Bruker MS Research Articles

Особливості діагностики та лікування жінок з синдромом полікістозних яєчників в умовах підвищеного рівня експонування нікелем

Abstract. Polycystic ovary syndrome (PCOS) refers to endocrinopathies that women of reproductive age could have. These disorders can be caused by both endogenous (endocrine disorders, chronic infections, stress, etc.) and exogenous factors (environmental, smoking, etc.). Aim. Determination of serum nickel values in women with polycystic ovary syndrome and investigation of the mechanisms underlying the toxic effects of nickel on the reproductive system. Explore potential sources of nickel exposure and develop recommendations for the prevention and treatment of PCOS caused by increased levels of nickel exposure. Materials and Methods. The research was conducted on the bases of State Institution “O.M. Lukianova Institute of Pediatrics, Obstetrics and Gynecology of NAMS of Ukraine” 52 women who were diagnosed with PCOS were selected for the study. The control group consisted of 38 women of reproductive age without reproductive system pathology. Determination of serum nickel values in women with PCOS was performed using a Bruker MS 820 inductively coupled plasma mass spectrometer (Australia) using special ICP-MS Expert software. The study of indicators of environmental pollution with nickel was carried out by processing data published in open sources of information. Results. Women with PCOS showed elevated serum nickel values (median ‒ 0.036 mg/l, 95-percentile ‒ 0.194 mg/l), which significantly exceeded the control group (median ‒ 0.001 mg/l, 95-percentile ‒ 0.097 mg/l, p=0.0012). The study of exposure possible sources showed that elevated levels of nickel were found in drinking water from surface water sources. Contamination of surface waters and soils used in agriculture was also detected. The authors propose a set of measures for the prevention and treatment of complications caused by nickel exposure. Conclusions. Nickel exposure occurs mainly by alimentary and inhalation. When exposed to nickel, there is a violation of the function of the endocrine system, activation of inflammatory processes and oxidative stress. In women with PCOS with an increased level of nickel exposure, it is necessary to carry out a set of measures aimed at reducing the level of exposure, accelerating nickel excretion and treating complications that have occurred. Keywords: polycystic ovary syndrome, nickel, exposure, principles of prevention and treatment.

A-240 Rapid and Reliable Identification of Microorganisms from Early Growth of Positive Blood Cultures Using MALDI-TOF Mass Spectrometry

Abstract Background Rapid identification of organisms causing bloodstream infections is critical for treatment. Traditional workflows rely on 18–24 h growth of isolated colonies. Our objective was to evaluate identification of early growth using Bruker and VITEK® MS PRIME MALDI-TOF systems. Materials Positive blood cultures (BACT/ALERT® FA/FN Plus; bioMérieux VIRTUO®) were included if bottles were positive <8 h and had time-to-positivity of <40 h. Broths were sub-cultured to blood agar and incubated at 35°C/5% CO2. MALDI-TOF identification of 6–8 h growth (short incubation, SI) and 18–24 h growth (standard incubation, SDI) was performed by a high (HEU, >1 year) and low (LEU, <1 month) experience user. Each identification was performed using standard set-up for Bruker and VITEK MS PRIME, and target set-up using VITEK PICKMETM pen. Identification level and time-to-result (TTR) were measured and compared using Fisher’s exact and t-test. Results 53 organisms (36 Gram-positive, 15 Gram-negative, 2 yeast) were isolated from 50 blood cultures. The HEU achieved species-level identification for 87% on VITEK MS PRIME using routine set-up and PICKME after SI vs 74% on Bruker, while 66% and 70% of organisms were identified on VITEK MS PRIME vs 75% on Bruker by the LEU (Table). After SDI, ≥94%(HEU) and ≥87%(LEU) of organisms were identified, with no differences noted by set-up or user experience (P > 0.05). Species-level identification after SI was higher for Gram-negative (100%) vs Gram-positive (67–86%) organisms for the HEU. PICKME pen usage increased identifications to 100% from 67% for Gram-negatives for the LEU. For mixed cultures (n = 3), only 1 of 2 organisms was identified after SI. TTR was significantly longer(P < 0.05) for SI vs SDI and Bruker vs VITEK MS PRIME for the HEU. Conclusions Identification of microorganisms after short incubation on solid media achieves early, reliable identification for a majority of positive blood cultures without need for additional hands-on time or extraction.

