- Email: [email protected]
Application of the biocopolymer preparation system, rapid BACpro® II kit, for mass-spectrometry-based bacterial identification from positive blood culture bottles by the MALDI Biotyper system
Journal of Microbiological Methods 152 (2018) 86–91
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Application of the biocopolymer preparation system, rapid BACpro® II kit, for mass-spectrometry-based bacterial identification from positive blood culture bottles by the MALDI Biotyper system
T
Sachio Tsuchidaa, Syota Muratab, Akiko Miyabeb, Mamoru Satoha, Masaki Takiwakia, ⁎ Kazuho Ashizawac, Takashi Teradac, Daisuke Itoc, Kazuyuki Matsushitab,d, Fumio Nomuraa,d, a
Division of Clinical Mass Spectrometry, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Department of Clinical Laboratory, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan R&D Department, Nittobo Medical Co., Ltd, Japan d Division of Clinical Genetics, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacterial identification Blood culture Rapid BACpro® II kit Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove nonbacterial components and selectively collect microorganisms are a prerequisite for successful identification, and a variety of home-brew and commercial protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We recently developed a method to collect bacteria from positive blood culture bottles using a polyallylamine-polystyrene copolymer that has been used in wastewater processing. This pretreatment protocol is now commercially available as the rapid BACpro® II kit (Nittobo Medical Co., Tokyo, Japan). The operation time required for processing using this novel kit is approximately 10 min, and the entire procedure can be completed within a biosafety cabinet. Since the performance of the rapid BACpro® II kit has not been tested using the MALDI Biotyper system, we prospectively evaluated the performance of the rapid BACpro® II kit as compared with the Sepsityper® kit. Performance of the rapid BACpro® II kit was evaluated using a total of 193 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the rapid BACpro® II kit or the Sepsityper® Kit, and isolated cells were subjected to direct identification by MS fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with > 1.7 score and > 2.0 score obtained using the rapid BACpro® II kit were 99.5% and 80.8%, respectively, whereas those obtained using the Sepsityper® Kit were 89.1% and 68.4%, respectively (P < 0.05 for > 1.7 and P < 0.05 for > 2.0 by Pearsons's chi-square). In Gram-positive cases, the rapid BACpro® II kit gave identification rate of 100% with > 1.7 score and 69.4% with > 2.0 score, whereas there were 84.7% and 56.8%, respectively by the Sepsityper® Kit (P < 0.05 for > 1.7). These results are preliminary, but considering that this new kit is easy to perform and the identification rates are promising, the rapid BACpro® II kit deserves assessment in a larger-scale study with a variety of platforms for MS-based bacterial identification.
1. Introduction Prompt identification of the causative pathogen is critical for early initiation of appropriate antimicrobial therapy in blood stream infections (Angus and Wax, 2001 and Seifert, 2009). Indeed, it has been
reported that one hour of delay in proper antibiotic treatment for sepsis-related hypotensive patients decreases the chance of survival by 7.6% (Kumar et al., 2006). Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a quick and reliable method for
Abbreviations: MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MS, mass spectrometry ⁎ Corresponding author at: Division of Clinical Mass Spectrometry, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail address: [email protected] (F. Nomura). https://doi.org/10.1016/j.mimet.2018.07.017 Received 16 April 2018; Received in revised form 25 July 2018; Accepted 29 July 2018 Available online 01 August 2018 0167-7012/ © 2018 Elsevier B.V. All rights reserved.
Journal of Microbiological Methods 152 (2018) 86–91
S. Tsuchida et al.
between the rapid BACpro® II kit and the Sepsityper Kit. Positive monomicrobial isolates were subjected in parallel to direct identification by MS fingerprinting and conventional subculturing for reference identification.
identification of microorganisms; the role of this technique is rapidly growing in clinical diagnostic microbiology (Neville et al., 2011; Clark et al., 2013; Nomura, 2015; Angeletti, 2017; Prod'hom et al., 2010; Ferreira et al., 2011). Direct identification of bacteria from blood culture bottles is a promising application of MALDI-TOF MS, as has been described in several reviews (Angeletti, 2017; Prod'hom et al., 2010; Ferreira et al., 2011; Faron et al., 2017; Florio et al., 2018). Since blood culture bottle media contain nonbacterial proteins such as blood cells that may be obstacles for the interpretation of bacterial proteome profiles, pretreatment to substantially and selectively remove these host proteins is essential for successful identification of the causative organisms. A number of laboratory-developed or commercially available protocols for purification of bacteria have been reported, as reviewed by Florio et al. and Scott et al. (Florio et al., 2018 and Scott et al., 2016). Some representative examples of the laboratory-developed test protocols are as follows: (1) stepwise sedimentation of blood cells and microorganisms (Ferreira et al., 2011); (2) low-speed centrifugation to eliminate blood cells, followed by an additional lysis procedure (Prod'hom et al., 2010 and Monteiro et al., 2015); and (3) removal of blood cells using a serum separator tube (Moussaoui et al., 2010). The Sepsityper® kit (Bruker Daltonics, GmbH, Bremen, Germany) (Buchan et al., 2012) and the Vitek® MS Blood Culture Kit (bioMerieux, Inc., Marcy l'Etoile, France) (Fothergill et al., 2013) are the two major commercial purification kits. The overall concordance of the results obtained with the home-brew protocols and the commercial kits compared with those obtained by conventional direct colony methods ranges from 84% to 99% for the identification of Gram-negative bacteria and is approximately 80% for Gram-positive cases (Ferreira et al., 2011). The Sepsityper® kit is the most widely used commercial kit; a meta-analysis of the performance of the Sepsityper® kit indicated that this kit provided a reliable species-level identification for 80% of 3320 positive blood culture cases (Morgenthaler and Kostrzewa, 2015). In an attempt to further improve the identification rates and also to reduce the long hands-on time of the previous pretreatment protocols, we recently developed a method to collect bacteria in positive blood culture bottles using a polyallylamine-polystyrene copolymer that has been used in wastewater processing (Ashizawa et al., 2017). This pretreatment protocol is now commercially available as the rapid BACpro® II kit (Nittobo Medical Co., Tokyo, Japan). Since the performance of the rapid BACpro® II kit has not been tested using the MALDI Biotyper system, we prospectively evaluated the performance of the rapid BACpro® II kit compared with the Sepsityper® kit. Specifically, we evaluated these two kits when used for sample preparation of positive blood culture bottles for MALDI-TOF MS-based bacterial identification using the MALDI Biotyper system.
2.2. Conventional identification All solid media were incubated at 37 °C in 5% CO2 for 18–24 h. Each clinical specimen was plated on an appropriate solid medium (chocolate agar or trypticase soy agar II with 5% sheep blood, Nippon Becton Dickinson, Tokyo, Japan) depending on the sample and in accordance with our laboratory recommendations. Following Gram staining and determination of catalase and oxidase activities, the isolates were identified by one of three different phenotypic tests, including the Microscan Walkaway system (Siemens Healthcare Diagnostics, Deerfield, IL, USA), the BD Phoenix system (BD Diagnostics Systems, Sparks, MD, USA), and the API system (Sysmex Biomerieux, Lyon, France). The respective manufacturer's instructions for each system were followed. 2.3. Sample pretreatment methods for direct MALDI-TOF MS analysis 2.3.1. Rapid BACpro® II kit Workflow of the protocol using copolymer was per the manufacturer's instructions; this protocol was essentially as reported previously (Ashizawa et al., 2017), albeit with some modifications. In short, the manufacturer's protocol consisted of four steps (blood cell lysis, reaction with cationic particles, washing, and the final extraction of bacteria), as illustrated in Fig. 1. Briefly, a 1-mL aliquot of blood culture sample was added to a reaction tube containing 500 μL of lysis buffer (Nittobo Medical Co., Tokyo, Japan) and the mixture was centrifuged (Chibitan R; Merck Millipore, Billerica, MA) at 2000 ×g for 3 min. The supernatant was discarded and the pellet was resuspended in 800 μL of distilled water. The suspended sample was combined with 200 μL of cationic particle solution (Nittobo Medical Co.) and 200 μL of reaction buffer (Nittobo Medical Co.). The mixture then was transferred into another tube and centrifuged (Chibitan R; Merck Millipore) at 2000 ×g for 1 min. After removing the supernatant, the pellet was resuspended in 1000 μL of 70% ethanol (Nacalai Tesque, Kyoto, Japan), and the resulting suspension was centrifuged at 2000 ×g for 1 min. After the supernatant was removed, the pellet was resuspended in 30 μL of 70% formic acid (Wako, Tokyo, Japan) and 30 μL of 100% acetonitrile (Wako, Tokyo, Japan) and then centrifuged at 2000 ×g for 1 min. One microliter of the resulting supernatant then was transferred to a sample spot on a MALDI target plate and the spot was allowed to air dry. One microliter of an αcyano-4-hydroxycinnamic acid (CHCA) matrix solution, comprising a saturated solution of CHCA in 50% acetonitrile and 2.5% trifluoroacetic acid (Bruker Daltonics, Leipzig, Germany), was added to each spotted sample and the spots were allowed to air dry. Two spots on the MALDI target plate were used for each sample.
