Vibrio 속 16S rRNA 유전자 염기서열의 이질성 분석

기장서,Ki, Jang-Seu 한국미생물학회 2009 미생물학회지 Vol.45 No.4

세균 16S rRNA 유전자 염기서열은 분자계통분류, 진화역사 규명, 미생물 검출 등 다양한 목적으로 이용되어 왔다. 세균 제놈(genome)은 multiple rRNA 오페론을 갖고 있으며, 이들 유전자 염기서열은 일부 변이가 있는 것으로 알려져 있다. 본 연구에서는 Vibrio 속의 16S rRNA 유전자 염기서열을 이용하여 세포 내 16S rRNA의 이질성을 규명하였다. 분석은 GenBank 자료 중에서 제놈 염기서열 annotation이 완료된 V. cholerae, V. harveyi, V. parahaemolyticus, V. splendidus, V. vulnificus를 이용하여 실시하였다. Vibrio 속은 1번 염색체에 7~10개의 16S rRNA 유전자 copy를 갖고 있으며, 이들의 세포 내 유전자 변이는 0.9% 이하 상이성(99.1%이상 DNA 상동성)을 보였다. 2번 염색체에서는 16S rRNA 유전자가 1개 이하로 존재하였다. 유전체내 16S rRNA 유전형은 최소 5개(V. vulnificus #CMCP6)에서 최대 8개(V. parahaemolyticus #RIMD 2210633, V. harveyi #ATCC BAA-1116)로 조사되었다. 본 결과는 Vibrio 속의 16S rRNA 유전자 염기서열이 높은 이질성을 갖는 것을 제시해 준다. Bacterial 16S rRNA gene sequences have been widely used for the studies on molecular phylogeny, evolutional history, and molecular detections. Bacterial genomes have multiple rRNA operons, of which gene sequences sometimes are variable. In the present study, heterogeneity of the Vibrio 16S rRNA gene sequences were investigated. Vibrio 16S rRNA sequences were obtained from GenBank databases, considering the completion of gene annotation of Vibrio genome sequences. These included V. cholerae, V. harveyi, V. parahaemolyticus, V. splendidus, and V. vulnificus. Chromosome 1 of the studied Vibrio had 7~10 copies of the 16S rRNA gene, and their intragenomic variations were less than 0.9% dissimilarity (more than 99.1% DNA similarity). Chromosome 2 had none or single 16S rRNA gene. Intragenomic 16S rRNA genotypes were detected at least 5 types (V. vulnificus #CMCP6) to 8 types (V. parahaemolyticus #RIMD 2210633, V. harveyi #ATCC BAA-1116). These suggest that Vibrio has high heterogeneity of the 16S rRNA gene sequences.

그람 음성 간균에서 16S rRNA Methylase 생성 빈도와 생성 세균의 내성 양상

이경원,이혁민,고은미,김창기,염종화,정윤섭 대한임상미생물학회 2010 Annals of clinical microbiology Vol.13 No.1

Background: Recently a novel plasmid-mediated resistant mechanism that conferred high-level resistance to aminoglycoside via methylation of 16S rRNA was reported. The aims of this study were to determine the prevalence of the 16S rRNA methylase genes and to characterize the coresistance to other antibiotics in Gram-negative bacilli. Methods: Consecutive non-duplicate Gram-negative bacilli were isolated from clinical specimens at a Korean secondary- and tertiary-care hospital from July 2006to June 2007. The antimicrobial susceptibility was tested by the CLSI agar dilution method,and PCR was performed to detect the 16S rRNA methylase genes in the arbekacin-resistant isolates. Results: In Gram-negative bacilli, the proportions of 16S rRNA methylase gene-positive isolates were 5%(75/1,471) in the secondary-carehospital and 4%(48/1,251) in the tertiary-care hospital, and the positive rates by species were 1% Escherichiae coli 16% (10/1,062), Klebsiella pneumoniae 16% (75/460), K. oxytoca 2% (1/44), Citrobacter spp. 9% (7/82), Enterobacter spp. 2% (4/181), Serratia marcescens 6% (6/100), Proteus miriabilis 4% (2/57), Achromobacter xylosoxidans 20% (1/5), Pseudomonas aeruginosa <1% (1/505), Acinetobacter spp. 10% (11/112), and Stenotrophomonas maltophilia 2% (1/66),respectively. Among 16S rRNA methylase-positive isolates from secondary- and tertiary-care hospitals,93% (70/75) and 90% (43/48), respectively, were armA positive, and others, except one rmtA positive isolate, were positive for the rmtB gene, according to PCR results. The rates of ESBL-positive and cefoxitin-resistant K. pneumoniae were 59% and 92%,respectively. In addition, 91% of 16S rRNA methylase-producing K. pneumoniae were positive for qnrB. There were no MBL producers among 16S rRNA methylase-producing Pseudomonas and Acinetobacter species. Conclusion: The novel aminoglycoside-resistant mechanisms involving16S rRNA methylase were prevalent and widely distributed among Gram-negative bacilli in Korea, and other resistance mechanisms were commonly associated with 16S rRNA methylasemediated resistance in Korea. (Korean J Clin Microbiol 2010;13:19-26)

