Earticle

본 연구에서는 한국의 닭에서 분리된 E. coli 균주들로부터 퀴놀론계 항생제 내성을 나타내는 균주를 분리·동정하고 그 내성 기전과 유병률에 관하여 조사하였다. 또한 multilocus sequence typing (MLST)을 이용하여 E. coli 균주들의 분자생물학적 성상을 분석하였다. 항생제 감수성 테스트에서 63.5% (54/85) 의 E. coli 균주들에서 퀴놀론계 항생제 내성률을 보였다. 또한 퀴놀론계 항생제 내성을 보이는 54개 모두에서 gyrA 유전자의 sense mutations과 parC 유전자의 57th, 80th, or 84th residues에서 점돌연변이를 관찰할 수 있었다. MLST를 통한 분석에서 E. coli ST는 parE 유전자의 염기치환과 깊은 상관관계를 보이는 것으로 관찰되었다. 이 결과들을 바탕으로 우리가 먹는 가축 및 가금류에 대한 무분별한 항생제 사용은 항생제 내성균의 증가와 유전변이를 초래함을 알 수 있었다. 따라서 식용 동물에 대한 지속적인 감시와 모니터링을 통하여 항생제 내성균의 확산방지를 통제하는 것이 필요할 것으로 사료된다.

An aim of current study was to investigate the prevalence and the mechanism of quinolone-resistance in E. coli isolates obtained from chicken cecum in Korea. In addition, multilocus sequence typing (MLST) was also performed for the molecular characterization of E. coli isolates. In an antimicrobial susceptibility test by the disk diffusion method, the 63.5% (54/85) of E. coli isolates showed the resistance to quinolone group of antimicrobial agents. All of the 54 E. coli isolates showing resistant to quinolone group had sense mutations in gyrA gene and point mutations at the 57th, 80th, or 84th residues in parC gene were detected in 90.7% of the isolates. Interestingly, E. coli ST was closely related to amino acid substitutions in parE gene. Our results indicated that the long-term use of antimicrobial agents in food-producing animals was strongly associated with a prevalence of antimicrobial resistance in commensal Enterobacteriaceae, suggesting the need for continuous surveillance and monitoring of antimicrobial resistant determinants in bacterial isolates from food animals.

We compared rapid fingerprinting using repetitive sequencebased PCR (rep-PCR) for subtyping Campylobacter jejuni isolates to the widely used multilocus sequence typing (MLST). Representative C. jejuni isolates (n = 16) from broilers were analyzed using MLST and rep-PCR. Both techniques demonstrated an equal discriminatory power of 0.8917, and 9 subgroups were identified. Clonal identification of all 16 isolates was identical for both techniques. The rep-PCR as described in this study may be used as a rapid and cost-effective alternative for subtyping of C. jejuni isolates, or as an effective screening tool in large epidemiological studies.

Propionibacterium acnes, a gram-positive, anaerobic, and aerotolerant bacterium that is found frequently in the skin as part of the human microbiome causes inflammatory acne, shoulder infection, and the contamination of medical devices. The study goals were the antibiotic resistant and molecular epidemiological characterization of the P. acnes isolates in Korea. A total of 22 P. acnes isolates originated from diverse patients were obtained from three National Culture Collections for Pathogens in South Korea. The hemolytic properties and minimum inhibitory concentrations (MIC) of five antibiotics (tetracycline, doxycycline, clindamycin, erythromycin, and minocycline) were determined. Only one isolate showed high MIC values and resistance to all five antibiotics. Genotypic characterization was achieved by multilocus sequence typing (MLST) for eight loci (aroE, guaA, tly, camp2, atpD, gmk, lepA, and sodA) and repetitive-sequence-based PCR (rep-PCR) analysis using the DiversiLab kit. MLST revealed four phylogroups that were type $IA_1$ (27.3%), type $IA_2$ (18.2%), type IB (13.6%), and type II (40.9%). Rep-PCR results demonstrated three clusters that were cluster I (39.1%), cluster II (45.5%), and cluster III (13.6%). The isolates of cluster I were part of phylogroup type IA (both $IA_1$ and $IA_2$), and the isolates of cluster II belonged to phylogroup type II. All isolates of phylogroup type IB were hemolytic and belonged to cluster III. The results of rep-PCR clustering analysis showed a good correlation with those of MLST phylogroups, suggesting that rep-PCR could be an alternative method to track P. acnes subtype lineages.

