Pediococcus pentosaceus의 Multilocus Sequence Typing 분석에서 신규 Sequence Type의 확인

이설희 ( Sulhee Lee ),박영서 ( Young Seo Park ) 한국산업식품공학회 2015 산업 식품공학 Vol.19 No.4

The genus Pediococcus belongs to the lactic acid bacteria and includes 15 species which are used in the food industry as both starter and probiotic cultures. The importance of Pediococcus spp. is due to their use as starter cultures in fermented meat as well as to their presence as the natural microbiota in vegetables. The availability of P. pentosaceus in the food industry increases the need for reliable molecular techniques for strain identification. To date, the reliable molecular methods for definite identification at strain level of microorganisms used in food industry has not been developed. Molecular identification based on suitable marker genes could be a promising alternative to conventional molecular typing methods such as ribotyping. In this study, the applicability of seven housekeeping genes gyrB, pyc, pgm, leuS, glnA, and dalR in combination with the pgi gene in multilocus sequence typing of P. pentosaceus was assessed. Sequencing and comparative analysis of sequence data were performed on 6 strains isolated from various vegetables. In addition to 17 sequence types, two new sequence types were identified and these fortified sequence types and seven marker genes allowed for a clear differentiation of the strains analyzed, indicating their applicability in molecular typing.

Molecular Analysis of Eight American Type Culture Collection Gonococcal Strains by Neisseria gonorrhoeae Multiantigen Sequence Typing and PorB Sequence Typing

Yousun Chung,Minje Han,박지영,Sora Kang,Inhee Kim,Jung A Park,Jae-Seok Kim 대한임상검사정도관리협회 2019 Journal of Laboratory Medicine And Quality Assuran Vol.41 No.1

Background: Molecular epidemiological typing of Neisseria gonorrhoeae is crucial for monitoring the spread of resistant strains. As reference strains can be used for laboratory internal quality control, we genetically characterised the American Type Culture Collection (ATCC) gonococcal strains by Neisseria gonorrhoeae multiantigen sequence typing (NGMAST) and porB sequence typing using public multilocus sequence typing (PubMLST). Methods: Eight ATCC gonococcal reference strains (ATCC 19424, ATCC 31426, ATCC 35541, ATCC 43069, ATCC 43070, ATCC 49226, ATCC 49926, and ATCC 49981) from Culti-Loops (Thermo Fisher Scientific, USA) were cultured. After DNA extraction, porB and tbpB were amplified and sequenced. Sequence types (STs) and allele numbers were each determined by NG-MAST (http://www.ng-mast.net) and porB sequence typing using PubMLST (http://pubmlst.org/neisseria/porB/). Results: ATCC 19424 was identified as ST 266 by NG-MAST, and as Allele 946 by PubMLST. ATCC31426 was assigned a novel ST by NG-MAST, and was assigned Allele 958 with 1.2% mismatch by PubMLST. ATCC 35541 was identified as ST 12 by NG-MAST, and as Allele 624 by PubMLST. ATCC 43069 and ATCC 43070 were both identified as ST 681 by NGMAST, and as Allele 984 by PubMLST. ATCC 49226 was identified as ST 1572 by NG-MAST, and as Allele 2110 by PubMLST. ATCC 49926 and ATCC 49981 were both identified as ST 16496 by NG-MAST, and as Allele 928 by PubMLST. Conclusions: The ST data obtained for ATCC gonococcal reference strains by NG-MAST and porB sequence typing using PubMLST can be used for quality assurance of molecular epidemiological typing in clinical microbiological laboratories.

Multilocus Sequence Typing for Candida albicans Isolates from Candidemic Patients: Comparison with Southern Blot Hybridization and Pulsed-field Gel Electrophoresis Analysis

윤명하,신종희,이진솔,김수현,신명근,서순팔,양동욱 대한진단검사의학회 2011 Annals of Laboratory Medicine Vol.31 No.2

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. 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.

