Microbank vial

Acinetobacter baumannii NCTC 13304 was stored at −20°C in Microbank vials (Pro – Lab Diagnostics, Cheshire, UK) and subcultured overnight at 37°C on Muller–Hinton agar (MHA) (Oxoid Ltd., Basingstoke, UK).

This study utilised Candida albicans strain SC5314 and a series of routine patient anonymised clinical bloodstream isolates (n  =  30) collected under the approval of the NHS Scotland Caldicott Gaurdians, as part of candidaemia epidemiology surveillance study17 (link)36 (link). C. albicans clinical bloodstream isolates previously identified to have high biofilm formation (HBF [n = 15]) and low biofilm formation (LBF [n = 15]) were used throughout this study17 (link)36 (link). Isolates were stored in Microbank^® vials (Pro-Lab Diagnostics, Cheshire, UK) at −80 °C until sub-cultured onto Sabouraud’s dextrose agar (SAB [Sigma-Aldrich, Dorset, UK]). Isolates were propagated in yeast peptone dextrose (YPD) medium (Sigma-Aldrich, Dorset, UK), washed by centrifugation and re-suspended in RPMI-1640 (Sigma-Aldrich, Dorset, UK) to 1 × 10⁶ cells/mL, as described previously37 (link). Biofilms were grown in polystyrene plates or 75 cm² tissue culture flasks in RPMI for 24 h at 37 °C.

This study utilised clinical Candida albicans (n = 60) bloodstream isolates, collected under the approval of the NHS Scotland Caldicott Gaurdian's [3 (link),24 (link)]. Isolates were stored in Microbank® vials (Pro-Lab Diagnostics, Cheshire, UK) at −80 °C until sub-cultured onto Sabouraud's dextrose agar (SAB [Sigma-Aldrich, Dorset, UK]). C. albicans isolates were propagated in yeast peptone dextrose (YPD) medium (Sigma-Aldrich, Dorset, UK), washed by centrifugation and re-suspended in RPMI-1640 with 2% D-glucose (RPMI-1640, Sigma-Aldrich, Dorset, UK) to 1 × 10⁶ cells/mL, as described previously [19 (link)].

Reference P. aeruginosa strains PAO1, PA14, and PAK and 11 clinical P. aeruginosa isolates were used in this study. Clinical strains were selected from the CF Bacterial Repository at the National Heart and Lung Institute, Imperial College London; this collection consists of bacteria isolated from airway samples of people with cystic fibrosis at the Royal Brompton Hospital, London (Table 1). Bacteria were stored in Microbank vials (Pro-Lab Diagnostics) at −80°C. Clinical strains were selected with a range of antibiograms according to clinical lab results using EUCAST clinical breakpoints. Isolates were revived onto cetrimide agar (Merck) for confirmation of purity and grown overnight at 37°C followed by subculture onto LB agar (Merck). Single colonies were inoculated into Mueller-Hinton broth (Merck) and incubated overnight at 37°C with agitation at 200 rpm.

All organisms, including those identified as strains, such as P. aeruginosa PAO1, are referred to as isolates in this work for ease. The strains, namely S. aureus ATCC 25923, P. aeruginosa ATCC 27853 and E. coli ATCC 25922 are distinguished as indicator strains, which was noted by the American Type Culture Collection.
The Pseudomonas aeruginosa isolates and uropathogenic Escherichia coli CFTO73 were generous gifts from Dr. J. Harrison (University of Calgary). All bacterial stains and isolates were stored in Microbank™ vials at −80 °C as described by the manufacturer (ProLab Diagnostics, Richmond Hill, ON, Canada). Prior to the disk diffusion assay, the strains and isolates were streaked out on Luria-Bertani (LB) media agar (1.5%) plates and grown overnight at 37 °C. Our choice of growth medium is reflected in other works that have also used this medium to monitor the susceptibility of microorganisms to metals.

Candida albicans SC5314, 3153A and a series of routine patient anonymised clinical bloodstream isolates (n = 42) collected under the approval of the NHS Scotland Caldicott Gaurdians from the Royal Hospital for Sick Children (Yorkhill Division), Glasgow, UK, as part of candidaemia epidemiology surveillance study. All clinical isolates obtained during this period were independently identified using Colorex Candida chromogenic plates (E&O Laboratories Ltd, Bonnybridge, UK) and were stored in Microbank® vials (Pro-Lab Diagnostics, Cheshire, UK) at −80°C until further use. These isolates were sub-cultured onto Sabouraud’s dextrose agar (SAB [Sigma-Aldrich, Dorset, UK]). Plates were incubated at 30°C for 48 h and maintained at 4°C. Isolates were propagated in yeast peptone dextrose (YPD) medium (Sigma-Aldrich, Dorset, UK), washed by centrifugation and resuspended in the appropriate media (Sigma-Aldrich) to the desired concentration, as described previously [50 (link)].

From September 2011 to June 2012, a prospective observational study of community-onset severe sepsis and septic shock in adults was conducted at Skaraborg Hospital, a secondary hospital with 640 beds, in the western region of Sweden (Ljungström et al., 2019 (link)). All patients ≥18 years consecutively admitted to the emergency department for suspicion of community-onset sepsis were asked to participate in the study. The study was approved by the Regional Ethical Review Board of Gothenburg (376-11). As the present study only focused on bacterial isolates recovered from cultures included in the routine patient care, no individual written consent was needed. During 9 months, approximately 1,800 bacterial isolates from different sample types were recovered from the patients enrolled in the study. These isolates were cryopreserved at the time of recovery by transferring colonial material to Microbank™ vials (Pro-Lab Diagnostics, Ontario, Canada) stored at −80°C. For the present study, all isolates identified as Klebsiella spp. (n = 105) with routine microbiological methods based on cultures followed by MALDI-TOF MS (DB-4110) were selected. The isolates were recovered from 82 patients in samples collected from blood (n = 29), urine (n = 71), nasopharynx (n = 4), and wound (n = 1).