Biotyper System Research Articles

An update on non-aureus staphylococci and mammaliicocci in cow milk: unveiling the presence of Staphylococcus borealis and Staphylococcus rostri by MALDI-TOF MS

Non-aureus staphylococci and mammaliicocci (NASM) are the microorganisms most frequently isolated from milk. Given their numerosity and complexity, MALDI-TOF MS is one of the preferred species identification approaches. Nevertheless, reference mass spectra for the novel species Staphylococcus borealis were included only recently in the Bruker Biotyper System (MBT) library, and other species of veterinary interest such as S. rostri are still absent. This work provides an updated picture of the NASM species found in milk, gained by retrospectively analyzing the data relating to 21,864 milk samples, of which 6,278 from clinical mastitis (CM), 4,039 from subclinical mastitis (SCM), and 11,547 from herd survey (HS), with a spectrum library including both species. As a result, S. borealis was the second most frequently isolated NASM (17.07%) after S. chromogenes (39.38%) in all sample types, with a slightly higher percentage in CM (21.84%), followed by SCM (17.65%), and HS (14.38%). S. rostri was also present in all sample types (3.34%), reaching 8.43% of all NASM in SCM and showing a significant association (p < 0.01) with this condition. Based on our findings, the presence of S. borealis and S. rostri in milk and their potential association with mastitis might have been overlooked, possibly due to the difficulties in differentiating these species from other closely related NASM. Our results indicate that S. borealis could be a more frequent contributor to bovine udder infections than previously thought and that S. rostri should also not be underestimated considering its significant association with SCM.

Evaluation of MALDI-TOF mass spectrometry coupled with ClinProTools as a rapid tool for toxin-producing Clostridioides difficile

IntroductionThe performance of MALDI-TOF MS combined with analysis platform for identification of toxin-producing Clostridiodes difficile is yet to be known. MethodsBetween August 2018 and September 2020, 61 isolates from stool specimens of patients with C. difficile-associated diarrhea were analyzed using the MALDI Biotyper system. A C. difficile toxin-producer detection model was developed using ClinProTools. The model was validated using 28 known strains that differed from the isolates used to develop the model. ResultsThe sensitivity and specificity of the Genetic Algorithm (GA) model using isolates grown on Brucella with hemin and vitamin K (BHK) agar plates were 91.7% and 44.4%, respectively. When isolates grown on cycloserine-cefoxitin mannitol agar were analyzed by the model, sensitivity and specificity were 6.3% and 100%, respectively. The GA model using BHK medium showed the highest discriminatory performance in detection of toxin-producing C. difficile. However, a discrepancy in detection of toxin-producing C. difficile was observed in the results generated when the model was being developed and when the model was validated which suggests that incubation conditions may have affected the results. ConclusionMALDI-TOF analysis using ClinProTools has a potential to be a cost-effective tool for rapid diagnosis and contribute to antimicrobial stewardship by differentiating toxin-producing C. difficile from non-producers.

Performance assessment of the Bruker Biotyper MALDI-TOF MS for the identification of difficult-to-identify viridans group streptococci.

Differentiating Streptococcus pneumoniae among nonpneumococcal viridans group streptococci (VGS) is challenging in conventional laboratories. Therefore, we aimed to evaluate the performance of the latest Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system in identifying VGS by comparing the results to those of the specific gene sequencing approach. Clinical isolates were initially identified using the BD Phoenix system to identify Streptococcus species. The optochin test was used to distinguish nonpneumococcal VGS from S. pneumoniae. The species of individual reference strains and clinical isolates were determined by comparing the sequences of the 16S rDNA, gyrB, sodA, groESL, or coaE genes with those in the GenBank sequence databases. We evaluated the performance of the Bruker Biotyper MALDI-TOF MS in VGS identification using two different machines with three databases. We collected a total of 103 nonpneumococcal VGS and 29 S. pneumoniae blood isolates at a medical center in northern Taiwan. Among these isolates, only seven could not be identified at the species level by the specific gene sequencing approach. We found that none of the nonpneumococcal VGS isolates were misidentified as pneumococci by the latest Biotyper system, and vice versa. However, certain strains, especially those in the mitis and bovis groups, could still not be correctly identified. The latest Bruker Biotyper 4.1 (DB_10833) showed significant improvement in identifying VGS strains. However, a specific gene sequencing test is still needed to precisely differentiate the species of strains in the mitis and bovis groups.

