Streptococcus pneumoniae

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Streptococcus pneumoniae is a type of bacteria that is commonly found in the upper respiratory tract. It is a Gram-positive, catalase-negative, and alpha-hemolytic bacterium. Streptococcus pneumoniae can be used in laboratory settings for various research and testing purposes.

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34 protocols using streptococcus pneumoniae

Bacterial Strain Cultivation and Assay

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The following four bacterial standard strains from the American Type Culture Collection (ATCC, Manassas, VA, USA) were used: Haemophilus influenzae ATCC 49247, Staphylococcus aureus ATCC 29213, Streptococcus pneumoniae ATCC 49619, and Streptococcus pyogenes ATCC 19615. Cultivation and assay media (broth/agar) were Mueller–Hinton (MH) complemented by Haemophilus Tested Medium (H. influenzae), MH (S. aureus), and Brain Heart Infusion (S. pneumoniae and S. pyogenes). The pH of the broths was equilibrated to a final value of 7.6 using Trizma base (Sigma-Aldrich, Praha, Czech Republic). All microbial strains and cultivation media were purchased from Oxoid (Basingstoke, UK).
Stock cultures of bacterial strains were cultivated in broth medium at 37 °C for 24 h prior to testing. For the preparation of inoculum, the turbidity of the bacterial suspension was adjusted to 0.5 McFarland standard using a Densi-La-Meter II (Lachema, Brno, Czech Republic) to obtain a final concentration of 10⁸ CFU/mL.

Houdkova M., Chaure A., Doskocil I., Havlik J, & Kokoska L. (2021). New Broth Macrodilution Volatilization Method for Antibacterial Susceptibility Testing of Volatile Agents and Evaluation of Their Toxicity Using Modified MTT Assay In Vitro. Molecules, 26(14), 4179.

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Minimum Inhibitory Concentration Assay

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Broth microdilution minimum inhibitory concentration (MIC) testing was carried out according to Clinical and Laboratory Standards Institute (CLSI) guidelines M7-A7. MIC values were determined against a panel of bacteria that included E. coli (ATCC 25922), E. coli TolC mutant, Enterococcus faecalis (ATCC 29212), Haemophilus influenzae (ATCC 49766), Moraxella catarrhalis (ATCC 25238), P. aeruginosa (ATCC 47085), P. aeruginosa PAO200 (efflux-pump mutant), P. aeruginosa hypersensitive strain (ATCC 35151), Staphylococcus aureus (ATCC 29213), and Streptococcus pneumoniae (ATCC 49619).
Time-kill studies were performed using M. catarrhalis and S. aureus, based on the MIC assay results, according to the CLSI document M26-A. Growth media was Brain Heart Infusion and Trypticase Soy Broth from Remel (Lenexa, KS). The compound concentration was four times the MIC.

Hughes C.A., Gorabi V., Escamilla Y., Dean F.B, & Bullard J.M. (2020). Two Forms of Tyrosyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Discovery of Inhibitory Compounds. SLAS discovery : advancing life sciences R & D, 25(9), 1072-1086.

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Synergistic Antibacterial Combinations

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Antibacterial activity of combinations of ciprofloxacin with tempol, melatonin or pentoxifylline were evaluated against different reference bacteria including Escherichia coli ATCC 35218, Staphylococcus aureus ATCC29213, Pseudomonas aeruginosa ATCC 9027, Staphylococcus epidermidis ATCC 12228, Acinetobacter baumannii ATCC 17978, Proteus mirabilis ATCC 12459, Klebsiella pneumoniae ATCC 13883, methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300), and Streptococcus pneumoniae (ATCC 25923). The organisms were stored at −70 °C in trypticase-soy broth and 20% glycerol (Becton Dickinson, East Rutherford, NJ, USA). When ready for batch susceptibility testing, samples were thawed. Minimum inhibitory concentrations (MICs) were determined in accordance with the Clinical and Laboratory Standards Institute [24 ].

Masadeh M.M., Alzoubi K.H., Al-azzam S.I., Khabour O.F, & Al-buhairan A.M. (2016). Ciprofloxacin-Induced Antibacterial Activity Is Atteneuated by Pretreatment with Antioxidant Agents. Pathogens, 5(1), 28.

