In yeasts, this analysis has also been shown to be possible [ ] for several species [ , , ]. The main drawback of GC-MS is that it demands that the analytes are in a volatile form and as several metabolites are nonvolatile, time-consuming derivatization steps are required [ , , ]. In this case, two different GC columns are conjugated, increasing the metabolite detection coverage, and the speed of scanning rate TOF-MS with extra sensitivity for improved detection.
However, this method has high costs, so it is not yet routinely used. The connection of the flame ionization detector FID —GC-FID can also be applied for routine sample analysis, being rapid, very sensitive, and with an associated lower cost [ ]. It is based on the ionization of the microbial cells with short laser pulses and then accelerating the particles in a vacuum system using an electric field [ 56 , 66 ]. After the ionization, a molecular fingerprint in the form of a spectra profile is obtained, which is specific for each microorganism.
This spectrum is then compared to an existing database, resulting in its identification by an automated program. An alternative to MALDI-TOF which sometimes entails problems associated with the use of a chemical matrix mixed with the sample and the laser used to effect desorption and ionization of the analyte , is a technique called electrospray ionization ESI -MS that analyzes samples in a liquid state and the ionization is carried out at atmospheric pressure, without recurring to the same lasers as in MALDI-TOF-MS.
Due to this particular aspect, ESI-MS has a large spectra of applications regarding microbial identification [ 71 , , , ]. It is used in systems biology, being a nondestructive, very simple, and to some extent, precise approach, allowing vast amounts of information to be obtained in one measurement [ , ]. Fiber optics spectroscopy processes vibrations and rotations of molecular functional groups, which are outcomes from the energy shifted when radiation interacts with a sample, and originates electronic excitation, vibrational change, and rotational change.
The spectra will vary depending on the sample molecular groups, and therefore, they are linked to their chemical composition proteins, lipids, carbohydrates, membranes, pharmaceuticals, human tissues, among others reviewed in [ ].
Fluids, cells or tissues can be explored to find metabolic fingerprints, and in theory, any sample can be virtually analyzed by spectroscopy. With respect to the identification of microorganisms, these methods are of great value and complementary to molecular biology, because they do not normally need the destruction of the sample. In the recent past due to some limitations found, some caution was advised by some authors when using these methods, suggesting a careful validation of each procedure before its use.
Numerous techniques for spectroscopic analysis are accessible. The subdivision is not always easy, varying from the type of radiative energy, the nature of the interaction or the material below analysis. Recent advancements have been made especially in the application of new spectroscopic methods.
One of these methods, with great developments in several areas of microbiology, is the Fourier transform infrared spectroscopy FTIR. FTIR is versatile, fast, non-invasive, and it is easy to perform compared to other methodologies [ , , , , ].
This analytical technique is a chemical and label-free procedure which gives a clear elucidation about the chemical composition and the physical state of the entire sample where several biomolecules can be analyzed. With the use of only a minimal amount of sample, it is possible to obtain, in a single measurement, detailed information about the main biomolecules such as lipids, proteins, carbohydrates, and nucleic acids [ ].
Likewise, FTIR allows an economic biochemical characterization of complex biological systems, comprising the intact cells, tissues, and even whole-model organisms [ ]. The use of this technique to evaluate microorganisms as biological systems results in a very complex spectrum with the overlapping absorption bands of the principal compounds. Therefore, a proper multivariate statistical analysis is of crucial importance in order to extract from the spectra only the relevant information of the biological process under study [ ].
FTIR spectroscopy of biological systems provides a complex infrared absorption spectrum that should be preprocessed and then analyzed by applying resolution enhancement approaches. With respect to the operation mode of this technique, it comprises an IR energy source that emits a broad band of distinct wavelengths. After this, the radiation passes through an interferometer responsible for modulating the wavelength of IR. In the sample compartment, the resulting IR beam is absorbed in distinct and specific wavelengths by the organic or inorganic material.
The last step comprises the measurement of the intensity of the IR beam by a detector producing an interferogram, that is, subsequently, analyzed by a computer using Fourier transforms, giving rise to an IR spectrum.
