Microbial Identification Methods Research Articles

Typing and Characterization of Bacteria Using Bottom-up Tandem Mass Spectrometry Proteomics

Methods for rapid and reliable microbial identification are essential in modern healthcare. The ability to detect and correctly identify pathogenic species and their resistance phenotype is necessary for accurate diagnosis and efficient treatment of infectious diseases. Bottom-up tandem mass spectrometry (MS) proteomics enables rapid characterization of large parts of the expressed genes of microorganisms. However, the generated data are highly fragmented, making downstream analyses complex. Here we present TCUP, a new computational method for typing and characterizing bacteria using proteomics data from bottom-up tandem MS. TCUP compares the generated protein sequence data to reference databases and automatically finds peptides suitable for characterization of taxonomic composition and identification of expressed antimicrobial resistance genes. TCUP was evaluated using several clinically relevant bacterial species (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae), using both simulated data generated by in silico peptide digestion and experimental proteomics data generated by liquid chromatography-tandem mass spectrometry (MS/MS). The results showed that TCUP performs correct peptide classifications at rates between 90.3 and 98.5% at the species level. The method was also able to estimate the relative abundances of individual species in mixed cultures. Furthermore, TCUP could identify expressed β-lactamases in an extended spectrum β-lactamase-producing (ESBL) E. coli strain, even when the strain was cultivated in the absence of antibiotics. Finally, TCUP is computationally efficient, easy to integrate in existing bioinformatics workflows, and freely available under an open source license for both Windows and Linux environments.

Wine yeasts identification by MALDI-TOF MS: Optimization of the preanalytical steps and development of an extensible open-source platform for processing and analysis of an in-house MS database

Saccharomyces cerevisiae is the most important yeast species for the production of wine and other beverages. In addition, nowadays, researchers and winemakers are aware of the influence of non-Saccharomyces in wine aroma complexity. Due to the high microbial diversity associated to several agro-food processes, such as winemaking, developing fast and accurate methods for microbial identification is demanded. In this context, MALDI-TOF MS mass fingerprint provides reliable tool for fast biotyping and classification of microorganisms. However, there is no versatile and standardized method for fungi currently available. In this study, an optimized sample preparation protocol was devised for the biotyping of yeasts of oenological origin.Taking into account that commercially available reference databases comprise almost exclusively clinical microorganisms, most of them bacteria, in the present study a database of yeasts isolated from vineyards and wineries was created, and its accuracy was tested using industrial and laboratory yeast strains. In addition, the implementation of a program for MALDI-TOF MS spectra analysis has been developed as an extensible open-source platform for MALDI data processing and analysis with statistical techniques that has arisen from our previous experience working with MALDI data. The software integrates two R packages for raw MALDI data preprocessing: Continuous Wavelet Transform (CWT)-based algorithm and MassSpecWavelet. One of the advantages of the CWT is that it can be directly applied to a raw spectrum, without prior baseline correction.Mass fingerprints of 109 S. cerevisiae strains and 107 non-Saccharomyces isolates were generated by MALDI-TOF MS upon optimized sample preparation and instrument settings and analyzed for strain, species, and genus-level differentiation. As a reference method, for S. cerevisiae differentiation at strain level, the analysis of the polymorphism in the inter-delta region was chosen. The data revealed that MALDI-TOF MS can be used for the rapid and accurate identification of S. cerevisiae and non-Saccharomyces isolates at genus and species level. However, S. cerevisiae differentiation at strain level was not successfully achieved, and the differentiation among Metschnikowia species was also difficult.

