PCR detection, characterization, and distribution of virulence genes in Aeromonas spp.
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.
Kingombe
C I
CI
Microbiology Section, Swiss Federal Veterinary Office, Liebefeld-Bern, Switzerland.
Huys
G
G
Tonolla
M
M
Albert
M J
MJ
Swings
J
J
Peduzzi
R
R
Jemmi
T
T
D000333:Aeromonas / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity*; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics*; D002417:Cattle; D017931:DNA Primers; D019003:Deoxyribonuclease HpaII; D005060:Europe; D006460:Hemolysin Proteins / Q000235:genetics*; D008460:Meat / Q000382:microbiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D052899:Pore Forming Cytotoxic Proteins; D011200:Poultry; D017747:Seafood / Q000382:microbiology; D013552:Swine; D014675:Vegetables / Q000382:microbiology; D014774:Virulence / Q000235:genetics*
D001427:Bacterial Toxins; D017931:DNA Primers; D006460:Hemolysin Proteins; D052899:Pore Forming Cytotoxic Proteins; C032676:aerolysin; D019003:Deoxyribonuclease HpaII