Biotyper 3

Manufactured by Bruker

Sourced in Germany, United States

The Biotyper 3.1 software is a bacterial identification system developed by Bruker. It is designed to analyze mass spectra data obtained from microbial samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology. The software compares the mass spectra of unknown microbial samples to a comprehensive database of reference spectra, enabling the rapid and accurate identification of a wide range of microorganisms.

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67 protocols using biotyper 3

Gonococcal Isolates Analysis: 2006-2015

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All 116 N. gonorrhoeae isolates received by our research laboratory between 2006 and 2015 were studied. These isolates were sent to the LIMM immediately after their isolation and identification by public healthcare facilities and private diagnostic laboratories at their convenience but without any screening. Private laboratories provided 103 N. gonorrhoeae isolates. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS (Bruker Biotyper 3.1, Bruker Daltonics) was used to confirm isolate identification. Patient data included specimen type, gender, and age.
The most common specimen type was urethral (n = 81), followed by urine (n = 14), vaginal (n = 12), penile discharge (n = 3), cervical (n = 3), and rectal specimens (n = 2). The specimen type of one isolate was unknown. Ninety-seven patients were male, 15 were female, and four isolates were included in the study without gender identification. Patient ages varied between 13 and 70 years old. Patient sexual orientation is unknown.

da Costa-Lourenço A.P., Abrams A.J., dos Santos K.T., Argentino I.C., Coelho-Souza T., Caniné M.C., Ferreira A.L., Moreira B.M., Fracalanzza S.E., Trees D.L, & Bonelli R.R. (2017). Phylogeny and antimicrobial resistance in Neisseria gonorrhoeae isolates from Rio de Janeiro, Brazil. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 58, 157-163.

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MALDI-TOF MS for Bacterial Identification

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The isolates DBS 4 subjected to MALDI-TOF MS (Bruker Daltonik GmbH, German) analysis for the species level identification. Initially, the direct transfer of single colony forming unit (CFU) of the DBS4 to the MALDI target plate. Followed by the coating with the matrix solution 10 mg/mL of alpha-cyano-4-hydroxy cinnamic acid solution in acetonitrile: trifluoro-acetic acid (50:2.5%) and allowed them to dry. The mass spectral analysis was performed using the software Bruker BioTyper 3.1 (Germany). E. coli DH5 alpha used for external calibration of mass sectra. The MALDI target plate was placed inside the microflex LT MALDI-TOF mass spectrophotometer for the spectral analysis and data interpretation. The identification results represented in logarithmic score ranging from 0 to 3.0 by comparing with the peaks profile of the known organism available in the Bruker database. However, the MALDI Biotyper score ranging from 2.3 to 3.0 represent high probability species identification (+++), range between 2.0 and 2.299 genus-level identification, probable species identification (++), range 1.7–1.999 are probable genus identification (+) and ranges between 0 and 1.699 are considered as not reliable for identification (−).

Dawoud T.M., Alharbi N.S., Theruvinthalakal A.M., Thekkangil A., Kadaikunnan S., Khaled J.M., Almanaa T.N., Sankar K., Innasimuthu G.M., Alanzi K.F, & Rajaram S.K. (2019). Characterization and antifungal activity of the yellow pigment produced by a Bacillus sp. DBS4 isolated from the lichen Dirinaria agealita. Saudi Journal of Biological Sciences, 27(5), 1403-1411.

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Characterization of N. gonorrhoeae Isolates

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Between March 2014 and October 2017, 93 N. gonorrhoeae isolates were sent by public healthcare facilities and private clinical laboratories to our Laboratory of Investigation in Medical Microbiology (LIMM) at the Universidade Federal do Rio de Janeiro. No criteria have been applied for the selection of isolates. At LIMM, isolates were identified by MALDI-TOF MS (Bruker Biotyper 3.1, Bruker Daltonics, Germany) and those confirmed as N. gonorrhoeae were preserved in BHI broth supplemented with 20% glycerol at -170°C. Specimen types were urethral (n= 70), urine (n =14), vaginal (n = 7), eye discharge (n=1), and rectal (n = 1) types. Eighty-five patients were males and eight were females. Patient ages varied between 16 and 70 years (median 29). Patient sexual orientation is unknown.

Barros dos Santos K.T., Skaf L.B., Justo-da-Silva L.H., Medeiros R.C., Francisco Junior R.D., Caniné M.C., Fracalanzza S.E, & Bonelli R.R. (2019). Evidence for Clonally Associated Increasing Rates of Azithromycin Resistant Neisseria gonorrhoeae in Rio de Janeiro, Brazil. BioMed Research International, 2019, 3180580.

