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Abstract

Rapid detection and identification of microorganisms is a challenging and important aspect in a wide range offields, from medical to industrial, affecting human lives. Unfortunately, classical methods of microorganism identification are based on time-consuming and labor-intensive approaches. Screening techniques require the rapid and cheap grouping of bacterial isolates; however, modern bioanalytics demand comprehensive bacterial studies at a molecular level. Modern approaches for the rapid identification of bacteria use molecular techniques, such as 16S ribosomal RNA gene sequencing based on polymerase chain reaction or electromigration, especially capillary zone electrophoresis and capillary isoelectric focusing. However, there are still several challenges with the analysis of microbial complexes using electromigration technology, such as uncontrolled aggregation and/or adhesion to the capillary surface. Thus, an approach using capillary electrophoresis of microbial aggregates with UV and matrix-assisted laser desorption ionization time-of-flight MS detection is presented.

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... Rapid identification of bacterial and viral infections is a strategic stage in the fight for the patient's life because only a thorough understanding of the source of the disease allows implementation of a proper therapy. Unfortunately, despite their high accuracy, the methods currently used for this purpose (based on molecular biology) are expensive, time-consuming, and can be performed only in well-equipped laboratories with a high biosafety level (BSL 2 or 3) and qualified staff, access to which is limited in many regions of the world [13]. The accurate identification of pathogenic microorganisms is essential for scientists involved in many areas of applied research and industry, from clinical microbiology to food production. ...
... Importantly, phenotypic methods are not always suitable to identify microorganisms at the species level, or much more often at the strain level. One of the strategies to reduce time needed for microbial identification is the use of molecular biology techniques, which may also be supplemented with numerous molecular fingerprinting techniques [13]. Each of these methods has their strengths and weaknesses, and the most recent research approach involves the use of a compilation of multivariate techniques. ...
... Reliable and efficient identification of pathogens is a key tool enabling implementation of appropriate therapies, and thus saving lives. Unfortunately, conventional techniques used in diagnostic laboratories to identify pathogens based on biochemical tests that differentiate bacteria phenotypically are time consuming, work intensive and often inadequate to differentiate between phenotypically similar species [13]. ...
Article
Fast determination, identification and characterization of pathogens is a significant challenge in many fields, from industry to medicine. Standard approaches (e.g., culture media and biochemical tests) are known to be very time-consuming and labor-intensive. Conversely, screening techniques demand a quick and low-cost grouping of microbial isolates, and current analysis call for broad reports of pathogens, involving the application of molecular, microscopy, and electromigration techniques, DNA fingerprinting and also MALDI-TOF methods. The present COVID-19 pandemic is a crisis that affects rich and poor countries alike. Detection of SARS-CoV-2 in patient samples is a critical tool for monitoring disease spread, guiding therapeutic decisions and devising social distancing protocols. The goal of this review is to present an innovative methodology based on preparative separation of pathogens by electromigration techniques in combination with simultaneous analysis of the proteome, lipidome, and genome using laser desorption/ionization analysis.
... These methods include biotyping, serotyping, bacteriophage typing, evaluation of susceptibility profiles and protein analysis methods. Biotyping examines biochemical requirements, environmental conditions (pH, temperature, antibiotic resistance, susceptibility to bacteriocins) and physiological aspects (colony and cell morphology, cell walls and cell membrane composition such as fatty acid profiles) [100][101][102]. ...
... A further acceleration of the time of the detection and identification of microorganisms was possible thanks to the introduction of a new generation of automatic systems for the identification of bacteria based on biochemical properties. The identification of bacterial culture based on the principle of comparing the biochemical reaction profile with the database is most often performed using automated sets such as Analytical Profile Index API ® , BD Phoenix™ Automated Microbiology System or Vitek 2 Compact [100,104]. Commercial automated systems for the identification and determination of bacterial susceptibility are usually based on the same principles as conventional tests, as they use miniaturized versions of these tests. In addition, automated systems not only determine susceptibility but also indicate the likely mechanism of antibiotic resistance, such as extended-spectrum beta-lactamases (ESBL), methicillin resistance in Staphylococci, glycopeptide resistance or high-degree aminoglycosides resistance in Enterococci [100,107,108]. ...
... Commercial automated systems for the identification and determination of bacterial susceptibility are usually based on the same principles as conventional tests, as they use miniaturized versions of these tests. In addition, automated systems not only determine susceptibility but also indicate the likely mechanism of antibiotic resistance, such as extended-spectrum beta-lactamases (ESBL), methicillin resistance in Staphylococci, glycopeptide resistance or high-degree aminoglycosides resistance in Enterococci [100,107,108]. ...
Article
Full-text available
Antibiotics were initially natural substances. However, nowadays, they also include synthetic drugs, which show their activity against bacteria, killing or inhibiting their growth and division. Thanks to these properties, many antibiotics have quickly found practical application in the fight against infectious diseases such as tuberculosis, syphilis, gastrointestinal infections, pneumonia, bronchitis, meningitis and septicemia. Antibiotic resistance is currently a detrimental problem; therefore, in addition to the improvement of antibiotic therapy, attention should also be paid to active metabolites in the body, which may play an important role in exacerbating the existing problem. Taking into account the clinical, cognitive and diagnostic purposes of drug monitoring, it is important to select an appropriate analytical method that meets all the requirements. The detection and identification of the microorganism responsible for the infection is also an essential factor in the implementation of appropriate antibiotic therapy. In recent years, clinical microbiology laboratories have experienced revolutionary changes in the way microorganisms are identified. The MALDI-TOF MS technique may be interesting, especially in some areas where a quick analysis is required, as is the case with clinical microbiology. This method is not targeted, which means that no prior knowledge of the infectious agent is required, since identification is based on a database match.
... Since the microorganisms carry charged or chargeable groups on their outer surface, they can be easily separated using electrophoretic techniques [23][24][25][26][27] focusing (IEF) is suitable technique for both separation and pre-concentration of microorganisms [28][29][30]. The IEF separates amphoteric analytes according to their different isoelectric points (pI) and it also increases concentration of the separated analytes during focusing. ...
... The major problem regarding CIEF separations is that microbial cells have a strong tendency to form aggregates and to interact with the inner surface of the separation capillary. Nevertheless, several approaches based on the use of different additives (e.g., poly(ethylene oxide) or various detergents), either into the electrophoretic buffers or into the microbial samples, have been proposed to solve this problem [26,27,29]. Although CIEF represents an efficient analytical technique for analysis of various microorganisms, its wider application is limited by the very small volume of the sample injected into the separation capillary. ...
Article
This study describes a new method for simultaneous identification of uropathogens in the case of polybacterial urinary tract infections. The method utilizes recently developed preparative isoelectric focusing (IEF) in cellulose-based separation medium with a subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Preparative IEF was successfully used for both purification and separation of bacteria, Escherichia coli (pI 4.6) and Staphylococcus aureus (pI 3.4), in urine samples. The focused zones of bacteria, localized by the positions of focused colored pI markers, were easily collected from the separation media after the IEF analysis and then unambiguously identified by MALDI-TOF MS. The proposed method enables the identification of bacteria in urine specimens when the concentration of individual bacteria is ≥104 cells mL-1. Another benefit is the viability of bacteria extracted from the collected fractions after preparative IEF.
... Currently, automatic and semi-automatic systems are the most commonly used biochemical methods for identifying microorganisms. The most commonly used automated techniques are VITEK ® 2 Compact (bioMerieux, Marcy l'Etoile, France) and BD Phoenix (BD Diagnostics, Franklin Lakes, NJ, USA) [14]. ...
... The market offers numerous semi-automatic biochemical tests for the identification of Gram-negative bacteria, including MICROBACT™ (Thermofisher), The Microgen ™ (Microbiology Interational), Microgen Biochemical Identification Kits (Microgen Bioproducts), EnteroTest 24N (Erba-Lachema, Brno, Czech Republic) and API ® (bioMerieux, Marcy l'Etoile, France), of which the API ® system is the most popular test [14]. ...
Article
Full-text available
Enterobacterales is a prevalent order, which inhabits a variety of environments including food. Due to the high similarities between pathogenic and non-pathogenic species, their identification might be difficult and laborious, and therefore there is a need for rapid and precise identification. The aim of this study was to compare the effectiveness of the available methods of identifying order Enterobacterales strains isolated from fresh fish and shrimps (n = 62). The following methods were used in this study: biochemical, sequencing and identification using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For this purpose, biochemical identification was performed with the use of the EnteroTest 24N set, while the identification using the MALDI-TOF MS technology was operated on VITEK® MS. Results were compared with identification made by 16S rRNA sequencing. The results of the study showed that conventional identification methods might provide a false result. Identification by VITEK® MS to the species level was correct at 70.97%, and the accuracy of EnteroTest 24N identification did not exceed 50.0%. The genus identification reached 90.32% for the MALDI-TOF technique, while for EnteroTest 24N it was nearly 70.0%. Due to errors in identification, especially of pathogenic organisms, the use of each of these methods should be confirmed by another method, preferably sequencing.
