Mass spectrometry-based microbiological testing for blood stream infection.

BACKGROUND: The most successful application of mass spectrometry (MS) in laboratory medicine is identification (ID) of microorganisms using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in blood stream infection. We describe MALDI-TOF MS-based bacterial ID with particular emphasis on the methods so far developed to directly identify microorganisms from positive blood culture bottles with MALDI-TOF MS including our own protocols. We touch upon the increasing roles of Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) as well. MAIN BODY: Because blood culture bottles contain a variety of nonbacterial proteins that may interfere with analysis and interpretation, appropriate pretreatments are prerequisites for successful ID. Pretreatments include purification of bacterial pellets and short-term subcultures to form microcolonies prior to MALDI-TOF MS analysis. Three commercial protocols are currently available: the Sepsityper® kit (Bruker Daltonics), the Vitek MS blood culture kit (bioMerieux, Inc.), and the rapid BACpro® II kit (Nittobo Medical Co., Tokyo). Because these commercially available kits are costly and bacterial ID rates using these kits are not satisfactory, particularly for Gram-positive bacteria, various home-brew protocols have been developed: 1. Stepwise differential sedimentation of blood cells and microorganisms, 2. Combination of centrifugation and lysis procedures, 3. Lysis-vacuum filtration, and 4. Centrifugation and membrane filtration technique (CMFT). We prospectively evaluated the performance of this CMFT protocol compared with that of Sepsityper® using 170 monomicrobial positive blood cultures. Although preliminary, the performance of the CMFT was significantly better than that of Sepsityper®, particularly for Gram-positive isolates. MALDI-TOF MS-based testing of polymicrobial blood specimens, however, is still challenging. Also, its contribution to assessment of susceptibility and resistance to antibiotics is still limited. For this purpose, liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) should be more useful because this approach can identify as many as several thousand peptide sequences. CONCLUSION: MALDI-TOF MS is now an essential tool for rapid bacterial ID of pathogens that cause blood stream infection. For the purpose of assessment of susceptibility and resistance to antibiotics of the pathogens, the roles of liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) will increase in the future.

An in-house centrifugation and membrane filtration technique for identifying microorganisms from positive blood culture bottles with high identification rates using matrix-assisted laser desorption ionization-Time-of-flight mass spectrometry: A preliminary report.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most promising technologies for the identification of microbial pathogens directly from positive blood culture bottles. As blood culture bottle medium contains various nonbacterial proteins, including those derived from blood cells, pretreatment to effectively remove host cells is key for successful proteome-based identification of microorganisms. Although the Sepsityper® kit is the most widely used pretreatment protocol, its performance is not satisfactory, particularly for gram-positive isolates. We developed a new in-house protocol, the centrifugation and membrane filtration technique (CMFT), in which vacuum-filtration is coupled with differential centrifugation. We prospectively evaluated the performance of this novel method compared with that of the Sepsityper®. For gram-negative bacterial isolates, the species-level identification rates obtained with the CMFT and the Sepsityper® were comparable (98.8% vs 92.9%). By contrast, for gram-positive isolates, the performance of the CMFT was significantly better than that of the Sepsityper® (P < 0.05). Using our new protocol, 81 (95.3%) isolates were identified with a score >2.0, and 85 (100%) isolates were identified with a score >1.7, versus 46 (54.1%) and 69 (81.2%), respectively, for the Sepsityper®. These results are preliminary, but considering that this novel protocol provides notably high species-level identification rates for gram-positive isolates, it deserves assessment in a larger-scale study with a variety of platforms for MS-based identification of microorganisms.

External quality control survey on identification of microorganisms using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（ MALDI-TOF MS） is a bacterial typing tool that was approved as a medical device in 2011. However, external accuracy control examination of bacterial typing using mass spectrometry is still only performed on a small scale. In this study, E. faecium and S. maltophilia were selected and tested according to established procedures using Score Values at 228 institutions. The Score Values for MALDI Biotyper were 2.43±0.08 for E. faecium and 2.38±0.08 for S. maltophilia; and those for VITEK MS/PRIME were 99.9±0.0 for E. faecium and S. maltophilia. These results suggest that it is useful to evaluate external accuracy control with Score Values using the procedures we have developed.

