Molecular typing of Vibrio parahaemolyticus isolates originating from different sources by DNA microarray analysis Christin Eichhorn1, Kati Tschischkale1, Ralf Ehricht2, Peter Slickers2, Eckhard Strauch3, Florian Gunzer1 1TU
Dresden, Institute for Medical Microbiology and Hygiene, Faculty of Medicine Carl-Gustav-Carus, Dresden, Germany 2Alere Technologies, Jena, Germany 3Federal Institute for Risk Assessment, Department Biological Safety, Berlin, Germany Contact:
[email protected]
INTRODUCTION Vibrio parahaemolyticus is one of the 12 pathogenic species of the genus Vibrio. Its natural habitat is the marine environment and the pathogen is a leading cause of seafood borne gastroenteritis. In most cases, genes encoding the thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH) were found in clinical strains and are considered as typical virulence factors of this species. We were interested to identify additional virulence determinants that have impact on the pathogenic potential of this Vibrio species. The aim of this study was to describe parameters that allow us to distinguish between environmental V. parahaemolyticus isolates and pathogenic strains, which can cause human infections. To achieve this, we have analyzed more than 60 V. parahaemolyticus strains originating from different sources.
METHODS
RESULTS
The array enables us to detect non-cholerae Vibrio spp. and to investigate the presence of virulence determinants in a fast and easy way. The detection system uses simultaneous amplification and biotinylation of relevant target sequences by linear multiplex PCR. Positive hybridizations to specific DNA probes are visualized by an enzyme-mediated precipitation reaction. Analysis and evaluation of densitometry measurements is performed on an automated array scanner. The report of experimental data including a patternmatch, species affiliation and the genomic profile of each Vibrio parahaemolyticus strain (see Fig. 1).
More than 60 V. parahaemolyticus strains were analyzed (see Fig. 2). With the aid of 222 DNA probes, spotted in duplicate on the glass chip, 93 target genes of different Vibrio spp. can be detected, chosen from genes encoding adherence and invasion mechanisms, capsule synthesis, exoenzymes and toxins, type III secretion systems and resistance mechanisms. Based on the hybridization results, we established molecular fingerprints of human, environmental and food isolates of V. parahaemolyticus (see Fig. 3). According to DNA microarray data, there is a tendency that eight target genes were detected predominantly in human V. parahaemolyticus strains compared to environmental and food isolates (see Fig. 4). A
Vp VN-0003
B
Vp RIMD 2210633
Fig. 3: Scanning image of array hybridization signals obtained with biotin-labeld amplicons from two Vibrio spp. strains: (A) V. parahaemolyticus VN-0003 – environmental strain, isolated 1999 from seawater in Germany; (B) V. parahaemolyticus RIMD 2210633 – genome sequenced clinical reference type strain. 16 14
Fig. 1: Excerpt of an automated analysis report of a microarray experiment.
Number of isolates
12 10 8
Sample origin
6
Environmental Human Seafood
4 2 0
14 28
Environmental Human Seafood
20
Fig. 2: Sample origin of V. parahaemolyticus isolates used in this study.
Hybridization probes Fig. 4: Probes of Vibrio-microarray 2.0 with predominantly positive hybridization signals within the group of human V. parahaemolyticus isolates (ntotal = 62, see Fig. 2). Probes represented following products coding by target genes: tdh – Thermostable direct hemolysin; VP0226 – Putative rhamnosyl transferase from K antigen biosynthesis gene cluster; VP2134 – Uncharcterized protein of Vp island-4 (VPaI-4); VPA1255 – GGDEF family protein of genomic island VPaI6; VPA1268 – Uncharcterized protein of genomic island VPaI-6; VPA1327 – Exoenzyme T (T3SS2 secreted effector); VPA1333 – Transcriptional regulator; VPA1357 – Possible membrane protein of 1622 aa.
SUMMARY The investigation of a series of V. parahaemolyticus isolates with this newly developed DNA microarray allows for a very detailed characterization of these strains, based on molecular fingerprints. The results will help us to establish a risk matrix that can be used for pathotyping of individual V. parahaemolyticus isolates. TH 68
ANNUAL MEETING OF THE GERMAN SOCIETY OF HYGIENE AND MICROBIOLOGY (DGHM) ULM, 2016