2. Materials and methods 2.1. Samples and study design The clinical evaluation was conducted during working hours from June 2016 to July 2018 at the Chiba University Hospital, Chiba, Japan. During this study period, a total of 196 blood culture bottles from 196 patients were flagged as positive. BD BACTEC™ Plus Aerobic medium in glass culture vials (BD, Franklin Lakes, NJ, USA) and the BACTEC™ FX incubation system (BD, Franklin Lakes, NJ, USA) were used. Separate portions of positive culture bottles were Gram stained and subcultured. The subcultures were conducted on 5% sheep blood agar (SBA) and chocolate agar. Eosin methylene blue and anaerobic SBA were used for Gram-negative bacteria and anaerobic bottle subcultures, respectively. The blood cultures were held at room temperature following removal from the blood culture instrument and were analyzed within 8 h of culture positivity identification. None of the positive cultures failed to grow on subculturing, and 3 were polymicrobial. The remaining 193 monomicrobial cases were included in the comparative analysis
2.3.2. MALDI Sepsityper® kit One milliliter of the medium from each positive blood culture bottle was combined with 200 μL of the Sepsityper® lysis reagent (Bruker Daltonics). The mixture was vortexed for 10 s and then held at room temperature for 5 min before being centrifuged for 1 min at 13,000 ×g. Following centrifugation, the supernatant was discarded; the pellet was re-suspended in 1 mL of Sepsityper® Washing Buffer (Bruker Daltonics) and centrifuged again for 1 min at 13,000 ×g. The supernatant was discarded and the pellet was re-suspended in 300 μL MALDI-grade water and 900 μL 100% ethanol followed by centrifugation for 2 min at 13,000 ×g. The supernatant then was decanted and each sample recentrifuged for another 2 min at 13,000 ×g. Any residual ethanol then was removed and the pellet was air dried for 10 min at room temperature. Extractions were performed according to the manufacturer's 87
Journal of Microbiological Methods 152 (2018) 86–91
S. Tsuchida et al.
Fig. 1. Schematic representation of the rapid BACpro® II kit as a pretreatment for MALDI-TOF MS-based bacterial identification from positive blood culture bottles.
recommendations. In brief, 30 μL of 70% formic acid and 30 μL of 100% acetonitrile were added to the tube; the pellet was thoroughly re-suspended; and the sample was centrifuged for 2 min at 13,000 ×g. One microliter of the resulting supernatant then was transferred to a sample spot on a MALDI target plate and the spot was allowed to air dry. Two spots on the MALDI target plate were used for each sample. One microliter of α-CHCA matrix solution (Bruker Daltonics) was added to each spotted sample and the spots were allowed to air dry.
for peak acceptance was a signal-to-noise ratio of 10. Peaks with a massto-charge ratio difference (after alignment) of < 250 ppm were considered to be identical. The peak lists generated were used for matches against the reference library, by directly using the integrated patternmatching algorithm of the software. Results of the pattern-matching process were expressed as proposed by the manufacturer, with scores ranging from 0 to 3. Scores below 1.7 were regarded as unreliable identification; scores between 1.7 and 2.0 were regarded as genus-level identification; and scores > 2.0 were regarded as species-level identification, per the manufacturer's instructions. Visual evaluation of MALDI-TOF spectra was performed using FlexAnalysis 3.0 software (Bruker Daltonics).
2.4. MALDI-TOF MS analysis A Microflex LT (Bruker Daltonics) apparatus was used, with the measurement range set at 2 to 20 kDa. FlexControl (ver. 3.3; Bruker Daltonics), Flex Analysis (ver. 3.3; Bruker Daltonics), and Bio Typer (ver. 3.1; Bruker Daltonics) analytical software were used, and version 4.0.0.1 of the database (Bruker Daltonics) was used for identification, in accordance with manufacturer's instructions and as previously described (Sogawa et al., 2011). The spectra were calibrated externally with the Bruker Daltonics bacterial test standard (Escherichia coli extracts, including the additional proteins RNase A and myoglobin). Calibration masses were: RL 36, 4364.3 Da; RS 22, 5095.8 Da; RL 34,5380.4 Da; RL meth, 6254.4 Da; RL 32, 6315.2 Da; RL 29, 7273.5 Da; RS 19, 10,229.1 Da; RNase A, 13682.2 Da; and myoglobin, 16,952.5 Da. Automated analysis of the raw spectral data was performed by the MALDI BioTyper automation 3.1 software (Bruker Daltonics). A single colony was deposited directly on an MTP Big AnchorChip 384 TF target plate (Bruker Daltonics). The preparation was overlaid with 1 μL of αCHCA matrix solution (Bruker Daltonics). The spot then was air dried at room temperature to enable co-crystallization. The mass spectra obtained from each isolate were imported into the Biotyper software (version 3.1, Bruker Daltonics) and were analyzed by standard pattern matching. The whole process, from MALDI-TOF MS measurement to identification, was performed automatically without any user intervention. Briefly, the software generated a list of up to 100 peaks. The threshold
2.5. 16S rRNA sequencing and sequence analysis For clinical isolates not identified by the conventional reference methods, we conducted 16S rRNA sequencing and sequence analysis as described previously (Sogawa et al., 2011). 2.6. Statistical analysis We used the Persons's chi-square test to compare the results from the same specimens processed either using the rapid BACpro® II kit or using the Sepsityper Kit. We performed all statistical analyses using SPSS 18 software (version 18.0; SPSS, Chicago, IL, USA). P < .05 was considered statistically significant. 3. Results 3.1. Comparison of direct MALDI-TOF MS results based on two different pretreatment protocols Of 193 positive monomicrobial blood cultures, one clinical isolate was not identified by conventional methods but was identified as Candida albicans by 16S rRNA sequencing and analysis. Of the 193 88
Journal of Microbiological Methods 152 (2018) 86–91
S. Tsuchida et al.
Table 1 MALDI-TOF MS identification of bacteria from positive blood cultures using the improved biocopolymer preparation system and sepsityper kit. Organism
Gram-negative bacteria Bacteroides fragilis Citrobacter freundii Citrobacter koseri Edwardsiella tarda Enterobacter aerogenes Enterobacter cloacae Escherichia coli Haemophilus influenzae Klebsiella oxytoca Klebsiella pneumoniae Proteus mirabilis Pseudomonas aeruginosa Raoultella ornithinolytica Serratia ureilytica Stenotrophomonas maltophilia Subtotal Gram-positive bacteria Gram-positive bacteria Bacillus cereus Bacillus subtilis Corynebacterium striatum Clostridium perfringens Enterococcus aerogenes Enterococcus avium Enterococcus casseliflavus Enterococcus faecalis Enterococcus faecium Microbacterium sp Staphylococcus aureus Staphylococcus capitis Staphylococcus epidermidis Staphylococcus caprae Staphylococcus haemolyticus Staphylococcus hominis Streptococcus agalactiae Streptococcus anginosus Streptococcus dysgalactiae Streptococcus mitis Streptococcus oralis Yeast Candida albicans Subtotal Total
No. of isolates
Rapid BACpro®IIkit
Sepsityper® Kit
No.(%) correctly identified
No.(%) correctly identified
Species level (Score, > 2.0)
Genus level (Score, 1.7–1.99)
No. (%) not reliably identified (Score, < 1.7)
Species level (Score, > 2.0)
Genus level (Score, 1.7–1.99)
No. (%) not reliably identified (Score, < 1.7)
2 3 1 1 3 2 27 1 2 21 2 10 1 2 3
1 3 1 1 3 2 27 1 2 21 2 9 1 2 3
1 0 0 0 0 0 0 0 0 0 0 1 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1 2 1 0 3 2 25 1 2 20 1 5 1 2 3
0 1 0 1 0 0 2 0 0 1 1 3 0 0 0
1 0 0 0 0 0 0 0 0 0 0 2 0 0 0
81
79 (97.5)
2 (2.5)
0 (0)
69 (85.2)
9 (11.1)
3 (3.7)
6 1 2 3 1 1 1 13 1 1 32 10 17 5 2 6 1 1 4 1 2
3 0 2 3 1 1 0 13 1 1 29 8 3 3 0 3 1 0 3 1 1
3 1 0 0 0 0 1 0 0 0 3 2 14 2 2 3 0 1 1 0 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3 0 2 3 1 1 0 12 1 1 21 6 3 4 0 2 0 0 2 0 1
3 1 0 0 0 0 1 0 0 0 6 2 9 1 2 4 0 0 1 1 0
0 0 0 0 0 0 0 1 0 0 5 2 5 0 0 0 1 1 1 0 1
1 112 193
0 77 (68.8) 156 (80.8)
0 34 (30.3) 36 (18.7)
1 1 (0.9) 1 (0.5)
0 63 (56.3) 132 (68.4)
0 31 (27.7) 40 (20.7)
1 18 (16.0) 21 (10.9)
The overall MALDI-TOF MS-based identification rates with > 1.7 score and > 2.0 score were 99.5% and 80.8% respectively, using the rapid BACpro® II kit, whereas the rates were 89.1% and 68.4%, respectively, using the Sepsityper® kit. In Gram- positive cases, the rapid BACpro® II kit yielded > 1.7 score at 100% and > 2.0 score at 69.4%. In contrast, Gram-positive cases were identified using the Sepsityper® kit at 84.7% and 56.8%, respectively.