Evaluation of a novel sample preparation method for identifying Aspergillus fumigatus using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: Combining Yatalase and silica beads treatment.

Aspergillus spp. belong to filamentous fungi and sometimes cause invasive aspergillosis which has high mortality. Filamentous fungi are generally identified morphologically. However, morphologic identification is time consuming and requires advanced skills. It is difficult to train technicians and ensure a high level of quality. Therefore, an identification technique that is both accurate and relatively easy to learn is needed. In the present study, we focused on the effects of Yatalase and silica beads, which enable the efficient extraction of proteins via cell wall disruption of Aspergillus spp., and aimed to establish a novel sample preparation method using Yatalase and silica beads to enhance the efficiency of Aspergillus spp. identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The sample preparation method using the combination of Yatalase and silica beads showed higher accuracy for the identification of Aspergillus spp. compared with Yatalase or silica beads alone. The Yatalase/silica beads method also resulted in significantly higher identification scores compared with the conventional method for the identification of Aspergillus fumigatus (n = 33). These findings indicate that our novel Yatalase/silica beads method provides more reliable identification of A. fumigatus than does the conventional method.

Extension of bacterial rDNA sequencing for simultaneous methylation detection and its application in microflora analysis.

Although polymerase chain reaction (PCR) amplification and sequencing of the bacterial 16S rDNA region has numerous scientific applications, it does not provide DNA methylation information. Herein, we propose a simple extension for bisulfite sequencing to investigate 5-methylcytosine residues in the bacterial 16S rDNA region from clinical isolates or flora. Multiple displacement amplification without DNA denaturation was used to preferentially pre-amplify single-stranded bacterial DNA after bisulfite conversion. Following the pre-amplification, the 16S rDNA region was analyzed using nested bisulfite PCR and sequencing, enabling the simultaneous identification of DNA methylation status and sequence data. We used this approach (termed sm16S rDNA PCR/sequencing) to identify novel methylation sites and a methyltransferase (M. MmnI) in Morganella morganii and different methylation motifs among Enterococcus faecalis strains from small volumes of clinical specimens. Further, our analysis suggested that M. MmnI may be correlated to erythromycin resistance. Thus, sm16S rDNA PCR/sequencing is a useful extension method for analyzing the DNA methylation of 16S rDNA regions in a microflora, providing additional information not provided by conventional PCR. Given the relationship between DNA methylation status and drug resistance in bacteria, we believe this technique can be effectively applied in clinical sample testing.

Rapid identification of microorganisms by mass spectrometry: improved performance by incorporation of in-house spectral data into a commercial database.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used as a microbial diagnostic method for species identification of pathogens. However, MALDI-TOF identification of bacteria at the species level remains unsatisfactory, with the major problem being an incomplete database that still needs refinement and expansion. Augmentation of the original MALDI BioTyper 2.0 (Bruker) database by incorporating mass spectra obtained in-house from clinical isolates may increase the identification rate at the species level. We conducted a prospective study to assess whether the augmented database can improve the performance of MALDI-TOF MS for routine identification of species. Cluster analyses revealed distinct differences in MS spectral profiles of clinical isolates obtained in our hospital and those of ATCC strains in the Bruker database. In the first part of the study, which was performed over 3 weeks, 259 bacterial isolates were subjected to analysis by MALDI-TOF MS, and MS spectra of 229 successfully identified isolates (49 species) were incorporated into the original database to give the augmented Bruker-Chiba database. In a second separate analysis, the concordance of identification of 498 clinical isolates of the 49 species with conventional methods was 87.1% (434/498) with the commercial Bruker database and 98.0% (488/498) using the Bruker-Chiba database. These results indicate that refinement of a commercial database can be achieved relatively easy and effectively by incorporating MS spectra of clinical isolates obtained in a clinical laboratory.

Evaluation of Serotyping of Environmental and Clinical Isolates of Legionella pneumophila using MALDI-TOF MS.

