Potential for detection of microorganisms and heavy metals in potable water using electronic nose technology

Studies have been carried out to determine the potential for the detection of different microbial species (Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa), alone and in the presence of low concentrations of different heavy metals (As, Cd, Pb and Zn) in bottled, reverse osmosis (RO) and tap water, using an electronic nose. Studies show that it is possible to discriminate control water samples from water contaminated with 0.5 ppm of a mixture of metals. The presence of heavy metals may modify the activity of microorganisms and thus the volatile production patterns. Bacterial species at 10(2)-10(4) colony forming units (CFUs) ml(-1) could be detected after 24 h of incubation. Work is in progress to identify the limits of detection for a range of other microorganisms, including, fungi and cyanobacteria, and chlorinated phenols using electronic nose technology.     

Extensive set of mitochondrial LSU rDNA-based oligonucleotide probes for the detection of common airborne fungi

Fungi exist in every indoor and outdoor environment. Many fungi are toxigenic or pathogens that may cause various public health concerns. Rapid and accurate detection and identification of fungi require specific markers. In this study, partial mitochondrial large subunit rDNA was amplified and sequenced from 32 fungal strains representing 31 species from 14 genera. Based on the sequence variation pattern, 26 oligonucleotide probes were designed for their discrimination. The specificity of the probes was evaluated through homology search against GenBank database and hybridization examination on 38 fungal strains. The 26 probes were verified as highly specific to 20 fungal species. A two-step detection procedure through PCR followed by probe hybridization gave ten-fold increase in detection sensitivity than single-step PCR assay and would be a practical approach for environmental sample screening. The probes developed in this study can be applied in clinical diagnosis and environmental monitoring of fungal agents.     

DNA-based methods for monitoring invasive species: a review and prospectus

The recent explosion of interest in DNA-based tools for species identification has prompted widespread speculation on the future availability of inexpensive, rapid, and accurate means of identifying specimens and assessing biodiversity. One applied field that may benefit dramatically from the development of such technologies is the detection, identification, and monitoring of invasive species. Recent studies have demonstrated the feasibility of DNA-based tools for such important tasks as confirmation of specimen identity and targeted screening for known or anticipated invaders. However, significant technological hurdles must be overcome before more ambitious applications, including estimation of propagule pressure and comprehensive surveys of complex environmental samples, are to be realized. Here we review existing methods, examine the technical difficulties associated with development of more sophisticated tools, and consider the potential utility of these DNA-based technologies for various applications relevant to invasive species monitoring.     

Update on the Evolving Role of MALDI-TOF MS for Laboratory Diagnosis of Fungal Infections

MALDI-TOF MS is a recent technique, which revolutionized bacterial species identifications, due to its simplicity, accuracy and speed of analysis. The same efficiency of species identification for fungi is highly sought. This review aims to discuss the evolving role of MALDI-TOF MS in the laboratory diagnosis of fungal infections. Yeast identifications are increasingly being performed with MALDI-TOF MS in a routine setting with good results. A further extension of the libraries will further increase its potential. Direct identification of yeast in blood cultures and MALDI-TOF MS susceptibility testing are new promising applications. The identification of filamentous fungi on MALDI-TOF MS is still challenging, but knowledge and experience is taking huge leaps forward.     

Use of an electronic nose system for diagnoses of urinary tract infections

The use of volatile production patterns produced by bacterial contaminants in urine samples were examined using electronic nose technology. In two experiments 25 and 45 samples from patients were analysed for specific bacterial contaminants using agar culture techniques and the major UTI bacterial species identified. These samples were also analysed by incubation in a volatile generation test tube system for 4-5 h. The volatile production patterns were then analysed using an electronic nose system with 14 conducting polymer sensors. In the first experiment analysis of the data using a neural network (NN) enabled identification of all but one of the samples correctly when compared to the culture information. Four groups could be distinguished, i.e. normal urine, Escherichia coli infected, Proteus spp. and Staphylococcus spp. In the second experiment it was again possible to use NN systems to examine the volatile production patterns and identify 18 of 19 unknown UTI cases. Only one normal patient sample was mis-identified as an E. coli infected sample. Discriminant function analysis also differentiated between normal urine samples, that infected with E. coli and with Staphylococcus spp. This study has shown the potential for early detection of microbial contaminants in urine samples using electronic nose technology for the first time. These findings will have implications for the development of rapid systems for use in clinical practice.     