The differences between the rapid BACpro® II kit and the Sepsityper® kit were more apparent in the identification of Gram-positive bacteria. Using the rapid BACpro® II kit, the identification rates were 100% and 69.4% with scores of > 1.7 and > 2.0, respectively. In contrast, the identification rates using the Sepsityper® kit were 84.7% and 56.8%, respectively. Although 17 (15.3%) gram-positive isolates processed using the Sepsityper® kit could not be identified, all grampositive isolates processed using the rapid BACpro® II kit were identified. Notably, for representative Gram-positive bacteria, S. aureus, coagulase-negative staphylococci and streptococci, the number of identifications (scores > 1.7) in samples processed using the rapid BACpro® II kit was significantly higher than that in samples processed using the Sepsityper® kit (Table 2). For Gram-negative bacteria, the number of identifications (scores > 2.0) in samples processed using the rapid
positive blood cultures conventionally identified, 111 (57.5%) were Gram-positive and 81 (42.0%) were Gram-negative and 1(0.5%) was yeast. Table 1 summarizes the results of the MALDI-TOF MS identification for the 193 monomicrobial isolates using two different pretreatment protocols, the rapid BACpro® II kit and the Sepsityper® kit. With the rapid BACpro® II kit, 156 (80.8%) isolates were identified with a score of > 2.0 and 192 (99.5%) were identified with a score of > 1.7. One Candida isolate was not reliably identified. In contrast, the Sepsityper® kit identified 132 isolates (68.4%) with a score of > 2.0 and 172 isolates (89.1%) with a score of > 1.7. A total of 21 isolates, 18 of which were Gram-positive bacteria, were not reliably identified following preparation with the Sepsityper® kit. In fact, the rapid BACpro® II kit exhibited a 97.5% identification rate for Gram-negative bacterial isolates at the species level, whereas the rate exhibited by the Sepsityper® kit was 85.2%. 89
Journal of Microbiological Methods 152 (2018) 86–91
S. Tsuchida et al.
samples prepared using the rapid BAC Pro than in samples prepared using other representative home-brew methods (Ashizawa et al., 2017). This copolymer-based pretreatment protocol has now been commercialized as the rapid BACpro® II kit (Nittobo Medical Co., Tokyo, Japan), but the performance of this kit still needs to be evaluated under the conditions of a real-world clinical microbiology laboratory. With increasing regulatory demands for quality assurance of in vitro diagnostic products, the maintenance of the so-called “laboratory-developed test” is challenging for individual clinical microbiology laboratories. For this reason, clinical laboratories prefer to use commercially available diagnostic products, for which regulatory approval is supposed to be provided by the manufacturer, rather than the clinical laboratories' home-brew methods. At present, there are (to our knowledge) three commercial purification kits for MS-based identification of microorganisms in blood culture bottles: the Sepsityper kit (Bruker Daltonics) (Buchan et al., 2012), the Vitek MS blood culture kit (bioMerieux, Inc.) (Fothergill et al., 2013) and the rapid BACpro® II kit (Nittobo Medical Co.) (Ashizawa et al., 2017). In our laboratory, we use a MALDI Biotyper system (Sogawa et al., 2011). Since the Vitek MS blood culture kit is not accessible for nonVitek MS users in Japan, we evaluated the performance of the rapid BACpro® II kit compared to that of the Sepsityper kit in the MALDI Biotyper system. Using the rapid BACpro® II kit, we identified 156 (80.8%) out of 193 monomicrobial isolates with scores of > 2.0 and 192 (99.5%) monomicrobial isolates with scores of > 1.7 (Table 1). The rates obtained by the Sepsityper kit were 68.4% and 89.1%, respectively (Table 1). Although the manufacturer's recommended cut-off score for species-level identification of organisms from cultured bacterial colonies is 2.00, it has been suggested that cut-off values could be lowered to improve the rate of identification from positive blood culture bottles. Indeed, the results of several reports have indicated that lowering the cut-off levels for bacterial identification from blood culture bottles would increase the sensitivity of the test without remarkably decreasing the specificity (Scott et al., 2016; Buchan et al., 2012; Farina et al., 2015; Christner et al., 2010). In the present study, 36 isolates with scores between 1.7 and 2.0 were obtained for samples prepared using the rapid BACpro® II kit. The results for these 36 isolates agreed with those obtained by conventional subculturing. The Sepsityper® kit is the most widely used commercial purification kit in the MALDI-TOF MS context. The Sepsityper® kit involves the lysis of blood cells, followed by centrifugation and washing steps. Since the initial report on the use of this kit for MALDI-TOF MS (Tanner et al., 2017), a growing number of reports using this kit have been published, as reviewed by Morgenthaler et al. (Morgenthaler and Kostrzewa, 2015). Indeed, for the recent parallel evaluation of MALDI-TOF MS- and microarray-based tests for the rapid identification of Gram-negative bacilli from positive blood cultures, the MALDI Sepsityper® kit was used as a representative pretreatment protocol for the MALDI-TOF MS-based method (Schubert et al., 2011). The performance of the MALDI Sepsityper® kit is not necessarily satisfactory, particularly for the identification of Gram-positive cocci in positive blood culture bottles. A metaanalysis of the performance of the Sepsityper® kit indicated that this kit provided overall rates of identification at the species level for Grampositive cases of as low as 60% (Morgenthaler and Kostrzewa, 2015). The results of the present study indicated that the overall identification rates obtained using the rapid BACpro® II kit are greater than those obtained using the Sepsityper® kit. The 84.7% identification rate of Gram-positive cases with the 1.7 cut-off score (for samples prepared using the commercial kit) in the present study is comparable to or better than those previously reported (Buchan et al., 2012; Saffert et al., 2012), suggesting that the commercial kit was used appropriately in our hands. In particular, compared with the Sepsityper® kit, the samples processed with the rapid BACpro® II kit yielded significantly higher identification scores for Staphylococcus spp. (Table 2). The rapid BACpro® II kit has two basic advantages. First, the
Table 2 MALDI-TOF MS identifications of Gram-negative bacteria, Gram-positive bacteria, Staphylococcus aureus, coagulase-negative staphylococci (CNS), and Streptococcus spp. in samples processed using either the rapid BACpro® II kit or the Sepsityper® kit. Organism
Gram-negative bacteria (n = 81) Gram-positive bacteria (n = 111) Staphylococcus aureus (n = 32) Coagulase-negative staphylococci (CNS)(n = 40) Streptococcus spp.(n = 9)
Rapid BACpro®IIkit
Sepsityper® Kit
Correctly identified (n)
Correctly identified (n)
Score > 2.0
Score > 1.7
Score > 2.0
Score > 1.7
79⁎
81
69
78
77
111⁎
63
94
29⁎
32⁎
21
27
17
40⁎
15
33
6
9⁎
3
5
Results were analyzed for significance using the Person's chi-square test for comparisons. P < .05 was considered statistically significant. ⁎ P < .05.
BACpro® II kit was significantly higher than that in samples processed using the Sepsityper® kit processed samples (Table 2). Our study demonstrated that all rapid BACpro® II kit processed samples (156/193, species level, score > 2.0) generated significantly more accurate identifications than the Sepsityper® kit processed samples (132/193, species level, score > 2.0) (Pearsons's chi-square; P = .005).
