MALDI-TOF MS Method Research Articles

Best Practices for the Analytic Phase of Anaerobic Bacteriology

The analytic process for identification of anaerobic bacteria involves direct specimen Gram stains and workup of growth in culture. Direct Gram stains help to develop the initial differential, aiding in guiding both the extent of laboratory workup and clinician decision making for patient management. Workup of anaerobic growth should involve an aerotolerance test to assess aerobic growth and to compare growth rates in anaerobic versus aerobic environments. While matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has become a routine means of anaerobe identification for many clinical laboratories, spot biochemical tests, such as indole and catalase, should be available in every laboratory that identifies anaerobes. If MALDI-TOF MS and biochemical methods are unsuccessful at providing an identification, 16S rRNA gene PCR and/or sequencing may be performed if the information impacts clinical care. The extent of culture workup to the level of presumptive identification versus definitive identification depends upon several factors, including the specimen source, direct Gram stain results, and the presence or absence of mixed microbiota. In addition to discussing the current best practices for anaerobic culture workup, commonly encountered anaerobic organism Gram stains and culture growth are detailed.

Investigation of The Efficacy of Three Different Protein Extraction Protocols and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Identification of Nontuberculous Mycobacteria

There are more than 160 defined nontuberculous mycobacteria (NTM) species within Mycobacterium genus. In recent years, the number of NTM species associated with human infections and the infections caused by them have been reported at increasing rates. The identification of these species by phenotypic methods is difficult, laborious, and unlikely to obtain reliable results. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) MALDI-TOF MS has proven to be a good method for the identification of bacteria and yeasts in routine laboratory practices. However, Mycobacterium species differ from other bacteria by their cell wall structures, less ribosomal protein content, and slower growth rates. A standardized and efficient protein extraction protocol for MALDI-TOF MS analysis of mycobacteria is essential. The aim of our study was to investigate the efficacy of different protein extraction protocols and the MALDI-TOF MS method in the diagnosis of NTM species. A total of 73 NTM isolates, grown in both solid and liquid media and previously identified with line probe assay, were evaluated with MALDI-TOF MS (Bruker Daltonics GmbH and Co. KG, Germany). Stock isolates were homogenized and decontaminated by N-Acetyl L-cysteine (NALC)/Sodium hydroxide (NaOH) method. For solid media, isolates were inoculated on Löwenstein-Jensen medium and incubated at 35˚C in a normal atmosphere. For liquid media culture, BD BACTEC MGIT 960 automated system (Becton, Dickinson, Sparks, MD, USA) was performed according to the manufacturer's instructions. For the identification of all isolates by MALDI-TOF MS, the manufacturer's recommended protein extraction protocol (Protocol 1) was compared with the two other protocols, using a simplified extraction procedure (Protocol 2), and freezing temperature (Protocol 3). In the liquid media analysis, the rates of the isolates identified by MALDI-TOF MS (score≥ 2.0) for Protocol 1, 2, and 3 were found as 84.93% (n= 62), 63.01% (n= 46), and 43.83% (n= 32), respectively. In the solid media analysis, the rates of the isolates with an identification score of ≥ 2.0 for the protocols with the same order were determined as 87.67% (n= 64), 52.05% (n= 38), and 31.50% (n= 23), respectively. Isolates grown in both solid and liquid media were identified in the same species level in all three protocols, regardless of the identification values and misidentification was not presented. When the reliable identification score was evaluated as ≥ 2.0 in our study, the manufacturer's recommended MYCOEX IVD procedure was found to be the most effective method for the isolates grown in both liquid and solid media. In conclusion, MALDI-TOF MS has the potential to be a reliable, easy-to-use and fast method that can be used in routine practice for the identification of NTM species with its standardized protein extraction protocols.

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis.

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.

Direct Identification, Antimicrobial Susceptibility Testing, and Extended-Spectrum β-Lactamase and Carbapenemase Detection in Gram-Negative Bacteria Isolated from Blood Cultures.

