This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by González-Escalona, N.
Right arrow Articles by DePaola, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by González-Escalona, N.
Right arrow Articles by DePaola, A.
Agricola
Right arrow Articles by González-Escalona, N.
Right arrow Articles by DePaola, A.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, December 2006, p. 7925-7929, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01548-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Characterization of a Vibrio alginolyticus Strain, Isolated from Alaskan Oysters, Carrying a Hemolysin Gene Similar to the Thermostable Direct Hemolysin-Related Hemolysin Gene (trh) of Vibrio parahaemolyticus{triangledown}

Narjol González-Escalona,1,2* George M. Blackstone,2 and Angelo DePaola2

Department of Food Science, North Carolina State University, Raleigh, North Carolina,1 FDA Gulf Coast Seafood Laboratory, Dauphin Island, Alabama2

Received 5 July 2006/ Accepted 9 October 2006


arrow
ABSTRACT
 
A Vibrio strain isolated from Alaskan oysters and classified by its biochemical characteristics as Vibrio alginolyticus possessed a thermostable direct hemolysin-related hemolysin (trh) gene previously reported only in Vibrio parahaemolyticus. This trh-like gene was cloned and sequenced and was 98% identical to the trh2 gene of V. parahaemolyticus. This gene seems to be functional since it was transcriptionally active in early-stationary-phase growing cells. To our knowledge, this is the first report of V. alginolyticus possessing a trh gene.


arrow
INTRODUCTION
 
Vibrio parahaemolyticus is a gram-negative, estuarine bacterial species and is the leading cause of seafood-associated bacterial gastroenteritis worldwide (3, 4). During a V. parahaemolyticus outbreak associated with Alaskan oysters in 2004 (12), numerous V. parahaemolyticus strains were isolated from oysters. Many of these strains carried both thermostable direct hemolysin (tdh) and thermostable direct hemolysin-related hemolysin (trh) genes, which are associated with pathogenicity (1, 11, 13, 17). The tdh gene can be subdivided by sequence similarities into five subtypes (tdh1 through tdh5), sharing 96 to 98% identity (14). In contrast, there exists among the trh genes a significant nucleotide sequence variation, and they can be clustered into two main subgroups, trh1 and trh2, which share 84% identity (9).

During investigation of the Alaskan V. parahaemolyticus outbreak of 2004, two strains isolated from oysters tested positive for the presence of the trh gene but were negative for the presence of the tlh gene (thermolabile hemolysin), reportedly in all V. parahaemolyticus strains (2, 21, 22, 26). These strains were also urease positive, indicating the presence of the ure gene, which is genetically linked to the trh gene in V. parahaemolyticus (6, 7). The trh gene was detected using an alkaline phosphatase-labeled DNA probe designed specifically for the detection of trh (16). The presence of the trh but not the tlh gene in those isolates was very unusual, since the trh gene has been reported only in V. parahaemolyticus. These results suggest that vibrios other than V. parahaemolyticus could be a reservoir for trh in the environment.

In the present study, we described the characterization of a Vibrio alginolyticus strain isolated from Alaskan oysters that possesses and expresses a trh gene with 98% homology to the trh2 gene of V. parahaemolyticus.


arrow
Bacterial strains and phenotypic characterization.
 
V. parahaemolyticus strain 93A-5807 (clinical isolate) was obtained from the GCSL culture collection. This strain and those described below were originally isolated from thiosulfate citrate bile sucrose agar. Individual colonies were selected and streaked for purification on T1N1 agar as recommended in the FDA's Bacteriological Analytical Manual (BAM) (FDA/CFSAN; http://www.cfsan.fda.gov/~ebam/bam-9.html). Single-well-isolated colonies were selected for all phenotypic and genotypic assays. V. parahaemolyticus strains Vp 28 (AK 2228-1 0826), Vp 32 (AK 2162-1B 1054B), and Vp 33 (AK 2162-1A 1054A) were isolated from Alaskan oysters during the 2004 outbreak and were tdh, trh, tlh, and urease positive. The presence of tdh and tlh was determined using alkaline phosphatase-labeled DNA probe colony hybridization as described in the BAM. The presence of trh was determined using alkaline phosphatase-labeled DNA probe colony hybridization (16). This assay was conducted twice to confirm that there was no error in the detection of this gene. Urease production was determined as recommended in the BAM. V. alginolyticus ATCC 33787 was kindly provided by Marlene Janes of Louisiana State University. V. alginolyticus strains Va 29 (AK 1296-A2-1 1296) and Va 30 (AK 2208-1B 1073B) were also isolated from Alaskan oysters during the 2004 outbreak and were trh positive but tlh and tdh negative by the hybridization assay described in the BAM. They produced yellow colonies on thiosulfate citrate bile sucrose agar and grew in the presence of 10% NaCl, and biochemical characterization with API-20E (bioMérieux, Inc., Hazelwood, MO) (Table 1) indicated that these two isolates were V. alginolyticus. All strains were grown at 37°C in Luria-Bertani medium (LB).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Biochemical properties of V. alginolyticus ATCC 33787, Va 29, and Va 30


