Abstract
The Vitek GNI card was used to identify 212 isolates of 10 Yersinia species. Identification was correct for 96.3% of the isolates (156 of 162) to the genus level and for 57.4% of the isolates (93 of 162) to the species level for Yersinia spp. listed in the Vitek database. We recommend additional identification methods for isolates assigned to the genus Yersinia by the Vitek system.
The genus Yersinia belongs to the family Enterobacteriaceae and contains 11 species, three of which are pathogenic in humans. Yersinia pestis is the bacterial agent of plague and was not included in this study. Yersinia enterocolitica and Yersinia pseudotuberculosis can cause gastroenteritis and mesenteric lymphadenitis mimicking appendicitis, but the bacteria may also cause infections at other sites, such as wounds, joints, and the urinary tract, or invoke postinfectious sequelae such as reactive arthritis, urethritis, pleurisy, vasculitis, cholecystitis, and erythema nodosum. Y. enterocolitica and Y. pseudotuberculosis are the most important causes of gastroenteritis after Salmonella spp. and Campylobacter spp. (9). Other species include Yersinia aldovae, Yersinia bercovieri, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia mollaretii, and Yersinia rohdei, all of which were formerly considered to be biovars of Y. enterocolitica and can act as opportunistic pathogens (2, 5, 6, 8, 15, 16). Yersinia ruckeri causes redmouth disease in salmonids (10).
Yersinia species are relatively slow growers among the Enterobacteriaceae and display their biochemical characteristics most reliably at temperatures between 25 and 32°C (3). Traditional identification is based on a number of biochemical reactions often not incorporated in commercially available tests. In Y. enterocolitica, enteropathogenicity is restricted to members of some serovar-biovar combinations harboring the 64-kDa Yersinia virulence plasmid. Its presence is demonstrated by a positive autoagglutination test (1).
The Vitek system (bioMérieux Vitek, Inc., Hazelwood, Mo.) is an automated miniaturized biochemical test system operated at a fixed incubation temperature of 37°C. The GNI card is designed to identify members of the Enterobacteriaceae family and a select group of nonfermenting gram-negative bacteria. Six species of the genus Yersinia are listed in the Vitek database: Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. pestis, and Y. pseudotuberculosis. The Vitek system is routinely used for identification of Enterobacteriaceae in our laboratory. Rapid commercial identification systems generally favor the faster-growing and biochemically more active Enterobacteriaceae, which might result in a higher probability of misidentification for Yersinia spp.
The present study evaluated the ability of the Vitek Gram-Negative Identification (GNI) card—an automated rapid miniaturized biochemical identification system—to identify members of the genus Yersinia. Evaluation of the Vitek system has already been reported for various genera of the Enterobacteriaceae family, including Yersinia. The present study contains the largest collection of strains belonging to the genus Yersinia tested by the Vitek system.
A total of 212 strains belonging to the genus Yersinia was included in this study. Enteropathogenic and nonenteropathogenic strains of Y. enterocolitica and Y. pseudotuberculosis were isolated from humans. Other isolates included one reference strain of each species and field strains from human, animal, and environmental sources. Strains were provided by the Institute of Hygiene (Hamburg, Germany), G. Wolf (Munich, Germany), the Central Institute of FAF (Munich and Berlin, Germany), B. Niederwöhrmeier (Munster, Germany), and by the Institute for Medical Microbiology (Regensburg, Germany). Type strains (except Y. pestis) were obtained from the American Type Culture Collection (Rockville, Md.). Strains were maintained at 4°C on nutrient agar and subcultured twice on Columbia blood agar before testing (subculture on cefsulodin-Irgasan-novobiocin-agar [Oxoid, Wesel, Germany] did not influence identification results [data not shown]). Conventional biochemical tests were done as described by Bockemühl (7) by using the differentiation scheme of Aleksic and Bockemühl (1), including testing of enteropathogenicity by autoagglutination. Identification by the GNI card was carried out in accordance with the instructions of the manufacturer, including additional tests for melibiose, rhamnose, raffinose, and salicin (read after 48 h) when necessary. Manual reading of Vitek reactions was done in accordance with the table of color reactions provided by the manufacturer.
