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Journal of Clinical Microbiology, March 2003, p. 1048-1054, Vol. 41, No. 3
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.3.1048-1054.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Detection and Characterization of the Hemolysin Genes in Aeromonas hydrophila and Aeromonas sobria by Multiplex PCR

Gehua Wang,1* Clifford G. Clark,1 Chenyi Liu,1 Chad Pucknell,1 Cindy K. Munro,2 Tamara M. A. C. Kruk,1 Richard Caldeira,1 David L. Woodward,1 and Frank G. Rodgers1,{dagger}

National Laboratory for Enteric Pathogens,1 National Laboratory for Bacteriology, National Microbiology Laboratory, Winnipeg, Manitoba, Canada2

Received 13 June 2002/ Returned for modification 6 August 2002/ Accepted 28 November 2002

A multiplex PCR assay was designed to amplify the Aeromonas hydrophila and A. veronii bv. sobria hemolysin and aerolysin genes. The assay was evaluated by using 121 clinical isolates and 7 reference strains of Aeromonas spp., and these were divided into five genotypes on the basis of the results of the multiplex PCR. The five genotypes were characterized as type 1 for those carrying the ahh1 gene only (36% of isolates), type 2 for those carrying the asa1 gene only (8.5% of isolates), type 3 for those carrying both the ahh1 and the asa1 genes (4% of isolates), type 4 for those carrying the ahh1 gene and the A. hydrophila aerA (aerolysin) gene (37.5% of isolates), and type 5 for those in which no hemolysin genes were detected (14% of isolates). The most common single hemolysin gene carried among all the Aeromonas isolates examined was ahh1, with 99 of 128 (77%) of isolates testing positive for this gene either alone or in combination with other hemolysin genes. Phenotypic expression of toxins was evaluated in a Vero cell culture cytotoxicity assay. These results indicated that there is a statistically significant correlation between the cytotoxin titers and the hemolysin genotype. Isolates belonging to genotype 4 (carrying both the ahh1 gene and the aerolysin and hemolysin aerA genes) expressed higher cytotoxin titers than isolates of the other genotypes (P < 0.001). These isolates were more cytotoxic in cell culture and may have greater clinical significance.

* Corresponding author. Mailing address: Special Projects Unit, National Laboratory for Enteric Pathogens, National Microbiology Laboratory, Winnipeg, Manitoba R3E 3R2, Canada. Phone: (204) 789-6077. Fax: (204) 789-5012. E-mail: Gehua_Wang{at}hc-sc.gc.ca.

{dagger} Present address: Department of Microbiology, University of New Hampshire, Durham, NH 03824.

Journal of Clinical Microbiology, March 2003, p. 1048-1054, Vol. 41, No. 3
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.3.1048-1054.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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