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

Abstract

Staphylococcus epidermidis is a harmless human skin colonizer responsible for ~20% of orthopedic device-related infections due to its capability to form biofilm. Nowadays there is an interest in the development of anti-biofilm molecules. Marine bacteria represent a still underexploited source of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. Previous results have demonstrated that the culture supernatant of Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 impairs the formation of S. epidermidis biofilm. Further, evidence supports the hydrophobic nature of the active molecule, which has been suggested to act as a signal molecule. In this paper we describe an efficient activity-guided purification protocol which allowed us to purify this anti-biofilm molecule and structurally characterize it by NMR and mass spectrometry analyses. Our results demonstrate that the anti-biofilm molecule is pentadecanal, a long-chain fatty aldehyde, whose anti-S. epidermidis biofilm activity has been assessed using both static and dynamic biofilm assays. The specificity of its action on S. epidermidis biofilm has been demonstrated by testing chemical analogs of pentadecanal differing either in the length of the aliphatic chain or in their functional group properties. Further, indications of the mode of action of pentadecanal have been collected by studying the bioluminescence of a Vibrio harveyi reporter strain for the detection of autoinducer AI-2 like activities. The data collected suggest that pentadecanal acts as an AI-2 signal. Moreover, the aldehyde metabolic role and synthesis in the Antarctic source strain has been investigated. To the best of our knowledge, this is the first report on the identification of an anti-biofilm molecule form from cold-adapted bacteria and on the action of a long-chain fatty aldehyde acting as an anti-biofilm molecule against S. epidermidis.

Keywords: Pseudoalteromonas haloplanktis TAC125; Staphylococcus epidermidis; anti-biofilm; long fatty acid aldehyde; quorum sensing.

Figure 1

Anti-biofilm assay and identification by GC-MS analysis. (A) The anti-biofilm activity of different fractions obtained from reverse phase C₁₈ column. The fraction S eluted with 95% acetonitrile showed the highest inhibition activity. (B) GC-MS chromatogram of the fraction S. (C) Mass spectrum of compound A, and (D) compound B.

Figure 2

Relevant sections of ¹H-¹³C HSQC spectrum, recorded in CDCl₃ at 298K at 600 MHz. (A) The correlation at δ 9.79/203.1 ppm, clearly indicated the aldehydic functional group of the molecule. (B) The carbon signal at δ 203.1 ppm is in turn correlated in the HMBC experiment (data not shown), with the protons at δ 2.43 ppm (CH₂ C2). (C) The attribution of the aliphatic chain (C3–C15).

Figure 3

Pentadecanal anti-biofilm activity on S. epidermidis biofilm formation. (A) The effect of pentadecanal at different concentrations on biofilm formation of S. epidermidis O-47 and S. epidermidis RP62A. The data are reported as percentages of residual biofilm. Each data point is composed of three independent samples. (B) Biofilm formation of S. epidermidis O-47 in a BioFlux system in the presence of pentadecanal. Each image contains two channels: the bottom channel is the pentadecanal-treated sample and the top channel is the control. Bright-field microscopic images were collected at 1-min intervals. The images presented were taken from the complete set of 720 images taken at 40x magnification.

Figure 4

CLSM of S. epidermidis O-47 and S. epidermidis RP62A biofilms in the presence and absence of pentadecanal. (A) CLSM of S. epidermidis O-47 and S. epidermidis RP62A biofilms in BHI medium without pentadecanal and (C) with pentadecanal 100 μg/ml. The bacteria were grown in chamber slides for 20 h and then stained with LIVE/DEAD reagents. The green fluorescence (SYTO9) indicates viable cells PI and the red fluorescence (PI) indicates dead cells. (B) Z-stack analysis of S. epidermidis O-47 and RP62A biofilms without pentadecanal. (D) Z-stack analysis of S. epidermidis O-47 and RP62A biofilms treated with 100 μg/ml pentadecanal.

Figure 5

Anti-biofilm activity of different aldehydes and alcohols on S. epidermidis O-47. The anti-biofilm activity of different long-chain aldehydes and alcohols on S. epidermidis O-47. The data are reported as percentages of residual biofilm. Each data point is composed of four independent samples.

Figure 6

V. harveyi LuxS/AI-2 QS system responds to the presence of pentadecanal. Bioluminescence (solid lines) and growth curves (dotted lines) of the V. harveyi BB170 strain incubated for 20 h in the presence of only the AB medium (green lines), 12.5 μg/mL pentadecanal (black lines) and 200 μg/mL pentadecanal concentrations (red lines).

Figure 7

The cell-free supernatant of P. haloplanktis TAC125 grown in microaerobiosis has an anti-biofilm activity S. epidermidis O-47 biofilm formation after incubation with P. haloplanktis TAC125 cell-free supernatants obtained from sessile (SN-S) and planktonic growths (SN-P) and from microaerobiosis growth (SN-M). The data are reported as percentages of residual biofilm. Each data point represents the mean ± SD of at least three independent samples.