Evaluation of the Bruker MALDI Biotyper for Identification of Fastidious Gram-Negative Rods

Abstract

Matrix-assisted laser desorption ionization؊time of flight mass spectrometry (MALDI-TOF MS) has entered clinical laboratories, facilitating identification of bacteria. Here, we evaluated the MALDI Biotyper (Bruker Daltonics) for the identification of fastidious Gram-negative rods (GNR). Three sample preparation methods, direct colony transfer, direct transfer plus on-target formic acid preparation, and ethanol-formic acid extraction, were analyzed for 151 clinical isolates. Direct colony transfer applied with the manufacturer's interpretation criteria resulted in overall species and genus identification rates of 43.0% and 32.5%, respectively; 23.2% of the isolates were not identified, and two misidentifications (1.3%) were observed. The species identification rates increased to 46.4% and 53.7% for direct transfer plus formic acid preparation and ethanol-formic acid extraction, respectively. In addition, we evaluated score value cutoff alterations. The identification rates hardly increased by reducing the genus cutoff, while reducing the 2.0 species cutoff to 1.9 and to 1.8 increased the identification rates to up to 66.2% without increasing the rate of misidentifications. This study shows that fastidious GNR can reliably be identified using the MALDI Biotyper. However, the identification rates do not reach those of nonfastidious GNR such as the Enterobacteriaceae. In addition, two approaches optimizing the identification of fastidious GNR by the MALDI Biotyper were demonstrated: formic acid-based ontarget sample treatment and reductions in cutoff scores to increase the species identification rates. Fastidious Gram-negative rods (GNR) are slow-growing bacteria that belong to different genera, such as Neisseria, Pasteurella, and the HACEK organisms (Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, and Kingella). Many of these species are commensal organisms of the human or animal oral cavity. They are isolated from wound infections, e.g., after a human or animal bite, but are also associated with endocarditis, septicemia, and abscesses, particularly in immunocompromised and elderly patients (1-4).Traditionally, identification of fastidious GNR in diagnostic laboratories is based on morphological and biochemical criteria. Identification is particularly challenging and time-consuming as these bacteria usually do not grow on standard media and require supplemental nutrients and/or a CO 2 -enriched atmosphere for growth (1-3). 16S rRNA gene sequencing is widely used in addition to conventional identification methods (5-7). Matrix-assisted laser desorption ionizationϪtime of flight mass spectrometry (MALDI-TOF MS) has been suggested as an alternative to molecular methods for the identification of fastidious GNR and other bacteria that are difficult to culture (8-10).Numerous studies have compared MALDI-TOF MS-based bacterial identification with molecular or biochemical identification methods (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Most of these studies included only limited numbers of fastidious GNR ...

Direct Identification of Aerobic Bacteria by Matrix‐Assisted Laser Desorption Ionization Time‐of‐Flight Mass Spectrometry Is Accurate and Robust

“…Reduction of cutoff scores was particularly beneficial for the Gram‐positive organisms, with a 10.3% increase noted at ≥1.9 cutoff compared to 3.6% for the Gram‐negative organisms ( P = 0.0001). Of the discrepant results, 20 viridans group streptococci were misidentified as Streptococcus pneumonia , which is not surprising as similar findings have been previously reported in the literature . Thus, omission of these 20 isolates dramatically increased the species‐level identification rate of Gram‐positive organisms from 84.8% to 95.7% at a ≥1.9 cutoff.…”

“…While extraction of cellular content prior to analysis by MALDI‐TOF MS increases spectral scores and the rate of species‐level identification , it involves additional hands‐on steps, which can become laborious for the CLS performing batch setup of multiple isolates , increasing the preparation and identification time by up to fivefold per specimen . Previous studies have demonstrated that although the direct‐smear method is sufficient for Gram‐negative bacteria identification to the species level, the yield for species‐level identification of Gram‐positive is low and typically requires an extraction step . In this study, we successfully demonstrated that identification of aerobic Gram‐positive and Gram‐negative organisms could be performed accurately and efficiently by MALDI‐TOF MS and Bruker Biotyper system using the direct‐smear method alone and a decreased score of ≥1.9 for species‐level identification of both Gram‐negative and Gram‐positive organisms.…”

“…Using these parameters, studies have shown that approximately 95% of bacterial isolates in the clinical laboratory can be reliably identified to the species level . However, these cutoff scores may be considered conservative and the rate of bacterial isolates correctly identified to the species level can be increased by lowering the scoring threshold . Ford and Burnham found that all Gram‐negative isolates with a spectra score of ≥ 1.9 were correctly identified to the species level with an improvement of 6.75% in identification rate compared to the manufacturer's cutoff of ≥ 2.0.…”

Misidentification of <i>Neisseria cinerea</i> as <i>Neisseria meningitidis</i> by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS)

“…Nonetheless, there have been reports that some isolates are identified correctly by means of low Score Values, whereas some others are misidentified via a high Score Value (5-7). Lower species cutoffs that may offer an optimal balance between an increased number of correct identifications and a limited number of misidentifications have been suggested in some reports (8)(9)(10). In our case, however, N. cinerea was misidentified as N. meningitidis with the Score Value above 2.0 (i.e., 2.024).…”

High Prevalence of Fluoroquinolone-Resistant Campylobacter Bacteria in Sheep and Increased Campylobacter Counts in the Bile and Gallbladders of Sheep Medicated with Tetracycline in Feed

“…All isolates were identified to the species level. Sample preparation and analysis were done as described previously (56). Mass spectra were acquired and analyzed using a microflex LT mass spectrometer (Bruker Daltonics) in combination with research use only version of the MALDI Biotyper Compass software 4.1 and the reference database MBT 7311 MSP Library (no.…”