We investigated a total of 212 phenotypically characterized strains belonging to the genus Yersinia. For the species listed in the Vitek database, the GNI card correctly identified 96.3% (156 of 162) of all strains to the genus level and 57.4% (93 of 162 strains) to the species level. Correct identification to the species level was 44.9% (44 of 98 strains) for Y. enterocolitica, 95.5% (21 of 22 strains) for Y. pseudotuberculosis, 77.8% (14 of 18 strains) for Y. frederiksenii, 41.7% (5 of 12 strains) for Y. intermedia, and 75% (9 of 12 strains) for Y. kristensenii. The time to identification was 9.7 ± 2.7 h (mean ± standard deviation) without and 32.4 ± 24.4 h with the additional biochemical tests recommended in the Vitek manual. Ninety-two percent of pathogenic strains of Y. enterocolitica (24 of 26) were identified correctly to the species level, compared to 27% (17 of 63) of nonpathogenic strains (P < 0.001, chi-square test according to Pearson). Eighty-four percent of Y. enterocolitica strains (42 of 51) were misidentified as Y. frederiksenii, and 17.6% (9 of 51) were misidentified as Y. intermedia. For 18 isolates of Y. enterocolitica misidentified as Y. frederiksenii, Y. intermedia, or Hafnia alvei, the absolute calculated likelihood was ≥85%. A detailed analysis of the parameters associated with the identification is shown in Tables 1 and 2.
TABLE 1.
Performance of the Vitek GNI card in the identification of Yersinia spp.
| Yersinia spp. | No. of isolates | % Of isolates correctly identified
|
% Of isolates identified with additional tests | Time to identification (h [mean ± SD])
|
||
|---|---|---|---|---|---|---|
| Genus level | Species level | Without additional tests | With additional tests | |||
| All species | 212 | 90 | 10.7 ± 2.8 | |||
| In Vitek database | ||||||
| All | 162 | 96 | 57 | 32 | 9.7 ± 2.7 | 32.4 ± 24.4 |
| Y. enterocolitica | 98 | 98 | 45 | 35 | 10.5 ± 1.9 | 46.5 ± 19.9 |
| Y. pseudotuberculosis | 22 | 100 | 96 | 5 | 7.2 ± 2.7 | 7.2 ± 2.7 |
| Y. frederiksenii | 18 | 100 | 78 | 56 | 10 ± 2.5 | 40.6 ± 23.8 |
| Y. intermedia | 12 | 92 | 42 | 50 | 9 ± 0.0 | 28.2 ± 26.3 |
| Y. kristensenii | 12 | 75 | 75 | 0 | 11.3 ± 3.3 | 11.3 ± 3.3 |
| Not in Vitek databasea | ||||||
| All | 50 | 72 | 66 | 10.7 ± 3.0 | 44.3 ± 22.8 | |
| Y. aldovae | 12 | 75 | 41 | 10.6 ± 3.5 | 38.6 ± 25.1 | |
| Y. bercovieri | 12 | 75 | 92 | 10.6 ± 3.0 | 54.6 ± 13.4 | |
| Y. mollaretii | 11 | 82 | 54 | 9.9 ± 3.5 | 36.1 ± 26.7 | |
| Y. rohdei | 10 | 80 | 70 | 11.1 ± 2.6 | 44.7 ± 23.6 | |
| Y. ruckeri | 5 | 20 | 80 | 12.2 ± 1.8 | 50.6 ± 21.1 | |
For Yersinia spp. not listed in the Vitek database, the correct identification is evaluated only to the genus level.
TABLE 2.
