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PubMed2001

Simplified technique for identification of the aerobic spore-forming bacteria by phenotype.

The use of modern research approaches of genetics, biochemistry and molecular biology has led to progress in bacterial taxonomy. Systematic study of the aerobic spore-forming bacteria has resulted in the realignment of the genus Bacillus into several new genera. In the meantime, the identification process has become more difficult for the non-specialist in Bacillus taxonomy. This paper presents a key for the simplified phenotypic identification of the mesophilic, aerobic, spore-forming bacteria belonging to the genera Bacillus, Paenibacillus, Brevibacillus, Aneurinibacillus, Geobacillus and Virgibacillus. A total of 81 species were included and 115 morphological and physiological tests were analysed for their discriminative efficiency. This key is practical for rough but quick identification of aerobic spore-forming bacteria isolated from nature. Such preliminary identification will be helpful for the selection of reference strains and methods for more precise identification using the newest techniques. The reliability of the proposed identification key was tested on 100 cultures from the Ukrainian Collection of Microorganisms. The developed identification key is represented in interactive mode on a website (http://www/imv.kiev.ua/key/).

Bacillaceae↗
PubMed2002

Identification of coryneform bacteria and related taxa by Fourier-transform infrared (FT-IR) spectroscopy.

An extensive Fourier-transform infrared (FT-IR) spectroscopy database for the identification of bacteria from the two suborders Micrococcineae and Corynebacterineae (Actinomycetales, Actinobacteria) as well as other morphologically similar genera was established. The database consists of averaged IR spectra from 730 reference strains, covering 220 different species out of 46 genera. A total of 192 species are represented by type strains. The identity of 352 reference strains was determined by comparative 16S rDNA sequence analysis and, if necessary, strains were reclassified accordingly. FT-IR frequency ranges, weights and reproducibility levels were optimized for this section of high-G+C gram-positive bacteria. In an internal validation, 98.1% of 208 strains were correctly identified at the species level. A simulated external validation which was carried out using 544 strains from 54 species out of 16 genera resulted in a correct identification of 87.3% at the species level and 95.4% at the genus level. The performance of this identification system is well within the range of those having been reported in the literature for the identification of coryneform bacteria by phenotypical methods. Coryneform and related taxa display a certain degree of overlapping distribution of different taxonomical markers, leading to a limited differentiation capacity of non-genotypical identification methods in general. However, easy handling, rapid identification within 25 h starting from a single colony, a satisfactory differentiation capacity and low cost, render FT-IR technology clearly superior over other routine methods for the identification of coryneform bacteria and related taxa.

Actinomycetales↗
PubMed2007

Comparison of different biochemical and molecular methods for the identification of Vibrio parahaemolyticus.

AIMS: Multicentre evaluation of biochemical and molecular methods for the identification of Vibrio parahaemolyticus. METHODS AND RESULTS: For the biochemical identification methods, API 20E and API 20NE and Alsina's scheme were evaluated in intra- and interlaboratory tests in order to determine the accuracy and concordance of each method. Both in intra- and interlaboratory tests, the Alsina's scheme showed the highest sensitivity (86% of correct identifications in the interlaboratory test). False-positive results were obtained by all methods (specificity was 95% for API 20E, 73% for API 20NE and 84% for Alsina's scheme) and concordance varied from 65% of API 20NE to 84% of API 20E. For the molecular identifications, polymerase chain reaction (PCR) for the detection of toxR gene, tl gene and pR72H fragment were tested on 30 strains by two laboratories. The PCR for toxR showed the highest inclusivity (96%), exclusivity (100%) and concordance (97%). CONCLUSIONS: Among the biochemical identification methods tested, the Alsina's scheme gave more reliable results; however, in order to avoid false-positive results, all the biochemical identifications should be confirmed by means of molecular methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Availability of an efficient identification method of Vibrio parahaemolyticus to use in official control of fisheries products.

Bacterial Proteins↗
PubMed1996

Evaluation of the Rapid ID 32A system for the identification of the Bacteroides fragilis group.