... Generally, a biotool should be fast and accurate in identifying specific microbial species, even when a sample contains multiple different species. Moreover, pathogenic bacteria analysis is routinely performed via phenotypic tests, such as culture and growth characteristics, Gram staining, and biochemical patterns [4]. These methods are considered the gold standard molecular biology tools for the characterization and identification of microbial species with moderate sensitivity and specificity. ...
... Using these fingerprint signatures, bacteria can be accurately detected by the interpretation of protein profiles with reference data via various algorithms [17,23,24]. As a result, MALDI-TOF MS has been used for the generation of protein sequences/fingerprints from whole bacterial cells, such as Escherichia coli, Bacillus cereus, Staphylococcus aureus and Listeria species [4,[22][23][24]34]. Cain et al. first reported the utilization of MALDI-TOF MS for the simple and rapid identification of protein profiles in microorganisms [35]. ...
Article
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has achieved considerable progress in bacterial analysis, allowing the identification of pathogenic microbial species at both species and genus level. This review analyzes the recent progress and implications of MALDI-TOF MS as a promising biotool for the rapid analysis of multiple pathogenic microbes in environmental and clinical samples. Nanoparticle (NP)-based MALDI-TOF MS methods for accurate bacterial analysis are also discussed. This review also summarizes sample preparation protocols and used matrix solutions in MALDI-TOF MS for the analysis of microbial species. Furthermore, we discuss the currently established advantages and limitations of MALDI-TOF MS in identifying microbial species.
... Mossel medium culture, macroscopic analysis, microscopic analysis (Gram-positive bacilli), Gram-positive bacteria with 3% KOH, spore-forming bacteria test, and the positive catalase reaction had allowed the classification of the working bacterium as the Bacillus genus. A total of 155 pure cultured isolates were obtained from different raw material palm wine (30), Ntoba Mbodi (21), Mbala-mpinda (19), Pandé (12), banana (35), sugar cane wine (13), soil (15), and intestine of Guppy fish (10). All bacteria were spore-forming colonies, catalase positive and Gram-positive staining. ...
... It seems obvious that the use of molecular identification methods has more advantages. These methods are more reliable [35] compared to conventional one. Here, we exploited the polymorphism of the fibE gene. ...
Article
Full-text available
Fibrinolytic enzyme gene (fibE) is widely conserved among Bacillus spp. belonging to group I species. This is encoding a serine-like enzyme (FibE) secreted in extracellular medium. This present work aims to assess the molecular usefulness of this novel conserved housekeeping gene among group I Bacillus spp. to identify and discriminate some related strains in traditional fermented food and beverages in Republic of Congo. First of all 155 isolates have been screened for enzymatic activities using caseinolytic assays. PCR techniques and nested PCR method using specific primers and correlated with 16S RNA sequencing were used. Blotting techniques have been performed for deep comparison with molecular methods. As a result B. amyloliquefaciens (1), B. licheniformis (1), B. subtilis (1), B. pumilus (3), B. altitudinis (2), B. atrophaeus (1), and B. safensis (3) have been specifically identified among 155 isolates found in fermented food and beverages. Genetic analysis and overexpression of glutathione S-transferases (GSTs) fused to mature protein of FibE in Escherichia coli BL21 and TOP10 showed 2-fold higher enzymatic activities by comparison with FibE wild type one. Immunodetection should be associated but this does not clearly discriminate Bacillus belonging to group I.
... The identification of the microorganism is a tedious task [9]. There is always a need for an expert and a person who understands the nuances of taxonomy and other characteristics of microbes such as odor [10]. ...
... Recent studies [9,19,20] are focusing on automating the process of identification with the help of image processing and machine learning models. The purpose of image processing is to effectively isolate the microorganism body from the images. ...
Article
Full-text available
The accurate classification of microbes is critical in today’s context for monitoring the ecological balance of a habitat. Hence, in this research work, a novel method to automate the process of identifying microorganisms has been implemented. To extract the bodies of microorganisms accurately, a generalized segmentation mechanism which consists of a combination of convolution filter (Kirsch) and a variance-based pixel clustering algorithm (Otsu) is proposed. With exhaustive corroboration, a set of twenty-five features were identified to map the characteristics and morphology for all kinds of microbes. Multiple techniques for feature selection were tested and it was found that mutual information (MI)-based models gave the best performance. Exhaustive hyperparameter tuning of multilayer layer perceptron (MLP), k-nearest neighbors (KNN), quadratic discriminant analysis (QDA), logistic regression (LR), and support vector machine (SVM) was done. It was found that SVM radial required further improvisation to attain a maximum possible level of accuracy. Comparative analysis between SVM and improvised SVM (ISVM) through a 10-fold cross validation method ultimately showed that ISVM resulted in a 2% higher performance in terms of accuracy (98.2%), precision (98.2%), recall (98.1%), and F1 score (98.1%).
... On the other hand, molecular markers were successfully used for classification and identification of many microbial strains [24,25]. Recently, the positive and negative points of bacterial identification by molecular and other techniques have been reviewed, intensively [25][26][27]. ...
Article
Full-text available
S. gregaria is able to swarm and disseminate bacterial load over vast areas. Adult locusts were collected from Kingdom of Saudi Arabia (KSA), individual locust was divided into head, thorax and abdomen. External and internal cultivable bacteria were cultured, cell and colony characteristics were investigated. Pure colonies were sequenced using 16S rRNA. Sequences and phylogeny were analyzed. Using morphological and molecular results, 9 bacterial genera belonging to 5 families, 3 orders, 2 classes, and 2 phyla were recorded. Ribosomal Database Project (RDP) Classifier succeeded to confirm 8 out of 9 genera. The ninth isolate was designated as unclassified. To specify a genus to its closest species, 16S rRNA genes of the type species have been downloaded. Phylogenetic tree was generated using all available sequences. Similarity measures and phylogenetic trees were used to place each genus to its closest species. Six genera created similarities less than 98% to related species. Generally, analyses of 16S rRNA gene succeeded to identify 3 isolates to species level, 5 isolates to genus level and designated one isolate “unclassified”. Investigations are recommended to place all genera to their operational taxonomic units (OTU). Significance of our findings relies on the impact of locust-microbiota on heath of people who consumed insects as food. Bacterial species which were internally, isolated from abdomen were described beneficial commensals. Bacterial species which were internally, isolated from thorax and head were described non-pathogenic beneficial endosymbionts, except Bacillus sp. Salmonella sp. was isolated as external species and was described external contaminant of the locust.
... Such PCR-based DNA fingerprinting techniques are rapid, having moderate-to-high sensitivity, cost-effective compared to DNA sequencing and metagenomics and multiple samples can be analyzed at same time. The DNA fingerprinting approaches have revealed the microbial communities or differences between such microbial communities in one step; however, these may not provide phylogenetic information in some instances (Buszewski et al. 2017). These DNA fingerprinting techniques have the potential of replacing the morphological/phenotypic methods which are labor-intensive and time-consuming. ...
Article
Zinc plays a key role in plant nutrition at low levels; however, at higher concentrations Zn ions can be highly phytotoxic and plant growth-promoting rhizobacteria can be used to reduce such metal toxicity. In the present investigation we had reported the zinc biosorption and molecular characterization of plant growth-promoting zinc-tolerant bacteria. Initially, thirty bacteria having zinc solubilizing ability were screened for MIC against zinc ion and displayed high value of MIC ranging from 2.5 to 62.5 mM. Biochemically, all the 30 isolates showed significant difference in the 6 biochemical tests performed. The molecular diversity studies based on the repetitive DNA PCR viz, REP, ERIC and BOX elements showed significant genetic diversity among these 30 zinc-tolerant bacteria. These ZTB strains also showed multiple PGP activities and all ZTB strains were found positive for production of IAA, GA3 and ammonia, whereas 24 were found positive for ACC deaminase activity, 8 showed siderophore production and 9 ZTB isolates were positive for HCN production. Out of 30 isolates, 24 showed phosphorus solubilization activity, 30 showed potash solubilization, 15 showed silica solubilization and 27 showed phytase production activities. All the 30 ZTB stains showed zinc solubilization up to 0.25% insoluble ZnO in the medium, whereas at 2% ZnO in MSM only 12 isolates showed solubilization which were further selected for zinc biosorption and pot studies. The heavy metal removal studies revealed that ZTB stains were able to remove zinc ions effectively from the medium efficiently and the highest zinc biosorption (< 90%) was recorded with the bacterial strain Z-15. Further, the inoculation of ZTB strains under zinc stress conditions (pot containing 1000 mg/kg Zn) resulted in significant increase of shoot length, root length and total chlorophyll content in maize seedlings compared with the uninoculated control. The partial 16S rDNA sequence of the potential ZTB isolates viz. Z-15, Z-24, Z-28 and Z-29 revealed their identity as Serratia sp. The ability of these zinc-tolerant bacteria to tolerate the toxic level of zinc may serve as suitable candidates for developing microbial formulations for the growth of crop plants in Zn-contaminated areas.
