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

Abstract

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.

Detection of aerolysin gene in Aeromonas hydrophila isolated from fish and pond water

“…However, 22 isolates were positive for presence of 326 bp aerolysin gene by PCR amplifi cation. It is possible that some of the isolates though positive for aerolysin gene did not cause hemolysis on blood agar either due to disruption of gene or due to mutation in gene [13]. Another interesting fi nding is that there appears a positive correlation between the phenotypic production of aerolysin toxin and the pathogenicity of A. hydrophila.…”

“…In non-hemolytic A. hydrophila isolates (5 no. ), three were positive for hemolysin and remaining two for both genes [13]. Recently, specifi c primers based on β-hemolysin gene (208 bp) have been designed for the detection of pathogenic isolates of A. hydrophila of fi sh from China [15].…”

“…PCR is used to amplify a precise fragment of DNA from a complex mixture of starting material usually template genomic DNA. A number of reports are available for PCR amplifi cation of conserved aerolysin gene [11][12][13] and hemolysin gene of A. hydrophila [13][14][15]. Other virulence genes of A. hydrophila have also been detected by PCR.…”

Prevalence of Hemolytic and Enterotoxigenic Aeromonas Spp. in Healthy and Diseased Freshwater Food Fishes as Assessed by Multiplex PCR

“…3), which is very well conserved in Aeromonas spp. (Martinez-Murcia 1999) and reported to be positive for all isolates of Aeromonas (Wang et al, 2003). The selected toxin genes appeared in four different patterns viz.…”

Detection of toxigenic strains of Aeromonas species in foods by a multiplex PCR assay

“…Several virulence-associated genes have been targeted for the detection of potentially pathogenic aeromonads by PCR assays that include either cytolysin or hemolysin or both of these genes [11][12][13][14][15]. Recently Chu and Lu [14] reported a multiplex PCR (mPCR) to rapidly and specifi cally detect pathogenic A. hydrophila from symptomatic and asymptomatic fi sh targeting aerolysin and 16S rDNA genes.…”