The emergence of carbapenemase-producing Enterobacterales in hospitals: a major challenge for a debilitated healthcare system in Lebanon.

Carbapenem- and extended-spectrum cephalosporin-resistant Enterobacterales (CR-E and ESCR-E, respectively) are increasingly isolated worldwide. Information about these bacteria is sporadic in Lebanon and generally relies on conventional diagnostic methods, which is detrimental for a country that is struggling with an unprecedented economic crisis and a collapsing public health system. Here, CR-E isolates from different Lebanese hospitals were characterized. Non-duplicate clinical ESCR-E or CR-E isolates (N = 188) were collected from three hospitals from June 2019 to December 2020. Isolates were identified by MALDI-TOF, and their antibiotic susceptibility by Kirby-Bauer disk diffusion assay. CR-E isolates (n = 33/188) were further analyzed using Illumina-based WGS to identify resistome, MLST, and plasmid types. Additionally, the genetic relatedness of the CR-E isolates was evaluated using an Infrared Biotyper system and compared to WGS. Using the Kirby-Bauer disk diffusion assay, only 90 isolates out of the 188 isolates that were collected based on their initial routine susceptibility profile by the three participating hospitals could be confirmed as ESCR-E or CR-E isolates and were included in this study. This collection comprised E. coli (n = 70; 77.8%), K. pneumoniae (n = 13; 14.4%), Enterobacter spp. (n = 6; 6.7%), and Proteus mirabilis (n = 1; 1.1%). While 57 were only ESBL producers the remaining 33 isolates (i.e., 26 E. coli, five K. pneumoniae, one E. cloacae, and one Enterobacter hormaechei) were resistant to at least one carbapenem, of which 20 were also ESBL-producers. Among the 33 CR-E, five different carbapenemase determinants were identified: blaNDM-5 (14/33), blaOXA-244 (10/33), blaOXA-48 (5/33), blaNDM-1 (3/33), and blaOXA-181 (1/33) genes. Notably, 20 CR-E isolates were also ESBL-producers. The analysis of the genetic relatedness revealed a substantial genetic diversity among CR-E isolates, suggesting evolution and transmission from various sources. This study highlighted the emergence and broad dissemination of blaNDM-5 and blaOXA-244 genes in Lebanese clinical settings. The weak AMR awareness in the Lebanese community and the ongoing economic and healthcare challenges have spurred self-medication practices. Our findings highlight an urgent need for transformative approaches to combat antimicrobial resistance in both community and hospital settings.

Prediction of methicillin-resistant Staphylococcus aureus and carbapenem-resistant Klebsiella pneumoniae from flagged blood cultures by combining rapid Sepsityper MALDI-TOF mass spectrometry with machine learning

