Direct identification of pure Penicillium species using image analysis

This paper presents a method for direct identification of fungal species solely by means of digital image analysis of colonies as seen after growth on a standard medium. The method described is completely automated and hence objective once digital images of the reference fungi have been established. Using a digital image it is possible to extract precise information from the surface of the fungal colony. This includes color distribution, colony dimensions and texture measurements. For fungal identification, this is normally done by visual observation that often results in a very subjective data recording. Isolates of nine different species of the genus Penicillium have been selected for the purpose. After incubation for 7 days, the fungal colonies are digitized using a very accurate digital camera. Prior to the image analysis each image is corrected for self-illumination, thereby gaining a set of directly corresponding images with respect to illumination. A Windows application has been developed to locate the position and size of up to three colonies in the digitized image. Using the estimated positions and sizes of the colonies, a number of relevant features can be extracted for further analysis. The method used to determine the position of the colonies will be covered as well as the feature selection. The texture measurements of colonies of the nine species were analyzed and a clustering of the data into the correct species was confirmed. This indicates that it is indeed possible to identify a given colony merely by macromorphological features. A classifier (in the normal distribution) based on measurements of 151 colonies incubated on yeast extract sucrose agar (YES) was used to discriminate between the species. This resulted in a correct classification rate of 100% when used on the training set and 96% using cross-validation. The same methods applied to 194 colonies incubated on Czapek yeast extract agar (CYA) resulted in a correct classification rate of 98% on the training set and 71% using cross-validation.     

A rapid PCR-based approach for molecular identification of filamentous fungi

In this study, a novel rapid and efficient DNA extraction method based on alkaline lysis, which can deal with a large number of filamentous fungal isolates in the same batch, was established. The filamentous fungal genomic DNA required only 20 min to prepare and can be directly used as a template for PCR amplification. The amplified internal transcribed spacer regions were easy to identify by analysis. The extracted DNA also can be used to amplify other protein-coding genes for fungal identification. This method can be used for rapid systematic identification of filamentous fungal isolates     

Evaluation of a new chromogenic medium (Candida ID) for the isolation and presumptive identification of Candida albicans and other medically important yeasts

Candidiasis is a frequent human infection caused mainly by Candida albicans. However, other species are emerging as important pathogens, as Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei or Candida guilliermondii. Rapid identification of clinical isolates could facilitate diagnosis and treatment. Candida ID (bioMerieux, Spain) is a new medium for the isolation and presumptive identification of yeasts: C. albicans grows as blue colonies, and C. tropicalis, C. guilliermondii, Candida kefyr and Candida lusitaniae as pink ones. The utility of Candida ID was evaluated with more than 700 clinical isolates and type culture collection strains from different genera including Candida, Cryptococcus, Saccharomyces, and Rhodotorula. Presumptive identification was confirmed by germ tube test, microscopic morphology and chlamydoconidia production on corn meal agar and carbohydrate assimilation on API-ATB ID 32C or Vitek (bioMerieux). Growth on Candida ID was rapid (18-24 h) for most of the yeast strains tested. Sensitivity and specificity of identification of C. albicans was significantly high (>98%), since a very low number of isolates were found to be false negative or false positive. A better result was obtained for species growing as pink colonies (>99.5%). Detection of different species of medical important yeasts was easy with Candida ID, as perfectly distinct colors and textures of colonies were observed on this medium. Candida ID allowed the discrimination between C. glabrata (creamy and smooth) and C. krusei (rough and white) colonies. Other species showed different colony textures and colours, white being the predominant colour. Candida ID was very useful for the presumptive identification C. albicans isolates.     

