Isolation and structural characterisation of two antibacterial free fatty acids from the marine diatom, Phaeodactylum tricornutum

Abstract

One solution to the global crisis of antibiotic resistance is the discovery of novel antimicrobial compounds for clinical application. Marine organisms are an attractive and, as yet, relatively untapped resource of new natural products. Cell extracts from the marine diatom, Phaeodactylum tricornutum, have antibacterial activity and the fatty acid, eicosapentaenoic acid (EPA), has been identified as one compound responsible for this activity. During the isolation of EPA, it became apparent that the extracts contained further antibacterial compounds. The present study was undertaken to isolate these additional antibacterial factors using silica column chromatography and reverse-phase high-performance liquid chromatography. Two antibacterial fractions, each containing a pure compound, were isolated and their chemical structures were investigated by mass spectrometry and nuclear magnetic resonance spectroscopy. The antibacterial compounds were identified as the monounsaturated fatty acid (9Z)-hexadecenoic acid (palmitoleic acid; C16:1 n-7) and the relatively unusual polyunsaturated fatty acid (6Z, 9Z, 12Z)-hexadecatrienoic acid (HTA; C16:3 n-4). Both are active against Gram-positive bacteria with HTA further inhibitory to the growth of the Gram-negative marine pathogen, Listonella anguillarum. Palmitoleic acid is active at micro-molar concentrations, kills bacteria rapidly, and is highly active against multidrug-resistant Staphylococcus aureus. These free fatty acids warrant further investigation as a new potential therapy for drug-resistant infections.

Differential antibacterial activities of fusiform and oval morphotypes of Phaeodactylum tricornutum (Bacillariophyceae)

“…Mass of fatty acids attributable to each cell in methanol cell extracts from cultures enriched to contain either .71% fusiform cells or 100% oval cells showing that cells in the fusiform-enriched cultures contained significantly greater masses of C14:0, C16:2 n-4 and C16:3 n-4 (P , 0.05; Bonferroni correction). Moreover, extracts from fusiform-enriched cultures contained a significantly greater mass of fatty acids in total (P , 0.05), as well as a significantly greater total mass of three antibacterial fatty acids (EPA, HTA and PA) isolated from similar extracts previously (P , 0.05; Desbois et al, 2008Desbois et al, , 2009 …”

“…:2 n-4, C16:3 n-4 and C16:4 n-1 (P , 0.05; Bonferroni correction) but significantly lower proportions of C16:0, C20:4 n-6 and C20:6 n-3 (P , 0.05; Bonferroni correction). Extracts from enriched cultures did not differ significantly in the sum proportion of the three antibacterial fatty acids (EPA, HTA and PA) isolated from similar extracts in previous studies (Desbois et al, 2008(Desbois et al, , 2009 N ¼ 10; data given as mean +1 SD to two decimal places. Table 2.…”

“…The present study now shows that extracts from fusiform cells contain a greater quantity than the ovals of the fatty acids, EPA, HTA and PA, (42.7 and 36.6 Â 10 213 g, respectively). These are known to have strong antibacterial activity against our test bacterium, Staphylococcus aureus, as well as various marine bacteria (Desbois et al, 2008(Desbois et al, , 2009. Greater quantities of antibacterial free fatty acids in the extracts from fusiform cells may be due to fusiform cells containing more lipid as a proportion of cell composition than the oval cells or alternatively, the fusiform cells may be more fragile due to their shape and cell wall structure compared with the oval cell, which is more siliceous and rigid (Lewin et al, 1958;Borowitzka & Volcani, 1978;Francius et al, 2008).…”

“…Methanol extracts of laboratory grown cultures of P. tricornutum are known to have growth inhibitory effects in vitro against Gram-positive and Gram-negative bacteria of marine or non-marine origin (Duff et al, 1966;Kellam & Walker, 1989;Desbois et al, 2008Desbois et al, , 2009. We have shown that certain unsaturated free fatty acids are responsible for the bactericidal properties of P. tricornutum methanol extracts, namely eicosapentaenoic acid (C20:5 n-3; EPA) (Desbois et al, 2009), (6Z, 9Z, 12Z )-hexadecatrienoic acid (C16:3 n-4; HTA) and palmitoleic acid (C16:1 n-7; PA) (Desbois et al, 2008). In this paper, therefore, we compare the antibacterial activities and fatty acid compositions of cell extracts derived from cultures highly enriched with either oval or fusiform cells.…”

“…Antibacterial activity was studied because prokaryotes represent a major group of competitors for microalgae in terms of space and resources and could also pose a threat as intracellular or epibiont parasites or direct pathogens (Cole, 1982;Imai et al, 1993;Mayali & Azam, 2004). Methanol extracts of laboratory grown cultures of P. tricornutum are known to have growth inhibitory effects in vitro against Gram-positive and Gram-negative bacteria of marine or non-marine origin (Duff et al, 1966;Kellam & Walker, 1989;Desbois et al, 2008Desbois et al, , 2009. We have shown that certain unsaturated free fatty acids are responsible for the bactericidal properties of P. tricornutum methanol extracts, namely eicosapentaenoic acid (C20:5 n-3; EPA) (Desbois et al, 2009), (6Z, 9Z, 12Z )-hexadecatrienoic acid (C16:3 n-4; HTA) and palmitoleic acid (C16:1 n-7; PA) (Desbois et al, 2008).…”