Acinetobacter baumannii

Reference bacterial strains as the source for developing the PAC were derived from the American Type Culture Collection (ATCC): K. pneumoniae ATCC BAA-1706, S. aureus ATCC 25923, S. pneumoniae ATCC 49619, P. aeruginosa ATCC 27853, M. tuberculosis H37Rv, and H. influenzae ATCC 49247. Other standard reference strains used for the sensitivity and specificity evaluation in this study includes K. pneumoniae ATCC BAA-1705, S. aureus ATCC 33591, S. pneumoniae ATCC 51916, S. pneumoniae ATCC 700673, P. aeruginosa ATCC 9027, Mycobacterium bovis ATCC 35720, H. influenzae ATCC 49766, Acinetobacter baumannii ATCC 19606, Aeromonas hydrophila ATCC 7966T, Bacillus cereus ATCC 14579, Bacillus subtilis ATCC 6633, Enterobacter aerogenesATCC 13048, Enterobacter cloacae ATCC 13047, Escherichia coli ATCC 25922, E. coli O157 non-toxigenic NCTC 12900, Listeria monocytogenes ATCC 7644, Neisseria meningitidis ATCC 13090, Neisseria gonorrhoeae ATCC 43069, Proteus mirabilis ATCC 29245, Staphylococcus epidermidis ATCC 12228, Streptococcus viridians ATCC 36395, Streptococcus pyogenes ATCC 19615, Streptococcus mutans ATCC 35668, and Streptococcus sanguinis ATCC 10556. A total of 129 clinical isolates were acquired from the Department of Medical Microbiology and Parasitology, Universiti Sains Malaysia (USM), Malaysia.

The following pathogenic bacterial strains were used: The Gram-positive Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (HNCMO112011), Listeria monocytogenes (ATCC 19111) and the Gram-negative Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 8739), Salmonella enterica (ATCC 13076), Klebsiella pneumoniae (NCTC 13440), Acinetobacter baumannii (ATCC 17978) obtained from the ATCC (United States) and NCTC (National Collection of Type Cultures–England). For the antifungal experiment the Candida albicans W01 strain (Ördögh et al., 2014 (link)) was used. Antimicrobial activities of peptides against ∼10⁷ log phase bacteria were tested in Potassium-Phosphate Buffer (PPB, pH 7.4) as described by Jenei et al. (2020) (link) while anti-Candida assays were done according to Szerencsés et al. (2021) (link). The two-fold dilution series of peptides ranged from 25 μM to 0.125 μM, while that of the antibiotics ampicillin (Merck) and miconazole (Duchefa Biochemie) from 10.24 mM to 0.1 μM. The lowest concentration of the antimicrobial agents, which completely eliminated viable bacteria or C. albicans were considered as the minimal bactericidal concentration (MBC) and minimal fungicidal concentration (MFC).

For this analysis and for the preparation of microencapsulated plant extract, the alcohol was evaporated from the plant extracts in a Heidolph Rotavapor (Heidolph Instruments GmbH & Co, Schwabach, Germany) at a temperature of 40 °C and a pressure of 175 mbar.
The antimicrobial properties of extracts were tested against gram-positive bacteria (Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 11778, Enterococcus faecalis ATCC 19433, Geobacillus stearothermophilus ATCC 7953), gram-negative bacteria (Escherichila coli ATCC 25922, Acinetobacter baumannii ATCC^® BAA-747, Salmonella abony NCTC 6017), as well as one pathogenic fungus Candida albicans ATCC 10231. All test culture was purchased from American Type Culture Collection (ATCC), except Salmonela abony, which was obtained from The National Collection of Type Cultures (NCTC; UK). Bacterial cultures were pre-cultured in Mueller Hinton broth overnight at 37 °C. Each strain was adjusted to a concentration corresponding to the 0.5 McFarland standard [17 (link)]. The fungal inoculum was prepared from the 48-hour culture grown in Potato dextrose broth [18 ].