Macconkey

Upon clinical suspicion of systemic infection, at least two blood samples were injected into BD BACTEC™ Peds Plus TM/F culture vials (enriched Soybean Casein Digest Broth with CO₂) and upon arrival at the lab immediately processed in the BACTEC FX (Becton Dickinson, Heidelberg, Germany) blood culture system. Blood cultures were incubated for five days. On positivity, Gram-staining and sub-cultivation on agar plates were performed according to the standard techniques [21 ]. Positive samples were cultivated on Columbia Blood Broth, chocolate, MacConkey and Schaedler Anaerobic Agar culture media (all Becton Dickinson, Heidelberg, Germany) and incubated for 24 h at 37 °C in aerobic and 48 h in anaerobic conditions.
Species identification of positive cultures was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS, Bruker Daltonik, Bremen, Germany) using the reference Biotyper library v4.1 (Bruker Daltonik, Bremen, Germany). Antimicrobial susceptibility testing was performed according to NCCLS/CLSI guidelines until 2011 [22 ]. In 2011, Austrian microbiological laboratories switched their methodology to EUCAST (breakpoint tables for interpretation of MICs and zone diameters—2011–2021, Versions 1.3 to 11.0). Strains were classified as susceptible or resistant according to the breakpoints applied in the year of their isolation.

Participants hands were placed on trypticase soy agar with 5% sheep blood, 150 mm and the plates were incubated overnight at 35–37°C, then colony-forming units (CFUs) were counted. Potential pathogenic bacteria, Enterobacterales, Staphylococcus aureus, Enterococcus spp, Pseudomonas aeruginosa, and Acinetobacter spp were identified by first subculturing unique colonies onto trypticase soy agar with 5% sheep’s blood, MacConkey, and phenylethyl alcohol agar (Becton Dickenson, Sparks, MD). They were then worked up and identified using the Vitek or MALDI-TOF system.
We collected the gloves of participants after we sampled them, and we discarded gloves that were visibly soiled. We used a standardized approach to test the gloves for microperforations.¹⁸
We carefully turned gloves right-side out to avoid introducing new holes. We then poured water into each glove, stopping 1.27 cm (0.5 inches) below the top of the stretched glove. After 2 minutes, we inspected the outside surface of the glove for water accumulation.

The blue mussel flesh was weighed and diluted 1:10 by adding BPW-ISO to the Stomacher bag, prior to stomaching or shaking for 30 s to two minutes to homogenize. The samples were further serially diluted in BPW-ISO (aquarium experiment) or Peptone Saline (1 g peptone, 8.5 g NaCl/L) (heat treatment with inoculated blue mussel flesh) and 100 µL of the appropriate dilutions was plated out with a L-rod on TBX (Oxoid) or MacConkey (Becton Dickinson) supplemented with 1 mg/L cefotaxime (Sigma) (MaC-CO) for quantification of E. coli or ESBL-producing E. coli, respectively. In order to obtain a detection limit of 10 cfu/g, one mL of the initial homogenate was distributed equally on the surface of three plates. The TBX and MaC-CO plates were incubated at 37 ± 1 °C and 41.5 ± 1 °C, respectively. Typical colonies on the different agars were counted and a selection of colonies was further confirmed (Section 2.4.3).

After preparation, the samples were centrifuged at 200× g for 5 min at 4 °C. One loopful of the supernatant (0.01 mL) was taken and streaked onto to selective agar, namely MacConkey (pink/red colour; Becton Dickinson, Bergen, NJ, USA), XLT4 (black; Becton Dickinson, Bergen, NJ, USA), Baird–Parker (grey/black; Oxoid, Basingstoke, UK) and Kenner KF (purple; Becton Dickinson, Bergen, NJ, USA) agar to identify E. coli, Salmonella, S. aureus and Enterococcus, respectively. For quality control of each batch, all of the selective media were incubated at 38 °C for 24 h. As positive controls, the four bacteria were directly applied to the selective agar. The isolation and identification of pathogenic colonies were performed according to each manufacturer’s instruction manual.
Twenty isolated colonies from each selective agar were streaked onto the blood agar at 38 °C and incubated for 24 h. For Gram-positive bacteria, the incubation time was 24–36 h (FAO regional antimicrobial resistance monitoring and surveillance guidelines, 2019). The isolated colonies from the blood agar were taken for MALDI-TOF-MS analysis (Bruker, Germany) at the Bacteriology Laboratory of the Kimron Institute for confirmation of their identities [44 (link)].