M01033: A study to investigate and establish the best methods for detection of campylobacter in red meat production, and accurate methods for their enumeration from red meats

Study Duration: January 2004 to June 2005

Contractor: Campden and Chorleywood Food Research Association (CCFRA)

Campylobacter is frequently isolated from cattle, sheep and pig faeces as well as poultry. Data relating to the detection of the organism on red meat carcasses or from red meat is both limited and variable in comparison to data from poultry. In addition, Agency projects attempting to reduce levels of campylobacter in poultry are hampered by the inaccuracy of current methods for counting campylobacter.

An evaluation of sampling and recovery techniques for campylobacter from red meat carcasses, cuts and associated environmental samples will be undertaken to establish practical protocols that could be used in the food production environment.

The suitability of the draft International Organization for Standardization (ISO) culture procedure for detection of low numbers of thermotolerant Campylobacter spp. (C. jejuni, C. coli and C. lari) in red meat carcasses, cuts and associated environmental samples will be investigated. Critical factors will be identified with the aim of improving the recovery and performance. The effect on recovery and isolation rates of incorporating an immunomagnetic separation (IMS) step into the optimised campylobacter method will then be investigated.

An evaluation of enumeration procedures for the accurate quantification of campylobacter from poultry and, if appropriate, red meat and associated samples will be undertaken in an attempt to improve the current draft ISO procedure.

This study sought to practically assess the sensitivity and suitability of a range of campylobacter sampling, detection and enumeration procedures for meat samples.

• Sampling: maximum recovery diluent (MRD) was found to maintain the viability of Campylobacter for up to 24 hours on sponges stored at 2-8 ºC whereas sponges pre-moistened with sterile water required testing within 4 hours after sampling. Supplementing water soaked sponges after sampling with a small volume (10 ml) of Bolton broth (BB) containing haemin prolonged the viability of Campylobacter cells for up to 24 hours storage at 2-8 ºC.
• Detection:the best media and procedures for the detection of low numbers of Campylobacter in red meat samples resembled the ISO method (ISO/DIS 10272-1) with modifications described below:
• Aerobic incubation (instead of microaerobic incubation) of enrichment broths produced satisfactory growth of Campylobacter provided samples were enriched in containers with minimum headspace.
• Incubating plates for up to 5 days was recommended as plates inspected after 48 hours occasionally failed to yield visible Camplyobacter growth.
• The combination of Modified Charcoal Cefoperazone Desoxycholate agar (mCCDA) and Preston agar (PA) used in parallel provided good isolation of Campylobacter.
• Evaluating the recovery and isolation rates of Campylobacter using immunomagnetic separation (IMS) was not carried out in this project due to manufacturer difficulties.
• Enumeration: direct plating onto a suitable selective medium appeared to be a reasonably reliable and simple method for enumerating Campylobacter. However, this method may not be suitable for stressed or sub-lethally injured cells. Furthermore, practical difficulties identifying and counting Campylobacter colonies can arise if plates are not dried correctly or because of interference from competitor organisms. Ensuring that plates are sufficiently dry before use and incorporating the supplement triphenyltetrazolium chloride (TTC) into mCCDA can assist with colony recognition and confirmation.
• The MPN procedure was labour and material intensive and proved unreliable for Campylobacter enumeration in this study.
• The SimPlate Campylobacter test used generally showed good agreement with direct plate counts
• A semi-quantitative culture method (NMKL) and semi-quantitative real-time PCR test provided an acceptable estimate of Campylobacter numbers, but the development of quantitative real-time PCR approaches, not used in this study, could be investigated further.

The final report is available from the Agency’s Information Centre.
To obtain a copy, please contact the Enquiry Desk, Information Services, Food Standards Agency (tel: 020 7276 8181/8182 or email: infocentre@foodstandards.gsi.gov.uk)

For any enquiries concerning this research project, please contact the relevant Programme contact or email: science@foodstandards.gsi.gov.uk