Comparison of Autof ms1000 and Bruker Biotyper MALDI-TOF MS Platforms for Routine Identification of Clinical Microorganisms.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is widely used in clinical microbiology laboratories because it is cost-effective, reliable, and fast. This study is aimed at comparing the identification performance of the recently developed Autof ms1000 (Autobio, China) with that of the Bruker Biotyper (Bruker Daltonics, Germany). From January to June 2020, 205 preserved strains and 302 clinical isolates were used for comparison. Bacteria were tested with duplicates of the direct transfer method, and formic acid extraction was performed if the results were not at the species level. Fungi were tested with formic acid extraction followed by ethanol extraction methods. 16S rRNA or ITS region sequence analysis was performed on isolates that could not be identified by any of the instruments and on isolates that showed inconsistent results. The time to result of each instrument was also compared. Among preserved strains, species-level identification results were obtained in 202 (98.5%) strains by the Autof ms1000 and 200 (97.6%) strains by the Bruker Biotyper. Correct identification at the species/complex level was obtained for 200 (97.6%) strains by the Autof ms1000 and for 199 (97.1%) strains by the Bruker Biotyper. Among clinical isolates, species-level identification results were obtained in 301 (99.7%) strains and 300 (99.3%) strains by the Autof ms1000 and Bruker Biotyper, respectively. Correct identification at the species/complex level was achieved for 299 (99.0%) strains by the Autof ms1000 and for 300 (99.3%) strains by the Bruker Biotyper. The time to analyze 96 spots was approximately 14 min for the Autof ms1000 and approximately 27 min for the Bruker Biotyper. The two instruments showed comparable performance for the routine identification of clinical microorganisms. In addition, the Autof ms1000 has a short test time, making it convenient for use in clinical microbiology laboratories.

Evaluation of the Autof MS1000 mass spectrometer in the identification of clinical isolates.

BACKGROUND: To evaluate the accuracy and performance of the Autof MS1000 mass spectrometer in bacteria and yeast identification, 2342 isolates were obtained from microbial cultures of clinical specimens (e.g. blood, cerebrospinal fluid, respiratory tract samples, lumbar puncture fluid, wound samples, stool, and urine) collected in 2019 in Henan Provincial People's Hospital. Repetitive strains from the same patient were excluded. We tested the Autof MS1000 and Bruker Biotyper mass spectrometry systems and the classical biochemical identification system VITEK 2/API 20C AUX. Inconsistencies in strain identification among the three systems were identified by 16S rDNA and gene sequencing. RESULTS: At the species level, the Autof MS1000 and Bruker Biotyper systems had isolate identification accuracies of 98.9 and 98.5%, respectively. At the genus level, the Autof MS1000 and Bruker Biotyper systems were 99.7 and 99.4% accurate, respectively. The instruments did not significantly differ in identification accuracy at either taxonomic level. The frequencies of unreliable identification were 1.1% (26/2342) for the Autof MS1000 and 1.5% (34/2342) for the Bruker Biotyper. In vitro experiments demonstrated that the coincidence rate of the Autof MS1000 mass spectrometer in the identification of five types of bacteria was > 93%, the identification error rate was < 3%, and the no identification rate was 0. This indicates that the Autof MS1000 system is acceptable for identification. CONCLUSIONS: The Autof MS1000 mass spectrometer can be utilised to identify clinical isolates. However, an upgradation of the database is recommended to correctly identify rare strains.

Spoligotyping of Mycobacterium tuberculosis isolates using Luminex®-based method in Lebanon.

INTRODUCTION: Data about the genotypes of circulating Mycobacterium tuberculosis isolates (MTB) in Lebanon are scarce. This study was undertaken to reveal the spoligotypes of MTB isolates recovered from patients in Lebanon. METHODOLOGY: MTB isolates from 49 patients living in Lebanon were recovered and identified. The samples were heat killed and subjected to DNA extraction. Spoligotyping was performed using microbeads from TB-SPOL Kit and the fluorescence intensity was measured using Luminex 200®. Generated patterns were assigned to families using the SITVIT2 international database of the Pasteur Institute of Guadeloupe and compared. RESULTS: The spoligotyping of the 49 MTB isolates revealed that 31 isolates belonged to Lineage 4 (Euro-American, 63.3%), 12 to Lineage 3 (East- African Indian, 24.5%), 3 to Lineage 2 (East Asian, 6%) and 2 were unknown. Over half of the genotypes (16 of 30) harbored SIT127 supposed to belong to the L4.5 sublineage. One isolate belonging to the rare Manu-Ancestor SIT523 was recovered for the first time in Lebanon, being associated with highly virulent extensively drug-resistant (XDR) MTB phenotype. CONCLUSION: The application of the Spoligotyping Multiplex Luminex® method is an efficient, discriminatory and rapid method to use for first-lane genotyping of MTB isolates. Though humble numbers were tested, this study is one of the first to describe the genomic diversity and epidemiology of MTB isolates of Lebanon, and suggests an increasing prevalence of SIT127 in the country.