This study investigated combination of the Rapid Sepsityper Kit and a machine learning (ML)-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) approach for rapid prediction of methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Klebsiella pneumoniae (CRKP) from positive blood culture bottles. The study involved 461 patients with monomicrobial bloodstream infections. Species identification was performed using the conventional MALDI-TOF MS Biotyper system and the Rapid Sepsityper protocol. The data underwent preprocessing steps, and ML models were trained using preprocessed MALDI-TOF data and corresponding labels. The interpretability of the model was enhanced using SHapely Additive exPlanations values to identify significant features. In total, 44 S. aureus isolates comprising 406 MALDI-TOF MS files and 126 K. pneumoniae isolates comprising 1249 MALDI-TOF MS files were evaluated. This study demonstrated the feasibility of predicting MRSA among S. aureus and CRKP among K. pneumoniae isolates using MALDI-TOF MS and Sepsityper. Accuracy, area under the receiver operating characteristic curve, and F1 score for MRSA/methicillin-susceptible S. aureus were 0.875, 0.898 and 0.904, respectively; for CRKP/carbapenem-susceptible K. pneumoniae, these values were 0.766, 0.828 and 0.795, respectively. In conclusion, the novel ML-based MALDI-TOF MS approach enables rapid identification of MRSA and CRKP from flagged blood cultures within 1 h. This enables earlier initiation of targeted antimicrobial therapy, reducing deaths due to sepsis. The favourable performance and reduced turnaround time of this method suggest its potential as a rapid detection strategy in clinical microbiology laboratories, ultimately improving patient outcomes.

Improved identification of Streptococcus bovis-Streptococcus equinus-complex species and subspecies by MALDI-TOF MS using a novel library

ObjectivesTo develop an in-house matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) library for improved identification of species and subspecies of the Streptococcus bovis/Streptococcus equinus-complex (SBSEC). MethodsA total of 236 SBSEC isolates from blood stream infections and culture collections, determined by whole genome sequencing to subspecies level, were grown in brain heart infusion broth. Mass spectra were collected using the Bruker MALDI Biotyper system after ethanol-formic acid extraction. Main spectral profiles from 117 isolates were used to create the “SBSEC-CMRS library.” The remaining 119 spectra were used for evaluation of Bruker MALDI Biotyper (MBT) Compass Library Revision K (2022) and the SBSEC-CMRS library. ResultsThe Bruker library correctly identified species and subspecies in 72 of 119 (61 %) isolates, while the SBSEC-CMRS library identified 116 of 119 (97 %), using a cutoff score of ≥2.0. ConclusionsThe SBSEC-CMRS library showed sufficient diagnostic accuracy, and can be implemented in clinical practice for SBSEC species and subspecies identification.

Performance of Two Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) Systems for Identification of the Viridans Group Streptococci.

Due to similar colony morphology among viridans group streptococci (VGS), the differentiation of VGS species remains difficult in routine clinical microbiology. Recently, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been described as a fast method for identifying various bacteria at species level, and also for the VGS strains. A total of 277 VGS isolates were identified with the two MALDI-TOF MS systems (VITEK MS and Bruker Biotyper). The tuf and rpoB gene sequencing was used as the reference identification method for comparison. Based on tuf and rpoB gene sequencing, 84 isolates were S. pneumoniae and 193 strains were other VGS isolates including S. anginosus group (n=91, 47.2%), S. mitis group (n=80, 41.5%), S. bovis group (n=11, 5.7%), S. salivarius group (n=10, 5.2%), and S. mutans group (n=1, 0.5%). VITEK MS and Bruker Biotyper accurately identified 94.6% and 89.9% of all VGS isolates, respectively. VITEK MS showed better identification results than Bruker Biotyper for S. mitis group including S. pneumoniae and S. bovis group, but for other VGS isolates, two MALDI-TOF MS systems showed comparable identification performance. However, VITEK MS was able to identify S. gallolyticus to the subspecies level with high-confidence (S. gallolyticus ssp. pasteurianus), while the Bruker Biotyper system could not. While Bruker Biotyper system could be able to correctly differentiate the subspecies of S. salivarius from S. vestibularis, VITEK MS poorly identify. This study demonstrated that two MALDI-TOF MS systems allowed discrimination for most VGS isolates with different identification performance, but Bruker Biotyper could produce more misidentifications and VITEK MS system. It is crucial to be familiar with the performance of MALDI-TOF MS systems used in clinical microbiology.

Validation of an expanded, in-house library and an optimized preparation method for the identification of fungal isolates using MALDI-TOF mass spectrometry.

