246004; BD) was previously added, according to manufacturers’ guidelines. Since the inoculum of GPC in ID broth was shown to be almost 10 times lower than is standard in a 0.5 McFarland suspension, 250 μl of inoculated ID broth was added to AST broth for GPC, instead of the 25 μl in the manufacturers’ guidelines. For GNR, the Phoenix system panel NMIC/ID-75
(product no. 448087; BD) was used. For GPC, the PMIC-58 panel (product no. 448052; BD) was used. To calculate the original number of CFU/ml in the ID broth and to serve as purity control, dilutions of ID broth were also subcultured, using the Eddy Jet spiral plater (IUL, S.A., Barcelona, Spain). Routinely BKM120 manufacturer used inoculation For the routinely used method, a small volume of positive see more blood culture was inoculated on Columbia sheep blood agar plates and incubated at 35°C with 5% CO2. A standard inoculum in ID broth was prepared from the bacteria grown on the agar medium and inoculated into Phoenix panels, following the manufacturer’s recommendations. Identification of GPC Since the Phoenix system is not used for ID of GPC in routine diagnostics, ID by direct inoculation was not tested in this group.
To discern Staphylococcus species from other GPC, a catalase test was performed. For the identification of Staphylococcus species, selleck inhibitor catalase-positive strains were tested for coagulase and DNAse production. If both tests were negative, the strain was identified as a coagulase-negative Staphylococcus species (CoNS). To discern Enterococcus species from other catalase-negative GPC, bile esculin and tellur diagnostic tablets (Rosco Diagnostica, Taastrup, Denmark) were used, according to manufacturer’s guidelines. If both tests were positive, the strain was identified as Enterococcus faecalis, whereas in case of a positive bile esculin test but a negative tellur test, an API 20 Strep test (Biomérieux SA, Marcy l’Etoile,
France) was performed to further identify the strain. Results of identification were adjusted in the Phoenix results retrospectively for both the standard and direct method, after which the software automatically adjusted MIC cutoff Tolmetin values to those of the identified species. Discrepancy analysis To resolve differences in ID of GNR, the API system was used (API 20E for Enterobacteriaceae and API 20NE for non-fermenters (Biomérieux)). In case of discrepancies in AST between results of the direct method and the routinely used method for ceftazidime, ceftriaxone, cefuroxime, ciprofloxacin, clindamycin, levofloxacin, moxifloxacin, linezolid, penicillin, piperacillin, piperacillin-tazobactam, and tobramycin an E-test (Biomérieux) was performed according to manufacturer’s guidelines, and used as gold standard [20, 21]. Discrepancies for amoxicillin, amoxicillin-clavulanate, erythromycin, gentamicin, oxacillin, rifampin, tetracycline and trimethoprim-sulfamethoxazole were resolved using microbroth dilution, as described in the CLSI-guidelines .