Quantitative comparison of thermotolerant Campylobacter spp. growth in Bolton and Preston broth with and without growth supplement (FBP)oonoses Monitoring from 2016-2023 within the food production chain

Thermotolerant Campylobacter spp., the leading cause of foodborne bacterial diarrhoea in Europe, are fastidious in vitro. Thus, a cultural detection can be challenging. ISO 10272-1:2017/Amd 1:2023 recommends direct streaking or enrichment with either Bolton or Preston broth supplemented with growth supplement (FBP), depending on the expected number of C. spp. and microbiota. Here, we compared the growth performance of C. jejuni, C. coli and C. lari field isolates using Bolton broth, Preston broth and Preston broth with FBP. Media performance testing was investigated using 123 C. spp. isolates from food and animal matrices according to ISO 11133:2018. The presence of FBP supplement in Preston broth reduced false negative results in 100% of the C. jejuni, 60% of the C. coli, and 84% of the C. lari isolates on mCCDA. In comparison, 98% Bolton enrichments supported C. spp. detection. Qualitative growth was quantitatively confirmed by Real-time PCR (qPCR). In addition, chicken meat, chicken neck skin, frozen spinach and egg shells were pre-inoculated with representative C. spp., either in exponential growth phase (“fresh bacteria”) or after stress exposure upon incubation with matrix at cooling conditions before enrichment. Except for chicken neck skin, addition of matrix enhanced growth of “fresh” C. spp. in Preston for up to ~5.5log10 C. spp./ml and further addition of FBP had either no effect or a marginal extra growth enhancement of up to 1.5log10 C. spp./ml. Enrichment in Bolton (24 or 48h incubation) always showed highest detection ~8-9log10 C. spp./ml. Using naturally stressed C. spp. cells pre-incubated with matrix at cooling conditions, addition of FBP to Preston broth drastically enhanced growth of stressed C. spp. in raw milk from a level of <1log10 C. spp./ml to ~6log10 C. spp./ml. Stressed C. spp. in chicken meat, egg shell or iceberg lettuce reached comparable growth levels in Preston, Preston + FBP and Bolton after 24 h, while maximal growth levels of ~8log10 C. spp./ml were observed in Bolton after 48h incubation. Our data suggest that testing naturally stressed Campylobacter spp. in different matrices is important for performance evaluation of enrichment media. Whenever low matrix microbiota is expected, Bolton broth might be superior to Preston broth, which can partially be improved by addition of FBP supplement.