The goal of this study was to validate an optimized sample preparation method for filamentous fungal isolates coupled with the use of an in-house library for the identification of moulds using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) in a multicenter context. For that purpose, three Spanish microbiology laboratories participated in the identification of 97 fungal isolates using MALDI-TOF MS coupled with the Filamentous Fungi library 3.0 (Bruker Daltonics) and an in-house library containing 314 unique fungal references. The isolates analyzed belonged to 25 species from the genus Aspergillus, Fusarium, Scedosporium/Lomentospora, the Mucorales order and the Dermatophytes group. MALDI-TOF MS identification was carried out from hyphae resuspended in water and ethanol. After a high-speed centrifugation step, the supernatant was discarded and the pellet submitted to a standard protein extraction step. The protein extract was analyzed with the MBT Smart MALDI Biotyper system (Bruker Daltonics). The rate of accurate, species-level identification obtained ranged between 84.5% and 94.8% and the score values were1.8 for 72.2-94.9% of the cases. Two laboratories failed to identify only one isolate of Syncephalastrum sp. and Trichophyton rubrum, respectively and three isolates could not be identified in the third center (F. proliferatum, n=1; T.interdigitale, n=2). In conclusion, the availability of an effective sample preparation method and an extended database allowed high rates of correct identification of fungal species using MALDI-TOF MS. Some species, such as Trichophyton spp. are still difficult to identify. Although further improvements are still required, the developed methodology allowed the reliable identification of most fungal species.

Molecular Confirmation of an Indole-Negative Klebsiella oxytoca Isolated from a Patient with Cystitis.

Klebsiella oxytoca is an opportunistic pathogen that causes nosocomial infections. Here, we describe an unusual clinical strain of indole-negative K. oxytoca, GU175, isolated from the urine of a patient with cystitis. The GU175 strain was identified as K. pneumoniae with a probability of 99.40%, negative for indole production, and resistant to third-generation cephalosporins by using the MicroScan Walkaway 40 SI system with the Negative combo EN1 J panel. Biochemical characterization of this strain using lysine-indole motility medium was negative for indole production. However, identification tests using the MALDI Biotyper system and 16S rRNA gene sequence analysis revealed that GU175 is K. oxytoca. DNA sequence analysis of the tryptophanase operon comparing the GU175 strain with the revertant GU176 strain, which tested positive for indole, revealed a point mutation in the Shine-Dalgarno sequence upstream of tnaC in the GU175 strain. This is the first report of indole-negative K. oxytoca, which was attributed to a mutation in the DNA sequence of the tryptophanase operon isolated from a patient with a urinary tract infection. As indole-negative K. oxytoca can be misidentified as K. pneumoniae by biochemical characterization, clinical microbiologists should be aware of such misidentifications.

Identification of Francisella tularensis Subspecies in a Clinical Setting Using MALDI-TOF MS: An In-House Francisella Library and Biomarkers.

Francisella tularensis is a zoonotic bacterium that is endemic in large parts of the world. It is absent in the standard library of the most applied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems: the Vitek MS and the Bruker Biotyper system. The additional Bruker MALDI Biotyper Security library contains F. tularensis without subspecies differentiation. The virulence of F. tularensis differs between the subspecies. The F. tularensis subspecies (ssp.) tularensis is highly pathogenic, whereas the subspecies holarctica displays lower virulence and subspecies novicida and F. tularensis ssp. mediasiatica are hardly virulent. To differentiate the Francisellaceae and the F. tularensis-subspecies, an in-house Francisella library was built with the Bruker Biotyper system and validated together with the existing Bruker databases. In addition, specific biomarkers were defined based on the main spectra of the Francisella strains supplemented with in silico genome data. Our in-house Francisella library accurately differentiates the F. tularensis subspecies and the other Francisellaceae. The biomarkers correctly differentiate the various species within the genus Francisella and the F. tularensis subspecies. These MALDI-TOF MS strategies can successfully be applied in a clinical laboratory setting as a fast and specific method to identify F. tularensis to subspecies level.

