High-Resolution Genotyping of Campylobacter Strains Isolated from Poultry and Humans with Amplified Fragment Length Polymorphism Fingerprinting

Abstract

For epidemiological studies of Campylobacterinfections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing ofCampylobacter jejuni and Campylobacter colistrains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleasesHindIII and HhaI were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amplified fragments ranging from 50 to 500 bp long. The discriminatory power of AFLP was assessed with aC. jejuni strain, an isogenic flagellin mutant, and distinct C. jejuni strains having known pulsed-field gel electrophoresis and fla PCR-restriction fragment length polymorphism genotypes. Unrelated C. jejuni strains produced heterogeneous patterns, whereas genetically related strains produced similar AFLP patterns. Twenty-five Campylobacterstrains obtained from poultry farms in The Netherlands grouped in threeC. jejuni clusters that were separate from a C. coli cluster. The band patterns of 10 C. jejunistrains isolated from humans were heterogeneous, and most of these strains grouped with poultry strains. Our results show that AFLP analysis can distinguish genetically unrelated strains from genetically related strains of Campylobacter species. However, desirable genetically related strains can be differentiated by using other genotyping methods. We concluded that automated AFLP analysis is an attractive tool which can be used as a primary method for subtyping large numbers of Campylobacter strains and is extremely useful for epidemiological investigations.

Identification of taxonomic and epidemiological relationships among Campylobacter species by numerical analysis of AFLP profiles

“…These results indicated the method to be as sensitive as macrorestriction pro¢ling with each of four enzymes, and also suggested potential as an identi¢cation tool, since di¡erences in AFLP pro¢les between the two species were evident. Shortly after our study was published, Duim et al [9] showed cluster analysis of HindIII-HhaI-derived AFLP pro¢les delineated C. jejuni subsp. jejuni and C. coli to di¡erent clusters.…”

“…These results may be due to the presence of restriction modi¢cation systems that inhibit the action of one or both of the enzymes used. Such phenomena have been described in Campylobacter species before [6,9,10]. Because of these results, C. helveticus was not considered in any numerical analyses.…”

Scientific Opinion on Quantification of the risk posed by broiler meat to human campylobacteriosis in the EU

“…Most (47 of 64) human isolates in this study were in human-only clones. In a Dutch study, including only human and chicken isolates, 3 main clusters were identified, each of which included isolates of both human and chicken origin with no cluster restricted to either host (Duim et al, 1999). In a second Dutch study, including human, chicken and cattle isolates, again no convincing evidence was found for a hostassociation using AFLP .…”

“…The consequence is that it is not possible to combine the data from the above PFGE studies for joint analysis. Five AFLP studies have assessed host association and possible sources of human infection but there is little evidence for the capacity of this method to predict source (Duim et al, 1999;.…”

“…Pulsed-Field Gel Electrophoresis (PFGE) Yan et al, 1991)) or amplification of DNA (e.g. Randomly Amplified Polymorphic DNA (RAPD) ) or a combination of both such as Amplified Fragment Length Polymorphism (AFLP) (Duim et al, 1999). These approaches generate band patterns representing the genome wide distribution of the binding sites of the enzymes or primers used.…”