program should be continued for infection prevention.

Molecular and Epidemiological Characterization of Carbapenem-Resistant Acinetobacter baumannii in Non-Tertiary Korean Hospitals

PURPOSE: The increasing prevalence and global spread of carbapenem-resistant Acinetobacter baumannii (A. baumannii) has become a serious problem. The aim of this study was to investigate molecular and epidemiological characteristics of carbapenem-resistant A. baumannii isolates collected from Korean non-tertiary hospitals. MATERIALS AND METHODS: Thirty six non-duplicated carbapenem-resistant A. baumannii isolates were collected from 17 non-tertiary hospitals in Korea between 2004 and 2006. Isolates were typed by multilocus sequence typing and repetitive-sequence-based PCR (rep-PCR). Detection of genes encoding OXA carbapenemase and their relationship with ISAba1 was performed by PCR. RESULTS: Two clones were prevalent among 36 isolates: ST69 (17 isolates, 47.2%) and ST92 (19 isolates, 52.8%). Rep-PCR patterns were diverse and revealed that all isolates were clustered into eight band patterns. The ISAba1-activated blaOXA-23-like and ISAba1-activated blaOXA-51-like genes were prevalent among the carbapenem-resistant A. baumannii isolates. CONCLUSION: The class D beta-lactamase genes of A. baumannii were distributed nationwide in non-tertiary Korean hospitals.

Usefulness of the recombinant liver stage antigen-3 for an early serodiagnosis of Plasmodium falciparum infection

In order to develop tools for an early serodiagnosis of Plasmodium falciparum infection, we evaluated the usefulness of P. falciparum liver stage antigen-3 (LSA-3) as a serodiagnostic antigen. A portion of LSA-3 gene was cloned, and its recombinant protein (rLSA-3) was expressed in Escherichia coli and purified by column chromatography. The purified rLSA-3 and 120 test blood/serum samples collected from inhabitants in malaria-endemic areas of Mandalay, Myanmar were used for this study. In microscopic examinations of blood samples, P. falciparum positive rate was 39.1% (47/120) in thin smear trials, and 33.3% (40/120) in thick smear trials. Although the positive rate associated with the rLSA-3 (30.8%) was lower than that of the blood stage antigens (70.8%), rLSA-3 based enzyme-linked immunosorbent assay could detect 12 seropositive cases (10.0%), in which blood stage antigens were not detected. These results indicate that the LSA-3 is a useful antigen for an early serodiagnosis of P. falciparum infection.

The First Outbreak of Botulism in Korea / 대한임상미생물학회지

Botulism is a rare neuroparalytic disease caused by neurotoxins of Clostridium species. A ten-year-old girl and her mother were admitted to a hospital with symptoms of progressive dizziness, blurred vision, slurred speech, constipation and difficulty in swallowing. These characteristic manifestations and clinical course prompted an examination of the possibility of botulism. Mouse bioassay performed with mother's stool demonstrated type A botulinum toxin and culture of the mother's stool was positive for Clostridium botulinum type A. This is the first case of botulism in Korea.

Molecular Analysis of Clostridium difficile Isolates by Arbitrarily Primed-Polymerase Chain Reaction and Polymerase Chain Reaction-Ribotyping / 감염

BACKGROUND: Clostridium difficile is known as the major cause of nosocomially acquired diarrhea. Various phenotypic and genotypic methods have been used to subtype C. difficile strains. The purpose of the present study is to evaluate several typing methods which can be used as tools for subtyping C. difficile isolates for epidemiological studies. METHODS: In two Korean tertiary care hospitals, a total of 81 C. difficile isolates were collected from symptomatic, hospitalized patients in 1998. All isolates were examined for the release of toxin A and toxin B by PCR assay and cell culture assay. Also arbitrarily primed-PCR and PCR-ribotyping profiles were determined for the typing of C. difficile strains on a genetic level. RESULTS: The toxin B gene was detected in 65.4% (54/81) of isolates by both PCR assay and cell cultureassay. Nine types were identified with T-7 primer, and 13 types were identified with PG-05 primer in AP- PCR. Sixteen types were identified in PCR-ribotyping. When two typing methods were compared, reproducibility by PCR-ribotyping was 100%, while it was only 83% and 33% AP-PCR with primer T-7, and PG-05, respectively. The discrimination index was 0.88 for PCR-ribotyping, 0.82 for AP-PCR with primer T-7 and 0.81 with primer PG-05. CONCLUSION: These data suggest that PCR-ribotyping provides a reproducible, discriminatory, and simple alternative to conventional molecular approaches for typing strains of C. difficile.