Value of morphotyping for the characterization of Candida albicans clinical isolates

Until recently, morphotyping, a method evaluating fringe and surface characteristics of streak colonies grown on malt agar, has been recommended as a simple and unexpensive typing method for Candida albicans isolates. The discriminatory power and reproducibility of Hunter's modified scheme of Phongpaichit's morphotyping has been evaluated on 28 C. albicans isolates recovered from the oral cavity of asymptomatic human immunodeficiency virus-positive subjects, and compared to two molecular typing methods: randomly amplified polymorphic DNA (RAPD) fingerprinting, and contour clamped homogeneous electric field (CHEF) electrophoretic karyotyping. Morphological features of streak colonies allowed to distinguish 11 different morphotypes while RAPD fingerprinting yielded 25 different patterns and CHEF electrophoresis recognized 9 karyotypes. The discriminatory power calculated with the formula of Hunter and Gaston was 0.780 for morphotyping, 0.984 for RAPD fingerprinting, and 0.630 for karyotyping. Reproducibility was tested using 43 serial isolates from 15 subjects (2 to 6 isolates per subject) and by repeating the test after one year storage of the isolates. While genetic methods generally recognized a single type for all serial isolates from each of the subjects studied, morphotyping detected strain variations in five subjects in the absence of genetic confirmation. Poor reproducibility was demonstrated repeating morphotyping after one year storage of the isolates since differences in at least one character were detected in 92.9% of the strains.     

Genetic Diversity, Aggressiveness and Metalaxyl Sensitivity of Pythium spinosum Infecting Cucumber in Oman

A total of 24 isolates of Pythium spinosum from cucumber obtained from five regions in Oman were characterized for genetic diversity using amplified fragment length polymorphism (AFLP) fingerprinting and three isolates from the Netherlands, South Africa and Japan were included for comparison. Isolates from Oman were also characterized for aggressiveness on cucumber seedlings and sensitivity to metalaxyl. Identity of all isolates was confirmed using sequences of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), which showed more than 99% nucleotide similarity among all isolates. Using six primer‐pair combinations, AFLP fingerprinting resolved 295 AFLP markers of which 193 were polymorphic among isolates from other countries and only six were polymorphic among isolates of P. spinosum from Oman. Seven different AFLP phenotypes of P. spinosum were recovered in Oman; two of them were found to contain over 79% of isolates and one was recovered from all regions in Oman. Phenotypes from Oman showed very high (≥99%) levels of genetic similarity to each other compared to moderate (mean =53%) levels of genetic similarity with phenotypes from other countries. In addition, all isolates from Oman were found to be highly sensitive to metalaxyl and all were aggressive on cucumber seedlings at 25°C. The high genetic similarity among phenotypes of P. spinosum in Oman as well as recovering two major clones across regions may suggest that P. spinosum has been recently introduced in Oman via a common source.     

Genetic diversity of the natural strains of Streptococcus thermophilus

The method of RAPD-PCR and comparative analysis of the PCR fingerprinting profiles similarity was used to characterize interspecific diversity of natural isolates of the lactic acid bacteria Streptococcus thermophilus. The strain genetic diversity was demonstrated using three primer variants, designed for different bacterial genome regions. The resolution of RAPD-PCR technique with different primers for identification at the species level and for certification at the strain level, was examined relative to the commercially important cultures of S. thermophilus. The results provided conclusion on preferable usage of RAPD-PCR with the primer ERIC-1 for specific identification of S. thermophilus, and with the primer M13 for certification of natural isolates of this species at the strain level.     

M13-microsatellite PCR and rDNA sequence markers for identification of Trichoderma (Hypocreaceae) species in Saudi Arabian soil

Seven fungal isolates were identified as pan-global Hypocrea/Trichoderma species, from section Trichoderma, on the basis of their morphology. These species were H. lixii/T. harzianum and H. orientalis/T. longibrachiatum. PCR-based markers with primer M13 (core sequence of phage M13) and internal-transcribed spacer sequences of ribosomal DNA were used to confirm the identity of the two Trichoderma species. Sequence identification was performed using the TrichOKEY version 2.0 barcode program and the multilocus similarity search database TrichoBLAST. Sequences from the ribosomal DNA internal-transcribed spacer regions showed limited variation among the Trichoderma species. This analysis divided the isolates into two main groups. Grouping the isolates based on cluster analysis of their DNA profiles matched the grouping based on morphological taxonomy. Molecular data obtained from analyses of gene sequences are essential to distinguish phonetically cryptic species in this group and to establish phylogenetic relationships.     

