Microscan walkaway

One or several samples were obtained from tissue, aspiration fluid, synovial fluid, and the drain. All samples were sent to the microbiology laboratory. Standard culture was performed at first. If samples were not cultured using standard culture methods, an enrichment culture method was performed using broth culture medium for 5 days according to a previous study [18 ]. Briefly, standard culture was performed using 5% sheep’s blood agar plates containing peptone and sodium chloride. The enrichment culture comprised semi-solid Gifu anaerobic medium containing peptone, soy peptone, protease peptone, beef extract, yeast extract, liver extract, glucose, starch, l-tryptophan, l-cysteine hydrochloride, thioglycolic acid sodium salt, l-arginine, vitaminK1, hemin, potassium dihydrogen phosphate, and sodium chloride. The standard culture method was conducted for 24 h at 35°C/5% CO₂. Enrichment culture was performed for 5 days at 35°C in ambient air. When the enrichment culture was negative, it was identified as culture-negative. Bacteria were identified by analysis of biological properties using MicroScan WalkAway and a combo panel (Beckman Coulter, Brea, CA). The possibility of tuberculous, fungal, or viral infection was also investigated.

Antibiotic susceptibility testing was preliminary performed using the Vitek2® system (BioMérieux, Marcy l’Etoile, France) or MicroScan Walkaway (Beckman, United States) at the participating hospital laboratories, and subsequently by the broth microdilution method using commercially available microplates (MERLIN Diagnostika GmbH, Germany). Discrepant results were resolved by testing individual isolates with the disk diffusion method. Results were interpreted according to the EUCAST breakpoints (version 11.0, 2021; see footnote 1).

MIC values of TZP were determined by two methods: (i) the semiautomatic system MicroScan WalkAway (Beckman Coulter, USA), which is the routine method used in many hospitals for the determination of the susceptibility profile, and (ii) TZP gradient strips (Liofilchem, Italy) following the manufacturer’s instructions. All experiments were repeated in three separate experiments, using daily freshly prepared plates and inocula. Clinical breakpoints were established according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (24 ). Hence, isolates with TZP MICs of ≤8 mg/L were categorized as susceptible, while those with MICs of >8 mg/L were categorized as resistant.

All processes were performed by trained technicians using both systems, according to the same workflow after a BC bottle was flagged as positive: (i) Gram stain and subculture on blood agar and chocolate agar, (ii) identification directly from the pellet by MALDI-TOF mass spectrometry (Bruker, Germany) (25 (link)), (iii) rapid disc diffusion antibiogram, and (iv) final AST by a semiautomated microdilution system (MicroScan WalkAway; Beckman Coulter, USA). Gram stain, MALDI-TOF mass spectrometry, and AST results were communicated to the attending physician immediately if the microorganism was not considered to be a contaminant and if this was the first bacteremia episode for that patient.

Antimicrobial susceptibility was determined by minimal inhibitory concentrations by using the MicroScan WalkAway^® -automated system (BECKMAN COULTER, Inc., Brea, CA, United States) according to the manufacturer’s instructions. The antibiotics tested were ticarcillin, aztreonam, ceftazidime, cefepime, ampicillin–sulbactam, piperacillin–tazobactam, imipenem, meropenem, amikacin, gentamicin, tobramycin, levofloxacin, ciprofloxacin, trimethoprim–sulfamethoxazole, fosfomycin, colistin, minocycline, and tigecycline. Additionally, resistance to ampicillin, cefotaxime, chloramphenicol, and nalidixic acid was determined by disk diffusion assays (Becton Dickinson, Sparks, MD, United States). All results were interpreted according to the CLSI guidelines (Wayne, 2017 ). Multidrug resistance status was attributed to those isolates resistant to at least one agent of three or more different antimicrobial categories, including resistance to β-lactamase inhibitors (Magiorakos et al., 2012 (link)).