albicans was cultured alone, the count was 2.85×10 8 CFU/mL when cultured together with P. mirabilis or bacterial-cultured supernatants were cultured together for 72 hours, and C.
albicans biofilm formation decreased in proportion to the concentration of the bacterial-cultured supernatants (data not shown). To determine whether this effect was due to a depletion of nutrients in the medium or to the secretory products of the bacteria, we diluted the cultured supernatants and tested the concentration effect on C. albicans biofilm formation was reduced by 60–70%, compared to C. mirabilis were cultured for 72 hours, and remaining bacteria were removed by filtration. In order to determine if this reduction was due to the effect of the bacteria or of secretory products when cultured, P. mirabilis were co-cultured, biofilm formation of C. The ACT1 and PMA1 genes were used for this purpose. Real-time PCR data were normalized with the geometric mean of two reference genes.
Proteus vulgaris identification software#
Expression levels were analyzed using ABI 7900 System SDS software (Applied Biosystems). Dissociation curves were analyzed for all reactions to verify single peaks/products. Real-time PCR reactions were performed at 95℃ for 5 minutes, followed by 40 cycles of 15 seconds at 95℃ and 1 minute at 60℃. The PCR was run on MicroAmp® Optical 384-well reaction plates in an ABI 7900 Real-Time PCR system (Applied Biosystems).
Real-time polymerase chain reaction (PCR) contained 10 µL of Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), as well as forward and reverse primers (1 µL of each) ( Table 1) 22 and sterile water, at a final volume of 20 µL. Reactions were incubated at 42℃ for 50 minutes and then at 70℃ for 15 minutes.
To each reaction tube, 10 µL of the following mixture was added: 4 µL of 5x First-Strand Buffer, 2 µL of 10 mM MgCl 2, 2 µL of 0.1 M DTT, 1.4 µL of RNase inhibitor, and 1 µL of Superscript II. Reactions were incubated at 65℃ for 5 minutes and cooled on ice. Each reaction contained 1 µg of total RNA, 1 µL of 50 µM hexamer, and 1 µL of 10 mM dNTP in a final volume of 10 µL. RNA was treated with amplification grade DNase I (Epicentre Biotechnologies) and used for cDNA synthesis with random hexamer primer (Invitrogen Life Technologies, Carlsbad, CA, USA) using Superscript II reverse transcriptase reagents (Invitrogen Life Technologies). albicans cells using the MasterPure Yeast RNA Extraction kit (Epicentre Biotechnologies, Madison, WI, USA). A colorimetric change resulting from XTT reduction was measured using a microtiter plate reader (EMax, Molecular Devices, Sunnyvale, CA, USA) at 490 nm. The plates were then incubated in the dark for up to 3 hours at 37℃. Louis, MO, USA) and menadione (0.4 mM, Sigma) solution was added to each well containing the prewashed biofilm and the control well. A 200-µL aliquot of XTT (1 mg/mL, Sigma, St. A quantitative measure of biofilm formation was calculated using the XTT reduction assay. After biofilm formation, the medium was aspirated, and non-adherent cells were removed by thoroughly washing the biofilm three times with PBS. A volume of 200 µL of medium was added to each well, and the plate was then incubated for another 72 hours. After the initial adhesion phase, the cell suspensions were aspirated, and each well was washed twice with phosphate-buffered saline (PBS) to remove loose adherent cells. The plate was incubated for 90 minutes at 37℃ in an orbital shaker at 75 rpm. Microorganisms were prepared for each condition and transferred to selected wells of a microtiter plate. 23 Biofilms were formed on commercially available pre-sterilized, polystyrene, flat-bottomed, 96-well microtiter plates (Costar, Cambridge, MA, USA). Biofilm formation was quantified using the method developed by Ramage, et al.