A 3D-Printed Offline Nano-ESI Source for Bruker MS Instruments.

Nanoelectrospray ionization (nano-ESI) is a highly efficient and a widely used technique for the ionization of minute amounts of analyte. Offline nano-ESI sources are convenient for the direct infusion of complex mixtures that suffer from high matrix content and are crucial for the native mass spectrometric analysis of proteins. For Bruker instruments, no such source is readily available. Here we close this gap and present a 3D-printable nano-ESI source for Bruker instruments, which can be assembled by anyone with access to 3D printers. The source can be fitted to any Bruker mass spectrometer with an ionBooster ESI source and only requires minor, reversible changes to the original Bruker hardware. The general utility was demonstrated by recording high-resolution MS spectra of small molecules, intact proteins, as well as complex biological samples in negative and positive ion mode on two different Bruker instruments.

Clinical Origin and Species Distribution of Fusarium spp. Isolates Identified by Molecular Sequencing and Mass Spectrometry: A European Multicenter Hospital Prospective Study.

Fusarium spp. are widespread environmental fungi as well as pathogens that can affect plants, animals and humans. Yet the epidemiology of human fusariosis is still cloudy due to the rapidly evolving taxonomy. The Mass Spectrometry Identification database (MSI) has been developed since 2017 in order to allow a fast, accurate and free-access identification of fungi by matrix-assisted laser desorption ionization—time of flight (MALDI-TOF) mass spectrometry. Taking advantage of the MSI database user network, we aim to study the species distribution of Fusarium spp. isolates in an international multicenter prospective study. This study also allowed the assessment of the abilities of miscellaneous techniques to identify Fusarium isolates at the species level. The identification was performed by PCR-sequencing and phylogenic-tree approach. Both methods are used as gold standard for the evaluation of mass spectrometry. Identification at the species complex was satisfactory for all the tested methods. However, identification at the species level was more challenging and only 32% of the isolates were correctly identified with the National Center for Biotechnology Information (NCBI) DNA database, 20% with the Bruker MS database and 43% with the two MSI databases. Improvement of the mass spectrometry database is still needed to enable precise identification at the species level of any Fusarium isolates encountered either in human pathology or in the environment.

Comparison of traditional and commercial kefir microorganism compositions and inhibitory effects on certain pathogens

ABSTRACT The aim of this study was to compare traditional and commercial kefir products and their microbiological compositions along with their inhibitory activities against various pathogens. Different media and inhibitory conditions were used for identification of microbiological groups. Each microbiological group was subjected to gram and fluorescence staining. Biochemical identification was performed according to gram staining. Vitek 2 Compact system was used for biochemical identification. Identification cards; Gram-negative (GN), Gram-positive (GP), Anaerobe and Corynebacterium (ANC), Neisseria-Haemophilus (NH), Yeas-mold (YM) were selected according to gram staining. Bruker MS (16 s rRNA) was used for microorganism identification. The inhibitory effect onsome pathogens were examined. A diverse spectrum of bacterial genera were identified in kefir samples including: Lactobacillus (six species), Lactococcus (one species), Saccharomyces (one species) Streptococcus (one species), Bifidobacterium (two species). While traditional kefir had a greater inhibitory effect on Enterobacter cloacae and Escherichia coli pathogens than commercial kefir, commercial kefir had a greater inhibitory effect on Enterococcus faecalis pathogen than traditional kefir. In general, traditional kefir had most effective inhibitory effect on pathogens.

NaOH-HCl neutralized urine preparation for direct testing of uropathogens by Bruker MS.