4. Discussion Blood culturing remains the best approach to identifying the etiological agents of bloodstream infections because such cultures are highly sensitive and easy to perform (Opota et al., 2015). The introduction of the MALDI-TOF MS-based method has revolutionized the identification of microorganisms in clinical microbiology laboratories. The MALDI-TOF MS technology was first applied to bacterial colonies grown on agar plates; the ability to perform direct analysis of clinical specimens without prior culturing or subculturing has further increased the usefulness of this technology. Microorganisms in blood culture bottles are expected to be good targets for analysis by a MSbased approach, but for accurate identification, pretreatments to effectively remove a variety of non-bacterial components are needed along with concentration of bacteria prior to MS. A number of homebrew or commercial protocols have been reported, as reviewed by Florio et al. and Scott et al. (Florio et al., 2018 and Scott et al., 2016). We recently reported a new pretreatment protocol that provides efficient removal of blood cells and efficient collection of pathogens in blood culture bottles (Ashizawa et al., 2017). In this protocol, a polyallylamine-polystyrene copolymer is used to collect microorganisms in positive blood culture bottles. Since the majority of bacteria are adherent to positively charged polymer surfaces, the copolymer has been used in wastewater treatment (Nicolella et al., 2000). Bacterial aggregation via copolymerization of polyallylamine and polystyrene was shown to facilitate sedimentation of bacteria as macroscopically visible aggregates (Ashizawa et al., 2017). Generally, the quality of MALDI spectra obtained from bacteria grown in blood culture bottles is considered to be lower than those of spectra obtained directly from bacterial colonies on agar plates, most likely as a result of interference by blood-related components. In our previous report, we showed that interference by hemoglobin during MALDI-TOF MS analysis was lower in 90
Journal of Microbiological Methods 152 (2018) 86–91
S. Tsuchida et al.
operation time is as short as 10 min per sample, shorter than either the Sepsityper® kit or previous home-brew protocols. Secondly, the minimum centrifugal force required for effective bacterial collection is only 2000 ×g, enabling the entire protocol to be completed within a biosafety cabinet. This observation represents a significant advantage in terms of the biosafety of laboratory staff. In contrast, the Sepsityper® kit requires a high-speed centrifuge, necessitating the use of equipment outside of a biosafety cabinet. Despite the encouraging results obtained using the rapid BACpro® II kit, this report has some limitations. First, this work represents a preliminary study, performed with a limited number and distribution of clinical isolates. Therefore, a larger-scale study will be needed. A second limitation is that this work was performed solely with the MALDI Biotyper. Two major MS-based microbial identification systems are available in the global market: the MALDI Biotyper (Bruker Daltonics, Lepzig, Germany) and the VITEK MS plus (bioMerieux, Inc., Marcy l'Etoile. France). Performance of the rapid BACpro® II kit with the VITEK MS plus system remains to be determined. Despites these limitations, and taking into consideration that this new protocol is quick, easy, safe, and provides promising identification rates, we believe that the rapid BACpro® II kit deserves to be evaluated in a larger-scale study with a variety of platforms for MS-based bacterial identification.
Farina, C., Arena, F., Casprini, P., Cichero, P., Clementi, M., Cosentino, M., Degl'Innocenti, R., Giani, T., Luzzaro, F., Mattei, R., Mauri, C., Nardone, M., Rossolini, G.M., Serna Ortega, P.A., Vailati, F., 2015. Direct identification of microorganisms from positive blood cultures using the lysis-filtration technique and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF MS): a multicentre study. New Microbiol. 38, 245–250. Faron, M.L., Buchan, B.W., Ledeboer, N.A., 2017. Matrix-assisted laser desorption ionization-time of flight mass spectrometry for use with positive blood cultures: methodology, performance, and optimization. J. Clin. Microbiol. 55, 3328–3338. Ferreira, L., Sánchez-Juanes, F., Porras-Guerra, I., García-García, M.I., García-Sánchez, J.E., González-Buitrago, J.M., Muñoz-Bellido, J.L., 2011. Microorganisms direct identification from blood culture by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry. Clin. Microbiol. Infect. 17, 546–551. Florio, W., Morici, P., Ghelardi, E., Barnini, S., Lupetti, A., 2018. Recent advances in the microbiological diagnosis of bloodstream infections. Crit. Rev. Microbiol. 44 (3), 351–370. https://doi.org/10.1080/1040841X.2017.1407745. (May, Epub 2017 Nov 29). Fothergill, A., Kasinathan, V., Hyman, J., Walsh, J., Drake, T., Wang, Y.F., 2013. Rapid identification of bacteria and yeasts from positive-blood-culture bottles by using a lysis-filtration method and matrix-assisted laser desorption ionization-time of flight mass spectrum analysis with the SARAMIS database. J. Clin. Microbiol. 51, 805–809. Kumar, A., Roberts, D., Wood, K.E., Light, B., Parrillo, J.E., Sharma, S., Suppes, R., Feinstein, D., Zanotti, S., Taiberg, L., Gurka, D., Kumar, A., Cheang, M., 2006. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit. Care Med. 34, 1589–1596. Monteiro, J., Inoue, F.M., Lobo, A.P., Sugawara, E.K., Boaretti, F.M., Tufik, S., 2015. Fast and reliable bacterial identification direct from positive blood culture using a new TFA sample preparation protocol and the Vitek® MS system. J. Microbiol. Methods 109, 157–159. Morgenthaler, N.G., Kostrzewa, M., 2015. Rapid identification of pathogens in positive blood culture of patients with sepsis: review and meta-analysis of the performance of the sepsityper kit. Int. J. Microbiol. 2015, 827416. Moussaoui, W., Jaulhac, B., Hoffmann, A.M., Ludes, B., Kostrzewa, M., Riegel, P., Prévost, G., 2010. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identifies 90% of bacteria directly from blood culture vials. Clin. Microbiol. Infect. 16, 1631–1638. Neville, S.A., Lecordier, A., Ziochos, H., Chater, M.J., Gosbell, I.B., Maley, M.W., van Hal, S.J., 2011. Utility of matrix-assisted laser desorption ionization-time of flight mass spectrometry following introduction for routine laboratory bacterial identification. J. Clin. Microbiol. 49, 2980–2984. Nicolella, C., van Loosdrecht, M.C.M., Heijnen, J.J., 2000. Wastewater treatment with particulate biofilm reactors. J. Biotechnol. 80, 1–33. Nomura, F., 2015. Proteome-based bacterial identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS): a revolutionary shift in clinical diagnostic microbiology. Biochim. Biophys. Acta 1854, 528–537. Opota, O., Croxatto, A., Prod'hom, G., Greub, G., 2015. Blood culture-based diagnosis of bacteraemia: state of the art. Clin. Microbiol. Infect. 21, 313–322. Prod'hom, G., Bizzini, A., Durussel, C., Bille, J., Greub, G., 2010. Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets. J. Clin. Microbiol. 48, 1481–1483. Saffert, R.T., Cunningham, S.A., Mandrekar, J., Patel, R., 2012. Comparison of three preparatory methods for detection of bacteremia by MALDI-TOF mass spectrometry. Diagn. Microbiol. Infect. Dis. 73, 21–26. Schubert, S., Weinert, K., Wagner, C., Gunzl, B., Wieser, A., Maier, T., Kostrzewa, M., 2011. Novel, improved sample preparation for rapid, direct identification from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. J. Mol. Diagn. 13, 701–706. Scott, J.S., Sterling, S.A., To, H, Seals, S.R., Jones, A.E., 2016. Diagnostic performance of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry in blood bacterial infections: a systematic review and meta-analysis. Infect. Dis. (Lond). 48, 530–536. Seifert, H., 2009. The clinical importance of microbiological findings in the diagnosis and management of bloodstream infections. Clin. Infect. Dis. 48, S238–S245. Sogawa, K., Watanabe, M., Sato, K., Segawa, S., Ishii, C., Miyabe, A., Murata, S., Saito, T., Nomura, F., 2011. Use of the MALDI BioTyper system with MALDI-TOF mass spectrometry for rapid identification of microorganisms. Anal. Bioanal. Chem. 400, 1905–1911. Tanner, H., Evans, J.T., Gossain, S., Hussain, A., 2017. Evaluation of three sample preparation methods for the direct identification of bacteria in positive blood cultures by MALDI-TOF. BMC Res. Notes. 10, 48.
Acknowledgments The authors express their appreciation for the contributions of members of the bacterial laboratory of Chiba University, Chiba, Japan. Declarations of interests None. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Angeletti, S., 2017. Matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology. J. Microbiol. Methods 138, 20–29. Angus, D.C., Wax, R.S., 2001. Epidemiology of sepsis: an update. Crit. Care Med. 29, S109–S116. Ashizawa, K., Murata, S., Terada, T., Ito, D., Bunya, M., Watanabe, K., Teruuchi, Y., Tsuchida, S., Mamoru, S., Nishimura, M., Matsuhita, K., Sugama, Y., Nomura, F., 2017. Applications of copolymer for rapid identification of bacteria in blood culture broths using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. J. Microbiol. Methods 139, 54–60. Buchan, B.W., Riebe, K.M., Ledeboer, N.A., 2012. Comparison of the MALDI Biotyper system using Sepsityper specimen processing to routine microbiological methods for identification of bacteria from positive blood culture bottles. J. Clin. Microbiol. 50, 346–352. Christner, M., Rohde, H., Wolters, M., Sobottka, I., Wegscheider, K., Aepfelbacher, M., 2010. Rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting. J. Clin. Microbiol. 48, 1584–1591. Clark, A.E., Kaleta, E.J., Arora, A., Wolk, D.M., 2013. Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology. Clin. Microbiol. Rev. 26, 547–603.
