Reproducibility of three microdilution systems for identification of enterobacteriaceae,compared with API 20E and micro-ID test systems

Five systems for identifying Enterobacteriaceae were evaluated in terms of their ability to provide reproducible results. Frozen microdilution panels (Micro-Media Systems), prepared media ready for dispensing into microdilution trays (Dynatech laboratories), media prepared in our laboratories and dispensed into microdilution trays (UC media), the Micro-ID system (General Diagnostics), and the API 20E system (Analytab Products, Inc.) were all evaluated. Fifty selected isolates were tested with each system on three separate days. Although biotypes were some-what variable, the final identification was rarely affected. The API 20E system was the most variable and microdilution tests, with UC media, were the least variable. Precision of tests performed in microdilution trays was as good, if not better than, that obtained with the other two commercial products (Micro-ID and API 20E).     

Comparison of miniaturized multitest systems with conventional methodology for identification of Enterobacteriaceae from foods.

Four miniaturized multiple test systems were compared with tube methodology used to identify Enterobacteriaceae encountered in foods. Identification aids supplied with each system were used to assign names to isolates at the species level. For the 129 strains tested, the Minitek system demonstrated a 96.9 percent agreement with reactions in tubed media. The Inolex, Analytab, and PathoTec test systems exhibited 94.3, 93.8, and 92.7 percent agreement, respectively. Analytab identified 96.1 percent of the isolates to the species level, whereas the Minitek, PathoTec, and Inolex systems were able to identify 78.3, 32.6, and 27.1 percent, respectively. The results indicate that the Analytab and Minitek systems are acceptable substitutes for the tube methodology routinely employed in identifying enterics from foods. Although the PathoTec system might be used to screen isolates for their identity, neither the presently available PathoTec nor the Inolex systems should be substituted for current methodology when definitive identification of foodborne organisms is required.     

Comparison of miniaturized multitest systems with conventional methodology for identification of Enterobacteriaceae from foods.

Four miniaturized multiple test systems were compared with tube methodology used to identify Enterobacteriaceae encountered in foods. Identification aids supplied with each system were used to assign names to isolates at the species level. For the 129 strains tested, the Minitek system demonstrated a 96.9 percent agreement with reactions in tubed media. The Inolex, Analytab, and PathoTec test systems exhibited 94.3, 93.8, and 92.7 percent agreement, respectively. Analytab identified 96.1 percent of the isolates to the species level, whereas the Minitek, PathoTec, and Inolex systems were able to identify 78.3, 32.6, and 27.1 percent, respectively. The results indicate that the Analytab and Minitek systems are acceptable substitutes for the tube methodology routinely employed in identifying enterics from foods. Although the PathoTec system might be used to screen isolates for their identity, neither the presently available PathoTec nor the Inolex systems should be substituted for current methodology when definitive identification of foodborne organisms is required.     

Evaluation of Modified R-B System for Identification of Members of the Family Enterobacteriaceae

In a paired, double-blind study, the modified ("Beckford tube") R-B system was compared with conventional bacteriological procedures for the identification of members of the family Enterobacteriaceae from clinical isolates and stock cultures. The tests in the R-B system yielding positive reactions comparable to those predicted by Ewing's taxonomic classification of Enterobacteriaceae were production of hydrogen sulfide and presence of lysine and ornithine decarboxylasè activities. The test reactions in the R-B system found to be comparable to those in the conventional method were fermentation of glucose, hydrogen sulfide production, and lysine and ornithine decarboxylase activities. The production of gas from glucose was positive in the R-B system more often than in the conventional method; however, the motility test and the production of indole were positive less often in the R-B system. Adequate preliminary identification of the Enterobacteriaceae with the R-B system is enhanced if Simmons' citrate and Christensen's urea tests are used concomitantly. These findings emphasize the manufacturer's instructions that, in interpretation of results, colonial morphology and biochemical reactions must be used concurrently to make an accurate identification.     

Identification of staphylococci from bovine udders: evaluation of the API 20GP system

The API 20GP system (Analytab Products, Plainview, NY) correctly identified 56.1% of staphylococci isolated from the bovine mammary gland. The system identified 90.2% of Staphylococcus aureus strains, but failed to recognize strains of Staphylcoccus hyicus and others. Poor performance was attributed to the limited number of veterinary strains contained in the profile index data base. The API 20GP was determined to be an unacceptable method for identification of staphylococci isolated from the bovine mammary gland.     

