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Choi, Moon, Park, Kang, Kim, Ahn, Kim, Yun, Lim, Shin, Kook, Chang, and Jang: Comparison of the Utility of dnaJ and 16S rDNA Sequences for Identification of Clinical Isolates of Vibrio Species
Original Article

Lab Med Online 2018;8(1):7-14.

Published online: 19 January 2018

DOI: https://doi.org/10.3343/lmo.2018.8.1.7

Comparison of the Utility of dnaJ and 16S rDNA Sequences for Identification of Clinical Isolates of Vibrio Species

In-Sun Choi, M.D.1, Dae Soo Moon, M.D.1, Geon Park, M.D.1, Seong-Ho Kang, M.D.1, Choon-Mee Kim, Ph.D.2, Young-Joon Ahn, M.D.3, Dong-Min Kim, M.D.4, Na Ra Yun, M.D.4, Dong Hoon Lim, M.D.5, Sung Heui Shin, M.D.6, Joong-Ki Kook, D.D.S.7, Young-Hyo Chang, Ph.D.8, Sook-Jin Jang, M.D.1

1Department of Laboratory Medicine, College of Medicine, Chosun University, Gwangju, Korea

2Premedical Science, College of Medicine, Chosun University, Gwangju, Korea

3Departments of Medical Education, College of Medicine, Chosun University, Gwangju, Korea

4Departments of Internal Medicine, College of Medicine, Chosun University, Gwangju, Korea

5Departments of Urology, College of Medicine, Chosun University, Gwangju, Korea

6Departments of Microbiology, College of Medicine, Chosun University, Gwangju, Korea

7Korean Collection for Oral Microbiology and Department of Oral Biochemistry7, School of Dentistry, Chosun University, Gwangju, Korea

8ABS Research Support Center, Korea Research Institute of Bioscience & Biotechnology, Daejon, Korea

Corresponding author: Sook-Jin Jang Department of Laboratory Medicine, Chosun University College of Medicine, 309 Pilmun-daero, Dong-gu, Gwangju 61452, Korea Tel: +82-62-220-3259, Fax: +82-62-232-2063, E-mail: sjbjang@chosun.ac.kr

Received 22 May 2017       Revised 21 June 2017       Accepted 23 June 2017

Copyright © 2018 The Korean Society for Laboratory Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background:

Among the many Vibrio species that can cause infections in humans, several species can cause a fatal outcome. Therefore, accurate identification of Vibrio species is very important. Since some species show atypical phenotypic features, selecting an appropriate molecular method is necessary to avoid misdiagnosis.

Methods:

Vibrio clinical isolates (N=53) and reference strains (N=8) were used in this study. We analyzed the following sequences for identification: dnaJ gene, 16S rDNA, gyrase B (gyrB) V. vulnificus–specific sequence, gyrB V. navarrensis–specific sequence, and V. vulnificus hemolysin gene PCR (Vvh PCR). We performed phylogenetic analysis of the 16S rDNA, dnaJ, and gyrB sequences. Final identification was based on the combined results of all tests described above. Concordance of the 16S rDNA and dnaJ sequence analysis was measured using the Chi-square test.

Results:

The 61 Vibrio strains were identified as follows, in descending order: V. vulnificus (78.69%), V. parahaemolyticus (6.56%), V. navarrensis (4.92%), V. mimicus (1.64%), V. cholera (1.64%), V. furnissii (1.64%), V. alginolyticus (1.64%), and Grimontia hollisae (1.64%). The accuracy rates of the dnaJ gene and 16S rDNA sequence for identification were 91.80% and 86.89%, respectively. The 16S rDNA and dnaJ sequences showed a concordance rate of 0.45, which indicates moderate agreement.

Conclusions:

Our results suggest that analysis of the dnaJ sequence may be a useful method for the identification of clinical isolates of Vibrio species, especially for distinguishing between closely related Vibrio species.

Keywords: Vibrio, Identification, 16S rDNA, dnaJ gene, gyrB gene

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Table 1.
Primers used in this study
Target Primer Sequence (5´ to 3´) mer Tm (°C) GC (%) size (bp) Reference
dnaJ 805-VibrioMF2 TTT TAY GAA GTD YTD GGY GT 20 49.3 37.5 558 [8]
  806-VibrioMR GAC AVG TWG GAC AGG YYT GYT G 22 58.1 54.5   [8]
16S rDNA 27F AGA GTT TGA TCM TGG CTC AG 20 51 45 1,466 [9]
  1492R TAC GGY TAC CTT GTT ACG ACT T 22 52 41   [10]
gyrB Vvul-F gyrB AGC CAA ACC TAT CGT CAT GG 20 53.7 50 993 This study
  Vvul-R gyrB GAT CAG CGT TGC GAC TTC TT 20 54.2 50   This study
  Vnav-F gyrB GTG GCG GCC ATA TTC ATA CT 20 53.7 50 1,036 This study
  Vnav-R gyrB CTT CCT GCG AAG AGA GCA TC 20 53.8 55   This study
vvh Vvh-P5 CCG CGG TAC AGG TTG GCG C 19 65.1 73.7 519 [6]
  Vvh-P6 CGC CAC CCA CTT TCG GGC C 19 66.2 73.7   [6]

Abbreviations: gyrB, gyrase B; vvh, Vibrio vulnificus hemolysin gene.

