Antibiofilm activity of Brasilian propolis extracts against Staphylococcus aureus producers isolated from goat and sheep mastitic milk

The aim of this study was to investigate the action of inhibiting S. aureus biofilm formation, and the ability to eliminate formed biofilm, by alcoholic extracts of green, red and brown propolis from Brazil. Ten isolates of S. aureus have been tested, 8 field isolates, 1 MRSA and 1 ATCC 25923, by microplate quantitative method. For the evaluation of inhibitory action, the isolates were inoculated, in triplicate, in TSB 1% glucose in the presence of green (1), red (2) and brown (4) propolis extracts. Biofilm formation was evaluated by optical reading, compared to a negative control consisting of a mixture of TSB and extract. For biofilm elimination assay, extracts were added to plates with 24h cultures of the same isolates. Assays were repeated three times on three different days. Eight out of the 10 isolates produced less biofilm in the presence of the green propolis extracts, so the inhibitory effect is 80%. Brown propolis extracts inhibited the formation of biofilm in 10% to 70% of the isolates and the red extracts in 30% to 80%. Regarding the biofilm elimination activity, green propolis extract was positive for 9 out of the 10 isolates (90%), the brown propolis extracts were positive for 0% to 100% isolates and red extracts for 0% to 10% isolates.

Propolis components accountable for bactericidal accomplishment and antibiofilm activity

This study aimed at evaluating antimicrobial and antibiofilm activity of phenolic compounds present in propolis ethanol extracts (PEE). Seventy per cent ethanol extracts from seven types of propolis, one Green, two Red and four Brown collected in four Brazilian States were prepared and total phenolics, flavonoids, tannins and anthocyanins were assessed by high-performance liquid chromatography (HPLC). Minimum bactericidal concentration (MBC) and inhibitor effect on Staphylococcus aureus biofilm formation and capacity to disrupt established biofilms were assessed towards eight S. aureus isolates from milk of small ruminants with mastitis, one methicillin-resistant S. aureus (MRSA) and S. aureus ATCC 25923. To evaluate different propolis components accountability for bactericidal accomplishment and antibiofilm activity, the results were analysed by the non-parametric Spearman coefficient. Results of phenolic compounds were 216,21 to 312,08 gallic acid milligram equivalent per extract gram (mg EGA/g) of total phenolics, 55,08 to 140,6 quercetin milligram equivalent per extract gram (mg EQ/g) of flavonoids, 118,51 to 3766,16 catechin milligram equivalent per extract gram (mg EC/g) of tannins and 1,03 to 8,39 milligram per extract gram (mg/g) of anthocyanins. Red1 and Red2 showed higher tannin contents, while Red2 exhibited superior amount of anthocyanins and total phenolics. Brown3 presented higher flavonoid quantity. Green, Red1 and Red2 PEE showed the lowest levels of flavonoids, but the higher antimicrobial activity. Most PEE exhibit bactericidal activity at a concentration of 1.6 mg/mL. Brown4 PEE showed the worst capacity to inhibit S. aureus. Green PEE showed to be the most efficient in both preventing and disrupting biofilm. All PEE studied exhibited a better inhibitory activity prior-to than post-biofilm formation. According to non-parametric Spearman correlation analysis, there seems to be a significant negative correlation between the ability to disrupt biofilm and both tannins and anthocyanins contents.