... The MALDI-TOF MS technique is used in routine microbiological diagnostics to identify species of food-related microorganisms (Dušková et al., 2012;Król et al., 2018). In addition, attempts have been made to identify species of LAB both in clinical samples and in food (Buszewski et al., 2017;Król et al., 2018). The MALDI technique is also a promising tool in lipidomic research, in particular for imaging lipids from tissue preparations, as well as for the identification and classification of bacteria (Pulfer and Murphy, 2003). ...
Article
Full-text available
Analysis by MALDI-TOF mass spectrometry and gas chromatography-mass spectrometry was used to characterize the lipid profile of 3 lactic acid bacteria strains. By gas chromatography coupled with mass spectrometry, 23 fatty acids were identified. Dominant acids were palmitic (C16:0), oleic (C18:1), and α-linoleic acid (C18:3n-3) for Lactobacillus paracasei; for Lactococcus lactis they were palmitic (C16:0), gondoic (C20:1), myristoleic (C14:1), and eicosadienoic acid (C20:2), respectively; and in the case of Lactobacillus curvatus were C18:1, C18:2n-6, and C16:0, respectively. The effect of the medium on fatty acid composition was also determined. In addition, the fatty acid profile was also compared using MALDI MS analysis. The MALDI-TOF MS was used for qualitative analysis and identification of bacterial lipids. Phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, triacylglycerols, and ceramides were the most abundant species in lactic acid bacteria. One hundred different combinations of fatty acids in polar and nonpolar lipids have been identified, including 11 phospholipids (18 phosphatidylglycerol, 16 phosphatidylethanolamine, 10 phosphatidylinositol, 8 phosphatidylcholine, 4 lyso-phosphatidylethanolamine, 3 lyso-phosphatidylcholine, 3 phosphatidylserine, 1 lyso-phosphatidic acid, 1 lyso-phosphatidylglycerol, 1 lyso-phoshatidylinositol, and 1 phosphatidic acid), 23 triacylglycerols, 9 ceramides, and 2 sphingomyelin. The most abundant fatty acids identified were C16:0, C16:1, C18:0, and C18:3. Obtained lipid profiles allowed to distinguish the tested bacterial strains.
... The inspection authorities need reliable control methods. Therefore, it is essential to have valid methods to monitor freshness and safety in order to ensure quality, regardless of the point of view of the consumer, the industry, the inspection authority or the scientist (Nychas, 2011;Buszewski et al. 2017). ...
Article
Food is of vital importance for human life. Consumers prefer healthy, safe and quality food. In recent years, consumers are prone towards ready to eat, ready to serve and ready to cook foods. Therefore, to maintain the long shelf life of these foods without affecting their sensory attributes, the preservation of foods is a major challenge for producers. Food moves through many levels, from production to storage domains. During these phases, several physical, chemical and microbiological factors are responsible for deterioration in quality. Spoilage of food is a threat to food security. In this article, the role of different type of microbes in food processing, methods for detecting spoilage and preventive measures have been explored. The beneficial functions of microbes have also been highlighted in this article.
... Such PCR-based DNA fingerprinting techniques are rapid, having moderate-to-high sensitivity, cost-effective compared to DNA sequencing and metagenomics and multiple samples can be analyzed at same time. The DNA fingerprinting approaches have revealed the microbial communities or differences between such microbial communities in one step; however, these may not provide phylogenetic information in some instances (Buszewski et al. 2017). These DNA fingerprinting techniques have the potential of replacing the morphological/phenotypic methods which are labor-intensive and time-consuming. ...
Article
Zinc plays a key role in plant nutrition at low levels; however, at higher concentrations Zn ions can be highly phytotoxic and plant growth-promoting rhizobacteria can be used to reduce such metal toxicity. In the present investigation we had reported the zinc biosorption and molecular characterization of plant growth-promoting zinc-tolerant bacteria. Initially, thirty bacteria having zinc solubilizing ability were screened for MIC against zinc ion and displayed high value of MIC ranging from 2.5 to 62.5 mM. Biochemically, all the 30 isolates showed significant difference in the 6 biochemical tests performed. The molecular diversity studies based on the repetitive DNA PCR viz, REP, ERIC and BOX elements showed significant genetic diversity among these 30 zinc-tolerant bacteria. These ZTB strains also showed multiple PGP activities and all ZTB strains were found positive for production of IAA, GA3 and ammonia, whereas 24 were found positive for ACC deaminase activity, 8 showed siderophore production and 9 ZTB isolates were positive for HCN production. Out of 30 isolates, 24 showed phosphorus solubilization activity, 30 showed potash solubilization, 15 showed silica solubilization and 27 showed phytase production activities. All the 30 ZTB stains showed zinc solubilization up to 0.25% insoluble ZnO in the medium, whereas at 2% ZnO in MSM only 12 isolates showed solubilization which were further selected for zinc biosorption and pot studies. The heavy metal removal studies revealed that ZTB stains were able to remove zinc ions effectively from the medium efficiently and the highest zinc biosorption (< 90%) was recorded with the bacterial strain Z-15. Further, the inoculation of ZTB strains under zinc stress conditions (pot containing 1000 mg/kg Zn) resulted in significant increase of shoot length, root length and total chlorophyll content in maize seedlings compared with the uninoculated control. The partial 16S rDNA sequence of the potential ZTB isolates viz. Z-15, Z-24, Z-28 and Z-29 revealed their identity as Serratia sp. The ability of these zinc-tolerant bacteria to tolerate the toxic level of zinc may serve as suitable candidates for developing microbial formulations for the growth of crop plants in Zn-contaminated areas.
... Even though, some amount of mechanization and automation has been used in techniques like flow cytometry, enzyme-linked immunosorbent assay (ELISA), or polymerase chain reaction (PCR), we cannot consider these as fully automated as their outcomes cannot be used automatically for plant operation control (Sharpe Anthony 2018). Chemical sensors and biosensors (Poghossian et al. 2019), spectrometric methods, PCR-based methods, multivariate techniques (Buszewski et al. 2017), etc. are proposed as alternative faster methods. A detailed description on such rapid methods is given elsewhere (Chaps. ...
Chapter
Dairy engineering is the multidisciplinary field located at the intersection of “dairy” and “engineering.” It involves the utilization of insights in the areas like processing techniques, physical properties, chemistry, microbiology, engineering, economics, and management for fixing issues related with production, processing, testing, handling, and storage of milk and milk products. This chapter describes briefly about the advanced engineering interventions in milk production, testing, processing and associated activities, storage, and distribution.
... Several recently published studies have referred to the use of CE for the investigation of the bacterial metabolome through the monitoring of processes that take place in the extra-and intracellular environment, the detection and quantification of bacterial metabolites or endotoxins in various samples, and the study of target and non-targeted fingerprinting of microbiota in biological samples [45,46]. Although this analytical method has significant advantages, it also has disadvantages, mainly related to the low repeatability of retention times, low sensitivity, and the possible adhesion of basic compounds to the capillary walls; these disadvantages make it impossible to use this method for biological sample investigation. ...
Article
Full-text available
Contamination of surface waters with pathogens as well as all diseases associated with such events are a significant concern worldwide. In recent decades, there has been a growing interest in developing analytical methods with good performance for the detection of this category of contaminants. The most important analytical methods applied for the determination of bacteria in waters are traditional ones (such as bacterial culturing methods, enzyme-linked immunoassay, polymerase chain reaction, and loop-mediated isothermal amplification) and advanced alternative methods (such as spectrometry, chromatography, capillary electrophoresis, surface-enhanced Raman scattering, and magnetic field-assisted and hyphenated techniques). In addition, optical and electrochemical sensors have gained much attention as essential alternatives for the conventional detection of bacteria. The large number of available methods have been materialized by many publications in this field aimed to ensure the control of water quality in water resources. This study represents a critical synthesis of the literature regarding the latest analytical methods covering comparative aspects of pathogen contamination of water resources. All these aspects are presented as representative examples, focusing on two important bacteria with essential implications on the health of the population, namely Pseudomonas aeruginosa and Escherichia coli.
... The identification of bacteria based on the phenotype has been carried out, but this identification method needs more time and energy (López-Campos et al. 2012), different environmental conditions may decreases the validity (Tshikhudo et al. 2013), some bacteria difficult to be cultured so that identification cannot be done (Gugliandolo et al. 2011), and there are some morphological and physiological similarities that are difficult to be distinguished (Boivin-Jahns et al. 1995). A fast and reliable method is widely used to overcome this problem by analyzing the barcode 16S rRNA gene (Buszewski et al. 2017;Tshikhudo et al. 2013). ...