Additionally, with the use of second derivatives it is possible to promote a clear separation of the absorption components, thus helping to understand their variations throughout the biological process under study.
Subsequently, an adequate multivariate analysis should be implemented to validate the spectroscopic results, as well as to identify the main relevant bands of the process studied.
Finally, the interpretation and analysis of the spectral data should be coupled with other standard methods to ensure the reliability of FTIR spectroscopy analysis [ ]. Some of the principal advantages of this spectroscopic technique are related to: I the possibility of analyzing several compounds at the same time; II the facility of sample preparation, since it does not require cell lysis to release the biomolecules to be evaluated; III the association of an environmentally friendly role, as the toxic compounds are not implemented in this method; and IV the possibility of using this technique for real-time process monitoring and the accomplishment of high-throughput screenings [ ].
Other regions of the infrared spectrum can be used in combination with spectroscopy to analyze microbial diversity. In particular, considering the infrared spectral regions, the wavelength region between 0. However, the most relevant for spectroscopic purposes are the near-infrared, the mid-infrared MIR , and the far-infrared radiation [ ].
It discriminates itself from other systems by the manageability of use at a low cost, high speed, and an extensive report chemical composition, the structure, and interactions of biomolecules in the microorganisms [ , ].
In fact, this procedure uses vibrational, rotational, and other low-frequency modes in the system in order to produce a structural fingerprint by which molecules can be identified, providing complementary information to traditional spectroscopic methods, being many times, as will be described later, advantageous to combine several spectroscopic methods together.
The structural fingerprint obtained is then used to identify microorganisms, as this method is capable of correctly distinguish between species and strains within a few hours. Although this high specificity is attributed to Raman spectroscopy, its sensitivity is rather poor.
As in other spectroscopic characterizations, Raman spectroscopy depends on its interaction with the atoms and molecules, when light is incident on the matter. When atoms vibrate it will change the polarizability of functional groups, having nonpolar groups such as C-C and S-S intense Raman bands. Raman spectroscopy evaluates the inelastic scattering of radiation of monochromatic light, promoting a spectral shift, i.
Raman spectroscopy has been largely applied to microbial identification in recent years [ , , , , ]. Because vibrational spectroscopy discriminates microorganisms based on their biochemical composition, it is very useful for differentiating between minor differences among the same species. NMR spectroscopy is an alternative and potent technique for microorganism identification.
Strong magnetic fields and radio frequency pulses to the nuclei of the atoms are applied and in the case of atoms such as 1 H or 13 C, the magnetic field will cause a nuclear spin, absorbing the radio frequency energy low-energy to high-energy spin states , and the emission of radiation is detected [ ]. As compared with other methods, NMR can be performed in a non-invasive manner.
The term electrokinetics refers in science to the relative motion of a charged particle through a matrix. These methods make use of the differences in microbial composition to obtain different migration patterns, and in this way, without recurring to sample labelling, separate different microbial species.
It combines the separation process of electrophoresis with MS detection [ ]. In comparison with GC and LC, it includes better separation efficiencies, the use of very little sample volumes, speed, small reagent costs, and the possibility to separate cations, anions and uncharged molecules in a single run. This approach has been used to analyze the metabolome of numerous microorganisms, both for target and nontarget studies, having interesting outcomes in detection and quantification of several metabolite classes [ , ] e.
CE has deficient sensitivity related to the small sample volumes, particularly when attached to MS, has a restricted quantity of accessible commercial libraries, and reduced retention time reproducibility.
Armstrong et al. The uniqueness of this study was that it demonstrated that intact biological cells could be efficiently separated by employing techniques that are usually limited to macromolecules. Nowadays, another possibility used is the combination of CE with fluorescence, which can be used to observe the separation process, and in this way monitor the operational conditions and the microbial dynamics in terms of cell aggregation and focusing effects [ , , ]. The main advantage of these types of techniques which focus on electrokinetics is the possibility to exploit several microbial parameters, such as size, shapes, and charges, which are very advantageous to their separation and identification.
Electrical field-flow fractionation EIFFF is another technique that uses the ability of microorganisms to migrate in an electric field.
