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Journal of Clinical Microbiology, November 1998, p. 3366-3368, Vol. 36, No. 11
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Comparison of Systems for Identification and
Differentiation of Species within the Genus
Yersinia
Heinrich
Neubauer,1,*
Thomas
Sauer,2
Heinz
Becker,2
Stojanca
Aleksic,3 and
Hermann
Meyer1
Institute of Microbiology, Federal Armed
Forces Medical Academy, D-80937 Munich,1
Institute of Milk Hygiene, Ludwig-Maximilians University,
D-80539 Munich,2 and
Institute for
Hygiene, National Reference Center for Yersiniosis, D-29539
Hamburg,3 Germany
Received 3 April 1998/Returned for modification 2 June
1998/Accepted 18 August 1998
 |
ABSTRACT |
Of four tested identification systems (API 20E, API Rapid 32 IDE,
Micronaut E, and the PCR-based Yersinia enterocolitica
Amplification Set), API 20E is still the system of choice for
identifying pathogenic Yersinia isolates. It
provides the highest sensitivity both at the genus and at the species
level and has the best cost-effectiveness correlation.
| |
TEXT |
The genus Yersinia
consists of 11 species. Y. pseudotuberculosis and some
biovar-serovar combinations of Y. enterocolitica are
pathogenic for warm-blooded animals and humans. They cause a complex
clinical picture known as yersiniosis (1). Slide agglutination tests for the most prevalent pathogenic serovars in
combination with identification systems based on biochemical properties
are used frequently in routine diagnostic practice. The major
disadvantage of this technique is the presence of antigens O:3 and O:9
also in four nonenteropathogenic species, including Y. intermedia (1). Members of apathogenic species can be
isolated from meat and milk of animals and from the stool or blood of
symptomatic and asymptomatic humans (2, 4-7, 20, 21).
Hence, correct typing depends on the knowledge of the reliability of
the test system used.
In the present study, the identifications (by four systems)
of 118 phenetically typed strains (1, 8) from 10 Yersinia species (i.e., all except Y. pestis) were compared. API 20E (BioMerieux, Nürtingen, Germany), API Rapid 32 IDE (BioMerieux), and
Micronaut E (Merlin, Bornheim-Hersel, Germany) were used as instructed
by the manufacturers, except for the incubation of API 20E strips at
28°C (3). For use in the Yersinia
enterocolitica Amplification Set (Kreatech, Amsterdam, The
Netherlands), Yersinia strains were incubated in
Mueller-Hinton broth at 28°C for 12 h, and a 0.5-µl aliquot
was added to a 50-µl PCR mixture containing 5× PCR optimization buffer N (Invitrogen, DeShelp, The Netherlands), PCR Nucleotide Mix
(Roche Diagnostics, Boehringer, Mannheim, Germany), and
Taq polymerase (Applied Biosystems, Weiterstadt, Germany).
Optimization of cycling conditions resulted in initial
denaturation for 10 min at 94°C and 30 cycles each consisting of
denaturation (1 min, 94°C), annealing (1 min, 55°C), and elongation
(1 min, 72°C). Labeling and dot blot hybridization (stringent washing
at 45°C) were done as described previously (15, 18).
The API 20E system is considered the "gold standard" (9, 13,
14, 17) to which new identification systems have to be
compared. For this assay we found an overall sensitivity of 79% (Table
1). Differentiation at the genus level
was 91%. All pathogenic Y. enterocolitica strains
and 95% of apathogenic strains were correctly identified, resulting in
a sensitivity of 96% for the species Y. enterocolitica.
Sensitivity for Y. pseudotuberculosis was 90%. Of the
Y. intermedia strains, 70% were misidentified. The time
needed to get results was 25 h. Our results are in agreement with three preliminary investigations. Archer et al. reported that recording a negative result for the Voges-Proskaner test enhances the sensitivity for Yersinia spp. from 66 to
93% (3). Sharma et al. found a sensitivity of 90% for
Yersinia spp. (19). The sensitivity for Y. intermedia was considered to be unacceptable (19). In
both studies, only clinical isolates were examined and pathogenicity
was not investigated. O'Hara et al. tested 30 Yersinia
strains, resulting in a sensitivity of 70 or 94% when results were
read after 24 or 48 h, respectively (16). The API 20E
system in combination with slide agglutination tests is therefore suited for routine detection of pathogenic Yersinia
isolates. The overall sensitivity of the API Rapid 32 IDE was 86% at
the genus level but only 42% at the species level. It identified 92% of the pathogenic Y. enterocolitica strains and 85% of the
Y. pseudotuberculosis strains. An incubation temperature of
28°C increased the number of nonidentified isolates, as could be
expected on the basis of reaction kinetics. The low sensitivity might
be caused by the weak metabolic activities of the members of the genus.
Results were obtained after 5 h. The Micronaut E system is
comparable to API 20E in its sensitivity at the genus level (92%) and
at the species level (72%). Of the Y. intermedia strains, 94% were misclassified. The codes for Y. intermedia
biotypes have to be revised. Due to the computer program used, reading
of the results occurs after 24 h. Giving only "yes" or
"no" answers, the Yersinia enterocolitica Amplification
Set provides significantly less information than the other systems. The
sequences of the sets' primers and gene probe are not available. The
PCR product is approximately 400 bp long. The overall sensitivity of
85% mimics the actual sensitivity of 80% for the species Y. enterocolitica and Y. intermedia, causing
false-positive reactions. A total of 14 apathogenic Y. enterocolitica and 3 Y. intermedia strains were misidentified. The reason for this loss in sensitivity may be the
presence of various 16S rRNA genomospecies in the
investigated strains (10-12). Further drawbacks are the
recommended procedure of end labeling the provided oligonucleotide and
the lack of DNA of type strains for optimization. The test procedure is
time-consuming (two working days) and sophisticated and therefore not
suited for routine diagnosis. API 20E turned out to be the most
cost-effective test, followed by Micronaut E, API Rapid 32 IDE,
and the PCR assay. The reading and computing device for the Micronaut E
system is essential and has to be considered as an important cost
factor. Detection of pathogenic Yersinia isolates is a
problem not only for physicians and veterinarians but also for anyone
dealing with food and water hygiene. Although reclassification of the
genus Yersinia was completed in 1988, resulting in seven new
species (2, 4, 5, 7, 20, 21), this increase in number is not
reflected in the indices of the identification kits. The API 20E index
lists seven species, and the API Rapid 32 IDE and Micronaut E indices
list only six species each. A correction is overdue. The close
relationship of various Yersinia species with regard to
biochemical characteristics requires an optimal combination of key
reactions for differentiation at the species level. None of the four
kits tested in the present study solved this problem completely. The
most important reactions (Simmons citrate, sorbose, saccharose,
melibiose, and rhamnose) for distinguishing Yersinia species
are not present or are simply interpreted divergently. Additional tube
testing should be advised in the indices of the kits. Compared to the
API 20E or Merlin E system, there is no advantage in using diagnostic
PCR systems based on 16S rRNA gene sequences as long as no clear
definition of the connection between genomospecies and phenotypic
species exists. Still, seeking the expertise of a reference center is
advised in cases of doubt.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Mikrobiologie, Sanitätsakademie der Bundeswehr,
Neuherbergstr. 11, D-80937 Munich, Germany. Phone: 49-89-3168-3113. Fax: 49-89-3168-3829. E-mail: 320027945810{at}t-online.de.
| |
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Journal of Clinical Microbiology, November 1998, p. 3366-3368, Vol. 36, No. 11
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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