Efficiency of chemical versus mechanical disruption methods of DNA extraction for the identification of oral Gram-positive and Gram-negative bacteria.

OBJECTIVE: Clinical diagnostics often requires the detection of multiple bacterial species in limited clinical samples with a single DNA extraction method. This study aimed to compare the bacterial DNA extraction efficiency of two lysis methods automated with the MagNA-Pure LC instrument. The samples included five oral bacterial species (three Gram-positive and two Gram-negative) with or without human saliva background. METHODS: Genomic DNA (gDNA) was extracted from bacterial cultures by bead-beating lysis (BMP) or chemical lysis (MP), followed by automated purification and measurement by quantitative PCR. RESULTS: For pure bacterial cultures, the MP method yielded higher quantities of extracted DNA and a lower detection limit than the BMP method, except where the samples contained high numbers of Gram-positive bacteria. For bacterial cultures with a saliva background, no difference in gDNA extraction efficacy was observed between the two methods. CONCLUSIONS: The efficiency of a bacterial DNA extraction method is not only affected by the bacterial cell wall structure but also by the sample milieu. The MP method provided superior gDNA extraction efficiency when the samples contained a single bacterial species, whereas either of the BMP and MP methods could be applied with similar efficiencies to samples containing multiple species of bacteria.

Plasmonic Colloidosome-Based Multifunctional Platform for Bacterial Identification and Antimicrobial Resistance Detection.

Antimicrobial resistance (AMR) is an urgent threat to public health. Rapid bacterial identification and AMR tests are important to promote personalized treatment of patients and to limit the spread of AMR. Herein, we explore the utility of plasmonic colloidosomes in bacterial analysis based on mass spectrometry (MS) and Raman scattering. It is found that colloidosomes can provide a rigid micrometer-size platform for bacterial culture and analysis. Coupled with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, this platform enables bacterial identification at the species level with cell counts as low as 50, >100 times more sensitive than the standard method of MALDI-TOF MS based bacterial identification. Coupled with Raman scattering, it can distinguish single bacterial cells at the strain level and recognize AMR at the single-cell level. These reveal the broad potential of the platform for flexible and versatile bacterial detection and typing.

Bacterial identification using a SCIEX 5800 TOF/TOF MALDI research instrument and an external database.

In our current study we were identifying 26 bacterial isolates using a SCIEX 5800 TOF/TOF MALDI instrument and an external database. The results were compared with the results of a Vitek® MS system and in case of discrepancies at the species level 16s rRNA sequencing was performed for further verification.

Heptylmannose-functionalized cellulose for the binding and specific detection of pathogenic E. coli.

We developed a chemical method to covalently functionalize cellulose nanofibers and cellulose paper with mannoside ligands displaying a strong affinity for the FimH adhesin from pathogenic E. coli strains. Mannose-grafted cellulose proved efficient to selectively bind FimH lectin and discriminate pathogenic E. coli strains from non-pathogenic ones. These modified papers are valuable tools for diagnosing infections promoted by E. coli, such as cystitis or inflammatory bowel diseases, and the concept may be applicable to other life-threatening pathogens.

[Performance of MALDI-TOF MS for the identification of gram-negative bacteria grown on eosin methylene blue (EMB) agar: A simple method for improving the effectiveness of identification]. / MALDI-TOF MS'nin EMB agarda üreyen gram-negatif bakterileri tanimlama performansi: Tani etkinligini arttiran basit bir yöntem.

Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is a new method that is increasingly used in microbiology laboratories due to its ability of reliable and rapid identification (ID) of bacteria and fungi. However, some problems emerge in the routine clinical diagnosis since only one gram-negative selective medium has been suggested up to date. Though EMB agar is one of the traditional gram-negative selective media, there is no data in the scientific literature, about the ID performance of MALDI TOF MS with the gram-negative bacteria grown on this medium. In this study, we tested the ID performance of MALDI-TOF MS for gram-negative isolates on EMB agar and aimed to develop a rapid and easy sample preparation method for improving this performance. A total of 468 clinical isolates of gram-negative bacteria, consisting of 37 different species from 20 genera, were included in this study. The isolates were identified using the Vitek MS MALDI-TOF MS (Bio Mérieux, France) both directly from EMB agar, and also through a two-step colony washing (once with physiologic saline, and three times with 70% ethanol) method. The performances of these two IDs were compared. In the direct reading from EMB medium, 382 (81.6%) of 468 studied isolates were correctly identified at species level; no ID was detected for 80 (17%) isolates, and 6 (1.2%) isolates (four at the genus level) were misidentified Performance of MALDI-TOF MS directly from EMB agar was excellent (100%) for 14 species including Stenotrophomonas maltophilia, Klebsiella oxytoca, Salmonella spp., and Proteus mirabilis; and lowest for Providencia spp. (62.5%), Escherichia coli (70.5%) and Acinetobacter spp. (70.7%). Following the washing procedure which was performed about 20 min with simple laboratory equipment, 434 (92.7%) isolates were correctly identified at species level; 30 (6.4%) strains could not be identified, and four (0.85%) isolates (two at the genus level) were misidentified. Statistical analyses indicated that the washing procedure defined here significantly increased the overall ID performance of MALDI-TOF MS with EMB agar (p= 0.001), particularly with improving the IDs of those markedly dye-absorbing genera, such as E.coli and A.baumannii. In this study, EMB agar which has no data until today on its suitability for mass spectrometric identification has been shown to be useful for bacterial identification with MALDI-TOF MS. In addition, the unidentified gram-negative bacteria in the direct reading from the EMB medium have been shown to be identified after the colony washing method as described here. Determination of the different medium alternatives will contribute to effective usage of MALDI-TOF MS in microbiology laboratories.

A Simple DNAzyme-Based Fluorescent Assay for Klebsiella pneumoniae.

Pathogenic bacteria pose a serious threat to public health, and the rapid and cost-effective detection of such bacteria remains a major challenge. Herein, we present a DNAzyme-based fluorescent paper sensor for Klebsiella pneumoniae. The DNAzyme was generated by an in vitro selection technique to cleave a fluorogenic DNA-RNA chimeric substrate in the presence of K. pneumoniae. The DNAzyme was printed on a paper substrate in a 96-well format to serve as mix-and-read fluorescent assay that exhibits a limit of detection (LOD) 105  CFUs mL-1 . Evaluated with 20 strains of clinical bacterial isolates, the DNAzyme produced the desired fluorescence signal with the samples of K. pneumoniae, regardless of their source or drug resistance. The assay is simple to use, rapid, inexpensive, and avoids the complex procedures of sample preparation and equipment. We believe that this DNAzyme-based fluorescent assay has potential for practical applications to identify K. pneumoniae.

Limitations of routine MALDI-TOF mass spectrometric identification of Acinetobacter species and remedial actions.

A set of 204 taxonomically well-defined strains belonging to 17 Acinetobacter spp., including 11 recently described species (A. albensis, A. bohemicus, A. colistiniresistens, A. courvalinii. A. dispersus, A. gandensis, A. modestus, A. proteolyticus, A. seifertii, A. variabilis, and A. vivianii) and six species of the so-called haemolytic clade (A. beijerinckii, A. gyllenbergii, A. haemolyticus, A. junii, A. parvus, and A. venetianus), were subjected to MALDI-TOF mass spectrometric profiling. The identification outputs were evaluated using the current version (8.0.0.0) of the commercially available Bruker Daltonics, Biotyper database, which does not contain reference entries for six of the species tested. Up to 29% of the strains were falsely identified as different Acinetobacter spp. present in the Biotyper database, resulting mostly from the close phylogenetic relationship of species of the haemolytic clade. To obtain more reliable identification, extending the commercial database showed only partial improvement, while the use of an alternative MALDI matrix solution (strongly acidified ferulic acid) allowed correct identification of nearly all problematic strains.

Genotyping based on thermal denaturation of amplification products identifies species of the Mycobacterium tuberculosis complex.