Its use for microbial identification was confirmed in [ ]. It is based on the separation of sample components in a channel as a result of different layers fractionation of each group of components under the influence of various electrical fields. The EIFFF apparatus uses the two main walls of the channel to create a difference in the potential between the electrodes which leads to a separation between charges [ ].
Since its appearance in the early s, the microfluidics field of research has seen great and rapid developments [ ]. It is a technique that combines separation and detection of sample constituents by controlling the movement of fluids within microfluidic chips, without the need for special sample preparation or reaction [ , ].
These platforms are small portable devices that combine microchannels with dimensions from tens to hundreds of micrometers , pressure systems, and detection systems in the same piece.
Several reviews have been published over recent years about the plenitude of microfluidics application [ , , , , ], although, in light of this review, the development of microfluidics chips to detect microorganisms are the most relevant [ , , , ].
Detection of pathogenic bacteria and viruses using Chips is possible by recurring small sample volumes with great sensitivity, and it has huge applicability in food safety control, environmental monitoring, and clinical diagnosis. Recent approaches for microbial detection and identification are focusing on combinations of analytical standard techniques within microfluidics chips, without the need for labeling procedures. In particular, there are several reports of combinations of microfluidics devices with PCR [ , ], MS approaches [ , , ], spectrometry [ ], electrochemistry [ , ], among others.
These phylogenetic dyes have the ability to penetrate into cells without promoting their lysis, and within the cell they are able to form hybrids with the microbial ribosomal RNA.
Given that the ribosomes are distributed throughout the cell in prokaryotic organisms, the whole cell becomes fluorescent [ ]. These dyes are generally specific, reacting with only one species or a few related microbial species, as well they produce more generally and react with, in some cases, all cells of a given phylogenetic group.
It is important to highlight that the use of this technique allows the identification and search of an organism, or domain of interest, that is present in a natural sample [ ]. FISH technology can also use various phylogenetic probes. Thus, one can use a set of probes where each is designed to react with a particular organism or group of organisms, where each contains its own fluorescent dye.
With FISH it is possible to determine the phylogenetic amplitude of a single habitat in a single experiment, and by associating FISH with CLSM, it is possible to study microbial populations in greater detail and use it in biofilm study [ 30 ].
Additionally, FISH technique can be used to measure the gene expression of the organisms present in a natural sample. In this case, since the target corresponds to mRNA less abundant than the rRNA present in the ribosomes of a cell standard FISH techniques cannot be applied, and instead the target mRNA or fluorescence signal must be amplified [ ].
Previously, the identification and characterization of bacterial species was largely done by phenotypic and biochemical methods, which relied on preliminary isolation and culture. While these methods continue to hold place in certain settings, molecular-based techniques have provided unprecedented insights into bacterial identification and typing.
To name a few examples, genotypic methods have enabled the identification of a large diversity of previously unknown taxa, the characterization of uncultivable bacteria, and facilitated metagenomics studies on large and diverse bacterial communities [ ]. Both clinical and research setting have provided in depth insights into bacterial virulence, pathogenesis, antibiotic resistance, and epidemiological typing, as well as identification of novel, emerging, and re-emerging species [ ].
In addition, the widespread use and availability of molecular tools for bacterial genotyping has resulted in high throughput analysis, more sensitive and discriminatory results, and rapid turn-around-times, which are only likely to get better with automated tools and data analysis pipelines. Most molecular methods for bacterial identification are based on some variation of DNA analysis, either amplification or sequencing based. While the advantages and limitations of these approaches vary, the choice of the technology employed depends on several factors including sample type clinical or research, single-species or mixed-species , depth and accuracy of results generated, resources and cost factors, as well as the turn-around-times expected.
The rapid amplification of nucleic acid targets from relatively lower starting material, makes PCR one of the most sensitive techniques available for detection of bacterial targets. PCR-based identification of bacterial DNA through amplification and sequencing of the 16S rRNA gene has become a standard molecular method, both in the laboratory as well as in clinical settings.