Table 2.
Pattern of results of final identification of the 61 clinical and reference strains of Vibrio species, using analysis of dnaJ, 16S rDNA, gyrB V. vulnificus–specific, gyrB V. navarrensis–specific sequences, and V. vulnificus hemolysin gene PCR (Vvh PCR), and the result of Vitek 2 system
Final identidication No. (%) Pattern No. (%) dnaJ gene sequence 16S rDNA sequence VvulGyrB PCR sequence V VnavGyrB PCR sequence Vvh PCR Vitek 2
V. vulnificus 48 (78.69) A 26 (54.17) V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
B 18 (37.5) V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
C 2 (4.17) Negative V. vulnificus V. vulnificus Negative Positive V. vulnificus
D 1 (2.08) Vibrio species V. vulnificus V. vulnificus Negative Positive V. vulnificus
E 1 (2.08) Vibrio species V. vulnificus V. vulnificus Negative Positive NT
V. parahaemolyticus 4 (6.56) F 2 (50) V. parahaemolyticus V. alginolyticus/ V. campbellii V. parahaemolyticus Negative Negative V. parahaemolyticus
G 1 (25) V. parahaemolyticus V. parahaemolyticus V. parahaemolyticus Negative Negative NT
H 1 (25) V. parahaemolyticus V. parahaemolyticus NT Negative Negative NT
V. navarrensis 3 (4.92) I 1 (33.33) V. navarrensis V. vulnificus Negative V. navarrensis Negative V. vulnificus
J 1 (33.33) V. navarrensis Vibrio species Negative V. navarrensis Negative V. vulnificus
K 1 (33.33) Vibrio species V. navarrensis Negative V. navarrensis Negative NT
V. mimicus 1 (1.64) L 1 (100) V. mimicus V. cholerae/V. mimicus Negative Negative Negative V. mimicus
V. cholerae 1 (1.64) M 1 (100) V. cholerae V. cholerae/V. mimicus Negative Negative Negative V. cholerae
V. furnissii 1 (1.64) O 1 (100) V. furnissii NT V. furnissii Negative Negative NT
V. alginolyticus 1 (1.64) P 1 (100) V. alginolyticus V. alginolyticus/ V. campbellii V. alginolyticus V. alginolyticus Negative NT
Grimontia hollisae 1 (1.64) Q 1 (100) Grimontia hollisae Grimontia hollisae Negative Negative Negative NT
Vibrio species 1 (1.6) N 1 (100) V. parahaemolyticus Vibriospecies NT Negative Negative NT

Abbreviations: NT, not tested; VnavGyrB, gyrB V. navarrensis–specific; VvulGyrB, gyrB V. vulnificus–specific; vvh, V. vulnificus hemolysin gene.

Table 3.
Identification of 61 clinical and reference strains of Vibrio species as determined by analysis of dnaJ, 16S rDNA, gyrB V. vulnificus–specific gyrB V. navarrensis–specific sequences, and V. vulnificus hemolysin gene PCR (Vvh PCR), and the result of Vitek 2 system
Strain No. Final identification dnaJ sequence 16S rDNA sequence VvulGyrB PCR sequence VnavGyrB PCR sequence Vvh PCR Vitek 2
1 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
2 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
3 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
4 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
5 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
6 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
7 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
8 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
9 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
10 V. vulnificusT V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
11 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
12 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
16 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
17 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
18 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
21 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
27 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
30 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
31 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
32 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
34 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
35 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
36 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
37 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
38 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
19 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive V. vulnificus
45 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
46 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
47 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
48 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
49 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
50 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
52 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
53 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
54 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
55 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
56 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
57 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
58 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
59 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
60 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
62 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
63 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
64 V. vulnificus V. vulnificus V. vulnificus V. vulnificus Negative Positive NT
29 V. vulnificus Vibrio species V. vulnificus V. vulnificus Negative Positive V. vulnificus
51 V. vulnificus Vibrio species V. vulnificus V. vulnificus Negative Positive NT
39 V. vulnificus Negative V. vulnificus V. vulnificus Negative Positive V. vulnificus
28 V. vulnificus Negative V. vulnificus V. vulnificus Negative Positive V. vulnificus
66 V. parahaemolyticus V. parahaemolyticus V. parahaemolyticus V. parahaemolyticus Negative Negative NT
68 V. parahaemolyticus V. parahaemolyticus V. parahaemolyticus NT Negative Negative NT
20 V. parahaemolyticus V. parahaemolyticus V. alginolyticus/V. campbellii V. parahaemolyticus Negative Negative V. parahaemolyticus
25 V. parahaemolyticusT V. parahaemolyticus V. alginolyticus/V. campbellii V. parahaemolyticus Negative Negative V. parahaemolyticus
14 V. navarrensis V. navarrensis V. vulnificus Negative V. navarrensis Negative V. vulnificus
13 V. navarrensis V. navarrensis Vibrio species Negative V. navarrensis Negative V. vulnificus
65 V. navarrensisT Vibrio species V. navarrensis Negative V. navarrensis Negative NT
26 V. mimicusT V. mimicus V. cholerae/V. mimicus Negative Negative Negative V. mimicus
24 V. choleraeT V. cholerae V. cholerae/V. mimicus Negative Negative Negative V. cholerae
67 Vibrio species V. parahaemolyticus Vibrio species NT Negative Negative NT
41 V. furnissiiT V. furnissii NT V. furnissii Negative Negative NT
23 V. alginolyticusT V. alginolyticus V. alginolyticus/V. campbellii V. alginolyticus V. alginolyticus Negative NT
42 Grimontia hollisaeT Grimontia hollisae Grimontia hollisae Negative Negative Negative NT

Abbreviations: NT, not tested; T, type strain; VnavGyrB, gyrB V. navarrensis–specific; VvulGyrB, gyrB V. vulnificus–specific; vvh, V. vulnificus hemolysin gene.

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