최근임상에서 분리된 다제 내성 Enterobacter cloacae의 생물학적 유전학적 특성

김재중,구선회 대한진단검사의학회 2018 Laboratory Medicine Online Vol.8 No.3

배경: 2014년 1월부터 2015년 12월까지 중부지방 2곳의 종합병원에서 6개 계열 8종 항균제에 다제 내성을 나타내는 Enterobactercloacae 69주를 수집하였다. 방법: E-test법으로 최소억제농도(MIC)를 구하였고, TEM, SHV, CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, NDM, OXA, IMI, IMP, VIM, FOX, EBC, CIT, ACC, DHA, MOX 등 17개 유전자를 PCR 증폭 후 검출하였다. 균주 간 역학적 연관성은 REP-PCR과 multilocus sequence typing (MLST)법으로 확인하였다. 결과: E. cloacae 69주는 ESBL과 AmpC를 생성하며 cefotaxime, ceftazidime, aztreonam에 다제 내성을 나타냈다. ESBL SHV (N=12, 17.4%)는 VI type의 ST56로, CTX-M (N=11, 15.9%)은 I, IV, VI, VII type의 ST53, ST114, ST133, ST550로, carbapenemase NDM (N=1, 1.5%)은 II type의 ST24, OXA (N=1, 1.5%)는 III type의 ST668로 확인되었다. AmpC DHA (N=2, 2.89%)는 type이 동정되지 않은 ST134, EBC (MIR/ACT) (N=18, 26.1%)는 V, I, III, IV, VII, VI type의 ST53, ST24, ST41, ST114, ST422, ST466, ST484, ST550로 확인되었다. CTX-M-1은 blaCTX-M-3, blaCTX-M-22, CTX-M-9은 blaCTX-M-9, blaCTX-M-125, DHA는 blaDHA-1, EBC (MIR/ACT)는 blaMIR-7, blaACT-15,17,18,25,27,28 아형을 생성하였다. 결론: 유전자 생성과 내성발생 균주 간 역학적 연관성은 없었다. Background: From January 2014 to December 2015, 69 clones of Enterobacter cloacae showing multidrug resistance to six classes of antimicrobial agents were collected from two medical centers in Korea. Methods: Minimum inhibitory concentrations were determined using the E-test method, and 17 genes were detected using polymerase chain reaction (PCR). The epidemiological relatedness of the strains was identified using repetitive element sequence-based PCR and multilocus sequence typing. Results: The 69 E. cloacae clones produced extended spectrum β lactamase (ESBL) and AmpC and showed multidrug resistance to cefotaxime, ceftazidime, and aztreonam. We identified the following sequence types: ST56 of type VI for ESBL SHV (N=12, 17.4%), ST53, ST114, ST113, and ST550 of types I, IV, VI, and VII, respectively, for CTX-M (N=11, 15.9%), and ST668 of type III for the carbapenemase NDM gene (N=1, 1.5%). The AmpC DHA gene (N=2, 2.89%) was confirmed as ST134, although its type was not identified, whereas EBC (MIR/ACT, N=18, 26.1%) was identified as ST53, ST24, ST41, ST114, ST442, ST446, ST484, and ST550 of types V, I, III, IV, VII, and VI, respectively. The formed subclasses were blaCTX-M-3 and blaCTX-M-22 by CTX-M-1, blaCTX-M-9 and blaCTX-M-125 by CTX-M-9, blaDHA-1 by DHA, and blaMIR-7 and blaACT-15, 17, 18, 25, 27, 28 by EBC (MIR/ACT). Conclusions: There were no epidemiological relationships between the gene products and the occurrence of resistance among the strains.

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 Ch

전세미,임나라,권승직,심태선,박미선,김범준,김성한 질병관리본부 2014 Osong Public Health and Research Persptectives Vol.5 No.3

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, pulsedfield 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.

Multilocus Sequence Typing for <i>Candida albicans</i> Isolates from Candidemic Patients: Comparison with Southern Blot Hybridization and Pulsed-field Gel Electrophoresis Analysis

Myoung, Youn,Shin, Jong Hee,Lee, Jin Sol,Kim, Soo Hyun,Shin, Myung Geun,Suh, Soon Pal,Ryang, Dong Wook The Korean Society for Laboratory Medicine 2011 Annals of Laboratory Medicine Vol.31 No.2