OBJECTIVE: To evaluate the use of a rapid identification system, Rapid ID 32A (bioMérieux), for the identification of clinically important species in the B. fragilis group. METHODS: The use of Rapid ID 32A was validated on 249 clinical isolates, all of which were tested by conventional techniques, and in selected instances API 20A. Rapid ID 32A (and API 20A as appropriate) was then applied in a central laboratory to the identification of 1289 B. fragilis group clinical isolates from 22 laboratories in 15 European countries. RESULTS: Improvements in the initial database permitted the accurate identification of isolates of B. fragilis, B. thetaiotaomicron and B. vulgatus, but further tests, especially for catalase production, were required to distinguish between B. ovatus and B. uniformis, while an identification of B. distasonis could be accepted only after careful review of results. There were too few isolates of B. caccae, B. merdae and B. stercoris for us to reach satisfactory conclusions, but further tests are clearly necessary. CONCLUSIONS: The study emphasizes the importance of including sufficient numbers of isolates of different species in the validation of identification methods. Rapid ID 32A is a reliable system for the identification of the common species in the B. fragilis group, especially B. fragilis and B. thetaiotaomicron.

Journal Article↗
PubMed1999

Evaluation of the ID 32E for the identification of Gram-negative glucose-fermenting and glucose-non-fermenting bacilli.

OBJECTIVE: To evaluate the ID 32E bacterial identification system for accuracy in the identification of members of the family Enterobacteriaceae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii/Iwoffii. METHODS: Stock cultures of 497 Enterobacteriaceae and 27 commonly encountered non-enteric Gram-negative rods were tested in the ID 32E system. For each isolate, the resulting 11-digit profile number was converted to an identification using the APILAB Plus software (version 3.2.2). This identification was then compared to the reference identification obtained using conventional biochemicals. RESULTS: Of the 524 isolates tested, 405 (77.3%) were identified correctly; 52 (9.9%) were identified incorrectly. Sixty-seven (12.8%) identifications were either doubtful or unacceptable, and were not limited to any particular genus or species, with the exception of Ewingella americana and Serratia plymuthica, which did not grow well enough in the strip at 35 degrees C to be correctly identified. All five isolates of Acinetobacter Iwoffii were misidentified as Alcaligenes spp. CONCLUSIONS: With this challenge set of organisms, the ID 32E correctly identified 77.3% of the isolates tested. For commonly encountered isolates, the accuracy approached 90%. We conclude that the ID 32E offers an alternative for the identification of common clinical isolates.

Journal Article↗
PubMed2005

Ribosomal DNA sequencing: experiences from use in the Danish National Reference Laboratory for Identification of Bacteria.

Diagnostic tools for identification of bacteria have developed dramatically in the last decade. Sequencing of genes coding for rRNA has led to revolutionary insights into the phylogeny and taxonomy of bacteria, and to new demands on the service provided by national reference laboratories for identification of bacteria. At the Danish Reference Laboratory for Identification of Bacteria, partial 16S rDNA sequencing has been used since 2001 to identify "difficult" strains submitted for taxonomic elucidation. Experiences relating to phenotypic as well as 16S rDNA sequencing of the first 175 strains examined are presented. Approximately 2/3 of the strains were Gram-positive and 1/3 Gram-negative. One fifth of the strains were anaerobic, while 4/5 were either facultatively anaerobic or aerobic. Methodological agreement was seen for most strains at species and/or genus level. Methodological disagreement was relatively rare. In 1/6 of the strains valuable information was obtained from sequencing results, while for some strains identification was based primarily on the phenotypic results. Only a few strains could not be clearly identified by either method. A very large number of strains representing taxons ranging from facultatively anaerobic to aerobic and anaerobic species and genera, Gram-positive as well as Gram-negative, were successfully examined. Of the submitted strains many have only rarely been encountered as human pathogens. Thus, genotypic identification may result in recognition of hitherto seldom recognized or unrecognized bacteria as human pathogens, which will lead to a better understanding of the nature of human infections. It is self evident that we should focus on slowly growing, fastidious or 'difficult' organisms when using sequencing for national reference purposes. Short sequences (450-650 base pairs) seem sufficient for most identifications. Molecular bacterial identification is a powerful tool for national reference laboratories, enhancing both the speed and validity of examinations performed.