Background: Multilocus sequence typing (MLST) was designed to overcome the low discriminatory power and poor reproducibility of previous molecular typing schemes, and it is useful for inter-laboratory, inter-regional, and inter-national comparison of pathogenic clones. MLST includes labor-intensive sequencing processes and meticulous allelic/sequence type (ST) determination processes, often prone to error. We developed a free automated MLST determination program (MLST typer) based on the Visual Basic for Applications macro, which runs on Microsoft Excel. Methods: MLST typer imports sequence data in the FASTA format, converts reverse complement counterparts of the reverse sequences, assembles forward and reverse-complement converted sequences, and returns allelic numbers for each gene and ST of each isolate. To evaluate the performance of MLST typer, we tested the sequence data from 200 clinical isolates, each consisting of seven housekeeping gene sequences, with a total of 1,400 allelic number determinations. The results were compared with manual assessment. Results: MLST typer comprises three worksheets: the Main page, Result page, and Summary page. The Main page console operates the process according to user-specified parameters. The Result and Summary pages provide the allelic type and ST determinations. It took approximately 12 minutes to analyze the sequence data from 200 clinical isolates. Compared with manual assessment, the rate of correct identification was 97.2% (1,361/1,400). Conclusions: MLST typer can be widely used for epidemiological studies owing to its thoroughness in repetitive functions, good compatibility with FASTA type data files, and easyto- understand outputs for allelic and ST determinations.

Streptococcus suis is a swine pathogen that causes meningitis, septicemia, pneumonia, and endocarditis. The first case of human S. suis infection was reported in Denmark in 1968, and since then, this infection with has been reported in many countries, especially in Southeast Asia because of the high density of pigs in this region. We report the case of a patient with septic arthritis and bacteremia caused by S. suis. Cases in which S. suis is isolated from the joint fluid are very rare, and to the best of our knowledge, this is first case report of S. suis infection in Korea. The identity of this organism was confirmed by phenotypic characterization and 16S rRNA sequence analysis. An 81-yr-old Korean woman who presented with fever, arthralgia, and headache was admitted to a secondary referral center in Korea. Culture of aspirated joint fluid and blood samples showed the growth of S. suis biotype II, which was identified by the Vitek2 GPI and API 20 Strep systems (bioMerieux, USA), and this organism was susceptible to penicillin G and vancomycin. The 16S rRNA sequences of the blood culture isolates showed 99% homology with those of S. suis subsp. suis, which are reported in GenBank. The patient’s fever subsided, and blood and joint cultures were negative for bacterial growth after antibiotic therapy; however, the swelling and pain in her left knee joint persisted. She plans to undergo total knee replacement.

Background: We evaluated the efficacy of multilocus sequence typing (MLST) for assessing the genetic relationship among Candida albicans isolates from patients with candidemia in a hospital setting. Methods: A total of 45 C. albicans isolates from 21 patients with candidemia were analyzed. The MLST results were compared with results obtained by Southern blot hybridization (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE analysis included karyotyping and restriction endonuclease analysis of genomic DNAs using BssHII (REAG-B) and SfiI (REAG-S). Results: The 45 isolates yielded 20 unique diploid sequence types (DSTs) by MLST, as well as 12 karyotypes, 15 REAG-B patterns, 13 REAG-S patterns, and 14 C1 fingerprinting types. Microevolution among intra-individual isolates was detected in 6, 5, 3, 5, and 7 sets of isolates by MLST (1 or 2 allelic differences), REAG-B, REAG-S, C1 fingerprinting, and a combination of all methods, respectively. Among 20 DSTs, 17 were unique, and 3 were found in more than 1 patient. The results of 2 DSTs obtained from 9 patient isolates were in agreement with REAG and C1 fingerprinting patterns. However, the remaining DST, which was shared by 2 patient isolates, showed 2 different PFGE and C1 fingerprinting patterns. In addition, 3 sets of isolates from different patients, which differed in only 1 or 2 alleles by MLST, also exhibited different PFGE or C1 fingerprinting patterns. Conclusions: MLST is highly discriminating among C. albicans isolates, but it may have some limitations in typing isolates from different patients, which may necessitate additional analysis using other techniques.

