A probability matrix for the identification of species of Vibrio and related genera

A matrix for the probabilistic identification of species of Vibrio and related genera has been constructed using the data from 1091 strains collected throughout the world and classified. Thirty-eight phenons are included in the matrix, 31 of these represent previously identified species or biovars and seven represent phenons which could not be identified and may represent new species. The identification matrix incorporates 81 characters although a subset of 30 tests can be used to distinguish the 38 phenons from each other. The additional 51 tests were included to assist the identification of some strains for which the initial 30 tests were inadequate. No significant cluster overlap was found at the 5% level and the identification score for the Hypothetical Median Organism of each cluster exceeded 0.9999 in all cases.     

Numerical classification of species of Vibrio and related genera

Data from 1091 strains of the family Vibrionaceae collected in five different studies have been merged into a single data matrix and analysed in a taxonomic study. A set of 142 characters was selected to compare these data. Seventy-nine characters were common to all studies, but data for the other 63 characters were incomplete. Cultures of 90 strains, examined in more than one of the original studies, were used to estimate test error and inter-study variability. The data from these replicate strains also allowed the problem of merging data from different studies to be assessed. Taxonomic resemblance was estimated on the basis of 111 characters using the S_SM coefficient and UPGMA clustering. A taxonomic analysis based on 999 strains, which included most of the major species of the family Vibrionaceae, gave 59 clusters and 44 unclustered strains. A table of properties of these phenons was produced. The results showed that data obtained from studies carried out at different times and in different locations, but using standard techniques, could be combined and used to provide useful taxonomic information.     

A probability matrix for the identification of vibrios

A probability matrix for computer-assisted identification of vibrios has been constructed, based on the API 20E system. Data were gathered from 173 strains representing 31 taxa of vibrios and related organisms, from a variety of sources. The matrix was tested internally by four statistical programs. Program OVERMAT tested the separation and program MOSTTYP the discretion and homogeneity of the taxa. Most of the taxa were satisfactory but a few were less so; reasons for this are discussed. Program CHARSEP and program DIACHAR tested the separation and diagnostic values, respectively, of the characters used. The overall test error was 4–5%. The matrix was assessed externally by its performance in the identification of vibrio-like strains isolated from freshwater. Of 243 wild strains, 79–4% were identified with ten taxa, with a Willcox score of ζ 0–99.     

A probability matrix for the identification of vibrios

A probability matrix for computer-assisted identification of vibrios has been constructed, based on the API 20E system. Data were gathered from 173 strains representing 31 taxa of vibrios and related organisms, from a variety of sources. The matrix was tested internally by four statistical programs. Program OVERMAT tested the separation and program MOSTTYP the discretion and homogeneity of the taxa. Most of the taxa were satisfactory but a few were less so; reasons for this are discussed. Program CHARSEP and program DIACHAR tested the separation and diagnostic values, respectively, of the characters used. The overall test error was 4.5%. The matrix was assessed externally by its performance in the identification of vibrio-like strains isolated from freshwater. Of 243 wild strains, 79.4% were identified with ten taxa, with a Willcox score of greater than or equal to 0.99.     

A probability matrix for the identification of campylobacters, helicobacters and allied taxa

A probabilistic identification matrix for campylobacteria, comprising 67 phenotypic characters and 37 taxa, is described. The accuracy and integrity of the matrix was evaluated using established computer-assisted methods. Certain taxa (for example, Campylobacter concisus and Camp. gracilis ) demonstrated significant phenotypic diversity; previous data corroborated these findings. Differentiation between a few pairs of taxa proved difficult, although discriminatory characteristics were noted in each of these cases. The results indicate that most campylobacteria can be identified accurately and objectively with phenotypic tests when probabilistic methods of data assessment are employed.     

PCR-based identification of Vibrio cholerae and the closely related species Vibrio mimicus using the large chromosomal ori sequence of Vibrio cholerae

The bacterial chromosomal replication origin (ori) sequences are a highly conserved essential genetic element. In this study, the large chromosomal replication origin sequence of Vibrio cholerae (oriCIVC) has been targeted for identification of the organism, including the biotypes of serogroup O1. The oriCIVC sequence-based PCR assay specifically amplified an 890 bp fragment from all the V. cholerae strains examined. A point mutation in the oriCIVC sequence of the classical biotype of O1 serogroup led to the loss of a BglII site, which was utilized for differentiation from El Tor vibrios. Interestingly, the PCR assay amplified a similarly sized ori segment, designated as oriCIVM, from V. mimicus strains, but failed to produce any amplicon with other strains. Cloning and sequencing of the oriCIVM revealed high sequence similarity (96%) with oriCIVC. The results indicate that V. mimicus is indeed very closely related to V. cholerae. In addition, the BglII restriction fragment length polymorphism (RFLP) between oriCIVM and oriCIVC sequences allowed us to differentiate the two species. The ori sequence-based PCR-RFLP assay developed in this study appears to be a useful method for rapid identification and differentiation of V. cholerae and V. mimicus strains, as well as for the delineation of classical and El Tor biotypes of V. cholerae O1.     