91
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Application of the biocopolymer preparation system, rapid BACpro® II kit, for mass-spectrometry-based bacterial identification from positive blood culture bottles by the MALDI Biotyper system
T
Sachio Tsuchidaa, Syota Muratab, Akiko Miyabeb, Mamoru Satoha, Masaki Takiwakia, ⁎ Kazuho Ashizawac, Takashi Teradac, Daisuke Itoc, Kazuyuki Matsushitab,d, Fumio Nomuraa,d, a
Division of Clinical Mass Spectrometry, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Department of Clinical Laboratory, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan R&D Department, Nittobo Medical Co., Ltd, Japan d Division of Clinical Genetics, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacterial identification Blood culture Rapid BACpro® II kit Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove nonbacterial components and selectively collect microorganisms are a prerequisite for successful identification, and a variety of home-brew and commercial protocols have been reported. Although commercially available kits, mainly the Sepsityper Kit, are increasingly used, the identification rates reported often are not satisfactory, particularly for Gram-positive isolates. We recently developed a method to collect bacteria from positive blood culture bottles using a polyallylamine-polystyrene copolymer that has been used in wastewater processing. This pretreatment protocol is now commercially available as the rapid BACpro® II kit (Nittobo Medical Co., Tokyo, Japan). The operation time required for processing using this novel kit is approximately 10 min, and the entire procedure can be completed within a biosafety cabinet. Since the performance of the rapid BACpro® II kit has not been tested using the MALDI Biotyper system, we prospectively evaluated the performance of the rapid BACpro® II kit as compared with the Sepsityper® kit. Performance of the rapid BACpro® II kit was evaluated using a total of 193 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the rapid BACpro® II kit or the Sepsityper® Kit, and isolated cells were subjected to direct identification by MS fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with > 1.7 score and > 2.0 score obtained using the rapid BACpro® II kit were 99.5% and 80.8%, respectively, whereas those obtained using the Sepsityper® Kit were 89.1% and 68.4%, respectively (P < 0.05 for > 1.7 and P < 0.05 for > 2.0 by Pearsons's chi-square). In Gram-positive cases, the rapid BACpro® II kit gave identification rate of 100% with > 1.7 score and 69.4% with > 2.0 score, whereas there were 84.7% and 56.8%, respectively by the Sepsityper® Kit (P < 0.05 for > 1.7). These results are preliminary, but considering that this new kit is easy to perform and the identification rates are promising, the rapid BACpro® II kit deserves assessment in a larger-scale study with a variety of platforms for MS-based bacterial identification.
1. Introduction Prompt identification of the causative pathogen is critical for early initiation of appropriate antimicrobial therapy in blood stream infections (Angus and Wax, 2001 and Seifert, 2009). Indeed, it has been
reported that one hour of delay in proper antibiotic treatment for sepsis-related hypotensive patients decreases the chance of survival by 7.6% (Kumar et al., 2006). Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a quick and reliable method for
Abbreviations: MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MS, mass spectrometry ⁎ Corresponding author at: Division of Clinical Mass Spectrometry, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail address: [email protected] (F. Nomura). https://doi.org/10.1016/j.mimet.2018.07.017 Received 16 April 2018; Received in revised form 25 July 2018; Accepted 29 July 2018 Available online 01 August 2018 0167-7012/ © 2018 Elsevier B.V. All rights reserved.
Journal of Microbiological Methods 152 (2018) 86–91
S. Tsuchida et al.
between the rapid BACpro® II kit and the Sepsityper Kit. Positive monomicrobial isolates were subjected in parallel to direct identification by MS fingerprinting and conventional subculturing for reference identification.
identification of microorganisms; the role of this technique is rapidly growing in clinical diagnostic microbiology (Neville et al., 2011; Clark et al., 2013; Nomura, 2015; Angeletti, 2017; Prod'hom et al., 2010; Ferreira et al., 2011). Direct identification of bacteria from blood culture bottles is a promising application of MALDI-TOF MS, as has been described in several reviews (Angeletti, 2017; Prod'hom et al., 2010; Ferreira et al., 2011; Faron et al., 2017; Florio et al., 2018). Since blood culture bottle media contain nonbacterial proteins such as blood cells that may be obstacles for the interpretation of bacterial proteome profiles, pretreatment to substantially and selectively remove these host proteins is essential for successful identification of the causative organisms. A number of laboratory-developed or commercially available protocols for purification of bacteria have been reported, as reviewed by Florio et al. and Scott et al. (Florio et al., 2018 and Scott et al., 2016). Some representative examples of the laboratory-developed test protocols are as follows: (1) stepwise sedimentation of blood cells and microorganisms (Ferreira et al., 2011); (2) low-speed centrifugation to eliminate blood cells, followed by an additional lysis procedure (Prod'hom et al., 2010 and Monteiro et al., 2015); and (3) removal of blood cells using a serum separator tube (Moussaoui et al., 2010). The Sepsityper® kit (Bruker Daltonics, GmbH, Bremen, Germany) (Buchan et al., 2012) and the Vitek® MS Blood Culture Kit (bioMerieux, Inc., Marcy l'Etoile, France) (Fothergill et al., 2013) are the two major commercial purification kits. The overall concordance of the results obtained with the home-brew protocols and the commercial kits compared with those obtained by conventional direct colony methods ranges from 84% to 99% for the identification of Gram-negative bacteria and is approximately 80% for Gram-positive cases (Ferreira et al., 2011). The Sepsityper® kit is the most widely used commercial kit; a meta-analysis of the performance of the Sepsityper® kit indicated that this kit provided a reliable species-level identification for 80% of 3320 positive blood culture cases (Morgenthaler and Kostrzewa, 2015). In an attempt to further improve the identification rates and also to reduce the long hands-on time of the previous pretreatment protocols, we recently developed a method to collect bacteria in positive blood culture bottles using a polyallylamine-polystyrene copolymer that has been used in wastewater processing (Ashizawa et al., 2017). This pretreatment protocol is now commercially available as the rapid BACpro® II kit (Nittobo Medical Co., Tokyo, Japan). Since the performance of the rapid BACpro® II kit has not been tested using the MALDI Biotyper system, we prospectively evaluated the performance of the rapid BACpro® II kit compared with the Sepsityper® kit. Specifically, we evaluated these two kits when used for sample preparation of positive blood culture bottles for MALDI-TOF MS-based bacterial identification using the MALDI Biotyper system.
2.2. Conventional identification All solid media were incubated at 37 °C in 5% CO2 for 18–24 h. Each clinical specimen was plated on an appropriate solid medium (chocolate agar or trypticase soy agar II with 5% sheep blood, Nippon Becton Dickinson, Tokyo, Japan) depending on the sample and in accordance with our laboratory recommendations. Following Gram staining and determination of catalase and oxidase activities, the isolates were identified by one of three different phenotypic tests, including the Microscan Walkaway system (Siemens Healthcare Diagnostics, Deerfield, IL, USA), the BD Phoenix system (BD Diagnostics Systems, Sparks, MD, USA), and the API system (Sysmex Biomerieux, Lyon, France). The respective manufacturer's instructions for each system were followed. 2.3. Sample pretreatment methods for direct MALDI-TOF MS analysis 2.3.1. Rapid BACpro® II kit Workflow of the protocol using copolymer was per the manufacturer's instructions; this protocol was essentially as reported previously (Ashizawa et al., 2017), albeit with some modifications. In short, the manufacturer's protocol consisted of four steps (blood cell lysis, reaction with cationic particles, washing, and the final extraction of bacteria), as illustrated in Fig. 1. Briefly, a 1-mL aliquot of blood culture sample was added to a reaction tube containing 500 μL of lysis buffer (Nittobo Medical Co., Tokyo, Japan) and the mixture was centrifuged (Chibitan R; Merck Millipore, Billerica, MA) at 2000 ×g for 3 min. The supernatant was discarded and the pellet was resuspended in 800 μL of distilled water. The suspended sample was combined with 200 μL of cationic particle solution (Nittobo Medical Co.) and 200 μL of reaction buffer (Nittobo Medical Co.). The mixture then was transferred into another tube and centrifuged (Chibitan R; Merck Millipore) at 2000 ×g for 1 min. After removing the supernatant, the pellet was resuspended in 1000 μL of 70% ethanol (Nacalai Tesque, Kyoto, Japan), and the resulting suspension was centrifuged at 2000 ×g for 1 min. After the supernatant was removed, the pellet was resuspended in 30 μL of 70% formic acid (Wako, Tokyo, Japan) and 30 μL of 100% acetonitrile (Wako, Tokyo, Japan) and then centrifuged at 2000 ×g for 1 min. One microliter of the resulting supernatant then was transferred to a sample spot on a MALDI target plate and the spot was allowed to air dry. One microliter of an αcyano-4-hydroxycinnamic acid (CHCA) matrix solution, comprising a saturated solution of CHCA in 50% acetonitrile and 2.5% trifluoroacetic acid (Bruker Daltonics, Leipzig, Germany), was added to each spotted sample and the spots were allowed to air dry. Two spots on the MALDI target plate were used for each sample.