PurposeTo shorten the turnaround time for blood culture (BC) analyses, a rapid method was developed for the direct identification, antimicrobial susceptibility testing (AST), and multidrug resistance testing of bacteria-positive BCs.Materials and MethodsThe mixtures in BC bottles were treated with the multistep centrifugation method developed here and the conventional culture-based method. The bacterial sediment obtained after centrifugation was analyzed directly with MALDI-TOF MS and Vitek 2 Compact, and AST was performed directly with the Kirby–Bauer (K–B) disk diffusion, VITEK 2 Compact, and E-test methods. Extended spectrum lactamases (ESBLs) were detected with discs containing cefotaxime, cefotaxime/clavulanate, ceftazidime, and ceftazidime/clavulanate, and carbapenemase was detected with the modified carbapenem inactivation method (mCIM) and EDTA-mCIM (eCIM).ResultsAll the results of direct testing were compared to those of the conventional methods, to evaluate the accuracy of the direct methods. The accuracies of the direct Vitek 2 Compact and MALDI-TOF MS methods were 95.5% (214/224) and 90.2% (202/224), respectively. Direct AST with K–B, Vitek 2, and E-test showed category agreement of 96.0% (2611/2721), 96.1% (2614/2721), and 97.4% (2650/2721), respectively, and the major errors and very major errors were < 2% for all three methods. In the direct determination of ESBLs, the results for cefotaxime combined with cefotaxime/clavulanate were completely consistent with those after the standard isolation method. The carbapenemase detection rate with direct mCIM and eCIM was exactly the same as that with the standard method.ConclusionThese direct procedures based on multistep centrifugation are not only highly accurate but are appropriate for clinical laboratory use because the turnaround time is shorter.

Identification of Dominant Strains in Liu Shenqu by MALDI-TOF MS and DNA Sequencing Methods

Liu Shenqu has been widely used to treat the weakness of spleen and stomach, indigestion, etc. in China. As a fermented product, strains make an important role in the fermentation process, which will affect the quality of Liu Shenqu. Therefore, it is important to identify the dominant strains in the fermentation process of Liu Shenqu. In this study, we aim to identify the dominant strains in Liu Shenqu through Matrix-assisted laser desorption ionization time-of-flight mass spectrometry Biotyper (MALDI-TOF MS) combined with DNA sequencing methods. This research involves two parts: MALDI-TOF MS identified the dominant bacteria, and the Sanger sequencing method identified the dominant fungi. Total number of 21 bacterial species were identified by MALDI-TOF MS and 21 fungi species were identified by Sanger sequencing. We searched the types of enzymes in the identified strains above based on the GB2760-2014 National Food Safety Standard and Food Additives Use Standard. Compared the types of enzymes reported in Liu Shenqu with the types of enzymes retrieved in GB2760-2014 National Food Safety Standard and Food Additives Use Standard, Aspergillus oryzae and Rhizopus oryzae were finally determined to be the dominant strains in Liu Shenqu. This study showed that MALDI-TOF MS combined with DNA sequencing methods could be used for identification the dominant strains in Liu Shenqu. This strategy is promising for application to strains identification of other fermented products.

Bacterial counts of hospital linen and effectiveness of laundry process

Background: Health care linen is a source of healthcare-associated infections. Hospital laundry services play a vital role in linen decontamination. Effective laundry is essential in preventing hospital-acquired infections, especially in multidrug-resistant organisms. Aims and Objectives: The study aimed to check the microbial load in linen after the laundry process and the during the supply chain in the hospital. We investigated the bacterial load in linen before and after the laundering process and the linen from stored linen banks. Materials and Methods: Linens (n=125) from the hospital were collected in different stages of the laundry process [31 linen before decontamination; 94 linen after decontamination (Post washing = 30; Linen Bank = 34; Ward 30)]. A square inch of linen from each linen was vortexed in normal saline, and colony forming units (CFU)/inch of linen were calculated using appropriate media. The identification and susceptibility of organisms were performed using MALDI-TOF MS and CLSI disk diffusion methods, respectively. The difference in the load of the organism was analysed between different stages of the laundry and storage process. Results: We found that100% of pre-decontamination linen harbour viable organisms (22.59% including multidrug-resistant pathogenic organisms: MDROs), which decreased to 40% (no MDROs) post-decontamination. The overall load was reduced by a factor of 103.77. Conclusion: The microbial contamination of linen is common, and there is an urgent need to develop consensus on the microbial examination frequency and acceptable range of organisms on hospital linen worldwide.

