Maldi biotyper 3

To explore the growth of different bacteria in the water traps of sinks over time, environmental samples were collected with ESwabs (Copan Diagnostics Inc., Murrieta, CA, USA) from all 14 patient-associated sinks in the Burn Centre. The sampling took place at 8 a.m. every time, i.e., approximately 4 h after the last disinfection cycle. The swabs were inserted through the strainer and turned around. The collection of samples started directly after the installation of the self-disinfecting sinks in September 2019 and continued on a weekly basis until April 2020, for a total of 35 weeks. Records were kept concerning patient occupancy of each room upon sampling.
The samples were sent to the Department of Clinical Microbiology, Linköping University Hospital, and streaked onto three different types of media using the swabs: blood agar, hematin agar, and chromogenic urinary tract infection (UTI) agar (Thermo Fisher Scientific, Waltham, MA, USA). Discs (Thermo Fisher Scientific, Waltham, MA, USA) with imipenem (10 µg), trimethoprim–sulfamethoxazole (1.25–23.75 µg), and linezolid (10 µg) were placed on the plates, respectively. The plates were incubated at 35 °C for approximately 48 h. Bacteria were identified to the species level with a MALDI Biotyper 3.0 (Bruker Corporation, Karlsruhe, Germany).

Bacterial cells were spotted on a sample spot of a MALDI target plate (MSP 96 target, ground steel; Bruker Daltonics, Billerica, MA, USA) and were overlaid with HCCA matrix solution (saturated solution of α-4-cyano-hydroxycinnamic acid; Bruker Daltonics, Billerica, MA, USA) in 50% acetonitrile (Sigma-Aldrich, St. Louis, MO, USA) and 2.5% trifluoroacetic acid solution (Sigma-Aldrich, St. Louis, MO, USA). Mass spectra profiles were acquired using a Microflex spectrometer (Bruker Daltonics, Billerica, MA, USA). The molecular ions were measured automatically in linear positive ion mode with the instrument parameters optimized for a range of 2,000–20,000 m/z. The software packages flexControl 3.0 (Bruker Daltonics, Billerica, MA, USA) and flexAnalysis 3.0 (Bruker Daltonics, Billerica, MA, USA) were used for mass spectra recording and processing. Spectra identification and analysis were carried out using the MALDI Biotyper 3.0 (Bruker Daltonics, Billerica, MA, USA). The identification was performed by comparing the obtained spectra with those in the MALDI Biotyper 3.0 library (version 3.2.1.1).

Individual bacterial colonies were collected every day for 4 days, and then each colony was identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Microflex Spectrometer; Bruker Daltonics, Bremen, Germany) as previously described^{77 (link)}. The obtained MALDI-TOF MS spectra were imported into MALDI Biotyper 3.0 software (Bruker Daltonics) and analysed against the reference bacterial spectral database. The MALDI Biotyper RTC software interprets the results according to predefined values, i.e., values between 2.00 ≤ species identified ≤ 3.00; of 1.70 ≤ probably identified ≤ 1.99 and 0.00 ≤ no identification ≤ 1.69. The unidentified colonies (with values from 0.00 to 1.99) were sequenced using the complete 16S rRNA gene.

The obtained isolates were initially identified as Streptococcus dysgalactiae by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. MALDI-TOF MS sample preparation, data processing, and analysis were done as previously described by Hijazin et al.^{30 (link)}. Mass spectra were acquired by Microflex™ mass spectrometer (Bruker Daltonik), with a mass range of 2–20 kDa, using flexControl™ 3.0 software (Bruker Daltonik). Spectra were loaded into MALDI BioTyper™ 3.0 (Bruker Daltonik), using default settings, and compared with the manufacturer’s library. Standard Bruker interpretative criteria were applied; scores ≥ 2.0 were accepted for species assignment and scores ≥1.7 but ≤2.0 for identification at the genus level.

A total of 18 non-duplicated Enterobacteriaceae strains including one carbapenem susceptible E. coli isolate, ATCC 25922, eight E. coli isolates with carbapenem resistance or reduced susceptibility, one carbapenem susceptible K. pneumoniae isolate, ATCC 13883, and six K. pneumoniae isolates with carbapenem resistance or reduced susceptibility were selected in this study (Table 1). Species identification for the 18 isolates were initially performed with the Vitek 2 compact system (bioMérieux, Durham, NC, USA) and then confirmed by MALDI-TOF MS (Bruker Daltonik GmbH, Bremen, Germany; MALDI Biotyper 3.0).

For culture, clinical specimens were inoculated onto laboratory-prepared Regan-Lowe agar upon arrival at the facilities. Plates were incubated at 35°C in a humidified aerobic atmosphere and examined daily for suspect colonies for up to twelve days.
Suspect colonies were identified using the microflex LT MALDI-TOF platform and MALDI Biotyper 3.0 software (Bruker Daltonics GmbH, Leipzig, Germany) [22 (link), 23 (link)]. Identification to the species level was accepted when all matches with a log score above 1.7 belonged to the same species, or when the best match had a log score both above 2.0 and more than 0.200 higher than that of the other matched species.
When MALDI-TOF results were inconclusive, the species was determined based on biochemical characteristics: growth or absence of growth on charcoal agar, Haemophilus agar and MacConkey agar, presence of oxidase and presence of urease. Positive results for all characteristics indicated Bordetella bronchiseptica. Growth on charcoal agar and Haemophilus agar and presence of urease but not oxidase indicated Bordetella parapertussis. Growth on charcoal agar only, combined with the presence of oxidase but not urease indicated Bordetella pertussis [24 ].

The species of the isolates were confirmed by MALDI-TOF mass spectrometry (UltrafleXtreme MALDI-TOF, Bruker Daltonics, Hamburg, Germany) using a standard ethanol/formic acid extraction sample preparation procedure [40 (link)]. The mass spectra obtained from each isolate were processed with the MALDI Biotyper 3.0 software package (Bruker Daltonics, Hamburg, Germany), and 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).