Hcca matrix

The identification of all colony-types was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using a Bruker Biotyper with the in-house extraction method. Each colony type was suspended in 0.5 mL of sterile deionized water in a 2 mL centrifuge tube to make a turbid suspension, and 1 mL of pure ethanol was added to the tube. The suspension was vortexed at high speed for 2 min and then centrifuged for 2 min at 13,000 rpm. The supernatant was then carefully removed with a pipette. The tube was then re-centrifuged for 30 s and any remaining ethanol removed. Ten sterile glass beads were added to the tube containing the pellet and 50 µL of acetonitrile was added. The tube was then vortexed at high speed for 1 min. A 50 µL aliquot of 70% formic acid was then added and the tube was vortexed for a further 5 s. The tube was then centrifuged for 2 min at 13,000 rpm. Duplicate aliquots of 1.5 µL of supernatent were spotted onto a MALDI stainless steel target and both spots were covered with 1.5 µL of HCCA matrix (Bruker, Coventry, UK) and allowed to dry. Species identification was achieved by matching spectra to those available in the general database and mycobacteria were also matched to the Bruker Mycobacteria Library version 4.0.

The MALDI-TOF Biotyper system (Bruker, Bremen, Germany) was used for identification of the isolates at species level. Fresh cultures were prepared from the frozen stock by inoculation in the same media of isolation. A single colony was collected and applied to ground steel Maldi target plates (Bruker) using sterilized wooden toothpicks and then 1 µL 70% formic acid was added for on-plate direct extraction before applying 1 µL HCCA matrix (Bruker) and identification. Automated data analysis of the raw spectra was performed using the MALDI BioTyper 1.1 software (Bruker) with default settings to create a list of the most significant peaks of the spectrums. Each spectrum was automatically compared with the MALDI Biotyper database using the parameter of the pattern-matching algorithms. Identification of the bacterial species was considered only for results > 2.

Fungal identification was performed using MALDI-TOF MS on a Bruker Microflex instrument (Bruker Daltonics GmbH, Bremen, Germany). From the culture on SDA-C, a partial extraction procedure was performed by adding 1 μL of 70% formic acid to the sample on a target plate (MSP 96 BC ground steel target; Bruker Daltonics). Then, 1 μL of saturated cyano-4-hydroxycinnamic acid solution (HCCA matrix; Bruker Daltonics) was added. Each microorganism tested was spotted twice on the same target plate. Measurements were performed with the Flex control V3.4 software (Bruker Daltonics), using default settings and BD 8326 (version V. 9.0) as a reference library [15 ]. The following criterion was used for reliable identification of fungal species: an MS Score ≥ 1.5 and the 3 first results identical and consistent with the appearance of the colonies on agar (with some adjustments according to the scores profile for difficult cases)

The sweet apples were collected from Edremit in Türkiye. Tryptic soy agar and malt extract agar were purchased from Sigma-Aldrich, Germany. HCCA Matrix was obtained from Bruker, United States. Crystal violet stain was purchased from Fluka, US.

The hydrolysis assay was developed using a Microflex™ (Bruker Daltonics, Billerica, MA, USA) mass spectrometer. The parameters settings were: ion source 1, 19.0 kV; ion source 2, 17.2 kV; lens, 6.0 kV; detector gain, 2.5 kV. Spectra were recorded in the mass range of 0–1000 Da with 60 Hz laser frequency. Each spectrum was obtained from 240 laser shots. The polished steel target plate (Bruker Daltonics, Bremen, Germany) and HCCA matrix (2.5 mg α-cyano-4-hydroxycinnamic acid dissolved in 50% acetonitril, 47.5% HPLC-pure H₂O and 2.5% trifluoroacetic acid, (Bruker Daltonics)) was used. For calibration the Peptide calibration standard II (Bruker Daltonics) was used. The peaks employed for calibration were CCA [M + H]⁺ at 190.05 Da, CCA [2 M + H]⁺ at 379.09 Da and Bradykinin (1–7) peak [M + H]⁺ at 757.40 Da.
The analysis of MALDI-TOF MS spectra was performed with the Flexanalysis 3.3 software (Bruker Daltonics). The spectra were smoothed and baseline subtracted and then manually examined for the specific ertapenem related peak patterns in the mass range of 4–600 Da previously described [4 (link)]. To approve a spectrum as reliable at least one sum buffer peak of hydrolysed or unhydrolysed ertapenem had to have a minimum intensity of 10⁴. The high intensity proves the specificity of the peaks and guarantees that no unspecific background noise is misinterpreted as a significant peak.

Representative individual colonies recovered from quantification plating were identified using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis following the manufactures protocol. Both non-filamentous and filamentous fungal species were recovered during initial plating, and samples were processed to identify fungal species using MALDI-TOF as per manufacturer instructions (Bruker Daltonics, Billerica, MA, USA). In brief, fungal isolates were inoculated in Sabourand liquid broth (BD Life Sciences, Huntsville, AL, USA) and incubated for 24 h at 26 °C for growth. Fungal growth was collected and washed two times with HPLC-grade water (MilliporeSigma, Burlington, MA, USA) and once with molecular grade ethanol (ThermoFisher, Waltham, MA, USA). Washes consisted of re-suspending the fungal pellet in the wash medium, followed by a 2-min centrifugation at room temperature at 13,000 rcf. Following the ethanol wash, the samples were dried until all residual ethanol evaporated. The dried pellet was then re-suspended in 70% formic acid and acetonitrile. The sample was then centrifuged and the supernatant was spotted on a MALDI-TOF-MS target plate, allowed to air dry, and then covered with a 1 µL HCCA matrix (Bruker Daltonics Billerica, MA, USA) and analyzed on a MALDI-TOF-MS biotyper (Bruker Daltonics, Billerica, MA, USA).

Aliquots of 25 g were transferred to new sterile plastic bags and diluted 1:10 in Buffered Peptone Water (BPW) (VWR, USA), homogenised for 30 s and incubated aerobically at 37 °C for 18–24 h. After incubation, 10 µL of the enrichment cultures were streaked on Simmons Citrate Agar (Bio-Rad, Hercules, CA, USA) supplemented with 1% Myo-Inositol (Sigma-Aldrich, St. Louis, MO, USA) (SCAI), a highly selective media for the isolation of Klebsiella spp. and Raoultella spp. [29 (link)] and incubated aerobically at 37 °C for 48 h. Samples collected in 2016 had been enriched in BPW by the same protocol and stored at −80 °C in 20% glycerol. Before the samples were analysed, they were thawed in room temperature and approx. 1.5 mL transferred to 10 mL BPW and incubated at 37 °C over night. Yellow colonies representing presumptive Klebsiella spp. were sub-cultured to obtain pure cultures. The obtained isolates were cultured overnight on Plate Count Agar (PCA) (Oxoid, UK) at 37 °C. Colonies were transferred directly to disposable 96 spot targets (Bruker, Germany) and covered with 1 µL HCCA matrix (Bruker, Germany). The spots were air dried and the isolates were identified using Matrix Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF MS) (Bruker, Germany).