Phylogenetische Charakterisierung von Campylobacter-Isolaten aus der Geflügelschlachtung

Thermophylic Campylobacter spp. present one of the most important bacterial foodborne infection agents in Germany and Europe. To analyse phylogenetic relationship of Campylobacter-infections in poultry 382 Campylobacter-isolates were typed by means of Restrictions-Fragment-Length-Polymorphism (RFLP) using the enzymes Smal respectively Kpnl and totalgenomic DNA. The isolates originated from 12 slaughter-flocks from seven different farms in Northern-Germany and were collected in the years 2005 and 2006. They were stored at -80°C by using a microbank-system. In addition to the isolates from Germany 10 Campylobacter-isolates of hens from Italy and four reference strains were included in the study. The MLST-analysis was based on the sequencing of seven housekeeping-gene sections (aspartate A, glutamine synthetase, citrate synthase, serine hydroxymethyltransferase, phosphoglycomutase, transketolase, ATP synthase) which first had to be amplified using polymerase-chain-reaction (PCR). By using the online database PubMLST allele numbers were assigned to the housekeeping-gene sequences. Sequence types (STs) respectively clonal complexes (CC) were assigned to the isolates by means of their allelic profile. In addition an UPGMA-dendrogramm as well as a population model (minimum-spanning-tree) based on the allelic profile of the isolates typed by MLST was generated. Isolates within RFLP-clusters that were built with a cut-off value of ≥ 90 % similarity had identical STs. Therefore the isolates with a cut-off value ≥ 90 % similarity were merged into clusters flock-wise on the basis of their Smal restriction profile and at least one isolate per cluster was typed by means of Multilocus-Sequence-Typing (MLST). In that way 130 Campylobacter-isolates were analysed by MLST. In total 47 different STs were detected from which 17 STs appeared the first time within this study. Furthermore the allele glyA-303 was described the first time. For the isolates two till seven different STs were detected within the flocks. In comparison of the allele sequences of the housekeeping-genes some alleles stood out with a high rate of nucleotide substitutions. These alleles could be assigned to C. coli-isolates. In the UPGMA-dendrogram which was built on the basis of the allele profile it was obvious that identical STs appeared repeatedly in flocks from different farms. Within the minimum-spanning-tree two clearly separated groups could be distinguished. All the C. coli-isolates belonged to one group and all C. jejuni-isolates belonged to the other group. Only one clonal complex was detected in the group of C. coli-isolates to which 10 out of 15 STs belonged. The other five STs could not be assigned to a clonal complex (singletons). 15 STs were identified as singletons within the group of C. jejuni-isolates. The others were divided into seven clonal complexes. In comparison of the allele sequences as well as in the minimum-spanning-tree the higher genetic diversity of the C. jejuni-isolates compared to C. coli-isolates became clear. It could be demonstrated that different Campylobacter-genotypes appeared within one flock. By the UPGMA-dendrograms developed on the basis of allele profiles showed no farm specific Campylobacter-genotypes.