Microflex lt system

Bacteria were grown on plates containing trypticase soy agar with 5% sheep blood and incubated overnight at 35°C. All S. maltophilia isolates were stored at −70°C until further use. S. maltophilia ATCC 13637 was used as the reference strain.
Cultures were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS, Microflex LT system, Bruker Daltonics, Bremen, Germany) according to the manufacturer’s recommendations. One colony from an overnight culture grown on blood agar at 37°C under aerobic conditions was applied with a sterile wooden toothpick on a 96-spot stainless steel target plate (Bruker Daltonics, Bremen, Germany). After drying, 1 μL 70% formic acid was added and air-dried prior to adding 1 μL of matrix solution (10 mg/mL α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid [Sigma-Aldrich, Toluca, Mexico]). The spots were air-dried, and the target plate was introduced into the equipment. The MALDI Biotyper 3.0 software was used to match the spectra profile with the database. Classification was performed according to the manufacturer's recommended score identification criteria, in which a score within 2.0–3.0 range indicates reliable species-level identification.

The Klebsiella spp. isolates were initially identified using
conventional biochemical reactions at the RHMS clinical laboratory. Bacterial
identification was confirmed by matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) mass spectrometry using the Microflex LT System and
analysis by Biotyper 2.0 software (Bruker Daltonics, Germany) at the
Universidade Federal de São Paulo. The minimum inhibitory concentrations (MICs)
of carbapenems were determined using the agar dilution method as recommended by
the Clinical Laboratory Standards Institute (CLSI) guidelines (CLSI, 2011 ).

In order to identify the microbiome inhabiting the surface of natural teeth, samples were taken from 3 patients (Section 2.1) who had teeth without signs of caries, but with symptoms of gingivitis. Individual 0.4 cm² swabs were taken from the outer surface of the tooth enamel of each patient. The swabs were placed in sterile physiological saline (0.85% NaCl), shaken, and made into a stock. For quantitative testing, the suspensions from 10-fold dilutions were plated using the spread plate technique in triplicate on TSA (tryptic soy agar, Oxoid, UK) medium. The plates were incubated at 37 °C for 48 h. After incubation, the colony-forming units (CFU) were counted and the mean was calculated from triplicate replicates. The results are given as the mean number of microorganisms in CFU/0.4 cm². For identification of microorganisms, 0.3 mL of the prepared suspension was plated on Columbia blood agar (Oxoid, UK) and incubated at 37 °C for 48 h in aerobic conditions with 5% CO₂. Then, a pure culture on TSA broth was obtained from each of the grown colonies. The microorganisms were identified by MALDI-TOF MS (matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry), using the Microflex LT system (Bruker Daltonics, Bremen, Germany) and the IVD HCCA matrix (Bruker, Billerica, MA, USA).