A MALDI-TOF MS library for rapid identification of human commensal gut bacteria from the class Clostridia.

Microbial isolates from culture can be identified using 16S or whole-genome sequencing which generates substantial costs and requires time and expertise. Protein fingerprinting via Matrix-assisted Laser Desorption Ionization-time of flight mass spectrometry (MALDI-TOF MS) is widely used for rapid bacterial identification in routine diagnostics but shows a poor performance and resolution on commensal bacteria due to currently limited database entries. The aim of this study was to develop a MALDI-TOF MS plugin database (CLOSTRI-TOF) allowing for rapid identification of non-pathogenic human commensal gastrointestinal bacteria. We constructed a database containing mass spectral profiles (MSP) from 142 bacterial strains representing 47 species and 21 genera within the class Clostridia. Each strain-specific MSP was constructed using >20 raw spectra measured on a microflex Biotyper system (Bruker-Daltonics) from two independent cultures. For validation, we used 58 sequence-confirmed strains and the CLOSTRI-TOF database successfully identified 98 and 93% of the strains, respectively, in two independent laboratories. Next, we applied the database to 326 isolates from stool of healthy Swiss volunteers and identified 264 (82%) of all isolates (compared to 170 (52.1%) with the Bruker-Daltonics library alone), thus classifying 60% of the formerly unknown isolates. We describe a new open-source MSP database for fast and accurate identification of the Clostridia class from the human gut microbiota. CLOSTRI-TOF expands the number of species which can be rapidly identified by MALDI-TOF MS.

Fourier transform infrared spectroscopy; can it be used as a rapid typing method of Neisseria gonorrhoeae?

BackgroundTyping of Neisseria gonorrhoeae is necessary for epidemiologic surveillance, while time consuming and resource intensive. Fourier transform infrared (FTIR) spectroscopy has shown promising results when typing several bacterial species. This study investigates whether FTIR spectroscopy can be used as a rapid method for typing clinical N. gonorrhoeae isolates, comparing FTIR spectroscopy to multi locus sequence typing (MLST), N. gonorrhoeae multi antigen sequence typing (NG-MAST) and whole genome sequencing (WGS). MethodsSixty consecutive isolates from a venereology clinic and three isolates from an outbreak were included. Isolates were analysed with FTIR spectroscopy on the IR Biotyper system (Bruker Daltonik) with the IR Biotyper software (version 2.1) with default analysis settings (spectral range 1300–800 cm−1). Four technical replicates of each isolate were analysed in three different runs. The output was a hierarchical cluster analysis (HCA) presented as a dendrogram; a tree-like overview of how closely different isolates are related. FTIR spectroscopy was compared to MLST, NG-MAST and WGS to see if the FTIR spectroscopy-dendrogram grouped the isolates in the same clusters. ResultsFifty-one out of 60 isolates, and the three outbreak isolates, produced at least one spectrum in each run and were included. No agreement between FTIR spectroscopy and MLST or NG-MAST or WGS was shown. The FTIR spectroscopy-dendrogram failed to cluster the outbreak isolates. ConclusionFTIR spectroscopy (spectral range 1300–800 cm−1) is not yet suitable for epidemiologic typing of N. gonorrhoeae. Absence of a capsule as well as phase- and antigenic variation of carbohydrate surface structures of the gonococcal cell wall may contribute to our findings. Future studies should include analysis of a wider range of the spectrum recorded (4000-500 cm−1), and should also explore further mathematical analytic approaches of the similarity between spectra.

Comparison of MALDI-TOF mass spectrometry and rpoB gene sequencing for the identification of clinical isolates of Aeromonas spp.