배경：한국의 3차 진료기관에서 분리되는 황색포도구균의 70% 이상은 methicillin 내성으로 보고 된 바 있다. 최근 multilocus sequence typing (MLST) 기법은 환자에서 분리되는 methicillin-resistant Staphylococcus aureus (MRSA)에 대해 지역 및 국가간의 역학분석을 위해 흔히 사용되고 있다. 저자들은 동일한 병동에서 일정한 기간을 두고 분리된 MRSA 균주들을 대상으로 유전적 배경을 비교해보고자 하였다. 방법：한국의 1개 3차 진료기관의 중환자실에서 분리된 MRSA를 대상으로 하였다. 1996년에 분리된 16주, 2004년에 분리된 17주를 수집하여 8년간 중환자실에서 분리된 MRSA의 균형의 변화를 알아보고자 하였다. 디스크 확산법으로 항균제 감수성 검사를 시행하였고, MLST를 시행해 각각의 sequence type을 결정하였다. 결과：1996년 분리주 중 ST5(n=11, 68.7%)가 가장 흔한 형이었고, ST254 (n=3, 18.8%), ST1(n=2, 12.5%)의 형들이 분리되었다. 2004년에는 ST5은 6균주에서만 확인되었고 ST239(n=10, 58.8%)로 가장 흔한 형이 바뀌었다. 결론：중환자실에서 분리된 MRSA의 sequence type은 1996년에는 ST5가 가장 우세하였으나, 2004 년에는 ST239로 변화하였다.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) accounts for more than 70% of S. aureus isolates from tertiary-care hospitals in Korea. Recently, a multilocus sequence typing (MLST) scheme has been used to study the local and global epidemiologies of MRSA. The aim of this study is to compare the genetic background of MRSA strains isolated in the same ward during two different periods. Methods: To investigate clonal changes of endemic MRSA isolates between 1996 and 2004, we studied a total of 33 MRSA strains (16 from 1996 and 17 from 2004) isolated in the intensive care units of a tertiary-care hospital in Korea. The isolates were analyzed for their sequence types by MLST and for their antimicrobial susceptibilities by the disk diffusion method. Results: ST5 was the most frequent type (n=11, 68.7%) in 1996, followed by ST254 (n=3, 18.8%) and ST1 (n=2, 12.5%). In 2004, ST239 was the most frequent type (n=10, 58.8%), followed by ST5 (n=6, 35.3%). Conclusion: The major clone type of MRSA isolates from intensive care unit patients changed from ST5 in 1996 to ST239 in 2004.

Background: Multilocus sequence typing (MLST) is well-established for Pasteurella multo- cida but remains undeveloped for Pasteurella canis. We established MLST for P. canis us- ing isolates from humans and companion animals in Japan and Korea to gain insights into its population biology. Methods: We analyzed 39 and 22 isolates from companion animals and humans, respec- tively. We selected seven housekeeping genes?adk, aroA, deoD, gdhA, g6pd, mdh, and pgi?used in P. multocida MLST. Primer pairs for PCR amplification and sequencing were designed based on conserved sites in 10 whole-genome sequences. We determined frag- ment sequences, variable sites, allelic profiles, and sequence types (STs) of each isolate. A phylogenetic tree of concatenated sequences was constructed using the goeBURST algo- rithm to identify STs and clonal complexes (CCs). ompA, encoding outer membrane protein A, was genotyped for molecular characterization. Results: The sequenced fragment lengths and allele numbers of the seven genes were 424, 451, 483, 439, 429, 419, and 440 bp and 16, 13, 15, 18, 22, 19, and 18, respec- tively. ST1?ST47, including CC2, CC10, CC18, CC31, and CC33, were diversely distributed among the isolates from different hosts/countries. In the seven-gene phylogenetic tree, apart from P. multocida, all isolates clustered together. goeBURST diagrams revealed di- verse ST distributions among different hosts (animal/human) and countries (Japan/Ko- rea/others). We found clusters 1?4 in ompA genotyping, indicating that MLST discrimina- tion is higher than ompA typing discrimination. Conclusions: We established MLST for P. canis isolates from humans and companion ani- mals in Japan and Korea, thereby providing a robust tool for population biology studies.

