Software for the development and evaluation of probabilistic identification matrices

A number of programs are described for the development and evaluationof probabilistic identification matrices for use with computer-assistedidentification. The program BEST reads an initial matrix ofper cent probabilities for all the binary characters examinedduring a cluster analysis and determines the most useful setof tests for distinguishing taxa in the matrix. Program RESORTcreates from the initial matrix an identification matrix inwhich the order of the tests may be different or the numberof tests reduced. A printed version of a matrix can be producedby MATPRJNT, which creates tables giving the per cent probabilityof a positive test result and the test results presented as‘–’, ‘v’ and ‘+’ dependingon a user-specified threshold. Program IDSC evaluates an identificationmatrix by calculating the best identification score for eachtaxon in the matrix using the expected result for each test.The programs were written in FORTRAN 77 and can be run on anymicrocomputer using the PC/MS-DOS operating system. Received on April 27, 1990; accepted on November 5, 1990     

Characteristics of maltose transporter activity in an ale and larger strain of the yeast Saccharomyces cerevisiae

R.R. BEUMER, M.C. TE GIFFEL, M.T.C. KOK AND F.M. ROMBOUTS. 1996. All confirmation and identification methods used in this study can be used for the screening of suspected colonies on isolation media for Listeria spp. In traditional enrichment procedures the Microscreen Listeria latex test gives fast results. The DNA probes (Accuprobe and Gene-Trak) are very specific in detecting Listeria monocytogenes . For identification of Listeria spp. both tests (API and Micro-ID) performed equally well. Preference may be given to the API test, since differentiation of L. monocytogenes from L. innocua is based on the absence of arylamidase, through which tests for haemolytic activity and/or CAMP reactions can be omitted. However, the use of Enhanced Haemolysis Agar as isolation medium makes further testing essentially superfluous, since L. monocytogenes strains can be differentiated from L. innocua .     

Bacterial contaminants of micropropagated plant cultures

Bacterial contaminants of micropropagated plant cultures were isolated and characterized with standard bacteriological tests and appropriate API strips. Results obtained were analysed by the API identification software. Of 198 bacterial strains isolated from nine plant species, 90% were identified as Bacillus, Enterobacter, Micrococcus, Staphylococcus, Pseudomonas or Lactobacillus species. Possible sources of contamination are discussed.     

Mathematical Analysis of the API Enteric 20 Profile Register Using a Computer Diagnostic Model

Results for 21 biochemical tests using the API-20 Enteric kit were obtained from the manufacturer's files for 27,820 bacterial isolates. These isolates were identified by the API Profile Register and also by a computer diagnostic model which estimates the relative likelihoods of various identifications. The computer confirmed the identification in the API Profile Register for 99.36% of the isolates. The manufacturer has reviewed areas of the API Profile Register questioned by the computer analysis; a number of resulting modifications to the API Profile Register have been incorporated in an update letter. This computer model provides a convenient and powerful way to interpret a large number of test results for bacterial identification. This study also demonstrates the use of a large collection of isolates to refine the data matrix used by the diagnostic model.     

Isolation of Bacillus strains from the rhizosphere of cereals and in vitro screening for antagonism against phytopathogenic, food-borne pathogenic and spoilage micro-organisms

Bacillus strains were isolated from the rhizosphere of cereals in order to be used as natural biocontrol agents (BCAs). They were screened for antagonism in vitro against various test micro-organisms. The isolates showing antagonism were identified to species level. A combination of techniques was employed for the isolation of Bacillus species. Using the direct method, only one of the 25 isolates screened showed antagonistic properties. This strain (IFS-01) was identified by means of API test strips and the ATB Plus computer programme. It proved to be Bacillus subtilis and consequently has been designated as Bacillus subtilis IFS-01. This strain produced either a broad spectrum antimicrobial compound or several compounds with different activities. The fungi and Gram-positive bacteria were more sensitive to the antagonistic isolate than the Gram-negative bacteria. A Bacillus strain producing BCAs which can be used as biopesticides or organic preservatives has been isolated and identified.     

