API 20E System Research Articles

Evaluation of a new identification system, Crystal Enteric/Non-Fermenter, for gram-negative bacilli

A total of 505 fermentative and 201 nonfermentative gram-negative bacilli, identified by conventional methods, were tested by the Crystal Enteric/Non-Fermenter ID kit and by the API 20E or API 20NE identification system. The overall correct results for fermenters were 92.9% by the Crystal kit and 89.1% by the API 20E system. The false identifications (genus and species incorrect) accounted for 3.1 and 7.1% for the Crystal and API systems, respectively. For nonfermenters, figures for correct identifications by the two systems were comparable (Crystal, 75.9%; API 20NE, 75.3%) while the API 20NE system gave twice as many incorrect results (13.8%) as Crystal (6.3%); however, Crystal failed to precisely identify several species included in a "miscellaneous" group. The Crystal Enteric/Non-Fermenter system is an easy-to-use kit which compares favorably with other commercial systems.

Biochemical and toxigenic properties of Vibrio furnissii isolated from a European eel farm

The present study describes for the first time the isolation of Vibrio furnissii strains from a European eel culture system which are pathogenic for eels ( Anguilla anguilla) (LD 50 dose, 10 6 cfu/fish). Biochemical characterization of the isolates was performed by API 20E system and by classical numerical taxonomy. Growth at 4 °C, and with 6–8% (w/v) NaCl in a broth medium were necessary to differentiate Vibrio furnissii from Aeromonas species when the API 20E system was used. Virulence for eels of V. furnissii isolates was demonstrated by intraperitoneal injection of living cells. The extracellular products (ECPs) produced by V. furnissii were lethal to elvers and induced some of the pathological signs observed in the experimental infections so they could play a role in the pathogenicity of this species for fish. These ECPs displayed proteasic and haemolytic activities as well as cytotoxic effects on homoiothermic and fish cells lines which were inactivated after heating. Overall results show that Vibrio furnissii is a potential pathogen of European eel.

Comparison of Crystal Enteric/Nonfermenter system, API 20E system, and Vitek AutoMicrobic system for identification of gram-negative bacilli

A comparative evaluation of the Crystal Enteric/Nonfermenter system (Crystal; Becton Dickinson, Cockeysville, Md.), API 20E (API; bioMérieux Vitek, Inc., Hazelwood, Mo.), and the Vitek GNI card (Vitek; bioMérieux Vitek) was performed with 512 clinical isolates of gram-negative bacilli, including 381 members of the family Enterobacteriaceae and 131 nonenteric bacilli. With supplemental testing, API, Crystal, and Vitek correctly identified to the genus and species level 505 (98.6%), 489 (95.5%), and 494 (96.5%) of the 512 isolates, respectively. Supplemental testing, as specified by the manufacturer, was required to identify 119 (23.2%), 18 (3.5%), and 5 (1.0%) of the isolates with the three systems, respectively. Of the 381 isolates from the family Enterobacteriaceae, API and Crystal correctly identified 90.3 and 91.6% by 18 to 24 h without supplemental testing, respectively, and Vitek identified 92.4 and 96.1% following 10 and 18 h of incubation, respectively. Of the 131 nonenteric organisms, API and Crystal correctly identified 28.2 and 93.9% by 18 to 24 h without supplemental testing, respectively, and Vitek identified 84.0% by 10 h and 93.9% by 18 h. Errors in identification with each system were infrequent and appeared to be randomly distributed among the genera evaluated. The three systems were comparable in accuracy when either a weighted clinical laboratory profile of organisms or a group of selected isolates in a stress test sample was evaluated (P > 0.05). There were no significant differences between the three systems in their ability to identify either the isolates in the weighted group or those in the stress test (P > 0.05). Crystal compared favorably with API and Vitek, which have established track records in clinical laboratories, and is acceptable for the identification of members of the Enterobacteriaceae and nonenteric bacilli in a clinical microbiology laboratory.