Background: Enterococcus faecium has emerged as an important nosocomial pathogen worldwide, and this trend has been associated with the dissemination of a genetic lineage designated clonal complex 17 (CC17). In the present study, characterization of the glycopeptide resistance mechanism, genetic relatedness, and pathogenicity in isolates of vancomycin-resistant E. faecium in the Chungcheong area were investigated. Methods: A total of 37 consecutive, non-duplicate, vancomycin-resistant E. faecium were isolated at three university hospitals in the Chungcheong area. The mechanism of glycopeptide resistance and pathogenicity factors were studied using PCR, and the genetic relatedness was determined via multilocus sequence type and esp repeat profile analysis. Additionally, the quinolone resistance-determining regions of parC and gyrA were sequenced to identify mutations involved in ciprofloxacin resistance. Results: Two genotypes of VRE were confirmed: VanAphenotype vanA genotype VRE (25 isolates) and VanB-phenotype vanA genotype VRE (12 isolates). MLST analysis revealed five sequence types. A significant result was that ST414 and CNS4 (4-1-1-1-1-1-1) were considered as belonging to CC17. The esp and hyl genes were found in 100% and 86.4% of the isolates, respectively. A total of 37 isolates showed genetic mutations in parC and gyrA. Conclusion: All isolated strains in the present study belonged to one of the CC17 genotypes including ST414 and CNS4 (4-1-1-1-1-1-1), which were not previously detected in Korea. The combination of MLST and the esp gene repeat profiles can be useful for genetic characterization of VREF isolates with regard to the evolutionary process and epidemiology of the clones.

Background: Stenotrophomonas maltophilia has emerged as an important opportunistic pathogen, which causes infections that are often difficult to manage because of the inherent resistance of the pathogen to a variety of antimicrobial agents. In this study, we analyzed the expressions of SmeABC and SmeDEF and their correlation with antimicrobial susceptibility. We also evaluated the genetic relatedness and epidemiological links among 33 isolates of S. maltophilia. Methods: In total, 33 S. maltophilia strains were isolated from patients in a tertiary hospital in Daejeon. Minimum inhibitory concentrations (MICs) of 11 antimicrobial agents were determined by using agar dilution method and E-test (BioMerieux, France). Real-time PCR analysis was performed to evaluate the expression of the Sme efflux systems in the S.maltophilia isolates. Additionally, an epidemiological investigation was performed using multilocus sequence typing (MLST) assays. Results: The findings of susceptibility testing showed that the majority of the S. maltophilia isolates were resistant to β-lactams and aminoglycosides. Twenty-one clinical isolates overexpressed SmeABC and showed high resistance to ciprofloxacin. Moreover, a high degree of genetic diversity was observed among the S. maltophilia isolates; 3 sequence types (STs)and 23 allelic profiles were observed. Conclusions: The SmeABC efflux pump was associated with multidrug resistance in clinical isolates of S. maltophilia. In particular, SmeABC efflux pumps appear to perform an important role in ciprofloxacin resistance of S. maltophilia. The MLST scheme for S. maltophilia represents a discriminatory typing method with stable markers and is appropriate for studying population structures.

Background: We determined the epidemiological characteristics of erythromycin (EM)-resistant Streptococcus pyogenes (group A streptococci, GAS) strains isolated from Korea and Japan, using emm genotyping and multilocus sequence typing (MLST). Methods: Clinical isolates of GAS had been collected from 1992 to 2012 in Korea and from 2004 to 2009 in Japan. EM resistance was determined by the microdilution method, and resistance genotypes were assessed by PCR. The emm genotyping and MLST were performed by DNA sequencing. Results: The emm genotypes and sequence types (STs) were concordant in 143 (85.1%) of 168 EM-resistant GAS strains from Korea. ST36/emm12 (35.1%), ST52/emm28 (22.6%), and ST49/emm75 (16.1%) were the most common types. Most of the ST36 (93.9%) and ST52 (95.8%) strains harbored erm(B), whereas strains ST49, ST42, and ST15 contained mef(A). The concordance between emm genotypes and STs was 41 (93.2%) among 44 EM-resistant GAS strains from Japan. ST36/emm12 (34.1%), ST49/emm75 (18.2%), and ST28/emm1 (15.9%) were the major types. ST36 isolates harbored either erm(B) (56.3%) or mef(A) (37.5%), whereas isolates ST28, ST49, and ST38 carried only mef(A). The proportion of erm(B) and mef(A) was 66.1% and 33.3% in Korea and 22.7% and 68.2% in Japan, respectively. Conclusions: The common STs in Korea and Japan were ST36 and ST49, whereas ST52 was present only in Korea and ST28 only in Japan. Genotype erm(B) was predominant in Korea, whereas mef(A) was frequent in Japan. There were differences between Korea and Japan regarding the frequencies of emm genotypes, STs, and EM resistance genes among the EM-resistant GAS.