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Antimicrobial Evaluation of Pathogenic Strains

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The following strains from the University of Messina's in-house culture collection (Messina, Italy) were used for the antimicrobial testing: Staphylococcus aureus ATCC 6538P, Staphylococcus aureus MRSA ATCC 43300, Staphylococcus epidermidis ATCC 49134, Staphylococcus epidermidis ATCC 35984, Staphylococcus epidermidis ATCC 12228, Streptococcus pneumoniae ATCC 6003, Streptococcus pyogenes ATCC 19615, Streptococcus mutans ATCC 35668, Listeria monocytogenes ATCC 7644, Listeria monocytogenes ATCC 1392, Enterococcus hirae ATCC 10541, Moraxella catarrhalis ATCC 8176, Bacillus subtilis ATCC 8176, Enterococcus durans (wild-type strain), Escherichia coli ATCC 25922, Klebsiella pneumoniae (wild-type strain), Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa ATCC 9027, Pseudomonas aeruginosa ATCC 15442, Proteus mirabilis (wild-type strain), Serratia marcescens (wild-type strain), Salmonella typhi ATCC 13311, Candida albicans ATCC 10231, Candida parapsilosis ATCC 29947, and the fungus Aspergillus niger ATCC 16404.
Bacteria were grown in Mueller-Hinton Broth (MHB, Oxoid, CM0405) at 37°C (24 h), whereas yeasts were cultured in Sabouraud Liquid Medium (SLM, Oxoid, CM0147) at 30°C (48 h). For solid media, 1.5% (w/v) agar (Difco) was added. Aspergillus niger was grown in Sabouraud Dextrose Agar at 30°C for 7 days as previously reported [10 (link)].

Filocamo A., Bisignano C., Mandalari G, & Navarra M. (2015). In Vitro Antimicrobial Activity and Effect on Biofilm Production of a White Grape Juice (Vitis vinifera) Extract. Evidence-based Complementary and Alternative Medicine : eCAM, 2015, 856243.

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Antimicrobial Peptide Characterization

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Indolicidin (sequence: ILPWKWPWWPWRR-NH₂, molecular weight: 1,906.3 g/mol) and FMOC-WWRR-NH₂ (molecular weight: 924.1) were obtained from ChemPep (USA) as lyophilized powder and acetate salts with a purity >95%. Ethanol and Congo red were purchased from Sigma-Aldrich (Australia). Sodium chloride was purchased from Alfa Aesar (United Kingdom). The negative staining agent phosphotungstic acid was purchased from Proscitech (Australia). A Milipore filter system was utilized to obtain Milli-Q water. BD Difco Mueller Hinton Broth (MHB) and Sabouraud broth (SB) were purchased from Cell biosciences (Australia). Strains: E. coli NCTC 10418, P. aeruginosa ATCC 9721, E. faecalis ATCC 19433, S. aureus ATCC 25923, S. aureus ATCC BAA-1756 (methicillin resistant), Streptococcus pneumoniae ATCC 49619, Candida albicans ATCC 10231 and Candida auris DSM 21092 (Fluconazole resistant) were provided by the RMIT microbial culture collection. Bacterial strains were stored in 50% glycerol at −20 and −80°C.

Cardoso P., Appiah Danso S., Hung A., Dekiwadia C., Pradhan N., Strachan J., McDonald B., Firipis K., White J.F., Aburto-Medina A., Conn C.E, & Valéry C. (2023). Rational design of potent ultrashort antimicrobial peptides with programmable assembly into nanostructured hydrogels. Frontiers in Chemistry, 10, 1009468.

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Antimicrobial Evaluation of Bacterial and Fungal Strains

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All microorganism strains were obtained from the American Type Culture Collection (ATCC) for our study. They were identified at the Department of Microbiology and Immunology, Faculty of Dental Medicine, Ovidius University of Constanta. The Gram-positive bacteria were Staphylococcus aureus (ATCC 29213), Enterococcus casseliflavus (ATCC 700327), Streptococcus pyogenes (ATCC 19615), Streptococcus pneumoniae (ATCC 49619), and the group of Gram-negative bacteria included Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13883), and Pseudomonas aeruginosa (ATCC 27853). The antifungal activity evaluation was performed using Candida albicans (ATCC 14053) and Candida parapsilosis (ATCC 22019).
Mueller-Hinton agar with 5% defibrinated sheep blood (Thermo Fisher Scientific, GmbH, Dreieich, Germany) was used as a culture medium for both Streptococcus sp. [90 (link)]. The other bacterial strains were maintained in Mueller Hinton agar (Thermo Fisher, Dreieich, Germany). For both Candida sp., Sabouraud 4% Glucose Agar (Merk KGaA, Darmstadt, Germany) was selected as the culture medium.

Popovici V., Bucur L., Calcan S.I., Cucolea E.I., Costache T., Rambu D., Schröder V., Gîrd C.E., Gherghel D., Vochita G., Caraiane A, & Badea V. (2021). Elemental Analysis and In Vitro Evaluation of Antibacterial and Antifungal Activities of Usnea barbata (L.) Weber ex F.H. Wigg from Călimani Mountains, Romania. Plants, 11(1), 32.

