Bacteria that are normal flora are important symbionts of the human body, most of which cause no ill effects and some, which are actually beneficial to human health. In order to identify an unknown in the clinical laboratory, a sample must be collected from the patient. This could be a sample of urine, feces, saliva, or a swab of the throat or skin. Because the clinical samples will most likely contain many microorganisms, both normal flora and pathogens, it is important to isolate the pathogen in a pure culture using various types of selective and differential media.
Following isolation, one of the first steps in identifying a bacterial isolate is the Gram stain , which allows for the determination of the Gram reaction, morphology, and arrangement of the organism. Although this information provides a few good clues, it does not allow us to determine the species or even genus of the organism with certainty. To my disappointment, not much grew on my bathroom mirror plate. I later learned that despite the absence of microbes on my plate, there might still be microbes present on my bathroom mirror.
Not all microbes grow on the same type of nutrients, or at the same temperature. This student project has many parallels to what microbiologists have been doing for centuries. From identifying microbes by physical and functional characteristics to the adaptation of more modern techniques, microbiologists and future microbiologists are continually building a vast toolkit to uncover the identities of previously unknown microscopic life.
Antoni van Leeuwenhoek first saw microbes through a microscope in the s. However, it must be emphasized that no single method has emerged as the method of choice, and some methods perform better than others at different levels of resolution [ ]. The number of laboratories now using the relevant molecular testing is rapidly increasing, resulting in an obvious need for standardization. The application of the appropriate technique depends on factors such as financial budget, experienced personnel, and equipment but an important issue is still the lack of sufficient species-specific primers [ ].
Aforementioned PCR-based detection of fungal DNA sequences can be sensitive, rapid, specific [ , , ] and it permits both intraspecies differentiation and species identification of yeast isolates [ ].
Because fungal cell walls are strong and difficult to disrupt, DNA isolation requires effort to overcome this barrier. Hence, glass beads are used for mechanical disruption, sonication, and phenol-chloroform in order to promote enzymatic digestion in the lysis phase [ , ]. As previously indicated, PCR tests, as well as detection of specimen type whole blood, serum, and plasma , should be standardized. The choice of primers is another important factor that could alter the diagnostic performance of PCR tests.
On the other hand, multiplex PCR can detect a wide variety of fungi at once in the same sample [ ]. The 18S, 5. Because of these properties they provide establishment of phylogenetic relationships [ , ]. More rapidly evolved regions are internal transcribed spacer 1 and 2 ITS1 and ITS2, respectively and thus they may vary among various species within a genus.
The abovementioned conserved sequence of 18S-rRNA was used for primer design with the goal of detecting 25 fungal species, including Candida spp. A bp product was amplified successfully by PCR from all 78 strains and specificity was subsequently confirmed by Southern analysis [ ].
Primers were designed to not cross-react with the other species, AND compatibility of amplicon sizes of one target species with the rest of target species in the same multiplex PCR was required as well as melting temperature compatibility of primers within the same multiplex PCR.
Another one-step, multiplex PCR to detect and identify Candida spp. No cross-reaction with closely- and distantly-related yeast species, Aspergillus spp.
RT-PCR assay is an important tool for rapid detection of pathogens, and it offers superior accuracy and specificity over traditional methods.
In a study, real-time amplification of two genes, melting-point analysis and two-dimensional plotting of T m data were used as a broad-range method for the identification of clinical isolates of Candida spp. Conserved sequences DNA in Candida spp. Further, species-specific real-time PCR primer sets covering C. They could be potentially assembled into a single PCR array for the rapid detection of Candida spp.
In another study, real-time PCR assay demonstrated to rapidly detect, identify, and quantify Candida spp. A total of 50 strains, of C. HRMA was verified in order to categorize C. Furthermore, Asadzadeh et al. The amplification products were also analyzed by agarose gel electrophoresis to confirm RT-PCR results. Melting temperature Tm for reference strains of C.
Similarly, quantitative PCR assays to determine the relative Paracoccidioides brasiliensis load in lungs from infected mice were also developed. Comparable scores were acquired when real-time PCR was applied as an amplicon with a Tm [ ]. Electrophoretic karyotyping methods, which are based on differences in the genetic structure of an isolate, reveal sufficient variation for strain delineation [ ]. Pulsed field gel electrophoresis PFGE enables separation of fungal chromosomal DNAs according to their size up to several megabases in agarose gels, and it is a worthwhile tool for fungal karyotyping [ , ].
