Columbia blood agar

Aerobic culture swabs were plated on Columbia blood agar, Colistin, Naladixic Blood Agar (CNA), and MaConkey Agar (Remel, Thermo Fisher Scientific, Waltham, MA, USA) both at 37°C with 5% CO₂, and at 25°C in room air. Anaerobic culture swabs were plated on Brucella Agar, Laked blood w/ kanamycin, vancomycin (LKV Agar), and phenol ethyl alcohol agar (PEA) (Remel, Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in anaerobic gas mixture. Organisms were identified using gram negative identification plate (GNID) and/or gram positive identification plate (GPID) panels (VersaTREK and Sensititre, Thermo Fisher Scientific, Waltham, MA, USA). Fastidious and/or non-fermenting gram negative organisms were identified using GN2 microplate panels (Biolog, Hayward CA, USA). Anaerobes were identified by the Wadsworth Disk method [36 ] or anaerobic identification test panel microplates (Biolog, Hayward CA, USA).

Sputum samples were collected after patients rinsed their mouths out with sterile water. If spontaneous sputum was not possible, induced sputum was systematically performed according to international recommendations [31 (link), 32 ].
Extended culture analysis was performed on the sputum. After liquefaction by N-acetylcysteine, serial dilutions (1/1000, 1/10,000, and 1/100,000) were made and cultured in Columbia blood agar, chocolate agar, Schaedler agar, and Pseudomonas selective cetrimide agar (Thermo Fisher Scientific, USA), at 37 °C for 48 h for aerobic and 5% CO₂ cultures and 5 days for anaerobic cultures. All colonies that appeared to be morphologically distinct were quantified as colony-forming unit (CFU) per milliliter and identified by matrix-associated laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MALDI Biotyper®, Bruker Daltonics, Bremen, Germany). The α-diversity of the airway microbiota was evaluated with the Shannon index (a marker of intra-individual diversity).
Chronic P. aeruginosa infection was defined by the isolation of P. aeruginosa in two or more cultures, at least 3 months apart in a consecutive period of 12 months at a stable state [33 (link)].

Quantification of E. coli in feces samples was determined by the plate count method (Columbia blood agar with sheep blood medium, Thermo Fisher Scientific, Waltham, Massachusetts, USA) followed by aerobically incubation at 37 °C overnight. Using a manual colony counter, hemolytic bacteria were counted and expressed as CFU/g feces. The detection limit was 10⁴ CFU/g feces. Serotyping of F4 and F18 E. coli was accomplished using the slide agglutination test with type specific antisera (SSI Diagnostica A/S, Hillerød, Denmark) on five colonies per plate. The dry matter content of feces was determined by freeze drying the samples to a constant weight.

Strains of Aerococcus sanguinicola, Lactobacillus harbinensis, Bifidobacterium longum, Bifidobacterium breve, Clostridium difficile, and Clostridium perfringens were obtained from a collection at the University Hospital of Bordeaux. Strains were identified using matrix-assisted laser desorption ionization–time-of-flight (MALDI–TOF) mass spectrometry. All strains were pre-cultured for 24 h by inoculation on Columbia blood agar (Thermo Scientific, Waltham, MA, USA) under anaerobic conditions (5% H₂, 10% CO₂, and 85% N₂) at 35 °C. Pre-cultures were resuspended in sterile water at a concentration equivalent to the MC Farland 4 opacity standard for each strain. These solutions were mixed at 1:4 dilution with BH broth (Thermo Scientific) containing various concentrations of metformin (0, 5, 10, 20, 50, and 100 mM). Solutions were then inoculated into a 96-well microplate and incubated under anaerobic conditions. The effect of metformin on bacterial growth was analyzed in terms of the optical density at a wavelength of 600 nm (OD₆₀₀) using a Nanodrop microplate reader (BMG Labtech, Champigny-sur-Marne, France) after 24 h of incubation.

Six different VRE isolates defined by MLST (ST80, ST117, ST192, ST203, ST721, and ST1489) were used for MV preparation. The isolates originated from a surveillance sample collection of the University Hospital Münster, Germany, and all of them were derived from routine anal swab samples. Based on previous investigations by Correa-Martinez et al. addressing the spread of VRE in the hospital environment^{41 (link)}, representative isolates were selected for the respective STs. E. faecalis ATCC 29212 and E. faecium ATCC 6057 served as vancomycin-susceptible reference strains; E. faecium ATCC 6057 was also used as a potential vesiductant.
For general cultivation purposes, all bacteria were grown on Columbia blood agar (Thermo Scientific, Waltham, USA) and incubated at 37 °C for approximately 24 h.
They were further cultured either in LB (Carl Roth, Karlsruhe, Germany) or on LB agar (Carl Roth) to investigate vesiculation under stressful growth conditions^{22 (link)}.

Strain typing was performed using a MALDI-TOF Microflex (Bruker UK) according to the Bruker MALDI Biotyper protocol described previously (34 (link)). The 2014–2015 HCAI outbreak isolates (n = 18) were recovered from −80°C onto prepoured Columbia blood agar containing 5% horse blood (ThermoFisher Scientific) and incubated aerobically at 37°C for 24 h. Measurements were obtained using Bruker flexControl software (version 3.4). Triplicate spots of formic acid-extracted protein solution were overlaid with 1 μL fresh α-cyano-4-hydroxycinnamic acid (HCCA) matrix (Bruker UK), allowed to air dry, and measured in triplicate over three separate days to obtain 27 spectra per isolate using freshly cultured isolates each day. Spectra were recorded in positive linear mode within the range of 2 and 20 kDa. External calibration of each MALDI-TOF MS typing experiment was through measurement of bacterial test standard (BTS) solution (Bruker UK).