Article
Four bacterial isolates from Ranu Pani and Ranu Grati in east java had been revealed to be potentials to produce IAA (PIS isolate), phosphate solubilizer (GPS isolate), cellulose hydrolysis (PSS isolate) and, amylum hydrolysis (PAS), two dominant bacterial isolates from Rani Pani (PØD isolate) and Ranu Grati (GØD isolate) which were co-cultured with microalgae promoted microalgae growth, yet its taxonomical position has not been clearly known. The aim of this study was to identify those bacterial isolates using 16S rRNA barcode. This research conducted by gDNA isolation, the 16S rRNA sequence was amplified using 27F and 1492R primers. Reconstructed phylogenetic trees and genetic distance analysis showed that the isolate PIS and PSS identified as Bacillus cereus Group closely related to Bacillus paramycoides. PAS isolate identified as Bacillus subtilis Group closely related to Bacillus amyloliquefaciens, GPS isolate identified as novel species in genus Enterobacter, and two dominant isolates (PØD and GØD) identified as Enterobacter cloacae complex closely related to Enterobacter cloacae. The genomic approach and additional phenotypes-examination are required to clarify its taxonomical position.
... Temel olarak analiz edilecek bakteri kolonilerinin kısa dalga boylu lazer aracılığıyla iyonize edilip hızlandırılarak elektrik alanından geçirilmesi olarak açıklanabilir. Ortaya çıkan spektral profilin bilgisayar yardımıyla analizi sayesinde bakteri idantifikasyonuna gidilmektedir (39). Yüksek derecede duyarlılık ve verimle çalışan bu yöntemin başarısı pek çok çalışmayla ortaya konulmuştur. ...
Article
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Bacteria are organisms that have interacted with human beings for centuries. In this interaction, some species of bacteria have found their place in human microbiota, and have taken useful and important roles. Despite their usefulness, bacteria that cause disease in humans have been responsible for the loss of thousands of people for centuries and even in the age we live. After the discovery of antibiotics and their coming into use, our success rate has increased in this struggle. Besides, it is crucial that the correct antibiotic is given to the patient immediately. In order to achieve this, rapid identification of the pathogen is required. Classical diagnostic methods include time-consuming and labor-intensive procedures based on the cultivation and characterization of bacteria. Even though these processes are carried out close to the ideal, the time spent, leads to a delay in initiating therapy, increased morbidity, mortality and labor loss. Today's modern scientific world has enabled faster, more precise and specific identification of organisms through the development of different devices and approaches. By means of methods such as polymerase chain reaction and DNA sequence analysis, organisms can be identified genotypically. Moreover, with these techniques, species identification has become clearer. In this way, clinicians have been able to plan treatment with more accurate data. Methods such as matrix-assisted laser desorption/ioniza-tion time-of-flight mass spectrometry, which are gradually becoming a part of routine clinical microbiology procedures, made bacteria identifiable within hours, even those which used to take a long time to identify. This review will emphasize the use of these methods in clinical microbiology in particular, although it is certain that many of them will also have a great importance for the control of environmental samples, food and pharmaceutical products. Klimik Dergisi 2018; 31(3): 176-80.
... nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FTIR), ultraviolet-visible (UV-VIS) spectroscopy, or fluorescence based-methods, is the basic approach in identification of biologically active compounds. Its advantage is it has very high sensitivity, which in combination with other analytical techniques, such as liquid chromatography (LC-MS) or electrophoretic (e.g., capillary zone electrophoresis (CZE-MS) and two-dimensional gel electrophoresis (2D-GE)) approach, gives a powerful tool for analysis of very complex mixtures, e.g., peptides and proteins, such as casein fractions [1], or even proteins of microbial cells [2]. Despite the huge variety of mass spectrometry techniques, the construction design in all the instruments is similar. ...
Article
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In recent years, matrix-assisted laser desorption/ionization (MALDI) has become the main tool for the study of biological macromolecules, such as protein nano-machines, especially in the determination of their molecular masses, structure, and post-translational modifications. A key role in the classical process of desorption and ionization of the sample is played by a matrix, usually a low-molecular weight weak organic acid. Unfortunately, the interpretation of mass spectra in the mass range of below m/z 500 is difficult, and hence the analysis of low molecular weight compounds in a matrix-assisted system is an analytical challenge. Replacing the classical matrix with nanomaterials, e.g., silver nanoparticles, allows improvement of the selectivity and sensitivity of spectrometric measurement of biologically important small molecules. Nowadays, the nanostructure-assisted laser desorption/ionization (NALDI) approach complements the classic MALDI in the field of modern bioanalytics. In particular, the aim of this work is to review the recent advances in MALDI and NALDI approaches.
... Even though we could end up with 100% detection of dermatophyte DNA but still the assay needs to be better optimized. And moreover, we need to use different reagents than those which are previously used (36) . And also, the gold standard methods could only permit the correct identification, if at all carried by a qualified mycologist who is having a sound knowledge on the morphological features of dermatophytes micro-and macroconidia. ...
Article
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Athlete's foot (Tinea pedis) and toe nails infection (onychomycosis) are disease conditions caused by dermatophytes; both diseases are prevalent in adults, especially in farmers who often wear robber shoes during farming. Proper treatment of dermatophytes related skin diseases needs a proper understanding of susceptibility of the causative agents to the intended treatment option. This knowledge can only be derived from proper identification and characterization of the related fungi. In the present study, both traditional and molecular identification approaches were applied on cultured samples for detection and identification of Tinea pedis. From the PCR analysis, Microsporum canis and Trichophyton mentagrophytes were identified as the two commonest species implicated in Tinea pedis basing on their DNA banding patterns. From the fifty two identified dermatophyte isolates via the conventional identification methods, only 45 isolates were confirmed via molecular approach, with 25 isolated being confirmed as M. canis while 20 isolates were T. mentagrophytes. The same was confirmed with real time PCR quantification.
... Mass spectrometry (MS) based techniques are an important microbial-typing tool because of the rapidity, low expense, ease of use, and effectiveness of all kinds of bacteria, archaea, and fungi. Mass spectrometry can be associated with multiple ionization and separation methods, such as gas chromatography (GC) and liquid chromatography (LC) (Fox 2006), matrix-assisted laser desorption ionization timeof-flight mode (MALDI-TOF) (Jang and Kim 2018), electromigration techniques (Buszewski et al. 2017), or electrospray ionization (ESI) (Zhang et al. 2011). ...
Chapter
Pathogenic infections cause tremendous health threats and socioeconomic burdens worldwide. Conventional methods for bacteria detection are laborious, time-consuming, expensive, and require particular devices and highly qualified specialists. Sensitive, selective, inexpensive, quick, and user-friendly biosensors are in urgent demand to prevent and detect bacterial infections in many fields, e.g., healthcare, food industry, or terrorism prevention. Among biorecognition elements utilized in biosensors, bacteriophages are highly promising due to their numerous advantages, such as host specificity, cheap and simple production, resistance to external factors, and ease of immobilization. Here we reviewed currently used methods for bacteria detection, pointing their advantages and disadvantages. We paid particular attention to bacteriophage-based methods, including phage-based sensors and phage display method.
... These techniques are laborious, slow and unspecific in some cases. New methods for the identification of bacteria based on the principles of molecular biology have also been developed and specially polymerase chain reactions (PCR) based methods [20,21]. In the last decade, different methods based on the combination of sensitive techniques such as surface-enhanced Raman spectroscopy (SERS), electrochemical methods or fluorescence methods combined with the use of nanoparticles, especially metal nanoparticles, for rapid and sensitive identification of bacteria have been developed. ...
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Advances in nanoparticle-based systems constitute a promising research area with important implications for the treatment of bacterial infections, especially against multidrug resistant strains and bacterial biofilms. Nanosystems may be useful for the diagnosis and treatment of viral and fungal infections. Commercial diagnostic tests based on nanosystems are currently available. Different methodologies based on nanoparticles (NPs) have been developed to detect specific agents or to distinguish between Gram-positive and Gram-negative microorganisms. Also, biosensors based on nanoparticles have been applied in viral detection to improve available analytical techniques. Several point-of-care (POC) assays have been proposed that can offer results faster, easier and at lower cost than conventional techniques and can even be used in remote regions for viral diagnosis. Nanoparticles functionalized with specific molecules may modulate pharmacokinetic targeting recognition and increase anti-infective efficacy. Quorum sensing is a stimuli-response chemical communication process correlated with population density that bacteria use to regulate biofilm formation. Disabling it is an emerging approach for combating its pathogenicity. Natural or synthetic inhibitors may act as antibiofilm agents and be useful for treating multi-drug resistant bacteria. Nanostructured materials that interfere with signal molecules involved in biofilm growth have been developed for the control of infections associated with biofilm-associated infections.
... MALDI-TOF MS is a fast and accurate method based on the ionization of intact microorganism cells with short laser pulses and the subsequent acceleration of the particles in a vacuum by way of an electric field. Each microorganism has a specific spectrum profile [46]. ...
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Saliva is a fascinating biological fluid which has all the features of a perfect diagnostic tool. In fact, its collection is rapid, simple, and noninvasive. Thanks to several transport mechanisms and its intimate contact with crevicular fluid, saliva contains hundreds of proteins deriving from plasma. Advances in analytical techniques have opened a new era—called “salivaomics”—that investigates the salivary proteome, transcriptome, microRNAs, metabolome, and microbiome. In recent years, researchers have tried to find salivary biomarkers for oral and systemic diseases with various protocols and technologies. The review aspires to provide an overall perspective of salivary biomarkers concerning oral diseases such as lichen planus, oral cancer, blistering diseases, and psoriasis. Saliva has proved to be a promising substrate for the early detection of oral diseases and the evaluation of therapeutic response. However, the wide variation in sampling, processing, and measuring of salivary elements still represents a limit for the application in clinical practice.