Evaluation of the Enterotube System for Identification of Members of the Family Enterobacteriaceae

The Enterotube system was evaluated, in parallel with conventional bacteriological procedures for the identification of members of the family Enterobacteriaceae, by using bacterial strains from a variety of clinical specimens and from stock cultures. Excellent agreement between the two test systems was obtained with the following reactions: hydrogen sulfide, indole, Simmons' citrate, glucose, and lactose. Agreement was not as good (<85%) with the urea, phenylalanine deaminase, and dulcitol reactions. The Enterotube lysine decarboxylase test was unsatisfactory. The Enterotube method will correctly identify strains of the family Enterobacteriaceae approximately 50% of the time; if identification only as Klebsiella-Enterobacter-Serratia group is needed, the method will be correct 85% of the time. On the basis of this evaluation, the Enterotube system appears to be both simple and rapid for the presumptive identification of these bacteria. Because of the limited usefulness of the lysine decarboxylase test, the results obtained by this test system are less reliable than those obtained by conventional methods.     

Identification of Enterobacteriaceae with the Minitek system

A total of 417 strains (361 Enterobacteriaceae, 56 Vibrionaceae) was examined in all the available Minitek system tests. The results were processed through four successive identification schemes devised by the manufacturer and the proportion of strains correctly identified, not identified or incorrectly identified determined for each scheme. From the results, a probability matrix was constructed incorporating all 35 Minitek tests. Test results for each strain were then processed through this matrix to determine its success in identification. From the matrix the order of separating value of the tests was determined. Forty-three of the strains were each tested three times to assess the level of test reproducibility; the corrected error rate was 0.85%.     

Identification of Enterobacteriaceae with the Minitek system

A total of 417 strains (361 Enterobacteriaceae, 56 Vibrionaceae) was examined in all the available Minitek system tests. The results were processed through four successive identification schemes devised by the manufacturer and the proportion of strains correctly identified, not identified or incorrectly identified determined for each scheme. From the results, a probability matrix was constructed incorporating all 35 Minitek tests. Test results for each strain were then processed through this matrix to determine its success in identification. From the matrix the order of separating value of the tests was determined. Forty-three of the strains were each tested three times to assess the level of test reproducibility; the corrected error rate was 0.85%.     

Evaluation of the Minitek System for Identification of Enterobacteriaceae

Clinical isolates (869) and stock cultures (35) of Enterobacteriaceae were tested in parallel with the Minitek and conventional systems. The Minitek correctly identified 822 of 904 cultures. When a deoxyribonuclease plate was inoculated along with the Minitek, it was possible to speciate Enterobacteriaceae within 24 h. False-positive hydrogen sulfide reactions were the major fault with this system. Reactions were clear-cut and easy for technologists to read.     

Efficiency of a Multitest System (Enterotube) for Rapid Identification of Enterobacteriaceae

Enterotube, a multiple-test system which combines nine biochemical tests useful in the identification of members of the family Enterobacteriaceae, was tested and compared with the PathoTec test system in the identification of gram-negative bacilli isolated from clinical specimens. It was found that both the Enterotube and the PathoTec systems rapidly and accurately defined the position of the organisms in the major groups of the family Enterobacteriaceae. Discrepancies were noted between the results of citrate tests on Simmons' citrate-agar and in the Enterotube, as well as between Simmons' citrate-agar and the PathoTec citrate test. It was concluded that the Enterotube system provides a simple, reliable, and rapid method for the presumptive identification of Enterobacteriaceae. The major advantage of the Enterotube is that all tests are done simultaneously by inoculation from a single isolated colony.     

IMMEDIATE DIFFERENTIATION OF SALMONELLA-RESEMBLING COLONIES ON BRILLIANT GREEN AGAR

A rapid biochemical system (OBIS) based on immediate enzymatic differentiation of Citrobacter, Proteus, Providencia, Hafnia and Morganella spp. from Salmonella on brilliant green agar was evaluated. A total of 96 field isolates from various Salmonella serotypes, 18 Citrobacter freundii and 25 isolates of other Enterobacteriaceae were tested. All Salmonella isolates were identified correctly by the kit, and none of the Enterobacteriaceae isolates were identified as Salmonella. The results indicate complete specificity for Salmonella colonies on brilliant green agar.     