arrow
DNA extraction, PCR amplification, cloning, and sequencing of the trh-like gene.
 
In order to confirm the presence of structural trh genes in V. alginolyticus strains Va 29 and Va 30, we designed a pair of primers, trh_1F and trh_570R (Table 2), that amplified both trh subgroups (9) and hybridize at the 5' and 3' ends, respectively, of the trh genes reported in GenBank (http://www.ncbi.nlm.nih.gov). V. parahaemolyticus strains Vp 93A-5807, Vp 28, Vp 32, and Vp 33 were used as a positive control for the trh gene. Bacterial DNA was extracted using a DNeasy tissue kit (QIAGEN, Valencia, CA). Figure 1A shows the relative positions of the primers employed in this study. The PCR amplification and analysis of amplicons were performed as previously described (18).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Primers used in this study


Figure 1
View larger version (35K):
[in this window]
[in a new window]

  		FIG. 1. Polyacrylamide gel electrophoresis of the products obtained after PCR amplification of the trh genes from the different strains used in this study. (A) Schematic representation of the trh gene, with the relative positions of the primers employed indicated by arrows. (B) PCR amplification products for the trh genes obtained with primers trh_1F and trh_570R. Lanes: 1, H2O; 2, Vp 28; 3, Va 29; 4, Va 30; 5, Vp 32. (C) PCR amplification products for the partial trh genes obtained with primers trh_20F and trh_570R (6, Vp 28; 7, Va 29; 8, Va 30; and 9, Vp 32) and primers trh_1F and trh_292R (strains for lanes 10 to 13 are the same as those for lanes 6 to 9). Ld indicates a 100-bp size ladder.

For the two V. alginolyticus strains, only the Va 29 isolate yielded a positive reaction when these primers were employed (Fig. 1B), although the observed band was smaller than those obtained for Vp 28, Vp 31, Vp 32, and Vp 93A-5807. The trh PCR amplification was repeated five times with the same results, confirming the presence of the trh gene in Va 29 (results not shown). Additional primers (Table 2 and Fig. 1A) targeting other regions of reported trh sequences were also employed and indicated that strain Va 30 possesses a trh-like gene. The target sequence for the trh_570R primer located near the 3' end of the trh gene appears to be missing or modified in this strain (Fig. 1C). As only Va 29 appeared to carry the entire trh gene, it was selected for further study. The sequence of the trh-like gene for this isolate was determined. The trh amplification products of strains Vp 28, Vp 29, and Vp 93A-5807 were used for cloning. PCR products were purified with a QIAquick PCR purification kit (QIAGEN) and later cloned into the TOPO TA-cloning vector, according to the manufacturer's recommendations (Invitrogen, Carlsbad, CA). Clones were screened for the presence of plasmid with inserts by PCR as indicated above (Invitrogen), except that the primers used for PCR were trh_1F and trh_570R. Plasmids containing the fragment of interest were purified with a QIAprep Spin Miniprep kit (QIAGEN). Clones containing the trh genes from strains Vp 28, Va 29, and Vp 93A-5807 were selected and sequenced in both directions. The sequencing reactions were run by MCLAB (San Francisco, CA), employing primers M13F, M13R, trh_20F, and trh_292R (Table 2). DNA sequences were inspected individually and manually assembled. The alignments and sequence similarities were determined using BioEdit (5). Sequence analysis indicated that V. parahaemolyticus strains Vp 28 and Vp 93A-5807 carried a trh1 gene, which is typical of most V. parahaemolyticus isolates that also possess tdh (17). However, the trh-like gene of the Va 29 strain was 98% identical to a trh2 gene of V. parahaemolyticus reported in GenBank (Fig. 2).


