Identification of aquatic Burkholderia (Pseudomonas) cepacia by hybridization with species-specific rRNA gene probes.

Burkholderia (Pseudomonas) cepacia is a common environmental bacterium which can be pathogenic for plants and humans. In this study, four strategies were used to identify aquatic isolates: API test strips, hybridization with species-specific DNA probes for the 16S and 23S rRNA genes, fatty acid methyl ester (FAME) profiles, and growth on selective medium (TB-T agar [C. Hagedorn, W. D. Gould, T. R. Bardinelli, and D. R. Gustarson, Appl. Environ. Microbiol. 53:2265-2268, 1987]). Only 59% of the isolates identified as B. cepacia with the API test strips were confirmed as B. cepacia by using fatty acid profiles. The 23S rRNA probe generated a few false-positive results but dramatically underestimated the number of B. cepacia isolates (i.e., 40% of the colonies that did not hybridize to the probe were B. cepacia, as determined by FAME). The 16S rRNA probe generated more false-positive results than the 23S rRNA probe but was effective in identifying the majority of the B. cepacia isolates. The selective medium was only partially successful in recovering B. cepacia. Use of the B. cepacia-specific 16S rRNA probe was the most efficient and accurate way of identifying B. cepacia.     

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.     

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.     

Evaluation of Colilert-18 for detection of coliforms and Eschericha coli in subtropical freshwater

The accuracy of Colilert-18 as a test for coliforms and Escherichia coli in subtropical freshwater was evaluated by using API 20E strips and fatty acid methyl ester analysis. The false-positive and -negative rates of detection were 7.4 and 3.5%, respectively, for E. coli and 9.6 and 6.3%, respectively, for coliforms.     

Evaluation of Colilert-18 for Detection of Coliforms and Eschericha coli in Subtropical Freshwater

The accuracy of Colilert-18 as a test for coliforms and Escherichia coli in subtropical freshwater was evaluated by using API 20E strips and fatty acid methyl ester analysis. The false-positive and -negative rates of detection were 7.4 and 3.5%, respectively, for E. coli and 9.6 and 6.3%, respectively, for coliforms.     

A Taxonomic Study of the Micrococcaceae

Recent proposals for new species in the genera Staphylococcus and Micrococcus prompted the present numerical taxonomic survey. The API 50E and API 20E identification strips have been used together with a few conventional tests to examine the Micrococcaceae. Many of the proposed species formed good distinct clusters and one group could be the basis for a new genus.     

Evaluation of Colilert-18 for the detection of coliforms and Escherichia coli in tropical fresh water

AIMS: To evaluate the suitability of Colilert-18 in detecting Escherichia coli and total coliforms in tropical freshwater samples. METHODS AND RESULTS: Target organisms were isolated from yellow-fluorescent and yellow wells of Colilert-18/Quanti-Tray using m-TEC agar and m-ENDO LES agar respectively. All the selected isolates were first identified based on their fatty acid methyl ester profile. Isolates showing contradictory results to that of the Colilert-18 procedure were re-identified using API 20E strips. A total of 357 isolates, 177 from yellow-fluorescent wells and 180 from yellow wells, were identified. CONCLUSIONS: The false-positive and -negative rates for E. coli detection using Colilert-18 were 36.4% and 11%, respectively, while for coliform detection the false-positive rate was 10.3%. SIGNIFICANCE AND IMPACT OF THE STUDY: The high false-positive rate of Colilert-18, tempers its value for E. coli detection when used for tropical freshwater samples.     

Enumeration of Obesumbacterium proteus in brewery yeasts and characterization of isolated strains using Biolog GN microplates and protein fingerprinting

Of a range of media tested for enumeration of Obesumbacterium proteus in brewers' yeast, Universal Beer agar and Wallerstein Laboratories Differential medium were most effective. MacConkey agar (several types) and Membrane Lauryl Sulphate agar were least effective. Other media (Wort agar, YM agar) were of intermediate efficacy. Nine O. proteus strains from commercial yeast samples were characterized using the API 20E test kit, the Biolog GN microplate (BGNM) and by SDS-PAGE of their total soluble proteins. Both the BGNM and SDS-PAGE techniques allowed the strains to be differentiated from one another: the API 20E kit did not. All strains isolated from UK breweries belonged to O. proteus biogroup II. Four of these strains displayed a branching cell morphology not hitherto described in any member of the Enterobacteriaceae.     

Comparison of biochemical and molecular methods for the identification of bacterial isolates associated with failed loggerhead sea turtle eggs

Aims: Comparison of biochemical vs molecular methods for identification of microbial populations associated with failed loggerhead turtle eggs. Methods and Results: Two biochemical (API and Microgen) and one molecular methods (16s rRNA analysis) were compared in the areas of cost, identification, corroboration of data with other methods, ease of use, resources and software. The molecular method was costly and identified only 66% of the isolates tested compared with 74% for API. A 74% discrepancy in identifications occurred between API and 16s rRNA analysis. The two biochemical methods were comparable in cost, but Microgen was easier to use and yielded the lowest discrepancy among identifications (29%) when compared with both API 20 enteric (API 20E) and API 20 nonenteric (API 20NE) combined. A comparison of API 20E and API 20NE indicated an 83% discrepancy between the two methods. Conclusions: The Microgen identification system appears to be better suited than API or 16s rRNA analysis for identification of environmental isolates associated with failed loggerhead eggs. Significance and Impact of the Study: Most identification methods are not intended for use with environmental isolates. A comparison of identification systems would provide better options for identifying environmental bacteria for ecological studies.     