The 16S rRNA gene is highly specific to each bacterial species and this makes it an ideal target for identification. The standard method involves PCR amplification of the 16S rRNA gene, followed by sequencing and comparison to known databases for identification. PCR-based methods are not only faster than conventional culture-based methods but are also helpful in identification of bacteria that are difficult to grow in laboratory conditions.
In one study, universal primers for the 16S rRNA gene were designed to identify bacteria in the root canals of patients with necrotic pulp tissue [ ]. The primers included ten putative bacterial pathogens commonly found in root canals with necrotic pulp.
After DNA extraction from the necrotic pulp, a PCR was run using universal primers, as well as species specific primers, and the products were analyzed using gel electrophoresis. Twenty-two of the 24 specimens tested positive with the universal bacterial primers. As expected, certain bacterial species such as Fusobacterium spp. Of these, PCR analysis revealed two samples that showed a product with the universal primers, but not with any of the 10 species-specific primer sets tested.
Sequencing of these PCR products revealed the presence of a close relative of the Olsenella genus, previously not associated with such infections.
Though the 16S rRNA gene has emerged as a popular target for PCR-based identification, in cases where the 16S rRNA gene is identical in two closely related species, other conserved genes, such as rpoB, tuf, gyrA, gyrB, and heat shock proteins are used as targets [ , , ].
In research laboratories, PCR-based identification is a straightforward procedure with reliable results. However, when applied to clinical settings, various factors come into play that can influence PCR results. Clinical samples often have very few bacteria to begin with, they also require various preprocessing steps before the PCR is carried out, to remove PCR inhibitors and enable extraction of maximum bacteria from the sample without contamination [ , ].
Despite these concerns, PCR-based identification has been successfully and widely employed to detect and identify bacteria in clinical samples [ , , ]. RT-PCR can also be quantitative or semi-quantitative, using the Cq value cycle number at which fluorescence intensity rises above the detectable level to quantify the amount of DNA. It is also applied to numerous kinds of samples, right from identification of bacteria in milk, which are otherwise non-culturable [ ], to the identification of bacteria within soil ecosystems [ ].
Using universal primers that target conserved regions of 16S rRNA gene, an assay has been developed that can detect Acinetobacter baumannii , Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa [ ]. The primers were able to successfully detect less than genomic DNA copies. This real-time based 16S rRNA PCR has also been used to identify or quantify bacterial loads in clinical infections such as chronic wound tissue [ ] and gastrointestinal mucosal biopsies [ ], and has also been applied in forensic investigations of saliva specimens [ ].
In addition, high resolution melting HRM is a rapid, reliable, accurate, and cost-effective emerging tool for genotyping bacteria, such as from the Lactobacillus casei group and both Gram-positive and Gram-negative bacterial pathogens [ , ]. This results in amplification of random, repetitive regions of template DNA, thereby providing a unique profile for bacterial identification [ ].
RAPD-PCR reactions can start from isolated DNA or crude bacterial lysates, which are then subject to amplification in the presence of a RAPD primer or set of primers and low levels of magnesium to enhance non-specific annealing [ ]. Amplified products are then subjected to standard agarose gel electrophoresis to generate unique RAPD fingerprints. This means it can be used to identify and type a diverse range of bacterial species that have either not been identified or for which no prior sequence data is available.
Furthermore, it can be performed from whole bacteria directly, without the need for DNA isolation, and can be applied on Gram-positive and Gram-negative species [ ]. In India, 20 alkaline protease producing bacterial strains isolated from soil samples from various geographic regions were subjected to RAPD screening with a set of random primers [ ].
Analysis of the amplification pattern enabled the classification of isolates into distinct groups based on alkaline protease production. This underscores that RAPD typing using universal random primers are a viable alternative to gene specific molecular marker identification, especially when analyzing a large number of samples of diverse species and without any prior genetic information. Restriction fragment length polymorphism RFLP is a method for identifying bacterial strains using unique fingerprints which relies on the presence of variations polymorphisms in homologous DNA sequences.
As in RAPD, these different fragments are separated by agarose gel electrophoresis to generate a unique pattern of bands for each bacterial strain. If two strains are closely related, their banding patterns will be identical or very similar. On the other hand, differences in banding patterns indicate bacterial strain diversity.