突发疫情中炭疽芽孢杆菌的分离及鉴定

对疑似炭疽感染病牛牛肉标本和牛血污染土壤标本进行了病原菌分离,经菌落形态和菌体形态观察、血清学实验和生化鉴定,证明分离到的细菌为炭疽芽孢杆菌。为进一步了解其特性,分别用保护性抗原、水肿因子和荚膜基因特异性引物对2株菌进行PCR扩增。结果显示,这两株菌有两个毒力相关质粒pX01和pX02,为有毒株。序列测定表明,这两株菌基因间同源性达99%,这两株菌与GenBank中炭疽芽孢杆菌A2012株、Ames Ancestor株和A16R疫苗株同源性达99%。     

Comparison of rapid NFT and API 20E with conventional methods for identification of gram-negative nonfermentative bacilli from pharmaceuticals and cosmetics.

The accuracy of the Rapid NFT and the API 20E identification systems was evaluated by comparing them with conventional biochemical methods for the identification of gram-negative, nonfermentative bacilli. The organisms were recovered from preserved, nonsterile pharmaceutical and cosmetic products. A total of 123 test isolates that are commonly encountered in these products were used. By using the criteria of accurate and reliable identification without employing additional tests, Rapid NFT was found to be more accurate after 48 h of incubation than API 20E for characterizing isolates to the species level. Therefore, close agreement between NFT and conventional methods for identification of industrial gram-negative isolates provides evidence that the Rapid NFT system is an improved and rapid method for identifying these organisms to the species level with minimal use of supplementary tests.     

Comparison of rapid NFT and API 20E with conventional methods for identification of gram-negative nonfermentative bacilli from pharmaceuticals and cosmetics

The accuracy of the Rapid NFT and the API 20E identification systems was evaluated by comparing them with conventional biochemical methods for the identification of gram-negative, nonfermentative bacilli. The organisms were recovered from preserved, nonsterile pharmaceutical and cosmetic products. A total of 123 test isolates that are commonly encountered in these products were used. By using the criteria of accurate and reliable identification without employing additional tests, Rapid NFT was found to be more accurate after 48 h of incubation than API 20E for characterizing isolates to the species level. Therefore, close agreement between NFT and conventional methods for identification of industrial gram-negative isolates provides evidence that the Rapid NFT system is an improved and rapid method for identifying these organisms to the species level with minimal use of supplementary tests.     

Identification of enterococci isolated from cow's milk cheese: comparison of the classical methods and the API 20 STREP system (technical note)

A comparison of the results obtained using the classical methods with those of the API 20 Strep system was carried out in identifying 24 enterococci strains isolated from San Simón cow's milk cheese, a traditional Spanish variety. The results of both identification systems coincided exactly in 9 strains (37.5% of the strains studied). In one strain the results obtained using the classical methods did not coincide with those using the API 20 Strep method. 3 strains (12.5%) could not be identified using the API 20 Strep system. However, 11 strains (45%), that remained doubtful between both species E. faecalis and E. faecium on the basis of the classical methods, were identified using the API 20 Strep system. The API 20 Strep system does not include some biochemical tests of importance in identifying of foodborne enterococci and could not identify the atypical strains of Enterococcus. Moreover, this system is adapted to the identification of enterococci of clinical origin and their database does not include some species common in foods. However, it could have an application in combination with the classical methods in order to carry out a reasonably rapid and reliable identification of enterococci related to cheese.     

Identification of streptococci in a medical laboratory

A total of 965 cultures of streptococci received at a reference unit for identification were examined with API-20 Strep kits and also by established methods. The API method, although it needed to be supplemented with additional tests, largely overcame the difficulty that pyogenic streptococci are usually identified by their serological reactions and that biochemical tests are used for the identification of the other streptococci. Representatives of at least 24 established or possible species were identified.     

Identification of streptococci in a medical laboratory

A total of 965 cultures of streptococci received at a reference unit for identification were examined with API-20 Strep kits and also by established methods. The API method, although it needed to be supplemented with additional tests, largely overcame the difficulty that pyogenic streptococci are usually identified by their serological reactions and that biochemical tests are used for the identification of the other streptococci. Representatives of at least 24 established or possible species were identified.     

