A quantitative assay was used to study the extracellular proteolytic activity of 160 Aeromonas hydrophila isolates on six various substrates: elastin, albumin, casein, gelatin, fibrinogen and hemoglobin.  The alkaline reversion reaction of Triple Sugar Iron agar (TSI) slants indicating a mixed hydrolytic reaction on protein was also observed.

Statistical analysis was used to evaluate the relationship between observed proteolytic reactions and the virulence of these isolates in goldfish.  Nearly 100% of 160 A. hydrophila isolates produced hydrolytic activity on elastin, casein, gelatin, albumin and fibrinogen. Over 70% of these isolates hydrolyzed hemoglobin and showed the alkaline reversion of TSI.  Quantitatively, positive isolates showed different degrees of activity.

A distribution graph of the frequency of activity which was measured by "zone ratio" (R) indicates a normal curve in all reactions except alkaline reversion of TSI and hemoglobin hydrolytic reaction. Statistical analysis showed these different proteolytic reactions (including alkaline reversion of TSI) to be correlated with each other.

A pathogenicity study using 40 representative isolates from the 160 studied showed that 70% of the isolates were virulent in goldfish.  The degree of virulence of the isolates showed a strong correlation with alkaline reversion of TSI and elastin hydrolytic reactions and lesser correlation with fibrinogen, gelatin, casein, hemoglobin and albumin hydrolytic reactions.

A primary study to purify the proteases from A. hydrophila isolates showed that there are at least two groups of proteases which have different activity on protein substrates.