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Bacterial identification methods used in routine identification of pathogens in medical microbiology include a combination approach of biochemical tests, mass spectrometry or molecular biology techniques. Extensive publicly-available databases of DNA sequence data from pathogenic bacteria have been amassed in recent years; this provides an opportunity for using bacterial genome sequencing for identification purposes. Whole genome sequencing is increasing in popularity, although at present it remains a relatively expensive approach to bacterial identification and typing. Complexity-reduced bacterial genome sequencing provides an alternative. We evaluate genomic complexity-reduction using restriction enzymes and sequencing to identify bacterial isolates. A total of 165 bacterial isolates from hospital patients in the Australian Capital Territory, between 2013 and 2015 were used in this study. They were identified and typed by the Microbiology Department of Canberra Public Hospital, and represented 14 bacterial species. DNA extractions from these samples were processed using a combination of the restriction enzymes PstI with MseI, PstI with HpaII and MseI with HpaII. The resulting sequences (length 30–69 bp) were aligned against publicly available bacterial genome and plasmid sequences. Results of the alignment were processed using a bioinformatics pipeline developed for this project, Currito3.1 DNA Fragment Analysis Software. All 165 samples were correctly identified to genus and species by each of the three combinations of restriction enzymes. A further 35 samples typed to the level of strain identified and compared for consistency with MLST typing data and in silico MLST data derived from the nearest sequenced candidate reference. The high level of agreement between bacterial identification using complexity-reduced genome sequencing and standard hospital identifications indicating that this new approach is a viable alternative for identification of bacterial isolates derived from pathology specimens. The effectiveness of species identification and in particular, strain typing, depends on access to a comprehensive and taxonomically accurate bacterial genome sequence database containing relevant bacterial species and strains.
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Several non-culture molecular (multiplex polymerase chain reaction assays, DNA microarrays, massive parallel DNA sequencing, in situ hybridization, microbiome profiling, and molecular typing of pathogens) and analytical (electrophoresis, gel electrophoresis, surface-enhanced Raman scattering, and mass spectrometry) tools have been developed in recent years for the identification of bacteria and diagnosis of bacterial infections from clinical samples. Among mass spectrometric techniques, electrospray ionization (ESI) and rapid evaporative ionization (REI) mass spectrometric (MS) techniques have attracted much attention in the identification of microorganisms (bacteria, fungi, and virus), and in the diagnosis of various bacterial infections. This review highlights the developed ESI-MS-based methods including, polymerase chain reaction (PCR) combined with ESI-MS and capillary electrophoresis (CE) and liquid chromatography (LC) -ESI-MS for the identification of microorganisms (pathogenic bacteria, fungi, and virus) in various samples. Recent applications of ESI- and REI-MS in identifying pathogenic bacteria are depicted in tables, and some significant findings are summarized.
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Here we demonstrate a direct droplet digital PCR assay (dddPCR) for simultaneous identification and quantification of bacteria in a bacterial mixture. We encapsulate bacterial cells in droplets together with PCR reagents to amplify specific target genes. Our method introduces accurate and precise quantitation of bacteria in a bacterial mixture by digital PCR, while eliminating the errors and workload associated with extraction and purification of DNA. The method thus combines the precision and specificity of digital PCR with robustness of a direct PCR. We also show that dddPCR is more tolerant to inhibition by non-specific bacteria as compared to traditional direct real-time PCR.
Thesis
L’analyse microbiologique pour confirmer l’absence de bactéries dans des échantillons biologiques normalement sains, comme le sang, est une routine dans de nombreux laboratoires. En effet, la présence de bactéries dans le sang, appelée bactériémie, peut avoir des conséquences très graves, voire mortelles pour le patient. Le protocole standard pour la détection des bactériémies repose jusqu’ici sur l’enrichissement des échantillons sanguins prélevés sur les patients lors de l’hémoculture, afin d’obtenir une population suffisante pour analyse. La lenteur de ce procédé retarde ainsi de parfois plusieurs jours le diagnostic et donc l’adaptation du traitement antibiotique administré au patient. Ces dernières décennies, des techniques comme l’identification par spectrométrie de masse ou les analyses moléculaires, ont permis de diminuer le délai requis pour identifier les pathogènes en cause. Dans ce contexte, l’emploi de biocapteurs est également une alternative. Ce travail propose d’inclure des sondes à large spectre dans un capteur optique par imagerie SPR (résonance de plasmons de surface). Ce système est déjà développé pour la reconnaissance spécifique de pathogènes au cours de leur croissance dans le sang. Les nouveaux ligands proposés et évalués sont les peptides antimicrobiens (PAM). Ces courts peptides cationiques et amphiphiles, présentent l’avantage d’un large spectre d’interaction couplé à une haute stabilité (chimique, thermique et séchage) comparativement aux anticorps employés jusqu’ici. Leur immobilisation sur des prismes SPRI permet d’évaluer simultanément l’affinité de plusieurs PAM à la même souche bactérienne. Les biocapteurs ainsi préparés ont permis de détecter des souches pathogènes d’Escherichia coli et Staphylococcus aureus en milieu de culture simple, comme en plasma et en sang dilué au milieu d’hémoculture. Le système obtenu permet la détection des pathogènes présents à une concentration initiale de l’ordre de 1 UFC.ml-1, en moins de 24 heures et quel que soit le milieu. Enfin, la mise en place d’analyses statistiques multidimensionnelles a abouti à une classification cohérente des espèces ciblées en milieu simple, comme en sang. Ces résultats montrent le potentiel de ce système pour parvenir à développer un biocapteur à large spectre capable à la fois de détecter mais aussi d’identifier par affinité croisée des pathogènes bactériens.
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Rapid detection of microorganisms is a major challenge in the medical and industrial sectors. In a pharmaceutical laboratory, contamination of medical products may lead to severe health risks for patients, such as sepsis. In the specific case of advanced therapy medicinal products, contamination must be detected as early as possible to avoid late production stop and unnecessary costs. Unfortunately, the conventional methods used to detect microorganisms are based on time-consuming and labor-intensive approaches. Therefore, it is important to find new tools to detect microorganisms in a shorter time frame. This review sums up the current methods and represents the evolution in techniques for microorganism detection. First, there is a focus on promising ligands, such as aptamers and antimicrobial peptides, cheaper to produce and with a broader spectrum of detection. Then, we describe methods achieving low limits of detection, thanks to Raman spectroscopy or precise handling of samples through microfluids devices. The last part is dedicated to techniques in real-time, such as surface plasmon resonance, preventing the risk of contamination. Detection of pathogens in complex biological fluids remains a scientific challenge, and this review points toward important areas for future research.
Chapter
The detection, quantification, and isolation of microbes are routine processes in the dairy industry which are advisable for monitoring food quality from an early stage. The microbiological tests can be classified into culture-dependent and culture-independent techniques. The microorganism in milk and milk products can be detected either by the conventional culture-based approach (enumeration, isolation, characterization, and identification) or through a culture-independent approach (isolation, purification of nucleic acid, and molecular identification). The culture-dependent methods are considered as the basic gold standard technique in microbial detection. The culture-dependent techniques (traditional approaches) rely on the isolation procedure from complex food matrices and the growth of microbial colonies on selective agar to detect and quantify viable microbes and identify microbes mainly depending upon their morphology and/or biochemical characteristics. The culture-independent techniques circumvent the steps of enumeration and characterization of microbes rather based on nucleic acid-based identification. In culture-independent techniques, nucleic acid (deoxyribonucleic acid (DNA)/ribonucleic acid (RNA)) is isolated and purified from the dairy matrix, and different molecular approaches are applied to identify and quantify the pathogens. This chapter gives a brief overview of the latest rapid microbiological methods.
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Bacterial resistance is becoming a global issue due to its rapid growth. Potential new drugs as antimicrobial peptides (AMPs) are considered for several decades as promising candidates to circumvent this threat. Nonetheless, AMPs have also been used more recently in other settings such as molecular probes grafted on biosensors able to detect whole bacteria. Rapid, reliable and cost-efficient diagnostic tools for bacterial infection could prevent the spread of the pathogen from the earliest stages. Biosensors based on AMPs would enable easy monitoring of potentially infected samples, thanks to their powerful versatility and integrability in pre-existent settings. AMPs, which show a broad spectrum of interactions with bacterial membranes, can be tailored in order to design ubiquitous biosensors easily adaptable to clinical settings. This review aims to focus on the state of the art of AMPs used as the recognition elements of whole bacteria in label-free biosensors with a particular focus on the characteristics obtained in terms of threshold, volume of sample analysable and medium, in order to assess their workability in real-world applications.
