Proteus mirabilis

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.

The microdilution method was used to determine the antimicrobial activity according to the European Committee on Antimicrobial Susceptibility Testing [20 ]. MH broth and MH broth with 2% glucose were used to grow the bacteria and yeasts, respectively. The following microbial strains were tested: Bacillus subtilis ATCC 6633, Candida albicans ATCC 2091, Candida glabrata ATCC 90030, Candida parapsilosis ATCC 22019, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Micrococcus luteus ATCC 10240, Proteus mirabilis ATCC 12453, Pseudomonas aeruginosa ATCC 90271, Salmonella Typhimurium ATCC 14028, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC BAA-1707 (methicillin-resistant strain), and Staphylococcus epidermidis ATCC 12228. The minimum inhibitory concentration (MIC) was calculated and reported for each sample and strain. All experiments were performed in triplicate.

The standard reference strains used in this study were Escherichia coli (ATCC 8739), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 29212), Citrobacter freundii (ATCC 43864), Proteus mirabilis (ATCC 29906), Candida albicans (ATCC 18804) and Candida parapsilosis (ATCC 22019), which were obtained from Microbiologics (St. Cloud, Minnesota) or from the Culture Collection of the Institute of Health Sciences, Federal University of Bahia (Universidade Federal da Bahia–UFBA), located in Salvador, Brazil. The selection of test strains was based on studies of microorganisms commonly causing nosocomial infections, as well as on the recommendations of regulatory agencies for evaluating the efficacy of chemical disinfectants [57 (link), 59 (link)–65 (link)]. The suspensions of the test microorganisms were prepared by transferring cells from the pure culture to plates containing 15–20 mL of plate count agar: agar (9 g/L); dextrose (1 g/L); tryptone (5.0 g/L) and yeast extract (2.5 g/L). To evaluate the disinfection profile in the chamber against the test microorganisms, the inocula were prepared by suspending 1–5 colonies in 5 mL of 0.85% saline solution and the turbidity was adjusted to McFarland No. 0.5 tube [66 ].

Strains used included clinical isolates Escherichia coli KPC-positive ID N°.181244635 (link), Escherichia coli multiresistant ID N°.210112335 (link) and carbapenemase-producing Klebsiella pneumoniae 1825971 (KPC971), as well as reference strains Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 12953, Staphylococcus aureus ATCC 29213, Methicillin-Resistant Staphylococcus aureus ATCC 33591, Streptococcus pyogenes ATCC 19615, Escherichia coli ATCC 8739, Klebsiella pneumoniae ATCC 13885, Proteus mirabilis ATCC 25933, Pseudomonas aeruginosa ATCC 15442 and Salmonella enterica ATCC 14028. Bacteria were plated on brain heart infusion (BHI) agar (Himedia, India) from a frozen stock. Following 24 h incubation, three isolated colonies were transferred to 1 mL of BHI broth. The broth culture was incubated overnight (12–16 h) at 37 °C with shaking36 (link).

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).

The bacterial species used were Proteus mirabilis (ATCC 29906), Escherichia fergusonii (ATCC 35469), Escherichia coli HB101 (Caenorhabditis Genetics Center), Pseudomonas fluorescens (ATCC 13525), Micrococcus luteus (ATCC 4698), Staphylococcus hominis (ATCC 27844), Rhizobium leguminosarum (ATCC 14479), Raoultella ornithinolytica JUb54 and Raoultella sp. JUb38 (obtained from Samuel lab, Baylor College of Medicine), and Bacteroides thetaiotaomicron (ATCC 29148, obtained from Hsiao lab, UCLA). E. coli, E. fergusonii, P. mirabilis, P. fluorescens, JUb38, and JUb54 were maintained on Luria broth (LB) plates, grown in LB, and diluted in LB. S. hominis and M. luteus were maintained on tryptic soy plates, grown in tryptic soy media, and diluted in tryptic soy media. R. leguminosarum was maintained on Rhizobium X plates, grown in Rhizobium X media, and diluted in Rhizobium X media. B. thetaiotaomicron was cultured anaerobically in brain-heart infusion supplemented media (BHIS) as previously described [70 (link)], and diluted in BHIS. Growth temperatures were 37 °C for all strains except for the Raoultella strains, P. fluorescens, and R. leguminosarum (28 °C); and M. luteus (30 °C). Each species was diluted to 1 × 10⁸–1 × 10⁹ CFU/mL as measured by a Thermo Scientific™ NanoDrop™ One spectrophotometer before use.