PURPOSE: To develop a fast and inexpensive genotyping assay to identify the Mycobacterium tuberculosis complex (MTC) species most prevalent in human tuberculosis (TB), based on the thermal denaturation profiles of PCR products from mycobacterial 16S rDNA and three MTC genomic regions of difference (RD). METHODOLOGY: Genotypes were determined by the presence and thermal denaturation profiles of the amplicons generated in the 'preliminary' PCR mixture (16S rDNA), followed by those of the simultaneous D1 (RD9+, RD1-) and D2 (RD4+, RD4-) PCR mixtures. The 16S rDNA profile identifies the genus Mycobacterium; the absence of any additional RD profile identifies Mycobacterium non-tuberculous (MNT) strains; additional RD4+ and RD9+ profiles without RD1- identify M. tuberculosis; an additional RD4+ profile per se identifies M. africanum; an additional RD4- profile per se identifies Mycobaterium bovis; additional RD1- and RD4- profiles identify M. bovis BCG. RESULTS: Genotypes of a panel with 44 mycobacterial strains coincided in 16 MB and five non-MTC strains; in the remaining 23 MTC strains, 17 MTB and five MA concordant genotypes and one discordant MB genotype were resolved. The genotypes of 13 human and bovine MTC isolates coincided in all four MB and eight of the nine MTB isolates. CONCLUSION: Sensitivity, specificity and positive and negative predictive values of the method are 100 % for the genus Mycobacterium, which resolves MB, MTB and MA genotypes. Species/genotype agreement is 97.7 % for the panel and 92.3 % for the MTC isolates. This method may be advantageously used to identify the most prevalent MTC species in humans.

Use of the mtrR Gene for Rapid Molecular Diagnosis of Neisseria gonorrhoeae and Identification of the Reduction of Susceptibility to Antibiotics in Endocervical Swabs.

BACKGROUND: Neisseria gonorrhoeae is one of the main etiological agents of sexually transmitted diseases. The asymptomatic course of the infection and its resistance to antibiotics can lead to pelvic inflammatory disease and infertility. OBJECTIVES: We developed a polymerase chain reaction (PCR) test using the methyltetrahydrofolate homocysteine methyltransferase reductase (mtrR) gene to identify N. gonorrhoeae and detect reduced susceptibility to antibiotics. MATERIAL AND METHODS: We analysed 250 samples of endocervical exudate from infertile women with a negative diagnosis of N. gonorrhoeae. We designed NGmtr primers to detect N. gonorrhoeae and identify the antibiotic-resistant strain. RESULTS: Of the 250 samples, 60 (24%) tested positive for N. gonorrhoeae using real-time PCR. Our study was validated using the HO primers and the Seeplex STD6 ACE System, with a 100% correlation. Furthermore, the NGmtr primers are specific for N. gonorrhoeae and not for other species. Additionally, the curves generated by real-time PCR differed between wild and variant strains (10.93%). The dissociation temperatures for the wild and variant strains were 86.5 and 89 °C, respectively. CONCLUSIONS: The NGmtr primers enabled us to identify N. gonorrhoeae strains with or without reduction of susceptibility to antibiotics. Therefore, this work constitutes a tool that will facilitate the diagnosis of this infection for a low cost and improve patient quality of life.

A liquid bead array for the identification and characterization of fljB-positive and fljB-negative monophasic variants of Salmonella Typhimurium.

Salmonella1,4,[5],12:i:- accounts currently for one of the most common serotypes observed worldwide. These isolates do not express the FljB flagellin and mostly derive from Salmonella Typhimurium. They are therefore termed Salmonella Typhimurium monophasic variants (STMV) and are considered of comparable public health risk. Since serological identification of the somatic and flagellar antigens of STMV is not sufficient to demonstrate relatedness with Salmonella Typhimurium, additional assays detecting genetic markers unique to Salmonella Typhimurium are required. In addition, identification of the mutations affecting expression of the flagellar gene fljB can be useful to support the monophasic character observed phenotypically. Finally, genetic subtyping of the various mono- and biphasic Salmonella Typhimurium clonal groups can facilitate their epidemiological follow-up. Here, we present a home-made liquid bead array able to fulfill these requirements. This array confirmed the monophasic character of 240 STMV isolates collected in Belgium during 2014-2015 and identified 10 genetic subtypes. Microevolution in and around the fljB locus linked to IS26 insertions is probably one of the driven force accounting for STMV population diversity. Thanks to its open design, other genetic signatures could later be merged to the assay to subtype additional STMV clonal groups and to detect rare mutations.