We evaluated the analytical performance of a NaOH-HCl neutralization protocol for identifying uropathogens directly from urine samples by a Bruker MS system (Bruker Daltonics, German) and sought to establish a protocol for integrating the method with conventional screening. Among all urine samples requested for Gram staining, UF-1000i, and urine cultures by physicians, we selected samples that were positive by both Gram staining and UF-1000i testing. Urine was prepared by neutralization using 0.01M NaOH-HCl, and the samples were processed by Bruker MS. For the low detection limit, one strain each of Escherichia coli and Enterococcus faecium was inoculated in sterile deionized water and sterile urine specimens at sequential dilutions. In a total of 1270 urine samples, 125 samples (9.8%) were positive by both Gram staining and UF-1000i. Of 94 samples showing a single morphotype on agar plates, 82 samples had colony counts ≥105 CFU/mL and most uropathogens (95.1%, 78 of 82) had UF-1000i counts ≥106 bacteria/mL. Among them, Bruker MS correctly identified 86.6% (71/82) of all isolates, 89.2% (66/74) of Gram-negative bacteria (GNB), and 62.5% (5/8) of Gram-positive cocci (GPC) with higher average scores for GPC (mean score 2.013) and GNB (mean score 2.110). Bruker MS with urine preparation by NaOH-HCl neutralization provides a simple, cost-effective, and accurate method for identifying uropathogens directly from urine. Using Bruker MS when single morphotype Gram staining and the UF-1000i count as ≥106 bacteria/mL may improve the efficiency of bacterial identification in routine practice.

Identification and Antifungal Susceptibility Profiles of Candida nivariensis and Candida bracarensis in a Multi-Center Chinese Collection of Yeasts.

Candida nivariensis and C. bracarensis are two emerging cryptic species within the C. glabrata complex. Thirteen of these isolates from 10 hospitals in China were studied for their species identification and antifungal susceptibilities. Phenotypic and molecular [rDNA ITS sequencing, D1/D2 sequencing and ITS sequencer-based capillary gel electrophoresis (SCGE)] and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS identification methods were compared for their performance in species identification. Twelve of 13 (92.3%) isolates were identified as C. nivariensis and one as C. bracarensis using ITS sequencing as the reference method. Results obtained by D1/D2 sequencing and ITS SCGE were concordant with ITS sequencing results for all (100%) isolates. SCGE was able to subtype 12 C. nivariensis into four ITS SCGE length types. All isolates failed to be identified by the Vitek MALDI-TOF MS system (bioMérieux), whilst the Bruker MS system (Bruker Daltoniks) correctly identified all C. nivariensis isolates but using a lowered (≥1.700) cut-off score for species assignment; the C. bracarensis isolate was identified but with score <1.700. The Vitek 2 Compact system could not identify 11 C. nivariensis and one C. bracarensis isolate and misidentified the remaining C. nivarensis strain as “C. glabrata.” All isolates were susceptible-dose dependent to fluconazole [minimum inhibitory concentration (MIC) range 0.5–4 μg/mL] and were classed as susceptible to echinocandins (MICs ≤ 0.06 μg/mL). All 13 isolates had low MICs for other azoles (MICs ≤ 0.5 μg/mL), amphotericin B (MICs ≤ 2 μg/mL) and 5-flucytosine (MICs ≤ 0.25 μg/mL). Our results reinforce the need for molecular differentiation of species of C. nivarensis and C. bracarensis. The performance of MALDI-TOF may be improved by adding mass spectral profiles (MSPs) into the current databases. The antifungal susceptibility profile of isolates should be monitored.

Evaluation of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system Clin-TOF-II MS in identification of gram-negative bacteria

Objective To evaluate the performance of domestic matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system Clin-TOF-Ⅱ MS with BioExplorer V2.3 database (Clin-TOF MS system) on gram-negative bacteria identification. Methods This was a methodological comparison study. A total of 1 025 gram-negative strains of 32 genus, 56 species or species complex were included in this study from 1999 to 2000 and 2014 to 2016 in Peking Union Medical College Hospital.The Bruker Biotyper MS system (Bruker MS system), Bruker Autoflex Speed with Biotyper v3.1 database were used as control. Identification by both MALDI-TOF MS systems were parallel conducted by direct smear method. The 16S rDNA sequencing based was performed when either MALDI-TOF MS system gave unbelievable result or results from two systems were not consistent. Results Amongst the isolates studied, 98.05% (1 005/1 025) was correctly identified to species or species complex level by Clin-TOF MS system. Comparatively, 99.22% (1 017/1 025) was correctly identified by Bruker MS system. There were 17 isolates just identified to genus level and 2 isolates were no identification by Clin-TOF MS system, meanwhile 1 Pseudomonas monteilii misidentified as P. putida. There were only two 2 isolates identified to genus level and 3 isolates were no identification by Bruker MS system. But it misidentified all 3 Aeromonas hydrophila (2 isolates as A. caviae and 1 isolate as A. media). It′s noted that both MS systems identified 1 Chryseobacterium gleum and 1 C. bernardetii to genus level. Conclusion The capability of domestic Clin-TOF MS system was good on gram-negative bacteria.(Chin J Lab Med, 2017, 40: 41-45) Key words: Gram-negative bacteria; Spectrometry, mass, matrix-assisted laser desorption-ionization