Bacteria↗
PubMed1999

The distribution and identification of dangerously venomous Australian terrestrial snakes.

The identification of dangerous Australian snakes is important in instituting therapy for envenomation. Despite the availability of a number of identification guides with varying degrees of generality, identification can be problematic for several reasons. These include a diversity of common names, many of which are inappropriate or regionally applied to different species, identification keys that focus on variable features, intraspecific variation and interspecific convergence in colouration, and recent changes in scientific nomenclature of species and genera. Geographic distribution of the dangerously venomous species can be a useful aid to identification, by limiting the range of options in a region. However, delineation of the limits of distribution relies on fine scale mapping beyond the resolution of most identification guides. This article provides a summary of the geographic limits of the dangerously venomous Australian snakes, with particular emphasis on major population centres, and clarifies some problems in identification, particularly among brown-coloured snakes.

Animals↗
PubMed1995

Binaural advantage for sound pattern identification.

Listeners were trained to identify six patterns of eight sequentially presented 48-ms tone bursts. The variation in frequency forming the patterns was confined to a relatively narrow range around the nominal center frequency, which was either 500, 1000, or 3000 Hz, or was selected randomly on each presentation from a range of 450-3300 Hz. Detection (500 and 3000 Hz only) and identification of the six patterns masked by Gaussian noise was measured in two interaural presentation conditions: masker in-phase and signal in-phase (NoSo), and masker in-phase and signal pi rad out-of-phase (NoS pi). The differences in performance in the two interaural presentation conditions for detection and identification are referred to as "masking-level differences" (MLDs) and "identification-level differences" (IDLDs), respectively. At 500 Hz, MLDs and IDLDs were about 11-13 dB. At 3000 Hz, the MLDs and IDLDs were about 1-3 dB. For the random-center-frequency condition, the slopes of the identification-level functions were much shallower for the NoS pi condition than for the NoSo condition so the binaural advantage was large at low signal-to-noise ratios and declined as signal-to-noise ratio increased. This finding was due to the broad frequency range over which the information was distributed and the decline in the binaural advantage with increasing frequency, a conclusion consistent with that reported for the binaural advantage for speech intelligibility. A second experiment demonstrated that MLDs and IDLDs could be manipulated independently: A 500-Hz tone was added to each element of the 3000-Hz patterns. A large MLD was found--due to detection of the 500-Hz tone--while the identification-level functions were determined solely by the high-frequency information, which produced small IDLDs. Finally, the Gaussian noise masker was replaced by an informational masker comprised of eight randomly chosen eight-tone bursts played simultaneously with the signal-pattern elements which were centered at 1000 Hz. Large amounts of informational masking were found for identification. The slopes of the identification-level functions were much shallower than found for the Gaussian noise masker and a relatively small binaural advantage (about 5 dB) was observed.

Adult↗
PubMed1998

Language, context, and speaker effects in the identification and discrimination of English /r/ and /l/ by Japanese and Korean listeners.

Japanese and Korean listeners' identification and discrimination of English /r/ and /l/ were compared using a common set of minimal pair stimuli. The effects of speakers (two native speakers of Australian English), position of the contrast within the word (word initial, initial consonant cluster. and medial positions), and listening task (forced choice identification versus oddball discrimination) were examined, with a view to assessing the relative importance of language-specific and language-independent factors operating at the acoustic-phonetic and phonological levels of signal processing in "foreign sound" speech perception. Both prior phonological learning and the relative acoustic discriminability of the items affected subjects' performance on the identification test. Where both factors were engaged, phonological learning effects predominated over the effects of acoustic discriminability. The extent to which a speaker encoded critical acoustic cues for the /r-l/ distinction was found to affect /r-l/ identification. Dynamic spectral features known to be relevant for the /r-l/ contrast were effective in predicting (in a linear regression analysis) speaker-dependent differences in identification scores. Although the discrimination test may have been influenced by ceiling effects, the performance profiles on the identification and discrimination tests were quite different, indicating that the identification and discrimination tests imposed quite different task demands upon listeners and that phonological processing of the signal was more engaged by the former task.

