Klebsiella pneumoniae

Escherichia coli O157:H7 ATCC 35150, Enterococcus faecalis ATCC 29212, Listeria monocytogenes ATCC 7644, Klebsiella pneumoniae ATCC 13833, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 43387, Salmonella enteritidis ATCC 13076 and Candida albicans ATCC 14053 were the reference human pathogens used in this study (American Type Culture Collection, USA). All strains were maintained in Tryptic Soy Agar (TSA, Oxoid, Milan, Italy) at 37 °C, while C. albicans ATCC 14053 was grown in Sabouraud Dextrose Agar (SDA, Oxoid). All stock cultures were kept at −80 °C in nutrient broth with glycerol 15%.

The antibacterial activity of all extracts was tested against four strains of Gram-positive bacteria (Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 11778, and Listeria monocytogenes NIPH-NIH 17/11) and eight strains of Gram-negative bacteria (Yersinia enterocolitica O3 NIPH-NIH 383/11, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC 35659, Shigella sonnei NIPH-NIH, Salmonella enterica subsp. enterica serovar Enteritidis ATCC 13076, Enterobacter aerogenes ATCC 13048, and Escherichia coli ATCC 25922).
The strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and clinical isolates from the National Institute of Public Health—National Institute of Hygiene (NIPH—NIH, Warsaw, Poland).
The bacterial strains were cultured on nutrient agar for 24 h at 37 °C. The inocula were diluted to approximately 1 × 10⁸ cfu/mL using 0.85% NaCl (w/v).

The reference strains of microorganisms from American Type Culture Collection (ATCC), Manassas, VA, USA, were included. The representative Gram-positive bacteria were: Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 10240, Bacillus subtilis ATCC 6633 and Bacillus cereus ATCC 10876, while those of Gram-negative bacteria: Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiella pneumoniae ATCC 13883, Salmonella Typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 9027 and Bordetella bronchiseptica ATCC 4617. Moreover, the fungi belonging to yeasts (Candida albicans ATCC 2091, Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019, Candida glabrata ATCC 90030 and Candida krusei ATCC 14243) were used.

Antibacterial activity of the sea cucumber extracts were tested against five Gram-positive/negative bacterial strains: Staphylococcus aureus (ATCC 25923), Micrococcus luteus (ATCC 9341), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 10031) and Vibrio harveyi (ATCC 14126); which were obtained from the Pasteur Institute, Tehran, Iran.
Antibacterial screening was done by disc diffusion method (Vanden Berghe and Vlietinck 1991 ). For stock solutions, 10 mg of each extract of S.herrmanni were dissolved in 1 mL of DMSO. Mueller–Hinton Agar plates were inoculated with an overnight culture (12–16 h) of each bacterial strain. Whatman paper disks were placed on the agar surface and injected with 10 μL of each stock solution extract. Disks injected with DMSO were used as solvent control. Disks with standard antibacterial agent ampicillin (at 10 µg/disc) were used as positive control. After the 24 h-long incubation at 37 °C, The diameter of the inhibition zone (IZ) of bacterial growth was measured. No inhibitory effects due to the solvent were observed. The assays were repeated in triplicate.

The bacterial strain used for SELEX selection was P. aeruginosa 692 (PA692) (ATCC 14502) obtained from New Zealand Culture Collection (Porirua, NZ). Other bacterial strains used were: P.aeruginosa PAO1 (ATCC 15692), Salmonella enterica serovar Typhi (ATCC 19430), Klebsiella pneumoniae (ATCC 13883), Enterobacter cloacae (ATCC 13047), Listeria innocua (ATCC 33090) and Escherichia coli DH5α. Additional P. aeruginosa strains used in binding studies were clinical isolates PA1024 (catheter, urine), PA1205 (left thigh), PA1323 (cystic fibrosis sputum), PA1079 (tracheal aspirate), and PA1236 (blood) obtained from Environmental Science and Research Culture Collection (Porirua, NZ). All bacterial strains were cultured in standard Luria Broth (LB) medium at 37°C for 16 hours with aeration. After growth, the cells were diluted 1:100 in fresh LB and grown for 4 hours to allow them to reach exponential phase.

In the current research, all solvents used were of analytical grade and were supplied by Thermo Scientific (Illkirch, France). All reagents and standards were bought from Merck (Saint-Quentin-Fallavier, Lyon, France). Strains including Bacillus subtilis (ATCC 6633), Klebsiella pneumoniae (ATCC 13883), Staphylococcus epidermidis (ATCC 14490), Pseudomonas aeruginosa (ATCC 9721), Escherichia coli (ATCC 15224), Aspergillus flavus (ATCC 9643), Aspergillus fumigatus (FCBP 66), Fusarium solani (FCBP 434), Aspergillus niger (ATCC 1015) and Mucor species (FCBP 300) were acquired from Department of Biotechnology, QAU, Pakistan.

The examined bacterial and fungal isolates were obtained from American Type Culture Collection (ATCC), in addition to a clinically confirmed Methicillin-Resistant Staphylococcus aureus (MRSA). The selected species of microorganisms are frequently isolated at clinical settings in our region and some possess multidrug resistance. The isolates included three Gram-positive species: Staphylococcus aureus (ATCC 25923), MRSA a clinical strain, and Enterococcus faecium (ATCC 700221) and four Gram-negative species: Klebsiella pneumoniae (ATCC 13883) Proteus vulgaris (ATCC 8427), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 9027). Meanwhile, the fungal isolate is Candida albicans (ATCC 90028).