Microscan walkaway system

Isolates were submitted on Dorset transport media (Diagnostic Media Products, National Health Laboratory Service, South Africa) to the National Institute for Communicable Diseases (NICD), Johannesburg, South Africa. Each isolate was plated onto a 5% blood agar plate (Diagnostic Media Products, National Health Laboratory Service, South Africa) followed by organism identification and antimicrobial susceptibility testing using automated systems [(VITEK II system (bioMèrieux, Marcy-l'Etoile, France)/MALDI-TOF MS (Microflex, Bruker Daltonics, MA, USA) and MicroScan Walkaway system (Siemens, Sacramento, CA, USA), (Gram-positive panel PM33)], respectively. An isolate was phenotypically non-susceptible if it had an oxacillin MIC of >2 and a positive cefoxitin screening result. Interpretation of susceptibility was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [17 ].

We isolated H. haemolyticus (strain identification 2019-19) from the suction sputum and identified it as quinolone-resistant H. influenzae by routine laboratory testing using a MicroScan WalkAway system (Siemens, https://www.siemens.com). Because quinolone-resistant H. influenzae had never been isolated from a pediatric patient, we performed a detailed susceptibility test for 2019-19 by the broth microdilution method. For controls in the biochemical test, we used H. influenzae GTC 14202^T (DSM 4690^T) and H. haemolyticus GTC 15009^T (NCTC 10659^T) type strains purchased from Gifu University (https://www.gifu-u.ac.jp). In addition, we used H. influenzae ATCC 49247 and Rd as quality control strains for antimicrobial susceptibility testing. We cultured the isolates overnight on chocolate agar at 37°C in a 5% CO₂ atmosphere and stored them in 10% skim milk at −80°C until use. This study was approved by the research ethics committees at the Tokyo University of Pharmacy and Life Sciences (case no. 16-12).

Phenotypic confirmation of ESBL production was carried out by combination disk test using both cefotaxime and ceftazidime disks, alone and in combination with clavulanic acid (Condalab). Minimum inhibitory concentrations were obtained with the MicroScan WalkAway system (Siemens Healthcare Diagnostics Inc., West Sacramento, CA, USA) using the NEG-MIC Type 44 panel. PCR detection of blaTEM, blaSHV, and blaCTX-M genes was done using the primers and conditions described by Monstein et al. [20 (link)]. Subtyping of CTX-M group was carried out by sequencing and comparison with AMRFinderPlus tool available from the National Center for Biotechnology Information (NCBI) server [21 ].

The amniotic fluid samples were analyzed according to standard methods used by the Clinical Laboratory Center (certified ISO15189) at Toyama University Hospital. First, 1 mL amniotic fluid sample was centrifuged at 1,880×g for 15 min to spin down the microorganisms, and 800 μL of the supernatant fraction was carefully removed in order to not lose the pellet, leaving the pellet with 200 μL of supernatant. One drop of the resulting pellet with 200 μL of supernatant was placed on the appropriate agar media (PPLO agar for Mycoplasma, Brucella HK agar for anaerobic bacteria, blood agar, BTB agar and chocolate agar, respectively) and incubated aerobically or anaerobically until sufficient growth was present to proceed with testing (PPLO agar was incubated anaerobically at 35°C with 10% CO₂ for up to 7 days, Brucella HK agar was incubated anaerobically at 35°C with 10% CO₂ for up to 72 hours, blood agar and BTB agar was incubated aerobically at 35°C for up to 72 hours, and chocolate agar was incubated aerobically at 35°C with 5% CO₂ for up to 72 hours). For all samples, the specific identification methods differed according to the organism, although they included the MicroScan WalkAway system (Siemens Healthcare Diagnostics, IL, USA), RapID ANA II (Thermo Fisher SCIENTICIC, UK) and various latex agglutination and biochemical spot tests.