Figure 2
View larger version (42K):
[in this window]
[in a new window]

  		FIG. 2. Alignment of the trh gene sequences obtained in the present study along with two representatives of the trh gene sequences (trh1 and trh2) retrieved from GenBank. All the sequences contain their respective GenBank accession numbers. The sequences generated from this study are in italics. Nucleotide positions conserved relative to those in the sequence located at the top are indicated by dots. Gaps included to preserve the alignments are indicated by dashes.


arrow
Determination of V. parahaemolyticus species DNA-specific marker pR72H.
 
Previous results obtained by Ruimy et al. (20) showed that the 16S rRNA gene sequences of V. alginolyticus and V. parahaemolyticus are highly similar (99%) and consequently are unreliable phylogenetic markers for differentiating those species (20). To further exclude the possibility that this was an atypical V. parahaemolyticus strain, we examined Va 29 for an additional (other than tlh) V. parahaemolyticus species-specific DNA marker, pR72H (10). This test was previously validated using DNA-DNA hybridization between V. alginolyticus and V. parahaemolyticus (19). PCR was used to amplify a fragment (320 or 387 bp long) of an unknown function that is highly conserved in V. parahaemolyticus and absent in V. alginolyticus. Primers Vp33 and Vp32 (Table 2) were used under PCR conditions similar to those for the trh gene, except that 60°C was used as the annealing temperature. PCR products (10 µl each) were separated by agarose gel electrophoresis in a 2% agarose gel (Invitrogen) run at 75 V for 1.5 h in 1x Tris-acetate-EDTA. Amplification products were visualized by ethidium bromide staining. Figure 3 shows that only strains Vp 93A-5807, Vp 28, Vp 32, and Vp 33 possess this fragment, indicating that strains Va 29 and Va 30 were not V. parahaemolyticus.


Figure 3
View larger version (36K):
[in this window]
[in a new window]

  		FIG. 3. Agarose gel electrophoresis of the products obtained after PCR amplification of the pR72H fragment. The PCR products were separated in a 2% agarose gel in 1x Tris-acetate-EDTA. Lanes: 1, H2O; 2, Vp 93A-5807; 3, Vp 28; 4, Va 29; 5, Va 30; 6, Vp 32; 7, Vp 33. Ld indicates a 100-bp size ladder.


arrow
Determination of the transcriptional activity of the trh2 gene in Va 29.
 
To determine whether the trh2 gene of Va 29 was actively expressed, we isolated total RNA from early-stationary-phase (optical density at 600 nm = 1.0) cells of the Vp 28 and Va 29 strains. The presence of trh mRNA was determined by reverse transcription (RT)-PCR using primers trh_1F and trh_570R (Table 2) for the trh gene. Briefly, strains Vp 28 and Va 29 were incubated at 37°C in LB until reaching an optical density at 600 nm of 1.0. One milliliter of each culture was centrifuged for 5 min at 5,000 x g. The pellets were resuspended in 100 µl of Tris-EDTA buffer. RNA extraction was carried out using an RNeasy mini kit (QIAGEN). The RNA was eluted with 50 µl of nuclease-free water. Prior to RT-PCR, 10-µl portions of each RNA solution were treated with DNase I (Invitrogen) for 15 min at room temperature, according to the manufacturer's instructions. DNase I was then inactivated by adding 1 µl of 25 mM EDTA solution to each reaction mixture and heating it for 10 min at 65°C. Portions (3 µl) of each reaction mixture were used as a template for RT-PCR. RT-PCR was carried out with a OneStep RT-PCR kit as indicated by the manufacturer (QIAGEN), using primers trh_1F and trh_570R. The amplification products were examined by electrophoresis in polyacrylamide gels and visualized by staining them with silver nitrate as previously described (18). After the RT-PCR, a single band equal in size to the band observed after trh DNA PCR amplification using the same primers was observed for each strain (Fig. 1A, lanes 2 and 3, and 4, lanes 2 and 3). This indicates that the trh gene in each strain was actively expressed in early-stationary-phase cells. DNA contamination was ruled out, as PCR amplification of the same RNA samples used in the RT-PCR did not give any product (data not shown). While transcriptional activity does not always result in full translational expression and function, it is likely that a functional protein is produced in this case, because the two trh genes have 98% identical DNA sequences and differ at only two amino acid residues. Additionally, V. alginolyticus is the closest relative of V. parahaemolyticus, further increasing the likelihood that a functional trh2-like toxin will be produced in V. alginolyticus.