Evaluation of the MicroScan enzyme-based system for the identification of foodborne yeasts

Eighty-nine strains representing 36 species of foodborne yeasts isolated from fruit juice concentrates were identified using the Baxter MicroScan enzyme-based kit, conventional tests according to a simplified identification method (SIM), and the API 20C kit. Of the 15 test species included in the MicroScan database, only 40% were correctly identified; 13% gave scores of unacceptably low probabilities, 20% were misidentified, and 27% could not be identified. Of the 21 test species not in the MicroScan database, 38% were misidentified and 62% produced biocodes with between-species differences not larger than differences between strains within species. The reliability of the MicroScan enzyme-based system is questioned, in that different results were sometimes obtained upon retesting the same strains. The MicroScan enzyme-based system is rapid, providing results within 4 h. However, because of its restricted and specific database and unreliability, the system appears to be unsuited for the identification of foodborne yeasts.     

Use of the API rapid NFT system for identifying nonfermentative and fermentative marine bacteria.

Thirty-five American Type Culture Collection type strains of marine bacteria were used to evaluate the Rapid NFT system (API Analab Products, Plainview, N.Y.) for use in identifying heterotrophic marine bacteria. The 21 biochemical and assimilation tests on the Rapid NFT test strips were treated according to the manufacturer's protocol, which included use of AUX medium (provided with the Rapid NFT system) for preparing assimilation tests, and by substituting phenol red broth base (BBL Microbiology Systems, Cockeysville, Md.) with and without an oil overlay for the AUX medium. A seven-digit numerical profile was obtained for each NFT test strip from each of the three procedures and matched to its corresponding number in the Rapid NFT identification codebook. Also, all biochemical and assimilation test results were analyzed with SASTAXAN and SAS/GRAPH programs (SAS Institute, Inc., Cary, N.C.); similarity matrices were computed for all 35 strains. For comparison purposes, bacterial strains were grouped at a similarity level of 70%. The results indicated a low efficacy of identification for all three procedures. In addition, similarity matrix analysis showed more cohesive grouping based on results of phenol red broth base-treated strains than for the AUX medium provided by the manufacturer. However, none of the three treatments provided exclusive grouping of type strains at the genus level. Thus, the reliability of the data obtained from the NFT system and modifications thereof should be evaluated carefully when environmental isolates are characterized.     

Use of the API rapid NFT system for identifying nonfermentative and fermentative marine bacteria.

Thirty-five American Type Culture Collection type strains of marine bacteria were used to evaluate the Rapid NFT system (API Analab Products, Plainview, N.Y.) for use in identifying heterotrophic marine bacteria. The 21 biochemical and assimilation tests on the Rapid NFT test strips were treated according to the manufacturer's protocol, which included use of AUX medium (provided with the Rapid NFT system) for preparing assimilation tests, and by substituting phenol red broth base (BBL Microbiology Systems, Cockeysville, Md.) with and without an oil overlay for the AUX medium. A seven-digit numerical profile was obtained for each NFT test strip from each of the three procedures and matched to its corresponding number in the Rapid NFT identification codebook. Also, all biochemical and assimilation test results were analyzed with SASTAXAN and SAS/GRAPH programs (SAS Institute, Inc., Cary, N.C.); similarity matrices were computed for all 35 strains. For comparison purposes, bacterial strains were grouped at a similarity level of 70%. The results indicated a low efficacy of identification for all three procedures. In addition, similarity matrix analysis showed more cohesive grouping based on results of phenol red broth base-treated strains than for the AUX medium provided by the manufacturer. However, none of the three treatments provided exclusive grouping of type strains at the genus level. Thus, the reliability of the data obtained from the NFT system and modifications thereof should be evaluated carefully when environmental isolates are characterized.     

Numerical taxonomy of alpha-amylase producing Bacillus species

A total of 134 alpha-amylase producing Bacillus isolates and 21 reference strains were divided into 12 groups according to their similarities (% SSM). Phenotypic characteristics determined by the API 20E and API 50CHB galleries, other biochemical tests and morphological characteristics were used for the numerical analysis. The API Computer Service identified 45% of the isolates. The amylase yields of 16 alpha-amylase hyperproducing (AHP) isolates were compared with those of seven amylolytic reference and type strains. The AHP isolates were related to Bacillus subtilis, B. licheniformis and 'B. amyloliquefaciens'.     