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Identification of S. anginosus by MALDI-TOF MS

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The S. anginosus sputum isolate from sample 009–1 was identified by MALDI-TOF MS as described previously [32]. In brief, the isolate was cultured on blood agar at 37°C and 5% CO₂. Individual colonies were transferred in duplicate onto a stainless-steel MALDI target using a toothpick. Upon drying at room temperature, 1 μl of a matrix solution, composed of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/2.5% trifluoro-acetic acid (HCCA), was added to the first spot. For so-called on-target extraction, the second spot was overlaid with 1 μl of 70% formic acid prior to the addition of 1 μl HCCA matrix. The samples were then analyzed with a Bruker microflex MALDI-TOF MS system using the Biotyper 3.0 software (Bruker Daltonik, Bremen, Germany).

Seinen J., Dieperink W., Mekonnen S.A., Lisotto P., Harmsen H.J., Hiemstra B., Ott A., Schultz D., Lalk M., Oswald S., Hammerschmidt S., de Smet A.M, & van Dijl J.M. (2019). Heterogeneous antimicrobial activity in broncho-alveolar aspirates from mechanically ventilated intensive care unit patients. Virulence, 10(1), 879-891.

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Isolation and Identification of S. aureus from Blood Cultures

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S. aureus analyzed in this study were obtained from positive blood cultures after incubation in Bactec Plus Aerobic/F and Bactec Lytic/10Anaerobic/F blood culture bottles (Becton Dickinson, Sparks, MD, USA). The blood samples were collected from patients treated at different public hospitals in São Luis, Northeast Brazil, during a period of seven months. The isolation from positive blood cultures was performed using blood agar plates (BioMérieux, Marcy l’Etoile, France). Subsequently, the isolates were identified by MALDI-TOF (Matrix Assisted Laser-Desorption Ionization-Time of Flight) Mass Spectrometry (MS). The mass spectra acquired for each bacterial strain were compared to the mass spectra contained in the database using Biotyper 3.0 software (Bruker, Billerica, MA, USA). All isolates were kept in Luria-Bertani broth supplemented with 15% glycerol at −80 °C.

Monteiro A.D., Pinto B.L., Monteiro J.D., Ferreira R.M., Ribeiro P.C., Bando S.Y., Marques S.G., Silva L.C., Neto W.R., Ferreira G.F., Bomfim M.R, & Abreu A.G. (2019). Phylogenetic and Molecular Profile of Staphylococcus aureus Isolated from Bloodstream Infections in Northeast Brazil. Microorganisms, 7(7), 210.

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Identification of Staphylococci Isolates

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Staphylococci isolated from FTS were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) in a positive linear mode (2000–20,000 m/z range) as described previously [8 (link)]. The resulting spectra were compared with reference spectra using the Biotyper 3.0 software (Bruker Daltonics, Germany). Escherichia coli DH5α (Bruker Daltonics, Germany) was used as a standard for calibration and quality control. 16S rRNA genes of isolates recovered from HP were amplified and sequenced by Sangon Biotech (Shanghai Ltd.). The sequences were then compared with the NCBI database [9 (link)].

Liu Y., Chen L., Duan Y, & Xu Z. (2022). Molecular Characterization of Staphylococci Recovered from Hospital Personnel and Frequently Touched Surfaces in Tianjin, China. The Canadian Journal of Infectious Diseases & Medical Microbiology = Journal Canadien des Maladies Infectieuses et de la Microbiologie Médicale, 2022, 1061387.

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Pneumococcal Isolation and Identification

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Blood, CSF, synovial fluid, pericardial fluid, pleural fluid, and peritoneal fluid samples taken from IPD children were inoculated on blood-agar plates and incubated at 35°C, 5% CO₂ incubators for 24 hours. S. pneumoniae isolates were identified by colony morphology on blood agar and optochin test, and confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Microflex LT; Bruker, Billerica, MA, USA). For MALDI-TOF analysis, bacterial proteins from blood cultures were extracted using a MALDI Sepsityper kit (Bruker). Each purified blood-culture extract (1 µL) was transferred to an individual spot on the Bruker 96-spot target plate and covered with a 1 µL α-cyano-4-hydroxycinnamic acid matrix (Bruker). The target plate was then read and analyzed by the Bruker Microflex LT system. A protein profile of each specimen with m/z values of 3,000–15,000 was generated based on a minimum of 240 laser-shot measurements. Profiles were further analyzed using Biotyper 3.0 software (Bruker) in blood-culture mode according to the manufacturer’s recommendation.

