Microflex

Samples were mixed in equal volumes with matrix containing 100 mg ml^-1 (w/v) 2,5-dihydroxybenzoic acid in 1:1:0.02 (v/v/v) water/acetonitrile/dimethylaniline [39 (link)]. Mass spectra were collected on a BrukerMicroflex (Bruker Daltonics, Bremen, Germany) and evaluated with the software mMass [40 (link)].

MALDI-TOF MS spectra were obtained for each of the strains listed in Table 1 according to the manufacturer’s recommendation using the formic acid-ethanol extraction method that has previously been described (Khot et al., 2012 (link); Hille et al., 2020 (link)). Spectra were collected on a linear MALDI-TOF MS (Bruker microflex, Bruker Daltonik, Billerica, MA) using settings and calibrations as described previously (Hille et al., 2020 (link)). For the sheep’s blood media models, strains were spotted three times on the target plate and analyzed once for each spot well resulting in three spectra per strain. For the bovine blood models, the strains were spotted five times each and analyzed twice per well resulting in 10 spectra per strain. This yielded a total of 970 unique spectra being analyzed for this study.

The new minigastrin analogs DOTA-[(N-Me)Nle¹¹,1Nal¹³]PP-F11N (1) and DOTA-[(GABOB)₂,desBOC,(N-Me)Nle³,1Nal⁵]]-PG (2) were synthesized by standard solid phase peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids as described previously [24 (link)]. The following protective groups were used to protect the amino acids’ reactive side chains: tert-butyl ester for Asp and DGlu, tert-butyl ether for Tyr, and tert-butyloxycarbonyl (BOC) for Trp. For coupling tris(tert-butyl) protected DOTA, a 3-fold molar excess was used.
Purification was carried out using RP-HPLC on a GILSON UV/VIS-155D multi-wavelength UV detector, equipped with an Eurospher II 100 Å 5 µm C18 column, 250 mm × 8 mm (Knauer, Berlin, Germany), combined with an Eurosil Bioselect 300 Å 5 µm C18 precolumn, Vertex Plus A, 30 mm × 8 mm (Knauer, Berlin, Germany), using a water/0.1% TFA (A) and acetonitrile/0.1% TFA (B) gradient with a flow rate 2 mL/min: 0–4 min 20% B, 4–24 min 20–60% B, 24–26 min 60% B, 26–27 min 60–80% B, 27–28 min 80% B, 28–29 min 80–20% B, and 29–37 min 20% B. The synthesized peptide conjugates with confirmed purity were characterized by MALDI-TOF MS (Bruker Microflex^®, Bruker Daltonics, Bremen, Germany) lyophilized and stored at −20 °C for further use. Peptides were dissolved in water containing 20% EtOH or PBS (~1 µg/mL).

Bacterial species was identified using MALDI-TOF mass spectrometry (Microflex, Bruker daltonics, Bremen, Germany). Carbapenemase production was confirmed using immuno-chromatographic assay (NG Rapid Test CARBA-5, NG Biotech, Guipry, France) and multiplex PCR (Xpert Carba, Cepheid, Sunnywale, USA). Antibiotic susceptibility testing was performed by agar diffusion according to the 2020 EUCAST recommendations (www.eucast.org).

The number of thirty eight UPEC strains isolated from patients was analyzed simultaneously to urine samples (23 from immunocompetent hosts and 15 from kidney recipients). E. coli in urine samples was identified during routine microbiological diagnostics based on MALDI TOF spectrometry. (Microflex, Bruker, USA) Virulence factors were confirmed with polymerase chain reaction (PCR), and the following genes were included in the study: fimH, papC, csgA, flu (named further as Ag43), and tosA. Genomic bacterial DNA was isolated with GeneMATRIX Bacterial, and Yeasts Genomic DNA Purification Kit (EURx, Poland, cat. no. E3580-01), and the quality of isolated DNA was verified with NanoDrop 1000 Spectrophotometer (ThermoScientific, USA). Isolated DNA was stored at -20°C until further analyses. PCR reactions were performed on Applied Biosystem Veriti 96 Thermal Cycler (Upland, CA, USA). Specific primers and respective references are listed in Table 1 [15 (link), 32 (link), 33 (link)]. The primer for the csgA gene was self-designed using the online tool Primer 3 [34 ]. E. coli strain CFT073 (ATCC 700928) was used as a control.

The conjunctival swabs were transported and stored at room temperature. All the samples were tested immediately at the Institute of Medical Microbiology and Hygiene (University Hospital of Tübingen). Supplemented Columbia sheep blood agar plates (Oxoid GmbH, Wesel, Germany), Endo agar plates, and supplemented brain heart infusion (BHI) agar plates (Institute of Medical Microbiology and Hygiene) were incubated at 37°C to test for bacterial contamination. A liver broth was also used. Additionally, yeast-gentamicin plates (Institute of Medical Microbiology and Hygiene) were used to detect fungal contamination, and they were incubated at 30°C. The plates were incubated for 10 days. They were read after 24 and 48 hours and after 10 days.If there was cultural growth, the microorganisms were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Microflex; Bruker Corporation, Billerica, MA). Any proof of bacterial or fungal microorganisms was documented as contamination.