Application of current methods for isolation and identification of staphylococci in raw bovine milk

Samples of raw milk were examined for counts of somatic cells, total viable bacteria, staphylococci (Schleifer & Kramer's medium) and Staphylococcus aureus (Baird-Parker medium, Baird-Parker medium with pig plasma and Baird-Parker medium with additional antibiotics). For the isolation of staphylococci from raw milk, Schleifer & Kramer's medium was found to be very selective and in general performed satisfactorily. From the results obtained with the three remaining media the continued use of Baird-Parker medium for isolation of Staph. aureus from raw milk is recommended with the proviso that colonies selected for identification should include those that clear and do not clear the egg yolk and are not limited to colonies with diameters greater than 1 mm. Staphylococci isolated from raw milk were identified by key tests using a multipoint inoculation procedure. A selected number were also examined by the API STAPH system in conjunction with the API LAB computer programme for identification of staphylococci. Of the staphylococci examined, 90.0% were identified using the multipoint procedure. For strains identified as Staph. aureus, Staph. hyicus subsp. hyicus, Staph. epidermidis, Staph. simulans, Staph. xylosus or members of the Staph. hominis/Staph. warneri/Staph haemolyticus group, the API system provided confirmatory evidence. With strains identified by the multipoint procedure as Staph. hyicus subsp. chromogenes, Staph. sciuri subsp. sciuri and Staph. sciuri subsp. lentus the API system did not always provide concurring results. Several strains which could not be identified by the multipoint procedure could be identified by the API system. Staph. aureus, Staph. hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains isolated from milk were examined for production of enterotoxin A-E. Only 3.9% of Staph. aureus strains examined produced detectable enterotoxin (type C). None of the Staph. hyicus subsp. hyicus or Staph. hyicus subsp. chromogenes strains produced any of the known enterotoxins.     

Routine procedures for isolation and identification of enterococci and fecal streptococci.

Over the past 6 years, a revised classification of the streptococci and enterococci, based primarily on molecular techniques such as 16S rRNA sequencing and DNA-DNA hybridization, emerged. However, little attention was placed on routine physiological tests that could be used in food and clinical laboratories to differentiate between species of a new genus, Enterococcus, and fecal Streptococcus spp. The purpose of this study was to devise a convenient and reliable system to identify enterococci and fecal streptococci by using conventional procedures. Fifty-nine strains of 13 Enterococcus spp., including the type strains and many strains used by previous investigators, were characterized by using conventional tube tests, the API Rapid Strep system, and MicroScan Pos ID panels. Results were compared with each other and with previously published results. A comparison of conventional tube tests versus published tube test results yielded 17 discrepancies. Although not all tests were done with each of the three systems, 28 discrepancies between results obtained with the API system and those obtained with conventional tube tests were found. There were 24 discrepancies between results obtained with the MicroScan Pos ID panel and those obtained with conventional tube tests. There were 12 discrepancies between the results with the API Rapid Strep system and those with the MicroScan Pos ID panels. We devised flow charts of key tests that might be used to identify cultures without resorting to nucleic acid analysis and other labor- and equipment-intensive analyses.     

Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System,and Antibiotic Sensitivity Profiling

Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes.     

A Simple and Rapid Computer-assisted Technique for the Identification of some Selected Bacillus Species using Biochemical Tests

A computer-assisted probabilistic identification technique was developed to identify species of the genus Bacillus known to be potentially pathogenic and/or frequently found as contaminants in pharmaceutical preparations. An identification matrix on 18 species of the genus Bacillus was constructed. Twenty-four biochemical tests were used. The reaction profile of each species was quantified in probability terms and called up on a computer. The computer method was used on 30 test cultures. In each case, the most likely identification, according to the computer method, was the species to which the strain had already been assigned. In 29 of the 30 cases, the identification was with a probability level of one; in seven of these, however, more than one species tied for first choice. Only one strain was identified with a lower level of probability. The technique developed is comparatively easy to use and would seem to constitute a useful diagnostic tool within the pharmaceutical field.     

Numerical taxonomy of α-amylase producing Bacillus species

A total of 134 α-amylase producing Bacillus isolates and 21 reference strains were divided into 12 groups according to their similarities (% S_SM). Phenotypic characteristics determined by the API 20E and API 50CHB galleries, other biochemical tests and morphological characteristics were used for the numerical analysis. The API Computer Service identified 45% of the isolates. The amylase yields of 16 α-amylase hyperproducing (AHP) isolates were compared with those of seven amylolytic reference and type strains. The AHP isolates were related to Bacillus subtilis, B. licheniformis and 'B. amyloliquefaciens' .