Rapid and cost-effective identification and antimicrobial susceptibility testing in patients with Gram-negative bacteremia directly from blood-culture fluid.

Rapid pathogen identification (ID) and antimicrobial susceptibility testing (AST) in bacteremia cases or sepsis could improve patient prognosis. Thus, it is important to provide timely reports, which make it possible for clinicians to set up appropriate antibiotic therapy during the early stages of bloodstream infection (BSI). This study evaluates an in-house microbiological protocol for early ID as well as AST on Gram negative bacteria directly from positive monomicrobial and polymicrobial blood cultures (BCs). A total of 102 non-duplicated positive BCs from patients with Gram-negative bacteremia were tested. Both IDs and ASTs were performed from bacterial pellets extracted directly from BCs using our protocol, which was applied through the combined use of a MALDI-TOF MS and Vitek2 automated system. The results of our study showed a 100% agreement in bacterial ID and 98.25% categorical agreement in AST when compared to those obtained by routine conventional methods. We recorded only a 0.76% minor error (mE), 0.76% major error (ME) and a 0.20% very major error (VME). Moreover, the turnaround time (TAT) regarding the final AST report was significantly shortened (ΔTAT = 8-20 h, p < 0.00001). This in-house protocol is rapid, easy to perform and cost effective and could be successfully introduced into any clinical microbiology laboratory. A final same-day report of ID and AST improves patient management, by early and appropriate antimicrobial treatment and could potentially optimize antimicrobial stewardship programs.

Rapid detection of Haemophilus parasuis using cross-priming amplification and vertical flow visualization.

Haemophilus parasuis infection is of considerable economic importance in the swine industry due to high morbidity and mortality in naive swine populations. Accurate detection and identification of the causative agent are difficult, yet necessary, for disease control. In this study, a simple and rapid method of cross-priming amplification (CPA) with a vertical flow (VF) visualization strip was established to detect H. parasuis. The reaction can specifically identify 15 serovar reference strains and 57 clinically isolated strains of H. parasuis, with a detection limit of 14CFU. The performance of the CPA-VF assay was evaluated and compared with that of species-specific PCR by testing 62 clinical culture-positive specimens of H. parasuis. The entire process, from specimen processing to analysis of the results, can be completed in 2h without a complicated apparatus. The convenience and speed of the CPA-VF assay in this study make it a suitable choice for epidemiological investigation and point-of-care testing (POCT) for H. parasuis infection.

Analysis of isoniazid and rifampicin resistance in Mycobacterium tuberculosis isolates in Morocco using GenoType® MTBDRplus assay.

OBJECTIVES: The number of multidrug-resistant tuberculosis (MDR-TB) cases is rising worldwide. The present investigation aimed to evaluate, using the GenoType® MTBDRplus assay, the most common mutations associated with rifampicin (RIF) and isoniazid (INH) resistance among resistant strains in Morocco. METHODS: A total of 319 Mycobacterium tuberculosis isolates sent to the National Tuberculosis Reference Laboratory between 2013 and 2015 were subjected to GenoType® MTBDRplus for detecting M. tuberculosis and determination of drug susceptibility. Correlation of the minimum inhibitory concentrations (MICs) of INH with genotypic assay results was carried out for 97 MDR-TB strains. Various concentrations of INH were tested. RESULTS: The most frequent mutations observed were rpoBS531L (67.2%) and katGS315T1/2 (66.5%). Isolates with inhA gene mutation, katG gene mutation, and dual mutations in katG and inhA had MICs ranging from 0.5-1µg/mL, 2-10µg/mL and ≥12µg/mL, respectively. CONCLUSION: In Morocco, 66.5% and 76.7% of M. tuberculosis strains carried mutations causing high-level resistance to INH and RIF, respectively.

Identification of Brucella genus and eight Brucella species by Luminex bead-based suspension array.

Globally, unpasteurized milk products are vehicles for the transmission of brucellosis, a zoonosis responsible for cases of foodborne illness in the United States and elsewhere. Existing PCR assays to detect Brucella species are restricted by the resolution of band sizes on a gel or the number of fluorescent channels in a single real-time system. The Luminex bead-based suspension array is performed in a 96-well plate allowing for high throughput screening of up to 100 targets in one sample with easily discernible results. We have developed an array using the Bio-Plex 200 to differentiate the most common Brucella species: B. abortus, B. melitensis, B. suis, B. suis bv5, B. canis, B. ovis, B. pinnipedia, and B. neotomae, as well as Brucella genus. All probes showed high specificity, with no cross-reaction with non-Brucella strains. We could detect pure DNA from B. abortus, B. melitensis, and genus-level Brucella at concentrations of ≤5 fg/µL. Pure DNA from all other species tested positive at concentrations well below 500 fg/µL and we positively identified B. neotomae in six artificially contaminated cheese and milk products. An intra-laboratory verification further demonstrated the assay's accuracy and robustness in the rapid screening (3-4 h including PCR) of DNA.