Miniaturized tests for computer-assisted identification of motile Aeromonas species with an improved probability matrix

AIMS: To develop miniaturized tests for the phenotypic identification of motile Aeromonas species using an improved probability matrix. METHODS AND RESULTS: Conventional tests were miniaturized for use in 96-well plates, and their performance assessed using 60 aeromonads comprising type and reference strains as well as clinical, fish and water isolates. A revised probability matrix for Aeromonas hybridization groups 1-14, including A. allosaccharophila, A. bestiarum, A. encheleia and A. popoffii, was developed. Using 26 tests, all the reference strains were correctly identified with the revised probability matrix, and 80% of the isolates were correctly identified at a Willcox probability level of 95%. CONCLUSION: The compact test format, coupled with a robust identification matrix, provides a convenient basis for identifying motile aeromonads. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification system for identifying aeromonads will be of use to medical and veterinary laboratories undertaking disease diagnosis.     

A set of keys for biochemical identification of environmental Vibrio species

A set of biochemical keys which provide fast and presumptive identification for Vibrio spp. is presented. They have been specially designed for environmental isolates, and can be used for strains that are Gram-negative, give a positive oxidase test, grow on TCBS medium and are facultative anaerobes. The keys are constituted by 28 tests and a maximum of 10 tests are needed for the most complicated identification. They have been designed for routine purposes, especially for studies with a high number of isolates. Some tests are included in enzyme-activity based kits that could be used with these keys through certain results, principally for environmental isolates, should be confirmed by standard methods.     

A biochemical protocol for the isolation and identification of current species of Vibrio in seafood

AIMS: We report a biochemical method for the isolation and identification of the current species of vibrios using just one operative protocol. METHODS AND RESULTS: The method involves an enrichment phase with incubation at 30 degrees C for 8-24 h in alkaline peptone water and an isolation phase on thiosulphate-citrate-salt sucrose agar plates incubating at 30 degrees C for 24 h. Four biochemical tests and Alsina's scheme were performed for genus and species identification, respectively. All biochemical tests were optimized as regards conditions of temperature, time of incubation and media composition. The whole standardized protocol was always able to give a correct identification when applied to 25 reference strains of Vibrio and 134 field isolates. CONCLUSIONS: The data demonstrated that the assay method allows an efficient recovery, isolation and identification of current species of Vibrio in seafood obtaining results within 2-7 days. SIGNIFICANCE AND IMPACT OF THE STUDY: This method based on biochemical tests could be applicable even in basic microbiology laboratories, and can be used simultaneously to isolate and discriminate all clinically relevant species of Vibrio.     

Distinction of species in Aureobasidium and related genera by PCR-ribotyping

Taxonomic markers for differentiation of the anamorph genera Aureobasidium, Hormonema and Kabatiella were developed using PCR-ribotyping with the primers 5.8S-R and LR7 for amplification and the restriction enzymes Alul, DdeI, Hhal, MspI and RsaI for digestion. Aureobasidium and Hormonema are optimally differentiated with MspI; DdeI is particularly useful to distinguish aureobasidium, Kabatiella and Selenophoma. Relationships of the anamorph genera Aureobasidium, Hormoneng and Kabatiella with the teleomorph genera Pringsheimia and Dothiora are discussed.     

A revised probability matrix for the identification of gram-negative, aerobic, rod-shaped, fermentative bacteria

The results of the identification of 933 strains of Gram-negative, aerobic, rod-shaped, fermentative bacteria (Enterobacteriaceae, Pasteurellaceae, Vibrionaceae) by a probabilistic method, in a computer, are given. The identification rate on the matrix was 89.2%. Many of the strains were atypical and had caused difficulty in identification in medical diagnostic laboratories. The results are given for each taxon by genus and species.     

A novel multiplex PCR for the identification of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus

AIM: To establish a simple multiplex polymerase chain reaction (PCR) that will identify Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. METHODS AND RESULTS: A total of 429 Vibrio spp. from various origins were tested with the novel primers targeting toxR. The reverse primers were all designed to be species specific, while the forward primer was universal. The primers correctly identified all the V. parahaemolyticus, V. cholerae and V. vulnificus isolates tested. CONCLUSIONS: The toxR multiplex PCR works well when the initial colony morphology is known. If not, Vibrio alginolyticus might represent a diagnostic obstacle. SIGNIFICANCE AND IMPACT OF THE STUDY: The method provides a fast and reliable way of identifying the main Vibrio spp. involved in food-borne disease. The method could prove very useful for laboratories working with identification of these Vibrio spp.     