2. Materials and methods 2.1. Samples and study design The clinical evaluation was conducted during working hours from June 2016 to July 2018 at the Chiba University Hospital, Chiba, Japan. During this study period, a total of 196 blood culture bottles from 196 patients were flagged as positive. BD BACTEC™ Plus Aerobic medium in glass culture vials (BD, Franklin Lakes, NJ, USA) and the BACTEC™ FX incubation system (BD, Franklin Lakes, NJ, USA) were used. Separate portions of positive culture bottles were Gram stained and subcultured. The subcultures were conducted on 5% sheep blood agar (SBA) and chocolate agar. Eosin methylene blue and anaerobic SBA were used for Gram-negative bacteria and anaerobic bottle subcultures, respectively. The blood cultures were held at room temperature following removal from the blood culture instrument and were analyzed within 8 h of culture positivity identification. None of the positive cultures failed to grow on subculturing, and 3 were polymicrobial. The remaining 193 monomicrobial cases were included in the comparative analysis
2.3.2. MALDI Sepsityper® kit One milliliter of the medium from each positive blood culture bottle was combined with 200 μL of the Sepsityper® lysis reagent (Bruker Daltonics). The mixture was vortexed for 10 s and then held at room temperature for 5 min before being centrifuged for 1 min at 13,000 ×g. Following centrifugation, the supernatant was discarded; the pellet was re-suspended in 1 mL of Sepsityper® Washing Buffer (Bruker Daltonics) and centrifuged again for 1 min at 13,000 ×g. The supernatant was discarded and the pellet was re-suspended in 300 μL MALDI-grade water and 900 μL 100% ethanol followed by centrifugation for 2 min at 13,000 ×g. The supernatant then was decanted and each sample recentrifuged for another 2 min at 13,000 ×g. Any residual ethanol then was removed and the pellet was air dried for 10 min at room temperature. Extractions were performed according to the manufacturer's 87
Journal of Microbiological Methods 152 (2018) 86–91
S. Tsuchida et al.
Fig. 1. Schematic representation of the rapid BACpro® II kit as a pretreatment for MALDI-TOF MS-based bacterial identification from positive blood culture bottles.
recommendations. In brief, 30 μL of 70% formic acid and 30 μL of 100% acetonitrile were added to the tube; the pellet was thoroughly re-suspended; and the sample was centrifuged for 2 min at 13,000 ×g. One microliter of the resulting supernatant then was transferred to a sample spot on a MALDI target plate and the spot was allowed to air dry. Two spots on the MALDI target plate were used for each sample. One microliter of α-CHCA matrix solution (Bruker Daltonics) was added to each spotted sample and the spots were allowed to air dry.
for peak acceptance was a signal-to-noise ratio of 10. Peaks with a massto-charge ratio difference (after alignment) of < 250 ppm were considered to be identical. The peak lists generated were used for matches against the reference library, by directly using the integrated patternmatching algorithm of the software. Results of the pattern-matching process were expressed as proposed by the manufacturer, with scores ranging from 0 to 3. Scores below 1.7 were regarded as unreliable identification; scores between 1.7 and 2.0 were regarded as genus-level identification; and scores > 2.0 were regarded as species-level identification, per the manufacturer's instructions. Visual evaluation of MALDI-TOF spectra was performed using FlexAnalysis 3.0 software (Bruker Daltonics).
2.4. MALDI-TOF MS analysis A Microflex LT (Bruker Daltonics) apparatus was used, with the measurement range set at 2 to 20 kDa. FlexControl (ver. 3.3; Bruker Daltonics), Flex Analysis (ver. 3.3; Bruker Daltonics), and Bio Typer (ver. 3.1; Bruker Daltonics) analytical software were used, and version 4.0.0.1 of the database (Bruker Daltonics) was used for identification, in accordance with manufacturer's instructions and as previously described (Sogawa et al., 2011). The spectra were calibrated externally with the Bruker Daltonics bacterial test standard (Escherichia coli extracts, including the additional proteins RNase A and myoglobin). Calibration masses were: RL 36, 4364.3 Da; RS 22, 5095.8 Da; RL 34,5380.4 Da; RL meth, 6254.4 Da; RL 32, 6315.2 Da; RL 29, 7273.5 Da; RS 19, 10,229.1 Da; RNase A, 13682.2 Da; and myoglobin, 16,952.5 Da. Automated analysis of the raw spectral data was performed by the MALDI BioTyper automation 3.1 software (Bruker Daltonics). A single colony was deposited directly on an MTP Big AnchorChip 384 TF target plate (Bruker Daltonics). The preparation was overlaid with 1 μL of αCHCA matrix solution (Bruker Daltonics). The spot then was air dried at room temperature to enable co-crystallization. The mass spectra obtained from each isolate were imported into the Biotyper software (version 3.1, Bruker Daltonics) and were analyzed by standard pattern matching. The whole process, from MALDI-TOF MS measurement to identification, was performed automatically without any user intervention. Briefly, the software generated a list of up to 100 peaks. The threshold
2.5. 16S rRNA sequencing and sequence analysis For clinical isolates not identified by the conventional reference methods, we conducted 16S rRNA sequencing and sequence analysis as described previously (Sogawa et al., 2011). 2.6. Statistical analysis We used the Persons's chi-square test to compare the results from the same specimens processed either using the rapid BACpro® II kit or using the Sepsityper Kit. We performed all statistical analyses using SPSS 18 software (version 18.0; SPSS, Chicago, IL, USA). P < .05 was considered statistically significant. 3. Results 3.1. Comparison of direct MALDI-TOF MS results based on two different pretreatment protocols Of 193 positive monomicrobial blood cultures, one clinical isolate was not identified by conventional methods but was identified as Candida albicans by 16S rRNA sequencing and analysis. Of the 193 88
Journal of Microbiological Methods 152 (2018) 86–91
S. Tsuchida et al.
Table 1 MALDI-TOF MS identification of bacteria from positive blood cultures using the improved biocopolymer preparation system and sepsityper kit. Organism
Gram-negative bacteria Bacteroides fragilis Citrobacter freundii Citrobacter koseri Edwardsiella tarda Enterobacter aerogenes Enterobacter cloacae Escherichia coli Haemophilus influenzae Klebsiella oxytoca Klebsiella pneumoniae Proteus mirabilis Pseudomonas aeruginosa Raoultella ornithinolytica Serratia ureilytica Stenotrophomonas maltophilia Subtotal Gram-positive bacteria Gram-positive bacteria Bacillus cereus Bacillus subtilis Corynebacterium striatum Clostridium perfringens Enterococcus aerogenes Enterococcus avium Enterococcus casseliflavus Enterococcus faecalis Enterococcus faecium Microbacterium sp Staphylococcus aureus Staphylococcus capitis Staphylococcus epidermidis Staphylococcus caprae Staphylococcus haemolyticus Staphylococcus hominis Streptococcus agalactiae Streptococcus anginosus Streptococcus dysgalactiae Streptococcus mitis Streptococcus oralis Yeast Candida albicans Subtotal Total
No. of isolates
Rapid BACpro®IIkit
Sepsityper® Kit
No.(%) correctly identified
No.(%) correctly identified
Species level (Score, > 2.0)
Genus level (Score, 1.7–1.99)
No. (%) not reliably identified (Score, < 1.7)
Species level (Score, > 2.0)
Genus level (Score, 1.7–1.99)
No. (%) not reliably identified (Score, < 1.7)
2 3 1 1 3 2 27 1 2 21 2 10 1 2 3
1 3 1 1 3 2 27 1 2 21 2 9 1 2 3
1 0 0 0 0 0 0 0 0 0 0 1 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1 2 1 0 3 2 25 1 2 20 1 5 1 2 3
0 1 0 1 0 0 2 0 0 1 1 3 0 0 0
1 0 0 0 0 0 0 0 0 0 0 2 0 0 0
81
79 (97.5)
2 (2.5)
0 (0)
69 (85.2)
9 (11.1)
3 (3.7)
6 1 2 3 1 1 1 13 1 1 32 10 17 5 2 6 1 1 4 1 2
3 0 2 3 1 1 0 13 1 1 29 8 3 3 0 3 1 0 3 1 1
3 1 0 0 0 0 1 0 0 0 3 2 14 2 2 3 0 1 1 0 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3 0 2 3 1 1 0 12 1 1 21 6 3 4 0 2 0 0 2 0 1
3 1 0 0 0 0 1 0 0 0 6 2 9 1 2 4 0 0 1 1 0
0 0 0 0 0 0 0 1 0 0 5 2 5 0 0 0 1 1 1 0 1
1 112 193
0 77 (68.8) 156 (80.8)
0 34 (30.3) 36 (18.7)
1 1 (0.9) 1 (0.5)
0 63 (56.3) 132 (68.4)
0 31 (27.7) 40 (20.7)
1 18 (16.0) 21 (10.9)
The overall MALDI-TOF MS-based identification rates with > 1.7 score and > 2.0 score were 99.5% and 80.8% respectively, using the rapid BACpro® II kit, whereas the rates were 89.1% and 68.4%, respectively, using the Sepsityper® kit. In Gram- positive cases, the rapid BACpro® II kit yielded > 1.7 score at 100% and > 2.0 score at 69.4%. In contrast, Gram-positive cases were identified using the Sepsityper® kit at 84.7% and 56.8%, respectively.