Aeromonas spp., widely present in rivers and soil, cause mild gastroenteritis, severe septicemia, and soft tissue infections in humans. Treatment of these infections require accurate identification of pathogenic Aeromonas spp. However, identification at the species level using conventional methods is highly challenging. In this study, we aimed to compare the accuracy of two different approaches developed for bacterial identification: (i) housekeeping gene sequencing (rpoB) in conjunction with phylogenetic analysis and (ii) matrix-assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF MS) (MALDI Biotyper and VITEK MS), for differentiating Aeromonas spp. We analyzed 58 Aeromonas isolates recovered from patients at different medical institutions in Japan using both identification methods. The rpoB sequencing method was the most accurate, identifying all Aeromonas isolates at the species level. Meanwhile, the MALDI Biotyper system correctly identified 53 (91.4%) isolates at the genus level and an additional 30 (51.7%) at the species level. The VITEK MS system correctly identified 58 (100%) isolates at the genus level and an additional 34 (58.6%) at the species level. Thus, MALDI Biotyper and VITEK MS accurately identified isolates at the genus level, but differences were found in the accuracy of identification of species. However, the low cost and ease of analysis make MALDI-TOF MS-based methods strong candidates for use in clinical laboratories that require easy-to-use identification methods.

The challenges in the identification of Escherichia coli from environmental samples and their genetic characterization

Escherichia coli bacteria are an essential indicator in evaluations of environmental pollution, which is why they must be correctly identified. This study aimed to determine the applicability of various methods for identifying E. coli strains in environmental samples. Bacterial strains preliminary selected on mFc and Chromocult media as E. coli were identified using MALDI Biotyper techniques, based on the presence of genes characteristic of E. coli (uidA, uspA, yaiO), as well as by 16S rRNA gene sequencing. The virulence and antibiotic resistance genes pattern of bacterial strains were also analyzed to investigate the prevalence of factors that may indicate adaptation to unsupportive environmental conditions and could have any significance in further identification of E. coli. Of the strains that had been initially identified as E. coli with culture-based methods, 36–81% were classified as E. coli with the use of selected techniques. The value of Cohen’s kappa revealed the highest degree of agreement between the results of 16S rRNA gene sequencing, the results obtained in the MALDI Biotyper system, and the results of the analysis based on the presence of the yaiO gene. The results of this study could help in the selection of more accurate and reliable methods which can be used in a preliminary screening and more precise identification of E. coli isolated from environmental samples.

Machine learning-based typing of Salmonella enterica O-serogroups by the Fourier-Transform Infrared (FTIR) Spectroscopy-based IR Biotyper system

BackgroundSalmonella enterica is among the major burdens for public health at global level. Typing of salmonellae below the species level is fundamental for different purposes, but traditional methods are expensive, technically demanding, and time-consuming, and therefore limited to reference centers. Fourier transform infrared (FTIR) spectroscopy is an alternative method for bacterial typing, successfully applied for classification at different infra-species levels. AimThis study aimed to address the challenge of subtyping Salmonella enterica at O-serogroup level by using FTIR spectroscopy. We applied machine learning to develop a novel approach for S. enterica typing, using the FTIR-based IR Biotyper® system (IRBT; Bruker Daltonics GmbH & Co. KG, Germany). We investigated a multicentric collection of isolates, and we compared the novel approach with classical serotyping-based and molecular methods. MethodsA total of 958 well characterized Salmonella isolates (25 serogroups, 138 serovars), collected in 11 different centers (in Europe and Japan), from clinical, environmental and food samples were included in this study and analyzed by IRBT. Infrared absorption spectra were acquired from water-ethanol bacterial suspensions, from culture isolates grown on seven different agar media. In the first part of the study, the discriminatory potential of the IRBT system was evaluated by comparison with reference typing method/s. In the second part of the study, the artificial intelligence capabilities of the IRBT software were applied to develop a classifier for Salmonella isolates at serogroup level. Different machine learning algorithms were investigated (artificial neural networks and support vector machine). A subset of 88 pre-characterized isolates (corresponding to 25 serogroups and 53 serovars) were included in the training set. The remaining 870 samples were used as validation set. The classifiers were evaluated in terms of accuracy, error rate and failed classification rate. ResultsThe classifier that provided the highest accuracy in the cross-validation was selected to be tested with four external testing sets. Considering all the testing sites, accuracy ranged from 97.0% to 99.2% for non-selective media, and from 94.7% to 96.4% for selective media. ConclusionsThe IRBT system proved to be a very promising, user-friendly, and cost-effective tool for Salmonella typing at serogroup level. The application of machine learning algorithms proved to enable a novel approach for typing, which relies on automated analysis and result interpretation, and it is therefore free of potential human biases. The system demonstrated a high robustness and adaptability to routine workflows, without the need of highly trained personnel, and proving to be suitable to be applied with isolates grown on different agar media, both selective and unselective. Further tests with currently circulating clinical, food and environmental isolates would be necessary before implementing it as a potentially stand-alone standard method for routine use.

