Effect of incubation temperature, time, and freezing on diversity of Campylobacter spp. recovered from naturally contaminated retail beef livers

Retail beef livers are often contaminated with Campylobacter species and represent a potential transmission route for human infections. The aim of this study was to evaluate the effect of incubation temperature and time on the isolation rate of different Campylobacter species from retail bovine liver juice samples, and the effect of freezing on the recovery of Campylobacter spp. from the naturally contaminated liver samples. Fresh retail beef liver packages (N = 20), packed in modified atmosphere, were purchased from local stores and examined using direct culture on mCCDA. All colonies grown under microaerobic conditions at either 37 °C or 41.5 °C were identified to genus and species levels using real-time colony polymerase chain reaction, and a representative collection of the Campylobacter isolates was further characterized using whole-genome sequencing by Illumina. The plates were inspected after incubation of 24, 48, and 72 h. Liver pieces were stored in the freezer at -20 °C and re-examined after thawing using both direct culture and enrichment in parallel. Campylobacter spp. were detected from 17 (85 %) of the fresh beef liver juice samples, compared with four (20 %) after freezing (P < 0.0001). C. jejuni was the most common species detected in all 17 Campylobacter-positive samples, followed by C. fetus detected in 6 samples prior to freezing. C. coli, C. lari, and C. hyointestinalis were not detected in any of the samples. The isolation rate of C. fetus from beef liver juice improved by lowering the incubation temperature from 41.5 °C to 37 °C and extending the incubation time from 48 to 72 h. Also, C. jejuni was isolated more often at 37 °C than at 41.5 °C. C. fetus subsp. fetus sequence type (ST)-6 was the most common finding from fresh liver juice samples (5/20), followed by C. jejuni ST-48 (4/20) and ST-58 (4/20). Several samples were contaminated with multiple STs or species. Freezing caused a 0.6-1.4 log reduction in the Campylobacter spp. count but did not completely eliminate the risk of transmission, particularly for samples starting with high-level natural contamination.