An insight into anti-biofilm and anti-quorum sensing activities of the selected anthocyanidins: the case study of Pseudomonas aeruginosa PAO1

Abstract Anti-biofilm activity of three anthocyanidins (pelargonidin, cyanidin and delphinidin) was evaluated for the first time at in vitro conditions. All the compounds reduced the formation of Pseudomonas aeruginosa PAO1 biofilm at low sub-MIC (0.125 MIC) with delphinidin (c 56.25 μg/mL) being the most active (43%). In comparison, ampicillin (c 93.75 μg/mL) and streptomycin (c 21.25 μg/mL) (used as positive controls) were considerably less effective at the same sub-MIC (8 and 12%, respectively). Furthermore, at 0.5 MIC (c 225 μg/mL) this anthocyanidin molecule partly reduced the bacterial protrusions. However, no any of the aforementioned compounds inhibited the production of pyocyanin by the bacterial strain P. aeruginosa PAO1. Taken all together, the delphinidin scaffold could be taken into consideration for the design of the novel and more effective anti-biofilm agents inspired by the anthocyanidins.


Introduction
Anthocyanins are water-soluble pigments that colour the fruit and flowers of many plants. They are flavonoids, formed by phenylpropanoid metabolism. Three major anthocyanidins are pelargonidin, cyanidin and delphinidin. There is mounting evidence that consumption of anthocyanin-rich food promotes health (Glover & Martin 2012).
Majority of all infectious diseases are related with the bacteria capable of forming biofilms (Husain et al. 2013). Pseudomonas aeruginosa is a bacterium which forms biofilms in the lungs, urinary tract and kidneys. Indeed, the role of its biofilm in disease development is well documented (Donlan 2011;Lee, Wu et al. 2013).

Results and discussion
The anthocyanidin compounds exhibited a weak antibacterial activity against P. aeruginosa ATCC 27853 with the MIC and MBC values in the range of 0.450-1.350 mg/mL (Table S1). At some extent delphinidin and pelargonidin showed higher antibacterial activity than cyanidin.
As for the literature data, (+)-catechin has been recently reported to exhibit no any P. aeruginosa anti-biofilm activity at the aforementioned sub-MICs (Pejin, Ciric, Markovic et al. 2015). While the hydroxylation of the B-ring of the compounds screened herein may affect P. aeruginosa biofilm formation at some extent, the chemical nature of the ring C and stereochemistry are likely to be much more important for this particular activity. Furthermore, caffeic acid, another flavonoid compound possessing simpler chemical structure, significantly inhibited biofilm formation (75.75%) at 0.125 MIC (c 28.13 μg/mL). Finally, quercetin displayed profound activity only at the highest sub-MIC tested (0.5 MIC, c 85 μg/mL) showing . This compound has also been found to be one of the major anti-biofilm compounds (Staphylococcus aureus) in the extract of Alnus japonica (Lee, Park et al. 2013). More recent study focussing on the flavonoid compounds from red wines has actually confirmed such an observation (Cho et al. 2015). P. aeruginosa biofilm formation also depends on the flagellum (swimming motility) and type IV pili (twitching motility) (Henrichsen 1972). The anthocyanidin compounds screened (0.5 MIC) reduced the twitching motility of the respective bacterial strain (Figure 1; Table S2). In addition to this, the colour of the colony ranged from white to light green: more precisely, only cyanidin changed it into white.
Finally, no any of the selected anthocyanidin compounds (0.5 MIC) exhibited pyocyanin production inhibitory activity (Table S3). Unlike cyanidin and delphinidin which practically had no influence on the production of pyocyanin, pelargonidin (c 225 μg/mL) significantly increased it (48.08%). In comparison, diterpene alcohol phytol (c 9.50 μg/mL) efficiently reduced the production of pyocyanin (51.94%), i.e. as much as streptomycin (used as a positive control) did .
Pioppino mushroom, a popular edible mushroom in southern europe and, especially in Italy, has been used in traditional Chinese herbal medicine. It's worth mentioning that Pioppino's methanol extract (0.5 MIC) displayed a potent anti-biofilm activity (84.24%; P. aeruginosa PAO1) at in vitro conditions (Petrović et al. 2014). This finding has confirmed the importance of nutritional bioactives as possible leading compounds in the fight with bacterial resistance. Further studies covering natural products with similar chemical structures such as europinidin, hirsutidin and malvidin should be undertaken for the better understanding of the anti-biofilm activity observed.

Experimental
See Supplementary Material.

Conclusion
In summary, this study has indicated that delphinidin possesses a moderate anti-biofilm activity (towards P. aeruginosa PAO1) at low sub-MIC for the first time. Therefore, its chemical scaffold may inspire the design of the novel and possibly more effective anti-biofilm agents targeting the aforementioned resistant bacterium.

Disclosure statement
No potential conflict of interest was reported by the authors.