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Diagnosis of fungal infection in lung parenchyma is relatively difficult. Bronchoscopy with bronchoalveolar lavage is very useful in its diagnosing. Therefore, a method for rapid on-line concentration and analysis of Aspergillus conidia in bronchoalveolar lavage fluid using combination of transient isotachophoresis (tITP) and micellar electrokinetic chromatography (MEKC) with subsequent off-line identification of the separated conidia by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is described in this study. In the proposed procedure, conidia were first dynamically adhered onto the roughened part of the inner surface of fused silica capillary prepared by etching with supercritical water. Then the adhered conidia were desorbed, concentrated, and separated using combination of tITP and MEKC. Finally, the fractions containing the separated conidia were collected from the capillary and analyzed by MALDI-TOF MS. Adhesion efficiency under the optimized experimental conditions was about 80 %. This rapid diagnosis will contribute to timely initiation of therapy and increase the patient's chances of survival.
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Objective In the present investigation, Trichoderma spp., isolated from rhizospheric soil, has been identified by Internal Transcribed Spacer (ITS) region sequencing technique and its antagonistic activity was evaluated against A. niger. Methods The sequencing analysis was done with its ITS1 region of the rRNA gene. Using the ITS1 amplified products for all isolated fungi, a bi-directional DNA sequencing was done with high quality bases (>98% - 100%). Antagonistic activity was done using dual culture technique. Results All of the ITS1 nucleotide sequences obtained in this study matched 97% - 100% with the published sequence of Trichoderma spp. The results confirmed the strains as T. asperellum and T. viride with gene bank accession no. (ZTa); MK937669 and (ZTv); MK503705, respectively. When phylogenetic analysis was done for the isolates, the optimal tree with the sum of branch length = 0.69585023 and 0.10077756 for T. asperellum and T. viride, respectively, was observed. There were a total of 678 and 767 for T. asperellum and T. viride positions in the final dataset, respectively. Antagonistic activity was done for the isolated strains of Trichoderma spp. against A. niger, and it was found that T. asperellum showed maximum antagonistic activity (79.33±7.09%). Conclusion The findings prolong the genome availability for relative investigations pointing out phenotypic variances to compare with Trichoderma genetic diversity. The present investigation delivered the Bases of future studies for better knowledge in understanding the complicated connections of Trichoderma spp. to be used as an effective biocontrol agent.
Article
Introduction. One of the main tasks of medical care for infectious diseases is the rapid identification of an infectious agent. The purpose of the study is to evaluate the effectiveness of the MALDI mass spectrometry for identification bacteria for microbiological control of wastewater quality. Materials and methods. Samples of wastewater samples at the treatment stage from the Kuryanovskaya aeration station were analyzed in accordance with MU 2.1.5.800-99 “Management of state sanitary and epidemiological supervision of wastewater disinfection” by the identification method in accordance with MUK 4.2.1884-04 “Sanitary-microbiological and sanitary - parasitological water analysis of surface water bodies” with application MALDI-TOF MS and 16S rRNA gene sequencing. Results. 5 strains of museum reference cultures and 22 bacterial isolates from wastewater samples grown on selective media of Endo, Enterococcus and Chromococcus coliform agar were studied, identified by MALDI-TOF MS, and confirmed by sequencing of specific regions of the 16S rRNA gene in bacteria of the genus Salmonella by MALDI-TOF MS identified only gender. Conclusion. In the routine practice of microbiological research, the identification of microorganisms is based on the determination of their cultural, tinctorial properties, and biochemical activity, the determination of which requires large financial and time costs. The use of the MALDI-TOF MS method makes it possible to reduce the time of identification of a microorganism when visible growth of microorganisms appears.
Article
Bacteria, bacterial metabolites and cell components can have positive as well as negative impact on human health. Therefore, there is a strong need for the determination of bacteria and bacterial metabolites in various samples, including food, natural objects, and biological fluids. Capillary electrophoresis (CE) is a powerful tool for the identification and separation of ionic compounds, characterized by high efficiency, low background electrolyte consumption, low sample volume and simplified sample preparation. Here we review recent applications of electrokinetic methods in combination with various detection systems and on-line concentration techniques for the analysis of bacterial metabolites, cell components, and bacteria. The modification of electrophoretic systems aiming to achieve superior analytes resolution and application of bacteria as a modifier of both background electrolyte and capillary walls are discussed. Therefore, this review provides a thorough analysis of applications and offers practical recommendations on the CE implementation for the bacterial samples.
Chapter
Polymerase chain reaction (PCR) is a popular molecular tool for detection of bacteria. PCR allows millions of copies of a target segment of DNA to be produced. The DNA is extracted from overnight grown cultures of pure bacterial isolates using either the organo-solvent method or a commercial DNA extraction kit. The quality and purity of the DNA is determined by performing gel electrophoresis on 0.8% agarose gel. The DNA is amplified by performing PCR assay. Bands of approximately 1.5 kb in size are obtained from the amplified products of DNA. The PCR products run on 1.5% agarose gel are visualized with UV light and imaged by gel documentation system. This chapter outlines the protocol for isolation and amplification of DNA from cellulolytic bacteria. Cellulolytic bacteria are considered a potential source of cellulases for pretreatment of crop residues during biogas production. PCR is considered a very powerful, sensitive, specific, fast, and reliable tool in molecular detection and diagnostics.
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Miniaturized electrokinetic methods have proven to be robust platforms for the analysis and assessment of intact microorganisms, offering short response times and higher integration than their bench scale counterparts. The present review article discusses three types of electrokinetic‐based methodologies: electromigration or motion‐based techniques, electrode‐based electrokinetics and insulator‐based electrokinetics. The fundamentals of each type of methodology are discussed and relevant examples from recent reports are examined, in order to provide the reader with an overview of the state‐of‐the‐art on the latest advancements on the analysis of intact cells and viruses with microscale electrokinetic techniques. The concluding remarks discuss the potential applications and future directions. This article is protected by copyright. All rights reserved
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We have developed a planar chip utilizing divergent geometry of separation channel capable of vertical free-flow electrophoresis of particles at flows of lower hundreds of microliters per minute. The divergent flow isoelectric focusing (DF-IEF) chip consists of two sheets of clear polystyrene glass which serve as a base with working channels and a top cover sealing the separation channel. Optimization showed that the chip is capable to form pH gradient within 1 h and separation is completed in 5 or more minutes depending on the sample volume. The vertical position of the chip enabled analysis of sedimenting particles including microorganisms. Four different common bacteria species inactivated with H2O2 vapors were analyzed in a series of experiments. Isoelectric points were determined with capillary isoelectric focusing with following fractionation using DF-IEF with intact cell matrix-assisted laser desorption/ionization mass spectrometry detection. The DF-IEF chip fractionation proved promising for bacterial sample preparation from complex matrices for subsequent identification of whole cells by mass spectrometry.
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Despite being commensal bacterium involved in the maintenance of healthy skin, Cutibacterium acnes is also associated with inflammatory diseases. Since inflammatory and immunogenic properties vary between C. acnes phylotypes, reliable classification of clinical C. acnes isolates is important for determining their pathogenicity. Combination of optimized separation methods, polymer-enhanced transient isotachophoresis and sweeping of the charged bacterial cells in micellar electrokinetic chromatography in the roughened fused silica capillary, was used for the separation of twenty clinical C. acnes isolates. Their correct classification into the individual phylotypes was achieved in twenty minutes at laboratory temperature. In addition, decrease in the separation temperature to 15 °C led to the separation of the individual isolates of some phylotypes. Relative standard deviations of migration times of both intra- and inter-day analyses did not exceed 1.7%. Linearity of the proposed method in the concentration range from 5×10⁵ to 1×10⁷ cells mL⁻¹ was characterized by the coefficient of determination R² = 0.9985. Limit of detection of 5×10⁵ cells mL⁻¹ (50 cells in 100 nL of the injected sample) was determined for all the examined bacteria.
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Human microbiome contains billions of microorganisms that play important roles in biological system and different diseases. Due to its complexity, conventional culture-independent technology may underestimate the value of low abundant bacteria, which calls for highly efficient method for its enrichment and comprehensive analysis. In this study, we developed a recycling free-flow isoelectric focusing (RFFIEF) method based electrophoresis method to separate salivary microbiome. First, we used E. coli (DH5α) as a model for RFFIEF method development, which was focused in a narrow pH range (0.38 pH unit). The recovery rate was 80.81% with 5.85% relative standard deviation (n=5). The optimized method was then adopted to separate the human salivary microbiome into 32 fractions, followed by 16S rRNA gene sequencing and metaproteomics analysis. After RFFIEF fractionation, we identified 508 bacterial genera, which increased by 225% on average (n=3) when compared to the results of before-fractionation. We further compared the compositional change of microbiome in the saliva of lung cancer group (n=22) and control group (n=21) through RFFIEF. Quantitative results demonstrated six bacterial genera were upregulated dramatically in lung cancer group, while two genera were downregulated. Through qPCR verification in an independent sample set (n = 48), we confirmed that genus Granulicatella was significantly upregulated in lung cancer group, whereas Pseudomonas was remarkably downregulated (p<0.001). RFFIEF is an efficient and reproducible technology to fractionate the microbiome for its comprehensive analysis, which can be further applied to the in-depth study of the complex microbiomes and contribute to the discovery of disease associated bacteria.