The development of gene expression systems for filamentous fungi

Filamentous fungi have been used for decades in the commercial production of enzymes, antibiotics, and specialty chemicals. Traditionally, improving the yields of these products has involved either mutagenesis and screening or modification of fermentation conditions. Generally, selective breeding of strains has not been successful, because most of the commercially important fungal species lack a sexual cycle. For a few species, strain improvements have been made possible by employing the parasexual cycle for genetic crosses (30). The recent development of DNA-mediated transformation systems for several industrially important fungal species has spawned a flurry of research activity directed toward the development of gene expression systems for these microorganisms. This technology is now a viable means for novel and more directed approaches to improving existing fungal strains which produce enzymes or antibiotics. In addition, fungal expression systems are now being tested for the production of heterologous gene products such as mammalian pharmaceutical proteins. The goal of this review is to present a summary of the gene expression systems which have recently been developed for some filamentous fungi of commercial importance. To insure that the most recent developments are presented we have included data from not only scientific papers, but also from personal communications, abstracts, symposia, and our own laboratory.     

Diversity and Taxonomy of Endophytic Xylariaceous Fungi from Medicinal Plants of Dendrobium (Orchidaceae)

Dendrobium spp. are traditional Chinese medicinal plants, and the main effective ingredients (polysaccharides and alkaloids) have pharmacologic effects on gastritis infection, cancer, and anti-aging. Previously, we confirmed endophytic xylariaceous fungi as the dominant fungi in several Dendrobium species of tropical regions from China. In the present study, the diversity, taxonomy, and distribution of culturable endophytic xylariaceous fungi associated with seven medicinal species of Dendrobium (Orchidaceae) were investigated. Among the 961 endophytes newly isolated, 217 xylariaceous fungi (morphotaxa) were identified using morphological and molecular methods. The phylogenetic tree constructed using nuclear ribosomal internal transcribed spacer (ITS), large subunit of ribosomal DNA (LSU), and beta-tubulin sequences divided these anamorphic xylariaceous isolates into at least 18 operational taxonomic units (OTUs). The diversity of the endophytic xylariaceous fungi in these seven Dendrobium species was estimated using Shannon and evenness indices, with the results indicating that the dominant Xylariaceae taxa in each Dendrobium species were greatly different, though common xylariaceous fungi were found in several Dendrobium species. These findings implied that different host plants in the same habitats exhibit a preference and selectivity for their fungal partners. Using culture-dependent approaches, these xylariaceous isolates may be important sources for the future screening of new natural products and drug discovery.     

Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for the identification of clinical filamentous fungi

Infections caused by filamentous fungi have become a health concern, and require rapid and accurate identification in order for effective treatment of the pathogens. To compare the performance of two MALDI-TOF MS systems (Bruker Microflex LT and Xiamen Microtyper) in the identification of filamentous fungal species. A total of 374 clinical filamentous fungal isolates sequentially collected in the Clinical Laboratory at the Beijing Tongren Hospital between January 2014 and December 2015 were identified by traditional phenotypic methods, Bruker Microflex LT and Xiamen Microtyper MALDI-TOF MS, respectively. The discrepancy between these methods was resolved by sequencing for definitive identification. Bruker Microflex LT and Xiamen Microtyper had similar correct species ID (98.9 vs. 99.2%), genus ID (99.7 vs. 100%), mis-ID (0.3 vs. 0%) and no ID (0 vs. 0). The rate of correct species identification by both MALDI-TOF MS (98.9 and 99.2%, respectively) was much higher compared with phenotypic approach (91.9%). Both MALDI-TOF MS systems provide accurate identification of clinical filamentous fungi compared with conventional phenotypic method, and have the potential to replace identification for routine identification of these fungi in clinical mycology laboratories. Both systems have similar performance in the identification of clinical filamentous fungi.     

DNA条形码分析方法研究进展

DNA条形码是一段短的、标准化的DNA序列,DNA条形码技术通过对DNA条形码序列分析实现物种的有效鉴定.随着生物DNA条形码序列的大量测定,DNA条形码分析方法得到迅速发展,推动了其在生物分子鉴定中的应用.2003年以来,DNA条形码技术已广泛应用于动物、植物和真菌等物种的鉴定,并有力地推动了生物分类学、生物多样性和生态学等学科的发展.本文在综述DNA条形码技术的基础上,总结了5类主要的DNA条形码分析方法,即基于遗传距离的分析、基于遗传相似度的分析、基于系统发育树的分析、基于序列特征的分析和基于统计分类法的分析,并进一步展望了DNA条形码技术的发展与应用.     

