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Applied and Environmental Microbiology, December 1999, p. 5293-5302, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Cesar Isigidi Bin Kingombe,1,* Geert Huys,2 Mauro Tonolla,3 M. John Albert,4 Jean Swings,2 Raffaele Peduzzi,3 and Thomas Jemmi1

Laboratorium voor Microbiologie, Universiteit Gent, Gent, Belgium2; Microbiology Section, Swiss Federal Veterinary Office, Liebefeld-Bern,1 and Istituto Cantonale Batteriosierologico, Lugano,3 Switzerland; and International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDRB), Dhaka-2, Bangladesh4

Received 20 May 1999/Accepted 22 September 1999

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.

* Corresponding author. Mailing address: Microbiology Section, Swiss Federal Veterinary Office, Schwarzenburgstrasse 161, CH-3003 Liebefeld-Bern, Switzerland. Phone: 41 31 322 22 63. Fax: 41 31 323 85 70. E-mail: cesar.kingombe{at}bvet.admin.ch.

Applied and Environmental Microbiology, December 1999, p. 5293-5302, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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