Bacillus cereus

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.

In this work, we used Bacillus cereus ATCC14579, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, and Staphylococcus aureus ATCC25923 (ATCC, USA) as gut pathogenic representatives for screening of antibacterial activity of the extracts using disc-diffusion and broth microdilution assays. For disc diffusion assay, a single colony of each bacteria was selected, sub-cultured into 5 mL NB (HiMedia, India), and incubated for 24 h at 37 °C. Each culture was then added into NSS until the turbidity reach McFarland’s standard No. 0.5. The inoculated NSS (approximately 1.5 × 10⁸ cells/mL) was swabbed on freshly prepared NA. Sterile 6 mm diameter filter paper discs (Whatman, UK) were impregnated with 2 and 5 mg extracts before placing on the bacterial swabbed NA. After the incubation at 37 °C for 24 h, the antibacterial activity was evaluated by measuring the IZD. We used ampicillin (10 μg/disc), ceftriaxone (30 μg/disc), chloramphenicol (30 μg/disc), and rifampicin (5 μg/disc) (Oxoid, UK) as positive controls for inhibition of Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus, respectively.

The effects of EOs were evaluated on 13 bacterial strains and 5 yeasts: Staphylococcus aureus (209 PCIP 53156), S. aureus (ATCC 29213), Micrococcus luteus (ATCC381), Bacillus cereus (ATCC 14579), Escherichia coli (ATCC 8739), E. coli (ATCC 35214), Pseudomonas aeruginosa (DSM 50090), P. aeruginosa (ATCC 27853), Klebsiella pneumoniae (CIP 104727), K. pneumoniae (clinical isolates), Enterococcus faecalis (ATCC 29212), Listeria monocytogenes (ATCC 19115), Salmonella enteritidis (DMB 560), Candida albicans (CCMM L4), C. krusei (CCMM L10), C. glabrata (CCMM L7), C. parapsilosis (CCMM L18) and Aspergilus niger (CCMM M100). The bacterial and yeast strains were provided by the Center of Biotechnology, Borj Cedria, Tunisia.

The disc diffusion assay was used to assess the antimicrobial properties of the fungal extracts against six human pathogenic bacteria- the three Gram-positive bacteria Bacillus cereus (ATCC 10876), Enterococcus faecalis (ATCC 29212) and Staphylococcus aureus (ATCC 29213) and the three Gram-negative bacteria Enterobacter cloacae (ATCC 13047), Escherichia coli (ATCC 25922) and Salmonella typhimurium (ATCC 14028). The bacteria were allowed to grow overnight in Mueller Hinton Broth (MHB) at 37°C. The cultures were adjusted to the 0.5 McFarland standard with the bacterial suspension being of 1.5 × 10⁸ colony-forming unit (CFU) (absorbance of 0.08–0.1 at 600 nm). One hundred microlitres of bacteria inoculum was spread onto Mueller-Hinton Agar (MHA). Sterile filter paper discs (6 mm) containing 10 µl of the fungal extracts were placed onto the agar plates. The plates were incubated at 37°C for 24 h and the zone of inhibition was measured to the nearest mm (Subramaniam et al. 2020 (link)).

The antimicrobial activity of Z. rhoifolium (extracts, isolated alkaloids and chelerythrine derivative) was tested against seven Gram-positive bacteria: Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 33019, Staphylococcus aureus ATCC 25923. Staphylococcus epidermidis ATCC 12228, Streptococcus pyogenes ATCC 19615, Enterobacter aerogenes ATCC 13048, Enterococcus spp. ATCC 6589; eight Gram-negative bacteria: Escherichia coli ATCC 25922, Klebsiella pneumonia ATCC 13883, Pseudomonas aeruginosa ATCC 27853, Enterobacter cloacae ATCC 1304, Shigella sonnei ATCC 25931, Salmonella typhimurium ATCC 14028, Burkholderia cepacia ATCC 17759, Morganella morganii ATCC 25829; and seven yeasts: Candida albicans ATCC 10231, Candida tropicalis ATCC 18803 Candida krusei ATCC 6258, Candida parapslosis ATCC 22018, Sacharomyces cerevisae ATCC 2601, Cryptococcus neoformans ATCC 28952, Cryptococcus gatti ATCC 2601.

Gram positive strains methicillin-resistant S. aureus (MRSA, NCTC 13616), Bacillus subtilis (ATCC 6633), Bacillus cereus (ATCC 14579) and gram negative strains Klebsiella pneumoniae (ATCC 43816), Escherichia coli (ATCC 8739), Proteus vulgaris (ATCC 13315) were procured from American type culture collection, USA. Methicillin-resistant S. aureus was purchased from culture collections, UK. All bacterial strains stored at −80 °C were streaked on Luria–Bertani (LB) agar plates (Hi-media Laboratories, Mumbai, India) and incubated at 37 °C for 20 to 24 h. A few isolated colonies were selected from each plate and suspended in 5 mL of LB broth in sterile culture vessel. The vessel was plugged with cotton and incubated with gentle shaking (140 rpm) at 37 °C for 20 h.