Cai K., Wang Y., Guo Z., Xu X., Li H, & Zhang Q. (2018). Clinical characteristics and antimicrobial resistance of pneumococcal isolates of pediatric invasive pneumococcal disease in China. Infection and Drug Resistance, 11, 2461-2469.

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Bacterial Identification by MALDI-TOF MS

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For bacterial identification by MALDI-TOF MS, the colonies were subjected to ribosomal protein extraction using the protocol described by Hijazin et al.^{42 (link)}. The Microflex™ mass spectrophotometer (Bruker Daltonik) from the São Paulo State Environmental Company (CETESB) was used. To capture the protein spectra, 1.0 µl of protein suspension was transferred to a 96-well stainless-steel plate and, after drying at room temperature, 1.0 µl of the polymer matrix (α-cyano-4-hydroxycinnamic acid) was added. Each strain was distributed in three wells and for each plate, two readings were performed with the FlexControl™ software (Bruker Daltonik) using the MTB_autoX method. Finally, the BioTyper™ 3.0 software (Bruker Daltonik) compared the captured spectra for each strain with the manufacturer's library for bacterial identification. By comparing the presence/absence of specific peaks, a log (score) value was obtained. The criteria for interpreting the standards used in this study were those of the manufacturer Bruker Daltonik: scores ≥ 2.0 were accepted for species attribution, and scores ≥ 1.7 and < 2.0 were used only for genus identification.

Poor A.P., Moreno L.Z., Monteiro M.S., Matajira C.E., Dutra M.C., Leal D.F., Silva A.P., Gomes V.T., Barbosa M.R., Sato M.I, & Moreno A.M. (2022). Vaginal microbiota signatures in healthy and purulent vulvar discharge sows. Scientific Reports, 12, 9106.

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Bacterial Identification by MALDI-TOF MS

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The selected colonies were identified by MALDI-TOF MS, and a bacterial culture was subjected to ribosomal protein extraction using the protocol described by Hijazin et al. [27 (link)]. Mass spectra were acquired using a Microflex™ mass spectrometer (Bruker Daltonics, Billerica, MA, USA) and α-cyano matrix (10 mg α-cyano-4-hydroxycinnamic acid mL⁻¹ in 50% acetonitrile/2.5% trifluoroacetic acid). Each strain was distributed in three wells; for each plate, two readings were performed with the FlexControl™ software (Bruker Daltonics, Billerica, MA, USA) using the MTB_autoX method. Finally, the BioTyper™ 3.0 software (Bruker Daltonics) compared the captured spectra for each strain with the manufacturer’s library for bacterial identification. Standard Bruker interpretative criteria were applied; scores ≥ 2.0 were accepted for species assignment, and scores ≥ 1.7 but ≤2.0 were accepted for genus identification.

Monteiro M.S., Matias D.N., Poor A.P., Dutra M.C., Moreno L.Z., Parra B.M., Silva A.P., Matajira C.E., de Moura Gomes V.T., Barbosa M.R., Sato M.I, & Moreno A.M. (2022). Causes of Sow Mortality and Risks to Post-Mortem Findings in a Brazilian Intensive Swine Production System. Animals : an Open Access Journal from MDPI, 12(14), 1804.

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Staphylococci Identification Protocol

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All isolates were initially screened using Gram staining, catalase and coagulase tests. Those that demonstrated potential staphylococci characteristics were identified by Matrix-assisted laser desorption ionization time flight mass-spectroscopy (MALDI-TOF-MS, Microflex LT, Bruker Daltonics, Coventry, UK) in a positive linear mode (2000–20,000 m/z range) as described previously [12 (link)]. The resulting spectra were compared with reference spectra by using the Biotyper 3.0 software (Bruker Daltonics, Coventry, UK). Escherichia. coli DH5α (Bruker Daltonics, Coventry, UK) was used as a standard for calibration and quality control.

Xu Z., Shah H.N., Misra R., Chen J., Zhang W., Liu Y., Cutler R.R, & Mkrtchyan H.V. (2018). The prevalence, antibiotic resistance and mecA characterization of coagulase negative staphylococci recovered from non-healthcare settings in London, UK. Antimicrobial Resistance and Infection Control, 7, 73.

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