Microscan system

The causative MDR/XDRAB pathogen was defined as an isolate from the blood, quantitative culture (≥10⁴ cfu/mL) of a bronchoalveolar lavage (BAL) specimen, semiquantitative culture (moderate or heavy growth) of a bronchoscopic aspirate or BAL specimen, quantitative culture (≥10⁵ cfu/mL) of an endotracheal aspirate, and semiquantitative culture (moderate or heavy growth) of an endotracheal aspirate with white blood cells >25/high power field (HPF) on Gram stain. Bacterial identification was performed using standard methods. Susceptibility testing was done using the Microscan system (Dade Behring, West Sacramento, CA, USA), and results were interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines published in 2012 [20 ]. The minimum inhibitory concentrations (MICs) of tigecycline and colistin were determined using the broth microdilution method, and in this study, isolates with MIC ≤2 mg/L were considered to be susceptible to tigecycline and colistin. The dose of tigecycline was a 100 mg loading, followed by 50 mg every 12 hours. The dose of colistin was 5 mg/kg colistin base activity loading, followed by 150 mg colistin base activity every 12 hours in patients with normal renal function.

Patients infected with colistin-resistant A. baumannii were identified at the Asan Medical Center, Seoul, South Korea, between January 2010 and December 2012. Colistin susceptibility testing was performed on all blood and some sputum isolates at the request of the treating physician. A colistin MIC of >2 mg/liter indicated resistance (14 ). Nine representative colistin-resistant A. baumannii isolates from different patients were included in this study. The clinical data of these patients were collected from electronic medical records, and A. baumannii was identified using a MicroScan system (Dade Behring, Deerfield, IL, USA) and/or a Vitek 2 system (bioMérieux Inc., La Balme les Grottes, France). The following 12 antimicrobial agents were selected based on previous studies suggesting their antimicrobial efficacy against MDR A. baumannii: colistin, ampicillin-sulbactam, amikacin, azithromycin, aztreonam, ceftazidime, meropenem, rifampin, tigecycline, trimethoprim-sulfamethoxazole, vancomycin, and teicoplanin (15 (link)– (link)27 (link)).

Identification of microorganisms and susceptibility testing were performed at each clinical microbiology laboratory. The minimum inhibitory concentration (MIC)s of the Enterobacterales were determined using the Vitek 2 (bioMérieux Vitek, Hazelwood, MO, USA) or MicroScan system (Siemens, Sacramento, CA, USA). According to Clinical and Laboratory Standards Institute (CLSI) guideline²³ , carbapenem nonsusceptibility was determined by MIC > 1 mg/L for meropenem and imipenem or MIC > 0.5 mg/L for ertapenem. Among the CRE, the phenotypic detection of CP-CRE was conducted by modified Hodge test and carbapenemase inhibition test in-house. After the CRE isolates were sent to the Institute of Health and Environment, a carbapenemase gene polymerase chain reaction (PCR) test was performed to detect the presence of carbapenemase.

The phenotypic confirmation of ESBL production was performed by combination disk test and double-disk synergy test. The antimicrobial susceptibility to additional antibiotics and minimum inhibitory concentrations were obtained in MicroScan^® system (Siemens AG, Munich, Germany). Following Clinical And Laboratory Standards Institute recommendations ESBL-producing isolates were categorized as sensitive and resistant (including “intermediate resistant” and “totally resistant”) [20 ].
The presence of β-lactamase genes (bla_TEM, bla_SHV, bla_OXA and bla_CTX-M) was detected by using a modification of two multiplex PCR [21 (link),22 (link)]. DNA amplification was performed in a DNA thermal cycler GeneAmp^® PCR system 2700 (Applied Biosystems Division, Foster City, CA, USA). In order to identify the bla genes a bidirectional DNA sequence analysis was performed by the Macrogen EZ-Seq purification service (Macrogen Europe, Amsterdam, The Netherlands). Each sequence was compared with the sequences included in GenBank and Lahey Clinic web [23 ].

Enterococcal isolates from all samples (except for clinical specimens) were obtained using mE agar. All isolates were divided into four groups based on pigmentation and motility. The isolates were identified to the genus level based on growth in BHI with 6.5% NaCl, growth at 45°C, esculin hydrolysis, catalase, and PCR amplifying of the Tuf gene [33 (link)]. Species level identification was done by a double digestion of the PCR product of the ATP synthase α subunit gene in combination with a restriction fragment length polymorphism (RFLP) assay (paper in preparation). Clinical strains obtained from a local hospital were identified using MicroScan system (Siemens Health Care). Up to 12 isolates each of E. faecalis or E. faecium were randomly selected per sample for virulence gene analysis.