Vitek ms

Overnight cultures of bacterial isolates were identified with MALDI-TOF apparatus (VITEK^® MS (Biomerieux, Nuertingen, Germany)) according to the manufacturer’s instructions. In summary, bacterial strains were grown on MacConkey agar plates for 24 h. A thin smear of the strains were made on MALDI plate and overlaid with 1 µL of matrix solution (saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid) and air dried at room temperature. The plates were put in VITEK^® MS (Biomerieux, Nuertingen, Germany) where the organisms were identified by comparing their mass spectra with reference spectra of the manufacturer database. Data were interpreted with scores of ≥2 considered as reliable species level identification. All identified Klebsiella pneumonia, Morganella morganii, Leclercia adecarboxylata and Citrobacter freundii were selected for further studies.

Homogeneity tests were performed for all samples with the methodology process of the study (Figs S1 and S2). For each sample, homogeneity was tested by PCR on three aliquots and by plating four aliquots in duplicate on CHROMID^® Colistin R (bioMérieux, Marcy‐L'Etoile, France) randomly selected from the positive test samples and by plating three aliquots in duplicate randomly selected from the negative test samples. The identity of sub‐cultured relevant isolates was confirmed by MALDI‐TOF (VITEK‐MS, bioMérieux, Marcy‐L'Etoile, France). The stability was tested using the same methodology on three aliquots randomly chosen among positive test samples. Plating was performed in duplicate on the three selective chromogenic media CHROMID^® Colistin R (bioMérieux,Marcy‐L'Etoile, France), CHROMagar^TM COL‐APSE (Mast Diagnostic, Amiens, France) and COLISTIGRAM (Kitvia, Labarthe Inard, France). The identity of sub‐cultured relevant isolates was confirmed by MALDI‐TOF (VITEK‐MS, bioMérieux, BrukerMarcy‐L'Etoile, France). Stability was assessed on the day of shipment (data obtained in the homogeneity study) and 1 day after reception and analysis of the samples by the participating laboratories. The results of stability testing were compared with those from the homogeneity tests (day 0). Results are presented in Table S2.

The sterility of the nanoparticles was assessed by the Department of Microbiology at the University Hospital Virgen de la Arrixaca (Murcia, Spain). Samples of 100 µL of the suspension of nanoparticles were directly inoculated, under aseptic conditions, into BACT/ALERT PF PLUS culture bottles (Ref. 410853, BioMérieux España S.A, Madrid, Spain) and incubated at 32.5 ± 2.5 °C for 14 days while aerobic and facultative anaerobic microorganisms were looking for by using the BACT/ALERT^® 3D Microbial Detection System (BioMérieux España S.A., Madrid, Spain). Negative and positive controls were included. Positive bottles were subcultured into 5% sheep’s blood agar. Samples of the grown colonies were then identified in a MALDI-TOF (Matrix Assisted Laser Desorption Ionization Time-of-Flight) mass spectrometry microbial identification system (VITEK^® MS, BioMérieux España S.A, Madrid, Spain) including the VITEK^® MS IVD and VITEK^® MS RUO, for the clinically relevant species database and the broad research database for microorganisms, respectively.

All non-duplicate PA isolates were selected with respect to their antimicrobial resistance profile based on microdilution antimicrobial susceptibility tests, choosing the most resistant isolates. All isolates were identified by MALDI-TOF MS using the VITEK MS (bioMérieux, Marcy l’Etoile, France) following the manufacturer’s instructions. The resistance profile was expressed by the different values of the minimum inhibitory concentration (MIC) determined by broth microdilution using the Micronaut S MDR MRGN-Screening 3 panel (MERLIN Diagnostika GmbH). PA isolates were considered non-susceptible to P/T, cefepime, ceftazidime, meropenem, amikacin, gentamicin, ciprofloxacin, colistin, C/T, and CZA if their MIC value was greater than the current EUCAST 2018 clinical breakpoint (susceptibility of PA to C/T and CZA for MIC values of ≤4 and ≤8 μg/dL, respectively).

Three reference strains (C. striatum ATCC 6940, C. propinquum DSM 44285, and C. simulans DSM 44415) and 99 clinical isolates were used in this study (Additional file 1). All clinical isolates were recovered from pulmonary specimens (80 sputum samples, 14 tracheal aspirates, and 5 bronchoalveolar lavage fluid samples) of patients clinically suspected of having respiratory tract infections, and were collected from 2016 to 2018 in the Department of Clinical Laboratory Medicine, Peking University People’s Hospital, Beijing, China. The organisms were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using the Vitek MS (bioMérieux, France) system and the results were confirmed by 16S rRNA gene sequencing.

Per sample, twelve grams of bean sprouts were enriched in 15 mL tryptic soy broth (TSB). After overnight incubation, 100 μL of the TSB was transferred to a selective TSB, containing cefotaxime (0.25 mg/L) and vancomycin (8 mg/L) (TSB-VC). After overnight incubation, 10 μL of the TSB-VC was subcultured on an ESBL screening agar, EbSA (AlphaOmega, ‘s-Gravenhage, the Netherlands), consisting of a split McConkey agar plate containing cloxacillin (400 mg/L), vancomycin (64 mg/L) and either cefotaxime or ceftazidime (1 mg/L). Species identification (VITEK-MS, bioMérieux, Marcy l’Etoile, France) and antibiotic susceptibility testing (VITEK2, bioMérieux, Marcy l’Etoile, France) were performed for all oxidase-negative Gram-negative isolates that grew on the EbSA. Minimal inhibitory concentrations (MIC) are given in mg/L. The production of ESBL was phenotypically confirmed with the combination disk diffusion method for cefotaxime (30 μg), ceftazidime (30 μg) and cefepime (30 μg). All with and without clavulanic acid (10 μg) (Rosco, Taastrup, Denmark). Test results were considered positive if the diameter of the inhibition zone was ≥5 mm larger for the disk with clavulanic acid as compared to the disk without clavulanic acid [25 ,26 ]. For interpretation of the phenotypic susceptibility testing EUCAST clinical breakpoints–bacteria (v 7.1) was used [27 ].

Different anatomical sites were sampled and sent for microbiological analysis (culture and isolation of S. aureus or S. pyogenes) in order to identify etiology and source of infection. Strains were identified by biochemical tests, such as clumping factor, the coagulase test or Lancefield group agglutination (bioMérieux, Marcy l'Etoile, France), and with Phoenix® strips (BD, Pont de Claix, France) or mass spectrometry (VITEK MS®, BioMérieux, France). Antibiotic susceptibilities were determined using a Phoenix 100® instrument (BD) or by the disc diffusion method (SIRSCAN® I2A, Peyrols, France) following CA-SFM guidelines.