Feto-Maternal Trafficking of Exosomes in Murine Pregnancy Models.

Timing and initiation of labor are well-orchestrated by signals communicated between the fetal and maternal compartments; however, how these signals are communicated is not completely understood. Fetal exosomes, intercellular signaling vesicles, may play a key role in the process. The objective of this study was to evaluate exosome trafficking in vivo from fetal to maternal compartments. Pregnant CD-1 mice were intra-amniotically injected on gestational day 16 and 17 with exosomes isolated from primary human amnion epithelial cells fluorescently labeled with the lipophilic dye 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR). All our analyses were performed on samples collected on Day 18. After 24 h, mice were imaged using Bruker MS FX PRO In vivo Imager and tissues were collected. In vivo imaging of mouse showed fluorescence in the uterus, on the exosome-injected side whereas the uterine tissues from the uninjected side and saline and dye alone injected animals remained negative. Histological analysis of placenta showed exosome migration from the fetal to the maternal side of the placenta. Fluorescence released from exosomes was seen in maternal blood samples as well as in maternal uterus and kidneys. This study demonstrates that exosomal cargo can be carried through systemic route from the fetal to the maternal side of the uterine tissues during pregnancy, supporting the idea that fetal signals can be delivered via exosomes.

Identification and Antifungal Susceptibility Profiles of Candida haemulonii Species Complex Clinical Isolates from a Multicenter Study in China

Candida haemulonii complex (Candida haemulonii, Candida haemulonii var. vulnera, and Candida duobushaemulonii) consists of emerging pathogens. Thirty-one isolates from 14 hospitals in China were studied for their species classification and antifungal susceptibilities. Performances of molecular (i.e., ribosomal DNA [rDNA] internal transcribed spacer [ITS] sequencing, D1/D2 sequencing, and ITS sequencer-based capillary gel electrophoresis [SCGE]) and phenotypic identification methods in species identification were compared. Twenty-six (83.9%) of 31 isolates were identified as C. haemulonii and 5 isolates were identified as C. duobushaemulonii by ITS sequencing as the reference method; results obtained by D1/D2 sequencing and ITS SCGE were concordant with those obtained by ITS sequencing for all (100%) of the isolates. All 31 isolates were identified as C. haemulonii by the Vitek matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system (bioMérieux, France), whereas the Bruker MS system (Bruker Daltoniks, Germany) correctly provided species identification for 77.4% and 100% of isolates using cutoff scores for species of ≥2.0 and ≥1.70, respectively. The Vitek 2 compact (bioMérieux) only identified 9 (29%) of 31 isolates. All isolates showed high MICs for amphotericin B (range, 2 to >8 μg/ml) and fluconazole (≥128 μg/ml) but low MICs (≤0.5 μg/ml) for the echinocandins. Our results reinforce the need for MALDI-TOF MS and/or molecular differentiation of species within the C. haemulonii complex. The multiresistant antifungal susceptibility profile of these isolates represents a challenge to therapy.