16S rRNA 유전자 염기서열 분석에 의해 확인된 Acinetobacter spp. 가성요로감염 유행

김수연,김진용,강지혜,박신영,이희승,박윤수,서일혜,조용균 대한감염학회 2007 감염과 화학요법 Vol.39 No.4

목적 : 본 연구는 일개 대학병원의 한 병동에서 16SrRNA 유전자 염기서열 분석을 통해 확인된 Acinetobacter spp. 가성요로감염 집단 발생에 대한 조사이다. 재료 및 방법 : 일개 대학병원의 일반병동에서 2005년 9월 23일부터 26일까지 5명의 환자에서 Bordetelta bronchiseptica 세균뇨가 동시에 분리되었다. 해당 환자들에 대한 입원 진료 기록을 확인하고, 이학적 검사를 시행하였고, 의료진 면담 등의 역학적 조사와 의심되는 전파원의 환경 감시배양을 시행하였다. 또한 다섯 균주들의 상동성 확인을 위해 pulsed field gel electrophoresis (PFGE)를 하였고, 정확한 균 동정을 위해 16S rRNA 유전자 염기서열 분석을 하였다. 결과 : VITEK system에 의해 B. bronchiseptica로 보고된 다섯 균주들은 거의 유사한 항생제 감수성을 가지고 있었다. 유행조사에서 요로감염의 증상이나 균혈증을 보인 환자는 없었고, 환경 감시배양에서 공통의 전파원은 증명되지 않았다. 또한 PFGE와 16S rRNA 유전자 염기서열분석에서 상동성을 가진 동일 Acinetobacter spp.로 확인되어 이에 의한 가성요로감염의 유행으로 결론지었다. 결론 : 역학적 조사와 함께 PFGE와 16s rRNA 유전자염기서열 분석과 같은 분자생물학적인 조사를 시행하는 것은 희귀한 균에 의한 병원감염 유행조사에 도움이 될 것이다. Background : Acinetobacter spp. is increasingly implicated in hospital-acquired infections. We experienced a pseudooutbreak of Bordetella bronchiseptica bacteriuria identified with biochemical tests, that was later identified as Acinetobacter spp. by using 16S rRNA gene sequence analysis. Materials and Methods : Five in-ward patients were found to have B. bronchiseptica bacteriuria without symptoms of urinary tract infection between September 23 and 26 of 2005. We conducted pulsed field gel electrophoresis (PFGE) of the bacteria and epidemiological investigation of this pseudooutbreak. In addition, 16S rRNA gene sequence analysis was performed for the verification of the strains. Results : All 5 isolates were identified as B. bronchiseptica with similar antibiogram by VITEK system. There was no evidence of any symptom or sign of urinary tract infection. The source of this pseudooutbreak was not detected even after performing environmental culture and interviews with healthcare workers. We could not get the appropriate results from the first PFGE with XbaI restriction enzyme. B. bronchiseptica is an unusual organism in human so we conducted 16S rRNA gene sequence analysis for verification. The analysis of 16S rRNA gene sequence with 5 isolates demonstrated 99-100% similarity to a sequence of Acinetobacter spp. (AU1523). According to the results of 16S rRNA gene sequence analysis, we performed the second PFGE with SmaI restriction enzyme, which showed indistinguishable pattern among the all 5 isolates. Conclusion : This investigation suggests that the combined method of 16s rRNA gene sequence analysis and PFGE would be helpful for investigation of outbreak caused by unusual organisms.

