Analysis of gyrB and toxR gene sequences of Vibrio hollisae and development of gyrB- and toxR-targeted PCR methods for isolation of V. hollisae from the environment and its identification

Abstract

Isolation of Vibrio hollisae strains, particularly from the environment, is rare. This may be due, in part, to the difficulty encountered when using conventional biochemical tests to identify the microorganism. In this study, we evaluated whether two particular genes may be useful for the identification of V. hollisae. The two genes are presumed to be conserved among the bacterial species (gyrB) or among the species of the genus Vibrio (toxR). A portion of the gyrB sequence of V. hollisae was cloned by PCR using a set of degenerate primers. The sequence showed 80% identity with the corresponding Vibrio parahaemolyticus gyrB sequence. The toxR gene of V. hollisae was cloned utilizing a htpG gene probe derived from the V. parahaemolyticus htpG gene, which is known to be linked to the toxR gene in V. hollisae. The coding sequence of the cloned V. hollisae toxR gene had 59% identity with the V. parahaemolyticus toxR coding sequence. The results of DNA colony hybridization tests using the DNA probes derived from the two genes of V. hollisae indicated that these gene sequences could be utilized for differentiation of V. hollisae from other Vibrio species and from microorganisms found in marine fish. PCR methods targeting the two gene sequences were established. Both PCR methods were shown to specifically detect the respective target sequences of V. hollisae but not other organisms. A strain of V. hollisae added at a concentration of 1 to 10(2) CFU/ml to alkaline peptone water containing a seafood sample could be detected by a 4-h enrichment incubation in alkaline peptone water at 37 degrees C followed by quick DNA extraction with an extraction kit and 35-cycle PCR specific for the V. hollisae toxR gene. We conclude that screening of seafood samples by this 35-cycle, V. hollisae toxR-specific PCR, followed by isolation on a differential medium and identification by the above htpG- and toxR-targeted PCR methods, can be useful for isolation from the environment and identification of V. hollisae.

FIG. 1

DNA colony hybridization test with the V. hollisae gyrB gene probe for strains of V. hollisae, other Vibrio species, and E. coli. (A) Location of the inoculated strains (numbers correspond to dot positions in panel B): 1 to 5, V. hollisae strains 89A 1961, 89A 1960, 89A 4206, 91A 2120, and RIMD 2221011, respectively; 6, V. cholerae O139 MO45; 7, V. cholerae O1 NIH 35A3; 8, V. anguillarum PT-87050; 9, V. alginolyticus AM2; 10, V. mimicus RIMD 2218001; 11, V. vulnificus RIMD 2219009; 12, V. fluvialis RIMD 2220002; 13, V. furnisii RIMD 2223001; 14, V. damsela RIMD 2222001; 15, V. parahaemolyticus WP1; 16, E. coli MC1061. (B) Hybridization signals of the test strains detected on the X-ray film.

FIG. 2

DNA colony hybridization test with the V. hollisae toxR gene probe for strains of V. hollisae and other Vibrio species. (A) Location of the inoculated strains (numbers correspond to dot positions in panel B): 1, V. cholerae O1 NIH41; 2, V. cholerae O1 NIH41; 3, V. cholerae O139 MO45; 4, V. cholerae non-O1, non-O139 AM21; 5, V. hollisae 525-82; 6, V. anguillarum PT-87050; 7, V. alginolyticus 219; 8, V. alginolyticus 220; 9, V. mimicus RIMD 2218001; 10, V. vulnificus RIMD 2219009; 11, V. fluvialis RIMD 2220002; 12, V. furnisii RIMD 2223001; 13, V. damsela RIMD 2222001; 14, V. metchnikovii RIMD 2208006; 15 to 24, V. hollisae strains 85A 7503, 89A 1961, 89A 1960, 89A 4206, 91A 2120, 93A 5688, SJ 90, FO 93, CDC 9039-81, and 4047, respectively. (B) Hybridization signals of the test strains detected on the X-ray film.

FIG. 3

Detection of the V. hollisae gyrB gene by PCR. Primer pair HG-F1 and HG-R2 and the amplification conditions and gel electrophoresis method described in Materials and Methods were employed. Lanes: 1, molecular weight markers (φX 174 phage DNA digested with HaeIII); 2 to 9, V. hollisae strains 89A 1961, 89A 1960, 89A 4206, 91A 2120, 93A 5688, SJ 90, FO 93, and RIMD 2221011, respectively; 10, V. anguillarum PT-87050; 11, V. cholerae O1 NIH41; 12, V. fluvialis RIMD 2220002; 13, V. furnisii RIMD 2223001; 14, V. parahaemolyticus WP1; 15, V. vulnificus RIMD 2219009; 16, V. alginolyticus AM2; 17, V. damsela RIMD 2222001. The position of the specific amplicons (363 bp) is indicated by the solid triangle.

FIG. 4

Detection of the V. hollisae toxR gene by PCR. Primer pair HT-F3 and HT-R2 and the amplification conditions and gel electrophoresis method described in Materials and Methods were employed. Lanes 1 and 2, V. hollisae strains 89A 1961 and 89A 1960, respectively; 3, Vibrio mediterranei ATCC 43341; 4, Vibrio orientalis ATCC 33934; 5, Vibrio diazotrophicus ATCC 33466; 6, V. fluvialis NCTC 11327; 7, Vibrio gazogenes ATCC 29988; 8, Vibrio proteolyticus NCMB 1326; 9, Vibrio nigripulchritudo ATCC 27043; 10, Vibrio cincinnatiensis ATCC 35912; 11, Vibrio navarrensis ATCC 51183; 12, Vibrio mytili ATCC 51288; 13, Vibrio ordalii ATCC 33509; 14, Vibrio ichthyoenteri IFO 15847; 15, Vibrio penaeicida IFO 15640; 16, Vibrio splendidus ATCC 33125; 17, molecular weight markers (φX 174 phage DNA digested with HaeIII). The position of the specific amplicons (306 bp) is indicated by the solid triangle.