Porcine haemophili and actinobacilli: characterization by means of API test strips and possible taxonomic implications

Thirty Haemophilus strains and six Actinobacillus strains, all of porcine origin, were examined for their biochemical reactivity on API 20E and API ZYM test strips using dense cell suspensions (supplemented with NAD as appropriate) as strip inocula. When combined with a test for V-factor dependency, the use of both strips allowed adequate differentiation of closely related organisms. Numerical taxonomic analysis of the data demonstrated that the majority of the haemophili and actinobacilli studied could be placed in one of four major clusters; these clusters contained, respectively, the H. pleuropneumoniae--A. pleuropneumoniae strains, the H. parasuis strains, strains belonging to Haemophilus taxon "minor group," and strains belonging to an unusual group of mannitol-positive, urease-negative haemophili. A representative of Haemophilus species taxon C and an unusual Actinobacillus isolate appeared to be comparatively unrelated to organisms in the four major clusters. Although it may, on occasion, be difficult to place an unusual isolate in any one particular group, owing to the uncertain taxonomy of some of these organisms, it is concluded that API test strips can serve as useful tools for the characterization and differentiation of porcine haemophili and actinobacilli.     

Comparison of a commercial biochemical kit and an oligonucleotide probe for identification of environmental isolates of Vibrio vulnificus

A. DALSGAARD, I. DALSGAARD, L. HØI AND J.L. LARSEN. 1996. Methods for the identification and isolation of environmental isolates of Vibrio vulnificus were evaluated. Alkaline peptone water supplemented with polymyxin B and colistin-polymyxin B-cellobiose agar were employed for the isolation of suspected V. vulnificus from water, sediment and shellfish samples. When comparing the identification of putative V. vulnificus obtained with the API 20E assay and an oligonucleotide probe, 29 API 20E profiles were obtained with only four profiles (representing 20 isolates) reaching the identification threshold of V. vulnificus among a total of 66 isolates hybridizing with the probe. The results indicated that, compared with colony hybridization, the API 20E assay was not adequate for the identification of environmental isolates of V. vulnificus .     

Fatty acid methyl ester (FAME) profiles as measures of soil microbial community structure

Analysis of fatty acid methyl ester (FAME) profiles extracted from soils is a rapid and inexpensive procedure that holds great promise in describing soil microbial community structure without traditional reliance on selective culturing, which seems to severely underestimate community diversity. Interpretation of FAME profiles from environmental samples can be difficult because many fatty acids are common to different microorganisms and many fatty acids are extracted from each soil sample. We used principal components (PCA) and cluster analyses to identify similarities and differences among soil microbial communities described using FAME profiles. We also used PCA to identify particular FAMEs that characterized soil sample clusters. Fatty acids that are found only or primarily in particular microbial taxa-marker fatty acids-were used in conjunction with these analyses. We found that the majority of 162 soil samples taken from a conventionally-tilled corn field had similar FAME profiles but that about 20% of samples seemed to have relatively low, and that about 10% had relatively high, bacterial:fungal ratios. Using semivariance analysis we identified 21:0 iso as a new marker fatty acid. Concurrent use of geostatistical and FAME analyses may be a powerful means of revealing other potential marker FAMEs. When microbial communities from the same samples were cultured on R2A agar and their FAME profiles analyzed, there were many differences between FAME profiles of soil and plated communities, indicating that profiles of FAMEs extracted from soil reveal portions of the microbial community not culturable on R2A. When subjected to PCA, however, a small number of plated communities were found to be distinct due to some of the same profile characteristics (high in 12:0 iso, 15:0 and 17:1 ante A) that identified soil community FAME profiles as distinct. Semivariance analysis indicated that spatial distributions of soil microbial populations are maintained in a portion of the microbial community that is selected on laboratory media. These similarities between whole soil and plated community FAME profiles suggest that plated communities are not solely the result of selection by the growth medium, but reflect the distribution, in situ, of the dominant, culturable soil microbial populations.     