Detailed MALDI-TOF Mass spectrometry analysis for the discrimination of Candida haemulonii complex species

Introduction Candida haemulonii is now considered to be a complex of two species and one variety : C. haemulonii , Candida duobushaemulonii and the variety Candida haemulonii var. vulnera . The correct identification (ID) of these species is clinically relevant, since resistance to azole derivatives has been reported and amphotericin B has poor in vitro activity against C. duobushaemulonii isolates. Initial evaluation using MALDI-TOF MS for the ID of the C. haemulonii complex species provided promising results, but the discrimination of C. haemulonii from the variety vulnera was problematic, and the performance of the VITEK MS remains unevaluated. Objectives Evaluation of the MALDI-TOF MS performance for the ID of C. haemulonii species ID with two platforms and its’ databases and softwares. Methods 14 C. haemulonii sensu stricto , 9 C. haemulonii var. vulnera and 10 C. duobushaemulonii from hospitals belonging to the Medical school from the University of Sao Paulo were analyzed (ID by ITS1 sequence analysis). A set of reference strains from the CBS-KNAW collection was also included : C. haemulonii CBS5149 T, C. duobushaemulonii CBS7798 T, C. duobushaemulonii CBS7799, and for specificity control, the close related species Candida pseudohaemulonii (CBS10004 and CBS12370). The isolates/strains were cultured on Sabouraud plates and incubated for 48 h at 30 °C before MALDI-TOF MS analysis. Protein extraction protocol with ethanol and formic acid 70 % was carried out according to the Bruker's recommendations. Measurements were performed on a Microflex LT and its standard database (database 3.3.1), and VITEK MS instrument equipped with both IVD and RUO (SARAMIS) databases. To differentiate C. haemulonii (target group) from the variety vulnera (control group), mass spectra analysis models were created with the ClinProTools 3.0 software (Bruker). For each model, the recognition capability (RC) and cross validation (CV) percentage was generated. For single peak analysis, the AUC of each peak for the discrimination of target group from control group was directly obtained from the ClinProTools software. For the peaks with the highest AUC, the detection performances were checked using FlexAnalysis 3.4 (Bruker Daltonics). The signal-to-noise ratios (SN) of the peaks were exported to SPSS 18.0, ROC curves were constructed, and their optimal cut-off values were determined. Results All C. haemulonii sensu stricto isolates/strain had correct species assignment by both Bruker and VITEK MS IVD databases. For the species C. duobushaemulonii, the Bruker database gave correct species ID for 75 % of the isolates/strains. VITEK MS IVD analysis misidentified all C. duobushaemulonii and C. pseudohaemulonii isolates/strains as C. haemulonii (99 % of confidence level). The VITEK MS SARAMIS was unable to give genus/species ID for all isolates/strains. Discrimination of the isolates belonging to the C. haemulonii sensu stricto and to the variety vulnera was not possible with all systems and databases. The ClinProTools models showed values of CV and RC above ≥90 % for the discrimination of target group from control group. The most discriminative peaks were 5107, 6878 and 13,750 m/z (AUC >0,9), with sensibility and specificity of 88,6 %, 80,8 %, 85,8 %, and 96,6 %, 92,9 %, 93,8 %, respectively. The SN cut-off values from the peaks 5107, 6878 and 13,750 m/z for the discrimination of the target group from the control group were 2,5, 3,9 and 6,3, respectively.

Microbiological diagnosis of Eggerthella lenta blood culture isolates in a Swedish tertiary hospital: Rapid identification and antimicrobial susceptibility profile

Eggerthella lenta is a Gram-positive anaerobic bacillus. Improved diagnostics and increased awareness of rare pathogens have revealed its potential to cause serious invasive infections. In this study, 18 clinical E. lenta isolates derived from positive blood cultures were included. Underlying problems of the patients were in the majority of cases related to the gastrointestinal tract. The performance of two MALDI-TOF MS systems, i.e. Bruker and Vitek MS, in identification of E. lenta was analyzed. In addition, the minimal inhibitory concentrations for clinically relevant antimicrobial agents were determined by routine procedures using E-test. 17 of the 18 E. lenta isolates investigated in this study were correctly identified to species level by the Bruker MS system, while the Vitek MS system identified all 18 isolates. Antimicrobial sensitivity towards the tested agents was in general good. However, high resistance rates were observed for penicillin G and piperacillin-tazobactam based on EUCAST breakpoints.

