Molecular Epidemiologic Analysis of Community-Onset Extended Spectrum Beta-Lactamase (ESBL) Producing Escherichia coli Using Infrequent-Restriction-Site Polymerase Chain Reaction (IRS-PCR) with Comparison by Pulsed-Field Gel Electrophoresis (PFGE) / 감염과화학요법

BACKGROUND: We evaluated the ability of infrequent restriction site-polymerase chain reaction (IRS-PCR) to perform molecular epidemiologic analysis of Community-Onset Extended Spectrum Beta-Lactamase (ESBL) producing Escherichia coli, and also assessed the use of PFGE as an alternative method. MATERIALS AND METHODS: IRS-PCR assay was performed using combinations of adaptors for XbaI and HhaI restriction sites on clinical isolates of E. coli (n=51). We compared the discriminatory power, quality and efficiency of IRS-PCR to PFGE. RESULTS: In E. coli, PFGE discriminated 39 (76.4%) and IRS-PCR discerned 41 (80.3%) of the total 51 strains. It took much less time to complete IRS-PCR (one day) than PFGE (at least 4 days). CONCLUSIONS: IRS-PCR is a more sensitive and rapid alternative to PFGE for molecular epidemiologic analysis of E. coli.

Evaluation of Infrequent-Restriction-Site PCR for Epidemiological Typing of Candida tropicalis / 대한임상미생물학회지

BACKGROUND: We evaluated the usefulness of a newly developed molecular typing method of infrequent restriction site polymerase chain reaction (IRS-PCR) as an epidemiological DNA fingerprinting tool for Candida tropicalis. METHODS: Thirty-two strains of C. tropicalis comprising eight sporadic strains and 24 clonal strains belonging to six clones, of which clonal type were previously confirmed by pulsed-field gel electrophoresis (PFGE), were tested by IRS-PCR to evaluate the usefulness of this technique. Twenty strains of Candida species, including C. glabrata, C. krusei, C. albicans, and C. parapsilosis, were also tested to assess the ability of IRS-PCR to discriminate among species of Candida. RESULTS: Using the IRS-PCR assay, sporadic strains of C. tropicalis could not be differentiated from clonal strains. Most strains belonging to the same clones were classified as different IRS-PCR types or clusters, and some different sporadic strains were classified as the same IRS-PCR types. When pattern variation was examined for different strains of C. tropicalis using IRS-PCR, pairwise similarity measured by the Dice coefficient was 75.4~100%. In contrast, pairwise similarity among isolates of five different species of Candida was 25~69.2%. Therefore, five different species of Candida were easily differentiated. CONCLUSION: The IRS-PCR typing assay appears to be an inadequate tool for the epidemiological typing of C. tropicalis, because the typing result of IRSPCR is not comparable to that of PFGE. To our knowledge, this is the first evaluation study for IRSPCR as an epidemiological typing tool for C. tropicalis.

Molecular Epidemiologic Analysis of Legionella pneumophila by Infrequent-Restriction-Site Polymerase Chain Reaction / 대한임상미생물학회지

BACKGROUND: The frequent outbreak of legionellosis makes it critical to identify infection sources for the prevention and blockade of transmission of the disease. METHODS: Thirty-one strains of Legionella pneumophila isolated from the cooling towers of big buildings in Busan and Gyungsangnamdo Province areas, 12 strains of L. pneumophila from patients in Japan, and one type strain (L. pneumophila ATCC 33152) were used for molecular strain typing by using an infrequent-restriction-site polymerase chain reaction (IRS-PCR). RESULTS: Each strain revealed to have 7-16 bands of 200-1000 bp size. All 44 strains showed band patterns different from each other, except two strains sharing 90% homology. CONCLUSION: The molecular typing of Legionellaby IRS-PCR is an excellent and rapid method for discriminating strains; therefore, it should be useful in demonstrating the identity of possible outbreak strains.