Adolescent↗
PubMed2003

Direct identification of cytochrome P450 isozymes by matrix-assisted laser desorption/ionization time of flight-based proteomic approach.

The main targets of our investigation were cytochrome P450 isozymes (P450), the key enzymes of the hepatic drug-metabolizing system. Current research approaches to the identification of individual P450 forms include specific P450 inhibitors or substrates, antibody-based identification, and mRNA-based expression profiling. All of these approaches suffer from one common disadvantage-they all are indirect methods. On the other hand, current developments in mass spectrometry provide a direct and reliable approach to protein identification with sensitivity in the femtomole or low picomole range. In this study we have used high-accuracy, matrix-assisted laser desorption/ionization time of flight (MALDI TOF)-based peptide mapping to perform direct identification of distinct P450 isozymes in various rat and rabbit liver microsomes. For the first time, the P450 isozyme composition of clofibrate-induced rat and phenobarbital-induced rabbit liver microsomes was determined by peptide mass fingerprinting (PMF). Application of MALDI TOF-based PMF allows differential identification of such highly homologous P450s as CYP2B1 and CYP2B2. We have found that CYP2A10 previously reported only in rabbit olfactory and respiratory nasal mucosa is present in phenobarbital (PB)-induced rabbit liver microsomes. Two other rabbit P450s, earlier identified only by screening a cDNA library, were found to be present in PB-induced rabbit liver microsomes. In summary, direct identification of P450s by proteomic technique offers advantages over other methods with regard to identification of distinct P450 isozymes and should become a standard approach for characterizing microsomes.

Animals↗
PubMed1998

Use of enzyme tests in characterization and identification of aerobic and facultatively anaerobic gram-positive cocci.

The contribution of enzyme tests to the accurate and rapid routine identification of gram-positive cocci is introduced. The current taxonomy of the genera of aerobic and facultatively anaerobic cocci based on genotypic and phenotypic characterization is reviewed. The clinical and economic importance of members of these taxa is briefly summarized. Tables summarizing test schemes and kits available for the identification of staphylococci, enterococci, and streptococci on the basis of general requirements, number of tests, number of taxa, test classes, and completion times are discussed. Enzyme tests included in each scheme are compared on the basis of their synthetic moiety. The current understanding of the activity of enzymes important for classification and identification of the major groups, methods of testing, and relevance to the ease and speed of identification are reviewed. Publications describing the use of different identification kits are listed, and overall identification successes and problems are discussed. The relationships between the results of conventional biochemical and rapid enzyme tests are described and considered. The use of synthetic substrates for the detection of glycosidases and peptidases is reviewed, and the advantages of fluorogenic synthetic moieties are discussed. The relevance of enzyme tests to accurate and meaningful rapid routine identification is discussed.

Animals↗
PubMed2006

Comparative study using various methods for identification of Staphylococcus species in clinical specimens.

Coagulase-negative staphylococci (CNS) play a predominant role in nosocomial infections. Rapid, reliable identification of these organisms is essential for accurate diagnosis and prompt effective treatment of these infections. Quite recently, the VITEK 2 g-positive (gram-positive [GP]) identification card (bioMérieux) has been redesigned for greater accuracy in the identification of gram-positive cocci. We compared the BD Phoenix (Becton Dickinson) and VITEK 2 (bioMérieux) automated microbiology systems, using their respective update version cards, and the API ID32 STAPH test. The glyceraldehyde-3-phosphate dehydrogenase (gap) gene-based T-RFLP (terminal restriction fragment length polymorphism) method was used for verifying the results. In total, 86 clinical isolates of CNS and 27 reference strains were analyzed. The results show that for identification of CNS, the automated identification methods using the newest VITEK 2 and BD Phoenix identification cards are comparable. However, API ID32 STAPH revealed more correct results compared to both automated microbiology systems. Despite the increased performance of the phenotypic automated identification systems compared to the former versions, molecular methods, e.g., the gap-based T-RFLP method, still show superior accuracy in identifying Staphylococcus species other than Staphylococcus aureus.

Bacterial Typing Techniques↗
PubMed2026

Comparison of culture and culture-free methods for comprehensive identification of mycobacteria: a single-center prospective study.