Heterogeneity of<Emphasis Type="Italic">Escherichia coli</Emphasis> derived from artiodactyla animals analyzed with the use of rep-PCR fingerprinting

Genetic polymorphism of 83 isolates of E. coli, derived from 4 species of artiodactyla animals living in a relatively close contact on the grounds of a theme park ZOO Safarii Swierkocin (Poland) was determined using the rep-PCR fingerprinting method, which utilizes oligonucleotide primers matching interspersed repetitive DNA sequences in PCR reaction to yield DNA fingerprints of individual bacterial isolates based on repetitive extragenic palindrome (REP) primers. The fingerprint patterns demonstrated the essential polymorphism of distribution of REP sequences in genomes of the examined isolates. The arithmetic averages clustering algorithm (UPGMA) statistical analysis of fingerprints with the use of the Jaccard similarity coefficient differentiated E. coli isolates into three similarity groups containing various numbers of isolates. The groups comprised isolates derived from two, three and four species of the source animals. The isolates derived from each source segregated in the dendrogram in a different way, both within the similarity groups and among them, indicating an individual repertoire of E. coli in the examined species of animals. The similarity relations among E. coli derived from the same source, illustrated in a dendrogram with a number of subclusters of a low mutual similarity (< or = 20%), indicated an essential interstrain differentiation in terms of the distribution of REP sequences. Our results confirmed the hypothesis of the oligoclonal characters of populations obtained from particular sources. The rep-PCR fingerprinting method with REP primers is simple and highly differentiating and can be recommended for use in explorations of large groups of animals and monitoring the variability of strains.     

Evaluation of DNA Fingerprinting, Aggression Tests, and Morphometry as Tools for Colony Delineation of the Formosan Subterranean Termite

Multilocus DNA fingerprinting, aggression tests, and morphometry were compared to evaluate their potential for the delineation of colonies of Coptotermes formosanus Shiraki (Isoptera; Rhinotermitidae) in Hawaii. DNA fingerprinting segregates the termites from all collection sites and allows the assignment of all individuals to their original collection site. The genetic similarity of termites from different collection sites approaches the population's genetic background similarity, consequently collection sites represent independent colonies. Aggression between colonies is comparatively low and does not provide reliable colony delineation. Morphometry allows a 79% classification rate of termites to their colony of origin. No correlation among genetic similarities, aggression levels, and morphometric distances is found. Of the three investigated methods, we conclude that the genetic approach is the most useful tool for colony delineation in C. formosanus.     

Genetic Analysis of the Drifting of Drones in Apis mellifera Using Multilocus DNA Fingerprinting

The presence of non-native drones in colonies of Apis mellifera was studied using multilocus DNA fingerprinting. Drones revealing a fingerprinting DNA banding pattern that did not correspond to the queen's genotype were classified as non-native animals. As previously reported for the drifting of workers, the position of the hive in relation to neighbouring colonies and the orientation of the flight entrance towards the sun showed significant correlations to the number of drifted drones in a colony. The frequency of non-native drones was low in central colonies (13 %) but high in marginal colonies (24 %). Furthermore, there was a significant correlation (r = 0.56) between the band-sharing coefficient of the non-native drones and the queen, and the number of drifted drones in the colony, which might indicate that genetically based nestmate recognition is involved in the drifting and/or acceptance of foreign drones in the colony.     