MALDI-TOF MS for rapid detection and differentiation between Tet(X)-producers and non-Tet(X)-producing tetracycline-resistant Gram-negative bacteria

ABSTRACT The extensive use of tetracycline antibiotics has led to the widespread presence of tetracycline-resistance genes in Gram-negative bacteria and this poses serious threats to human and animal health. In our previous study, we reported a method for rapid detection of Tet(X)-producers using MALDI-TOF MS. However, there have been multiple machineries involved in tetracycline resistance including efflux pump, and ribosomal protection protein. Our previous demonstrated the limitation in probing the non-Tet(X)-producing tetracycline-resistant strains. In this regard, we further developed a MALDI-TOF MS method to detect and differentiate Tet(X)-producers and non-Tet(X)-producing tetracycline-resistant strains. Test strains were incubated with tigecycline and oxytetracycline in separate tubes for 3 h and then analyzed spectral peaks of tigecycline, oxytetracycline, and their metabolite. Strains were distinguished using MS ratio for [metabolite/(metabolite+ tigecycline or oxytetracycline)]. Four control strains and 319 test strains were analyzed and the sensitivity was 98.90% and specificity was 98.34%. This was consistent with the results obtained from LC-MS/MS analysis. Interestingly, we also found that the reactive oxygen species (ROS) produced by tetracycline-susceptible strains were able to promote the degradation of oxytetracycline. Overall, the MALDITet(X)-plus test represents a rapid and reliable method to detect Tet(X)-producers, non-Tet(X)-producing tetracycline-resistant strains, and tetracycline-susceptible strains.

Novel members of bacterial community during a short-term chilled storage of common carp (Cyprinus carpio)

This work aimed to identify the key members of the bacterial community growing on common carp (Cyprinus carpio) fillets during chilled storage with next-generation sequencing (NGS) and cultivation-dependent methods. Carp fillets were stored for 96 h at 2 °C and 6 °C with and without a vacuum package, and an additional frozen-thawed storage experiment was set for 120 days. Community profiles of the initial and stored fish samples were determined by amplicon sequencing. Conventional microbial methods were used parallelly for the enumeration and cultivation of the dominant members of the microbial community. Cultivated bacteria were identified with 16S rRNA sequencing and the MALDI-TOF MS method. Based on our results, the vacuum package greatly affected the diversity and composition of the forming microbial community, while temperature influenced the cell counts and consequently the microbiological criteria for shelf-life of the examined raw fish product. Next-generation sequencing revealed novel members of the chilled flesh microbiota such as Vagococcus vulneris or Rouxiella chamberiensis in the vacuum-packed samples. With traditional cultivation, 161 bacterial strains were isolated and identified at the species level, but the identified bacteria overlapped with only 45% of the dominant operational taxonomic units (OTUs) revealed by NGS. Next-generation sequencing is a promising and highly reliable tool recommended to reach a higher resolution of the forming microbial community of stored fish products. Knowledge of the initial microbial community of the flesh enables further optimization and development of processing and storage technology.

Association of COMT Polymorphisms with Multiple Physical Activity-Related Injuries among University Students in China.

The catechol-O-methyltransferase (COMT) is a candidate gene to provide promising evidence of psychiatric disorders, but there is a knowledge gap between the genetic factor and multiple physical activity-related injuries (PARIs). The aim of this study was to explore the contribution of COMT to the risk of PARIs among university students in the Chinese Han population. We can further search for the intrinsic risk factors for the occurrence of multiple physical activity injuries and provide a scientific basis for early screening and precise intervention for the high-risk group of college students with multiple PARIs. A 1:1 matched case-control study of 61 PARIs cases and 61 healthy controls were carried out. DNA samples of the participants were isolated from saliva and genotyped on eight SNPs of the COMT gene (rs9265, rs4680, rs6269, rs4818, rs4633, rs165655, rs165656, and rs165722) using the MALDI-TOF MS method. We found that rs6269 and rs4818 were significantly associated with PARIs, and rs6269-GG and rs4818-GG contributed to the reduced risk of PARIs. Further haplotype analysis showed a four-marker C-G-C-G haplotype (rs165722-rs6269-rs4633-rs4818) acted with a protective role in the development of PARIs (p = 0.037; OR: 0.474, 95% CI: 0.269 to 0.834). However, the interactions between club membership and rs6269 or rs4818 would significantly increase the risk of PARIs (both p < 0.001, OR: 5.121 and 4.977, respectively). This is the first study to find the contribution of COMT to PARIs occurrence, suggesting that the COMT polymorphisms and the gene–environment interactions may alter the risk of PARIs.

Current Scenario and Challenges in the Direct Identification of Microorganisms Using MALDI TOF MS.

