Densicheck

An outline of the experimental procedure for Phase III of the iSSC validation study can be found in Figure 2. S. paucimobilis, R. pickettii, and C. basilensis, bacterial species isolated from the ISS potable water, were kept in glycerol stocks at −80 °C for preservation [2 (link),10 (link),11 (link)]. For each experiment, isolates were streaked onto tryptic soy agar TSA) from the glycerol stocks and were incubated at 30 °C for 48 h. After incubation, a bacterial suspension was prepared in a sterile phosphate saline buffer (PBS, pH 7.4). The suspension density was measured using Densicheck (bioMérieux, St. Louis, MO, USA) and then diluted to densities of 10⁵, 10⁴, 10³, and 10² CFU/mL. The bacterial suspensions, also referred to as the “inoculum”, were added to 1-L volumes of sterile PBS in triplicate. This resulted in unconcentrated samples with densities of 10⁵, 10⁴, 10³, and 10² CFU/L.

There were 62 fungal isolates used in this study. These strains were collected from the national surveillance program for invasive fungal infections (the CHIF-NET study, Table 1, Supplement A). The isolates were stored at −80°C until use at Peking Union Medical College Hospital, Beijing, China (PUMCH). The strains were identified by morphological analysis with CHROMagar^TM Candida medium (Difco Laboratories, Detroit, MI, USA) and sequencing of the rDNA internal transcribed spacer (ITS) and secondary alcohol dehydrogenase (SADH) [19 (link),20 (link)].
The simulated samples were composed of pure-cultured fungi mixed in defined proportions (Table 1, Supplement A and B). The cultured fungi were suspended in sterile saline solution and measured by Densicheck (Biomerieux, l’Etoile, Marcy l’Etoile, France). The suspensions were adjusted to a McFarland 0.5 turbidity standard and mixed in defined proportions.
This study was approved by the ethics committee of the National Institute for Communicable Disease Control and Prevention (ICDC) and is consistent with the guidelines of the Declaration of Helsinki.

Drug susceptibility was assessed by the disc diffusion method according to EUCAST recommendations [61 ,62 ]. For this purpose, several colonies of similar morphology were suspended in 0.9% NaCl solution with a sterile swab until an inoculum with a density of 0.5 McFarland was obtained. The density of the suspension was measured with a DensiCheck plus photometric device (Biomerieux, Crappone, France). Then, within 15 min of preparation of the inoculum, the prepared suspension was inoculated onto the MHA using a new, sterile cotton swab. Two antibiotic discs, ertapenem 10 µg (ETP10) and temocillin 30 µg (TEM30), were applied within 15 min of inoculation and incubated at 35 ± 1 °C for 18 ± 2 h. Performing an additional disc diffusion test was necessary due to the inability to determine MIC for ETP and TEM using VITEK2 AST cards.

Two different types of bacteria were cultured to determine antimicrobial activity. S. aureus (ATCC 25923), a Gram-positive aerobe, was grown at 37 °C overnight in Columbia Blood agar (BioMérieux, Marcy l’Etoile, France), while P. gingivalis (ATCC 33277), a Gram-negative anaerobe, was grown in an anaerobic system at 37 °C for 7 days in Brain Heart Infusion (BHI) agar (Oxoid, Hampshire, UK). After incubation, a single colony of both bacteria were transferred intoto BHI broth (Oxoid, UK), and incubated either at 37 °C for 1 hour (S. aureus) or in an anaerobic at 37 °C for 4 days (P. gingivalis), to obtain bacteria in the mid-logarithmic phase of growth. The suspensions were centrifuged at 2,100 rpm for 10 min, and microbial concentration adjusted to a McFarland standard of 0.5 (1.5 × 10⁸ CFU/ml) using a Densicheck (BioMérieux, Marcy l′Etoile, France). Suspensions were used for antimicrobial activity testing.