Emission of volatile compounds by Erwinia amylovora: biological activity in vitro and possible exploitation for bacterial identification

Several analytical techniques such as gas chromatography–mass spectrometry, proton transfer reaction–mass spectrometry and laser photoacoustic detection, were used to characterize the volatiles emitted by Erwinia amylovora and other plant-pathogenic bacteria. Diverse volatiles were found to be emitted by the different bacterial species examined. The distinct blend of volatiles produced by bacteria allowed their identification using an electronic nose (e-nose). The present study reports the discrimination of E. amylovora, the fire blight pathogen, from other plant-associated bacteria using an e-nose based on metal oxide semiconductor sensors. Two different approaches were used for bacterial identification. The first one was the direct comparison of the odorous profiles of unknown bacterial isolates with four selected reference species. The second approach was the use of previously developed databases representing the odorous variability among several bacterial species. Using these two strategies, the e-nose successfully identified the isolates in 87.5 and 62.5% of the cases, respectively. Finally, the profiling of the volatiles emitted by E. amylovora lead to identify some metabolic markers with a potential biological activity in vitro.     

Fungal molecular diagnostics: a mini review

Conventional methods to identify fungi have often relied on identification of disease symptoms, isolation and culturing of environmental organisms, and laboratory identification by morphology and biochemical tests. Although these methods are still fundamental there is an increasing move towards molecular diagnostics of fungi in all fields. In this review, some of the molecular approaches to fungal diagnostics based on polymerase chain reaction (PCR) and DNA/RNA probe technology are discussed. This includes several technological advances in PCR-based methods for the detection, identification and quantification of fungi including real-time PCR which has been successfully used to provide rapid, quantitative data on fungal species from environmental samples. PCR and probe based methods have provided new tools for the enumeration of fungal species, but it is still necessary to combine the new technology with more conventional methods to gain a fuller understanding of interactions occurring in the environment. Since its introduction in the mid 1980's PCR has provided many molecular diagnostic tools, some of which are discussed within this review, and with the advances in micro-array technology and real-time PCR methods the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual fungal species but also on whole communities.     

MALDI‐TOF‐MS‐based species identification and typing approaches in medical mycology

MALDI‐TOF MS‐based species identification has found its place in many clinical routine diagnostic laboratories over the past years. Several well‐established commercial systems exist and these allow precise analyses not only among bacteria, but also among clinically important yeasts. This methodology shows higher precision than biochemical and microscopic methods at significantly reduced turnaround times. Furthermore, the differentiation of different filamentous fungi including most dermatophytes and zygomycetes has been established. The direct identification of yeasts from blood culture bottles will be possible in a routine fashion with new standardized procedures. In addition to species identification, the MALDI‐TOF MS technology offers several further possibilities, like assays to detect or predict resistance phenotypes in fungi as well as subtyping approaches to detect clinically relevant subgroups. The differences between the commercial systems are discussed with respect to fungi and an overview of their performances provided. Factors influencing outcome of MALDI‐TOF‐based species identification are discussed.     

Recent advances in electronic and bioelectronic noses and their biomedical applications

Significant effort has been made in the development of an artificial nose system for various applications. Advances in sensor technology have facilitated the development of high-performance electronic and bioelectronic noses. Numerous articles describe the advantages of artificial nose systems for biomedical applications. Recent advances in the development of electronic and bioelectronic noses and their biomedical applications are reviewed in this article.     

Wood decay under the microscope

Many aspects of the interactions between host wood structure and fungal activity can be revealed by high resolution light microscopy, and this technique has provided much of the information discussed here. A wide range of different types of decay can result from permutations of host species, fungal species and conditions within wood. Within this spectrum, three main types are commonly recognised: brown rot, white rot and soft rot. The present review explores parts of the range of variation that each of these encompasses and emphasizes that degradation modes appear to reflect a co-evolutionary adaptation of decay fungi to different wood species or the lignin composition within more primitive and advanced wood cell types. One objective of this review is to provide evidence that the terms brown rot, white rot and soft rot may not be obsolete, but rigid definitions for fungi that are placed into these categories may be less appropriate than thought previously. Detailed knowledge of decomposition processes does not only aid prognosis of decay development in living trees for hazard assessment but also allows the identification of wood decay fungi that can be used for biotechnology processes in the wood industry. In contrast to bacteria or commercial enzymes, hyphae can completely ramify through solid wood. In this review evidence is provided that wood decay fungi can effectively induce permeability changes in gymnospermous heartwood or can be applied to facilitate the identification of tree rings in diffuse porous wood of angiosperms. The specificity of their enzymes and the mild conditions under which degradation proceeds is partly detrimental for trees, but also make wood decay fungi potentially efficient biotechnological tools.     

