Ivd maldi biotyper

Escherichia coli isolation was performed with a direct inoculation of the samples with a swab in MacConkey agar (Scharlau, Sentmenat, Spain) at 37 °C for 24 h. Presumptive colonies were subcultured in tryptic soy agar (Scharlau, Sentmenat, Spain) at 37 °C for 24 h.
Samples were directly inoculated with a swab in Slanetz and Bartley agar (Oxoid, Basingstoke, UK) at 37 °C for 48 h for Enterococcus spp., isolation. Dark pink colonies were subcultured in bile esculin azide agar (Merck, Darmstadt, Germany) at 44 °C for 4 h for Enterococcus confirmation. Presumptive colonies were subcultured in brain heart infusion (BHI) agar (Merck, Darmstadt, Germany) at 37 °C for 24 h.
E. coli and Enterococcus presumptive isolates were confirmed and characterized at species level, respectively, with MALDI-TOF mass spectrometry using the IVD MALDI Biotyper (Bruker Daltonik, Bremen, Germany) and following the manufacturer’s standard protocols.

Buffered peptone water (4.5 mL) (BPW, Haibo Biotechnology, Qingdao, China) was added to each sample (0.5 g) for pre-enrichment, according to a previously described method (18 (link)). After incubation at 37°C for 8–12 h, 0.5 mL of each pre-enriched culture was incubated in 4.5 ml of Tetrathionate Broth Buffer (TTB, Haibo Biotechnology) at 37°C for 24 h. After selective enrichment, one loopful of each broth culture was streaked onto xylose lysine tergitol 4 (XLT4, Haibo Biotechnology) agar and the plates were incubated at 37°C for 24–36 h. The presumptive Salmonella colonies were identified by polymerase chain reaction (PCR) assays using invA primers (19 (link)). The invA gene has a conserved sequence in Salmonella species and can therefore be used to detect and validate Salmonella strains (20 (link)). Here, invA primers (19 (link)) (F: 5′-ACAGTGCTCGTTTACGACCTGAAT-3′, R: 5′-AGACGACTGGTACTGATCGATAAT-3′) were used for PCR. The PCR cycling conditions were as follows: 1 denaturation cycle at 95°C for 5 min, 30 cycles of denaturation at 95°C for 30 s, followed by annealing at 56°C for 30 s and elongation at 72°C for 1 min, and a final 10 min elongation cycle at 72°C. Positive colonies were subsequently confirmed using a microbial mass spectrometer (IVD MALDI Biotyper, Bruker Bremen, Germany).

To explore the possible target genes of licochalcone A in E. faecalis, the licochalcone A non-sensitive E. faecalis isolates with possible mutations were induced and screened in vitro according to previous studies (Zheng et al., 2021 (link); Liu et al., 2022 (link)). E. faecalis isolates (16C51, 16C106) were subcultured serially in TSB containing licochalcone A. The initial inducing concentration of licochalcone A was 1/2 × MIC; the concentration was then increased successively to high concentrations. E. faecalis isolates in each concentration of licochalcone A were cultured for three to five passages before being inoculated and passaged to the next generation. E. faecalis isolates from the last passage of each concentration of licochalcone A were collected and subcultured on tryptic soy agar plates without licochalcone A for three passages, identified again by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (IVD MALDI Biotyper, Bruker, Bremen, Germany), and the MIC of licochalcone A was measured again. Finally, the licochalcone A non-sensitive E. faecalis isolates were kept frozen at −80°C in glycerol containing (35%) TSB.

Inductions were performed as detailed previously [26 (link)]. S. aureus, E. coli, and K. pneumoniae strains were subcultured serially in TSB containing AMXT-1501 (initial concentration of 1/4× MIC, then increased successively), and were subcultured serially in TSB containing linezolid or tigecycline as control groups. Samples were cultured for 3∼5 passages before being exposed to the next concentration. Control samples were subcultured serially in plain TSB. Isolates from the last passage of each concentration were picked and cultured on tryptic soy agar plates without AMXT-1501 for two passages. Clones were selected and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (IVD MALDI Biotyper, Bruker, Bremen, Germany). MICs were re-determined and then the samples were stored at −80 °C in glycerol containing (35%) TSB.

The identification of all Staphylococcus strains was carried out using mass spectrometry MALDI-TOF MS using the IVD MALDI Biotyper (Bruker Daltonik, Bremen, Germany) as described by Marek et al. [21 (link)].
The susceptibility of 11 antibiotics was tested using standard disc diffusion method on Mueller–Hinton agar plates (CM0337B, Oxoid, UK) using a bacterial suspension with the turbidity adjusted to a 0.5 McFarland standard. The susceptibility of bacteria was determined for the following agents (Oxoid, England): amoxicillin 25 µg (AML25); amoxicillin + clavulanic acid 20 + 10 µg (AMC30); ampicillin 10 µg (AMP10); penicillin G 10 units (P10); cefoxitin 30 µg (FOX30); clindamycin 2 µg (DA2); chloramphenicol 30 µg (C30); erythromycin 15 µg (E15); gentamicin 10 µg (CN10); tetracycline 30 µg (TE30); trimethoprim–sulphamethoxazole 1:19, 25 µg (SXT25). The categories susceptible, intermediate resistant or resistant were assigned on the basis of the Guidelines for Susceptibility Testing [20 ]. The Minimum Inhibitory Concentrations (MIC) for oxacillin were additionally evaluated by the broth microdilution method [20 ]. For oxacillin, S. aureus strains showing MIC of ≥ 4 µg/ml were determined to be MRSA. For quality control, S. aureus ATCC 25923, Escherichia coli ATCC 25922 and E. faecalis ATCC 29212 were used in the microdilution tests.

The swabs were inoculated onto blood agar medium (Blood LAB-AGAR; Biocorp, Warsaw, Poland) and Chapman’s selective medium (Mannitol Salt LAB-AGAR; Biocorp) and incubated in aerobic conditions at 37 °C for 24–48 h, depending on the bacterial growth rate. Single colonies were transferred to blood agar to isolate pure bacterial cultures, and preliminary bacteriological characterisation of the isolated bacteria was performed, including Gram staining, microscopic examination of cell morphology and motility, and determination of the type of haemolysis. No quantitative colony measurement was performed. Isolated bacteria were stored at −85 °C in 50% (v/v) glycerol in brain heart broth (BHI; Sigma-Aldrich, St. Louis, MO, USA) until further study.
The species of the isolates were confirmed by matrix-assisted laser desorption/ionisation (MALDI)–time-of-flight mass spectrometry using the IVD MALDI Biotyper (Bruker Daltonik, Bremen, Germany), as described by Marek et al. (2016) [15 (link)]. The mass spectra of each isolate were processed with the MALDI Biotyper 3.0 software package (Bruker Daltonics, Hamburg, Germany). The results were shown as the top 10 identification matches along with confidence scores ranging from 0.000 to 3.000, according to the manufacturer’s criteria (www.bruker.com; accessed on: 19 September 2021).