Alteration of virulence factors and rearrangement of pAsa5 plasmid caused by the growth of Aeromonas salmonicida in stressful conditions

Aeromonas salmonicida, a fish pathogen, is the causative agent of furunculosis. It was already shown that growing this bacterium in stressful conditions such as temperature above 22°C might lead to virulence attenuation. Unfortunately, many veterinary microbiology services and reference centers still routinely cultivate A. salmonicida at 25°C. Here we tested the presence of virulence factors by growth on specific medium as well as the integrity of the pAsa5 plasmid, which bears an important virulence factor, the type III secretion system (TTSS), by PCR analysis in twenty strains, most of which were grown at 25°C in their laboratory of origin. The analysis revealed that strains, which encountered the more stressful growth conditions displayed the most frequent absence of A-layer protein and secreted proteolytic activity. Moreover, many strains had lost parts of the pAsa5 plasmid in which the TTSS region was almost always affected. To confirm the effect of stressful growth conditions on the plasmid, three strains with an intact pAsa5 were cultured at 25°C for two weeks. A low but significant fraction of the tested colonies displayed pAsa5 rearrangements. The rearrangement always affected the TTSS region and led to a loss of virulence in the Dictyostelium discoideum co-culture assay. These results demonstrate that the instability of pAsa5 did not lead to its complete loss as previously proposed but to a more complex rearrangement phenomenon and emphasizes the necessity to grow A. salmonicida in appropriate conditions to preserve the complete virulence of the bacterium.