As evident, this technique is highly relevant in investigating the molecular epidemiology of infectious outbreaks, where it is important to establish whether multiple cases or patients belong to the same outbreak, to track the source of the outbreak, and to determine single or multiple bacterial strains involved in the outbreak.
Further, in a smaller group of patients, the highly discriminatory nature of PCR-RFLP analysis was able to correctly identify differences in a cluster of MRSA strains, thereby ruling out the possibility of an outbreak [ ].
Amplified fragment length polymorphism AFLP is similar to RFLP, in that it employs restriction enzymes usually a pair to fragment genomic DNA, but then amplifies a subset of restriction fragments using ligated adaptors. This amplification is achieved by using primers that are complementary to the adaptor sequences but also have certain unique nucleotides. Therefore, only a small number of restriction fragments are selectively amplified.
As evident, AFLP offers high specificity and discriminatory potential in the absence of any prior genome sequence knowledge. The advantages of AFLP as a DNA fingerprinting tool were leveraged in an outbreak investigation of Pseudomonas aeruginosa in an intensive care unit [ ]. During a period of one year, 23 P. Fluorescent amplified PCR fragments were separated by gel electrophoresis and analyzed. Notably, this outbreak strain was also isolated from the wash basin, water tap, and connection pieces from suction tubes, pointing to the possible source of the outbreak [ ].
Complete elimination of the outbreak was achieved after sterilization of the ICU equipment. Pulsed-field gel electrophoresis PFGE is a method of separating large fragments of DNA and is particularly useful for characterizing and typing bacteria for epidemiological studies. The agarose plugs are then incubated with restriction enzymes, which cut at specific sites to generate a limited number of DNA fragments.
The plugs are then subjected to electric current and alternate rotations in a magnetic field which enhances the movement of large DNA fragments , leading to the size separation of DNA fragments and emergence of a banding pattern [ ].
In an outbreak investigation of cholera over seven years, fifty isolates of Vibrio cholerae were subject to molecular typing by PFGE [ ]. Analysis revealed that over the years, the outbreak involved 15 different pulsotypes of V. Notably, PFGE typing revealed the chronological emergence of new types, which subsequently replaced the earlier pulsotype. Ribotyping is a method for bacterial identification and characterization that, unlike certain previously described molecular typing methods, employs rRNA based phylogenetic analysis.
Given that that rRNA genes such as 16S rRNA are highly conserved within a bacterial species, identifying 16S rRNA gene polymorphisms is a reflection of the evolutionary lineage of the bacterial species, and can shed light on bacterial classification, taxonomy, epidemiological investigation, and population biology [ ]. Ribotyping typically involves a multi-step process starting with restriction enzymes that target the genomic sequence of interest, followed by southern blot transfer and hybridization with probes, and analysis of ribotype RFLP bands.
However, with advances in molecular tools and knowledge of genomic sequences, several modifications to this technique have been published [ ]. It is important to note that for the purpose of primer and probe design, ribotyping requires some prior knowledge of the genome sequence under study.
In one study, PCR-ribotyping was employed to characterize 99 strains of Clostridium difficile isolated from patients with nosocomial diarrhea. The banding pattern revealed 41 different PCR-ribotypes with high reproducibility and discriminatory power.
In a modification of this method, PCR-ribotyping was directly employed on stool samples for detection and typing of C. Primer modifications targeting both, the 16SS rRNA intergenic spacer region and 16S and 23S genes itself, resulted in increased specificity for direct typing. With these new primers, PCR-ribotype could be detected directly from stool samples in 86 out of 99 cases, with a high degree of concordance with PCR-ribotyping done from isolated colonies.
Whole-genome sequencing WGS has recently become a highly accessible and affordable tool for bacterial genotyping. Analysis of the entire bacterial genome not only provides unprecedented insights into bacterial typing and evolutionary lineages but has also revolutionized our approach to understanding antimicrobial resistance and outbreak investigations. Advances in WGS technologies and analysis pipelines have rapidly increased output and analysis speed, while reducing overall costs [ ]. In spite of reservations from clinicians related to experimental protocols and cost factors, WGS-based approaches are being evaluated for the pathogen identification and antimicrobial resistance typing.
