Seong-Han Kim

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.

High prevalence of multiresistance in levofloxacin-nonsusceptible Streptococcus pneumoniae isolates in Korea

Korea exhibits the highest rates of multidrug resistance among Streptococcus pneumoniae. The increasing use of levofloxacin has raised concern about the dissemination of levofloxacin resistance in dominant multidrug-resistant (MDR) clones of our pneumococcal population. A total of 50 levofloxacin-nonsusceptible S. pneumoniae (MIC, ≥4 μg/mL) collected from a multihospital network from 1996 to 2006 were analyzed for serotype, antibiotic resistance profile, quinolone resistance-determining region mutation, and multilocus sequence type. Most levofloxacin-nonsusceptible S. pneumoniae (94.0%) exhibited an MDR phenotype. This phenotype was closely associated with a limited number of epidemic MDR clones that are well-known key agents of the global spread of antimicrobial resistance in S. pneumoniae. However, the clonal dissemination of levofloxacin-nonsusceptible S. pneumoniae was rare. Levofloxacin-nonsusceptible clones with nonvaccine serotypes increased during the post-vaccine era in this study. This result suggests that Korean clinicians must be aware of the levofloxacin resistance trend and need to be more prudent for the first choice of fluoroquinolone for empiric treatment of respiratory tract infections in clinical setting. Moreover, the emergence of new clones and their variations may be more frequently associated with resistance under this selective pressure, such as the introduction of a 7-valent pneumococcal conjugate vaccine into our community.

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.

Prevalence of Antibiotic Resistance in Escherichia coli Fecal Isolates From Healthy Persons and Patients With Diarrhea

Objectives This study aimed to investigate the prevalence of antibiotic resistance in fecal Escherichia coli isolates from healthy persons and patients with diarrhea. Methods E. coli isolates (n = 428) were obtained from fecal samples of apparently healthy volunteers and hospitalized patients with diarrhea. Susceptibility patterns of isolates to 16 antimicrobial agents were determined by agar disc diffusion. Results Most E. coli isolates exhibited less than 10% resistance against imipenem, cefotetan, aztreonam, cefepime, cefoxitin, amikacin and netilamicin, although greater than 65% were resistant to ampicillin and tetracycline. No significant difference in resistance rates for all tested antibiotics was found between isolates from the healthy-and diarrheal-patient groups, including for multi-drug resistance (p = 0.22). The highest number of resistant antibiotics was 12 antibiotics. No significant differences in antibiotic resistance were found among the sex and age strata for isolates from healthy individuals. However, antibiotic resistance rates to cefoxitin, cefotaxime, amikacin, and netilamicin were significantly higher in the isolates of men than those of women (p < 0.05) in isolates from patients with diarrhea. Furthermore, isolates from patients with diarrhea older than 40-years of age showed higher resistance to cefepime and aztreonam (p < 0.05). Conclusion High resistance to the antibiotics most frequently prescribed for diarrhea was found in isolates from patients with diarrhea and apparently healthy individuals without any significant difference.

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.

Evaluation and Comparison of Molecular and Conventional Diagnostic Tests for Detecting Tuberculosis in Korea, 2013

Objectives A fast and accurate diagnosis is necessary to control and eliminate tuberculosis (TB). In Korea, TB continues to be a serious public health problem. In this study, diagnostic tests on clinical samples from patients suspected to have TB were performed and the sensitivity and specificity of the various techniques were compared. The main objective of the study was to compare various diagnostic tests and evaluate their sensitivity and specificity for detecting tuberculosis. Methods From January 2013 to December 2013, 170,240 clinical samples from patients suspected to have TB were tested with smear microscopy, acid-fast bacilli culture, and real-time polymerase chain reaction (PCR). The test results were compared and data were analyzed. Results A total of 8216 cultures tested positive for TB (positive detection rate, 4.8%). The contamination rate in the culture was 0.6% and the isolation rate of nontuberculous mycobacteria was 1.0%. The sensitivity and specificity of smear microscopy were 56.8% and 99.6%, respectively. The concordance rate between the solid and liquid cultures was 92.8%. Mycobacterium isolates were not detected in 0.4% of the cases in the liquid culture, whereas no Mycobacterium isolates were detected in 6.8% of the cases in the solid culture. The sensitivity and specificity of real-time PCR for the solid culture were 97.2% and 72.4%, respectively, whereas the corresponding data for the liquid culture were 93.5% and 97.2%. Conclusion The study results can be used to improve existing TB diagnosis procedure as well as for comparing the effectiveness of the assay tests used for detecting Mycobacterium tuberculosis isolates.

Prevalent Multidrug-resistant Nonvaccine Serotypes in Pneumococcal Carriage of Healthy Korean Children Associated with the Low Coverage of the Seven-valent Pneumococcal Conjugate Vaccine

Objectives Our previous longitudinal multicenter-based carriage study showed that the average carriage rate of Streptococcus pneumoniae was 16.8% in 582 healthy children attending kindergarten or elementary school in Seoul, Korea. We assessed serotype-specific prevalence and antimicrobial resistance among colonizing pneumococcal isolates from young children in the era of low use of the seven-valent pneumococcal conjugate vaccine (PCV7). Methods Serotypes were determined by an agglutination test with specific antisera or by a multiplex polymerase chain reaction (PCR) assay. An antimicrobial susceptibility test was performed with broth microdilution in Korean 96-well panels from Dade-MicroScan (Sacramento, CA, USA). Results Pneumococcal colonization patterns were dynamic and longterm persistent carriage was rare, which indicated a sequential turnover of pneumococcal strains. Of the 369 pneumococci (except for 23 killed isolates), 129 (34.9%) isolates were PCV7 vaccine serotypes (VTs); 213 (57.8%) isolates were nonvaccine serotypes (NVTs); and the remaining 27 (7.2%) isolates were nontypable (NT). The highest rates of multidrug resistance (MDR) were observed in VTs (86.0%; 111/129 isolates) and NVTs (70.0%; 149/213 isolates). Conclusion This study overall showed the frequent carriage of VTs and NVTs with MDR in healthy children attending kindergarten or elementary school. Efforts should be directed toward reducing the extensive prescription of antibiotics and using new broader vaccines to reduce the expansion of MDR strains of NVTs in our community.