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To isolate fungal communities from coastal areas of the Red Sea in Saudi Arabia and identify and classify them by molecular techniques. Samples were collected from the seaside of the Red Sea in Jeddah, Saudi Arabia during March 2012 and stored in sterile screw cap bottles for further analysis. Phenotypic and genotypic characterization of fungal isolates were done using standard techniques. Eight fungal genera including Aspergillus, Penicillium, Thielavia, Fusarium, Emericella, Cladosporium, Scytalidium and Alternaria. Most isolated fungi showed significant growth on petroleum media and were thus considered capable of biodegradation of crude oil based substances. The fungal genera isolated from the Red Sea had 97–100% similarity with the related fungi recorded in the GenBank in which they were deposited. The morphological and molecular structure of these marine fungal isolates closely resembles their terrestrial counterparts in the GenBank. The capabilities of these fungal species to utilize petroleum as a source of carbon speaks to future applications in which marine fungi may be utilized in the breakdown of petroleum-based waste in an ecologically efficient manner.
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We studied the diagnostic performance of the IRIDICA PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS) assay applied on bronchoalveolar lavage (BAL) samples, from 51 mechanically ventilated patients with suspected pneumonia, in a prospective study. In 32 patients with X-ray verified pneumonia, PCR/ESI-MS was positive in 66% and BAL culture was positive in 38% (p = 0.045), and either of the methods was positive in 69%. The following BAL result combinations were noted: PCR/ESI-MS+/culture+, 34%; PCR/ESI-MS+/culture-, 31%; PCR/ESI-MS-/culture+, 3.1%; PCR/ESI-MS-/culture-, 31%; kappa 0.36 (95% confidence interval (CI), 0.10-0.63). In pneumonia patients without prior antibiotic treatment, optimal agreement was noted with 88% PCR/ESI-MS+/culture+ and 12% PCR/ESI-MS-/culture- (kappa 1.0). However, in patients with prior antibiotic treatment, the test agreement was poor (kappa 0.16; 95% CI, -0.10-0.44), as 10 patients were PCR/ESI-MS+/culture-. In 8/10 patients the pathogens detected by PCR/ESI-MS could be detected by other conventional tests or PCR tests on BAL. Compared with BAL culture, PCR/ESI-MS showed specificities and negative predictive values of ≥87% for all individual pathogens, an overall sensitivity of 77% and positive predictive value (PPV) of 42%. When other conventional tests and PCR tests were added to the reference standard, the overall PPV increased to 87%. The PCR/ESI-MS semi-quantitative level tended to be higher for PCR/ESI-MS positive cases with pneumonia compared with cases without pneumonia (p = 0.074). In conclusion, PCR/ESI-MS applied on BAL showed a promising performance and has potential to be clinically useful in mechanically ventilated patients with suspected pneumonia. The usefulness of the method for establishment of pneumonia etiology and selection of antibiotic therapy should be further studied.
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Molecular assays have not yet been able to replace time-consuming culture-based methods in clinical mycobacteriology. Using 6875 clinical samples and a study period of 35 months we evaluated the use of PCR-based assays to establish a diagnostic workflow with a fast time-to-result of 1–2 days, for 1. detection of Mycobacterium tuberculosis complex (MTB), 2. detection and identification of nontuberculous mycobacteria (NTM), and 3. identification of drug susceptible MTB. MTB molecular-based detection and culture gave concordant results for 97.7% of the specimens. NTM PCR-based detection and culture gave concordant results for 97.0% of the specimens. Defining specimens on the basis of combined laboratory data as true positives or negatives with discrepant results resolved by clinical chart reviews, we calculated sensitivity, specificity, PPV and NPV for PCR-based MTB detection as 84.7%, 100%, 100%, and 98.7%; the corresponding values for culture-based MTB detection were 86.3%, 100%, 100%, and 98.8%. PCR-based detection of NTM had a sensitivity of 84.7% compared to 78.0% of that of culture-based NTM detection. Molecular drug susceptibility testing (DST) by line-probe assay was found to predict phenotypic DST results in MTB with excellent accuracy. Our findings suggest a diagnostic algorithm to largely replace lengthy culture-based techniques by rapid molecular-based methods.
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Objective: Early identification of causative microorganism(s) in patients with severe infection is crucial to optimize antimicrobial use and patient survival. However, current culture-based pathogen identification is slow and unreliable such that broad-spectrum antibiotics are often used to insure coverage of all potential organisms, carrying risks of overtreatment, toxicity, and selection of multidrug-resistant bacteria. We compared the results obtained using a novel, culture-independent polymerase chain reaction/electrospray ionization-mass spectrometry technology with those obtained by standard microbiological testing and evaluated the potential clinical implications of this technique. Design: Observational study. Setting: Nine ICUs in six European countries. Patients: Patients admitted between October 2013 and June 2014 with suspected or proven bloodstream infection, pneumonia, or sterile fluid and tissue infection were considered for inclusion. Interventions: None. Measurements and main results: We tested 616 bloodstream infection, 185 pneumonia, and 110 sterile fluid and tissue specimens from 529 patients. From the 616 bloodstream infection samples, polymerase chain reaction/electrospray ionization-mass spectrometry identified a pathogen in 228 cases (37%) and culture in just 68 (11%). Culture was positive and polymerase chain reaction/electrospray ionization-mass spectrometry negative in 13 cases, and both were negative in 384 cases, giving polymerase chain reaction/electrospray ionization-mass spectrometry a sensitivity of 81%, specificity of 69%, and negative predictive value of 97% at 6 hours from sample acquisition. The distribution of organisms was similar with both techniques. Similar observations were made for pneumonia and sterile fluid and tissue specimens. Independent clinical analysis of results suggested that polymerase chain reaction/electrospray ionization-mass spectrometry technology could potentially have resulted in altered treatment in up to 57% of patients. Conclusions: Polymerase chain reaction/electrospray ionization-mass spectrometry provides rapid pathogen identification in critically ill patients. The ability to rule out infection within 6 hours has potential clinical and economic benefits.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially.
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The production of β-lactamases is the most important mechanism of Gram-negative rod resistance to β-lactams. Resistance to ceftazidime and cefepime in clinical isolates of Enter-obacteriaceae (especially ESβL-positive E. coli and K. pneumoniae) and P. aeruginosa is life-threatening. However, all strains of the above mentioned species possess chromosom-ally encoded RND efflux pump systems in addition to β-lactamase production. The main goal of this study was to assess the role of efflux pump systems in cefepime and/or ceftazi-dime resistant phenotypes of ESβL-positive clinical strains of Enterobacteriaceae and P. aeruginosa. The influence of the efflux pump inhibitor PAβN on the minimum inhibitory concentration (MIC) values of tested cephalosporins was species-dependent. Generally, a significant reduction (at least four-fold) of β-lactam MICs was observed in the presence of PAβN only in the case of P. aeruginosa clinical isolates as well as the ESβL-producing transformant PAO1161 ΔampC. The usage of this agent resulted in the restoration of susceptibility to cefepime and/or ceftazidime in the majority of the P. aeruginosa ESβL-positive strains with low and moderate resistance to the above cephalosporins. Moreover, an outer membrane permeabilizing effect in the presence of PAβN was identified. Strain-dependent β-lactamase leakage upon PAβN or β-lactam treatment was demonstrated. The most important observation was the restoration of susceptibility of P. aeruginosa WUM226 to cefe-pime (MIC decrease from 32 to 4 mg/L) and ceftazidime (MIC decrease from 128 to 4 mg/L) in the presence of PAβN, which occurred despite an almost complete lack of β-lactamase leakage from bacterial cells. In conclusion, these data indicate that RND efflux pumps can modify the susceptibility to β-lactams in Gram-negative rods producing ESβLs. However, this phenomenon occurs only in P. aeruginosa strains and was not observed among E. coli and K. pneumoniae strains, representing the Enterobacteriaceae family.
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Diarrhea is a common disease across the world. According to WHO, every year about two billion cases of diarrhea are reported in the world. It occurs mainly in the tropical regions and is a main cause of morbidity and mortality, particularly in young children and adults. One of the major causes of diarrheal diseases is bacteria; detection of pathogenic bacteria is a global key to the prevention and identification of food-borne diseases and enteric infections (like diarrhea). Therefore, development of rapid diagnostic methods with suitable sensitivity and specificity is very important about this infectious disease. In this review, we will discuss some of the important diagnostic methods.
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Nucleic acid amplification is an essential process in biological systems. The in vitro adoption of this process has resulted in powerful techniques that underpin modern molecular biology. The most common tool is polymerase chain reaction (PCR). However, the requirement for a thermal cycler has somewhat limited applications of this classic nucleic acid amplification technique. Isothermal amplification, on the other hand, obviates the use of a thermal cycler because reactions occur at a single temperature. Isothermal amplification methods are diverse, but all have been developed from an understanding of natural nucleic acid amplification processes. Here we review current isothermal amplification methods as classified by their enzymatic mechanisms. We compare their advantages, disadvantages, efficiencies, and applications. Finally, we mention some new developments associated with this technology, and consider future possibilities in molecular engineering and recombinant technologies that may develop from an appreciation of the molecular biology of natural systems.