Misidentification of Yersinia spp. by Vitek, biopattern, and absolute calculated likelihood
| Yersinia spp. | Total no. of isolates | GNI identification
|
Biopattern | Absolute calculated likelihood (%) | |
|---|---|---|---|---|---|
| Species | No. of isolates | ||||
| In Vitek database | |||||
| Y. enterocolitica | 54 | Y. enterocolitica, Y. frederiksenii, Y. intermedia | 1 | 7010364032 | 99 |
| Y. frederiksenii | 1 | 7341735260 | 96 | ||
| Y. intermedia | 2 | 7340634262 | 96 | ||
| Y. frederiksenii | 1 | 7341731262 | 95 | ||
| Y. frederiksenii | 1 | 7361735062 | 95 | ||
| Hafnia alvei | 1 | 3000700176 | 92 | ||
| Y. frederiksenii | 8 | 7341735262 | 92 | ||
| Y. intermedia | 2 | 7340630262 | 92 | ||
| Y. frederiksenii | 1 | 7741735262 | 91 | ||
| Y. frederiksenii | 1 | 7340774360 | 89 | ||
| Y. frederiksenii | 5 | 7341775262 | 79 | ||
| Y. frederiksenii | 1 | 7340735262 | 79 | ||
| Y. intermedia | 1 | 7341624262 | 73 | ||
| Y. intermedia | 1 | 7341674262 | 73 | ||
| Y. intermedia | 2 | 7341670262 | 67 | ||
| Y. frederiksenii | 20 | 7341735062 | 66 | ||
| Y. frederiksenii | 1 | 4370730362 | 61 | ||
| Y. frederiksenii | 1 | 7341770262 | 55 | ||
| Y. intermedia | 1 | 7341734262 | 55 | ||
| Y. frederiksenii | 1 | 7341675262 | 47 | ||
| Unidentified | 1 | 3341275262 | |||
| Y. pseudotuberculosis | 1 | Y. enterocolitica | 1 | 7010260030 | 99 |
| Y. frederiksenii | 4 | Y. enterocolitica | 1 | 7010240020 | 97 |
| Y. intermedia | 1 | 7610360232 | 92 | ||
| Y. intermedia | 1 | 7341670262 | 67 | ||
| Y. enterocolitica | 1 | 7341670062 | 39 | ||
| Y. intermedia | 7 | Y. frederiksenii | 1 | 7610360032 | 99 |
| Y. frederiksenii | 1 | 7610360030 | 99 | ||
| Y. frederiksenii | 1 | 7210360232 | 99 | ||
| Y. frederiksenii | 1 | 7210360032 | 99 | ||
| Y. frederiksenii | 1 | 7610764432 | 92 | ||
| Y. frederiksenii | 1 | 7634370230 | 76 | ||
| Unidentified | 1 | 7672364073 | |||
| Y. kristensenii | 3 | Actinobacillus ureae | 1 | 30406200000 | 99 |
| Hafnia alvei | 1 | 6002700033 | 68 | ||
| Unidentified | 1 | 6436324012 | |||
| Not in Vitek database | |||||
| Y. aldovae | 12 | Y. kristensenii | 2 | 7010324022 | 99 |
| Y. intermedia | 1 | 7030274030 | 99 | ||
| Hafnia alvei | 1 | 6040600433 | 99 | ||
| Actinobacillus ureae | 1 | 60102000000 | 99 | ||
| Actinobacillus ureae | 1 | 6010200000 | 99 | ||
| Y. enterocolitica, Y. frederiksenii, Y. intermedia | 1 | 7014360032 | 98 | ||
| Y. enterocolitica, Y. frederiksenii, Y. intermedia | 1 | 3041735262 | 97 | ||
| Y. kristensenii | 1 | 7014304020 | 95 | ||
| Y. kristensenii | 1 | 7014220032 | 89 | ||
| Y. kristensenii | 1 | 7010621032 | 77 | ||
| Y. kristensenii | 1 | 7014224032 | 76 | ||
| Y. bercovieri | 12 | Y. enterocolitica | 1 | 7014360032 | 99 |
| Y. enterocolitica | 1 | 7010360032 | 99 | ||
| Actinobacillus ureae | 1 | 6010300000 | 99 | ||
| Y. enterocolitica | 1 | 7410360072 | 98 | ||
| Y. enterocolitica | 1 | 7010364030 | 98 | ||
| Y. enterocolitica | 1 | 7010360032 | 98 | ||
| Y. enterocolitica, Y. frederiksenii, Y. intermedia | 3 | 7010360030 | 95 | ||
| Y. enterocolitica | 1 | 7040735260 | 93 | ||
| Pasteurella haemolytica | 1 | 6000360000 | 92 | ||
| Unidentified | 1 | 66143642101 | |||
| Y. mollaretii | 11 | Y. enterocolitica, Y. frederiksenii, Y. intermedia | 1 | 7014364032 | 99 |
| Y. enterocolitica | 1 | 7610260032 | 99 | ||
| Salmonella spp. | 1 | 6020724533 | 99 | ||
| Y. enterocolitica, Y. frederiksenii, Y. intermedia | 1 | 7410360032 | 98 | ||
| Y. enterocolitica | 1 | 7000360032 | 97 | ||
| Y. kristensenii | 1 | 7000721062 | 95 | ||
| Y. kristensenii | 1 | 7010320032 | 94 | ||
| Y. enterocolitica | 1 | 7014764232 | 91 | ||
| Y. enterocolitica, Y. frederiksenii, Y. intermedia | 1 | 7000340032 | 74 | ||
| Acinetobacter lwoffi | 1 | 4052000000 | 73 | ||
| Y. frederiksenii | 1 | 7614365232 | 67 | ||
| Y. rohdei | 10 | Y. intermedia | 1 | 7010370032 | 99 |