Identification and Typing of Vibrio anguillarum: A Comparison of Different Methods

Summary The majority (91%) of 260 isolates initially identified as Vibrio anguillarum , that were obtained from a wide range of hosts, habitats and geographical locations, were recovered in a single cluster based on the ribotype and were pathogenic to Atlantic salmon. A significant proportion of isolates (78% of the total) were allocated to 15 serogroups (O1–O10 and five previously undescribed groups referred to as VaNT1, VaNT2, VaNT4, NaNT5 and VaNT7). A minority of isolates (6%) reacted with more than one antiserum or were self-agglutinating, and the remainder did not react with any of the antisera tested. Good correlation was noted between serogroups and lipopolysaccharide profiles, particularly with respect to isolates belonging to serogroups O1, 02 and 04ߝ010. Plasmids were recognized in some serogroups. especially O1, which contained the 67 kb plasmid associated with virulence. However, the 19 profiles based on outer membrane protein patterns did not correspond to the results obtained with the other typing methods. Generally, the isolates were heterogeneous in their biochemical characteristics; 117 profiles were obtained with the API 20E system, and 9 and 32 clusters recognised from the results of BIOLOG fingerprinting and Biotype-100 biotyping methods, respectively. Three dominant dusters were defined from fatty acid methyl esters profiles.

Comparison of the Vitek, API 20E and Gene-trak® systems for the identification of Yersinia enterocolitica.

The current improvements in nucleic acid hybridization technology provide new techniques for the identification of micro-organisms. One such technique is the Gene-trak® DNA hybridization system (Framingham, MA, USA), which was introduced in 1983. The objective for this study was to evaluate the new Gene-trak®Yersinia enterocolitica kit in comparison with the API 20E and Vitek systems. A total of 101 strains including 18 reference non-Yersinia strains from the authors' stock culture collection and 83 suspected positive isolates from CIN agar were tested. Of these 83 isolates, 40 were identified as Y. enterocolitica after incubation at 37°C for 24 with the API 20E system; 37 strains were identified at 30°C for 48 h. The Gene-trak® method gave positive results with 39 strains. The Vitek system gave positive results with 27 strains. With the Gene-trak® method, Y. enterocolitica was detectable in mixed cultures provided that the numbers of cfu ml-1 were equal to or above 106 Y. enterocolitica ml-1 . Although enrichment procedures are still needed, the system provides a quick detection of these food-borne pathogens.

Usefulness of the API-20E system for the identification of bacterial fish pathogens

A total of 223 isolates belonging to motile Aeromonas, A. salmonicida, Vibrio anguillarum, Pasteurella piscicida and Yersinia ruckeri species were tested by the API-20E system and the results compared with those obtained with standard biochemical tube and plate tests. Depending on the species, the API-20E yielded false negative and positive reactions for fermentation of different sugars, lysine decarboxylase (LDC), Voges Proskauer (VP), citrate and gelatinase reactions. Thirteen of 32 (41%) A. salmonicida strains and 9 of 53 (17%) Y. ruckeri isolates were correctly identified using the API index. Forty-five of 69 (65%) motile Aeromonas tested (including 34 A. hydrophila, 10 A. sobria and 1 A. caviae strains) were identified as A. hydrophila. The low discrimination profiles generated by 9 Yersinia and 2 motile Aeromonas isolates were avoided using the supplementary tests cited in the API index. In the case of V. anguillarum and P. piscicida, which are not at present included in the API-20E system, 35 of 53 strains of V. anguillarum were wrongly identified as A. hydrophila, and all the P. piscicida isolates were incorrectly identified as Pseudomonas fluorescens/Ps. putida. A large number of isolates, including some reference strains, produced uncoded profiles. From these results, we consider that the API-20E system will be a useful tool for field diagnosis of bacterial fish diseases if its database is expanded to include V. anguillarum, P. piscicida, A. sobria and A. caviae species and the new profiles generated by strains of species already considered. In addition, some reactions necessary to discriminate between strains which share the same profile number are proposed.

Enterobacteria associated with grass and silages

Enterobacteria were isolated from grass and silages and identified by the API 20E system. The effects of various ensiling treatments on their numbers and types were determined. The enterobacteria appear to follow a pattern of succession. Erwinia herbicola and Rahnella aquitilis dominated on fresh grass but after ensiling were rapidly superseded by Hafnia alvei, and finally Escherichia coli. Serratia fonticola may appear early on if the pH falls very rapidly.