Genomic analysis of Vibrio vulnificus by Infrequent Restriction Site-Polymerase Chain Reaction / 대한임상미생물학회지

BACKGROUND: Vibrio vulnificus sepsis is one of the notifiable disease(Class 3) in Korea. It is usually acquired through the consumption of raw or undercooked seafood in summer. We studied the clinical findings of V. vulnificus septicemia and the genomic patterns of V. vulnificus isolates. METHODS: Seven patients with V. vulnificus septicemia were admitted to Hanyang University hospital from 1998 to 2002. We analysed the clinical findings and the genomic patterns by infrequent restriction site-polymerase chain reaction(IRS-PCR). RESULTS: All patients were over forty years old, and five were male. The patients had underlying diseases;five with liver cirrhosis, two with DM, and four patients with heavy alcoholism. Five of seven patients had history of ingesting raw fish and four had tissue necrosis with bullae or vesicles in their extremities. Four patients who died showed disseminated intravascular coagulation symptoms. We applied IRS-PCR to 6 isolates from blood and 2 isolates from wound. The six isolates from blood showed various genomic patterns that were all different from one another, while the two isolates from wound showed IRS-PCR patterns that were identical to the blood isolates of the same patients. CONCLUSIONS: The genomic patterns of IRS-PCR are quite different in 6 cases of V. vulnificus isolates in Korea.

Genotyping of Vibrio parahaemolyticus by Infrequent Restriction Site Polymerase Chain Reaction / 대한임상미생물학회지

BACKGROUND: Infrequent restriction site PCR (IRS-PCR) is a recently described DNA fingerprinting technique based on selective amplification of restriction endonuclease-cleaved fragments. We applied of IRS-PCR to clinical isolates of Vibrio parahaemolyticus associated with diarrhea. METHODS: IRS-PCR assay was performed with adaptors for XbaI and HhaI restriction sites. A total of 35 strains of V. parahaemolyticus which were isolated from clinical specimens of patients with diarrhea were analyzed. The isolates were collected from different geographic areas of Seoul (n=12), Incheon (n=21) and Gwangju (n=2) during 1998-2000 in Korea. RESULTS: In IRS-PCR, amplifed DNA fragments between 50 and 400 bp were found to be the most reproducible in this study. When V. parahaemolyticus isolates were amplified with AH1 and PX-G as primers, 35 isolates could be grouped into five IRS-PCR patterns: A (n=16), B (n=4), C (n=6), D (n=5) and E (n=4). The patterns were subdivided into 15 subtypes: A1, A2, B1, B2, B3, B4, C1, C2, C3, D1, D2, D3, E1, E2 and E3. The IRS-PCR patterns of V. parahaemolyticus did not show any relationship with serotype or geographic origin, but the isolates from same outbreak produced a same pattern(A1). CONCLUSION: The results provide evidence of the discriminatory power of the IRS-PCR method as it applies to V. parahaemolyticus.

Comparison of Pulsed-field Gel Electrophoresis, Amplified Fragment Length Polymorphism, and Infrequent Restriction Site-Polymerase Chain Reaction for Molecular Typing of Escherichia coli and Staphylococcus aureus Strains / 감염

BACKGROUND: Pulsed-field gel electrophoresis (PFGE) has been regarded a standard method for genotyping in epidemiologic studies. However, it is tedious and time-consuming to perform. Two alternative genotyping methods have recently been developed using the polymerase chain reaction (PCR):amplified fragment length polymorphism (AFLP) and infrequent restriction site-polymerase chain reaction (IRS-PCR). These methods have not yet been applied yet to common pathogens such as Staphylococcus aureus. The purpose of this study was to determine the applicability of AFLP and IRS-PCR for the genotyping of E. coli and S. aureus isolates. METHODS: We performed PFGE, AFLP, and IRS-PCR on clinical isolates of E. coli (n=27) and S. aureus (n=30). We assessed each method in terms of discriminatory power, quality, and efficiency. RESULTS: In E. coli, the discriminatory powers of IRS-PCR and AFLP were comparable to that of PFGE. PFGE discerned 24 (88.8%) out of 27 strains, IRS-PCR discerned 22 (81.5%) out of 27, and AFLP discerned 25 (92.6%) out of 27. In the case of S. aureus, PFGE discerned 27 (90%) out of 30 strains, while both IRA-PCR and AFLP discerned 12 (40%) out of 30. The test-ing took four days to complete with PFGE, two days with AFLP, and was completed within one day with IRS-PCR. IRS-PCR showed better resolution than both PFGE and AFLP. CONCLUSION: In cases of E. coli, AFLP and IRS-PCR could be good alternatives for epidemiologic typing, as they offer better efficiency and comparable discriminatory power to PFGE. On the other hand, IRS-PCR and AFLP do not seem to be suitable for the strain-to-strain differentiation of S. aureus.