Sphingomonas chungbukensis DJ77의 16S rRNA 염기서열과 이차구조

이관영,권해룡,이원호,김영창,Lee Kwan-Young,Kwon Hae-Ryong,Lee Won-Ho,Kim Young-Chang 한국미생물학회 2005 미생물학회지 Vol.41 No.2

S. chungbukensis DJ77로부터 16S rRNA유전자의 염기서열을 분식하였다. 염기서열은 총 1,502 bp로 2000 년에 등록된 부분 서열(1,435 bp)보다 5' 방향과 3' 방향으로 29 bp와 37 bp 길이만큼 각각 확장하였으며, 1 bp가 추가로 삽입되었다. E. coli의 16S rRNA유전자를 모델로 이차구조를 제작하였으며, 네 부위가 특이적임을 발견하였다. Sphnigomonas spp.의 16S rRNA 서열과 S. chungbukensis DJ77의 다중서열검색 결과, Sphingomonas종에서만 나타나는 보존부위와 가변부위를 발견할 수 있었다. 특히, Campylobacter jejuni에서만 나타나는 것으로 알려진 긴 stem loop구조가 서열은 조금 다르지만 구조적 일치를 보이는 유사한 구조를 S. chungbukensis DJ77에서도 발견하였다. 결과적으로, 다중서열검색을 통해 제작한 계통수와 nucleotide signatures분석에 근거하여 S. chugukensis DJ77을 cluster II (Sphingobium)로 분류하였다. A 16S ribosomal RNA gene from S. chungbukensis DJ77 has been sequenced. This sequence had a length of 1,502 bp and was extended for 29 bp at 5' and for 37 bp at 3' from the partial sequence (1,435 bp) registered in 2000 year. Besides, 1 bp was newly added near to the 3' end. We made the secondary structure of the 16S rRNA based on E. coli model and found four specific regions. We found constant and variable regions in genus Sphingomonas as the result of multiple alignment of 16S rRNA gene sequences from Sphingomonas spp. and S. chungbukensis DJ77. We found a stem loop structure in S. chungbukensis DJ77, which was only discovered in C. jejuni to date. It showed the structural agreement despite the difference of the sequences from the both organisms. Finally, S. chungbukensis DJ77 belonged to cluster II (Sphingobium) group, after the classification using phylogenetic analysis and nucleotide signature analysis.

16S rRNA 유전자를 이용한 Neisseria meningitidis, Haemophilus influenzae,Streptococcus pneumoniae 및 Streptococcus agalactiae의 검출

이채훈,김경동,전효진,전동석,김재룡 대한임상병리학회 2000 대한임상병리학회지 Vol.20 No.3

Development of Broad-range and Specific 16S rRNA PCR for Use in Routine Diagnostic Clinical Microbiology

Hyun-Chul Kim(김현철),Yun-Tae Kim(김윤태),Hyogyeong Kim(김효경),Sanghoo Lee(이상후),Kyoung-Ryul Lee(이경률),Young-Jin Kim(김영진) 한국생명과학회 2014 생명과학회지 Vol.24 No.4

16S rRNA gene PCR법은 환자 검체로부터 병원성 미생물을 검출 및 동정에 사용되어진다. 본 연구는 대량의 임상미생물 진단을 위해 bacterial 16S rRNA 부위 유전자 서열을 이용하여 광대한 범위와 높은 특이도를 가지는 primer을 포함한 PCR법을 개발하였다. 10개 표준 균주 16S rRNA 보존 부위의 유전자 서열을 기반으로 primer set를 구축하였다. 98명 환자 검체에서 임상 미생물을 분리하였다. 98개 균주는 phenotypic 방법을 이용하여 확인하고, 개발된 primer set와 universal primer set를 이용한 PCR법으로 확인하였다. 획득한 PCR 산물은 forward primer, reverse primer, 그리고 자동화 DNA 분석기를 이용하여 각 균주의 16S rRNA 유전자 서열을 분석 및 확인하였다. 본 연구에서 개발된 primer set와 universal primer set의 임상미생물 검출에 대한 효율성을 평가하였고, 또한 phenotypic 방법과 분자생물학적 방법을 비교했다. 분리된 98개 균주를 대상으로 개발된 primer set로 16S rRNA PCR을 진행하여 778 bp 크기의 단일밴드로 증폭 되었음을 확인했다. 총 98개중 94개 균주(95.9%)는 phenotypic 결과와 동일함을 확인했다. 새로 개발된 primer set를 이용한 결과는 universal primer set를 이용한 98개 균주(100%)의 결과와 동일함을 확인하였다. 개발된 16S rRNA gene PCR법은 임상미생물 검출 및 동정에서 신속성, 정확성, 그리고 검사 비용 절감의 장점을 가진다. 개발된 primer set는 병원성 미생물 동정에서 효율성을 확인했다. Broad-range and specific 16S rRNA gene PCR is used for detection and identification of bacterial pathogens in clinical specimens from patients with a high suspicion for infection. We describe the development of a broad-range and specific PCR primer, based on bacterial 16S rRNA, for use in routine diagnostic clinical microbiology services. The primers were designed by using conservative regions of 16S rRNA sequences from 10 strains. Ninety-eight clinical strains were isolated from clinical patient specimens. A total of 98 strains of bacteria were identified by phenotypic methods; PCR with newly designed primers and universal primers. All purified PCR products were sequenced using both forward and reverse primers on an automated DNA analyzer. In this study, we evaluated the usefulness of the newly designed primers and the universal primers for the detection of bacteria, and both these techniques were compared with phenotypic methods for bacteria detection. When we also tested 98 strains of clinical isolates with newly designed primers, about 778 bp DNA fragments were amplified and identified from all strains. Of the 98 strains, 94 strains (95.9%) correspond in comparison with phenotypic methods. The newly designed primers showed that the identities of 98 (100%) strains were the same as those obtained by universal PCR primers. The overall agreement between the newly designed primers and universal primers was 100%. The primer set was designed for rapid, accurate, and cheap identification of bacterial pathogens. We think the newly designed primer set is useful for the identification of pathogenic bacteria.

