Enterobacter aerogenes

The antimicrobial activities of the tested substances were carried out on fifteen bacteria made up of two strains of Gram positive bacteria: Staphylococcus aureus (ATCC 25922) and Enterococcus faecalis (ATCC 10541), nine strains of Gram negative bacteria: Pseudomonas aeruginosa (PA01), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 8739), Escherichia coli (ATCC 10536), Escherichia coli (ATCC 11775), Enterobacter aerogenes (ATCC 13048), Klepsiella pneumoniae (ATCC13883), Providencia stuartii (ATCC 29916), Salmonella typhi (ATCC 6539) and four clinical isolates of gram negative bacteria: Salmonella parathyphi A, Salmonella paratyphi B, Shigella flexneri and Proteus mirabilis.The references strains ATCC and the clinical isolates were obtained from the American Type Culture Collection (Rockville, MD, USA) and the Laboratory of Bacteriology and Mycology of the “Centre Pasteur” of Yaoundé-Cameroon respectively. These microorganisms were maintained on agar slant in refrigerator at 4 °C.

The bacterial strain E. coli ATCC 25922 (American Type Culture Collection, Georgetown, DC, USA) was maintained in Luria-Bertani (LB) medium. Whole bacterial cells to be use as targets for selection were cultured at 37°C to a OD₆₀₀ of 0.3 (equivalent to ~2.4 x 10⁸ bacteria/mL), washed twice with PBS (NaCl₂ 137 mM, 2.7 mM KCl, 4.3 mM Na₂HPO₄.7H₂O, 1.5 mM KH₂PO₄) and diluted in selection buffer (PBS containing 1.4 mM MgCl₂).
The following bacterial strains were used in binding assays: Klebsiella pneumonia ATCC 27853, Enterobacter aerogenes ATCC 13048, Proteus mirabilis ATCC 00557, Pseudomonas aeruginosa ATCC 700603, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212, from ATCC; and Enterobacter cloacae, Proteus vulgaris, Morganella morganii, Citrobacter freundii, Acinetobacter baumannii, Enterococcus faecalis, Enterococcus faecium, from the bacterial collection of the Instituto Adolfo Lutz (São Paulo, SP, Brazil). The MNEC clinical isolates used here were obtained from the Clinical Hospital of the Federal University of Paraná (HC, Curitiba, PR, Brazil).

Extracted DNA from the strains Escherichia coli K-12 MG1655 (700926), Bacillus cereus str. Frankland and Frankland (10876), Vibrio parahaemolyticus (17802D-5), and Clostridioides difficile (9689D-5) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The additional bacteria and their sources used in specificity testing included Bacillus megaterium, Bacillus subtilis, Bacillus thuringiensis, Citrobacter freudii, Enterobacter aerogenes, E. coli (ATCC 25922), Klebsiella oxytoca, Listeria monocytogenes, Micrococcus luteus, Salmonella enterica, Serratia macrescens, Shigella flexneri, Staphylococcus capitis, and Staphylococcus saprophyticus, as listed in Table 1. B. cereus and E. coli bacterial cultures were also obtained from the Carolina Biological Supply Company (Burlington, NC, USA). E. coli and B. cereus were inoculated in Luria–Bertani (LB) medium and grown at 37 °C. A hemocytometer and a compound light microscope were used to obtain cell counts. Human HL-60 DNA was also tested in the specificity studies and was obtained from ATCC.

These experiments used E. coli MC4100 (F- [araD139]B/r Δ(argF-lac)169* &lambda- e14- flhD5301 Δ(fruK-yeiR)725 (fruA25)‡ relA1 rpsL150(strR) rbsR22 Δ(fimB-fimE)632(::IS1) deoC1) carrying plasmids E3350 (pUC18T-mini-Tn7-Gm-eyfp, accession DQ493879) or E3322 (pUC18T-mini-Tn7-Gm-DsRedExpress, accession DQ493880) [53 (link)]. E. coli MC4100 was obtained from the E. coli Genetic Stock Center (CGSC #6152). For multispecies colonization experiments, Enterobacter aerogenes (ATCC 13048) and Serratia marcescens (ATCC 13880) were obtained from ATCC.
Bacterial strains were grown in individual cultures in LB + 30 μg/mL gentamycin for selection where necessary. For feeding assays, E. coli were grown overnight at 37°C, and E. aerogenes and S. marcescens were grown overnight at 30°C. Bacterial cultures were acclimated briefly (~1 h) to room temperature before feeding. To construct feeding cultures, E. coli were centrifuged for 1 min at 9,000 RPM to pellet, washed once in S medium, then resuspended in S medium + 30 μg/mL gentamycin. As S. marcescens is highly motile and very difficult to pellet through centrifugation, E. aerogenes and S. marcescens were chilled for 30–60 min before centrifugation, then centrifuged for 1 min at 10,000 RPM to pellet before washing and resuspension.

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).