Semi Jeon

Escherichia coli O104:H4 from 2011 European Outbreak and Strain from South Korea

To the Editor: Beginning in early May 2011, an outbreak caused by Shiga toxin–producing Escherichia coli O104:H4 was reported in Germany and other countries in Europe. In this outbreak, the number of hemolytic uremic syndrome (HUS) cases has been unusually high (1). As of June 9, 2011, a total of 722 cases of HUS, 19 deaths, and 2,745 cases of enterohemorrhagic E. coli (EHEC) infection were reported (2). A case of HUS caused by E. coli O104:H4 was first reported in South Korea in 2004 (3). Because infections caused by E. coli O104:H4 have been reported rarely, interest has arisen in the E. coli O104:H4 strain from South Korea. We characterized the E. coli O104:H4 strain isolated in South Korea (EC0417119) in 2004 and compared it with the E. coli O104:H4 strain associated with the current EHEC outbreak in Europe. The serotype EC0417119, isolated from a patient with HUS in 2004, was reconfirmed as E. coli O104:H4. The strain was positive for stx1 and stx2 by PCR (4) but negative for aggR by PCR (5). In the antimicrobial drug susceptibility test using VITEK 2 AST-N169 test kit (bioMerieux, Marcy L’Etoile, France), the strain was resistant to ampicillin, ampicillin/sulbactam, and trimethoprim/sulfamethoxazole but susceptible to ceftriaxone, cefotaxime, nalidixic acid, and tetracycline. We also performed pulsed-field gel electrophoresis (PFGE) for EC0417119, according to the PulseNet standard protocol (6), and compared its PFGE profile with that of the current outbreak strain E. coli O104:H4, which was obtained from the PulseNet Asia Pacific network. PFGE profiles resolved by either XbaI or BlnI did not match each other. The percentage similarity of XbaI- and BlnI-digested PFGE profiles of the 2 isolates was 75% and 66.7%, respectively, as shown in the Figure. Figure Clustering of A) XbaI- and B) BlnI-digested DNA fragments by pulsed-field gel electrophoresis (PFGE) for Escherichia coli O104:H4 2011 outbreak strain in Europe and isolate obtained in South Korea in 2004. Infections with the EHEC O104 strain were reported several times worldwide. In Europe, such occurrence was rare, and before the current outbreak, the EHEC O104:H4 strain was documented only once in South Korea. For this reason, it was logical to examine the possible relatedness of the EC0417119 strain and the strain causing the current outbreak. However, the EC0417119 strain has many different characteristics compared with the current outbreak strain: not possessing enteroaggregative E. coli determinant, not producing extended-spectrum β-lactamases, and not showing indistinguishable PFGE patterns. In conclusion, there is no evidence that the E. coli O104:H4 strain isolated in South Korea in 2004 is related to the strain that has a caused the massive and unprecedented EHEC outbreak in Europe.

Analysis of species and intra-species associations between the Mycobacterium abscessus complex strains using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST).

PFGE and MLST showed that the strains of M. massiliense hsp65 II-1 were clearly separated from the strains of M. massiliense hsp65 I or II-2 as well as the strains of M. abscessus or M. bolletii; thus, M. massiliense hsp6 5II-1 might represent an additional subspecies of M. massiliense.

Multiplex Real-time Polymerase Chain Reaction Assays for Simultaneous Detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus.

Objectives A multiplex real-time polymerase chain reaction (RT-PCR) method was developed for the identification of three Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. Methods Specific primers and probes targeting the hlyA, tlh, and vvhA genes were selected and used for multiplex real-time PCR to confirm the identification of V. cholerae, V. parahaemolyticus, and V. vulnificus, respectively. This method was applied to screen Vibrio species from environmental samples and combining it with a culture-based method, its effectiveness was evaluated in comparison with culture-based methods alone. Results Specific PCR fragments were obtained from isolates belonging to the target species, indicating a high specificity of this multiplex real-time PCR. No cross-reactivity with the assay was observed between the tested bacteria. The sensitivity of the multiplex real-time PCR was found to have a lower limit of 104 colony-forming units/reaction for all three Vibrio species. The combination strategy raised the isolation ratio of all three Vibrio species 1.26- to 2.75-fold. Conclusion This assay provides a rapid, sensitive, and specific technique to detect these three Vibrio species in the environment.