The genus Mycobacterium, including Mycobacterium tuberculosis and over 200 nontuberculous mycobacteria (NTM), shows wide variability in clinical outcomes and drug susceptibility. Although culture-based identification remains the gold standard, slow mycobacterial growth delays diagnosis and treatment. In this study, we evaluated a novel culture-free method for subspecies-level identification directly from sputum. In this single-center prospective cohort study at Osaka Toneyama Medical Center, we analyzed 125 sputum samples from 115 patients with NTM pulmonary disease and 10 with non-NTM respiratory conditions. Samples were decontaminated using N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) or succinic acid. We compared the reference culture method (mycobacterial culture plus whole-genome sequencing) and a culture-free direct target capture sequencing method. Core genome multi-locus sequence typing identified subspecies in both workflows, covering 186 mycobacterial species, including M. tuberculosis. The 115 NTM cohort specimens yielded 57 smear-positive and 93 culture-positive results. The identified subspecies included 48 Mycobacterium avium subsp. hominissuis, 22 Mycobacterium intracellulare subsp. intracellulare, 5 subsp. chimaera, 7 Mycobacterium abscessus subsp. abscessus, 5 subsp. massiliense, 1 M. tuberculosis, and 5 other NTM species. The culture-free method showed a high identification rate for smear-positive specimens (75.4%) but a low identification rate for smear-negative specimens (13.9%). NALC-NaOH pretreatment resulted in higher accuracy (90.5%) than did succinic acid pretreatment (66.7%). Thus, our culture-free subspecies-level identification method achieved high accuracy, especially in alkaline-treated smear-positive sputum samples, achieving rates above 90%. This method is recommended in clinical practice for patients who require rapid diagnosis and timely initiation of appropriate treatment, bypassing time-consuming culture steps.IMPORTANCEAccurate identification of Mycobacterium species and subspecies is crucial for effective treatment, as drug susceptibility and clinical outcomes vary significantly among them. However, conventional diagnosis relies on culture-based methods that can take several weeks, critically delaying appropriate therapy. This study validates a novel culture-free method using target capture sequencing for the comprehensive, subspecies-level identification of over 186 mycobacterial species directly from sputum specimens. Our findings revealed the high accuracy of this approach for smear-positive specimens, especially with alkaline pretreatment. This rapid method is applicable in clinical settings and enables timely and precise treatment decisions, greatly benefiting patients who require urgent intervention.

Humans↗
PubMed1979

Evaluation of Micro-Media Quad Panels for identification of the Enterobacteriaceae.

The Micro-Media Quad Enteric Panel and coding system were evaluated by comparing the identifications of 193 stock and 216 clinical isolates, representing 31 species of Enterobacteriaceae, with those provided by conventional methods. The results corresponded for 91% of the organisms if only the single or most probable identification was accepted and for 93% if the correct identification was listed as one of several possible, but not the most probable, identification by the coding system. With 73% of the isolates, the single identification provided by the coding system was correct, and in 20% of instances the correct identification was listed as the first of several choices in descending order of probability but requiring additional tests for differentiation. In most of the latter instances, these additional tests consisted of serological confirmation of salmonellae or shigellae. Discrepancies consisted of incorrect identifications (4%) and organisms with code numbers not appearing in the coding system (3%).

Bacteriological Techniques↗
PubMed1979

Rapid identification of Enterobacteriaceae from blood cultures with the Micro-ID system.

Micro-ID is a new test system designed to identify members of the family Enterobacteriaceae in 4 h. It consists of 15 biochemical tests on reagent-impregnated paper disks; each test is in its own compartment in a molded plastic tray. Based on the pattern of positive and negative biochemical reactions, a five-digit octal code number is calculated. A computer-generated identification manual accompanies the product, and for each octal code listed there is a numerical value that represents the unknown isolate's degree of fit to a typical organism (LFR), a second numerical value that represents its separation from other organisms (PNOR), and a verbal description of the quality of identification. Only one reagent is added to the system. Manufacturer's directions were modified in this laboratory to allow identification from a turbid blood culture bottle within 4 h. Based on 330 routine clinical cultures tested, there was a 96.1% agreement with conventional identification to the genus and species level: 1.2% yielded first two choices possible, with one being the correct choice; 1.2% provided a correct genus, but no species identification; and 1.5% produced an incorrect identification. The Micro-ID is an accurate, facile system for the rapid identification of Enterobacteriaceae from blood cultures.