Collaborative evaluation of the Radiometer Sensititre AP80 for identification of gram-negative bacilli.

A multicenter trial of the Sensititre AP80 panel read on the Sensititre AutoReader (Radiometer America, Westlake, Ohio) for the automated identification of gram-negative bacilli was conducted with 1,023 clinical isolates (879 members of the family Enterobacteriaceae plus 144 nonenteric organisms). Assignment of taxa was based on the computer-assisted interpretation of the results of a series of reactions with fluorogenic enzyme substrates after 5 h of incubation, with an incubation interval of approximately 18 h used when indicated. Accuracy was determined initially by comparison with the results obtained with the API 20E or Rapid NFT system (Analytab Products, Plainview, N.Y.). Isolates showing discrepancies were identified by using conventional biochemical profiles. Identifications were available after 5 h of incubation for 918 isolates (90%). Agreements with reference results for members of the family Enterobacteriaceae were 95.3 and 92.5% at the genus and species levels, respectively, and for the nonmembers of the family Enterobacteriaceae, the agreements with reference results were 95.1 and 84.7%, respectively. The Sensititre AP80 panel was found to be simple and convenient to use, allowed for the testing of three isolates per panel, required minimal supplementary testing for completion of identification, performed in a reproducible fashion, and demonstrated an accuracy of same-day identification comparable to that reported for other automated systems. The AP80 panel appears well suited for routine use in the clinical microbiology laboratory as an automated means of identifying both members of the family Enterobacteriaceae and nonenteric gram-negative bacilli.

Culture media for the differentiation of isolates of Yersinia ruckeri, based on detection of a virulence factor

Strains of the bacterial fish pathogen Yersinia ruckeri were identified with the API 20E system and distinguished on the basis of whole cell agglutination with antisera, sorbitol fermentation and polymyxin B sensitivity. Strains which were shown to possess the virulence-associated heat-sensitive factor (HSF) were shown to grow preferentially on culture media containing sodium dodecyl sulphate (SDS) and to produce a creamy deposit around the colonies. By contrast, strains lacking this factor (HSF-) grew poorly and without forming a deposit. Enhancement of the differentiation between the two types was shown by the incorporation of Coomassie brilliant blue dye into agar containing 1% SDS, and the uptake of Coomassie blue and Congo red was shown to be temperature-dependent. Most strains tested were shown to belong to serotype I, and were sensitive to polymyxin and did not ferment sorbitol. With the medium developed most serotype I strains but not those of other serotypes were shown to possess HSF. It is suggested that the medium is used in epidemiological studies of Y. ruckeri.

Evaluation of the API 20E system for identification and discrimination of Vibrio vulnificus biotypes 1 and 2

Evaluation of the API 20E system for identification and discrimination of <i>Vibrio vulnificus</i> biotypes 1 and 2

Gram-negative enteric bacteria in the oral cavity of leukemia patients

We examined changes in the bacterial flora in hospitalized patients with leukemia. This study placed special emphasis on enteric microorganisms and their relation to the general status of the patient. One hundred thirty bacterial cultures from 16 leukemia patients and 16 control subjects, were obtained. The organisms were isolated on MacConkey agar and identified by the API-20E system. Enteric microorganisms were isolated from 62.2% of the leukemia patients as compared with 28% from the control group ( p < 0.001). The enteric positive cultures were identified as Klebsiella (42.7%), Enterobacter (18.8%), and Pseudomonas (15.6%). In contrast to the negative cultures (1342), enteric microorganisms were cultured from 2948 specimens ( p < 0.005).

Coliform Bacteria in Drinking Water from South Bavaria: Identification by the API 20E-System and Resistance Patterns