Resistance to Fluoroquinolone by a Combination of Efflux and Target Site Mutations in Enteroaggregative Escherichia coli Isolated in Korea

Objectives Enteroaggregative Escherichia coli (EAEC) was recently reported as a major diarrheagenic pathogen in infant and adult travelers, both in developing and developed countries. EAEC strains are known to be highly resistant to antibiotics including quinolones. Therefore in this study we have determined the various mechanisms of quinolone resistance in EAEC strains isolated in Korea. Methods For 26 EAEC strains highly resistant to fluoroquinolone, minimal inhibitory concentrations for fluoroquinolones were determined, mutations in the quinolone target genes were identified by PCR and sequencing, the presence of transferable quinolone resistance mechanism were identified by PCR, and the contribution of the efflux pump was determined by synergy tests using a proton pump inhibitor. The expression levels of efflux pump-related genes were identified by relative quantification using real-time PCR. Results Apart from two, all tested isolates had common mutations on GyrA (Ser83Leu and Ser87Gly) and ParC (Ser80Gln). Isolates EACR24 and EACR39 had mutations that have not been reported previously: Ala81Pro in ParC and Arg157Gly in GyrA, respectively. Increased susceptibility of all the tested isolates to ciprofloxacin and norfloxacin in the presence of the pump inhibitor implies that efflux pumps contributed to the resistance against fluoroquinolones. Expression of the efflux pump-related genes, tolC, mdfA, and ydhE, were induced in isolates EACR 07, EACR 29, and EACR 33 in the presence of ciprofloxacin. Conclusion These results indicate that quinolone resistance of EAEC strains mainly results from the combination of mutations in the target enzyme and an increased expression of efflux pump-related genes. The mutations Ala81Pro in ParC and Arg157Gly in GyrA have not been reported previously the exact influence of these mutations should be investigated further.

A Contribution of MdfA to Resistance to Fluoroquinolones in Shigella flexneri.

In this study, we measured the drug resistance conferred by mdfA mutations in two Shigella flexneri strains. A mutant in mdfA genes was constructed by polymerase chain reaction–based, one-step inactivation of chromosomal genes. The antimicrobial susceptibility of parent and mutant strains to fluoroquinolones was determined by minimal inhibitory concentration (MICs). The △mdfA mutants were somewhat more susceptible to fluoroquinolones than the parent strains. The low level changes in MICs of the △mdfA mutants suggest that mdfA contributed the fluoroquinolone resistance in S flexneri. This finding found that the increased expression level of an MdfA efflux pump mediated fluoroquinolone resistance, but it is not likely a major effecter of higher resistance levels.

Molecular Typing of Mycobacterium intracellulare Using Pulsed-Field Gel Electrophoresis, Variable-Number Tandem-Repeat Analysis, Mycobacteria Interspersed Repetitive-Unit-Variable-Number Tandem Repeat Typing, and Multilocus Sequence Typing: Molecular Characterization and Comparison of Each Typing Methods

Objectives Mycobacterium intracellulare is the major causative agent of nontuberculous mycobacteria-related pulmonary infections. The strain typing of M. intracellulare is important for the treatment and control of its infections. We compared the discrimination capacity and effective value of four different molecular typing methods. Methods Antibiotic susceptibility testing, hsp65 and rpoB sequencing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), mycobacteria interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR), and VNTR assay targeting 44 M. intracellulare isolates obtained from patients with pulmonary infections were performed. Results All the antibiotic susceptibility patterns had no association with the molecular and sequence types tested in this study; however, the molecular and sequence types were related with each other. PFGE gave best results for discriminatory capacity, followed by VNTR, MLST, and MIRU-VNTR. Conclusion The high discriminatory power of PFGE, VNTR, and MLST is enough for differentiating between reinfection and relapse, as well as for other molecular epidemiological usages. The MLST could be regarded as a representative classification method, because it showed the clearest relation with the sequence types.

Multiplex Real-Time Polymerase Chain Reaction-Based Method for the Rapid Detection of gyrA and parC Mutations in Quinolone-Resistant Escherichia coli and Shigella spp.

Two real-time polymerase chain reaction assays were developed to detect mutations in codons 83 and 87 in gyrA and in codons 80 and 91 in parC, the main sites that causes quinolone resistance in pathogenic Escherichia coli and Shigella spp. isolates. These assays can be employed as a useful method for controlling infections caused by quinolone-resistant E coli and Shigella isolates.

