Detection and isolation of Campylobacter spp. from raw meat

Authors: He, Yiping; Capobianco, Joseph; Armstrong, Cheryl; Chen, Chinyi; Counihan, Katrina; Lee, Joseph - Joe; Reed, Sue; Tilman, Shannon

Submitted to: The Journal of Visualized Experiments (JoVE)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/23/2023
Publication Date: 2/23/2024
Citation: He, Y., Capobianco Jr, J.A., Armstrong, C.M., Chen, C., Counihan, K.L., Lee, J., Reed, S.A., Tilman, S.M. 2024. Detection and isolation of Campylobacter spp. from raw meat. The Journal of Visualized Experiments (JoVE). http://dx.doi.org/10.3791/66462.
DOI: https://doi.org/10.3791/66462

Interpretive Summary: Campylobacter is the most common foodborne pathogen with the CDC attributing an estimated 1.5 million cases of infections to it annually in the United States. This pathogen is common to raw poultry products and despite establishments enacting measures to reduce the prevalence throughout their facilities, contaminated products consistently reach consumers. A method for effective isolating and detecting Campylobacter spp. from raw poultry was developed which based upon the high motility feature of this organism and utilizes rapid molecular diagnostics to enhance sensitivity, enable species identification and strain typing. The technique is congruent with current sampling protocols used by inspection program personnel (IPP) from the Food Safety and Inspection Service (FSIS) and addresses limitations of existing methods for isolating and subsequent characterization of the pathogens. The implementation can contribute to efforts to harvest strains for epidemiological studies while also potentially improving public health by reducing the incidence of Campylobacter infections associated with poultry consumption.

Technical Abstract: This article presents a rapid yet robust protocol for isolating Campylobacter spp., specifically focusing on Campylobacter jejuni and Campylobacter coli, from raw meats. The protocol builds upon established methods, ensuring compatibility with the prevailing techniques employed by regulatory bodies such as the Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) in the USA, as well as the International Organization for Standardization (ISO) in Europe. Central to this protocol is the collection of a rinsate, which is concentrated and resuspended in Bolton Broth media containing horse blood. This medium has been proven to facilitate the recovery of stressed Campylobacter cells and reduce the required enrichment duration by 50%. The enriched samples are then transferred onto nitrocellulose membranes on brucella plates. To improve the sensitivity and specificity of the method, 0.45 and 0.65 µm pore-size filter membranes were evaluated. Data revealed a 29-fold increase in cell recovery with the 0.65 µm pore-size filter compared to the 0.45 µm pore-size, without impacting specificity. The highly motile characteristics of Campylobacter allow cells to actively move through the membrane filters towards the agar, which enables effective isolation of pure Campylobacter colonies. To identify the isolates at the species level, the protocol incorporates the use of multiplex real-time quantitative polymerase chain reaction (mqPCR) assay. This molecular technique offers a reliable and efficient means of species identification. Investigations conducted over the past 12 years involving retail meats have demonstrated the ability of this method to enhance recovery of naturally contaminated samples compared to current reference methods. Furthermore, this protocol boasts reduced preparation and processing time. As a result, it presents a promising alternative for the efficient recovery of Campylobacter from meat. Moreover, this protocol can be seamlessly integrated with DNA-based methods, facilitating rapid screening of positive samples alongside whole-genome sequencing analysis.