*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: ivo@sci.muni.cz

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