Tiziana Nicola (Kulmbach / DE), Sonja Lick (Kulmbach / DE), Dagmar Adeline Brüggemann (Kulmbach / DE), Jan Felix Evers (Heidelberg / DE), Sebastian Knorr (Kulmbach / DE)
Introduction
Campylobacteriosis is the most common food-borne disease caused by bacteria. During slaughtering, chicken skin can be contaminated by Campylobacter spp. due to the evisceration process. Although the number of Campylobacter spp. on broilers is controlled by process hygiene criteria that are constantly being improved, the number of human infections still remains high. Under stress conditions, Campylobacter spp. can enter a viable but non-culturable (VBNC) state, which is poorly understood. VBNC Campylobacter spp. cannot be detected with the DIN EN ISO 10272-2:2017 method and therefore an underestimation of the number of live Campylobacter spp. on chicken might be the result.
Goals
The aim was to develop a quantification method for live Campylobacter spp. cells in chicken meat samples with fluorescence microscopy, combining live-dead and species-specific antibody staining.
Methods
Campylobacter jejuni cells were embedded in agarose pads prepared either with Bolton broth or unsterile chicken rinse. They were either stained directly or incubated at 42 °C for 22 h under microaerobic conditions until microcolonies (µCFU) were formed. For live cell staining, tetrazolium salt CTC, an indicator for metabolic activity, was used. In addition, a specific fluorophore-tagged antibody was applied. Using CLSM, 3D-scans of a defined volume were taken. Campylobacter live numbers were automatically counted where CTC and antibody signals were co-localized.
Results
Live-dead discrimination could be achieved quantitatively in serial tenfold dilutions of dead and living C. jejuni cells. Absolute quantification was linear for µCFU formation within a range of 3x 101-105 µCFU/mL and for single cells in a range of 103-106 cells/mL, both in Bolton broth and chicken rinse.
Summary
A new 3D-scanning microscopy method has been developed for quantitative detection of low Campylobacter spp. numbers in a complex agarose-based growth matrix, using fluorophore-tagged Campylobacter-specific antibodies and CTC for live-dead discrimination. The method requires little time and sample preparation and allows simultaneous detection of reproducing cells (µCFU), VBNCs and dead cells.