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Sulfamethoxazole with fluconazole (FQ) is a popular treatment regimen for S. aureus infection or fungal infection. Because the fluconazole acts against multiple organisms, it has a broad spectrum of anti-fungal activity with minimal adverse effects on healthy skin [1]. However, a substantial fraction of S. aureus is resistant to even the highest concentrations of FQ in vitro, and the resistance may persist throughout life [15]. It is thus unclear whether fluconazole will be as effective in the treatment of S. aureus infections. To determine whether fluconazole can be used effectively in a clinical setting with S. aureus as an additional risk, this study evaluated the susceptibility of S. aureus to fluconazole alone and with a range of other antibacterial agents. The antimicrobial susceptibility of S. aureus was determined with a panel of common agents that have been investigated as alternatives to FQ for treating infections caused by S. aureus [2, 16, 17]. This study also evaluated the effect of antimicrobial agent on the growth and survival of S. aureus in a 3% agar gradient culture with and without 10% (v/v) glycerol as an acidifying agent. MATERIALS AND METHODS Patients Sigmund and Jürgen were screened for S. aureus by using blood culture and sensitivity as described [18] (Table 1). These were screened using serum and cerebrospinal fluid cultures. In addition, a 3x106 colony-forming units (CFU) S. aureus isolate was obtained from each patient via blood agar. After culture, the specimens were collected and stored in liquid nitrogen until assay. The antimicrobial susceptibility of S. aureus was determined by a combination of molecular techniques. One method has previously been suggested for the detection of S. aureus in blood as well other body fluids [18-20]. For this technique, a blood sample is collected for each Venlaf 80 Pills 100mg $321 - $4.01 Per pill patient through a venipuncture. The blood sample is then diluted in 1 ml of sample buffer and subsequently in a 96-well plate (BD HOBOTONE 1.4% protein, 50 mm centrifugation, 2 EDTA, 1.25 mol dL−1 glutamine, and 3.35 mmol NaCl, pH 6–6.8, with 1 mM MgSO 4 ). The dilutions are then used in 1 : 5 dilution. One such dilution, containing approximately 5–10 CFU, was used for each specimen to ensure sufficient numbers of S. aureus to generate an optical density reading (OPRD). This method was used as previously described [18, 20], and was adapted to S. aureus increase sensitivity growth on the plates. For OPD assay, a 3x103 S. aureus-like strain was obtained by isolating S. aureus from an isolate a patient who had clinical symptoms for 3 years but had no identifiable infection. This sample was further diluted in 1 ml of a serum agar dilution, and plates were coated with the dilution. plates were incubated for 15 min at 37°C to allow for OPD fluorescence detection. After detection of for 1 hour (or until plates were stained with H 2 O to stain for S. aureus growth), the plates were rinsed with phosphate–buffered saline. The plates were placed on a polypropylene filter, to allow for growth and development of colonies on the plate. Laboratory Procedure For each sample of a patient's cerebrospinal fluid, 100 μL of blood and 500 the dilution used for OPD fluorescence detection were treated for 15 min at 37°C to allow for fluorescence on both surfaces. A second 1.0 ml of the dilution was placed in each plate and washed three times for 5 min with 2 ml of 2% nonessential amino acid medium (R&D Systems, Minneapolis, MN, USA). After treatment, 5 μL of this medium was added to each well with 1 mL of phosphate–buffered saline solution to provide 0.1–4.0 times the OPD sensitivity of test medium. A microchip was placed inside the filter, which was incubated for 2 h on ice (prepared in PBS with 0.2% Tween-20 solution and 0.5% sodium deoxycholate; the chip was then washed with Tween and allowed to incubate for an additional 5–7 h.) and then placed on ice for further processing. Finally, the sample was diluted 1:4 in phosphate–buffered saline solution before being placed under a microscope and fixed in 50 μL of 1% formaldehyde (Sigma-Aldrich Pharmaceuticals, St. Louis, MO, USA) for 2 hr. The plates were fixed once more.

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