Legionella pneumophila (L. pneumophila) is responsible for most Legionnaire's disease cases diagnosed worldwide. The species includes 16 serogroups, but most Legionnaire's disease cases (85.7% in Europe, 87.0% in Japan) are caused by L. pneumophila serogroup 1. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be used to identify the L. pneumophila serogroup. In this study, we compared three sample preparation methods that are compatible with MALDI-TOF MS: the direct colony transfer method (DCTM), on-target extraction method (OTEM), and in-tube extraction method (ITEM). The aim was to improve the low identification rates for L. pneumophila, and establish and validate a simple, rapid and robust MALDI-TOF MS-based method for routine use in microbiological laboratories for assignment of L. pneumophila isolates to serogroups and identification of reliable peak biomarkers. Using ITEM, 100.0% (29/29) of hot spring water samples and clinical isolates were correctly identified at the species level. Augmented reference spectra correctly identified all 29 strains at the species level and 29 isolates at the serogroup level, displaying sensitivity, specificity and accuracy of 100.0% for serogroup assignment. MALDI-TOF MS is a relatively inexpensive method for assignment of L. pneumophila serogroups that can serve as a first-line tool for rapid prospective typing.

Examination of Conditions for External Quality Control in Identification of Microorganisms using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was approved for medical use in 2011 and is currently used as a rapid, accurate and lowcost technique for bacterial identification. External quality control for medical analysis is monitored using tests of the Japanese Association of Medical Technologists and Prefectural Association of Clinical Laboratory Technologists and through user surveys of reagent and equipment manufacturers. However, external quality control of bacterial typing using MS is not performed. Therefore, we examined procedures for evaluating quality control of bacterial typing using an identification reliability index at 38 facilities.

Use of the MALDI BioTyper system with MALDI-TOF mass spectrometry for rapid identification of microorganisms.

In a clinical diagnosis microbiology laboratory, the current method of identifying bacterial isolates is based mainly on phenotypic characteristics, for example growth pattern on different media, colony morphology, Gram stain, and various biochemical reactions. These techniques collectively enable great accuracy in identifying most bacterial isolates, but are costly and time-consuming. In our clinical microbiology laboratory, we prospectively assessed the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify bacterial strains that were routinely isolated from clinical samples. Bacterial colonies obtained from a total of 468 strains of 92 bacterial species isolated at the Department of Clinical Laboratory at Chiba University were directly placed on target MALDI plates followed by addition of CHCA matrix solution. The plates were then subjected to MALDI-TOF MS measurement and the microorganisms were identified by pattern matching with the libraries in the BioTyper 2.0 software. Identification success at the species and genus levels was 91.7% (429/468) and 97.0% (454/468), respectively. MALDI-TOF MS is a rapid, simple, and high-throughput proteomic technique for identification of a variety of bacterial species. Because colony-to-colony differences and effects of culture duration on the results are minimal, it can be implemented in a conventional laboratory setting. Although for some pathogens, preanalytical processes should be refined, and the current database should be improved to obtain more accurate results, the MALDI-TOF MS based method performs, in general, as well as conventional methods and is a promising technology in clinical laboratories.

Effect of humidity during sample preparation on bacterial identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a highly reliable and efficient technology for the identification of microbial pathogens. We previously found that 40% humidity was the optimal condition for the preparation of samples (co-crystallization of the sample and matrix) for serum peptidomic analysis via MALDI-TOF MS profiling. This optimum temperature was applied to obtain the highest reproducibility and throughput and greatest number of peaks. We therefore hypothesized that humidity control was also essential for MALDI-TOF MS bacterial identification. In this study, we constructed a simple sample preparation device that enables humidity control and used it for co-crystallization of the sample and matrix. Identification scores for five Gram-negative bacteria and six Gram-positive bacteria were determined using the MALDI BioTyper® system at three humidity ranges (10-20%, 30-40%, and 50-60%). As a result, higher identification scores were obtained at 30-40% humidity than at 10-20% or 50-60% humidity. At 30-40% humidity, 517/550 (94.0%) isolates scored greater than 2.0, indicating the success of species-level identification. Similarly, 537/550 (97.6%) isolates scored greater than 1.7, indicating the success of genus-level identification. Thus, 30-40% humidity generated optimal MALDI-TOF MS identification scores and the highest percentage of correct identifications. These results could lead to further improvements in the accuracy of MALDI-TOF MS bacterial identification.