The differences between the rapid BACpro® II kit and the Sepsityper® kit were more apparent in the identification of Gram-positive bacteria. Using the rapid BACpro® II kit, the identification rates were 100% and 69.4% with scores of > 1.7 and > 2.0, respectively. In contrast, the identification rates using the Sepsityper® kit were 84.7% and 56.8%, respectively. Although 17 (15.3%) gram-positive isolates processed using the Sepsityper® kit could not be identified, all grampositive isolates processed using the rapid BACpro® II kit were identified. Notably, for representative Gram-positive bacteria, S. aureus, coagulase-negative staphylococci and streptococci, the number of identifications (scores > 1.7) in samples processed using the rapid BACpro® II kit was significantly higher than that in samples processed using the Sepsityper® kit (Table 2). For Gram-negative bacteria, the number of identifications (scores > 2.0) in samples processed using the rapid
positive blood cultures conventionally identified, 111 (57.5%) were Gram-positive and 81 (42.0%) were Gram-negative and 1(0.5%) was yeast. Table 1 summarizes the results of the MALDI-TOF MS identification for the 193 monomicrobial isolates using two different pretreatment protocols, the rapid BACpro® II kit and the Sepsityper® kit. With the rapid BACpro® II kit, 156 (80.8%) isolates were identified with a score of > 2.0 and 192 (99.5%) were identified with a score of > 1.7. One Candida isolate was not reliably identified. In contrast, the Sepsityper® kit identified 132 isolates (68.4%) with a score of > 2.0 and 172 isolates (89.1%) with a score of > 1.7. A total of 21 isolates, 18 of which were Gram-positive bacteria, were not reliably identified following preparation with the Sepsityper® kit. In fact, the rapid BACpro® II kit exhibited a 97.5% identification rate for Gram-negative bacterial isolates at the species level, whereas the rate exhibited by the Sepsityper® kit was 85.2%. 89
Journal of Microbiological Methods 152 (2018) 86–91
S. Tsuchida et al.
samples prepared using the rapid BAC Pro than in samples prepared using other representative home-brew methods (Ashizawa et al., 2017). This copolymer-based pretreatment protocol has now been commercialized as the rapid BACpro® II kit (Nittobo Medical Co., Tokyo, Japan), but the performance of this kit still needs to be evaluated under the conditions of a real-world clinical microbiology laboratory. With increasing regulatory demands for quality assurance of in vitro diagnostic products, the maintenance of the so-called “laboratory-developed test” is challenging for individual clinical microbiology laboratories. For this reason, clinical laboratories prefer to use commercially available diagnostic products, for which regulatory approval is supposed to be provided by the manufacturer, rather than the clinical laboratories' home-brew methods. At present, there are (to our knowledge) three commercial purification kits for MS-based identification of microorganisms in blood culture bottles: the Sepsityper kit (Bruker Daltonics) (Buchan et al., 2012), the Vitek MS blood culture kit (bioMerieux, Inc.) (Fothergill et al., 2013) and the rapid BACpro® II kit (Nittobo Medical Co.) (Ashizawa et al., 2017). In our laboratory, we use a MALDI Biotyper system (Sogawa et al., 2011). Since the Vitek MS blood culture kit is not accessible for nonVitek MS users in Japan, we evaluated the performance of the rapid BACpro® II kit compared to that of the Sepsityper kit in the MALDI Biotyper system. Using the rapid BACpro® II kit, we identified 156 (80.8%) out of 193 monomicrobial isolates with scores of > 2.0 and 192 (99.5%) monomicrobial isolates with scores of > 1.7 (Table 1). The rates obtained by the Sepsityper kit were 68.4% and 89.1%, respectively (Table 1). Although the manufacturer's recommended cut-off score for species-level identification of organisms from cultured bacterial colonies is 2.00, it has been suggested that cut-off values could be lowered to improve the rate of identification from positive blood culture bottles. Indeed, the results of several reports have indicated that lowering the cut-off levels for bacterial identification from blood culture bottles would increase the sensitivity of the test without remarkably decreasing the specificity (Scott et al., 2016; Buchan et al., 2012; Farina et al., 2015; Christner et al., 2010). In the present study, 36 isolates with scores between 1.7 and 2.0 were obtained for samples prepared using the rapid BACpro® II kit. The results for these 36 isolates agreed with those obtained by conventional subculturing. The Sepsityper® kit is the most widely used commercial purification kit in the MALDI-TOF MS context. The Sepsityper® kit involves the lysis of blood cells, followed by centrifugation and washing steps. Since the initial report on the use of this kit for MALDI-TOF MS (Tanner et al., 2017), a growing number of reports using this kit have been published, as reviewed by Morgenthaler et al. (Morgenthaler and Kostrzewa, 2015). Indeed, for the recent parallel evaluation of MALDI-TOF MS- and microarray-based tests for the rapid identification of Gram-negative bacilli from positive blood cultures, the MALDI Sepsityper® kit was used as a representative pretreatment protocol for the MALDI-TOF MS-based method (Schubert et al., 2011). The performance of the MALDI Sepsityper® kit is not necessarily satisfactory, particularly for the identification of Gram-positive cocci in positive blood culture bottles. A metaanalysis of the performance of the Sepsityper® kit indicated that this kit provided overall rates of identification at the species level for Grampositive cases of as low as 60% (Morgenthaler and Kostrzewa, 2015). The results of the present study indicated that the overall identification rates obtained using the rapid BACpro® II kit are greater than those obtained using the Sepsityper® kit. The 84.7% identification rate of Gram-positive cases with the 1.7 cut-off score (for samples prepared using the commercial kit) in the present study is comparable to or better than those previously reported (Buchan et al., 2012; Saffert et al., 2012), suggesting that the commercial kit was used appropriately in our hands. In particular, compared with the Sepsityper® kit, the samples processed with the rapid BACpro® II kit yielded significantly higher identification scores for Staphylococcus spp. (Table 2). The rapid BACpro® II kit has two basic advantages. First, the
Table 2 MALDI-TOF MS identifications of Gram-negative bacteria, Gram-positive bacteria, Staphylococcus aureus, coagulase-negative staphylococci (CNS), and Streptococcus spp. in samples processed using either the rapid BACpro® II kit or the Sepsityper® kit. Organism
Gram-negative bacteria (n = 81) Gram-positive bacteria (n = 111) Staphylococcus aureus (n = 32) Coagulase-negative staphylococci (CNS)(n = 40) Streptococcus spp.(n = 9)
Rapid BACpro®IIkit
Sepsityper® Kit
Correctly identified (n)
Correctly identified (n)
Score > 2.0
Score > 1.7
Score > 2.0
Score > 1.7
79⁎
81
69
78
77
111⁎
63
94
29⁎
32⁎
21
27
17
40⁎
15
33
6
9⁎
3
5
Results were analyzed for significance using the Person's chi-square test for comparisons. P < .05 was considered statistically significant. ⁎ P < .05.
BACpro® II kit was significantly higher than that in samples processed using the Sepsityper® kit processed samples (Table 2). Our study demonstrated that all rapid BACpro® II kit processed samples (156/193, species level, score > 2.0) generated significantly more accurate identifications than the Sepsityper® kit processed samples (132/193, species level, score > 2.0) (Pearsons's chi-square; P = .005).
