Large-scale biofilm cultivation of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 for physiologic studies and drug discovery

Abstract

Microbial biofilms are mainly studied due to detrimental effects on human health but they are also well established in industrial biotechnology for the production of chemicals. Moreover, biofilm can be considered as a source of novel drugs since the conditions prevailing within biofilm can allow the production of specific metabolites. Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 when grown in biofilm condition produces an anti-biofilm molecule able to inhibit the biofilm of the opportunistic pathogen Staphylococcus epidermidis. In this paper we set up a P. haloplanktis TAC125 biofilm cultivation methodology in automatic bioreactor. The biofilm cultivation was designated to obtain two goals: (1) the scale up of cell-free supernatant production in an amount necessary for the anti-biofilm molecule/s purification; (2) the recovery of P. haloplanktis TAC125 cells grown in biofilm for physiological studies. We set up a fluidized-bed reactor fermentation in which floating polystyrene supports were homogeneously mixed, exposing an optimal air-liquid interface to let bacterium biofilm formation. The proposed methodology allowed a large-scale production of anti-biofilm molecule and paved the way to study differences between P. haloplanktis TAC125 cells grown in biofilm and in planktonic conditions. In particular, the modifications occurring in the lipopolysaccharide of cells grown in biofilm were investigated.

Anti-Biofilm Activity of a Long-Chain Fatty Aldehyde from Antarctic Pseudoalteromonas haloplanktis TAC125 against Staphylococcus epidermidis Biofilm

“…Starting from the idea that the production of an anti-biofilm compound might result from the selective pressure exerted on bacteria living in oligotrophic and extreme environments like Antarctica, cold-adapted marine bacteria have been investigated as a possible source of anti-biofilm molecules. Previous papers (Klein et al, 2011 ; Papa et al, 2013b , 2015 ; Parrilli et al, 2015 , 2016 ; Sun et al, 2015 ) have confirmed that cold adapted bacteria represent an untapped reservoir of biodiversity able to synthesize a broad range of potentially valuable bioactive compounds (Klein et al, 2011 ), including anti-biofilm molecules (Papa et al, 2013b ; Parrilli et al, 2015 , 2016 ).…”

“…The culture was carried out at 15°C for 48 h, or at 4°C for 96 h, in aerobic conditions using airflow of 6 L/h, without stirring. After incubation, supports were removed from the supernatant and the adherent cells were recovered by sonication as previously described (Parrilli et al, 2016 ). In parallel the supernatant was recovered and further separated from cells by centrifugation at 13,000 rpm, sterilized by filtration through membranes with a pore diameter of 0.22 μm and stored at 4°C until use.…”

“…As reported in the previous paper (Parrilli et al, 2016 ), P. haloplanktis TAC125 supernatant deriving from sessile growth was dialyzed, and the permeated water (2 g) was then purified on a gel-filtration column (Biorad, Biogel P-2) eluted with MilliQ water. The active fractions were collected and fractionated on a C 18 reverse phase column (Sigma, 30 mL, 40 × 0.5 cm, fraction volume 4 mL), eluted with CH 3 CN:H 2 O ranging from 10 to 95% of CH 3 CN.…”

“…In particular, the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 ( P. haloplanktis TAC125), when grown with a sessile life-style, proved to be able to secrete an anti-biofilm molecule capable of inhibiting S. epidermidis biofilm formation (Papa et al, 2013b ; Parrilli et al, 2015 , 2016 ). This anti-biofilm compound impairs biofilm development and disaggregates the mature biofilm of S. epidermidis , even in dynamic conditions, without affecting the bacterial viability, showing that its action is specifically directed against biofilm (Papa et al, 2013b ; Parrilli et al, 2015 , 2016 ).…”

“…In this research study the anti-biofilm molecule produced by P. haloplanktis TAC125 has been purified and characterized. In detail, a P. haloplanktis TAC125 biofilm cultivation in automatic bioreactor (Parrilli et al, 2016 ) has been used to obtain a cell-free supernatant in a sufficient amount to purify and characterize the anti-biofilm molecule. A suitable purification protocol was developed using an activity-guided fractionation strategy.…”