Comparing the identification of Clostridium spp. by two Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry platforms to 16S rRNA PCR sequencing as a reference standard: A detailed analysis of age of culture and sample preparation

We compared the identification of Clostridium species using mass spectrometry by two different Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) platforms (Bruker MS and Vitek MS) against 16S rRNA sequencing as the reference standard. We then examined the impact of different sample preparations and (on one of those platforms) age of bacterial colonial growth on the performance of the MALDI-TOF MS systems.We identified 10 different species amongst the 52 isolates by 16S rRNA sequencing, with Clostridium perfringens the most prevalent (n = 30). Spectrometric analysis using Vitek MS correctly speciated 47/52 (90.4%) isolates and was not affected by the sample preparation used. Performance of the Bruker MS was dependent on sample preparation with correct speciation obtained for 36 of 52 (69.2%) isolates tested using the Direct Transfer [DT] protocol, but all 52 (100%) isolates were correctly speciated using either an Extended Direct Transfer [EDT] or a Full Formic Extraction [EX] protocol. We then examined the effect of bacterial colonial growth age on the performance of Bruker MS and found substantial agreement in speciation using DT (Kappa = 0.62, 95% CI: 0.46–0.75), almost perfect agreement for EDT (Kappa = 0.94, 95% CI: 0.86–1.00) and exact agreement for EX (Kappa = 1.00) between different days.

Comparing the identification of clostridium SPP. from blood culture using phenotypic, molecular, and two matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry methods: a detailed analysis including the impact of culture age and sample preparation on the peformance on MALDI-TOF systems

Introduction We compared the identification of 52 Clostridium species from 52 incident bacteraemic patients using phenotypic analysis, two MALDI-TOF mass spectrometry platforms and molecular sequencing. We also examined the impact of different sample preparations and (on one platform) days of colonial growth on the performance of MALDI-TOF systems. Result 16S rRNA PCR accurately identified 10 Clostridium species amongst the 52 isolates. Combination phenotypic methods identified 44/52 (84.6%) to species level. Spectrometric analysis using Vitek MS yielded 47/52 (90.4%) species using direct transfer (DT), whilst analysis using Bruker MS yielded correct identification dependent on sample preparation [69.2% for DT protocol, 100% for extended direct transfer (EDT) protocol and 100% for full formic extraction (EX) protocol]. Sample preparation didn’t impact the Vitek MS. There was at least moderate agreement in performance for DT, EDT and EX between different days for the Bruker MS. Conclusion Phenotypic identification required numerous methods to accurately identified Clostridium species. Vitek MALDI-TOF MS performed better with the DT protocol compared to the Bruker MS, but the latter yielded correct identification in all samples tested with EDT and EX protocol. Sample preparations did not impact the Vitek MS. On the Bruker MS, the testing cycle can begin upon view of colonial growth.

Comparison of MALDI-TOF MS and VITEK 2 system for laboratory diagnosis of Granulicatella and Abiotrophia species causing invasive infections

Granulicatella and Abiotrophia spp. were known as nutritionally variant streptococci (NVS). Such strains have caused major diagnostic difficulties due to fastidious culturing and unspecific colony morphology. The present study is aimed at comparing the performance of laboratory available diagnostic methods for NVS isolates and determining the antimicrobial susceptibility of these isolates. Fourteen clinical invasive isolates, consisting of 10 Granulicatella adiacens, 1 Granulicatella elegans, and 3 Abiotrophia defectiva were in parallel analyzed by 2 matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) systems, i.e., Bruker MS and Vitek MS, as well as Vitek 2 for the species determination. 16S rRNA gene sequencing was applied as a reference method. The Vitek MS gave correct identification for all 14 isolates. The Bruker MS could correctly identify 8/10 G. adiacens, 0/1 G. elegans, and 3/3 A. defectiva isolates at the first analysis occasion, and all 14 isolates became identifiable after repeated tests. The Vitek 2 system could identify 6/10 G. adiacens, 1/1 G. elegans, and 2/3 A. defectiva isolates at the species level. Antimicrobial susceptibilities of 11 antibiotics were determined by Etest. Resistance against ciprofloxacin, ceftriaxone, rifampicin, and tetracycline were observed in 4, 10, 4, and 1 isolates, respectively. In conclusion, MALDI-TOF MS is a useful tool for the rapid diagnosis of NVS. Phenotypic testing by Vitek 2 is only partially effective for the accurate identification of such strains. The emergence of resistant NVS isolates indicates the necessity of monitoring antimicrobial susceptibilities of such uncommon pathogens.

