Variability in lag-duration of Campylobacter spp. during enrichment after cold and oxidative stress and its impact on growth kinetics and reliable detection

Abstract

Campylobacter jejuni and Campylobacter coli continue to be the leading cause of zoonotic gastroenteritis in the European Union, making reliable detection in food important. Low storage temperatures and atmospheric oxygen concentrations during food production can cause sub-lethal damage or transient non-culturability which is why ISO 10272-1:2017 includes an enrichment step to repair cell damage and increase cell concentrations, thereby supporting detection of campylobacters from foods.The aim of this study was to assess the variability in lag-duration of C. jejuni and C. coli during enrichment after different food-relevant stress treatments and evaluate its impact on growth kinetics and reliability of detection outcomes. Therefore, 13 C. jejuni and 10 C. coli strains were subjected to cold stress during refrigerated and frozen storage. Refrigerated storage did not significantly reduce culturability, but frozen storage reduced cell concentrations by 1.6 ± 0.1 log10cfu/ml for both species.Subsequently, cells were enriched following ISO 10272-1:2017-A and cell concentrations were determined over time and lag-duration and growth rate were determined by fitting the Baranyi-model. Without prior stress treatment, mean lag-duration for C. jejuni and C. coli was 2.5 ± 0.2 h and 2.2 ± 0.3 h, respectively. Refrigerated storage increased lag-duration for C. jejuni to 4.6 ± 0.4 h and for C. coli to 5.0 ± 0.4 h and frozen storage increased lag-duration to 5.0 ± 0.3 h and 6.1 ± 0.4 h for C. jejuni and C. coli, respectively.Comparison of strain- and biological variability showed that differences in recovery after cold stress can be attributed mainly to strain variability since strain variability after refrigeration and freeze stress increased respectively 3-fold and 4-fold while biological variability remained constant.A subset of strains was also subjected to oxidative stress that reduced cell concentrations by 0.7 ± 0.2 log10 cfu/ml and comparison of recovery patterns after oxidative and freeze stress indicated that recovery behaviour was also dependent on the stress applied. A scenario analysis was conducted to evaluate the impact of heterogeneity in outgrowth kinetics of single cells on the reliability of detection outcomes following ISO protocol 10272-1:2017. This revealed that a ‘worst-case’-scenario for successful detection by a combination of the longest lag-duration of 7.6 h and lowest growth rate of 0.47 h−1 still resulted in positive detection outcomes since the detection limit was reached within 32.5 h. This suggests that other factors such as competitive microbiota can act as a causative factor in false-negative outcomes of tested food samples.