Rapid and Accurate Identification of Nontuberculous Mycobacteria Directly from Positive Primary MGIT Cultures by MALDI-TOF MS.

Over the last years, nontuberculous mycobacteria (NTM) have emerged as important human pathogens. Accurate and rapid mycobacterial species identification is needed to successfully diagnose, treat, and manage infections caused by NTM. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS, was demonstrated to effectively identify mycobacteria isolates subcultured from solid or liquid media rather than new positive cultures. The present study aims to develop a new extraction protocol to yield rapid and accurate identification of NTM from primary MGIT cultures by MALDI-TOF MS. A total of 60 positive MGIT broths were examined by the Bruker Biotyper system with Mycobacteria Library v. 2.0 (Bruker Daltonics GmbH & Co. KG., Bremen, Germany). The results were compared with those obtained by the molecular method, line probe assay GenoType Mycobacterium CM/AS/NTM-DR. All samples were concordantly identified by MALDI-TOF MS and the molecular test for all the tested mycobacteria. Fifty-seven (95%) MGIT positive cultures for NTM from clinical samples had a MALDI-TOF MS analysis score S ≥ 1.8. Although a small number of strains and a limited diversity of mycobacterial species were analysed, our results suggest that MALDI-TOF MS could represent a promising routine diagnostic tool for identifying mycobacterial species directly from primary liquid culture.

Performance of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Scedosporium, Acremonium-Like, Scopulariopsis, and Microascus Species.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a powerful microorganism identification tool. Research on MALDI-TOF MS identification of rare filamentous fungi is still lacking. This study aimed to evaluate the performance of MALDI-TOF MS in the identification of Scedosporium, Acremonium-like, Scopulariopsis, and Microascus species. Sabouraud broth cultivation and formic acid/acetonitrile protein extraction were used for MALDI-TOF MS identification by a Bruker Biotyper system. An in-house database containing 29 isolates of Scedosporium, Acremonium-like, Scopulariopsis, and Microascus spp. was constructed. A total of 52 clinical isolates were identified using the Bruker Filamentous Fungi Library v1.0 (FFL v1.0) alone, and Filamentous Fungi Library v1.0 plus the in-house library, respectively. The mass spectrum profile (MSP) dendrograms of the 28 Scedosporium isolates, 26 Acremonium-like isolates, and 27 Scopulariopsis and Microascus isolates were constructed by MALDI Biotyper OC 4.0 software, respectively. The correct species identification rate significantly improved when using the combined databases compared with that when using FFL v1.0 alone (Scedosporium spp., 75% versus 0%; Acremonium-like spp., 100% versus 0%; Scopulariopsis and Microascus spp., 100% versus 62.5%). The MSP dendrograms differentiated Acremonium-like species, Scopulariopsis and Microascus species clearly, but cannot distinguish species in the Scedosporium apiospermum complex. In conclusion, with an expanded database, MALDI-TOF MS is an effective tool for the identification of Scedosporium, Acremonium-like, Scopulariopsis, and Microascus species.

