Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis of Gram-Positive, Catalase-Negative Cocci Not Belonging to the Streptococcus or Enterococcus Genus and Benefits of Database Extension

Christensen, Jens Jørgen; Dargis, Rimtas; Hammer, Monja; Justesen, Ulrik Stenz; Nielsen, Xiaohui Chen; Kemp, Michael

doi:10.1128/jcm.06339-11

Cited by 70 publications

(51 citation statements)

References 10 publications

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“…However, the extraction method significantly increases the processing time (14) and seems not suitable for high-throughput applications as required in clinical diagnostic laboratories. An adapted direct transfer method with on-target formic acid treatment leading to in situ cell lysis promises an increased identification rate without the time-consuming extraction method (10,12). To date, few studies have systematically compared different sample preparation methods for the identification of bacteria with MALDI-TOF MS (11,15).…”

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confidence: 99%

“…MALDI-TOF MS promises to be a fast, reliable, and cost-effective alternative to currently used methods (4,6,8). Apart from the species coverage of the reference database and the identification algorithm of the software (9,10), the sample preparation procedure seems to be critical for the performance of the system (11)(12)(13). Most studies evaluating the Bruker MALDI-TOF MS system used a direct transfer protocol, measuring whole bacterial cells prepared directly from freshly grown colonies.…”

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confidence: 99%

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Identification of Gram-Positive Cocci by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Comparison of Different Preparation Methods and Implementation of a Practical Algorithm for Routine Diagnostics

et al. 2013

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This study compared three sample preparation methods (direct transfer, the direct transfer-formic acid method with on-target formic acid treatment, and ethanol-formic acid extraction) for the identification of Gram-positive cocci with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A total of 156 Gram-positive cocci representing the clinically most important genera, Aerococcus, Enterococcus, Staphylococcus, and Streptococcus, as well as more rare genera, such as Gemella and Granulicatella, were analyzed using a Bruker MALDI Biotyper. The rate of correct genus-level identifications was approximately 99% for all three sample preparation methods. The species identification rate was significantly higher for the direct transfer-formic acid method and ethanol-formic acid extraction (both 77.6%) than for direct transfer (64.1%). Using direct transfer-formic acid compared to direct transfer, the total time to result was increased by 22.6%, 16.4%, and 8.5% analyzing 12, 48, and 96 samples per run, respectively. In a subsequent prospective study, 1,619 clinical isolates of Gram-positive cocci were analyzed under routine conditions by MALDI-TOF MS, using the direct transfer-formic acid preparation, and by conventional biochemical methods. For 95.6% of the isolates, a congruence between conventional and MALDI-TOF MS identification was observed. Two major limitations were found using MALDI-TOF MS: the differentiation of members of the Streptococcus mitis group and the identification of Streptococcus dysgalactiae. The Bruker MALDI Biotyper system using the direct transfer-formic acid sample preparation method was shown to be a highly reliable tool for the identification of Gram-positive cocci. We here suggest a practical algorithm for the clinical laboratory combining MALDI-TOF MS with phenotypic and molecular methods.

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Identification of Gram-Positive Cocci by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Comparison of Different Preparation Methods and Implementation of a Practical Algorithm for Routine Diagnostics

et al. 2013

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“…Therefore, it is likely that SSBA cultures can be incorrectly, and unknowingly, identified by the software as a species-level match to a BSL-2 near-neighborspecies profile. The importance of using the Biotyper SR library has been described (18), and a comparison of experimental sample mass spectral profiles to a database supplemented with locally generated MSPs provides higher and more-confident match scores than those with the standard Biotyper database (Table 1) (6,19,20). In this method verification, we employed a locally generated custom MSP database, which contributes to the development of a high-quality curated database for high-consequence SSBA pathogens using the Bruker guidelines for library creation (21).…”

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Verification of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Diagnostic Identification of High-Consequence Bacterial Pathogens