Evaluation of the multiplex PCR Allplex-GI assay in the detection of bacterial pathogens in diarrheic stool samples.

Rapid and accurate detection of the pathogens that cause gastrointestinal infection is important for appropriate therapy and proper infection control. This study assesses the performance of a new molecular assay for simultaneous detection of 13 different gastrointestinal bacteria in stool specimens. Using the Allplex GI-Bacteria (AGI-BI/AGI-BII) assay, a total of 394 stool samples were tested and the results were compared with culturing on selective differential followed by identification by mass spectroscopy. Discordant results were analyzed by a different multiplex PCR method, the Fast-Track Diagnostics Bacterial gastroenteritis (FTD-BG). The routine method (RM) detected 109 (27.7%) positive samples and the Allplex-GI assay, 261 (66.2%). Analysis of discordant results revealed that the molecular assay detected 44 pathogens that were not detected by the RM, including 23 Campylobacter spp., 11 Salmonella spp, 3 Y. enterocolitica, 2 EIEC/Shigella spp, 2 E. coli 0157, 2 C. difficile and 1 Aeromonas spp. Five cases not detected by the molecular method were detected by the RM (3 Aeromonas spp, 1 Salmonella spp and 1 Y. enterocolitica). For all targets, the percentages of sensitivity and specificity were >95%, except for Aeromonas spp., which were 81% and 99% respectively. This study suggests that Allplex-GI multiplex PCR is a sensitive and specific assay that enables a rapid and accurate diagnosis of bacterial gastrointestinal infections.

The Evolution of Strain Typing in the Mycobacterium tuberculosis Complex.

Tuberculosis (TB) is a contagious disease with a complex epidemiology. Therefore, molecular typing (genotyping) of Mycobacterium tuberculosis complex (MTBC) strains is of primary importance to effectively guide outbreak investigations, define transmission dynamics and assist global epidemiological surveillance of the disease. Large-scale genotyping is also needed to get better insights into the biological diversity and the evolution of the pathogen. Thanks to its shorter turnaround and simple numerical nomenclature system, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing, based on 24 standardized plus 4 hypervariable loci, optionally combined with spoligotyping, has replaced IS6110 DNA fingerprinting over the last decade as a gold standard among classical strain typing methods for many applications. With the continuous progress and decreasing costs of next-generation sequencing (NGS) technologies, typing based on whole genome sequencing (WGS) is now increasingly performed for near complete exploitation of the available genetic information. However, some important challenges remain such as the lack of standardization of WGS analysis pipelines, the need of databases for sharing WGS data at a global level, and a better understanding of the relevant genomic distances for defining clusters of recent TB transmission in different epidemiological contexts. This chapter provides an overview of the evolution of genotyping methods over the last three decades, which culminated with the development of WGS-based methods. It addresses the relative advantages and limitations of these techniques, indicates current challenges and potential directions for facilitating standardization of WGS-based typing, and provides suggestions on what method to use depending on the specific research question.

Traditional approaches versus mass spectrometry in bacterial identification and typing.

Biochemical methods such as metabolite testing and serotyping are traditionally used in clinical microbiology laboratories to identify and categorize microorganisms. Due to the large variety of bacteria, identifying representative metabolites is tedious, while raising high-quality antisera or antibodies unique to specific biomarkers used in serotyping is very challenging, sometimes even impossible. Although serotyping is a certified approach for differentiating bacteria such as E. coli and Salmonella at the subspecies level, the method is tedious, laborious, and not practical during an infectious disease outbreak. Mass spectrometry (MS) platforms, especially matrix assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS), have recently become popular in the field of bacterial identification due to their fast speed and low cost. In the past few years, we have used liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approaches to solve various problems hindering serotyping and have overcome some insufficiencies of the MALDI-TOF-MS platform. The current article aims to review the characteristics, advantages, and disadvantages of MS-based platforms over traditional approaches in bacterial identification and categorization.