Evaluation of ribosomal RNA gene restriction patterns for the classification of Bacillus species and related genera

AIMS: To identify Bacillus species and related genera by fingerprinting based on ribosomal RNA gene restriction patterns; to compare ribosomal RNA gene restriction patterns-based phylogenetic trees with trees based on 16S rRNA gene sequences; to evaluate the usefulness of ribosomal RNA gene restriction patterns as a taxonomic tool for the classification of Bacillus species and related genera. METHODS AND RESULTS: Seventy-eight bacterial species which include 42 Bacillus species, 31 species from five newly created Bacillus-related genera, and five species from five phenotypically related genera were tested. A total of 77 distinct 16S rRNA gene hybridization banding patterns were obtained. The dendrogram resulting from UPGMA analysis showed three distinct main genetic clusters at the 75% banding pattern similarity. A total of 77 distinct 23S and 5S rRNA genes hybridization banding patterns were obtained, and the dendrogram showed four distinct genetic clusters at the 75% banding pattern similarity. A third dendrogram was constructed using a combination of the data from the 16S rRNA gene fingerprinting and the 23S and 5S rRNA genes fingerprinting. It revealed three distinct main phylogenetic clusters at the 75% banding pattern similarity. CONCLUSIONS: The Bacillus species along with the species from related genera were identified successfully and differentiated by ribosomal RNA gene restriction patterns, and most were distributed with no apparent order in various clusters on each of the three dendrograms. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data indicate that ribosomal RNA gene restriction patterns can be used to reconstruct the phylogeny of the Bacillus species and derived-genera that approximates, but does not duplicate, phylogenies based on 16S rRNA gene sequences.     

A probability matrix for the identification of gram-negative, aerobic, non-fermentative bacteria that grow on nutrient agar

Results of the identification of 621 strains of Gram-negative, aerobic, non-fermentative bacteria by a computer-based probabilistic method are given. Although many of the strains were atypical and have caused difficulty in identification in the medical diagnostic laboratory, the identification rate on this matrix was 91.5%.     

Distinction of species in Aureobasidium and related genera by PCR-ribotyping

Taxonomic markers for differentiation of the anamorph genera Aureobasidium, Hormonema and Kabatiella were developed using PCR-ribotyping with the primers 5.8S-R and LR7 for amplification and the restriction enzymes Alul, DdeI, Hhal, MspI and RsaI for digestion. Aureobasidium and Hormonema are optimally differentiated with MspI; DdeI is particularly useful to distinguish aureobasidium, Kabatiella and Selenophoma. Relationships of the anamorph genera Aureobasidium, Hormoneng and Kabatiella with the teleomorph genera Pringsheimia and Dothiora are discussed.     

Improvement and update of a set of keys for biochemical identification of Vibrio species

M. ALSINA AND A.R. BLANCH. 1994. Two biochemical keys for fast and presumptive identification of certain Vibrio species are presented. They constitute a new improved version of a set of keys previously described, which were specially designed for environmental and clinical isolates. They may be used for Gram-negative, oxidase-positive, facultative anaerobes that grow on TCBS agar. The revised set of biochemical keys consists of 29 tests and a maximum of 10 tests is still sufficient for the most complicated identification. The new keys maintain the same criteria and characteristics of the original set of keys.     

Flavonoids in the species of Cyrtomium (Dryopteridaceae) and related genera

Flavonoids in 19 Cyrtomium, three Cyrtogonellum and two Phanerophlebia taxa were surveyed. Major flavonoids were flavonol O-glycosides based on kaempferol, quercetin, and sometimes myricetin, and C-glycosylflavones, such as isovitexin, vitexin, isoorientin, orientin and their O-glycosides. The C-methylflavanones, farrerol and cyrtominetin, and their 7-O-glucosides were isolated from Cyrtomium devexiscapulae and Cyrtomium laetevirens. Flavanones have been reported from Cyrtomium falcatum sensu lato. Though C. falcatum sensu lato is divided into four taxa, i.e. C. falcatum subsp. falcatum, C. falcatum subsp. australe, C. falcatum subsp. littorale, and C. devexiscapulae, the occurrence of the flavanones was restricted to C. devexiscapulae, and they did not occur in C. falcatum sensu stricto.