Identification of fungal pathogens associated with grapevine trunk using fluorescent-labelled ribosomal DNA probe

Detection and identification of fungal pathogens associated with grapevine trunk diseases (GTDs) are often difficult and laborious. The aim of the study is to develop a simple and time-saving protocol for the identification of a broad range of fungal species causing GTDs by fragment length analysis of the internal transcribed spacer (ITS) regions 1 and 2 and the large subunit D2 hypervariable region of the ribosomal DNA (rDNA). A total of 32 fungal isolates from declining vines and six type strains, representing 19 different species was included in this study. The majority of the fungi had unique species-specific PCR products ranging from 212 to 370 bp (ITS1), 332 to 428 bp (ITS2) and 320 to 325 bp (LSU-D2), and the combination of three regions identifies all pathogenic fungi tested. The protocol proposed here provides a highly sensitive, reliable and rapid identification method for a broad range of significant grapevine fungal trunk pathogens.     

Diagnostic characterization of Penicillium palitans, P. commune and P. solitum

A total of 98 isolates of Penicillium commune and P. solitum were analysed and it was shown that these isolates produced three unique combinations of secondary metabolites. Penicillium commune produced cyclopiazonic acid, cyclopaldic acid and rugulovasine A and B; P. solitum produced compactin and a new group including the ex-type culture of P. palitans produced cyclopiazonic acid and fumigaclavine A. Isolates in all three groups were able to produce cyclopenin, cyclopenol and viridicatin. An optimal thin layer chromatography system to detect the secondary metabolites from P. commune, P. palitans and P. solitum was developed using an agar plug method. The results were confirmed by high performance liquid chromatography with diode array detection. The chemotaxonomic allocations were backed up by differences in conidial colour on Czapek yeast autolysate agar, reverse colour on yeast extract sucrose agar and origin of the isolates. Even though P. palitans previously has been considered synonymous with P. commune or P. solitum it was concluded that P. palitans is a distinct species.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

Diversity among clinical isolates of Histoplasma capsulatum detected by polymerase chain reaction with arbitrary primers.

Clinical isolates of the fungal respiratory and systemic pathogen Histoplasma capsulatum have been placed in several different classes by using genomic restriction fragment length polymorphisms (RFLPs), but in general have not been distinguished further. We report here that a polymerase chain reaction (PCR)-based DNA fingerprinting method that has been termed arbitrary primer or random amplified polymorphic DNA (RAPD) PCR can distinguish among isolates in a single RFLP class. In this method, arbitrarily chosen oligonucleotides are used to prime DNA synthesis from genomic sites that they fortuitously match, or almost match, to generate strain-specific arrays of DNA fragments. Each of 29 isolates of RFLP class 2, the group endemic in the American Midwest, was distinguished by using just three arbitrary primers. In contrast, laboratory-derived S and E colony morphology variants of two strains were not distinguished from their R parents by using 18 such primers. Thus, the clinical isolates of H. capsulatum are quite diverse, but their genomes remain stable during laboratory culture. These outcomes suggest new possibilities for epidemiological analysis and studies of fungal populations in infected hosts.     

Use of RAPD-PCR fingerprinting to detect genetic diversity of soil Streptomyces isolates

Random amplified polymorphic DNA (RAPD) was used for identification and assessment of genetic diversity between isolates of Streptomyces from soil. Genomic DNA from 18 Streptomyces isolates and 2 reference strains were amplified using four different 10-mer primers. Different DNA fingerprinting patterns were obtained for all the isolates. Electrophoretic and cluster analysis of the amplification products revealed incidence of polymorphism among the isolates and none of them was identical to the reference strains although there were some common amplification bands. Two highly divergent groups were determined among the isolates. The results indicate that RAPD is an efficient method for discriminating and studying genetic diversity of Streptomyces isolates.     