16S rRNA와 16S-23S rRNA Gene Sequence Analysis을 이용한 물김치와 동치미 유산균의 분리 동정

설수연,김태운,김성언,김해영 경희대학교식량자원개발연구소 2007 硏究論文集 Vol.26 No.1

This study was carried out to identify lactic acid bacteria in Mul-Kimchi and Dongchimi using by 16S rRNA and 16S-23S gene sequence analysis. Diluted Mul-Kimchi and Dongchimi soup were plated on the MRS agar media and colonies obtained were used for 16S rRNA and 16S-23S rRNA gene sequence analysis. Genomic DNAs were extracted from the isolated lactic acid bacteria and used as a template for PCR amplification. Universal primer sets based on the 16S rRNA and 16S-23S intergenic spacer region genes were used for amplification. From the sequencing of PCR products, Weissella kimchii, Weissella cibaria, Leuconostoc lactis, Leuconostoc citreum and Leuconostoc garlicum were identified.

Genetic Similarity Between Jujube Witches¡?Broom and Mulberry Dwarf Phytoplasmas Transmitted by Hishimonus sellatus Uhler

Cha, Byeongjin,Han, Sangsub The Korean Society of Plant Pathology 2002 Plant Pathology Journal Vol.18 No.2

Using phytoplasma universal primer pair Pl and P7, a fragment of about 1.8 kb nucleotide sequences of 16S rRNA gene and 16S-23S rRNA intergenic spacer region, and a portion of 23S rRNA gene of jujube witches'broom (JWB) and mulberry dwarf(MD) phytoplasmas were determined. The nucleotide sequences of JWB and MD were 1,850 bp and 1,831 bp long, respectively. The JWB phytoplasma sequence was aligned with the homologous sequence of MD phytoplasma. Twenty-eight base insertions and nine base deletions were found in the JWB phytoplasma sequence compared with that of MD phytoplasma. The similarity of the aligned sequences of JWB and MD was 84.8%. The near-complete 16S rRNA gene DNA sequences of JWB and MD were 1,529 bp and 1,530 bp in length, respectively, and revealed 89.0% homology. The 16S-23S rRNA intergenic spacer region DNA sequences were 263 bp and 243 bp in lengths respectively, while homology was only 70% and the conserved tRNA-lle gene of JWB and MD was located into the intergenic space region between 16S-23S rRNA gene. The nucleotide sequences were 77 bp long in both JWB and MD, and showed 97.4% sequence homology. Based on the phylogenetic analysis of the two phytoplasmas, the JWB phytoplasma belongs to the Elm yellow phytoplasma group (16S rV), whereas, the MD phytoplasma belongs to the Aster yellow group (16S rI).