<P><B>Background</B></P><P>We evaluated the efficacy of multilocus sequence typing (MLST) for assessing the genetic relationship among <I>Candida albicans</I> isolates from patients with candidemia in a hospital setting.</P><P><B>Methods</B></P><P>A total of 45 <I>C. albicans</I> 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 <I>Bss</I>HII (REAG-B) and <I>Sfi</I>I (REAG-S).</P><P><B>Results</B></P><P>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.</P><P><B>Conclusions</B></P><P>MLST is highly discriminating among <I>C. albicans</I> isolates, but it may have some limitations in typing isolates from different patients, which may necessitate additional analysis using other techniques.</P>

소아 암 환자에서 발생한 Bacillus cereus 균혈증의 분자역학 분석에 관한 연구

김종민 ( Jong Min Kim ),박기섭 ( Ki-sup Park ),이병기 ( Byung-kee Lee ),김수진 ( Soo Jin Kim ),강지만 ( Ji-man Kang ),김양현 ( Yanghyun Kim ),유건희 ( Keon Hee Yoo ),성기웅 ( Ki Woong Sung ),구홍회 ( Hong Hoe Koo ),이남용 ( Nam Yo 대한소아감염학회 2016 Pediatric Infection and Vaccine Vol.23 No.3

목적: Bacillus cereus 는 암환자들에서 기회감염을 일으킬 수 있다. 2013년에서 2014년 기간 동안 삼 성서울병원 소아 암 병동에서 B. cereus 균혈증의 급격한 증가가 관찰되었다. 이에 증가된 B. cereus 균혈증에 대해 분자역학적 연구를 시행하였다. 방법: 2001년 1월부터 2014년 6월까지의 기간 동안 B. cereus 균혈증이 발생한 소아 암 환자들을 확 인하였다. 이번 연구에서 B. cereus 균혈증은, 오염여부와는 상관없이, 혈액배양검사에서 적어도 한 번 이상 B. cereus 가 확인된 경우로 정의하였다. 획득 가능한 균주들에 대해 multilocus sequence typing (MLST) 분석을 시행하였고, 후향적 챠트 리뷰를 실시하였다. 결과: 연구 기간 동안 총 19명의 B. cereus 균혈증 환자가 확인되었다. 그러나, 2013년도에는 B. cereus 균혈증 환자가 급격하게 증가하였다. 또한, 응급실 공사 중이던 2013년 7월의 1주, 2013년 10월의 한 주 동안 각각 3명의 환자가 발생하였다. 그러나 MLST 분석상 일정한 패턴이 없는, 다양한 sequence types (STs)들로 확인되었다. 2013년 이전의 5개의 균주들의 ST는 ST18, ST26, ST177, ST147-like type, ST219-like type이었고, 2013년도의 균주들의 ST는 ST18, ST73, ST90, ST427, ST784, ST34-like type, ST130-like type으로 확인되었다. 고찰: MLST 분석상 B. cereus 균주들의 다양한 ST 분포가 확인되었다. 이번 연구에서 단일 ST의 B. cereus 에 의한 균혈증 발생의 가능성은 낮아보인다. Purpose: Bacillus cereus has been reported as the cause of nosocomial infections in cancer patients. In our pediatric cancer ward, a sudden rise in the number of patients with B. cereus bacteremia was observed in 2013 to 2014. This study was performed to investigate the molecular epidemiology of increased B. cereus bacteremia cases in our center. Methods: Pediatric cancer patients who developed B. cereus bacteremia were identified from January 2001 to June 2014. The B. cereus bacteremia in this study was defined as a case in which at least one B. cereus identified in blood cultures, regardless of true bacteremia. Available isolates were further tested by multilocus sequence typing (MLST) analysis. A retrospective chart review was performed. Results: Nineteen patients developed B. cereus bacteremia during the study period. However, in 2013, a sudden increase in the number of patients with B. cereus bacteremia was observed. In addition, three patients developed B. cereus bacteremia within 1 week in July and the other three patients within 1 week in October, respectively, during emergency room renovation. However, MLST analysis revealed different sequence types without consistent patterns. Before 2013, five tested isolates were ST18, ST26, ST177, and ST147-like type, and ST219-like type. Isolates from 2013 were ST18, ST73, ST90, ST427, ST784, ST34-like type, and ST130-like type. Conclusions: MLST analyses showed variable ST distribution of B. cereus isolates. Based on this study, there was no significant evidence suggesting a true outbreak caused by a single ST among patients who developed B. cereus bacteremia.

