Maldi tof

Microbiological specimens were cultured according to the standard of care, e.g., Gram-stain and microscopy. Culture was performed on selective and diagnostic agar including antibiotic susceptibility testing. Before 2011 the isolate of Achromobacter spp. was identified using analytical profile index with 20 miniature biochemical tests for identification of Gram-negative non-Enterobacteriaceae (API 20NE) (bioMérieux,Marcy-l’SEtoile, France). After 2011 a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) (Bruker, Billerica, MA, USA) technique was used for identification [20 (link)]. Since 2020 Whole genome sequencing (WGS) for typing and identification has been done on all first Achromobacter spp. since MALDI-TOF typing is not accurate for Achromobacter spp. level typing [8 (link),20 (link)].

Transmission electronic microscopy (TEM) and scanning electron microscopy (SEM) were used to observe the morphology, elemental mapping and element distribution of nanoparticles. The hydrodynamic diameter and zeta potential were measured on Malvern Zetasizer (Nano ZS-90, Malvern, UK). X-ray photoelectron spectroscopy (XPS) was utilized to determine the surface chemistry of HMnO₂ and HMP. The UV-Vis absorbance was measured via a spectrophotometer (UH5700, HITACHI, Japan). Mass spectra was recorded by MALDI-TOF (UltrafeXtreme, Bruker, Germany). The amount of TMZ and Mn was investigated using a microplate reader (Multiskan Sky, Thermo, USA) at 328 nm and inductively coupled plasma (ICP), respectively, while the drug loading (DL) and encapsulation efficiency (EE) of TMZ were calculated using the following formulas.

Briefly, the sample material was plated on URI-Select agar plates with vancomycin (Bio-Rad, Hercules, CA, USA) and on ChromID ESBL chromogenic agar plates (bioMérieux, Marcy-l’Étoile, France) and incubated at 37°C overnight. Two antimicrobial susceptibility discs containing ceftazidime (10 μg/ml; Oxoid, Basingstoke, UK) and meropenem (10 μg/ml; Oxoid) were added to the URI-Select agar plates. Colonies of presumptive EPE were subcultivated on horse blood agar (HBA) or URI-Select agar and typed to bacterial species using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF; Bruker Daltonics, Bremen, Germany).
The phenotype of EPE was characterized by susceptibility to cloxacillin (AmpC) or clavulanic acid (ESBL) using the MAST test (Mast Group Ltd., Liverpool, England). All EPE strains were tested for susceptibility against the following antimicrobial agents: piperacillin-tazobactam, cefotaxime, ceftazidime-avibactam, ceftazidime, ceftolozane-tazobactam, imipenem, meropenem, gentamicin, tobramycin, amikacin, trimethoprim-sulfamethoxazole, ciprofloxacin, and temocillin.

After performing the three methods, all collected liquid samples were immediately processed (but not later than 4 h after collecting) at the microbiology laboratory. To detect the bacterial load (colony-forming units (CFU)/ml) at each method, a serial dilution method was performed according to the Clinical and Laboratory Standards Institute (appendix 27). After incubation at 37 °C over 24 h, the S. aureus–specific colonies were counted. S. aureus–specific colonies were defined according to the manufacturer’s instructions at mannitol salt agar (MSA; Becton Dickinson GmbH, Germany) as medium-sized yellow colonies with yellow surrounding medium and at S. aureus chromID (SAID; bioMérieux, France) green colonies. MSA were used for samples from all patients (n = 30) and SAID selection agar for samples from the 10^th patient on continually (n = 21). The amount of bacterial load was indicated in CFU per cm² of the investigated AD skin area. All S. aureus strains were routinely confirmed on species level by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF, Bruker Daltonics, Bremen, Germany).

Only isolates resistant to ertapenem (via MIC determination and via the carbapenemase-production test, MHT), were identified to the species level using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) (Bruker Daltonics, Bremen, Germany) and subjected to whole genome sequencing (WGS). WGS was performed by MicrobesNG Service (https://microbesng.com/) using the Illumina Miseq short-read technology (2 x 250 paired-end). The assembled and annotated draft genome provided by MicrobesNG Service was further analysed using free online bioinformatic tools at the Center for Genomic Epidemiology (CGE) (https://www.genomicepidemiology.org/). This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JAULJM000000000.