A total of 256 coliform strains (132 from central, 124 from decentralized water supplies) as defined by the German drinking water regulation were characterized by the API 20E-system and by their susceptibility to 15 antimicrobial agents. Alltogether 22 different species from 10 genera could be differentiated, whereas 14 strains were unidentifiable by the applied system. The frequency distribution of the strains isolated from central water supplies yielded 48.5 % for Citrobacter freundii, 17.4 % for Klebsiella pneumoniae, 6.8 % for Buttiauxella agrestis and 6.1 % for C. diversus. In decentralized water supplies C. freundii was found in 33.9 %, K. pneumoniae in 14.5 %, Serratia fonticola in 8.9 % and K. oxytoca in 6.5 %. About one third (73 strains) were fully susceptible to all used antibiotics. 42 strains, however, revealed complete or moderate resistance against three or more agents (multiresistant strains). Within this group a seasonal variation was evident. The proportion of multiresistant isolates was found to be relatively high in the first half of the year (21.4 % in January- March and 29.4 % in April-June) and relatively low in the second half-year (7.9 % in July-September and 11.3 % in October-December). This different distribution may indicate a conservation of R-factor bearing bacteria in the environment.

Spontaneous Bacterial Peritonitis due to Aeromonas Hydrophila

Spontaneous Bacterial Peritonitis due to Aeromonas Hydrophila

Genomic groups and biochemical profiles of clinical isolates of Enterobacter cloacae

A collection of 123 clinical strains presumptively identified as Enterobacter cloacae and 12 type and reference strains of Enterobacter spp. were genotypically investigated by a quantitative bacterial dot method for DNA-DNA hybridization, giving an estimate of delta Tm (difference in thermal denaturation midpoint between homologous and heterologous duplexes). The API 20E system was used for phenotypic characterization. Using discontinuities in the values of delta Tm as criterion, five genomic groups of E. cloacae could be demonstrated, eleven ungrouped strains representing at least one additional group. Nine API profiles were found, the ideal phenotype of E. cloacae (i.e. the phenotype showing the most common reaction for the species in all tests studied) being the most frequently found. The type strain of E. cloacae and the reference strain CDC 1347-71 represented rather small genomic groups of three and eight strains respectively, most of them inositol positive. A majority of 98 isolates formed one single genomic group, biochemically dominated by the ideal phenotype. The genomic groups could not be differentiated phenotypically. At present there seems to be no reason for an attempt to split E. cloacae into two or more species. The type strain of E. dissolvens showed itself to be closely related to the type strain of E. cloacae (delta Tm 2.3 degrees C), indicating that the two species may be regarded as subjective synonyms.

Ecology and seasonal distribution ofVibrio parahaemolyticusin aquatic environments of a temperate region

An environmental survey was done to study the ecology and distribution of Vibrio parahaemolyticus in 5 selected stations in Okayama Prefecture, which included fresh, brackish, and marine aquatic environments. Water and plankton samples were collected monthly for quantitative and qualitative analyses during the period October, 1987 to October, 1989 for V. parahaemolyticus. The pathogen was not detected from fresh water environments. A seasonality of the organism was observed in brackish and marine environments where average salinity ranged between 0.39 and 1.28%.Plankton samples yielded higher densities of V. parahaemolyticus compared with water samples. By applying several enrichment techniques, the pathogen was detected quite frequently during the winter months in the environments with temperatures ranging between 10 and 14°C. The identification following conventional tests, by the API 20E system and by serological methods reveal that the API 20E system is satisfactory to identify V. parahaemolyticus and further confirms that the serological method could be a simpler and more rapid procedure for V. parahaemolyticus identification.

Ecology and seasonal distribution of Vibrio parahaemolyticus in aquatic environments of a temperate region

An environmental survey was done to study the ecology and distribution of Vibrio parahaemolyticus in 5 selected stations in Okayama Prefecture, which included fresh, brackish, and marine aquatic environments. Water and plankton samples were collected monthly for quantitative and qualitative analyses during the period October, 1987 to October, 1989 for V. parahaemolyticus. The pathogen was not detected from fresh water environments. A seasonality of the organism was observed in brackish and marine environments where average salinity ranged between 0.39 and 1.28%.Plankton samples yielded higher densities of V. parahaemolyticus compared with water samples. By applying several enrichment techniques, the pathogen was detected quite frequently during the winter months in the environments with temperatures ranging between 10 and 14°C. The identification following conventional tests, by the API 20E system and by serological methods reveal that the API 20E system is satisfactory to identify V. parahaemolyticus and further confirms that the serological method could be a simpler and more rapid procedure for V. parahaemolyticus identification.

