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 of Aeromonas 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 from Aeromonas 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) with HpaII 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.

FIG. 1

PCR-RFLP maps of the 232-bp amplicon showing the three types of HpaII endonuclease restriction fragment patterns. The upper map represents PCR-RFLP 1 with two fragments of 66 and 166 bp. The second map represents PCR-RFLP 2 with three fragments of 18, 66, and 148 bp. The third map represent PCR-RFLP 3 with fragments of 7, 20, 66, and 139 bp. The lower map represents the scaled 232-bp amplicon. The arrows flanking the PCR-RFLP 1 map represent primers which may be AHCF1, -2, or -3 and AHCR1, -2, -3, -4, or -5.

FIG. 2

PCR amplicon of 232 bp and PCR-RFLP types of 232-bp amplicons of different HGs of reference strains of Aeromonas spp. PCR amplicon and PCR-RFLP types of reference and wild-type strains of Aeromonas spp. on 4% Metaphor agarose gel (Bioconcept, Allschwil, Switzerland) stained with ethidium bromide at 0.5 μg/ml (Bio-Rad). Lanes: 1 and 15, DNA size marker VIII (Roche Diagnostics AG; catalog no. 1336 045); 2, a PCR-RFLP 2 of A17 (ATCC 7966), a reference strain of HG1 isolated from canned milk from the United States; 3, PCR-RFLP 2 of A307 (ATCC 51108), a reference strain of HG2 isolated from a fish in France; 4, PCR-RFLP 2 of A14 (LMG 13674), a reference strain of HG3 that is an environmental isolate from Germany; 5, PCR-RFLP type 1 of A914 (CDC 9179), a reference strain of HG6 (source unknown); 6, PCR-RFLP 1 of A312 (ATCC 43979), a reference strain of HG7 that is a fish isolate from France; 7, PCR-RFLP 1 of A10 (CCUG 30356), a reference strain of HG8 that is an environmental isolate from Germany; 8, PCR-RFLP 1 of A901 (ATCC 35624), a reference strain of HG10 that is a clinical isolate (sputum) from the United States; 9, PCR-RFLP 1 of A926 (CDC 3136), a reference strain of HG11 that is a water isolate from the United States; 10, PCR-RFLP 3 of A1645 (ATCC 49659), a reference strain of HG13 that is a clinical isolate from the United States; 11, uncut 232-bp amplicon of strain A1645; 12, PCR-RFLP 2 of a wild-type strain (BP 3) of HG1 that is a clinical isolate of HG1 from Bangladesh; 13, PCR-RFLP 2 of a wild-type strain (CBK 45) of HG3 that is a pork meat isolate from Switzerland; 14, PCR-RFLP 1 of a wild-type strain (BE 6) of HG8 that is an environmental isolate from Bangladesh.