Figure 4
View larger version (55K):
[in this window]
[in a new window]

  		FIG. 4. Polyacrylamide gel electrophoresis of the products obtained after one-step RT-PCR amplification of the trh genes (with primers trh_1F and trh_570R) from the total RNA extracted from Vp 28 and Va 29 from early-stationary-phase growing cells. Lanes: 1, H2O; 2, RNA Vp 28; 3, RNA Va 29. Ld indicates a 100-bp size ladder.


arrow
Concluding remarks.
 
Being an autochthonous marine bacterium, V. alginolyticus is probably subjected to a high level of recombination with the diverse, closely related bacterial strains populating marine environments. Marine environments provide a habitat where vibrios can be exposed to high levels of gene transfer by transduction (8), and consequently, putative transfers of virulence factor genes like trh can occur between marine bacteria.

This is the first report of Vibrio spp. other than V. parahaemolyticus possessing and expressing the pathogenicity marker trh of V. parahaemolyticus. The tdh gene has been reported in vibrios other than V. parahaemolyticus (26), and some authors (15, 23) have suggested that tdh and possibly trh were acquired by ancestral V. parahaemolyticus strains from another organism. The presence of a trh2 gene actively expressed in a V. alginolyticus strain supports the hypothesis that this gene is transferred among vibrios. Xie et al. (25) showed that V. alginolyticus often possessed homologues of virulence genes (other than trh) of V. parahaemolyticus and V. cholerae, suggesting that V. alginolyticus can be a reservoir for these genes in the aquatic environment. The practical implications of these results are that detection of the trh gene in mixed cultures, such as broth enrichments or nucleic acid extracts of seafood or environmental samples, does not always imply that pathogenic V. parahaemolyticus is present.


arrow
Nucleotide sequence accession numbers.
 
The trh gene sequences have been deposited in GenBank under accession numbers DQ359748 (trh1 Vp 93A-5807), DQ359749 (trh1 Vp GCSL28), and DQ359750 (trh2 Va GCSL29).


arrow
ACKNOWLEDGMENTS
 
We thank J. L. Nordstrom for technical assistance and K. Calci for his assistance with the preparation of the manuscript. We thank the Alaska Department of Environmental Conservation for providing strains.

This study was supported by a grant from the U.S. Department of Agriculture, National Research Initiative, Competitive Grants Program, Epidemiological Approaches to Food Safety, project no. 2004-35212-14882.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: FDA Gulf Coast Seafood Laboratory, 1 Iberville Dr., Dauphin Island, AL 36528. Phone: (251) 690-3074. Fax: (251) 694-4477. E-mail: narjol{at}gmail.com. Back

