Aeromonas hydrophila

The bacterial strain P. syringae pv. syringae (DSM 21482) was purchased from Leibniz-Institute DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmmH (Braunschweig, Germany). P. syringae pv. actinidiae strains (CRA-FRU 8.43, 12.54, and 14.10) were purchased from Culture Collection of C.R.A.—Centro di Ricerca per la Frutticoltura (Roma, Italy). Pseudomonas aeruginosa (ATCC 27853), Aeromonas hydrophila (ATCC 7966), Salmonella enterica serovar Typhimurium (ATCC 13311 and ATCC 14028), Escherichia coli (ATCC 25922 and ATCC 13706), and Vibrio parahaemolyticus (DSM 27657) were purchased from ATCC and DSM culture collections, respectively. The other bacterial strains used in this study were isolated in previous research works from water samples collected in Ria de Aveiro (Aveiro, Portugal) [37 (link),38 (link)].
All bacteria were grown in Tryptic Soy Broth (TSB, Roseto degli Abruzzi (Te), Italia). The bacterial strains were stored at −80 °C in 10% glycerol. Before each assay, a stock culture of each bacteria was aseptically inoculated in 30 mL of TSB and was grown during 18 h at 25 °C with orbital shaking set at 120 rpm stirring. Then, an aliquot (300 µL) of each bacterial culture was transferred to 30 mL of fresh TSB and grown during 18 h at 25 °C under orbital shaking (120 rpm). The viable cell density was approximately 10⁹ colony-forming units (CFUs)/mL.

Test microorganism strains that were used in this study include Bacillus cereus ATCC 14579, Bacilus subtilis ATCC 8188, Enterococcus faecalis ATCC 700802, Enterococcus faecalis ATCC 29212, Enterococcus faecalis JH-22, Staphylococcus aureus ATCC 700699, Staphylococcus aureus ATCC 43300, Staphylococcus aureus ATCC 6538P, Staphylococcus aureus ATCC 29213, Aeromonas hydrophila ATCC 49140, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 10031, Proteus mirabilis ATCC 49140, Proteus vulgaris (Institute of Medical Research, Malaysia), Pseudomonas aeruginosa ATCC 10145, Pseudomonas aeruginosa ATCC BAA-47, Salmonella Typhimurium ATCC 14028, and Shigella flexneri ATCC 12022. Strains were cultured on Muller Hinton borth (MHB) (Oxoid, UK) at 37°C and maintained at −80°C in MHB with 25% (v/v) glycerol.

Fresh leaf samples of all five species were obtained from Selangor, Malaysia, within a 10 km radius. All plants were grown under similar conditions: a monthly mean minimum temperature from 20.8 °C to 25.0 °C, with a monthly mean maximum temperature from 29.6 °C to 32.8 °C; grown on soil; exposed to sunlight (mean daily solar radiation of 19.70 MJ/m²) and rain (300–400 mm/month) [29 ]; and with no fertilizer. For the antioxidant tests, for each species, leaves of similar size were collected from three different individual plants (n = 3).
Bacterial isolates were obtained from American Type Culture Collection (ATCC), with the exception of Proteus vulgaris, which was obtained from the Institute of Medical Research (IMR), Malaysia. A total of 12 strains of bacteria were used: six Gram-positive bacteria (Bacillus cereus (ATCC 14579), Bacillus subtilis (ATCC 8188), Enterococcus faecalis (ATCC 29212), Micrococcus luteus (ATCC 4698), methicillin-resistant Staphylococcus aureus (ATCC 33591), and Staphylococcus epidermidis (ATCC 12228)) and six Gram-negative bacteria (Aeromonas hydrophila (ATCC 49140), Enterobacter aerogenes (ATCC 13048), Pseudomonas aeruginosa (ATCC 10145), Proteus mirabilis (ATCC 12453), Proteus vulgaris (clinical), and Salmonella enterica Typhimurium (ATCC 14028)). All bacteria were grown on nutrient agar at 37 °C.