Detection of the Causative Agents of Traveler's Diarrhea Using a Real-Time PCR Screening Method

Background: The incidence of infectious diarrheal disease in Korea has decreased over the past decade, but travelers diarrhea (TD) is increasing in frequency. We therefore investigated the distribution of the causative agents of TD. Methods: A total of 132 rectal swab specimens were acquired from TD patients who entered the country via Gimhae International Airport. The specimens were screened for 12 bacterial pathogens by real-time PCR, and target pathogens were isolated from the PCR positive specimens using conventional microbiological isolation methods. Results: A total of 93 specimens (70.5%) showed positive PCR screening results, and of these specimens, nine species and 50 isolates (37.9%), including Vibrio parahaemolyticus (18 isolates) and ETEC (17 isolates), were isolated. No specimens were PCR positive for Listeria monocytogenes or Campylobacter jejuni, and no pathogenic Bacillus cereus were isolated. Conclusion: Even though viruses and EAEC were not included as target pathogens, the high isolation rate of these pathogens in this study provides indirect evidence that most cases of pathogen-negative TD are caused by undetected bacterial agents. Furthermore, our study results confirm the effectiveness of real-time PCR-based screening methods. This study is the first report in Korea to demonstrate that ETEC and V. parahaemolyticus are the major causative pathogens of TD, and this knowledge can be used to help treat and prevent TD. (Korean J Clin Microbiol 2009;12:186-192)

Genetic Characteristics and Relatedness of Imported Vibrio cholerae O1 Biotype El Tor in Korea

Background: Cholera is a representative water-borne disease that is caused by V. cholera ctx (+). V. cholera El Tor was previously the primary pathogen, but after the seventh pandemic outbreak, it was replaced by a V. cholera El Tor variant with a classical phenotype and genotype. In this study, we investigated the genotypic and phenotypic characteristics of imported V. cholerae El Tor in Korea. Methods: Forty-nine V. cholerae O1 El Tor strains isolated from 2004 to 2011 were used in this study. Polymerase chain reaction amplification of the ctxB and rstR genes was used for biotype determination. An antimicrobial susceptibility test was performed for phenotypic analysis, and pulse field gel electrophoresis (PFGE) was used for analysis of genetic relatedness. Results: Classical ctxB genes were found in all of the isolates, while classical, El Tor, and combined rstR genes were found. Twenty strains showed antimicrobial resistance against streptomycin, sulfamethoxazole/ trimethoprim, nalidixic acid, and ciprofloxacin. Based on PFGE, all isolates were grouped as cluster B. The country of origin and resistance pattern were highly related, although the time of influx and serogroup were not. Conclusion: Isolates of V. cholera El Tor imported since 2004 were hybrids of V. cholera El Tor, which has the classical ctxB gene and is considered to be a CTX prophage. The SXT element plays an important role in antimicrobial resistance. PFGE patterns, which can be used for analysis of imported V. cholera, revealed the relatedness of the resistant isolates. (Ann Clin Microbiol 2013;16:25-32)

A molecular epidemiological analysis of tuberculosis trends in South Korea

Molecular epidemiological data are needed to assess tuberculosis (TB)-management policy outcomes in South Korea. IS6110 restriction fragment-length polymorphism (IS6110-RFLP) and mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) analyses are major molecular epidemiological tools for investigating the transmission or reactivation of active TB. Here, we determined trends in the clustering rate (i.e., the prevalence of Mycobacterium tuberculosis isolates with identical genotype patterns) of active TB and related differences between the 1990s and 2000s in Korea. M. tuberculosis isolates (1,007) of nationwide origins were analyzed by IS6110-RFLP and 24-locus standardized MIRU-VNTR genotyping. The clustering rate was measured by IS6110-RFLP, 24-locus MIRU-VNTR, and both analytical methods in combination. IS6110-RFLP, 24-locus MIRU-VNTR typing, and the combined method revealed 882, 754, and 983 distinct profiles; 809, 651, and 961 unique isolates; and 198, 356, and 46 clustered isolates grouped into 73, 103, and 22 clusters, respectively. In addition, we confirmed that the clustering rates in the 2000s decreased by 11.2%, 2.1%, and 3.1% relative to that in the 1990s using the three methods, respectively. Furthermore, in multivariate analysis, the younger-age group (<30) clustered more frequently than the older-age group (>50), based on all the three methods. Our study is the first report to provide nationwide molecular epidemiological information on TB in Korea.