2016

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bWe examined the utility of a single matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry method for the identification of security-sensitive biological agents (risk group 3 bacterial pathogens). The goal was 2-fold: to verify a method for inclusion into our scope of accreditation, and to assess the biological safety of extractions. We developed our sample flow to include a tube-based chemical extraction, followed by filtration, before processing on MALDI-TOF MS instruments in a containment level 2 laboratory. Matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) is a rapid, robust, and cost-effective technology, which is emerging as a routine bacterial identification method in clinical diagnostic laboratories (1-3). Despite its utility, there are concerns with the safety of sample processing and the quality of spectral databases for the identification of security-sensitive biological agents (SSBAs). As our laboratory predominately works with SSBA bacterial pathogens (the risk group 3 [RG-3] pathogens: Francisella tularensis, Yersinia pestis, Bacillus anthracis, Brucella species, and Burkholderia pseudomallei), our goal was to verify the usefulness and safety of a MALDI-TOF MS method for inclusion into our scope of accreditation (ISO 17025). We chose to pursue a full-tube-based protein extraction method involving cellular disruption and filtration, rather than the direct-colony plate (smear) method (4, 5), in an effort to mitigate the risk of viable organisms.Over a period of 1 year, 146 bacterial samples (including 57 SSBA samples; Table 1) were processed as previously described (6). The bacteria were previously identified by standard genotypic and phenotypic methods. In brief, all cultures were chemically extracted in a microcentrifuge tube using 70% ethanol, 70% formic acid, and acetonitrile, and all SSBA bacteria were extracted in a biosafety level 3 (BSL-3) laboratory. SSBA extracts from sporeforming bacteria were filtered through a 0.1 M microcentrifuge filtration unit (EMD Millipore, Etobicoke, Ontario, Canada), as recommended by Dauphin and Bowen (7). The viability of the extract was performed by inoculating 10% of the extract onto both sheep's blood agar (SBA) (or cysteine heart agar [CHA] for F. tularensis) and into 2 ml of tryptic soy broth (TSB), and incubating at 35°C and 5% CO 2 for 48 h. After 48 h, the TSB was further subcultured to SBA or CHA and monitored for an additional 72 h. Any bacterial growth was tested with agent-specific assays to rule out the SSBA, indicating a contaminant. After a lack of growth was confirmed, extracts were removed from the BSL-3 laboratory, and 1.5 l of the extract was applied (at a minimum of four replicate spots) to an MSP-96 MALDI target plate (Bruker Daltonics, East Milton, Ontario, Canada), with an overlay of 1.5 l of ␣-cyano-4-hydroxycinnamic acid (CHCA) matrix solution (50% acetonitrile and 2.5% trifluoroacetic acid). Mass spectra were acquired with the FlexControl software (version 3.0...

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“…Aerococci are easily misidentified as streptococci, enterococci, or staphylococci and correct identification can be achieved with genetic methods [3] or more conveniently through matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) [4,5].…”

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confidence: 99%

Clinical and microbiological features of infective endocarditis caused by aerococci

et al. 2015

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Aerococci are increasingly recognized as causative agents in human infective endocarditis (IE). Information on the clinical presentation and the prognosis of IE caused by aerococci is limited. We employed the Swedish Registry of Infective Endocarditis (SRIE) to identify cases of aerococcal IE. The medical records were analysed and comparisons with cases of IE caused by other pathogens reported to the SRIE were made. Available bacterial isolates were reanalysed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) and the aetiology was confirmed to be Aerococcus urinae in fourteen cases and Aerococcus sanguinicola in two cases. The bacteria were sensitive to penicillin and Etest-based synergy testing and time-kill experiments suggested synergy between penicillin and gentamicin towards some isolates. The patients had a median age of 79 years, which was significantly higher than that of patients with IE caused by streptococci or Staphylococcus aureus. Most patients with IE caused by aerococci had underlying urinary tract diseases and many presented with symptoms suggesting a urinary tract focus of the infection. Despite the fact that many patients with aerococcal IE presented with severe sepsis, ICU treatment was needed only in one patient and there was no fatality. Valve exchange surgery was needed in four of sixteen patients with aerococcal IE and embolization was seen in three patients. This report is by far the largest on aerococcal IE and suggests that the prognosis of aerococcal IE is relatively favourable despite the fact that the patients are old and have significant comorbidities.

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Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis of Gram-Positive, Catalase-Negative Cocci Not Belonging to the Streptococcus or Enterococcus Genus and Benefits of Database Extension

Cited by 70 publications

References 10 publications

Identification of Gram-Positive Cocci by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Comparison of Different Preparation Methods and Implementation of a Practical Algorithm for Routine Diagnostics

Identification of Gram-Positive Cocci by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Comparison of Different Preparation Methods and Implementation of a Practical Algorithm for Routine Diagnostics

Verification of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Diagnostic Identification of High-Consequence Bacterial Pathogens

Clinical and microbiological features of infective endocarditis caused by aerococci

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