Microbial Diversity in Daqu during Production of Luzhou Flavored Liquor

Yeast, bacterial, and mold strains in different stages during the daqubased production of Luzhou-flavored liquor were isolated and identified by analyzing the 26S rRNA, 16S rRNA, and internal transcribed spacer region sequences, respectively. In total, 113 yeast, 63 bacteria, and 128 mold strains were identified, with the majority identified to the species level and the remaining strains identified to the genus level. In the final daqu product (day 90), the dominant bacteria, yeast, and mold strains were Bacillus subtilis subsp. inaquosorum, Saccharomycopsis fibuligera, and Lichtheimia ramosa, respectively. Thus, in the finished product, the daqu microflora mainly comprised bacteria and molds, mostly consisting of heat-resistant spore-forming Bacillus spp. and filamentous fungi. Corresponding changes in the enzyme activity patterns during daqu production revealed that Mucor circinelloides f. circinelloides was strongly correlated with protease, whereas saccharifying enzyme activity was mainly correlated with Rhizopus oryzae. A relatively strong correlation was found between saccharifying and liquefied enzyme activities and Staphylococcus saprophyticus subsp. saprophyticus, Bacillus tequilensis, Weissella cibaria, and Enterobacter mori. These results indicate the benefits of molecular methods for detailed microbial isolation and identification and provide basic information for subsequent studies to reveal the functional microorganisms of daqu during the production of Luzhou-flavored liquor.

Dynamic changes of the respiratory microbiota and its relationship to fecal and blood microbiota in healthy young cats.

Advances in the field of metagenomics using culture-independent methods of microbial identification have allowed characterization of rich and diverse communities of bacteria in the lungs of healthy humans, mice, dogs, sheep and pigs. These data challenge the long held belief that the lungs are sterile and microbial colonization is synonymous with pathology. Studies in humans and animals demonstrate differences in the composition of airway microbiota in health versus disease suggesting respiratory dysbiosis occurs. Using 16S rRNA amplicon sequencing of DNA extracted from rectal and oropharyngeal (OP) swabs, bronchoalveolar lavage fluid (BALF), and blood, our objective was to characterize the fecal, OP, blood, and lower airway microbiota over time in healthy cats. This work in healthy cats, a species in which a respiratory microbiota has not yet been characterized, sets the stage for future studies in feline asthma in which cats serve as a comparative and translational model for humans. Fecal, OP and BALF samples were collected from six healthy research cats at day 0, week 2, and week 10; blood was collected at week 10. DNA was extracted, amplified via PCR, and sequenced using the Illumina MiSeq platform. Representative operational taxonomic units (OTUs) were identified and microbial richness and diversity were assessed. Principal component analysis (PCA) was used to visualize relatedness of samples and PERMANOVA was used to test for significant differences in microbial community composition. Fecal and OP swabs provided abundant DNA yielding a mean±SEM of 65,653±6,145 and 20,6323±4,360 sequences per sample, respectively while BALF and blood samples had lower coverage (1,489±430 and 269±18 sequences per sample, respectively). Oropharyngeal and fecal swabs were significantly richer than BALF (mean number OTUs 93, 88 and 36, respectively; p < 0.001) with no significant difference (p = 0.180) in richness between time points. PCA revealed site-specific microbial communities in the feces, and upper and lower airways. In comparison, blood had an apparent compositional similarity with BALF with regard to a few dominant taxa, but shared more OTUs with feces. Samples clustered more by time than by individual, with OP swabs having subjectively greater variation than other samples. In summary, healthy cats have a rich and distinct lower airway microbiome with dynamic bacterial populations. The microbiome is likely to be altered by factors such as age, environmental influences, and disease states. Further data are necessary to determine how the distinct feline microbiomes from the upper and lower airways, feces and blood are established and evolve. These data are relevant for comparisons between healthy cats and cats with respiratory disease.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass-Spectrometry (MALDI-TOF MS) Based Microbial Identifications: Challenges and Scopes for Microbial Ecologists.

Matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) based biotyping is an emerging technique for high-throughput and rapid microbial identification. Due to its relatively higher accuracy, comprehensive database of clinically important microorganisms and low-cost compared to other microbial identification methods, MALDI-TOF MS has started replacing existing practices prevalent in clinical diagnosis. However, applicability of MALDI-TOF MS in the area of microbial ecology research is still limited mainly due to the lack of data on non-clinical microorganisms. Intense research activities on cultivation of microbial diversity by conventional as well as by innovative and high-throughput methods has substantially increased the number of microbial species known today. This important area of research is in urgent need of rapid and reliable method(s) for characterization and de-replication of microorganisms from various ecosystems. MALDI-TOF MS based characterization, in our opinion, appears to be the most suitable technique for such studies. Reliability of MALDI-TOF MS based identification method depends mainly on accuracy and width of reference databases, which need continuous expansion and improvement. In this review, we propose a common strategy to generate MALDI-TOF MS spectral database and advocated its sharing, and also discuss the role of MALDI-TOF MS based high-throughput microbial identification in microbial ecology studies.

