A4. Development of rapid methods for identification and quantification of microbiological contaminants in food

Over the last 15 years there has been an increased interest in Campylobacter spp. within the Food Industry. The number of reported infections caused by the intake of Campylobacter spp. by food increases every year. In several developed countries the incidence of campylobacteriosis exceeds even the combined infection rates of Salmonella and Shigella. Campylobacters are commensal microflora of the intestinal tracts of warm-blooded animals for example cattle, swine, sheep and poultry. During the slaughtering process and further processing steps, campylobacters can easily be spread from the intestinal contents to the carcasses. Reported prevalence studies have shown that up to 100% of all poultry products sold at retail are contaminated with campylobacters. The consumer can become infected by inadequate cooking practices or by cross-contamination; moreover, the minimum dose of infection for Campylobacter is very low (approximately 500 cells).

In a screening test for the presence of Campylobacter spp. on a large number of chicken and poultry products, sampled in slaughterhouses, wholesale and retail stores, more than 30% of the samples were found positive for Campylobacter spp. Most of the strains were identified as C. jejuni. The conventional detection methods for Campylobacter spp. are time-consuming and labour-intensive and because of this two commercially available DNA testkits for the detection of Campylobacter in foods were evaluated on speed, specificity and sensitivity. Both kits showed a specificity of almost 100%. To obtain a positive signal on the other hand, the presence of more than 10⁷ Campylobacter cells per ml seemed to be necessary, which means the need of a prolonged enrichment stage. In this way, the high speed of the detection kit (45 min. to 3 h) is disposed by an enrichment period of 2 days. Above all, an adequate technique to shorten this enrichment stage is necessary. In this research project we chose the application of the immunomagnetic separation (IMS) technique. In this technique microorganisms are concentrated and isolated out of a food sample by binding to specific antibodies coated on magnetic particles. By the application of a magnetic field, these particles together with the bounded organisms are separated and detected by plating or by using them in a rapid detection method. In this research project we used the commercially available Dynabeads© M-280 tosylactivated and coated them with the commercially available goat anti-Campylobacter antibodies. The tests established of a good binding between Dynabeads and the antibody on the one hand and between the antibody and the antigen (Campylobacter) on the other hand.

In tests with pure cultures of Campylobacter spp. in liquid media we tried in the first place to optimize the IMS technique by examining the most important process parameters. By the determination of the process sensitivity and specificity with pure cultures, recovery values up to 35% were achieved, while under optimal conditions a detection limit of approximately 200 Campylobacter cells was obtained. By using the IMS technique in mixed cultures, the occurence of cross-reactivity and the formation of non specific binding with other organisms were established. Finally, the IMS technique has been applied to 90 naturally contaminated poultry samples. After a preenrichment and enrichment (7 h) period, the IMS technique was performed in parallel with the conventional detection methode for Campylobacter. After separation the beads were streaked onto a number of selective isolation media for Campylobacter spp. on the one hand and applied in a specific PCR assay on the other hand. Screening of the 90 poultry samples with the conventional method resulted in 44 positive samples (49%). With the combination of IMS and plating only 12% of the samples were found positive, while the combination of IMS with PCR gained 57 positive samples (63%). A specific concentration technique (IMS) in combination with a sensitive and very specific identification method (PCR) not only detected more positive samples, moreover the results were obtained in a shorter time. While the conventional detection methode gives a presumptive result at the earliest after 72 h, the combination of IMS - PCR gives a specific result within 30 h and under optimal conditions even within 12 h.

The conventional methods used for the detection and identification of campylobacters are time demanding, tedious, and sometimes even unreliable. An alternative fast and sensitive detection method is the polymerase chain reaction or PCR. Our first approach was a method based on 16S rDNA. On the basis of available and new 16S rDNA sequences determined at our laboratory, universal PCR primers and group specific oligonucleotide probes were developed. PCR amplification and subsequent hybridisation, results in a group specific detection of entheropathogenic campylobacters. Because the 16S rDNA did not allow us to develop species specific detection methods, we switched to the use of 23S rDNA.

Based on the 23S rDNA, a method was developed for the specific detection of Campylobacter and Arcobacter. On the basis of approximately 40 sequences of a variable area of the 23S rDNA, determined at our laboratory, specific PCR primers were developed for the (a) group specific detection of thermophilic campylobacters, (b) species specific detection of thermophilic campylobacters, (c) species specific detection of nonthermophilic campylobacters, (d) group specific detection of arcobacters, and (e) species specific detection of arcobacters.

Using the thermophilic and nonthermophilic Campylobacter specific PCR primers, several clinical isolates that could not be identified with other methods, could be identified.

The PCR assay for the group specific detection of thermophilic campylobacters was used for the detection of Campylobacter spp. in enriched food samples. 144 poultry samples were investigated, of which approximately 46% gave a positive result with the PCR assay and of which approximately 43% gave a positive result with the conventional methods. Contradictory results were obtained for approximately 34% of the food samples. Further investigations showed that these discrepancies were not the result of the detection of dead Campylobacter cells by the PCR assay or of a difference of the detection limit between both methods.