배경: Multilocus sequence typing (MLST) 분석은 유전자의 장기적 역학 변동을 반영하고 실험자와 실험실 간의 오차 없이 동일 균종들의 역학적 기원 및 진화적 배경들을 추정할 수 있는 방법이다. 이에 본 연구는 국내 일개 대학병원에서 9년간 분리된 VanA형 반코마이신 내성 Enterococcus faecium을 대상으로 MLST 분석을 시행하여 기간에 따른 유전자 변이를 알아보고자 하였다. 방법: 2007년부터 2015년까지 아주대학교병원에서 수집된 vanA 유전자를 지닌 E. faecium 44주를 대상으로 하였다. 수집된 균주는 VitekII system (bioMerieux, USA)을 이용하여 동정하였고, CLSI 기준에 따라 디스크확산법과 E-test 방법으로 항균제 감수성 검사를 실시하였다. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF M/S) 분석법과 MLST 분석을 이용하여 이들 균주의 유전적 연관성과 시간에 따른 진화에 대한 분석을 실시하였다. 결과: 모든 균주는 ampicillin, ciprofloxacin과 반코마이신에 대해 고도내성을 보였고, teicoplanin에 대해서는 내성, 중등도 내성 및 감수성 등 다양한 내성양상을 보였다. MALDI-TOF M/S를 이용한 44균주의 유전적 형별분석 결과에서는 유전적 다양성을 보여 균주 간 연관성은 없는 것으로 나타났다. MLST 분석상 6개의 sequence type (ST)을 보였다. ST17형이 19주로 가장 많았고 ST78형 13주, ST192형 6주, ST64형 4주, ST262형과 ST414형이 각각 1주였다. 결론: MLST 분석에서 모든 균주는 clonal complex 17에 속하였다. 이는 대형 병원 유행에서 흔히 관찰되는 결과이다. 본 연구에서 각각 1주씩 분리된 ST292형과 ST414형은 산발적 발생임을 알 수 있었다. MLST 분석은 VanA형 VRE의 유전자의 진화 양상을 추정할 수 있는 효과적인 방법이라 사료된다. [Ann Clin Microbiol 2017;20:67-73]

Background: Multilocus sequence typing (MLST) is useful in determining the long-term evolutionary process and minimizes differences in experimental results across individuals and laboratories. It is also useful in determining evolutionary origins and backgrounds of bacterial species. This study carries out MLST analysis on VanA-type vancomycin-resistant Enterococcus faecium isolated from patient specimens in a single university hospital over nine years in order to observe changes in genetic evolution over time. Methods: During the years from 2007 to 2015, 44 clinical isolates of vanA-containing E. faecium were collected from Ajou University Hospital in Korea. Species were identified by the VitekII system (bioMerieux, USA), and antibiotic susceptibility testing was performed by disk diffusion and E-test according to Clinical and Laboratory Standards Institute (CLSI) guidelines. To determine genetic relatedness, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF M/S) was employed. To characterize clonal diversity, MLST analysis was used. Results: All isolates were highly resistant to ampicillin, ciprofloxacin, and vancomycin but showed variable levels of resistance to teicoplanin. The 44 clinical isolates were genetically unrelated according to MALDI-TOF M/S analysis. MLST showed that the clinical isolates harbored 6 sequence types (ST), with ST17 (n=19) being the most common, followed by ST78 (n=13), ST192 (n=6), ST64 (n=4), ST262 (n=1), and ST414 (n=1). Conclusion: The MLST analysis showed that the sequence types of most isolates belonged to clonal complex 17 This is consistent with outbreaks in hospitals. We had single observations for ST262 and ST414, suggesting that they were random occurrences. MLST can be useful for speculating the genetic evolution of VanA-containing E. faecium isolates. (Ann Clin Microbiol 2017;20:-73)