Impact of MALDI-TOF in the Routine Diagnostic Microbiology Laboratory in Alexandria University, Egypt

Background: In the last few years, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) has emerged as a useful tool for identification of microorganisms. Identification of the various bacterial genera has been found to be challenging using the routine phenotypic tests or commercially available automated systems relying on the biochemical identification of bacteria. Objectives: In this study, we aimed to evaluate the possible role of the MALDI-TOF system with its Biotyper software as an accurate and rapid method of bacterial identification. Methodology: This study was conducted over a period of one month, where 299 isolates were retrieved from 523 clinical samples. Results: MALDI TOF identified 275 (92%) isolates to the species level, 287 isolates (96%) to the genus level and no reliable identification for 12 (4%) isolates using the direct formic acid application method. When protein extraction was applied, valid results for species and genus identification were obtained for 296 and 299 isolates respectively, so the overall rate of identification rose to 99% and 100% respectively. Discordant results between MALDI TOF and conventional methods were confirmed by Vitek 2 and 16s rRNA sequencing. Conclusions: MALDI-TOF is a rapid, accurate and inexpensive method for microbial identification that rivals other identification systems available. Its relative ease of use and rapid turnaround time makes it a powerful tool in the microbiology laboratory.

Routine identification of microorganisms by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Success rate, economic analysis, and clinical outcome.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used in microbial identification. This study evaluated the performance of MALDI-TOF MS and investigated the economic and medical impact of MALDI-TOF MS implementation. A total of 12,202 clinical isolates collected from April to September 2013 were identified using MALDI-TOF MS, and the success rates in identifying isolates were analyzed. The differences in the processing time, cost of consumables, weight of waste, and clinical impact between MALDI-TOF MS and biochemical reaction were compared. MALDI-TOF MS successfully identified 96% of 12,202 isolates, including 96.8% of 10,502 aerobes, 90.5% of 1481 anaerobes, 93.8% of 81 yeasts, and 90.6% of 138 nontuberculous mycobacteria at the genus level. By using MALDI-TOF MS, the processing time for aerobes decreased from 32.5hours to 4.1hours, and that for anaerobes decreased from 71.5hours to 46hours. For detection of aerobes and anaerobes, the cost of consumables was estimated to decrease by US$0.9 per isolate, thus saving US$94,500 in total annual isolation. Furthermore, the weight of waste decreased six-fold, resulting in a reduction of 350kg/month or 4.2 tons/year. MALDI-TOF MS also increased the percentage of correct antibiotics treatment for Escherichia coli and Klebsiella pneumonia from 56.1% to 75% and shortened the initiation time of the correct antibiotic action from 3.3hours to 2.5hours. MALDI-TOF MS is a rapid, reliable, economical, and environmentally friendly method for routine microbial identification and may contribute to early appropriate antibiotic treatment in clinical settings.

The use of propidium monoazide in conjunction with qPCR and Illumina sequencing to identify and quantify live yeasts and bacteria

Culture-independent methods of microbial identification have been developed, which allow for DNA extraction directly from environmental samples without subjecting microbes to growth on nutrient media. These methods often involve next generation DNA sequencing (NGS) for identifying microbes and qPCR for quantifying them. Despite the benefits of extracting all DNA from the sample, results may be compromised by amplifying DNA from dead cells. To address this short-coming, the use of propidium monoazide (PMA) has been used to deactivate DNA in non-viable cells. Nevertheless, its optimization has not been fully explored under a variety of conditions. In this study, we optimized the PMA method for both yeasts and bacteria. Specifically, we explored the effect different PMA concentrations and different cell densities had on DNA amplification (as part of next generation DNA sequencing) from both dead and viable bacterial and yeast cells. We found PMA was effective in eliminating DNA that was associated with dead yeast and bacterial cells for all cell concentrations. Nevertheless, DNA (extracted from viable yeast and bacterial cells) amplified most abundantly when PMA concentration was at 6μM and when yeast densities ranged between 106 to 107CFU/mL and bacterial densities were approximately 108CFU/mL.

Comparison among four proposed direct blood culture microbial identification methods using MALDI-TOF MS.

Matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry facilitates rapid and accurate identification of pathogens, which is critical for sepsis patients. In this study, we assessed the accuracy in identification of both Gram-negative and Gram-positive bacteria, except for Streptococcus viridans, using four rapid blood culture methods with Vitek MALDI-TOF-MS. We compared our proposed lysis centrifugation followed by washing and 30% acetic acid treatment method (method 2) with two other lysis centrifugation methods (washing and 30% formic acid treatment (method 1); 100% ethanol treatment (method 3)), and picking colonies from 90 to 180min subculture plates (method 4). Methods 1 and 2 identified all organisms down to species level with 100% accuracy, except for Streptococcus viridans, Streptococcus pyogenes, Enterobacter cloacae and Proteus vulgaris. The latter two were identified to genus level with 100% accuracy. Each method exhibited excellent accuracy and precision in terms of identification to genus level with certain limitations.

Rapid Identification and Characterization of Microbial Pathogens by Molecular Biology Techniques.

Microbial infections including bacterial and viral infections are potentially life-threatening and require rapid identification and characterization of the microorganisms. Rapid and accurate detection of foodborne microorganisms from food, environment and clinical samples requires characterization to the species and/or subspecies level as well as determining their antibiotic susceptibility. These steps are important for: 1) selecting appropriate antibiotics, 2) reducing morbidity and mortality, and 3) reducing health care costs. Traditional microbiological detection starts with an enrichment step usually using a nonselective enrichment that allows injured cells to repair and multiply. These enrichment cultures are then subcultured on selective and differential agars. Typical and suspected colonies are then stained by Gram staining and subsequent biochemical identification, and susceptibility testing are performed. These steps usually take 3-7 days. Identification of virulence factors requires additional time and involves expensive phenotypic and genotypic tests. Traditional microbiological identification methods often do not provide prompt results for optimal early patient diagnosis and treatment management. In recent years, more and more studies have demonstrated the value of molecular biologic techniques to rapidly identify and genotype bacteria or virus in food, environment, and clinical samples. Molecular biology techniques are methods which generally involve the analysis and handling of nucleic acid and other molecules. Compared to traditional microbial identification methods, molecular biology methods can be used to detect and identify microorganisms simultaneously, which results in quicker diagnoses and treatments. The other advantages of molecular methods are that the protocols are of higher sensitivity and specificity. The aim of this hot topic is to provide an overview of current exciting molecular biology techniques that are employed to rapidly identify and characterize important pathogenic bacteria and viruses. The topic would update knowledge on prompt bacterial and viral identification and characterization methods, which can speed up the diagnosis and treatment of infectious diseases, improve epidemiologic screening, and reduce their morbidity and mortality. Staphylococcus aureus causes a wide range of suppurative infections, food poisoning and toxic shock syndrome. Methicillin resistant S. aureus (MRSA) is resistant to most β-lactam antibiotics. MRSA infection is more difficult to treat with commonly used antibiotics and thus more dangerous. A recent study showed that about 47% of the meat and poultry in U.S. grocery stores were contaminated with S. aureus with about half of those bacteria being resistant to multiple antibiotics [1]. It is important to precisely and timely detect and identify the organism for epidemical and clinical investigation. Liu et al. outline different PCR-based approaches for MRSA detection in detailed and particular ways. Bacteroides fragilis is an anaerobic opportunistic pathogen, which cause bacteremia, abscess formation, diarrhea, and inflammatory bowel disease [2]. B. fragilis is the most commonly isolated bacteria in anaerobic infections, especially infections involving gastrointestinal flora. Next generation sequencing and analysis tools can be employed to detect quickly B. fragilis. The isolation, detection and characterization methods of Enterotoxigenic B. fragilis are clearly outlined by Drs. Fathi and Wu. Francisella tularensis is a small, nonmobile, Gram-negative bacterium. It is highly infectious, causing life-threatening tularemia in animals and humans [3]. It is considered by Center for Disease Control and Prevention to be a category A agent [4]. F. tularensis grows slowly, so it requests molecular biology assays to detect rapidly. A review article written by Lai et al. detailed summarize the up-to-date developments of these assays. Noroviruses are now recognized as the most common cause of nonbacterial acute gastroenteritis and foodborne viral illness worldwide. Human noroviruses account for over 267 million infections worldwide annually [5]. Noroviruses are extremely infectious; a few viral particles can initiate an infection. A major impediment of the identification and diagnosis of norovirus infection is the lack of an effective cell culture system or small animal models. Molecular techniques have been developed to detect the viruses. The molecular identified and diagnostic methods of noroviruses are meticulously outlined by Drs. Chen and Hu. The value of molecular biology technique has been demonstrated in identifying and characterizing bacteria and viruses in food, environmental and clinical specimens, and in hastening diagnostic and epidemiological screening procedures, which circumvent the problems of traditional culture methods. Thus, these review articles will updated knowledge on microorganisms, and provide useful and practicable guides for rapid identification and characterization of the bacteria and viruses.