Challenges in Laboratory Detection of Fungal Pathogens in the Airways of Cystic Fibrosis Patients

Study of the clinical significance of fungal colonization/infection in the airways of cystic fibrosis (CF) patients, especially by filamentous fungi, is challenged by the absence of standardized methodology for the detection and identification of an ever-broadening range of fungal pathogens. Culture-based methods remain the cornerstone diagnostic approaches, but current methods used in many clinical laboratories are insensitive and unstandardized, rendering comparative studies unfeasible. Guidelines for standardized processing of respiratory specimens and for their culture are urgently needed and should include recommendations for specific processing procedures, inoculum density, culture media, incubation temperature and duration of culture. Molecular techniques to detect fungi directly from clinical specimens include panfungal PCR assays, multiplex or pathogen-directed assays, real-time PCR, isothermal methods and probe-based assays. In general, these are used to complement culture. Fungal identification by DNA sequencing methods is often required to identify cultured isolates, but matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is increasingly used as an alternative to DNA sequencing. Genotyping of isolates is undertaken to investigate relatedness between isolates, to pinpoint the infection source and to study the population structure. Methods range from PCR fingerprinting and amplified fragment length polymorphism analysis, to short tandem repeat typing, multilocus sequencing typing (MLST) and whole genome sequencing (WGS). MLST is the current preferred method, whilst WGS offers best case resolution but currently is understudied.     

Genotyping of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> by Box elements

PCR-based DNA fingerprinting techniques were evaluated to genotype eight diseased, particularly normal and environmental isolates of Aeromonas hydrophila. PCR-based fingerprinting method has an advantage of having repetitive sequence also called Box elements that are interspersed throughout the genome in diverse bacterial species. The BOX-PCR fingerprinting technique was evaluated for the discrimination of different isolates of A. hydrophila. All the studied isolates have shown major banding patterns ranged from 500–3000 bp. These finding could be advantageous to investigate the strain level specific fingerprints of A. hydrophila as potential genotypic markers.     

Heteroduplex Mobility Assay for Identification and Phylogenetic Analysis of Anthracnose Fungi

Colletotrichum spp . are casual agents of anthracnose on various economically important crops. To cope with the pitfalls of identifying the fungi by morphotaxonomic criteria, the application of heteroduplex mobility assay (HMA) of internal transcribed spacer (ITS) regions as a biochemical tool was explored. The ITS regions of 29 Colletotrichum isolates including Colletotrichum gloeosporioides , Colletotrichum acutatum , Colletotrichum musae , Colletotrichum graminicola , Colletotrichum capsici , Colletotrichum dematium , Colletotrichum lindemuthianum and three unidentified species of Colletotrichum , were PCR amplified. Comparison of the ITS sequences from 15 Colletotrichum isolates revealed a greater DNA divergence within ITS1 region than that within ITS2. The DNA distance and sequence identity within intra-species ranged from 0.0 to 1.1% and from 98.9 to 100%, respectively; whereas those within inter-species ranged from 1.46 to 13.43% and 90.02 to 98.56%, respectively. From the correlation of DNA distance and relative heteroduplex mobility observed among 15 reference isolates, a formula for estimation of distances of a tested DNA sequence was developed for estimation of DNA distances of a compared strain. The phylogenetic analysis of ITS regions of 29 Colletotrichum isolates using DNA distance inferred from relative heteroduplex mobility divided them into 5 distinctive species groups, namely CG, CA, CC, CM and CL, similar to that assembled based on DNA sequences analysis. Our results show that HMA of ITS regions is a relatively rapid and convenient method for species-specific identification of Colletotrichum spp. The potential use of the established techniques for identification of anthracnose and even other fungal diseases are discussed.     

Genetic diversity of the natural strains of <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis>

The method of RAPD-PCR and comparative analysis of the PCR fingerprinting profiles similarity was used to characterize interspecific diversity of natural isolates of the lactic acid bacteria Streptococcus thermophilus. The strain genetic diversity was demonstrated using three primer variants, designed for different bacterial genome regions. The resolution of RAPD-PCR technique with different primers for identification at the species level and for certification at the strain level, was examined relative to the commercially important cultures of S. thermophilus. The results provided conclusion on preferable usage of RAPD-PCR with the primer ERIC-1 for specific identification of S. thermophilus, and with the primer M13 for certification of natural isolates of this species at the strain level.