{triangledown} Published ahead of print on 20 October 2006. Back


arrow
REFERENCES
 
    1
  1. Ansaruzzaman, M., M. Lucas, J. L. Deen, N. A. Bhuiyan, X. Y. Wang, A. Safa, M. Sultana, A. Chowdhury, G. B. Nair, D. A. Sack, L. von Seidlein, M. K. Puri, M. Ali, C. L. Chaignat, J. D. Clemens, and A. Barreto. 2005. Pandemic serovars (O3:K6 and O4:K68) of Vibrio parahaemolyticus associated with diarrhea in Mozambique: spread of the pandemic into the African continent. J. Clin. Microbiol. 43:2559-2562.[Abstract/Free Full Text]
    2. 2
  2. Bej, A. K., D. P. Patterson, C. W. Brasher, M. C. Vickery, D. D. Jones, and C. A. Kaysner. 1999. Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh. J. Microbiol. Methods 36:215-225.[CrossRef][Medline]
    3. 3
  3. DePaola, A., C. A. Kaysner, J. Bowers, and D. W. Cook. 2000. Environmental investigations of Vibrio parahaemolyticus in oysters after outbreaks in Washington, Texas, and New York (1997 and 1998). Appl. Environ. Microbiol. 66:4649-4654.[Abstract/Free Full Text]
    4. 4
  4. Gonzalez-Escalona, N., V. Cachicas, C. Acevedo, M. L. Rioseco, J. A. Vergara, F. Cabello, J. Romero, and R. T. Espejo. 2005. Vibrio parahaemolyticus diarrhea, Chile, 1998 and 2004. Emerg. Infect. Dis. 11:129-131.[Medline]
    5. 5
  5. Hall, T. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41:95-98.
    6. 6
  6. Iida, T., K. S. Park, O. Suthienkul, J. Kozawa, Y. Yamaichi, K. Yamamoto, and T. Honda. 1998. Close proximity of the tdh, trh and ure genes on the chromosome of Vibrio parahaemolyticus. Microbiology 144:2517-2523.[Abstract/Free Full Text]
    7. 7
  7. Iida, T., O. Suthienkul, K. S. Park, G. Q. Tang, R. K. Yamamoto, M. Ishibashi, K. Yamamoto, and T. Honda. 1997. Evidence for genetic linkage between the ure and trh genes in Vibrio parahaemolyticus. J. Med. Microbiol. 46:639-645.[Abstract/Free Full Text]
    8. 8
  8. Jiang, S. C., and J. H. Paul. 1998. Gene transfer by transduction in the marine environment. Appl. Environ. Microbiol. 64:2780-2787.[Abstract/Free Full Text]
    9. 9
  9. Kishishita, M., N. Matsuoka, K. Kumagai, S. Yamasaki, Y. Takeda, and M. Nishibuchi. 1992. Sequence variation in the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus. Appl. Environ. Microbiol. 58:2449-2457.[Abstract/Free Full Text]
    10. 10
  10. Lee, C. Y., S. F. Pan, and C. H. Chen. 1995. Sequence of a cloned pR72H fragment and its use for detection of Vibrio parahaemolyticus in shellfish with the PCR. Appl. Environ. Microbiol. 61:1311-1317.[Abstract]
    11. 11
  11. Matsumoto, C., J. Okuda, M. Ishibashi, M. Iwanaga, P. Garg, T. Rammamurthy, H. C. Wong, A. DePaola, Y. B. Kim, M. J. Albert, and M. Nishibuchi. 2000. Pandemic spread of an O3:K6 clone of Vibrio parahaemolyticus and emergence of related strains evidenced by arbitrarily primed PCR and toxRS sequence analyses. J. Clin. Microbiol. 38:578-585.[Abstract/Free Full Text]
    12. 12
  12. McLaughlin, J. B., A. DePaola, C. A. Bopp, K. A. Martinek, N. P. Napolilli, C. G. Allison, S. L. Murray, E. C. Thompson, M. M. Bird, and J. P. Middaugh. 2005. Outbreak of Vibrio parahaemolyticus gastroenteritis associated with Alaskan oysters. N. Engl. J. Med. 353:1463-1470.[Abstract/Free Full Text]
    13. 13
  13. Nair, G. B., and J. C. Hormazabal. 2005. The Vibrio parahaemolyticus pandemic. Rev. Chil. Infectol. 22:125-130.
    14. 14
  14. Nishibuchi, M., and J. B. Kaper. 1990. Duplication and variation of the thermostable direct haemolysin (tdh) gene in Vibrio parahaemolyticus. Mol. Microbiol. 4:87-99.[CrossRef][Medline]
    15. 15
  15. Nishibuchi, M., and J. B. Kaper. 1995. Thermostable direct hemolysin gene of Vibrio parahaemolyticus: a virulence gene acquired by a marine bacterium. Infect. Immun. 63:2093-2099.[Medline]
    16. 16