MALDI-TOF

Abstract: Among different mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) has become popular in infectious disease management, infection control and antimicrobial stewardship especially in the field of bacterial and fungal diseases, due to its simple sample pre-treatment, rapid identification of microorganism, and future scope of detecting biomarkers in infectious diseases, antimicrobial susceptibility testing, epidemiological typing, microbial virulence factors and its use in the field of virology and parasitology as well. The technique has the potentiality for point-of-care diagnosis and screening of large number of samples simultaneously. Many laboratories across globe use this technique in routine microbiology laboratory due to its efficiency and cost-per-analysis point of view. It outcompetes the classical microbial identification methods. FDA approved both VITEK MS system (BioMérieux) and MALDI_TOF MS Biotyper (Bruker Daltonics) for microbial identification. The identification system requires pure culture (∼108microbial cells) of a single species bacteria, mycobacteria or fungi from liquid or solid media. The mixed microbial species are hard to identify unless the ratio between the species is not lower than 1:5. For Mycobacteria, Nocardia, and fungi a cell destruction or extraction step is required. The majority studies reported correct identification >95% isolates. However, for better identification, periodic update of database is essential. Optimization of the distinction of few species still remains problematic like E. coli and Shigella species. The other applications of MALDI that may have impact on management of infectious diseases include antimicrobial susceptibility testing and epidemiological typing. Antimicrobial susceptibility testing targets a) modification and degradation product of antimicrobial after exposure to microbe, b) proteomic changes in resistant versus susceptible microbial species, c) shift of protein peak after growth of microbe in presence of antimicrobials, d) semi-quantitative estimation of intracellular/extracellular antimicrobials. The techniques require further standardization before getting regulatory approval. In case of microbial typing, the technique compares unique isolate peaks, whereas the identification targets universally conserved peaks. Modification of MALDI-TOF like SELDI-TOF (Surface-enhanced laser desorption) or ESI-MS (Electron spray ionization) are currently explored for their clinical potential.

"Nanofiltration" Enabled by Super-Absorbent Polymer Beads for Concentrating Microorganisms in Water Samples.

Detection and quantification of pathogens in water is critical for the protection of human health and for drinking water safety and security. When the pathogen concentrations are low, large sample volumes (several liters) are needed to achieve reliable quantitative results. However, most microbial identification methods utilize relatively small sample volumes. As a consequence, a concentration step is often required to detect pathogens in natural waters. Herein, we introduce a novel water sample concentration method based on superabsorbent polymer (SAP) beads. When SAP beads swell with water, small molecules can be sorbed within the beads, but larger particles are excluded and, thus, concentrated in the residual non-sorbed water. To illustrate this approach, millimeter-sized poly(acrylamide-co-itaconic acid) (P(AM-co-IA)) beads are synthesized and successfully applied to concentrate water samples containing two model microorganisms: Escherichia coli and bacteriophage MS2. Experimental results indicate that the size of the water channel within water swollen P(AM-co-IA) hydrogel beads is on the order of several nanometers. The millimeter size coupled with a negative surface charge of the beads are shown to be critical in order to achieve high levels of concentration. This new concentration procedure is very fast, effective, scalable, and low-cost with no need for complex instrumentation.

An Alternative Approach to "Identification of Unknowns": Designing a Protocol to Verify the Identities of Nitrogen Fixing Bacteria.

