Multiplex PCR Method for Detection of Three Aeromonas Enterotoxin Genes

Abstract

A novel multiplex PCR method using three sets of specific primers was developed for the detection of the cytotoxic (act), heat-labile (alt), and heat-stable (ast) enterotoxin genes in Aeromonas spp. This assay was used to characterize 35 reference strains as well as 537 food-borne isolates. A total of seven gene pattern combinations were encountered, including act, alt, act/alt, act/alt/ast, act/alt/148-bp amplicon, alt/ast, and alt/148-bp amplicon. The alt gene was detected with 34 reference strains (97%) and occurred singly in 14% of these strains. The frequency of occurrence of the act/alt, act/alt/ast, and alt/ast gene patterns in reference strains was 14 (40%), 2 (6%), and 2 (6%), respectively. An unpredicted amplicon was detected in 11 reference strains (31%). Characterization of this amplicon showed that its size was 148 bp, as generated by the AHLF and AHLR primers, and that it uniquely aligned with the Aeromonas salmonicida A449 genome sequence (GenBank accession number CP000644). This amplicon was named Aeromonas salmonicida subsp. salmonicida hypothetical protein amplicon (AssHPA). In the 537 food-borne isolates, the act and alt genes were most dominant and were detected in 349 (65%) and 452 (84%) isolates, respectively, either alone or in combinations. The act and alt genes occurred singly in 30 (6%) and 128 (24%) of these strains, respectively. The act/alt gene pattern occurred in 315 isolates (59%), whereas the ast gene was always linked to strains exhibiting the act/alt/ast and alt/ast gene combinations in 4 (0.7%) and 5 (0.9%) isolates, respectively. The uniplex amplification of three enterotoxin genes separately confirms the specificity of the unique selected primers. This multiplex PCR is rapid and simple and can detect the presence of three Aeromonas enterotoxin genes in a single assay.Aeromonas spp. are widely distributed in aquatic environments and are isolated from a wide range of food of animal and plant origin (6). Aeromonas spp. can produce several putative virulence factors, including extracellular enzymes, siderophores, cytotoxic and cytotonic enterotoxins, Shiga-like toxins, endotoxins, invasins, and adhesins (18). Aeromonas spp. can grow and produce toxins in foods at refrigeration temperatures (17). The recent isolation of enterotoxigenic aeromonads from drinking water in the United States (21) reiterates the potential human health hazard of waterborne Aeromonas spp. The observation that strains harboring the alt and ast genes were more prevalent in children with diarrhea than in healthy controls underlines the importance of enterotoxins in the pathogenicity of aeromonads (3).Exotoxins are major virulence factors of Aeromonas spp. that include a cytotoxic heat-labile enterotoxin (Act), also known as aerolysin/hemolysin (8); a cytotonic heat-labile enterotoxin (Alt), also known as lipase, extracellular lipase, or phospholipase (4, 10, 24); and a cytotonic heat-stable enterotoxin (Ast) (9). These toxins are encoded by the genes act (GenBank accession number M84709), alt (GenBank acces...

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

“…hydrophila, A. sobria, A. caviae, A. jandaei, A. veronii, A. schubertii and A. trota and the dominant species was found to be A. hydrophila (48%), followed by A. sobria (15%) and A. caviae (15%) ( Tables 3 and 4). In agreement with this, Bin Kingombe et al (2010) also reported that the majority of the food-borne isolates belonged to A. caviae complex, A. hydrophila complex and A. sobria complex. Ottaviani et al (2011) also reported that A. hydrophila and A. sobria have been frequently isolated from food and environmental samples, which supported our findings.…”

“…The overall prevalence of the alt, act/hlyA/aer and ast genes was recorded to be 89.8%, 72.9% and 20%, respectively. In disagreement with some of the earlier reports (Sen and Rodger, 2004;Balsalobre et al, 2009) but in accordance with the others (Pablos et al, 2009;Bin Kingombe et al, 2010;Ottaviani et al, 2011;Pinto et al, 2012), the alt gene was found to be dominant among the MPCR positive isolates of Aeromonas spp. Balsalobre et al (2009) reported that 70.7%, 97.6% and 26.8% of the A. hydrophila strains tested were positive for act/hlyA/aer, ast and alt genes, respectively, and 2.4% and 36.6% of the A. jandaei strains were positive for act/hlyA/aer complex and alt genes.…”

“…and always in combination with alt and act/hlyA/aer genes. Bin Kingombe et al (2010) reported the same for the food borne isolates of Aeromonads.…”

“…Balsalobre et al (2009) reported that 70.7%, 97.6% and 26.8% of the A. hydrophila strains tested were positive for act/hlyA/aer, ast and alt genes, respectively, and 2.4% and 36.6% of the A. jandaei strains were positive for act/hlyA/aer complex and alt genes. Bin Kingombe et al (2010) reported that 36 % of isolates of food borne Aeromonas spp. as A. caviae complex, 38% as A. hydrophila complex and 24% as A. sobria complex, and 23 % as other aeromonads by a multiplex PCR targeting ast, alt and act enterotoxin genes.…”

“…(Pinto et al, 2012) and primers AHS and AHL amplify a 536 bp and 361 bp fragment of the heat-stable enterotoxin (ast) gene and the heat-labile enterotoxin (alt) gene, repectively for Aeromonas spp. (Bin Kingombe et al, 2010). The primer act/hlyA/aer complex amplifies 400 bp fragment of cytotoxic enterotoxin, aerolysin and hemolysin gene of Aeromonas spp.…”