| Y. intermedia | 1 | 7010274020 | 99 | ||
| Y. intermedia | 1 | 7010260032 | 99 | ||
| Y. enterocolitica, Y. frederiksenii, Y. intermedia | 1 | 7030270230 | 99 | ||
| Y. enterocolitica, Y. frederiksenii, Y. intermedia | 1 | 7030260230 | 99 | ||
| Y. enterocolitica, Y. frederiksenii, Y. intermedia | 1 | 7010370032 | 99 | ||
| Y. enterocolitica | 1 | 7030260030 | 99 | ||
| Actinobacillus ureae | 1 | 60102000000 | 99 | ||
| Y. enterocolitica | 1 | 7010374232 | 92 | ||
| Unidentified | 1 | 7060771260 | |||
| Y. ruckeri | 5 | Y. intermedia | 1 | 7010370032 | 99 |
| Hafnia alvei | 1 | 6040200022 | 63 | ||
| Shigella spp. | 1 | 3000600022 | 23 | ||
| Unidentified | 1 | 6042200022 | |||
| Unidentified | 1 | 2000200022 | |||
A number of authors have discussed the shortcomings associated with commercially available miniaturized tests for identifying members of the genus Yersinia (see overview in Table 3). Because of their slow growth, growth optimum at 25 to 32°C, and high biochemical similarity, Yersinia spp. present a special challenge to standard biochemical test systems. However, conventional biochemical testing, which was used as the “gold standard” in this study, might fail to correctly identify some isolates (11). From a clinical point of view, correct identification of Y. enterocolitica and Y. pseudotuberculosis (Y. pestis not investigated) and separation of nonenteropathogenic strains from Y. enterocolitica and Y. pseudotuberculosis are most important. In the present study, correct identification of Y. enterocolitica to the species level was achieved in only 44.9% of strains and in 18 of 98 (18.4%) cases, the absolute calculated likelihood was >85%. When the absolute calculated likelihood is ≥85%, the result is not likely to be questioned by the reader. Two of four isolates of Y. frederiksenii were misidentified as Y. enterocolitica, as were 1 of 12 Y. bercovieri isolates, 1 of 11 Y. mollaretii isolates, and 1 of 10 Y. rohdei isolates. However, pathogenic strains of Y. enterocolitica were correctly identified to the species level significantly more often than were nonpathogenic strains, presumably because of their higher metabolic activity. We made several attempts to improve inconclusive results by modifying the test procedure. Using an inoculum equivalent to a McFarland turbidity standard of no. 2 instead of no. 1 failed to influence profile generation (data not shown).
TABLE 3.
Results of studies evaluating identification of Yersinia spp. by commercial systems
| Test | Organism | No. of isolates | % Of isolates correctly identified
|
Reference | |
|---|---|---|---|---|---|
| Genus level | Species level | ||||
| API 20E | Y. enterocolitica | 15 | 100 | 100 | 13 |
| Crystal | Y. enterocoliticay | 14 | 100 | 100 | 13 |
| Crystal | Yersinia spp. | 24 | 88 | 12 | |
| Vitek GNI+ | Yersinia spp. | 75 | 12 | ||
| API 20E | Yersinia spp. | 105 | 93 | 4 | |
| API 20E | Yersinia spp. | 183 | 90 | 14 | |
| Vitek GNI | Yersinia spp. | 196 | 96 | 57 | Present study |
Unfortunately, incubation of Vitek cards in an external incubator at 28°C is not possible. We therefore stored cards with inconclusive results at room temperature for another 24 h and read the reactions manually, but these readings were no more conclusive (data not shown). Since the Vitek GNI card attributed 96.3% of all strains to the genus Yersinia within 9.7 ± 2.7 h, we conclude that the system can be used effectively to screen for Yersinia spp. among Enterobacteriaceae. Other methods such as traditional macroscale biochemical testing or sequencing of 16S rRNA should be used for definitive species identification.
Acknowledgments
This work was supported in part by bioMérieux Vitek, Inc.
We gratefully acknowledge the technical assistance given by Marianne Eitel.
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