Assessment of conventional and commercial methods for identification of clinical isolates of cysteine-requiring strains of Escherichia coli and Klebsiella species

Cysteine-requiring strains of the family Enterobacteriaceae that are auxotrophic for this amino acid because of defects in the sulfur assimilatory pathway account for about 1.5% of urinary tract isolates of Escherichia coli and Klebsiella species. Forty Escherichia and eight Klebsiella cysteine-requiring strains were used to test the ease with which various test systems identified clinical isolates of cysteine auxotrophs. In a preliminary experiment, the growth yield of 10 cysteine-requiring E. coli in 10 solutions of commercially available peptones was in each instance less than that of prototrophic control and showed that these sources of nutrients were suboptimal for these strains. A significant proportion of the cysteine-requiring strains were not adequately identified by growth-dependent tests which used various peptones as a nutrient source. Problems were encountered with all test systems examined, which were as follows: conventional methods; the API 20E, Microbact, and Vitek systems; and two rapid methods for the identification of E. coli, the Rapidec coli and the beta-D-glucuronidase tests. The performance of the test systems was only partly improved when inocula were derived from appropriately supplemented media. However, the problems of the growth-dependent tests were resolved when a cysteine-supplemented suspension was used to inoculate each test system.

Agreement between visual and automated UniScept API readings

The UniScept API system was evaluated for agreement of visual versus automated readings of both its identification panels and its antimicrobial susceptibility panels. The biochemical responses of 340 oxidase-negative and oxidase-positive fermentative bacterial cultures were read both visually and automatically in the UniScept API 20E system. Automated and visual readings agreed with 99.3% of the biochemicals. Of the 45 tests that disagreed, the tests for indole and citrate were most often in disagreement. A total of 470 fermentative and nonfermentative cultures were used in the UniScept MIC system to compare visual and automated readings of susceptibility results with 17 antimicrobial agents. Agreement within +/- 1 dilution occurred with 94.1% of the enteric fermenters and with 91.7% of the other cultures. Comparison of visual and automated readings resulted in very major discrepancies in 0.95% of the readings, with the largest percentage of discrepancies associated with glucose nonfermenters (1.8%). It was felt that an automated reading is an acceptable alternative to a visual reading of the biochemicals but that 0.95% was just within the acceptable range of the 1% allowable very major discrepancies in the automated reading of susceptibilities.

Ornithine decarboxylating strains of Klebsiella pneumoniae demonstrated by DNA‐DNA hybridization

Genotypic relatedness was assessed to clarify the taxonomic position of strains phenotypically behaving like K. pneumoniae, but for the ornithine reaction. Using DNA-DNA hybridization it could be shown that 25 non-motile ornithine decarboxylating strains showed high genotypic relatedness to the type strain of K. pneumoniae. Thus, it is proposed that they be considered as ornithine decarboxylating strains of the species K. pneumoniae. The API 20E system was used for phenotypic characterization, but the API code obtained by these strains was not registered in the API Profile Index. However, except for the ornithine reaction the isolates behaved as typical K. pneumoniae. Three ornithine negative strains of E. aerogenes were identified as K. pneumoniae by the API 20E System, but they showed high genotypic relatedness to the type strain of E. aerogenes.

Comparison of Airborne Microflora Collected by the Andersen Sieve Sampler and RCS Sampler in a Dairy Processing Plant

The performance of a Reuter centrifugal air sampler (RCS sampler) was compared with that of an Andersen 6-stage sieve sampler (Andersen impactor) to determine the suitability of the RCS sampler for monitoring airborne microflora in dairy processing plant environments. The types of microorganisms recovered from non-mold colony forming particles using the Andersen impactor and the RCS sampler were different at each location and trial. When selected Gram positive cocci and Gram negative rods were classified using Staph track and API 20E System, respectively, results indicated that the type of bacteria collected using each sampler was similar, though the percentage distributions were different. In addition, bacterial types were not consistently associated with different size particles. The RCS sampler was able to recover most bacterial types since these types were distributed over various particle sizes. When airborne fungi were collected, the RCS sampler recovered significantly lower amounts than that of Andersen impactor. The RCS sampler is suitable for use when approximate concentration of non-mold airborne particles, or relative degree of air cleanliness information is needed.