Bacteriological Techniques↗
PubMed1982

Rapid automated identification of gram-negative bacilli from blood cultures with the AutoMicrobic system.

Automated identification of gram-negative bacilli directly from blood culture bottles by using the AutoMicrobic System (AMS) was evaluated with a modified procedure for the AMS Enterobacteriaceae-plus nonfermenter identification card. A total of 150 strains were tested (44 clinical and 106 seeded) and compared with a conventional identification procedure. These strains included 107 Enterobacteriaceae and 43 oxidase-positive or glucose-nonfermenting, or both, organisms. AMS identifications on one of these strains were not interpretable owing to equal probability AMS identification values. Of the remaining 149 strains, 138 (92.6%) were correctly identified within 8 to 13 h of the first reading. Of 69 identifications analyzed after 6 h of incubation, 91% were correct. This procedure was found to be rapid, convenient, and nonlabor intensive and is recommended for presumptive identification of gram-negative bacilli in blood cultures.

Bacteria↗
PubMed1983

Comparison of three methods for anaerobe identification.

In this study we evaluated the ability of three commercial methods, API 20A (Analytab Products, Plainview, N.Y.), Minitek (BBL Microbiology Systems, Cockeysville, Md.), and Anaerobe-Tek (Flow Laboratories, Inc., McLean, Va.), to accurately identify 165 recent clinical and 38 stock isolates of anaerobic bacteria without supplemental tests or gas-liquid chromatography. Strains included 89 Bacteroides spp., 12 fusobacteria, 10 gram-positive, nonsporing rods, 43 Clostridium spp., 15 Streptococcus intermedius, 18 peptococci, 6 peptostreptococci, 3 Staphylococcus saccharolyticus, and 7 Veillonella spp. The methods used were those of manufacturers, without supplemental tests. API 20A correctly identified 70.0% of strains to species and 6.4% to genus only, with 17.2% as part of a spectrum of identifications and 6.4% incorrect. Minitek, according to the current code book, yielded 69.5% correct identifications to species, 16.8% spectrum identifications, and 13.8% incorrect. Anaerobe-Tek correctly identified 64.0% of strains to species, 21.2% spectrum identifications, and 14.8% incorrect. Thirteen strains were misidentified by API 20A, 28 were misidentified by Minitek, and 30 were misidentified by Anaerobe-Tek. For laboratories without gas-liquid chromatography support and where identification of clinically significant Bacteroides fragilis and Clostridium perfringens is desired, any of the three systems would provide accurate information. For more extensive anaerobe identification, including the less frequently isolated, more unusual organisms, API 20A and Minitek are preferred at this time. All systems have identification schemes associated with a percentage of misidentifications, the most recently introduced Anaerobe-Tek system being associated with the highest error rate.

Bacteria, Anaerobic↗
PubMed1984

Diagnostic probability matrix for identification of slowly growing mycobacteria in clinical laboratories.

A probability matrix is presented for identification of slowly growing mycobacteria that are likely to be encountered in clinical laboratories. The matrix includes 23 features that are useful for identifying members of 14 species or species complexes. The computer program identifies strains as a function of the ID (identification) score, which measures the discrimination among possible alternative identifications, and the R (ratio) score, which measures the degree of fit to the most likely taxa. It is not necessary to employ all 23 tests when initiating an identification; the program will suggest additional tests to perform when a partial data set fails to yield a definitive identification. Two independent sets of cultures comprising a total of 1,212 strains were used to test the matrix. Correct diagnoses were based on clustering behavior in numerical taxonomic analysis with larger numbers of features. The probable efficiencies with the two sets were 94.2 and 83.4%, respectively, and the accuracy of the definitive identifications for both sets exceeded 95%. A discussion is presented of situations when it may be appropriate to override an R score that has caused the rejection of an identification and to thereby enhance the efficiency.

Catalase↗