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Microbial Culture Protocols for Antimicrobial Screening

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All cultures were handled according to NIH Biosafety Level 2 guidelines, with culture manipulations restricted to biosafety cabinets or an anaerobic chamber. Bacterial strains for high-throughput screening and counter-screens were obtained from ATCC. ATCC catalog numbers are as follows: Clostridium difficile, BAA1382; Acinetobacter baumanii, BAA-1605; Escherichia coli, ATCC 25922; Klebsiella pneumoniae, ATCC 4352; Pseudomonas aeruginosa, ATCC 27853; Enterococcus faecium, ATCC 6569; Staphylococcus aureus, ATCC 43300; Streptococcus pneumoniae, ATCC 49619. C. difficile cultures were grown in Bacto brain–heart infusion (BHI) broth containing 2% Oxyrase in an anaerobic chamber. A. baumanii, E. coli, K. pneumoniae, P. aeruginosa, and S. aureus were cultured in cation-adjusted Mueller–Hinton (MH2) broth, and S. pneumoniae was cultured in MH2 broth containing 2.5% lysed horse blood. E. faecium was cultured in BBL BHI broth. All strains other than C. difficile were cultured aerobically.

Duvall J.R., Bedard L., Naylor-Olsen A.M., Manson A.L., Bittker J.A., Sun W., Fitzgerald M.E., He Z., Lee MD I.V., Marie J.C., Muncipinto G., Rush D., Xu D., Xu H., Zhang M., Earl A.M., Palmer M.A., Foley M.A., Vacca J.P, & Scherer C.A. (2017). Identification of Highly Specific Diversity-Oriented Synthesis-Derived Inhibitors of Clostridium difficile. ACS infectious diseases, 3(5), 349-359.

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Nephrotoxicity Assay with Kidney Cell Lines

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Human embryonic kidney (HEK293) cells and Madin-Darby Canine Kidney (MDCKII) cells were selected to perform the nephrotoxicity assay. Each cell line was cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified incubator containing 5% CO₂. HEK293 and MDCKII cells were grown as an adherent monolayer. Target bacterial strains, Escherichia coli (ATCC 25922, ATCC 4157, ATCC 12435, and ATCC 10798), P. aeruginosa (ATCC 27853), Haemophilus influenzae (ATCC 49247), Staphylococcus epidermidis (ATCC 12228), Sta. aureus (ATCC 12600), Enterococcus faecalis (ATCC 29212), and Streptococcus pneumoniae (ATCC 49619), were purchased from ATCC. All bacterial strains were grown in LB medium at 37°C in an incubator, with shaking at 200 rpm.

Kong J., Wu Z.X., Wei L., Chen Z.S, & Yoganathan S. (2020). Exploration of Antibiotic Activity of Aminoglycosides, in Particular Ribostamycin Alone and in Combination With Ethylenediaminetetraacetic Acid Against Pathogenic Bacteria. Frontiers in Microbiology, 11, 1718.

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Microdilution Assay of Bacterial Strains

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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).

Ye J., Chu A.J., Lin L., Yang X, & Ma C. (2019). First-In-Class Inhibitors Targeting the Interaction between Bacterial RNA Polymerase and Sigma Initiation Factor Affect the Viability and Toxin Release of Streptococcus pneumoniae. Molecules, 24(16), 2902.

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Antimicrobial Activity Screening Protocol

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Several strains were used in the research: bacteria – Bacillus sp. (ATCC 51912), Enterobacter aerogenes (ATCC 29009), Enterococcus faecalis (ATCC 33186), Escherichia coli (ATCC 25922), Haemophilus influenzae (DSM 4690), Neisseria meningitidis (ATCC 53414), Proteus mirabilis (DSM 4479), Pseudomonas aeruginosa (DSM 13626), Serratia marcescens (DSM 50904), Staphylococcus aureus (ATCC 33497), Staphylococcus epidermidis (ATCC 35983), Staphylococcus haemolyticus (DSM 20263), Streptococcus agalactiae (DSM 2134), Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes (DSM 20565), Streptococcus salivarius (DSM 20617), fungi – Aspergillus fumigatus (ATCC 14110), Candida albicans (ATCC 10231), Candida glabrata (DSM 11950), Candida parapsilosis (DSM 5784), Candida tropicalis (ATCC 20115).

Gosiewski T., Jurkiewicz-Badacz D., Sroka A., Brzychczy-Włoch M, & Bulanda M. (2014). A novel, nested, multiplex, real-time PCR for detection of bacteria and fungi in blood. BMC Microbiology, 14, 144.

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