Phoenix automated microbiology system

Species identification and susceptibility tests were performed in the clinical laboratory using an automated microdilution method (BD Phoenix™ Automated Microbiology System; Becton, Dickinson and Co., Tokyo, Japan). The pathogens were considered resistant to ampicillin or vancomycin if they exceeded the Clinical and Laboratory Standards Institute breakpoints (MIC of ≥16 μg/mL for ampicillin and ≥ 32 μg/mL for vancomycin).

P. aeruginosa was identified by the BD Phoenix
Automated Microbiology System (Becton Dickinson). The system is intended
for the rapid identification (ID) and antimicrobial susceptibility
testing (AST) of clinically significant bacteria. Tests used in the
Phoenix ID panels comprise a 45-well ID side that includes tests for
fermentation, oxidation, degradation, and hydrolysis of various substances
and an 85-well side containing dried antimicrobial agents in coordination
with the hospital formulary as QC and growth wells. The technique
involves exposing bacteria to decreasing concentrations of antimicrobial
agents. The Phoenix panels were inoculated according to the manufacturer’s
instructions as follows. A standardized inoculum of 0.5 McFarland
was prepared in Phoenix ID Broth, gently vortexed, and measured using
the BD PhoenixSpec nephelometer to ensure the correct inoculum; then,
25 μL of the inoculum was added to the Phoenix AST broth after
the addition of one drop of the Phoenix AST indicator solution. Once
inoculated with the solutions from both tubes, the panels were placed
in the Phoenix instrument and continuously incubated at 35 °C.
The instrument reads the panels every 20 min for ≤16 h. The
final ID and AST results are transferred via the EpiCenter to the
LIS.

Respiratory samples, included sputum culture and tracheal aspirates when on mechanical ventilation were collected. They were inoculated, first directly, then after a 100-fold dilution, onto Columbia agar + 5% sheep blood, Chocolate blood agar and 2 additional selective media; MacConkey agar and Columbia agar + 5% sheep blood + Colistin + Nalidixic acid.
S. maltophilia species were identified using the BD phoenix automated microbiology system (Becton Dickinson, Sparks, Md., USA). S. maltophilia species counts were determined after 24 h of growth at 37 °C and expressed as colony-forming unit per milliliter (CFU/mL). Antimicrobial susceptibility testing technique consisted of a determination of the minium inhibitory concentrations (MICs) to TMP-SMX, levofloxacin, ceftazidime and ticarcillin-clavulanic acid, using the same BD phoenix automated system.

Swab cultures (eSwab sterile liquid collection kit, Becton-Dickinson, Sparks, MD) were plated on blood agar plates in the microbiology laboratory and were incubated for growth up to 48 hours. For positive cultures, determination of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) status was performed using the BD PHOENIX Automated Microbiology System (Becton-Dickinson) by oxacillin susceptibility. The mupirocin minimum inhibitory concentration (MIC) was obtained using mupirocin E-test strips (0.064–1024 µg/mL; AB Biodisk, Solna, Sweden). The break point for mupirocin susceptibility was ≤1.0 µg/mL.¹³

Identification of each organism and antimicrobial susceptibility test were performed at our institution. Blood cultures were inoculated into BacT/ALERT® 3D bottles (SYSMEX bioMérieux Co., Ltd., Tokyo, Japan). M. catarrhalis, S. pneumoniae and H. influenzae were identified using standard methods (BD Phoenix™ Automated Microbiology System, Becton, Dickinson and Company, NJ, USA). Antimicrobial susceptibility was determined according to the criteria of the Clinical and Laboratory Standards Institute [11 ].