MALDI TOF MS-based microbial identification significantly lowers the operational costs because of minimal requirements of substrates and reagents for extraction. Therefore, it has been widely used in varied applications such as clinical, food, military, and ecological research. However, the MALDI TOF MS method is laced with many challenges including its limitation of the reference spectrum. This review briefly introduces the background of MALDI TOF MS technology, including sample preparation and workflow. We have primarily discussed the application of MALDI TOF MS in the identification of microorganisms. Furthermore, we have discussed the current trends for bioaerosol detection using MALDI TOF MS and the limitations and challenges involved, and finally the approaches to overcome these challenges.

MALDI-TOF MS as a method for rapid identification of Phytophthora de Bary, 1876.

The number of described species of the oomycete genus Phytophthora is growing rapidly, highlighting the need for low-cost, rapid tools for species identification. Here, a collection of 24 Phytophthora species (42 samples) from natural as well as anthropogenic habitats were genetically identified using the internal transcribed spacer (ITS) and cytochrome c oxidase subunit I (COI) regions. Because genetic identification is time consuming, we have created a complementary method based on by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Both methods were compared and hypothesis that the MALDI-TOF MS method can be a fast and reliable method for the identification of oomycetes was confirmed. Over 3500 mass spectra were acquired, manually reviewed for quality control, and consolidated into a single reference library using the Bruker MALDI Biotyper platform. Finally, a database containing 144 main spectra (MSPs) was created and published in repository. The method presented in this study will facilitate the use of MALDI-TOF MS as a complement to existing approaches for fast, reliable identification of Phytophthora isolates.

Microorganism idenitfication – stability and correctness of mass spectrometry method (MALDI TOF MS)

Introduction: MALDI TOF MS method is increasingly used in routine microbiological diagnostics to identify clinical strains. The result of the identification test is based on the measurement of the mass, charge and flight time of the protein ions. This makes it possible to monitor and supervise the method using a numerical score developed with statistical techniques. Aim: The study aimed to determine the stability and correctness of the mass spectrometry method. Materials and methods: To evaluate the characteristics of the method, microbial identification tests were performed using reference strains. All tests were performed as part of the MALDI TOF MS internal quality control system. Results: All reference strains (100%) were correctly identified to the species level, although the score values were not always within the reliability criteria of the results established by the producer. It was found that the mean values of the score were from 2.000 – 2.299 (49.2%) and 2.300 – 3.000 (50.0%). The coefficient of variation for control tests performed in the consecutive years ranged from 5.18 – 6.56%, which evidence of the high stability of the method. For individual species, reproducibility precision over the 8 years ranged from 2.89% (n = 13) for Enterococcus faecalis ATCC 51299 to 7.02% (n = 28) for Klebsiella pneumoniae ATCC 700603, which proves the high precision of measurements. Conclusions: The mass spectrometry method is characterized by very high stability and correctness. The intra-laboratory quality control system using reference strains is a useful tool for monitoring and supervising the method and laboratory personnel competency performing identification tests during routine microbiological work.

Study of Cases of Bacteremia with Gram-Negative Pathogens with Multiple and Extreme Antibiotic Resistance in Real Clinical Practice

Aim:To study cases of bacteremia caused by multidrug-resistant (MDR) and extremely-resistant (XDR) gram-negative bacteria in the departments of a multidisciplinary hospital.Material and Methods.Since 2017, a retrospective epidemiological study has been conducted to investigate cases of infections with bacteremia caused using gram-negative bacteria with multiple or extreme antibiotic resistance. The pathogens were identified using the MALDI-TOF MS method, the sensitivity to antibacterial drugs was determined using the automatic Phoenix system, the beta-lactamase genes were detected using real-time PCR on a GeneXpert analyzer.Results.In 2017, bacteremia caused by MDR and XDR pathogens was detected in 42 and 76 patients, in 2018 — in 57 and 81 patients, in 2019 — in 65 and 111 patients, respectively. During three years of observation, the most frequent XDR microorganisms were, in descending order,Klebsiella pneumoniae,Acinetobacter baumannii,Pseudomonas aeruginosa. In 2019, among 11K.pneumoniaestrains, class D carbapenemase (OXA-48) was detected in 5 cases (45.5%), 3 strains produced NDM metallo-carbapenemase, and 3 strains had a combination of NDM and OXA-48 enzymes. Infections caused by MDR and XDR pathogens were characterized by high mortality. Thus, the relative risk of death in patients with XDR infection was 1.33 times higher than in patients from the MDR group (95% CI 1.04-1.69, P&lt;0.05) in 2019. The duration of hospital stay also increased: in the XDR group — up to 29.5 days, in the MDR group – up to 16.4 days, with an average length of hospitalization of 6.0 bed-days in 2019.Conclusions.K.pneumoniaeandA.baumanniiwith extreme antibiotic resistance are the main causative agents of severe late nosocomial infections in immunosuppressed patients. Nosocomial infections with bacteremia caused by gram-negative bacteria with the XDR phenotype and resistant to carbapenems are characterized by a high mortality rate (from 72 to 80%), and increase the duration of hospitalization by more than 4 times.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Antimicrobial Susceptibility Testing.