Comparison of two matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry methods and API 20AN for identification of clinically relevant anaerobic bacteria

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid microbial diagnosis at relatively low cost and can be considered an alternative to conventional biochemical and molecular identification systems in clinical microbiological laboratories, including anaerobe laboratories. Two commercially available MALDI-TOF MS systems, Bruker Microflex MS and bioMérieux VITEK MS, were evaluated for the identification of 274 isolates of clinically significant anaerobic bacteria recovered from routine cultures of clinical specimens in parallel with blinded comparison with conventional biochemical (API 20AN) or molecular methods. All were recovered cultures obtained from patients attending the Mubarak Al Kabir Hospital, Kuwait, during a 6 month period. Discrepant results after two attempts at direct colony testing had failed to provide acceptable MALDI-TOF identification were resolved by gold-standard 16S gene sequencing. The VITEK MS gave high-confidence identification of the 274 isolates, all of which were correctly identified. The Bruker Microflex MS system also gave high-confidence identification for 272 of the 274. After discrepancy testing, the Bruker MS results agreed with biochemical or molecular methods for 89.1 % of the isolates at species level and 10.2 % at genus level (0.72 % were misidentified). The level of agreement with the VITEK MS was 100 % at both species and genus level; no isolates were misidentified. Our data suggest that implementation of MALDI-TOF MS as a first step for identification will shorten the turnaround time and reduce the cost in the anaerobe laboratory.

Evaluation of species-specific PCR, Bruker MS, VITEK MS and the VITEK 2 system for the identification of clinical Enterococcus isolates

The purpose of this investigation was to compare the performance of species-specific polymerase chain reaction (PCR), matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and phenotypic identification systems for the identification of Enterococcus species. A total of 132 clinical isolates were investigated by the following: (1) a multiplex real-time PCR assay targeting ddl Enterococcus faecium, ddl Enterococcus faecalis, vanC1 and vanC2/C3 genes, and a high-resolution melting (HRM) analysis of the groESL gene for the differentiation of Enterococcus casseliflavus and Enterococcus gallinarum; (2) Bruker MS; (3) VITEK MS; and (4) the VITEK 2 system. 16S rRNA gene sequencing was used as a reference method in the study. The 132 isolates were identified as 32 E. faecalis, 63 E. faecium, 16 E. casseliflavus and 21 E. gallinarum. The multiplex PCR, Bruker MS and VITEK MS were able to identify all the isolates correctly at the species level. The VITEK 2 system could identify 131/132 (99.2%) and 121/132 (91.7%) of the isolates at the genus and species levels, respectively. The HRM-groESL assay identified all (21/21) E. gallinarum isolates and 81.3% (13/16) of the E. casseliflavus isolates. The PCR methods described in the present study are effective in identifying the enterococcal species. MALDI-TOF MS is a rapid, reliable and cost-effective identification technique for enterococci. The VITEK 2 system is less efficient at detecting non-faecalis and non-faecium Enterococcus species.

Comparison of two matrix-assisted laser desorption ionisation-time of flight mass spectrometry methods for the identification of clinically relevant anaerobic bacteria

Two commercially available MALDI-TOF MS systems, Bruker MS and Shimadzu MS, were compared for the identification of clinically relevant anaerobic bacteria. A selection of 79 clinical isolates, representing 19 different genera, were tested and compared with identification obtained by 16S rRNA gene sequencing. Correct genus identification was achieved for 71% of isolates by Shimadzu MS and for 61% by Bruker MS. Correct identification at the species level occurred in 61% and 51%, respectively. Shimadzu showed markedly better results for identification of Gram-positive anaerobic cocci. In contrast, the Bruker system performed better than Shimadzu for the Bacteroides fragilis group. When strains not present in the database were excluded from the analyses for each database, both systems performed equally well, with 76.7% and 75.0% correct genus identification for Shimadzu and Bruker, respectively. Similarly, when the most recently updated Bruker database was applied, no difference was observed. We conclude that the composition and quality of the database is crucial for a correct identification. The databases currently available for both systems need to be optimized before MS can be implemented for routine identification of anaerobic bacteria.