Phylogeny of Flavobacteria Group Isolated from Freshwater Using Multilocus Sequencing Analysis

Mun, Seyoung,Lee, Jungnam,Lee, Siwon,Han, Kyudong,Ahn, Tae-Young Korea Genome Organization 2013 Genomics & informatics Vol.11 No.4

Sequence analysis of the 16S rRNA gene has been widely used for the classification of microorganisms. However, we have been unable to clearly identify five Flavobacterium species isolated from a freshwater by using the gene as a single marker, because the evolutionary history is incomplete and the pace of DNA substitutions is relatively rapid in the bacteria. In this study, we tried to classify Flavobacterium species through multilocus sequence analysis (MLSA), which is a practical and reliable technique for the identification or classification of bacteria. The five Flavobacterium species isolated from freshwater and 37 other strains were classified based on six housekeeping genes: gyrB, dnaK, tuf, murG, atpA, and glyA. The genes were amplified by PCR and subjected to DNA sequencing. Based on the combined DNA sequence (4,412 bp) of the six housekeeping genes, we analyzed the phylogenetic relationship among the Flavobacterium species. The results indicated that MLSA, based on the six housekeeping genes, is a trustworthy method for the identification of closely related Flavobacterium species.

Phylogeny of Flavobacteria Group Isolated from Freshwater Using Multilocus Sequencing Analysis

문세영,이정남,이시원,한규동,안태영 한국유전체학회 2013 Genomics & informatics Vol.11 No.4

Sequence analysis of the 16S rRNA gene has been widely used for the classification of microorganisms. However, we havebeen unable to clearly identify five Flavobacterium species isolated from a freshwater by using the gene as a single marker,because the evolutionary history is incomplete and the pace of DNA substitutions is relatively rapid in the bacteria. In thisstudy, we tried to classify Flavobacterium species through multilocus sequence analysis (MLSA), which is a practical andreliable technique for the identification or classification of bacteria. The five Flavobacterium species isolated from freshwaterand 37 other strains were classified based on six housekeeping genes: gyrB, dnaK, tuf, murG, atpA, and glyA. The genes wereamplified by PCR and subjected to DNA sequencing. Based on the combined DNA sequence (4,412 bp) of the sixhousekeeping genes, we analyzed the phylogenetic relationship among the Flavobacterium species. The results indicatedthat MLSA, based on the six housekeeping genes, is a trustworthy method for the identification of closely relatedFlavobacterium species.

Clonal Expansion of Macrolide-Resistant Sequence Type 3 <i>Mycoplasma pneumoniae</i> , South Korea

Lee, Joon Kee,Lee, Joon Ho,Lee, Hyunju,Ahn, Young Min,Eun, Byung Wook,Cho, Eun Young,Cho, Hwa Jin,Yun, Ki Wook,Lee, Hoan Jong,Choi, Eun Hwa U.S. Department of Health and Human Services * Cen 2018 Emerging Infectious Diseases Vol.24 No.8

<P>To investigate the genetic background for the emergence of macrolide resistance, we characterized the genetic features of <I>Mycoplasma pneumoniae</I> using multilocus sequence typing. Of the 146 <I>M. pneumoniae</I> strains collected during the 5 consecutive outbreaks of <I>M. pneumoniae</I> pneumonia during 2000–2016 in South Korea, macrolide resistance increased from 0% in the first outbreak to 84.4% in the fifth. Among the 8 sequence types (STs) identified, ST3 (74.7%) was the most prevalent, followed by ST14 (15.1%). Macrolide-susceptible strains comprised 8 different STs, and all macrolide-resistant strains were ST3 (98.3%) except 1 with ST14. The proportion of macrolide-resistant strains in ST3 remained 2.2% (1/46) until the 2006–2007 outbreak and then markedly increased to 82.6% (19/23) during the 2010–2012 outbreak and 95.0% (38/40) during the 2014–2016 outbreak. The findings demonstrated that clonal expansion of ST3 <I>M. pneumoniae</I> was associated with the increase in macrolide resistance in South Korea.</P>