PCR Detection, Characterization, and Distribution of Virulence Genes in Aeromonas spp

Abstract

We found 73.1 to 96.9% similarity by aligning the cytolytic enterotoxin gene of Aeromonas hydrophila SSU (AHCYTOEN; GenBank accession no. M84709 ) against aerolysin genes ofAeromonas spp., suggesting the possibility of selecting common primers. Identities of 90 to 100% were found among the eight selected primers from those genes. Amplicons obtained fromAeromonas sp. reference strains by using specific primers for each gene or a cocktail of primers were 232 bp long. Of hybridization group 4/5A/5B (HG4/5A/5B), HG9, and HG12 or non-Aeromonas reference strains, none were positive. PCR-restriction fragment length polymorphism (PCR-RFLP) withHpaII yielded three types of patterns. PCR-RFLP 1 contained two fragments (66 and 166 bp) found in HG6, HG7, HG8, HG10, and HG11. PCR-RFLP 2 contained three fragments (18, 66, and 148 bp) found in HG1, HG2, HG3, and HG11. PCR-RFLP 3, with four fragments (7, 20, 66, and 139 bp), was observed only in HG13. PCR-amplicon sequence analysis (PCR-ASA) revealed three main types. PCR-ASA 1 had 76 to 78% homology with AHCYTOEN and included strains in HG6, HG7, HG8, HG10, and HG11. PCR-ASA 2, with 82% homology, was found only in HG13. PCR-ASA 3, with 91 to 99% homology, contained the strains in HG1, HG2, HG3, and HG11. This method indicated that 37 (61%) of the 61 reference strains were positive with the primer cocktail master mixture, and 34 (58%) of 59 environmental isolates, 93 (66%) of 141 food isolates, and 100 (67%) of 150 clinical isolates from around the world carried a virulence factor when primers AHCF1 and AHCR1 were used. In conclusion, this PCR-based method is rapid, sensitive, and specific for the detection of virulence factors of Aeromonas spp. It overcomes the handicap of time-consuming biochemical and other DNA-based methods.

Potential virulence and antimicrobial susceptibility of Aeromonas popoffii recovered from freshwater and seawater

“…Our study demonstrated that the occurrence of the aerolysin/hemolysin genes in A. popo¤i (92%) is similar to that in common clinical species (e.g. A. veronii bt sobria), and also in the ¢sh pathogen A. salmonicida [13]. In the original description, the isolates of A. popo¤i were tested in sheep blood at 37³C and considered non-L-hemolytic [3].…”

“…Sequences from GenBank (GB) and European Molecular Biology Laboratory (EMBL) were used to design primers (Primer Designer 3 software, Scienti¢c Educational Software, Durham, NC, USA) for each group of genes ( Table 2) after alignment with a CLUSTAL W program [12]. The gene accession numbers for aerolysin/hemolysin were those of the 10 sequences used by Kingombe et al [13], while the others were: serine protease, GB AF159142 [14] and EMBL X67043 [15]; GCAT, EMBL X70686 [16] and X07279 [17]; lipases, GB U63543 [18], S65123 [19], AF092033 [20] and U14011 [21]; DNase, GB AF004392 (unpublished), L78266 [22] and M99491 [23]. PCR ampli-¢cations were performed with 1.2^2 Wg of DNA as previously described [11] and using conditions shown in Table 2.…”

Chironomid egg masses harbour the clinical speciesAeromonas taiwanensisandAeromonas sanarellii

“…The genes that encode the flagella (fla), lateral flagella (lafA), elastase (ahpB), cytotoxic and cytotonic enterotoxins (act, ast, alt), lipase (pla/lipH3/apl-l/lip), aerolysin/haemolysin (aerA) and serine protease (serine) were screened for all strains of both species using the conditions and primers described previously (Kingombe et al, 1999;Chacó n et al, 2004;Sen & Rodgers, 2004;Aguilera-Arreola et al, 2005). The TTSS genes ascF-G and ascV and the genes encoding the toxins delivered by this system, that is, AexT (aexT) and AopP (aopP), were investigated using conditions and primers previously described (Braun et al, 2002;Chacó n et al, 2004;Fehr et al, 2006).…”

Detection of three virulence genes alt, ahp and aerA in Aeromonas hydrophila and their relationship with actual virulence to zebrafish

“…An interesting approach for the direct detection of potentially pathogenic Aeromonas spp. isolates is the use of virulence determinants as genetic markers (Kingombe et al 1999).Analysis of pathogenic mechanisms associated with A. hydrophila has identified various virulence factors including toxins with haemolytic, cytotoxic and enterotoxic activities Singh et al 2009), protease (Cascó n et al 2000a,b), and structural features associated with adhesion, cell invasion and resistance to phagocytosis, such as S-layer proteins (Murray et al 1988). Heuzenroeder et al (1999) reported that the direct detection of the haemolytic genes aerA and hlyA was a reliable approach for identifying potentially pathogenic Aeromonas strains.…”