Characterization of a Potential Pathogenicity Island of Enterotoxigenic Bacteroides fragilis (ETBF) Strains

Enterotoxigenic B. pagilis (ETBF) strains which produce a 20 kDa zinc metalloprotease toxin (BFI) have been associated with diarrheal disease of animals and young children. Using B. pngilis toxin gene (bfi) from strain 86-5443-2-2 (piglet isolate) as a probe, the gene was identified in 74/77 human and animal ETBF strains but only 2/97 non-toxigenic B. fragilis (NTBF) strains. The region flanking bp was mapped with several restriction enzymes and 8 resriction fragments aacent to bft were used to probe colony blots of 77 KTBF and 97 NTBF strains. All 74 bft-positive ETBF strains hybridized to the 8 probes spanning a ca. 18 kb chromosomal region; however, this 18 kb region was absent in the 3 ETBF strains lacking p, and 47 of the 97 (48%) NTBF strains lacked the entire 18 kb region. Of note, the 2 NTBF strains containing btf did not have a ca. 12 kb region upstream of btfp. A ca. 9 kb fragment flanking the btf gene has been sequenced. Analysis of this data revealed several open reading frames (ORF) of which 3 are of particular interest (ORFs 1, 2 and 3). ORF1 and ORF3 encode proteins with significant homology to mobilization proteins, and ORF2 encodes a protein with significant homology to metalloprotease proteins, but only 50% similarity and 30% identity to BFf. These results suggest: 1) the btf genes are flanked by at least 18 kb of DNA largely unique to ETBF strains indicating a putative pathogenic island, 2) another metalloprotease protein present in ETBF strains may contribute to the pathogenicity and variable virulence of these diarrheagenic strains and 3) the pathogenic island may be mobiTized among different Bacteroides strains, and possibly among different species of intestinal bacteria.

The Detection of Enterotoxin Gene from Bacteroides fragilis Isolates in Korea by Polymerase Chain Reaction

Bacteroides fragilis is a Gram negative nonsporulating anaerobic rod bacterium that makes up about 1 to 2% of the norrnal human colonic microflora. In 1984, Myer et al. reported that some strains of B. fragilis produce enterotoxin and cause diarrheal disease in cattle and human. Since then it has been termed enterotoxigenic B. fragilis (ETBF). In this study, we tried to detect enterotoxin gene from 37 B. fragilis strains, isolated in Korean patients, to confirm the existence of ETBF and usefulness of PCR as a rapid diagnosis method. By this method, we identified 9 ETBF strains and confirmed their pathogenesis by cytotoxicity test. No significant cross- reactivity with other anaerobes or aerobes was observed. Thus, the PCR method may be considered useful for the sensitive and rapid detection of anaerobic infections. And the entire amplified PCR mixture was ligated into a pT7Blue T-vector and transformed into E. coli. When the nucleotide sequences of cloned PCR products were compared with reported enterotoxin gene, pBF529 inserted DNA sequence was nearly in good agreement with it but pBF570 inserted DNA sequence showed some difference at nucleotide 270-300. A search for nucleotide sequence homologies revealed that pBF529 exhibited 99%, but pBF570 indicated only 90% identity with reported enterotoxin gene. According to these results, it was suggested that ETBF toxin can be differentiated into at least 2 subtypes.