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The reference method for the diagnosis of bloodstream infections is blood culture followed by biochemical identification and antibiotic susceptibility testing of the isolated pathogen. This process requires 48 to 72 hours. The rapid administration of the most appropriate antimicrobial treatment is crucial for the survival of septic patients; therefore, a rapid method that enables diagnosis directly from analysis of a blood sample without culture is needed. A recently developed platform that couples broad-range PCR amplification of pathogen DNA with electrospray ionization mass spectrometry (PCR/ESI-MS) has the ability to identify virtually any microorganism from direct clinical specimens. To date, two clinical evaluations of the PCR/ESI-MS technology for the diagnosis of bloodstream infections from whole blood have been published. Here we discuss them and describe recent improvements that result in an enhanced sensitivity. Other commercially available assays for the molecular diagnosis of bloodstream infections from whole blood are also reviewed. The use of highly sensitive molecular diagnostic methods in combination with conventional procedures could substantially improve the management of septic patients.
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Detection and enumeration of Legionella bacteria in drinking water is regulated in Germany by ISO 11731–2. The mandatory method for species identification employs parallel subculturing of suspicious colonies on selective media requiring the handling of a large number of cultivation plates. After changes to the drinking water quality regulation in Germany in 2012 the demand for Legionella contamination testing increased drastically. A more reliable, faster and less laborious method for species identification is therefore desirable. Matrix-assisted laser desorption ionization followed by time of flight detection mass spectrometry (MALDI-TOF MS) promises an accelerated identification of bacteria with high reliability and reduced expenditure. Our study shows that MS-based species identification results are in full concordance with cultural and biochemical detection and differentiation and that valuable additional information can be gained, even though the ISO regulation demands an extended incubation period for primary bacterial cultures that is actually in contrast to the prerequisites of the MALDI Biotyper system. In addition, the established identification algorithm is very economical and improves time-to-result. Based on our findings, the amendment of MALID-TOF MS identification to ISO11731–2 as an alternative identification method should be taken into consideration.
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Recently, a novel method for differentiation of bacteria and fungi entered clinical microbiology laboratories, the identification by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This differentiation relies on the exact measurement of species-specific protein spectra, mainly of abundant ribosomal proteins. It is even more accurate than conventional biochemical differentiation methods. However, most importantly it provides results within minutes compared to several hours when using biochemical assays. In addition to differentiation of bacteria and yeasts grown on agar plates, direct identification is feasible from positive blood cultures as well as from urine samples of patients suffering from urinary tract infections. Future developments of MALDI-TOF MS for clinical microbiological diagnostic include the detection of β-lactamase and carbapenemase-activity, phenotypic antibiotic resistance testing as well as genotyping of bacteria below the species level.
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Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a technique of choice for quick classification of bacterial cells and precise characterization of microorganisms. The evaluated MALDI MS procedure was confronted with automated biochemical method using the VITEK 2 Compact system employed as a reference method. Moreover, the investigation aimed at describing and optimization of the environmental conditions having an impact on reproducibility and quality of one-dimensional intact-cell MALDI TOF spectra. The work presents off-line combination of capillary electrophoresis of microbial clumping with spectrometric detection.
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Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.
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We evaluated the performance of two MALDI-TOF MS systems for the identification of clinically important Gram-positive cocci. Vitek MS and Microflex LT correctly identified 97.2% and 94.7%, respectively. Both systems offer reliable and rapid identification of clinically important Gram-positive cocci isolated in clinical laboratories, including staphylococci, streptococci, and enterococci. Expanding the databases, especially of coagulase-negative staphylococci and viridans streptococci, would enhance performance. Copyright © 2015. Published by Elsevier B.V.
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The chemical composition, molecular structure and physicochemical properties of five Gram-positive bacterial strain: Bacillus cereus, Bacillus subtilis, Sarcina lutea, Staphylococcus aureus, Micococcus luteus were investigated by Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), NMR spectroscopy and intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (IC MALDI TOF MS). An analysis of FTIR spectra as a function of pH revealed the three major types of cell wall functional groups – carboxyl group, amino group and phosphate group. An analysis of XPS spectra was determinate the major surface components of bacterial cell. 13C NMR and IC MALDI TOF MS spectra of six bacterial species were registered. Our findings indicate that chemical and structural differences in the cell composition of Gram-positive bacteria can be detected. The obtained results also demonstrate that the combination of FTIR, XPS and NMR spectroscopy with IC MALDI TOF MS technique yields useful information and complements other biochemical and physical methods of microbial cells characteristics.
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When a bloodstream infection (BSI) is suspected, most of the laboratory results - biochemistry, and haematology - are available within the first hours after hospital admission of the patient. This is not the case for diagnostic microbiology that generally takes a longer time because blood culture, which is to date the gold standard for the documentation of the BSI microbial agents, relies on bacterial or fungal growth. The microbial diagnosis of BSI directly from blood has been proposed to speed the determination of the etiological agent but was limited by the very low number of circulating microbes during these paucibacterial infections. Thanks to recent advances in molecular biology, including the improvement of nucleic acid extraction and amplification, several PCR-based methods for the diagnosis of BSI directly from whole blood have emerged. In the present review we will discuss the advantages, but also the limitations of these new molecular approaches that at best complement the culture-based diagnosis of BSI. Copyright © 2015. Published by Elsevier Ltd.
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PLEX-ID uses polymerase chain reaction-electrospray ionization/mass spectrometry for rapid identification of infectious agents in clinical samples. We evaluated its concordance with our centre's standard methods (SM) for bacterial and fungal detection in bronchoalveolar lavage (BAL) fluid in a prospective observational cohort study. The primary outcome was concordance (%) between SM and PLEX-ID. Secondary outcomes included concordance when excluding commensal oral flora, detection of resistance genes, and PLEX-ID's potential impact on clinical management, as determined by two independent reviewers. Included were 101 specimens from 94 patients. BALs were performed primarily for suspected pneumonia (76/101, 75%) and lung transplant work-ups (12/101, 12%). Most specimens yielded at least one organism by either method (92/101, 91%). Among all microorganisms detected (n = 218), 83% and 17% were bacterial and fungal, respectively. Overall concordance between SM and PLEX-ID was 45% (45/101). Concordance increased to 66% (67/101) when discordance for commensal flora was excluded. PLEX-ID failed to detect 21% of all 183 SM-identified organisms, while SM did not identify 28% of the 191 PLEX-ID-identified organisms (p <0.001). There was low concordance for mecA detection. Two infectious-disease specialists' analyses concluded that in most of the 31 discordant, non-commensal cases, PLEX-ID results would have had little or no impact on patient management; in eight cases, however, PLEX-ID would have led to ‘wrong decision-making’. The tested version of PLEX-ID concurred weakly with standard methods in the detection of bacteria and fungi in BAL specimens, and is not likely to be useful as a standalone tool for microbiological diagnosis in suspected respiratory infections.
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In this study, food-borne yeast isolates (n=96), comprising at least 33 species, were identified using MALDI-TOF MS and conventional methods (API ID 32 C and Phoenix Yeast ID). Discrepancies of both methods were resolved by sequencing the ITS1-5.8S-rRNA-ITS2 region. For ten isolates, mainly classified to Rhodotorula and Trichosporon species, no clear final species identification was possible. 62 isolates were correctly identified to species level using either MALDI-TOF MS or conventional tests. 15 isolates were misidentified when applying conventional assays. In contrary, no species misidentifications were observed after MALDI-TOF MS based classification. In return, 16 isolates were not identifiable after matching their protein fingerprints against MALDI Biotyper 4.0.0.1 library. MALDI TOF MS in-house database update clearly improved the identification. In conclusion, the presented data suggest that MALDI-TOF MS is an appropriate platform for reliable classification and identification of food-borne yeast isolates.
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Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is demonstrated as a means for rapidly differentiating bacteria based upon their water-soluble-protein profiles. The procedure involves release of cell contents by breaking cell membranes using a sonication method, and is performed with minimal sample preparation. Minimal purification of cell contents is required for the procedure. Protein profile signals obtained are found to be enhanced greatly by the use of a Nafion substrate.
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In this study we evaluated MALDI-TOF MS and FilmArray methods for the rapid identification of yeast -positive blood cultures. FilmArray correctly identified 20/22 of yeast species, while MALDI-TOF MS identified 9/22. FilmArray is a reliable and rapid identification system for the direct identification of yeasts from positive blood cultures.
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Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically.
For some time, mass spectrometry (MS) has been an important diagnostic tool in the field of clinical chemistry; however, the technology is now making inroads in the clinical microbiology laboratory. In part I of this series, we discuss how MS coupled with PCR has shown great promise for direct-from-specimen identification and multiplexed detection of microorganisms. These and other promising MS technologies could serve as important new tools to meet the clinical diagnostic challenges of the 21st century.