  16. Nordstrom, J. L., R. Rangdale, M. C. L. Vickery, A. M. B. Phillips, S. L. Murray, and A. DePaola. Evaluation of an alkaline phosphatase-labeled oligonucleotide probe for the detection and enumeration of the thermostable-related hemolysin (trh) gene of Vibrio parahaemolyticus. J. Food Prot., in press.
    17. 17
  17. Okuda, J., M. Ishibashi, E. Hayakawa, T. Nishino, Y. Takeda, A. K. Mukhopadhyay, S. Garg, S. K. Bhattacharya, G. B. Nair, and M. Nishibuchi. 1997. Emergence of a unique O3:K6 clone of Vibrio parahaemolyticus in Calcutta, India, and isolation of strains from the same clonal group from Southeast Asian travelers arriving in Japan. J. Clin. Microbiol. 35:3150-3155.[Abstract]
    18. 18
  18. Pizarro, J., E. Jedlicki, O. Orellana, J. Romero, and R. T. Espejo. 1996. Bacterial populations in samples of bioleached copper ore as revealed by analysis of DNA obtained before and after cultivation. Appl. Environ. Microbiol. 62:1323-1328.[Abstract]
    19. 19
  19. Robert-Pillot, A., A. Guenole, and J. M. Fournier. 2002. Usefulness of R72H PCR assay for differentiation between Vibrio parahaemolyticus and Vibrio alginolyticus species: validation by DNA-DNA hybridization. FEMS Microbiol. Lett. 215:1-6.[Medline]
    20. 20
  20. Ruimy, R., V. Breittmayer, P. Elbaze, B. Lafay, O. Boussemart, M. Gauthier, and R. Christen. 1994. Phylogenetic analysis and assessment of the genera Vibrio, Photobacterium, Aeromonas, and Plesiomonas deduced from small-subunit rRNA sequences. Int. J. Syst. Bacteriol. 44:416-426.[Abstract/Free Full Text]
    21. 21
  21. Taniguchi, H., H. Hirano, S. Kubomura, K. Higashi, and Y. Mizuguchi. 1986. Comparison of the nucleotide sequences of the genes for the thermostable direct hemolysin and the thermolabile hemolysin from Vibrio parahaemolyticus. Microb. Pathog. 1:425-432.[CrossRef][Medline]
    22. 22
  22. Taniguchi, H., H. Ohta, M. Ogawa, and Y. Mizuguchi. 1985. Cloning and expression in Escherichia coli of Vibrio parahaemolyticus thermostable direct hemolysin and thermolabile hemolysin genes. J. Bacteriol. 162:510-515.[Abstract/Free Full Text]
    23. 23
  23. Terai, A., K. Baba, H. Shirai, O. Yoshida, Y. Takeda, and M. Nishibuchi. 1991. Evidence for insertion sequence-mediated spread of the thermostable direct hemolysin gene among Vibrio species. J. Bacteriol. 173:5036-5046.[Abstract/Free Full Text]
    24. 24
  24. Vickery, M. C. L., G. M. Blackstone, J. L. Nordstrom, and A. DePaola. 2003. Detection and quantification of total and potentially virulent Vibrio parahaemolyticus using a 4-channel multiplex real-time PCR targeting the tl, tdh, and trh genes and a novel PCR internal control, abstr. Q-083, p. 529. Abstr. 103rd Gen. Meet. Am. Soc. Microbiol. American Society for Microbiology, Washington, D.C.
    25. 25
  25. Xie, Z. Y., C. Q. Hu, C. Chen, L. P. Zhang, and C. H. Ren. 2005. Investigation of seven Vibrio virulence genes among Vibrio alginolyticus and Vibrio parahaemolyticus strains from the coastal mariculture systems in Guangdong, China. Lett. Appl. Microbiol. 41:202-207.[CrossRef][Medline]
    26. 26
  26. Zhang, X. H., and B. Austin. 2005. Haemolysins in Vibrio species. J. Appl. Microbiol. 98:1011-1019.[CrossRef][Medline]

Applied and Environmental Microbiology, December 2006, p. 7925-7929, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01548-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Nordstrom, J. L., Vickery, M. C. L., Blackstone, G. M., Murray, S. L., DePaola, A. (2007). Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters. Appl. Environ. Microbiol. 73: 5840-5847 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by González-Escalona, N.
Right arrow Articles by DePaola, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by González-Escalona, N.
Right arrow Articles by DePaola, A.
Agricola
Right arrow Articles by González-Escalona, N.
Right arrow Articles by DePaola, A.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»