Dataset Information

Cloning, sequencing, and role in virulence of two phospholipases (A1 and C) from mesophilic Aeromonas sp. serogroup O:34.

ABSTRACT: Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Escherichia coli DH5alpha. Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively. Defined insertion mutants of A. hydrophila AH-3 defective in either pla or plc genes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic. A. hydrophila AH-3 plc mutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plc single gene on these mutants abolished this effect, suggesting that Plc protein is a virulence factor in the mesophilic Aeromonas sp. serogroup O:34 infection process.

SUBMITTER: Merino S

PROVIDER: S-EPMC96688 | biostudies-literature | 1999 Aug

REPOSITORIES: biostudies-literature

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Cloning, sequencing, and role in virulence of two phospholipases (A1 and C) from mesophilic Aeromonas sp. serogroup O:34.

Merino S S Aguilar A A Nogueras M M MM Regue M M Swift S S Tomás J M JM

Infection and immunity 19990801 8

Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Escherichia coli DH5alpha. Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative ...[more]

PMID: 10417167

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Project description:Aeromonas spp. are ubiquitous bacteria that have received increasing attention as human pathogens because of their widespread occurrence in food, especially seafood and vegetables. The aim of this work was to assess the species identity and phylogenetic relationship of 118 Aeromonas strains isolated from fresh retail sushi from three producers, and to characterize the isolates with respect to genetic and phenotypic virulence factors. We also evaluate the potential hazard associated with their presence in ready-to-eat seafood not subjected to heat treatment. Mesophilic Aeromonas salmonicida was most prevalent (74%), followed by A. bestiarum (9%), A. dhakensis (5%), A. caviae (5%), A. media (4%), A. hydrophila (2%), and A. piscicola (1%). All isolates were considered potentially pathogenic due to the high prevalence of genes encoding hemolysin (hlyA) (99%), aerolysin (aerA) (98%), cytotoxic enterotoxin (act) (86%), heat-labile cytotonic enterotoxin (alt) (99%), and heat-stable cytotonic enterotoxin (ast) (31%). The shiga-like toxins 1 and 2 (stx-1 and stx-2) were not detected. Moreover, there was heterogeneity in toxin gene distribution among the isolates, and the combination of act/alt/hlyA/aerA was most commonly detected (63%). β-hemolysis was species-dependent and observed in 91% of the isolates. All A. media and A. caviae strains were non-hemolytic. For isolates belonging to this group, lack of hemolysis was possibly related to the absence of the act gene. Swimming motility, linked to adhesion and host invasion, occurred in 65% of the isolates. Partial sequencing of the gyrB gene demonstrated its suitability as a genetic marker for Aeromonas species identification and for assessment of the phylogenetic relationship between the isolates. The gyrB sequence divergence within a given species ranged from 1.3 to 2.9%. A. bestiarum, A. salmonicida, and A. piscicola were the most closely related species; their sequences differed by 2.7-3.4%. The average gyrB sequence similarity between all species was 93%, demonstrating its acceptable taxonomic resolution. The presence of multiple species of potential pathogenic Aeromonas in fresh retail sushi raises new food safety issues related to the increased consumption of ready-to-eat food composed of raw ingredients.

Project description:Cases of Aeromonas diarrhea have been described all over the world. The genus Aeromonas includes ca. 30 species, of which 10 have been isolated in association with gastroenteritis. The dominating species that account for ca. 96% of the identified strains are Aeromonas caviae, A. veronii, A. dhakensis, and A. hydrophila. However, the role of Aeromonas as a true enteropathogen has been questioned on the basis of the lack of outbreaks, the non-fulfillment of Koch's postulates and the low numbers of acute illnesses in the only existing human challenge study. In the present study we reassess the enteropathogenicity of Aeromonas using dose response models for microbial infection and acute illness. The analysis uses the data from the human challenge study and additional data from selected outbreak investigations where the numbers exposed and the dose were reported, allowing their inclusion as "natural experiments". In the challenge study several cases of asymptomatic shedding were found (26.3%, 15/57), however, only 3.5% (2/57) of those challenged with Aeromonas developed acute enteric symptoms (i.e., diarrhea). The "natural experiments" showed a much higher risk of illness associated with exposure to Aeromonas, even at moderate to low doses. The median dose required for 1% illness risk, was ~1.4 × 104 times higher in the challenge study (1.24 × 104 cfu) compared to natural exposure events (0.9 cfu). The dose response assessment presented in this study shows that the combined challenge and outbreak data are consistent with high infectivity of Aeromonas, and a wide range of susceptibility to acute enteric illness. To illustrate the outcomes, we simulate the risk associated with concentrations of Aeromonas found in different water and food matrices, indicating the disease burden potentially associated with these bacteria. In conclusion this study showed that Aeromonas is highly infectious, and that human susceptibility to illness may be high, similar to undisputed enteropathogens like Campylobacter or Salmonella.

Dataset Information

Cloning, sequencing, and role in virulence of two phospholipases (A1 and C) from mesophilic Aeromonas sp. serogroup O:34.

Publications

Cloning, sequencing, and role in virulence of two phospholipases (A1 and C) from mesophilic Aeromonas sp. serogroup O:34.

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