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*Ivo Sedláček, Tereza Gelbíčová, Eva Kroupová, Pavel Švec
Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
*Corresponding author: [email protected]
Acknowledgements This study was supported by project CEB (CZ.1.07/2.3.00/20.0183).
ReferencesALTWEGG, M. Aeromonas and Plesiomonas. In Murray, PR. et al. Manual of Clinical Microbiology. 7th ed. Washington, D.C.: ASM Press, 2005. p. 507-516.GONZÁLEZ-REY, C., SVENSON, SB., BRAVO, L., ROSINSKY, J., CIZNAR, I., KROVACEK, K. Specific detection of Plesiomonas shigelloides isolated from aquatic environments, animals and human diarrhoeal cases by PCR based on 23S rRNA gene. FEMS Immunol Med Microbiol, 2000, vol. 29, no. 2, p. 107-113.HERRERA, FC., SANTOS, JA., OTERO, A., GARCÍA-LÓPEZ, ML. Occurrence of Plesiomonas shigelloides in displayed portions of saltwater fish determined by a PCR assay based on the hugA gene. Int J Food Microbiol, 2006, vol. 108, no. 2, p. 233-238.
Fig 2. Dendrogram based on cluster analysis of ribotype patterns of P. shigelloides strains.
Fig 1. Dendrogram based on cluster analysis of Biolog results from P. shigelloides strains.
Material and methods
Bacterial strains A set of 45 P. shigelloides strains isolated from different human and veterinary materials worldwide (mainly faeces) was analysed. Reference strains P. shigelloides (CCM 1991, CCM 1996, CCM 5860 and CAPM 5861) were obtained from the Czech Collection of Microorganisms (http://www.sci.muni.cz/ccm/) and from the Collection of Animal Pathogenic Microorganisms (Brno, Czech Republic).
Biotyping The bacteria were classified to the species level by using two commercial identification systems, ENTEROtest 24 (Erba Lachema) and by Biolog GN2 Micro Plate kits (Biolog). Moreover, several conventional tube and plate tests (e.g. catalase, oxidase, OF test, gas from glucose, motility, DNase etc.) were additionally carried out to confirm the genus determination.
Genotyping Multiplex PCR of 23S rRNA gene (González-Rey et al. 2000) and hugA gene (Herera et al. 2006) was used for a species confirmation of strains at the molecular level. The automated ribotyping performed with EcoRI restriction endonuclease using the RiboPrinter system (DuPont Qualicon) was used for a more detailed characterization of the strains.
Results
The phenotypic variability found among the investigated isolates by ENTEROtest 24 was low; all analysed strains revealed almost coherent results mostly corresponding with the species description. Surprisingly, the majority of the isolates (91 %) showed positive DNase activity. In contrast, the biotyping with the commercial kit GN2 Micro Plate identified only 27 strains as P. shigelloides and 11 strains were misidentified as members of the genus Vibrio. However, these strains were confirmed as P. shigelloides by a multiplex PCR technique. Remaining 7 strains were unidentified using the Biolog GN2 kit, but the most closest entry offered by the Biolog identification database was P. shigelloides in all cases.
Similarly, the genetic heterogeneity revealed by the automated ribotyping within the investigated group was rather high. Numerical analysis of the ribotype patterns separated all strains into a few clusters and a few strains formed clearly differentiated singletons. Automated identification provided by the RiboPrinter system identified only strain P2268 as P. shigelloides; remaining strains were not assigned to the species level. No correlation between the biotyping and the automated ribotyping results was found (Fig 1. and Fig 2.).
Classification of Plesiomonas shigelloides
Introduction
Plesiomonas shigelloides represents Gram-negative, motile, facultative anaerobic and oxidase positive bacteria. Plesiomonads are very important enteric bacteria and only one valid species is recognized in the genus Plesiomonas at present. The species P. shigelloides was described as a member of the family Vibrionaceae but it was later reclassified as an enteric pathogen belonging to the family Enterobacteriaceae. Waters with low salinity are predominant sources of plesiomonads, but they are also occurring in different poikilothermic as well as homoiothermic animals and may be responsible for serious gastrointestinal and extra-intestinal infections of animals as well as humans. The alimentary way is crucial for the infection transmission. The aim of our study was a phenotypic (ENTEROtest 24, Biolog system) and genotypic (automated ribotyping) identification and classification of P. shigelloides strains isolated from animals, humans and the environment.
Czech Collection of Microorganismshttp://www.sci.muni.cz/ccm/
Conclusions
Our results confirmed that P. shigelloides strains are phenotypically very diverse. These data clearly showed inconvenience of Biolog GN2 kits to reliably identify plesiomonads – of 45 strains only 27 (60%) were identified correctly.
The biotyping by Biolog system misidentified eleven P. shigelloides strains as members of the genus Vibrio. A multiplex PCR technique based on 23S rRNA and hugA genes was used for the confirmation of the misidentified strains as P. shigelloides (e.g. P2282, P2284, P2287, P2291, P2302, P2303).
The majority of strains (91 %) showed an ability to hydrolyse DNA. This trait represents a putative virulence factor, but P. shigelloides is generally known as a DNase negative species (Altwegg, 2005).
Automated ribotyping revealed high genetic heterogeneity within the analysed strains which implies this technique as a useful tool for typing of plesiomonads but showed ribotyping as unsuitable for their identification.
The investigated strains originate from different sources and geographically distant areas. They could probably represent various biotypes and/or ecotypes of P. shigelloides.
Similarity (%)Biolog identification
100
90
80
70
60
50
40
30
20
Kb
P2273
P2275
P2281
P2284
P2269
P2303
P2268
P2296
CCM 1991
P2283
P2278
P2280
P2301
CCM 5860
P2305
P2294
P2293
P2277
P2297
P2304
P2299
P2300
CAPM 5861
P2271
P2287
P2286
P2302
P2292
CCM 1996
P2288
P2289
P2274
P2279
P2270
P2285
P2266
P2290
P2276
P2306
P2272
P2295
P2298
P2267
P2282
P2291
P. shigelloides
P. shigelloides
No ID
Vibrio sp.
P. shigelloides
Vibrio sp.
No ID
P. shigelloides
P. shigelloides
P. shigelloides
P. shigelloides
P. shigelloides
P. shigelloides
P. shigelloides
P. shigelloides
P. shigelloides
No ID
P. shigelloides
Vibrio sp.
P. shigelloides
P. shigelloides
P. shigelloides
P. shigelloides
No ID
Vibrio tubiashii
No ID
Vibrio sp.
Vibrio sp.
P. shigelloides
P. shigelloides
P. shigelloides
P. shigelloides
P. shigelloides
P. shigelloides
P. shigelloides
P. shigelloides
P. shigelloides
Vibrio sp.
P. shigelloides
Vibrio sp.
No ID
No ID
Vibrio tubiashii
Vibrio sp.
Vibrio sp.
0%
5%
10%
15%
20%
25%
P2306
P2305
P2286
CC
M 5
860
P2298
CC
M 1
991
P2292
P2289
P2278
P2291
P2268
P2273
CC
M 1
996
P2303
P2304
P2272
P2282
P2269
P2299
P2275
P2290
P2300
P2
27
0P
22
93
P2287
P2294
P2284
P2283
P2301
P2280
P2271
P2285
P2281
P2295
P2277
P2296
P2288
P2297
P2279
P2302
P2276
P2274
P2267
P2266
CA
MP
5861