Accuracy of ASTA MicroIDSys, a new matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system, for the identification of Korean reference and clinical bacterial and yeast strains

This study compared the accuracy of a new MALDI-TOF mass spectrometry system, ASTA MicroIDSys system, with that of MALDI Biotyper system for the identification of reference and clinical bacterial and yeast strains. The identification accuracy of the 2 systems was compared using a total of 406 strains comprising 142 aerobic and 180 anaerobic bacterial strains and 84 yeast strains. The genus and species identification rates were 98.0% and 89.4% using MicroIDSys and 96.1% and 89.4% using Biotyper, respectively. The species identification rates of MicroIDSys and Biotyper for aerobic bacteria were 93.0% and 97.2%, respectively, and those for anaerobic bacteria were 85.6% and 81.7%, respectively. The accuracy of yeast identification at the species level was 91.7% using MicroIDSys and 92.9% using Biotyper. These findings indicate that MicroIDSys could be useful for the accurate identification of bacteria and yeast in clinical microbiology laboratories.

Proteomics-based screening and antibiotic resistance assessment of clinical and sub-clinical Brucella species: An evolution of brucellosis infection control.

Brucellae are intracellular sneaky bacteria and they can elude the host’s defensive mechanisms, resulting in therapeutic failure. Therefore, the goal of this investigation was to rapid identification of Brucella species collected from animals and humans in Saudi Arabia, as well as to evaluate their resistance to antibiotics. On selective media, 364 animal samples as well as 70 human blood samples were cultured. Serological and biochemical approaches were initially used to identify a total of 25 probable cultured isolates. The proteomics of Brucella species were identified using the MALDI Biotyper (MBT) system, which was subsequently verified using real-time polymerase chain reaction (real-time PCR) and microfluidic electrophoresis assays. Both Brucella melitensis (B. melitensis) and Brucella abortus (B. abortus) were tested for antimicrobial susceptibility using Kirby Bauer method and the E-test. In total, 25 samples were positive for Brucella and included 11 B. melitensis and 14 B. abortus isolates. Twenty-two out of 25 (88%) and 24/25 (96%) of Brucella strains were recognized through the Vitek 2 Compact system. While MBT was magnificently identified 100% of the strains at the species level with a score value more than or equal to 2.00. Trimethoprim-sulfamethoxazole, rifampin, ampicillin-sulbactam, and ampicillin resistance in B. melitensis was 36.36%, 31.82%, 27.27%, and 22.70%, respectively. Rifampin, trimethoprim-sulfamethoxazole, ampicillin, and ampicillin-sulbactam resistance was found in 35.71%, 32.14%, 32.14%, and 28.57% of B. abortus isolates, correspondingly. MBT confirmed by microfluidic electrophoresis is a successful approach for identifying Brucella species at the species level. The resistance of B. melitensis and B. abortus to various antibiotics should be investigated in future studies.

Medically important Candida spp. identification: an era beyond traditional methods.

Candida infections are gaining more attention for the last few decades so diagnostic tools are very important for early diagnosis. Conventional identification of yeasts is time-consuming, molecular methods are more complicated and relatively expensive gold-standard methods. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) was put into the market due to its speed and high accuracy. The aim of this study was to evaluate the performance of corn meal tween-80 agar (CMTA), CHROMagar Candida medium, and MALDI-TOF MS and to compare the obtained results with DNA sequencing. The CHROMagar Candida medium, CMTA, and MALDI-TOF MS Biotyper System were used to test 416 isolates. The isolates with discrepant results by at least one of the three methods were subjected to sequence analysis. The identification results of the 351 (%84.4) were compatible with all three methods. When compared to the sequencing results, the most accurate results were obtained by the MALDI-TOF MS, especially for rare Candida species. MALDI-TOF MS is found to be the most accurate identification tool for clinically important Candida strains. CMTA alone should not be used for the final identification of Candida species and the chromogenic medium should always be considered presumptive.