Bacteriostatic anti-Vibrio parahaemolyticus activity of Pseudoalteromonas sp. strains DIT09, DIT44 and DIT46 isolated from Southern Chilean intertidal Perumytilus purpuratus

Abstract

We characterised the anti-Vibrio parahaemolyticus (anti-V. parahaemolyticus) marine bacteria DIT09, DIT44 and DIT46 isolated from the intertidal mussel Perumytilus purpuratus. The 16S rRNA gene sequences identify a Pseudoalteromonas sp. that form a clade with P. prydzensis and P. mariniglutinosa. The strains produced bacteriostatic anti-V. parahaemolyticus agents during the exponential growth phase, which were also active against V. cholerae and V. anguillarum, but not on other Gram positive and Gram negative bacteria. Bacteriostatic agents could be permeated by analytic ultra-filtration with 3.5 kDa cut-off, partially precipitated with 70 and 90 % ammonium sulphate, but not extracted with ethyl acetate. Reverse-phase HPLC revealed the production of a set of 5–6 active compounds by each strain (elution from 20 to 40 % acetonitrile), with similar but non identical HPLC patterns. Additionally, V. parahaemolyticus was able to progressively overcome the inhibition of antibiotics in trypticase soy agar with Fe(III) 0.5 up to 2 mM, suggesting the involvement of a set of novel siderophore or active molecules targeted at different Fe-siderophore uptake systems. The overall findings suggest that Pseudoalteromonas sp. DIT strains produce a putatively novel class of bacteriostatic and probably amphiphilic anti-Vibrio agents, indicating the need for further studies with chemical purification followed by their structural and functional characterization. Finally, the crude cell-free extracts, as well as the strains incubated at 10³ and 10⁵ c.f.u./mL, did not cause mortality in Artemia franciscana nauplii, suggesting that these bacteria are serious candidates for further probiotic evaluations with shellfish and fish cultures.