Evaluation of the PathoTec ?Rapid I-D System”

The 10 biochemical test strips included in the PathoTec Rapid I-D System were evaluated for accuracy as compared to standard tests and for efficacy in identification of 193 gram-negative bacilli. The test agreement was 100% for oxidase and phenylalanine deaminase, 99% for indole, nitrate, and Voges-Proskauer, 98% for malonate, 97% for lysine decarboxylase, 90% for urease, 84% for H(2)S, and 75% for esculin hydrolysis. Most of the commonly isolated Enterobacteriaceae were identified correctly within 4 h. Errors in identification of Proteus morganii and P. rettgeri occurred because of positive H(2)S tests on the PathoTec strips with these organisms.     

Micromethod System for Identification of Anaerobic Bacteria

A micromethod multitest system prepared by Analytab Products, Inc. and conventional tests employed at the Center for Disease Control for identification of anaerobes were compared. All procedures were conducted in an anaerobic glove box. A total of 104 cultures, including 18 reference strains and 86 diagnostic cultures, were examined. Ninety-one percent of the total tests performed with the two systems were in agreement. Greater than 90% agreement between the two systems was obtained with 12 of the 17 differential tests compared. The tests for nitrate reduction and H(2)S production gave the poorest agreement, 77.8 and 80.8%, respectively. Only 66% of the 86 diagnostic cultures could be presumptively identified with the micromethod system supplemented only with microscopy and colonial characteristics. However, when appropriate supplementary tests and gas-liquid chromatography were used with the micromethod system, 85% of the 86 strains could be identified. When Ehrlich reagent, instead of Kovac reagent, was used with the micromethod to test for indole, the agreement in identification was raised to 93%.     

Oligonucleotide probes specific for the genus Salmonella and for Salm. typhimurium

The Crystal Enteric/Nonfermenter (E/NF) identification kit (Becton Dickinson Microbiology Systems, USA) was evaluated using water-derived bacterial isolates and results compared to those obtained by the API 20E system (BioMérieux, UK). Both the E/NF and 20E systems correctly identified 93% of the Enterobacteriaceae reference cultures. Both systems agreed in the identification of 64·9% of environmental isolates. The E/NF system gave a positive identification to 88·0% of isolates and the 20E to 79·5% of isolates. The principal tests which gave differing reactions between the two systems were arginine dihydrolase, lysine decarboxylase, urease and citrate utilization.     

The Examination of Foods for Enterobacteriaceae using a Test of the Type Generally Adopted for the Detection of Salmonellae

S ummary : In countries that have recently increased their bacteriological inspection of foods, many products have shown considerable improvement in microbiological quality; simultaneously, however, discrepancies between salmonella and coli-aerogenes tests, especially on dehydrated and frozen foods, have tended to become more frequent. These discrepancies have been eliminated by applying the following measures: incorporating glucose in culture media so as to reveal all Enterobacteriaceae; placing reliance on growth rather than gas formation so as to avoid missing anaerogenic organisms, and, especially, examining quantities of foods commensurate with those used in salmonella tests.
For this purpose a procedure is recommended in which 10 g of well homogenized food are enriched in 100 ml of buffered brilliant green-bile-glucose broth, with no attention being paid to gas formation; the enrichment cultures are then streaked on to MacConkey's glucose agar. Single colonies so obtained are tested for fermentative attack on glucose and may be further examined for other characteristics. The same enrichment fluid can be used for the so-called 'nonselective pre-enrichment' of samples of food containing salmonellae impaired by periods spent in conditions of low water activity, low pH, etc.     

Use of a computer for the purpose of identifying bacteria of the family Enterobacteriaceae and the determination of the minimal set of differential tests]

Algorhythm and a program for identification of bacteria of the Enterobacteriaceae family, based on Edwards and Ewing's diagnostic scheme, were worked out. Use of this program permitted to analyze different sets of abbreviated biochemical tests. To determine the genera and species of enterobacteria a minimal set of 11 tests is suggested, including indol formation, Voges-Proskauer's reaction, the presence of urease enzymes, gelatinase, lysine decraboxylase, phenylalanine deaminase, glucose fermentation (gas), or lactose, inosite, sorbit, arabinose, rhamnose. The program admits increase of both the biochemical tests, and toxonomic groups of bacteria, this permitting to consider several families. The presence of strains deviating by properties from this scheme points to the necessity of further improvement of diagnostic schemes for the enterobacteria identification.     

