Api 20c aux

A total of 91 yeast strains, collected between 1998 and 2015 in two tertiary hospitals located in Rio de Janeiro, Brazil, and preliminarily identified by the API 20C AUX (bioMérieux, France) as C. glabrata, were included in this study. Strains were isolated from several clinical specimens, such as gastric aspirate (n = 1); renal abscess secretion (n = 1); pleural fluid (n = 1); secretion of surgical drain (n = 1); secretion of postoperative wound (n = 1); ascitic fluid (n = 2); abdominal secretion (n = 3); peritoneal fluid (n = 4); sputum (n = 4); venous catheter (n = 4); bronchoalveolar lavage (n = 5); vaginal secretion (n = 7); feces (n = 9); tracheal secretion (n = 10); urine (n = 13); and blood (n = 25). Before the experiments, these clinical strains were recovered from storage (−20°C) and grown on Sabouraud Dextrose Agar and CHROMagar Candida medium (both at 37°C for 48 h) in order to evaluate their viability and purity, respectively. The phenotypic confirmation of the species after storage was achieved by a biochemical analysis with the Vitek 2 system (bioMérieux, Marcy-L'Etoile, France) using the YST card according to the manufacturer's guidelines. In addition, C. glabrata ATCC 2001 type strain was included as a control strain in all experiments.

In our centre, two central venous blood culture samples for aerobic and anaerobic cultures were taken from patients with fever under aseptic conditions and after disinfection of the central venous access device hub, in addition to a blood culture sample from peripheral veins. Fever was defined as a body temperature > 38.5 °C for at least 4 h or a body temperature > 39 °C once. Neutropenia was described as a total number of granulocytes < 0.5 × 10⁹/L or a total number of leukocytes < 1.0 × 10⁹/L without differential counts available [17 (link)].
Each blood culture bottle (taken from blood or ports) was placed in the BacT/ALERT 9240 automated system (bioMérieux, Marcy l’Etoile, France) [18 (link)]. The microorganisms were identified with a VITEK-2 compact system bioMérieux). Identification and antibiotic susceptibility tests of Gram-positive bacteria were performed using the automated VITEK-2 system with Gram-positive identification card ASTP592, a supplementary Etest (bio- Mérieux, Durham, NC, USA), and a disk diffusion test according to the manufacturer’s instructions [19 (link)]. This system was also used for the identification and antibiotic susceptibility tests of Gram-negative bacteria with Gram-negative identification cards AST-N325, AST-N326, and AST- N327 [20 (link)]. Yeast identification was performed using API 20 °C AUX (bioMérieux) [21 (link)].

Blood samples were cultured in the BacT/AlerT 3D system (Biomérieux). Chromo Agar medium was used for Candida species identification (Biomérieux) and API 20C AuX (Biomérieux) was used for confirmation.

The distinguished colonies on the incubated plates were picked and purified by repeated subculturing done by streaking on the appropriate media with a sterile loop (the strategy consisted of picking 1 colony to represent every visibly different morphology on each plate) using the streak method. Purified colonies were prepared in their respective broth: Mueller Hinton broth for coliforms and Sabouraud Dextrose broth for yeast and moulds. From these preparations, 0.5 ml of each was pipitted into 0.5 ml of glycerol and stored in a freezer at −5°C awaiting identification. All the bacterial cultures were subcultured prior to their use in further experiments and the obtained fresh cultures were used for biochemical tests.
By microscopic observation of each culture following incubation, the purity of isolates was confirmed and preliminary identifications were done according to Bergey's Manual [33 , 34 ]. Proper identification to species level was carried out on the basis of biochemical tests with API 20E (for identification of Enterobacteriaceae and other nonfastidious Gram-negative rods) and API 20 C AUX (for the identification of yeast) (bioMerieux, Marcy l'Etoile, France) according to the instructions of the manufacturer.

Fresh colonies were collected after culturing on PDA for 48 h at 28°C. The API 20 C AUX (bioMérieux, France) kit was used according to the manufacturer's instructions. Results were collected after 48 and 72 h, respectively. Results were analyzed manually following the manufacturer's instructions or using the apiweb software (bioMérieux, France).

API 20 C AUX and API 5 CH (both from bioMérieux, Marcy l’Étoile, France) were used to identify strains for the sugar (carbon) availability test according to the manufacturer’s protocols. These tests include 19 carbohydrate assimilation test strains along with a negative control and are based on turbidity measurements. Isolates were cultured on PDA and TSA plates. Colonies were collected and re-suspended in 0.85% NaCl to obtain a MacFarland turbidity of 2. A 100-μl aliquot of the turbid solution was thoroughly mixed with 7 ml API 20 C medium ampoule 2, and the resultant mixture was transferred to API 20 C AUX and API 5 CH cups using a sterile pipette. Strips were read after incubation for 48 h at 30 °C.