Comparison of culture-based methods to enumerate Escherichia coli in tropical and temperate freshwaters

Aims: The specificity of a method for the enumeration of Escherichia coli (chromocult agar, CC) was tested using freshwater samples from a tropical area (Cuba Island) by isolating colonies and identifying them with API (Appareillage et procédé d’identification) strips. Enumerations of E. coli by the most probable number (MPN) microplate method were compared with counts on chromogenic and fluorogenic agar media [CC, rapid E. coli (REC), fluorocult] in tropical and temperate freshwater samples. Methods and Results: A high percentage of specificity (95·7%) for the CC agar enumeration of E. coli was observed. High regression coefficients (log‐log linear regressions) were found between E. coli counts on agar media and by the MPN method. In the tropical environment, counts with REC medium were significantly different from those obtained with the other methods. MPN counts were found to be significantly higher than those obtained using the plate counts methods in the temperate environment. Conclusions: Escherichia coli enumeration methods based on glucuronidase activity appear to be suitable for the evaluation of microbiological quality in the tropical environment featured in this study. Significance and Impact of the Study: The methods for the enumeration of E. coli tested in this study should help improve the evaluation of microbiological contamination of Cuban freshwaters.     

Changes in Bacteria Recoverable from Subsurface Volcanic Rock Samples during Storage at 4°C

The abundance of viable microorganisms recovered from deep subsurface volcanic rock samples increased after rock perturbation and storage for 1 week at 4°C, while the diversity and evenness of recoverable heterotrophic bacterial communities generally decreased. One sample of each morphologically distinct colony type, recovered both before and after storage of U12n rock samples, was purified and characterized by fatty acid methyl ester (MIDI) and API rapid NFT strips. As determined by MIDI cluster analysis, the composition of the recoverable microbial communities changed with storage of rock samples; some groups of organisms were recovered only before, only after, or at both sample times. In general, the isolates recovered only after storage of rock samples had a greater ability to utilize the carbohydrates included in API test strips and had faster generation times than isolates recovered only on initial plating. The nutritional versatility and faster growth rates of organisms recovered in higher proportions after sample storage provide evidence that some microbial community changes may be due to the proliferation of a few bacterial types. However, because some new genera are recovered only after storage, the possibility also exists that dormant bacterial types are resuscitated during sample perturbation and storage.     

Differences in the API 20E biochemical patterns of clinical and environmental Vibrio parahaemolyticus isolates

Genetic differences in clinical and environmental strains of Vibrio parahaemolyticus have been widely used as criteria in identifying pathogenic isolates. However, few studies have been carried out to assess the differences in biochemical characteristics of V. parahaemolyticus isolates from human and environmental sources. We compared the biochemical profiles obtained by the characterization of V. parahaemolyticus isolates from human infections and the marine environment using the API 20E system. Environmental and clinical isolates showed significant differences in the gelatin and arabinose tests. Additionally, clinical isolates were correctly identified according to the API 20E profile using 0.85% NaCl diluent, but they presented nonspecific profiles with 2% NaCl diluent. In contrast, use of 2% NaCl diluent facilitated correct identification of the environmental isolates. Clinical isolates showed significant differences in up to five biochemical tests with respect to the API 20E database. The API 20E system is widely used in routine identification of bacteria in clinical laboratories, and this discrepancy in an important number of biochemical tests may lead to misidentification of V. parahaemolyticus infection.     

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'.     

Diluent composition for use of API 20E in characterizing marine and estuarine bacteria.

Nine chemically defined inoculation diluents, with compositions ranging from 0.85% NaCl to 35% marine salts, were used to evaluate the influence of diluent composition on the biochemical profiles of 30 marine and estuarine bacterial strains, including species of Vibrio, Aeromonas, Allomonas, and Photobacterium. Results demonstrated that a 20% marine salts diluent enabled the characterization of halophilic strains normally nonreactive by the API 20E system. Furthermore, the use of 20% marine salts showed that certain environmental isolates, identifiable as Vibrio parahaemolyticus by the recommended clinical inoculation procedure, were Vibrio vulnificus. An analysis of the profiles provided by the nine diluents indicates that the API 20E system, modified by the use of a diluent composed of 20% marine salts and incubated at 22 degrees C, can provide a reliable tool for the rapid characterization of marine and estuarine bacterial isolates.     

Spatial and Temporal Variation of Enterobacter Genotypes in Sediments and the Underlying Hyporheic Zone of an Agricultural Stream

Population studies of enteric bacteria in an agriculturally impacted stream (Ledbetter Creek, Murray, Kentucky, USA) were conducted over a period of 2 years. Total number of bacteria, cultivated heterotrophic aerobic bacteria, and enteric bacteria showed significant differences between winter and summer. The cultivated numbers of heterotrophic aerobic bacteria and enteric bacteria were significantly more abundant in summer than in winter. The abundance of enteric bacteria was 12.9% in an upwelling zone and 9.8% in a downwelling zone in summer. Most of the enteric bacterial strains isolated on MacConkey agar were assigned to Enterobacter cloacae and E. agglomerans by API 20E and an analysis of the restriction patterns produced by amplified DNA coding for 16S rRNA (ARDRA) with the enzyme Hpa II. E. cloacae and E. agglomerans genotypes isolated from three hyporheic and gravel bar depth intervals (0-10 cm, 15-25 cm, and 30-40 cm) in summer and fall showed significant spatial variation and were heterogeneously distributed along the stream. Temperature, inorganic nutrients, and occurrence of anoxic zones affected the distribution of enteric bacteria. These techniques can be used as a model to monitor shifts among different species in the stream ecosystem.