Endo agar

TMC was quantified using GTK-M agar plates (MILCOM a.s., Tábor, Czech Republic) by culturing at 30 °C for 72 h under the aerobic conditions, according to ČSN EN ISO 4833-1 [14 ], which is the Czech equivalent of ISO 4833-1.
Identification of bacteria and their count: pathogenic bacteria in milk were determined by the Veterinary Laboratory Vedia s.r.o. (Strakonice, Czech Republic). The presence of coliform bacteria, staphylococci, streptococci, and corynebacteria was monitored. Basic cultivation of bacteria occurred on the Columbia blood agar (Oxoid, UK). ENDO agar (Oxoid, UK) was used to cultivate coliform bacteria. Enterococcus Selective Agar-BAA (Oxoid, UK) was used to distinguish streptococci and enterococci, and Edwards agar (Oxoid, UK) was used for streptococcal culture, and for staphylococci, Baird-Parker agar (Oxoid, UK) and Staphylococcus agar (Sigma-Aldrich, USA) was applied. A coagulase test was used to distinguish between coagulase-negative and -positive staphylococci (Staphylase Test, Oxoid, UK).
When necessary, a microflex LT MALDI TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) with a microSCOUT ion source and a TOF flight time analyzer (Bruker Daltonics, Bremen, Germany) in conjunction with the MALDI Biotyper software system (Bruker Daltonics, Bremen, Germany) was used to identify bacterial species.

In vivo pathogenicity was analyzed in larvae of G. mellonella as described previously [26]. Survival of A. baumannii in G. mellonella larvae was investigated by injecting 1 × 10⁵ bacteria into the last left proleg, followed by incubation at 37°C. Immediately after injection and after 24, 48 and 72 h, larvae were homogenized and serial dilutions were plated onto Endo agar (Oxoid) for CFU determination. For quantification of hemocytes in G. mellonella, larvae were infected as described above and incubated for 72 h. For the indicated time points, larvae were cut with a scalpel and centrifuged to separate the hemolymph. Hemocytes were resuspended in trypsin-EDTA (0.05%, Gibco), stained with trypan blue and enumerated using a hemocytometer.

Anal and pharyngeal swabs are taken every week from all patients on the NICU of our hospital for screening of microbiological colonization according to German infection control guidelines. Screening samples as well as clinical samples used in this study were plated on Columbia sheep blood agar plates (Oxoid, Wesel, Germany), Endo agar (Oxoid, Wesel, Germany), or ESBL screening agar (bioMérieux, Marcy-l’Étoile, France). Interpretation was performed after incubation for 24 and 48 h at 37°C. Routine species identification was achieved by linear Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) on a MALDI Biotyper system (based on a Microflex LT/SH instrument; Bruker Daltonik, Bremen, Germany). 239 isolates identified as E. cloacae complex from 24 patients were collected during a 4-month period (July 2017 until October 2017; Supplementary Table S1). Isolates were stored at −80°C until analysis. During a 5-week outbreak period (October 2018–November 2018) 14 additional isolates from 12 patients were collected (Supplementary Table S1) and analyzed immediately.