Auxotab—a Device for Identifying Enteric Bacteria

A multitest system called the Auxotab that uses ten dehydrated reagents on a paper card has been evaluated with 417 known stock cultures of Enterobacteriaceae. In double-blind studies with the Auxotab, 87% of the strains tested were correctly identified. Results of this study indicate that there is a need for modification of the product in regard to ease of handling, time required for use, and accuracy of identification of enteric bacteria.     

Evaluation of the Redesigned Enterotube—a System for the Identification of Enterobacteriaceae

The redesigned Enterotube has been evaluated with 414 unknown Enterobacteriaceae cultures from the stock culture collection of the Center for Disease Control. When the Enterotube was used as recommended by the manufacturer, an average of 96.4% of these cultures were correctly identified. Only two groups (Salmonella and Edwardsiella) were identified with less than 90% accuracy (89.2 and 87.5%, respectively). The Enterotube now provides a convenient, rapid, and accurate test system for the identification of typically reacting enteric bacteria.     

Fate of salmonellas and competing flora in meat sample enrichments in buffered peptone water and in Muller-Kauffmann's tetrathionate medium

Studies have been carried out in which growth patterns of a Salmonella sp. and competing micro-organisms, especially other Enterobacteriaceae, were followed during pre-enrichment in buffered peptone water (BPw) and subsequent selective enrichment in tetrathionate broth (TBB). Pre-enrichment cultures were inoculated with minced meat and three reference samples containing nalidixic acid-resistant salmonellas. Irrespective of their initial numbers in BPw, Enterobacteriaceae increased to 10(8)/ml or more. During incubation in TBB at 43 degrees C, numbers of lactose-positive Enterobacteriaceae decreased in most enrichments which resulted in a positive salmonella isolation, but remained constant in the majority of those that did not. Levels of lactose-negative Enterobacteriaceae did not decrease in most salmonella-positive tests, but did so in half of the salmonella-negative ones. In the salmonella-positive tests the numbers of salmonellas had increased to 10(3)-10(7)/ml in BPw and after transfer to TBB slowly reached 10(4)/ml or more. In all cases the numbers of salmonellas exceeded those of the competing flora on brilliant green agar (BGA). In the salmonella-negative tests the numbers of salmonellas had increased less in BPw and decreased in most of the TBB enrichments. In none of these negative tests did the numbers of salmonellas exceed those of the competing flora on BGA. Escherichia coli dominated in most of the salmonella-negative tests. The results suggest more influence of lactose-positive than lactose-negative Enterobacteriaceae on the detection of salmonellas. The effect of competing microorganisms seems to depend not only upon their initial numbers, but also upon the types that can interact with salmonellas during selective enrichment.     

Fate of salmonellas and competing flora in meat sample enrichments in buffered peptone water and in Muller-Kauffmann's tetrathionate medium

Studies have been carried out in which growth patterns of a Salmonella sp. and competing micro-organisms, especially other Enterobacteriaceae, were followed during pre-enrichment in buffered peptone water (BPw) and subsequent selective enrichment in tetrathionate broth (TBB). Pre-enrichment cultures were inoculated with minced meat and three reference samples containing nalidixic acid-resistant salmonellas. Irrespective of their initial numbers in BPw, Enterobacteriaceae increased to 10⁸/ml or more. During incubation in TBB at 43C, numbers of lactose-positive Enterobacteriaceae decreased in most enrichments which resulted in a positive salmonella isolation, but remained constant in the majority of those that did not. Levels of lactose-negative Enterobacteriaceae did not decrease in most salmonella-positive tests, but did so in half of the salmonella-negative ones. In the salmonella-positive tests the numbers of salmonellas had increased to 10³–10⁷/ml in BPw and after transfer to TBB slowly reached 10⁴/ml or more. In all cases the numbers of salmonellas exceeded those of the competing flora on brilliant green agar (BGA). In the salmonella-negative tests the numbers of salmonellas had increased less in BPw and decreased in most of the TBB enrichments. In none of these negative tests did the numbers of salmonellas exceed those of the competing flora on BGA. Escherichia coli dominated in most of the salmonella-negative tests. The results suggest more influence of lactose-positive than lactose-negative Enterobacteriaceae on the detection of salmonellas. The effect of competing microorganisms seems to depend not only upon their initial numbers, but also upon the types that can interact with salmonellas during selective enrichment.