Microbiology courses often include a laboratory activity on the identification of unknown microbes. This activity consists of providing students with microbial cultures and running biochemical assays to identify the organisms. This approach lacks molecular techniques such as sequencing of genes encoding 16S rRNA, which is currently the method of choice for identification of unknown bacteria. A laboratory activity was developed to teach students how to identify microorganisms using 16S rRNA polymerase chain reaction (PCR) and validate microbial identities using biochemical techniques. We hypothesized that designing an experimental protocol to confirm the identity of a bacterium would improve students' knowledge of microbial identification techniques and the physiological characteristics of bacterial species. Nitrogen-fixing bacteria were isolated from the root nodules of Medicago truncatula and prepared for 16S rRNA PCR analysis. Once DNA sequencing revealed the identity of the organisms, the students designed experimental protocols to verify the identity of rhizobia. An assessment was conducted by analyzing pre- and posttest scores and by grading students' verification protocols and presentations. Posttest scores were higher than pretest scores at or below p = 0.001. Normalized learning gains (G) showed an improvement of students' knowledge of microbial identification methods (LO4, G = 0.46), biochemical properties of nitrogen-fixing bacteria (LO3, G = 0.45), and the events leading to the establishment of nitrogen-fixing symbioses (LO1&2, G = 0.51, G = 0.37). An evaluation of verification protocols also showed significant improvement with a p value of less than 0.001.

Differentiation of Bacillus pumilus and Bacillus safensis using MALDI-TOF-MS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) despite being increasingly used as a method for microbial identification, still present limitations in which concerns the differentiation of closely related species. Bacillus pumillus and Bacillus safensis, are species of biotechnological and pharmaceutical significance, difficult to differentiate by conventional methodologies. In this study, using a well-characterized collection of B. pumillus and B. safensis isolates, we demonstrated the suitability of MALDI-TOF-MS combined with chemometrics to accurately and rapidly identify them. Moreover, characteristic species-specific ion masses were tentatively assigned, using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and primary literature. Delineation of B. pumilus (ions at m/z 5271 and 6122) and B. safensis (ions at m/z 5288, 5568 and 6413) species were supported by a congruent characteristic protein pattern. Moreover, using a chemometric approach, the score plot created by partial least square discriminant analysis (PLSDA) of mass spectra demonstrated the presence of two individualized clusters, each one enclosing isolates belonging to a species-specific spectral group. The generated pool of species-specific proteins comprised mostly ribosomal and SASPs proteins. Therefore, in B. pumilus the specific ion at m/z 5271 was associated with a small acid-soluble spore protein (SASP O) or with 50S protein L35, whereas in B. safensis specific ions at m/z 5288 and 5568 were associated with SASP J and P, respectively, and an ion at m/z 6413 with 50S protein L32. Thus, the resulting unique protein profile combined with chemometric analysis, proved to be valuable tools for B. pumilus and B. safensis discrimination, allowing their reliable, reproducible and rapid identification.

The Importance of Multilocus Sequence Typing: Cautionary Tales from the Bacterium Xylella fastidiosa.

Microbial identification methods have evolved rapidly over the last few decades. One such method is multilocus sequence typing (MLST). MLST is a powerful tool for understanding the evolutionary dynamics of pathogens and to gain insight into their genetic diversity. We illustrate the importance of accurate typing by reporting on three problems that have arisen in the study of a single bacterial species, the plant pathogen Xylella fastidiosa. Two of these were particularly serious since they concerned contamination of important research material that has had detrimental consequences for Xylella research: the contamination of DNA used in the sequencing of an X. fastidiosa genome (Ann-1) with DNA from another X. fastidiosa strain, and the unrecognized mislabeling of a strain (Temecula1) distributed from a culture collection (ATCC). We advocate the routine use of MLST to define strains maintained in culture collections and emphasize the importance of confirming the purity of DNA submitted for sequencing. We also present a third example that illustrates the value of MLST in guiding the choice of taxonomic types. Beyond these situations, there is a strong case for MLST whenever an isolate is used experimentally, especially where genotypic differences are suspected to influence the outcome.

