Biotyper

A laboratory reference strain C. albicans SC5314 and six Candida clinical strains (C. albicans A14 and A69, C. tropicalis T18S and T38R, and C. parapsilosis P149 and P152) were used in the study. The six clinical strains were initially isolated from blood culture samples, retrieved from our archival laboratory collection and selected because of their strong biofilm-forming ability. All strains were identified with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) using a Bruker Biotyper (Bruker, United States) and the manufacturer’s database (MBT Compass Library DB-7311) with a score of ≥1.7 (Vlek et al., 2014 (link)). Prior to experiment, Candida cells were first subcultured from glycerol stock culture onto Sabouraud dextrose agar (SDA) and incubated at 35°C for at least 1 day to assess the purity and viability of the culture.
Type strains L. rhamnosus GG ATCC 53103 (LGG), L. plantarum ATCC 8014 (LP8014), and L. acidophilus ATCC 4356 (LA4356) were used. Stock cultures were prepared by resuspending bacterial cells in de Man, Rogosa and Sharpe (MRS) broth with 10% glycerol and stored at −80°C until use. Before experiments, the bacterial cells were first streaked from stock culture onto MRS agar and incubated at 37°C under microaerophilic conditions (5% O₂, provided by CampyGen, Thermo Fisher Scientific, United States) for 2 days.

Enterococcus strains were isolated from external ear canal swab samples collected from dogs with chronic otitis externa from animal hospitals in Seoul and Gyeongsangnam-do, South Korea. The swab samples were maintained in Amies transport medium (YUHAN LAB TECH, Seoul, Korea) until processing. The samples were placed in a 1.5 mL centrifuge tube containing phosphate-buffered saline (PBS) and vortexed vigorously. The supernatants were spread on Columbia blood agar (5% sheep blood; Oxoid, Hamspire, UK) and incubated overnight at 37 °C. Suspected Enterococcus colonies were subcultured on fresh tryptic soy agar (TSA; Difco, MI, USA) and incubated overnight at 37 °C. The subculture step on TSA was repeated thrice. After isolating the purified colonies, the isolates were identified via time-of-flight mass spectrometry using a matrix-assisted laser desorption/ionization Biotyper (Bruker Daltonics, Bremen, Germany), according to the manufacturer’s instructions. A total of 197 isolates belonged to the genus Enterococcus; however, only 89 strains were identified. The bacteria were stored at −70 °C in tryptic soy broth (Difco) containing 15% glycerol until biochemical tests were performed.

From calendar week (CW) 15/2018 to CW14/2019, fecal samples from six healthy individuals were screened for the presence of K. pneumoniae. Fecal samples of about 2 g were collected in sterile plastic containers once a week and processed in the laboratory within 24 h. Volunteers lived in six different households in Vienna (subject 1) and Graz (subjects 2–6). Subjects 2 and 4 often spent lunch breaks together, having their meals in various restaurants. Subject 1 was 60–65 years old, subjects 2 and 4 were aged 25–30, subject 3 was aged 40–45, and subjects 5 and 6 were aged 50–55 years. Subject 4 followed a gluten-free diet. Subject 6 was vegetarian but ate fish.
To detect K. pneumoniae, all feces samples were plated on Simmons Citrate Agar with 1% Innositol (SCAI) (BIO-RAD, Hercules, United States) and incubated for 48 h at 44°C. In addition, broth enrichment was performed (1 g feces in 9 ml LB medium with 10 μg/l ampicillin overnight at 37°C), followed by cultivation on an SCAI medium for 48 h at 44°C. Up to eight single colonies resembling K. pneumoniae morphologically were selected from each agar plate and subcultured for further processing. Species confirmation was carried out using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) Biotyper (Bruker, Billerica, MA, United States) according to the manufacturer’s instructions.

A total of 55 left-over stool samples were obtained from healthcare check-ups program at the Golden Jubilee Medical Center Mahidol University, Nakhon Pathom, Thailand, in 2022. Samples were cultured in MacConkey agar supplemented with 2 mg/L colistin. Citrobacter isolates were identified using traditional biochemical tests (Farmer et al., 1985 (link)) and species-level identification was confirmed by Biotyper (matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry) according to the manufacturer’s protocol (Bruker Daltonik, Leipzig, Germany). Colistin-resistant isolates were further confirmed by the gold standard broth-microdilution method defined by the Clinical and Laboratory Standards Institute (CLSI) (Clinical Laboratory and Standards Institute (CLSI), 2020 ). The presence of mcr-1 to mcr-10 was screened by multiplex PCR using the previously described protocols (Lescat et al., 2018 (link); Wang et al., 2020 (link); Borowiak et al., 2020 (link)), and the gene sequence was confirmed by Sanger DNA sequencing.

Candida species were identified based on matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (Biotyper; Bruker Daltonics, Billerica, MA, USA) with library version 4.0 or sequencing of the D1/D2 domains of the 26S rRNA gene [14 (link),16 (link),17 (link)]. In vitro antifungal tests for susceptibility to fluconazole, amphotericin B, and micafungin were performed using the CLSI M27-A3 broth microdilution method. The interpretative guideline in the CLSI document M60 ED1 was used to classify isolates using species-specific clinical break points (CBPs) [18 ]. If there was no CBP for an antifungal agent for the corresponding species, epidemiological cutoff values (ECVs) were used [19 ,20 (link)].