Its application allows for species or even strain specific profiles to be obtained. For example, the chromosomal DNAs of eight Candida spp. Delineating strains of C. Concisely, DNA extracted from isolates is split into fragments by specific DNA restriction enzymes, and the fragments are divided based on molecular size by gel electrophoresis. To spot alterations or matches in the fragments a staining of the gel with ethidium bromide with visualization under UV light or DNA hybridization with a specific DNA probe is done [ ].
Presently, there is a validated database with over clinical isolates ITS2 length and sequence polymorphisms for 34 yeast different species [ ]. In another study, Candida spp. Specifically, for the C. A great difference was found between these two methods. It may be argued that Msp I and Bln I restriction enzyme fragments can be used in the identification of medically important Candida spp.
Further studies are needed to develop this kind of restriction profile to be used in the identification of candidal strains [ ]. Restriction enzyme analysis of C.
The restriction digestion with MwoI was able to distinguish between five different species C. Mitochondrial DNA mtDNA can also be useful to distinguish closely related strains in hospital acquired infection outbreaks since, as compared to nuclear DNA, its higher mutational load and evolutionary rate readily reveals microvariants [ ]. Restriction endonuclease analysis of mtDNAs from 19 isolates representing seven Candida spp.
Rare shared restriction fragments were clear and there was no correspondence among the base compositions of nuclear and mitochondrial DNAs. Unbiased evolution, great variability, easy PCR isolation, and full length sequencing regions can lead to a novel outlook in molecular findings of C.
RAPD or restriction enzyme analysis REA are valuable to establish the source of an outbreak, nonetheless, further reproducible and discriminatory procedures may be a requisite e. Multiple Candida strains from nosocomial infections have been identified [ ]. In addition, the differentiation between C. Moreover, genetic profiles of 39 clinical isolates of C.
The identification of yeasts was set by nested-PCR which involved two amplification stages. Using CDC3 and HIS3 markers, microsatellite endorsed the observation of six and seven unlike alleles, respectively [ ]. In this methodology, the genomic DNA is digested with two restriction enzymes e. In a collection of clinical isolates catalogued as C.
About C. All previously described molecular techniques can be applied for detection of new fungal species as well as for routine laboratory identification.
For example, Candida milleri and Candida humilis are the most characteristic yeasts found in type I sourdough ecosystems. Genetic characterization, assimilation test of carbohydrates, and metabolome assessment by FTIR analysis exposed a high degree of intraspecific polymorphism and 12 distinctive genotypes were categorized [ ]. Several methods were shown to be useful to determine isogenicity among C.
The tools for the determining the identity of a microbial sample have been emerging in the last decades. Although having limitations, culture and microscopy are still two of the most utilized techniques. PCR and other genetic approaches are particularly important for nonculturable microorganisms and MS has been shown to be useful, quick, and easy for the identification of microbial samples and detection of microbial threats.
However, it is reserved for pure isolates and cannot be used for complex samples, since they may promote interference in the background. This may be simplified through the use of chromatography-based methods e. In the future, development of the detection limits for microorganisms will continue to be a key assignment in clinical microbiology.
The combination of these and possibly others methodologies and instrumentation will surely improve the skills for the detection of pathogens. All authors contributed to the manuscript; conceptualization C.
All the authors read and approved the final manuscript. National Center for Biotechnology Information , U. Journal List Microorganisms v. Published online May Find articles by Snehal Kadam. Karishma S.
Find articles by Karishma S. Find articles by Antonio Bevilacqua. Find articles by Maria Rosaria Corbo. Find articles by Hubert Antolak. Author information Article notes Copyright and License information Disclaimer. Received Apr 12; Accepted May 8. This article has been cited by other articles in PMC.
Abstract Fast detection and identification of microorganisms is a challenging and significant feature from industry to medicine. Introduction Microorganisms have always been extremely important for human life and bacteria, yeasts and molds have been known for both positive and negative reasons. Table 1 Methods used in the area of microorganism identification. May be used to identify specific microbes in a mixed population as well as identify non-culturable microbes. For example, microscopic techniques are powerful tools used in the identification of microorganisms by visualization of the characteristic structures and for organisms in the VBNC viable but not culturable state.