The advent of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology has dramatically improved the accuracy and speed of diagnostics. However, this progress has mainly been limited to the identification of microorganisms, whereas the practical improvement of antimicrobial susceptibility testing (AST) still lags behind. MALDI-TOF MS-based approaches include the detection of selected resistance mechanisms and the universal phenotypic AST. This minireview focuses on the discussion of those MALDI-TOF MS methods that allow universal growth-based phenotypic AST. The method of minimal profile change concentrations (MPCC) is based on detecting proteome modification in the presence of an antimicrobial. Using stable-isotope labeling, characteristic mass shifts in the presence of an antimicrobial indicate the incorporation of the isotopic labels, and thus the viability and resistance of the microorganism. For MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA), microorganisms are incubated with or without an antimicrobial, followed by cell lysis, protein extraction, and transfer of the cell lysate onto a MALDI target plate. Using the internal standard, peak intensities are correlated to the amount of microbial proteins, and the relative microbial growth is calculated. Most recent development in the field is the direct-on-target microdroplet growth assay (DOT-MGA). Here, incubation of microorganisms with antimicrobials takes place directly on spots of a MALDI target in the form of microdroplets. After incubation, nutrient medium is removed by dabbing with absorptive material. Resistant microorganisms grow despite the presence of antimicrobial, and their amplified biomass is detected by MALDI-TOF MS. Finally, an outlook is provided for further assay improvements.

Comparative Assessment of Matrix-assisted Laser Desorption Ionization-time of Flight Mass Spectrometry (MALDI-TOF-MS) and Conventional Methods in the Identification of Clinically Relevant Yeasts.

Background: Candida species are generally identified by conventional methods such as germ tube or morphological appearance on cornmeal agar (CMA), biochemical methods using API kits, and molecular biological techniques. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) has revolutionized the identification of fungi reducing the turnaround time of days to minutes.Purpose: To compare the performance of MALDI-TOF MS and conventional methods in the identification of clinically relevant yeasts.Materials and methods: In this study, Candida identifications on CMA are compared with the results obtained on MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). Discrepant results were confirmed by sequencing internal transcribed spacer (ITS) regions of rDNA.Results: A total of 114 clinical Candida species isolated from blood cultures were isolated and identified with conventional methods as well as with the MALDI-TOF-MS system. The agreement between the two test results were analyzed using Inter-rater reliability analysis (Cohen's Kappa) in SPSS Software Version 24 (IBM Corp., Armonk, NY). Overall, there was substantial agreement (Cohen's kappa=0.763) between the two methods. A value between 0.61 and 0.80 is classified under substantial. The most frequently isolated bloodstream Candida species included Candida albicans, C. tropicalis, C. parapsilosis, C.lusitaniae, C. glabrata which were accurately identified by MALDI-TOF-MS. When compared with conventional identification methods, MALDI-TOF-MS results are more reliable and rapid for Candida identification.

Prevalence of rare F5 variants in general population from Bosnia and Herzegovina

Human gene F5, encoding coagulation factor V, was previously reported to be highly polymorphic. Apart from FV Leiden, several other rare variants have been detected in clinical practice and associated with thrombotic events, especially in cases when patient's phenotype and FV Leiden genotype were not in agreement. In this study, the prevalence of 17 rare F5 variants has been studied on a sample of 130 healthy adult individuals from the general Bosnian-Herzegovinian population. DNA was isolated from buccal swab samples, while genotyping was performed using MALDI-TOF MS method. The results have shown that Asp2194Gly and Met2120Thr are polymorphic in the study population with minor allele frequencies of 0.077 and 0.073, respectively. Additionally, these two variants were mutually exclusive with FV Leiden and none of them was positively associated with participants' family history of cardiovascular or cerebrovascular diseases. While the obtained results are in agreement with previously reported data for the general Caucasian populations, it is worth noting that only two rare F5 variants were detected in the study population, albeit at considerable frequencies. Still, scientific information on rare F5 variants is rather scarce and further studies aiming to assess functional importance of these variants, as well as their role as prothrombotic factors are necessary.

