Enterobacter cloacae

The dried plant extracts were dissolved in water (10 mg/mL) and the antibacterial capacity was evaluated by the microdilution method, using the methodology previously described [64 (link)]. For the antibacterial assay, different Gram-positive bacteria were employed-Staphylococcus aureus (ATCC 6538), Bacillus cereus (clinical isolate), Micrococcus flavus (ATCC 10240), and Listeria monocytogenes (NCTC 7973)-as well as Gram-negative bacteria-Enterobacter cloacae (ATCC 35030) and Salmonella typhimurium (ATCC 13311). Minimum inhibitory concentration (MIC) and the minimum bactericidal (MBC) concentration, in mg/mL, were determined. Streptomycin was used as a positive control.
Antifungal activity was determined according to a previous protocol [65 (link)]. Five strains of fungi were employed: Aspergillus fumigatus (human isolate), Aspergillus versicolor (ATCC 11730), Aspergillus niger (ATCC 6275), Penicillium funiculosum (ATCC 26839), and Penicillium aurantiogriseum (ATCC 58604). MIC and minimal fungicidal concentration (MFC), in mg/mL, were calculated. Ketoconazole was used as a positive control.
The microorganisms employed for the analysis were deposited at the Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research “Sinisa Stanković”, University of Belgrade, Serbia.

The antibacterial and antifungal activities of the extracts were determined according to methods described in detail by Pires et al. (2018) [35 (link)] and Heleno et al. (2013) [46 (link)], respectively. Five Gram-negative bacteria (Enterobacter cloacae—ATCC 49741, Escherichia coli—ATCC 25922, Pseudomonas aeruginosa—ATCC 9027, Salmonella enterica—ATCC 13076, and Yersinia enterocolitica—ATCC 8610) and three Gram-positive bacteria (Bacillus cereus—ATCC 11778, Listeria monocytogenes—ATCC 19111 and Staphylococcus aureus—ATCC 25923), besides two fungi strains (Aspergillus fumigatus—ATCC 204305 and Aspergillus brasiliensis—ATCC 16404) purchased from Frilabo (Porto, Portugal) were used. All work was performed with sterile materials handled under laminar flow. Prior to the assays, microorganisms were incubated (37 °C ± 0.5 °C for 24 h for bacteria and 25 °C ± 0.5 °C for 72 h for fungi) with appropriate media for each strain to reach a state of exponential growth. Negative controls of the extract and culture medium were prepared, and the antibiotics streptomycin, methicillin, ampicillin and antifungal ketoconazole served as positive controls. The antimicrobial potential was assessed as the minimum concentration required to inhibit the microorganism growth (MIB) and to cause bacteria or fungi death (MBC or MFC, respectively).

Standard strains: Moraxella catarrhalis (GTC 01544), Klebsiella pneumoniae (ATCC 13883), Escherichia coli (K12), Salmonella typhimurium (ATCC 14028), Streptococcus pyogenes (ATCC 19615), Streptococcus agalactiae (ATCC 13813), Staphylococcus epidermidis (ATCC 12228), Neisseria lactamicus (ATCC 23970), Enterobacter cloacae, (ATCC 23355), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (209P), and Pseudomonas aeruginosa (IFO 3445).

Clinical strains: Moraxella catarrhalis, Bacillus cereus, Aeromonas hydrophila, Salmonella typhi, Vibrio cholerae, and Yersinia enterocolitica.

In addition to a sea urchin (Anthocidaris crassispina) derived Bacillus sp. which obtained from the Laboratory in Medical Plants Garden, Nagasaki University.(c)

Fungal strains: the fungal strains used were Aspergillus niger (NBRC 33023), Penicillium crustosum (NBRC 33015), Schizophyllum commune (NBRC 30749), Trichophyton concentricum (NBRC 31068), and Candida albicans (NBRC 10108).

The following bacterial strains were used in this study for the microdilution assay: Enterococcus faecalis ATCC 19433, Streptococcus pneumoniae ATCC 49619, Klebsiella pneumoniae ATCC 700603, Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa ATCC 27853, Enterobacter cloacae ATCC 13047, Escherichia coli ATCC 25922, Streptococcus pyogenes ATCC 19615, Streptococcus agalactiae ATCC 12386, Staphylococcus epidermidis ATCC 12228 and Staphylococcus saprophyticus ATCC 15305 (the American Type Culture Collection, Manassas, Virginia, United States). The antibiotic controls were purchased from Sigma-Aldrich (St. Louis, Missouri, United States).

Ten bacterial strains used in this study were obtained from Guangdong Microbial Culture Collection Center. E. coli MDR was a generous gift from Zhejiang Academy of Agricultural Sciences. The E. coli O157:H7 (ATCC 35 150), E. coli (ATCC 8739), E. coli MDR, Salmonella Typhimurium (ATCC 14 028), Klebsiella pneumoniae (ATCC 13 883), Acinetobacter baumannii (ATCC 17 978), Pseudomonas aeruginosa (ATCC 10 145), and Enterobacter cloacae (ATCC 13 047) were used as representative gram‐negative bacteria. The Enterococcus faecium (ATCC 19 434), Staphylococcus aureus (ATCC 25 923), and Staphylococcus aureus MRSA (ATCC 43 300) were used as representative gram‐positive bacteria. All strains were cultured in nutrient broth (NB) medium at 37 °C with shaking at 180 rpm before use.

Strains were routinely grown with Mueller−Hinton broth at 37 °C with shaking at 225 rpm. Bacterial strains were obtained through ATCC or BEI Resources or from academic laboratories. The strains used were the following: Staphylococcus aureus ATCC 29213, S. aureus NRS70, Enterococcus faecalis ATCC 33186, E. faecium ATCC 19434, Streptococcus pyogenes ATCC 700294, Streptococcus pneumoniae R6, Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa ATCC 15692, Klebsiella pneumoniae ATCC 700603, Proteus mirabilis ATCC 25933, Stenotrophomonas maltophilia ATCC 13637, Enterobacter cloacae ATCC 13047, Staphylococcus epidermidis ATCC 14990, E. coli K-12, and E. coli JW5503 (ΔtolC). The M. tuberculosis H37Rv strain was grown in Middlebrook 7H9 broth supplemented with 10% albumin-dextrose complex, 0.05% (v/v) Tween 80 at pH 7.4, (7H9/ADG pH 7.4) with shaking at 225 rpm at 37 °C.