Using an Illumina Miseq benchtop sequencer, WGS established that isolates from patient two and three differed from that of patient one only by a single, non-synonymous polymorphism, each pointing to ICU transmission.
In addition, the distinct SNPs in isolates from patient two and three also indicated two separate direct transmission events from patient one, rather than linear transmission from patient one to patient two to patient three. As expected, the isolates were shown to carry genes vanA conferring resistance to vancomycin. Therefore, the in-depth analysis offered by WGS was not only able to establish antibiotic resistance but could also infer transmission dynamics and evolutionary lineage of the outbreak strains.
In addition to advancements in genomics, proteomics-based approaches for bacterial identification and characterization have emerged. These methods are primarily based on mass spectrometry, which enables rapid and high-throughput analysis of biomolecular signatures produced by a bacterial strain [ ].
For this, the bacterial sample to be analyzed is mixed with organic matrices and ionized by a laser beam. As the resulting ions move towards the mass analyzer, the mass:charge ratio is obtained which creates a spectra pattern. This pattern is then compared with a known library of fingerprints. This not only underscores the relevance of mass spectrometry-based approaches for bacterial identification and typing, but also indicates that it could assist with clinical decisions such as the initiation of appropriate antibiotics for the treatment of S.
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 [ ].
Still, novel identified fungal species can differ in virulence and drug resistance. Culture-based identification methods have been the gold standard for the diagnosis of fungal infection [ ], but these classical phenotypic and biochemical assays are time consuming and are not suitable to accurately distinguish all the species belonging to a specific cryptic complex [ ]. Therefore, several molecular biology approaches have gained great potential, as they can be applied to detect the pathogen directly without prior cultivation to identify species and subspecies [ ] and they go further than biotype or serotype [ ].
Molecular methods are based on the detection of the nucleic acid sequence of a gene specific to an organism, and therefore they do not detect viable organisms, only indicate their presence [ ]. The principle of the probe-based identification is to obtain a double-strand hybrid as a result of binding the single stranded DNA or RNA of the organism to a complementary sequence.
For molecular detection of fungal pathogens, PCR is the most preferred method [ , ] and is regarded as a standard platform in many clinical laboratories, even in developing countries, due to its affordability and reproducibility [ , ].
RT-PCR assays with a short turnaround time can provide desirable alternatives for the rapid detection of microbes [ , ], and they are able to quantify the amount of amplified DNA in real time.
DNA fingerprinting methods have evolved as major tools for identification in fungal epidemiology. 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.
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.
Thus, microbiologists use characteristic biochemical activities to more specifically identify bacterial species. Knowledge of these key characteristics will enable the identification of unknown bacterial isolates. It is important to thoroughly understand the basis for each biochemical test and know the key physiological characteristics of the bacterial genera and species presented in these labs. Rachel Watson, M. Real-time PCR facilitates a rapid detection of low amounts of bacterial DNA accelerating therapeutic decisions and enabling an earlier adequate antibiotic treatment.
Microarrays combines the potential of simultaneous bacterial identification and speciation. This method is versatile and makes it possible to detect and discriminate different bacterial samples on a single slide. The rapid identification of the bacteria in clinical samples is important for patient management and antimicrobial therapy.
DNA microarray-based approach is used for the quick detection and identification of bacteria using species-specific oligonucleotide probes designed for specific regions of various targeted genes. Toggle navigation. Challenges in Bacterial Identification Traditional methods of bacterial identification rely on phenotypic identification of the causative organism using gram staining, culture and biochemical methods.
Real Time PCR Based Bacterial Identification Using a DNA based assay, one can easily detect bacterial strains directly from clinical samples or from small amounts of cultured bacterial cells, thus improving the sensitivity and decreasing the time required for bacterial identification. Microarray Based Bacterial Identification Microarrays combines the potential of simultaneous bacterial identification and speciation.
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