Open in a separate window. Figure 1. Historical Evolution of Microorganism Identification During the last decade, scientists have searched for the more prompt and effective means of microbial identification [ 1 ].
Identification Methods Using Chromogenic Media In these methods, the identification of microorganisms based on cultivation has the initial objective of obtaining pure culture. Microscopy Techniques The microscope is an essential identification tool for microorganisms present in a natural sample. Biochemical Analytical Methods to Detect Microorganisms 3.
Traditional Biochemical Methods In microbiology, traditional identification methods rely mainly on cultivation proceedings employing various media to enumerate, isolate, and identify specific microorganisms. Mass Spectrometry-Based Methods Research in microorganism identification has evolved mainly by following the strategy of reducing the time required for the identification of a particular microbial in routine diagnostics.
Liquid Chromatography: High Performance Liquid Chromatography HPLC -Based Methods The combination of liquid chromatography LC with MS LC-MS , despite initial hesitations, revolutionized analytical determination of metabolome, consequently, allowing microorganism identification, by enabling the analysis of non-volatile or thermally labile high molecular compounds where gas chromatography and mass spectrometry GC-MS approaches were not suitable [ 72 , 73 , 74 ].
Gas Chromatography—Mass Spectrometry GC coupled to MS has been extensively used in the identification of complex biological mixtures [ 92 , 93 , 94 ]. Infrared Spectroscopy FTIR Recent advancements have been made especially in the application of new spectroscopic methods. Electrokinetic Separation Methods The term electrokinetics refers in science to the relative motion of a charged particle through a matrix.
Microfluidic Chips Since its appearance in the early s, the microfluidics field of research has seen great and rapid developments [ ]. Ribotyping Ribotyping is a method for bacterial identification and characterization that, unlike certain previously described molecular typing methods, employs rRNA based phylogenetic analysis. Molecular Methods Used to Detect Yeasts Rapid and precise identification of pathogens from clinical specimens leads to appropriate therapeutic plans [ ], but the growing diversity of infectious species and strains makes the identification of clinical yeasts increasingly difficult [ ].
PCR Aforementioned PCR-based detection of fungal DNA sequences can be sensitive, rapid, specific [ , , ] and it permits both intraspecies differentiation and species identification of yeast isolates [ ]. DNA Fingerprinting Methods 5. Pulsed Field Gel Electrophoresis PFGE Electrophoretic karyotyping methods, which are based on differences in the genetic structure of an isolate, reveal sufficient variation for strain delineation [ ].
Conclusions The tools for the determining the identity of a microbial sample have been emerging in the last decades. Author Contributions All authors contributed to the manuscript; conceptualization C. Funding C. Conflicts of Interest The authors declare no conflict of interest.
References 1. Bisen P. Wiley-Blackwell; Oxford, UK: Prakash O. Polyphasic approach of bacterial classification—An overview of recent advances. Indian J. Manafi M. Fluorogenic and chromogenic substrates in culture media and identification tests. Food Microbiol. Ramamurthy T. Public Heal. Bochner B. Global phenotypic characterization of bacteria. FEMS Microbiol. Castro-Escarpulli G. Lincoln R. A Dictionary of Ecology, Evolution, and Systematics.
Foundations of Phylogenetic Systematics. Verlag Dr. Friedrich Pfeil; Munich, Germany: Trends in Taxonomy today: An overview about the main topics in Taxonomy. Godfray H. Challenges for taxonomy. Enghoff H. What is taxonomy? An overview with myriapodological examples. Soil Org. Donelli G. Phenotyping and genotyping are both essential to identify and classify a probiotic microorganism. Health Dis.
Yeung P. Dairy Sci. S 02 Lagier J. Skerman V. Steel K. Microbial Identification. Madigan M. Brock Biology of Microorganisms. Pearson; London, UK: Breitwieser F.
A review of methods and databases for metagenomic classification and assembly. Klenk H. Colwell R. Polyphasic taxonomy of the genus vibrio: Numerical taxonomy of Vibrio cholerae, Vibrio parahaemolyticus, and related Vibrio species.