Occurrence of Listeria monocytogenes in Artisanal Cheeses from Poland and Its Identification by MALDI-TOF MS

Listeria monocytogenes is a foodborne pathogen. A source of infection can be artisanal cheeses. Identification of the Listeria species is important for the protection of public health and the food industry. This study aimed to examine artisanal cheeses for the presence of L. monocytogenes and the effectiveness of the MALDI-TOF MS method in the identification of the L. monocytogenes isolates. A total of 370 samples of artisanal cheeses were examined. L. monocytogenes was found in 23 cheese samples (6.2%). The reliability of L. monocytogenes identification achieved by MALDI-TOF MS was varied, and the vast majority of the isolates (27/32) were identified only to the secure genus, probable species level. This study showed that (i) the occurrence of L. monocytogenes in the artisanal cheeses was at a higher level than that in the other EU countries, (ii) the standard of species identification of L. monocytogenes isolates from artisanal cheeses achieved by MALDI-TOF MS was not satisfactory and (iii) the presence of L. monocytogenes in artisanal cheeses remains a problem with regard to the food safety criterion and a potential public health risk.

Streptococcus mutans detection on mother-child pairs using matrix-assisted laser desorption ionization – time of flight mass spectrometry and polymerase chain reaction

Background: Streptococcus mutans (S. mutans) bacteria mainly cause dental caries in children. These bacteria are not considered oral indigenous bacteria since they are transmitted from people around children during their deciduous teeth eruption. The detection of these bacteria can be used for dental caries prevention in children. Purpose: To determine the strain and serotype of S. mutans by using matrix assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) and polymerase chain reaction (PCR) on dental plaque samples taken from mother-child pairs. Methods: Sixteen dental plaque samples of mother-child pairs were cultured on brain heart infusion broth (BHIB) and mitis salivarius bacitracin (MSB) media until S. mutans colony isolates were obtained. Next, the isolates of S. mutans colony were introduced into the target plates of MALDI-TOF MS, and then ionized to become peptide mass fingerprint (PMF). Afterwards, the colony isolates were detected by database software. The detected S. mutans DNA then was extracted by using conventional 727 bp PCR (serotype C). Results: Six strains of S. mutans were detected by MALDI-TOF MS method. Five samples were classified into UA159, two samples were 3SN1, two samples were NFSM1, two samples were 11A1, two samples were U138, two samples were 4SM1, and one sample was classified into another bacterium. Five out of 16 samples were detected by PCR as serotype C (UA159). Conclusion: Six strains of S. mutans were detected, namely UA159, 3SN1, NFSM1, 11A1, U138, and 4SM1, one of them (UA159) was detected as serotype C.

A MALDI-TOF MS database for fast identification of Vibrio spp. potentially pathogenic to marine mollusks

In mollusk aquaculture, a large number of Vibrio species are considered major pathogens. Conventional methods based on DNA amplification and sequencing used to accurately identify Vibrio species are unsuitable for monitoring programs because they are time-consuming and expensive. The aim of this study was, therefore, to develop the MALDI-TOF MS method in order to establish a rapid identification technique for a large panel of Vibrio species. We created the EnviBase containing 120 main spectra projections (MSP) of the Vibrio species that are potentially responsible for mollusk diseases, comprising 25 species: V. aestuarianus, V. cortegadensis, V. tapetis and species belonging to the Coralliilyticus, Harveyi, Mediterranei, and Orientalis clades. Each MSP was constructed by the merger of raw spectra obtained from three different media and generated by three collaborating laboratories to increase the diversity of the conditions and thus obtain a good technique robustness. Perfect discrimination was obtained with all of the MSP created for the Vibrio species and even for very closely related species as V. europaeus and V. bivalvicida. The new EnviBase library was validated through a blind test on 100 Vibrio strains performed by our three collaborators who used the direct transfer and protein extraction methods. The majority of the Vibrio strains were successfully identified with the newly created EnviBase by the three laboratories for both protocol methods. This study documents the first development of a freely accessible database exclusively devoted to Vibrio found in marine environments, taking into account the high diversity of this genus.Key points• Development of a MALDI-TOF MS database to quickly affiliate Vibrio species.• Increase of the reactivity when faced with Vibrio associated with mollusk diseases.• Validation of MALDI-TOF MS as routine diagnostic tool.

High-throughput analysis of high-molecular weight glutenin subunits in 665 wheat genotypes using an optimized MALDI-TOF-MS method.

The online version contains supplementary material available at 10.1007/s13205-020-02637-z.