Lactobacillus acidophilus ATCC 4356의 16S-23S rRNA intergenic spacer region sequence 특성과 균체 섭취에 의한 Salmonella enteritigis 감염시 쥐의 체중변화에 미치는 영향

배진성,윤영호 中央大學校 食糧資源硏究所 2002 食糧資源硏究所 論文集 Vol.14 No.1

Lactobacillus acidophilus의 16S-23S rRNA intergenic spacer region의 특성과 Lactobacillus spp. 급여에 의하여 Salmonella enteritidis 감염 이후 체중저하에 대한 보호효과를 주요 장기 중에 오염된 생균수 및 장기 중량 변화성향을 측정한 결과는 다음과 같다. Lactobacillus acidophilus의 16S-23S rRNA intergenic spacer region의 sequence를 측정하고 gene bank에 등록된 균주의 sequence와 비교한 결과 homology의 차이를 나타내었다 Lactobacillus spp. 급여 이후 Lactobacillus enteritidis를 challenge 이후 체중 변화는 4일 경과 후부터 체중 감소현상을 보였으며 체중 감소율이 가장 낮은 균주는 Lb. helveticus CU 631이었으며 균주간의 차이를 나타내었고, 간과 비장에 Salmonella enteritidis 오염 생균수는 Lactobacillus spp. 급여군이 대조군보다 낮은 성행을 보였고 간장 및 비장의 중량은 대조군보다 낮은 성향을 보였다. 16S-23S rRNA intergenic spacer region sequence and effects of Lactobacillus acidophilus ATCC 4356 against typical enteritis causing pathogen Salmonella enteritidis were determinded. Changes in the weight of body, spleen, liver and the contaminati on level of Salmonella enteritidis in the vital organ after challenge have been determined. The sequence of 16S-23S rRNA intergenic spacer region of Lactobacillus acidophilus could be utilized as a strain identification, those sequences comparison showed difference in homology. A rapid decline in body weight after seven days of challange with Salmonella enteritidis were observed in control mice, the protective effects from the decline in body weight by feeding Lactobacillus spp was most prominent in Lb. helveticus CU 631 and in Lb. acidophilus, more numerous viable number of Salmonella enteritidis in the tissues of spleen and liver and heavier average organ weight in control mice(unfed with Lactobacillus spp.) than lactobacillus spp. fed mice was observed.

Uridylate kinase를 이용한 원핵생물의 분류

이동근,김철민,김상진,하배진,하종명,이상현,이재화 한국생명과학회 2003 생명과학회지 Vol.13 No.6

원핵생물 (Procaryote)의 분류에 16S rRNA유전자가 많이 이용되어 있으나 제한된 해상력과 유전자의 수에 차이가 있는 등의 문제가 있어 이를 보완할 수 있는 새로운 생체분자를 찾고 그 분류 결과를 16S rRNA의 결과와 비교하였다. COG (Clusters of Orthologous of protein) 방법을 이용하여 43종의 미생물중에서 진핵생물을 제외한 42종의 원핵생물 (procaryote)에서만 발견되는 3종류의 COG인 Transcription elongation factor인 COG0195과 bacterial DNA primase인 COG0358 그리고 uridylate kinase인 COG0528를 구하였다. 이중 유사도와 유전자 수를 바탕으로 새로운 분류의 키로 uridylate kinase를 설정하여 분석한 결과, 같은 속 (genus)에 속하는 세균들은 아주 높은bootstrap value를 갖고 분류도에서 같은 위치에 분포하고 고세균 (Archaebacteria) 내부의 응집성이 높은 등의 유사성을 보였다. 한편 alpha와 epsilon 그룹의 Proteobacteria가 분류도에서 다르게 위치하고 진정세균 (Eubacteria)의 Spi-rochaetales에 속하는 Treponema pallidum (Tpa)와 Borrelia burgdorferi (Bbu)가 고세균과 유연관계가 높게 나타나는 등 차이점도 보였다. Uridylate kinase를 이용한 분류는, 아주 높은 보존성에 의해서 생기는 16S rRNA 유전자를 이용한 문제점을 보완하여 원핵생물의 정확한 분류에 기여할 수 있을 것으로 사료되었다. The 16S rRNA gene is the most common gene in the phylogenetic analysis of procaryotes. However very high conservative of 16S rRNA has limitation in the discrimination of highly related organisms, hence other molecule was applied in this study and the result was compared with that of 16S rRNA. Three COGs (Clusters of Orthologous of protein) were only detected in 42 procaryotes ; transcription elongation facto. (COG0195), bacterial DNA primase (COG0358) and uridylate kinase (COG0528). Uridylate kinase gene was selected because of the similarity and one single copy number in each genome. Bacteria, belong to same genus, and Archaebacteria were same position with high bootstrap value in phylogenetic tree like the tree of 16S rRNA. However, alpha and epsilon Proteobcteria showed different position and Spirochaetales of Eubarteria was grouped together with Archaebacteria unlike the result of 16S rRNA. Uridylate kinase may compensate the problem of very high conservative of 16S rRNA gene and it would help to access more accurate discrimination and phylogenetic analysis of bacteria.