Early detection of spoilage moulds in bread using volatile production patterns and quantitative enzyme assays

AIMS: Early detection of spoilage fungi (two Eurotium spp., a Penicillium chrysogenum species) in bread analogues over periods of 72 h at 25 degrees C and 0.95 water activity was evaluated using volatile production patterns, hydrolytic enzyme production, and changes in fungal populations. METHODS AND RESULTS: Using an electronic nose system it was possible to differentiate between uninoculated controls and samples contaminated with P. chrysogenum within 28 h. After 40-48 h it was possible to differentiate between the Eurotium spp., P. chrysogenum and the control using Principal Component Analysis (PCA). Cluster analyses could differentiate between the control, P. chrysogenum and the Eurotium spp. after 40 h. Of seven hydrolytic enzymes examined after 48 h, the specific activities of three were significantly different from uninoculated control bread. There were also differences between the mould species in production of three enzymes. Penicillium chrysogenum populations increased fastest, from about 10(3) cfu g(-1) to 10(6)-10(7) cfu g(-1) after 72 h. For the Eurotium spp. this increase was slower. CONCLUSIONS: Overall, this study suggests, for the first time, that an electronic nose system using a surface polymer sensor array is able to detect qualitative changes in volatile production patterns for the early detection of the activity of spoilage moulds in bakery products. SIGNIFICANCE AND IMPACT OF THE STUDY: Potential exists for application of such systems for microbial quality assurance in intermediate moisture food products.     

The Black Yeasts: an Update on Species Identification and Diagnosis

Purpose of review

Black yeast-like fungi are capable of causing a wide range of infections, including invasive disease. The diagnosis of infections caused by these species can be problematic. We review the changes in the nomenclature and taxonomy of these fungi, and methods used for detection and species identification that aid in diagnosis.

Recent findings

Molecular assays, including DNA barcode analysis and rolling circle amplification, have improved our ability to correctly identify these species. A proteomic approach using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has also shown promising results. While progress has been made with molecular techniques using direct specimens, data are currently limited.

Summary

Molecular and proteomic assays have improved the identification of black yeast-like fungi. However, improved molecular and proteomic databases and better assays for the detection and identification in direct specimens are needed to improve the diagnosis of disease caused by black yeast-like fungi.

     

Biotechnology offers revolution to fish health management

Biotechnology has many applications in fish health management. The application of monoclonal antibodies (mAbs) provides a rapid means of pathogen identification; antibodies to immunoglobulins from different fish species can be used to monitor the host response following vaccination; and mAbs also have the potential for screening broodstock for previous exposure to pathogens. Luminex technology exemplifies a novel antibody-based method that can be applied to both pathogen detection and vaccine development. Molecular technologies, such as the polymerase chain reaction (PCR), real time PCR and nucleic acid sequence-based amplification (NASBA), have enabled detection, identification and quantification of extremely low levels of aquatic pathogens, and microarray technologies offer a new dimension to multiplex screening for pathogens and host response. Recombinant DNA technology permits large-scale, low-cost vaccine production, moreover DNA vaccination, proteomics, adjuvant design and oral vaccine delivery will undoubtedly foster the development of effective fish vaccines in the future.     

Microplate-based, label-free detection of biomolecular interactions: applications in proteomics

This review describes a new type of label-free optical biosensor that is inexpensively manufactured from continuous sheets of plastic film and incorporated into standard format microplates to enable highly sensitive, high-throughput detection of small molecules, proteins and cells. The biosensor and associated detection instrumentation are applied to review two fundamental limiting issues for assays in proteomics research and drug discovery: requirement for quantitative measurement of protein concentration and specific activity, and measurements made with complex systems in highly parallel measurements. SRU BIosystems, Inc.'s BIND label-free detection will address these issues using data examples for hybridoma screening, epitope binning and mapping, small-molecule screening, and cell-based functional assays. The review describes several additional applications that are under development for the system, and the key issues that will drive adoption of the technology over the next 5 years.