Yang F. Identification of microorganisms producing lactic acid during solid-state fermentation of Maotai flavour liquor. Dige I. In situ identification of streptococci and other bacteria in initial dental biofilm by confocal laser scanning microscopy and fluorescence in situ hybridization. Oral Sci. Bayraktar B. Feature extraction from light-scatter patterns of Listeria colonies for identification and classification.
Rajwa B. Discovering the unknown: Detection of emerging pathogens using a label-free light-scattering system. Obara B. Bacterial cell identification in differential interference contrast microscopy images. BMC Bioinformatics. Ahmed W. IEEE J. Cardinale M. Microbiome analysis and confocal microscopy of used kitchen sponges reveal massive colonization by Acinetobacter, Moraxella and Chryseobacterium species. Schuler B. Beier B. Raman microspectroscopy for species identification and mapping within bacterial biofilms.
AMB Express. Amann R. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Sabnis R. Handbook of Fluorescent Dyes and Probes. Nam H. Noble R. Pierini F. Xie C. Props R. Measuring the biodiversity of microbial communities by flow cytometry.
Methods Ecol. Buszewski B. Identification of Microorganisms by Modern Analytical Techniques. AOAC Int. Juste A. Recent advances in molecular techniques to study microbial communities in food-associated matrices and processes.
Engvall E. Sutton S. How do you decide which microbial identification system is best? Forum News. Smith P. Washington J. Sandle T. In: Batt C. Encyclopedia of Food Microbiology. Varettas K. Funke G. Puttaswamy S. Ligozzi M. Evaluation of the VITEK 2 system for identification and antimicrobial susceptibility testing of medically relevant gram-positive cocci.
Jorgensen J. Klingler J. Evaluation of the Biolog automated microbial identification system. Fox A. In: Pezzati L. Identification of Microorganisms by Mass Spectrometry. Sauer S. Mass spectrometry tools for the classification and identification of bacteria. Matsuo T. Introduction to Modern Biological Mass Spectrometry. Mass Spectrom. Petrotchenko E.
Proteomics in Biomedicine and Pharmacology. Volume Elsevier Inc. Sandrin T. Characterization of microbial mixtures by mass spectrometry. Claydon M. The rapid identification of intact microorganisms using mass spectrometry. Senes C. Fernando W. Identification and use of potential bacterial organic antifungal volatiles in biocontrol.
Soil Biol. Schauer N. GC-MS libraries for the rapid identification of metabolites in complex biological samples. FEBS Lett. Mass spectrometry for species or strain identification after culture or without culture: Past, present, and future. Jang K. Haiko J. Rahi P. Microbiological Identification Strategy for Pharmaceutical Microbiology. Dierig A. Dingle T. Biswas S. Electrokinetic detection and characterization of intact microorganisms.
Zhang J. Rapid direct lipid profiling of bacteria using desorption electrospray ionization mass spectrometry. Vaidyanathan S. Lattanzio V. MASS Spectrom. Meredith S.
Warren C. A liquid chromatography—mass spectrometry method for analysis of intact fatty-acid-based lipids extracted from soil.
Soil Sci. Van der Werf M. Microbial metabolomics: Toward a platform with full metabolome coverage. Bakhtiar R. Verhoeckx K. Characterization of anti-inflammatory compounds using transcriptomics, proteomics, and metabolomics in combination with multivariate data analysis. Smilde A. Fusion of mass spectrometry-based metabolomics data. Bajad S. Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry.
Edwards J. Effect of decreasing column inner diameter and use of off-line two-dimensional chromatography on metabolite detection in complex mixtures. Bruce S. Recent developments in liquid chromatography-mass spectrometry and related techniques. Preparation of monolithic silica columns for high-performance liquid chromatography. Heideloff C. Drug Monit. Food Chem. Kadi A. Heinisch S. Sense and nonsense of high-temperature liquid chromatography.
Teutenberg T. Potential of high temperature liquid chromatography for the improvement of separation efficiency—A review. Cunliffe J. Plotas P. Mounier J. Application of denaturing high-performance liquid chromatography DHPLC for yeasts identification in red smear cheese surfaces. Kind T. Seven Golden Rules for heuristic filtering of molecular formulas obtained by accurate mass spectrometry.
Lommen A. An untargeted metabolomics approach to contaminant analysis: Pinpointing potential unknown compounds. Franco-Duarte R.
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