4. Discussion Blood culturing remains the best approach to identifying the etiological agents of bloodstream infections because such cultures are highly sensitive and easy to perform (Opota et al., 2015). The introduction of the MALDI-TOF MS-based method has revolutionized the identification of microorganisms in clinical microbiology laboratories. The MALDI-TOF MS technology was first applied to bacterial colonies grown on agar plates; the ability to perform direct analysis of clinical specimens without prior culturing or subculturing has further increased the usefulness of this technology. Microorganisms in blood culture bottles are expected to be good targets for analysis by a MSbased approach, but for accurate identification, pretreatments to effectively remove a variety of non-bacterial components are needed along with concentration of bacteria prior to MS. A number of homebrew or commercial protocols have been reported, as reviewed by Florio et al. and Scott et al. (Florio et al., 2018 and Scott et al., 2016). We recently reported a new pretreatment protocol that provides efficient removal of blood cells and efficient collection of pathogens in blood culture bottles (Ashizawa et al., 2017). In this protocol, a polyallylamine-polystyrene copolymer is used to collect microorganisms in positive blood culture bottles. Since the majority of bacteria are adherent to positively charged polymer surfaces, the copolymer has been used in wastewater treatment (Nicolella et al., 2000). Bacterial aggregation via copolymerization of polyallylamine and polystyrene was shown to facilitate sedimentation of bacteria as macroscopically visible aggregates (Ashizawa et al., 2017). Generally, the quality of MALDI spectra obtained from bacteria grown in blood culture bottles is considered to be lower than those of spectra obtained directly from bacterial colonies on agar plates, most likely as a result of interference by blood-related components. In our previous report, we showed that interference by hemoglobin during MALDI-TOF MS analysis was lower in 90
Journal of Microbiological Methods 152 (2018) 86–91
S. Tsuchida et al.
operation time is as short as 10 min per sample, shorter than either the Sepsityper® kit or previous home-brew protocols. Secondly, the minimum centrifugal force required for effective bacterial collection is only 2000 ×g, enabling the entire protocol to be completed within a biosafety cabinet. This observation represents a significant advantage in terms of the biosafety of laboratory staff. In contrast, the Sepsityper® kit requires a high-speed centrifuge, necessitating the use of equipment outside of a biosafety cabinet. Despite the encouraging results obtained using the rapid BACpro® II kit, this report has some limitations. First, this work represents a preliminary study, performed with a limited number and distribution of clinical isolates. Therefore, a larger-scale study will be needed. A second limitation is that this work was performed solely with the MALDI Biotyper. Two major MS-based microbial identification systems are available in the global market: the MALDI Biotyper (Bruker Daltonics, Lepzig, Germany) and the VITEK MS plus (bioMerieux, Inc., Marcy l'Etoile. France). Performance of the rapid BACpro® II kit with the VITEK MS plus system remains to be determined. Despites these limitations, and taking into consideration that this new protocol is quick, easy, safe, and provides promising identification rates, we believe that the rapid BACpro® II kit deserves to be evaluated in a larger-scale study with a variety of platforms for MS-based bacterial identification.
Farina, C., Arena, F., Casprini, P., Cichero, P., Clementi, M., Cosentino, M., Degl'Innocenti, R., Giani, T., Luzzaro, F., Mattei, R., Mauri, C., Nardone, M., Rossolini, G.M., Serna Ortega, P.A., Vailati, F., 2015. Direct identification of microorganisms from positive blood cultures using the lysis-filtration technique and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF MS): a multicentre study. New Microbiol. 38, 245–250. Faron, M.L., Buchan, B.W., Ledeboer, N.A., 2017. Matrix-assisted laser desorption ionization-time of flight mass spectrometry for use with positive blood cultures: methodology, performance, and optimization. J. Clin. Microbiol. 55, 3328–3338. Ferreira, L., Sánchez-Juanes, F., Porras-Guerra, I., García-García, M.I., García-Sánchez, J.E., González-Buitrago, J.M., Muñoz-Bellido, J.L., 2011. Microorganisms direct identification from blood culture by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry. Clin. Microbiol. Infect. 17, 546–551. Florio, W., Morici, P., Ghelardi, E., Barnini, S., Lupetti, A., 2018. Recent advances in the microbiological diagnosis of bloodstream infections. Crit. Rev. Microbiol. 44 (3), 351–370. https://doi.org/10.1080/1040841X.2017.1407745. (May, Epub 2017 Nov 29). Fothergill, A., Kasinathan, V., Hyman, J., Walsh, J., Drake, T., Wang, Y.F., 2013. Rapid identification of bacteria and yeasts from positive-blood-culture bottles by using a lysis-filtration method and matrix-assisted laser desorption ionization-time of flight mass spectrum analysis with the SARAMIS database. J. Clin. Microbiol. 51, 805–809. Kumar, A., Roberts, D., Wood, K.E., Light, B., Parrillo, J.E., Sharma, S., Suppes, R., Feinstein, D., Zanotti, S., Taiberg, L., Gurka, D., Kumar, A., Cheang, M., 2006. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit. Care Med. 34, 1589–1596. Monteiro, J., Inoue, F.M., Lobo, A.P., Sugawara, E.K., Boaretti, F.M., Tufik, S., 2015. Fast and reliable bacterial identification direct from positive blood culture using a new TFA sample preparation protocol and the Vitek® MS system. J. Microbiol. Methods 109, 157–159. Morgenthaler, N.G., Kostrzewa, M., 2015. Rapid identification of pathogens in positive blood culture of patients with sepsis: review and meta-analysis of the performance of the sepsityper kit. Int. J. Microbiol. 2015, 827416. Moussaoui, W., Jaulhac, B., Hoffmann, A.M., Ludes, B., Kostrzewa, M., Riegel, P., Prévost, G., 2010. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identifies 90% of bacteria directly from blood culture vials. Clin. Microbiol. Infect. 16, 1631–1638. Neville, S.A., Lecordier, A., Ziochos, H., Chater, M.J., Gosbell, I.B., Maley, M.W., van Hal, S.J., 2011. Utility of matrix-assisted laser desorption ionization-time of flight mass spectrometry following introduction for routine laboratory bacterial identification. J. Clin. Microbiol. 49, 2980–2984. Nicolella, C., van Loosdrecht, M.C.M., Heijnen, J.J., 2000. Wastewater treatment with particulate biofilm reactors. J. Biotechnol. 80, 1–33. Nomura, F., 2015. Proteome-based bacterial identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS): a revolutionary shift in clinical diagnostic microbiology. Biochim. Biophys. Acta 1854, 528–537. Opota, O., Croxatto, A., Prod'hom, G., Greub, G., 2015. Blood culture-based diagnosis of bacteraemia: state of the art. Clin. Microbiol. Infect. 21, 313–322. Prod'hom, G., Bizzini, A., Durussel, C., Bille, J., Greub, G., 2010. Matrix-assisted laser desorption ionization-time of flight mass spectrometry for direct bacterial identification from positive blood culture pellets. J. Clin. Microbiol. 48, 1481–1483. Saffert, R.T., Cunningham, S.A., Mandrekar, J., Patel, R., 2012. Comparison of three preparatory methods for detection of bacteremia by MALDI-TOF mass spectrometry. Diagn. Microbiol. Infect. Dis. 73, 21–26. Schubert, S., Weinert, K., Wagner, C., Gunzl, B., Wieser, A., Maier, T., Kostrzewa, M., 2011. Novel, improved sample preparation for rapid, direct identification from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. J. Mol. Diagn. 13, 701–706. Scott, J.S., Sterling, S.A., To, H, Seals, S.R., Jones, A.E., 2016. Diagnostic performance of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry in blood bacterial infections: a systematic review and meta-analysis. Infect. Dis. (Lond). 48, 530–536. Seifert, H., 2009. The clinical importance of microbiological findings in the diagnosis and management of bloodstream infections. Clin. Infect. Dis. 48, S238–S245. Sogawa, K., Watanabe, M., Sato, K., Segawa, S., Ishii, C., Miyabe, A., Murata, S., Saito, T., Nomura, F., 2011. Use of the MALDI BioTyper system with MALDI-TOF mass spectrometry for rapid identification of microorganisms. Anal. Bioanal. Chem. 400, 1905–1911. Tanner, H., Evans, J.T., Gossain, S., Hussain, A., 2017. Evaluation of three sample preparation methods for the direct identification of bacteria in positive blood cultures by MALDI-TOF. BMC Res. Notes. 10, 48.
Acknowledgments The authors express their appreciation for the contributions of members of the bacterial laboratory of Chiba University, Chiba, Japan. Declarations of interests None. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Angeletti, S., 2017. Matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology. J. Microbiol. Methods 138, 20–29. Angus, D.C., Wax, R.S., 2001. Epidemiology of sepsis: an update. Crit. Care Med. 29, S109–S116. Ashizawa, K., Murata, S., Terada, T., Ito, D., Bunya, M., Watanabe, K., Teruuchi, Y., Tsuchida, S., Mamoru, S., Nishimura, M., Matsuhita, K., Sugama, Y., Nomura, F., 2017. Applications of copolymer for rapid identification of bacteria in blood culture broths using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. J. Microbiol. Methods 139, 54–60. Buchan, B.W., Riebe, K.M., Ledeboer, N.A., 2012. Comparison of the MALDI Biotyper system using Sepsityper specimen processing to routine microbiological methods for identification of bacteria from positive blood culture bottles. J. Clin. Microbiol. 50, 346–352. Christner, M., Rohde, H., Wolters, M., Sobottka, I., Wegscheider, K., Aepfelbacher, M., 2010. Rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting. J. Clin. Microbiol. 48, 1584–1591. Clark, A.E., Kaleta, E.J., Arora, A., Wolk, D.M., 2013. Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology. Clin. Microbiol. Rev. 26, 547–603.
91