Examination of conditions for regular internal quality control in identification of microorganisms using MALDI-TOF MS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was approved for medical use in 2011, and is currently used as a rapid, accurate and low-cost technique for bacterial identification. Microbiological testing and internal accuracy control in Japan are mainly implemented in accordance with the standards of the Clinical and Laboratory Standards Institute (CLSI). However, few facilities perform internal accuracy control of bacterial identification by MALDI-TOF MS. Therefore, we examined the procedures for internal accuracy control of bacterial identification using MALDI-TOF MS in daily work at clinical laboratories in the seven hospitals.

An improved in-house lysis-filtration protocol for bacterial identification from positive blood culture bottles with high identification rates by MALDI-TOF MS.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is now a well-established method for identification of microorganisms from positive blood cultures. Pretreatments to effectively remove non-bacterial proteins are a prerequisite for successful identification, and a variety of 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 developed a new, in-house lysis-filtration protocol and prospectively evaluated its performance compared to the Sepsityper kit. The in-house protocol consists of three simple steps: lysis by ammonium chloride, aspiration with a syringe fitted with a 0.45-µm membrane, and centrifugation to collect microbes. The novel protocol requires only 20 min. Performance of the in-house protocol was evaluated using a total of 117 monomicrobial cases of positive blood culture. Medium from blood culture bottles was pretreated by the in-house protocol or the commercial kit, and isolated cells were subjected to direct identification by mass spectrometry fingerprinting in parallel with conventional subculturing for reference identification. The overall MALDI-TOF MS-based identification rates with score > 1.7 and > 2.0 obtained using the in-house protocol were 99.2% and 85.5%, respectively, whereas those obtained using the Sepsityper Kit were 85.4% and 61.5%, respectively. For Gram-positive cases, the in-house protocol yielded scores >1.7 and > 2.0 at 98.5% and 76.1%, respectively, whereas the commercial kit yielded these scores at 76.1% and 43.3%, respectively. Although these are preliminary results, these values suggest that this easy lysis-filtration protocol deserves assessment in a larger-scale test.

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.

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 non-bacterial 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.

Rapid Discrimination between Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus Using MALDI-TOF Mass Spectrometry.

　Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens responsible for nosocomial infections. The presence of MRSA in a hospital is detrimental to patients and to hospital management. Thus, rapid identification of MRSA is needed. Here, we report on a prospective method to rapidly discriminate of MSSA from MRSA using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and support vector machine (SVM) analysis in 160 clinical isolates of S. aureus. The predictive model was tested using 100 S. aureus isolates (50 MSSA and 50 MRSA). The identification rates were 90.0% for MSSA and 87.5% for MRSA in a 10-fold cross-validation SVM. In blind test sets, 60 S. aureus isolates (30 MSSA and 30 MRSA) were correctly classified, with identification rates of 93.3% for MSSA and 86.7% for MRSA. The method proposed in this study using the predictive model enables detection of one colony in 5 minutes, and thus is useful at clinical sites at which rapid discrimination of MRSA from MSSA is required.

Applications of copolymer for rapid identification of bacteria in blood culture broths using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be used to identify pathogens in blood culture samples. However, sample pretreatment is needed for direct identification of microbes in blood culture bottles. Conventional protocols are complex and time-consuming. Therefore, in this study, we developed a method for collecting bacteria using polyallylamine-polystyrene copolymer for application in wastewater treatment technology. Using representative bacterial species Escherichia coli and Staphylococcus capitis, we found that polyallylamine-polystyrene can form visible aggregates with bacteria, which can be identified using MALDI-TOF MS. The processing time of our protocol was as short as 15min. Hemoglobin interference in MALDI spectra analysis was significantly decreased in our method compared with the conventional method. In a preliminary experiment, we evaluated the use of our protocol to identify clinical isolates from blood culture bottles. MALDI-TOF MS-based identification of 17 strains from five bacterial species (E. coli, Klebsiella pneumoniae, Enterococcus faecalis, S. aureus, and S. capitis) collected by our protocol was satisfactory. Prospective large-scale studies are needed to further evaluate the clinical application of this novel and simple method of collecting bacteria in blood culture bottles.