The unseen world: environmental microbial sequencing and identification methods for ecologists

Microorganisms inhabit almost every environment, comprise the majority of diversity on Earth, are important in biogeochemical cycling, and may be vital to ecosystem responses to large‐scale climatic change. In recent years, ecologists have begun to use rapidly advancing molecular techniques to address questions about microbial diversity, biogeography, and responses to environmental change. Studies of microbes in the environment generally focus on three broad objectives: determining which organisms are present, what their functional capabilities are, and which are active at any given time. However, comprehending the range of methodologies currently in use can be daunting. To provide an overview of environmental microbial sequence data collection and analysis approaches, we include case studies of microbiomes ranging from the human mouth to geothermal springs. We also suggest contexts in which each technique can be applied and highlight insights that result from their use.

MALDI-TOF MS in the clinical microbiology laboratory

Traditional methods for microbial identification are often very laborious and time consuming. A new mass spectrometry based technique, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), has been described as a rapid, practical and low-cost method for this purpose. In this article, primary and possible future applications of this tool are briefly discussed.

Pathology Consultation on Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Microbiology

Traditional microbial identification methods are based on morphology (both micro- and macroscopic) and biochemical tests that require long incubation periods and a good deal of technologist hands-on time. In addition, many of these methods have some degree of subjectivity. In comparison, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identifies microorganisms from colonies grown on solid media within minutes using very few reagents. A case-based approach was used to review the strengths and weaknesses of MALDI-TOF MS in clinical microbiology laboratories. MALDI-TOF MS has been proven to be an accurate and reliable method for organism identification including bacteria, yeast, molds, and mycobacteria. It is rapid, with results often 24 hours earlier than traditional methods, and inexpensive. There are no FDA-approved systems available currently. Susceptibility data are still reliant on conventional methods. There are genetically similar organisms that cannot be discriminated reliably with this method. MALDI-TOF MS is an exciting, innovative method for organism identification in the clinical microbiology laboratory.

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: a Fundamental Shift in the Routine Practice of Clinical Microbiology

Within the past decade, clinical microbiology laboratories experienced revolutionary changes in the way in which microorganisms are identified, moving away from slow, traditional microbial identification algorithms toward rapid molecular methods and mass spectrometry (MS). Historically, MS was clinically utilized as a high-complexity method adapted for protein-centered analysis of samples in chemistry and hematology laboratories. Today, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS is adapted for use in microbiology laboratories, where it serves as a paradigm-shifting, rapid, and robust method for accurate microbial identification. Multiple instrument platforms, marketed by well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based identification practices. This review summarizes the current position of MALDI-TOF MS in clinical research and in diagnostic clinical microbiology laboratories and serves as a primer to examine the "nuts and bolts" of MALDI-TOF MS, highlighting research associated with sample preparation, spectral analysis, and accuracy. Currently available MALDI-TOF MS hardware and software platforms that support the use of MALDI-TOF with direct and precultured specimens and integration of the technology into the laboratory workflow are also discussed. Finally, this review closes with a prospective view of the future of MALDI-TOF MS in the clinical microbiology laboratory to accelerate diagnosis and microbial identification to improve patient care.

An investigation of the presence of specific anaerobic species in necrotic primary teeth

Different microbial identification methods have shown that the microbial community profiles in endodontic infections are diverse and assorted. The aim of this study was to evaluate the frequency of selected endodontic pathogens in the pulp chambers (PCs) and root canals (RCs) of infected primary teeth using PCR methods. Paired PC and RC samples were collected from 15 subjects and analyzed by PCR for the presence of Filifactor alocis, Fusobacterium nucleatum, Parvimonas micra, Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Prevotella tannerae, Tanerella forsythia, Treponema denticola, and Treponema socranskii. The frequency of each species was determined in the PC and RC of each case. The species most frequently detected in PCs were P. nigrescens (86.7%), P. gingivalis (73.3%), and F. alocis (73.3%). Of the PC samples, 13.3% contained P. micra and T. denticola, and 6.7% contained T. forsythia. The species most frequently detected in RCs were P. gingivalis (100%) and P. nigrescens (93.3%). P. tannerae, P. micra, and T. denticola were found in 40% of the RC samples; T. forsythia was found in 26.7% of the RC samples. The "red complex", which comprises P. gingivalis, T. denticola, and T. forsythia, was not found in the PC of any tooth but was found in 30% of the RC samples. The detection of P. nigrescens in the PC was statistically associated with the presence of P. nigrescens in the RC (p = 0.04). The results suggest high heterogeneity among the samples, even among those from the same subject.