Cryogenic microbank vials

We used 97 isolates from the Enterobacterales order and non-fermenting bacteria: 93 from clinical samples in the period from 2008 to 2018 at the clinical microbiology laboratories of the University Hospital Basel, Switzerland; Cantonal Hospital Lucerne, Switzerland and laboratory Viollier in Allschwil, Switzerland (Additional file 6), and four reference strains: E. coli ATCC-25922, E. coli NCTC-13846 (mcr-1), P. mirabilis ATCC-25933 and E. coli KP-37 (mcr-2) [46 ]. All colistin resistant isolates tested with Vitek 2® (bioMérieux, Marcy’l Etoile, France) from the University Hospital Basel were included in the study. The strain collection included 43 colistin sensitive isolates.
Species identification was performed at the time of diagnosis with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS; Bruker, Bremen, Germany) by using the mass-spectrum library and the MALDI Biotyper 3 software (OC 3.1, Bruker Daltonics) at standard conditions. All bacterial isolates were frozen at − 70 °C in cryogenic Microbank™ vials (Pro-Lab Diagnostics, Birkenhead, UK). Prior to testing, the strains were cultured on Columbia agar supplemented with 5% sheep blood (BD Diagnostic Systems, Allschwil, Switzerland) with subsequent subculture after 24 h.

Six C. auris clinical blood isolates (CJ94, CJ97, CJ98, CJ99, CJ100, and CJ102) from an outbreak in the Hospital Universitario y Politécnico La Fe (Valencia, Spain) [30 (link)] were included in this study. Fungal strains were stored in vials with sterile distilled water at room temperature for up to 1 year, while commercially prepared cryogenic Microbank vials (Pro-Lab Diagnostics, USA) maintained at −70 °C were used for prolonged storage. Strains were cultivated in Sabouraud dextrose agar (SDA) for 24 h before every experiment. MIC determination at 24 h was performed following EUCAST guidelines [31 ]. MICs for isavuconazole, anidulafungin, caspofungin, and micafungin were 0.06, 0.125, 0.125, and 0.25 mg/L, respectively, for all isolates.

The neurotoxigenic C. butyricum type E strains LCL 063, LCL 155, KZ 1886 and KZ 1890 were a kind gift from Professor Shinichi Nakamura of Kanazawa University to Dr Paolo Aureli of the Istituto Superiore di Sanità. Strains were maintained at −80 °C in cryogenic microbank vials (Prolab Diagnostics, Round Rock, TX, USA). Prior to DNA extraction, stock cultures of the strains were transferred to egg yolk agar (EYA) plates and checked for purity after 48 h of incubation at 37 °C under anaerobic conditions (Gas Pack anaerobic jars and AnaeroGen atmosphere generation). The four neurotoxigenic C. butyricum type E strains were confirmed to be positive for the bont/e gene by PCR, as previously described [36 (link)].