Characterization of the Clostridium perfringens and Clostridium difficile hazards in the bovine, pig and poultry sectors in slaughterhouses – ClostAbat

The project methodology combined field sampling campaigns, microbiological and genomic analyses, as well as the experimental evaluation of control practices and technologies to investigate C. perfringens and C. difficile in the bovine, porcine, and poultry sectors.

Sampling was carried out over 18 months, through 8 to 12 visits per sector, in three slaughterhouses representative of each production system, in order to account for herd diversity, seasonality, and hygiene conditions. Samples included feces and carcasses along the slaughter line, meat cuts in processing plants, and elements of the production environment (equipment, machines, surfaces, air), along with measurements of temperature, humidity, and air flow to characterize physical conditions.

Each sample underwent an enrichment step followed by isolation on selective media specific to each pathogen, enabling the detection and recovery of representative colonies for the study of their genetic diversity and pathogenic potential. Confirmed isolates were characterized: for C. perfringens, molecular typing and detection of toxin-encoding genes were performed by PCR, while for C. difficile, toxin gene detection was carried out by PCR, complemented by ribotyping and antimicrobial resistance profiling. These data enabled the selection of representative strains for population structure analysis and source attribution.

Selected isolates were sequenced to characterize population structure and identify potential persistence of strains within slaughterhouses, while the diversity and composition of surface-associated bacterial communities were analyzed through 16S rDNA sequencing to assess correlations between pathogen presence and the abundance of other taxa. Genomic source attribution models and machine learning approaches, based on allelic variation and gene presence/absence, were applied to identify the origin of strains and genomic signatures of host adaptation.

Finally, risk practices in slaughterhouses were assessed and compared with recorded physical parameters to determine their impact on contamination, survival, and resistance of both vegetative cells and spores. The effectiveness of an LED-based light treatment was evaluated on contaminated surfaces and meat, including the analysis of induced cellular alterations.

This integrated approach provided a comprehensive understanding of pathogen contamination, diversity, and persistence, paving the way for concrete recommendations to improve meat safety.

The ClostAbat project aimed to improve understanding of the presence, spread, and persistence of two bacteria of public health concern, Clostridium perfringens and Clostridioides difficile, in French cattle, pig, and poultry slaughterhouses. These bacteria are naturally carried by animals, can contaminate meat, and in some cases cause human disease.

Between 2022 and 2023, nearly 1,900 samples were collected in nine representative slaughterhouses, including animal samples (faeces), carcasses and meat cuts, as well as the production environment (surfaces, equipment, and air). This comprehensive approach made it possible to track contamination throughout the slaughter process.

The results show that Clostridium perfringens is widely present across all sectors. It is frequently detected in animals and slaughterhouse environments, making it a “structural” hazard. However, most of the strains identified are weakly virulent for humans, and the observed contamination mainly results from repeated introduction of bacteria via incoming animals rather than from long‑term establishment inside slaughterhouses. Nevertheless, when control measures are insufficient, temporary dissemination to surfaces, air, and meat can occur.

In contrast, Clostridioides difficile was detected much less frequently. It was absent in the cattle sector, very rarely found in pigs, and mainly detected in the poultry sector. The identified strains showed high genetic diversity but no evidence of long‑term persistence in the slaughterhouses studied.

The project also demonstrated that contamination levels are not determined solely by animal carriage, but largely depend on hygiene practices, process organisation, and environmental conditions—particularly air humidity and surface management. Experimental studies showed that highly humid conditions favour bacterial survival, whereas innovative approaches such as light‑based treatments can effectively reduce vegetative forms of C. perfringens.

In conclusion, ClostAbat highlights that effective control of contamination in slaughterhouses primarily relies on good environmental management and professional practices. The results provide a strong scientific basis to improve meat safety, better protect consumers, and support food sectors within a prevention framework grounded in the “One Health” approach.

The ClostAbat project opens up several important scientific, methodological, and operational perspectives for improving the understanding and control of spore‑forming bacteria of public health concern in slaughterhouse environments and along the food chain.

From a scientific standpoint, the results highlight the need to further investigate the ecological mechanisms governing the persistence and dissemination of Clostridium perfringens and Clostridioides difficile, with a particular focus on their interactions with environmental microbiota, surfaces, air, and physicochemical parameters such as humidity. Future studies integrating multi‑scale approaches—combining genomics, microbiology, environmental monitoring, and process data—will be essential to better explain the strong variability observed between sectors and individual establishments.

From a methodological perspective, the tools and analytical frameworks developed within ClostAbat, notably genomic source attribution models, microbiota analyses of low‑biomass environments, and multivariate approaches to link contamination profiles with practices, offer strong potential for reuse and extension. These approaches could be applied to other pathogenic or spoilage bacteria and adapted to additional food production contexts beyond slaughterhouses. Improving sampling strategies and analytical sensitivity in low‑contamination environments also remains a key challenge and an important avenue for future research.

At an operational level, the project paves the way for the development and evaluation of integrated contamination control strategies that combine hygiene practices, environmental management, and innovative technologies. The promising results obtained with light‑based treatments, particularly against vegetative forms of C. perfringens, justify further work to assess their feasibility, effectiveness, and integration under real industrial conditions. In the longer term, ClostAbat contributes to the foundations required to build predictive models and decision‑support tools that can help food sector stakeholders and health authorities better anticipate risk situations and prioritise control measures.

Overall, these perspectives reinforce the relevance of a prevention strategy grounded in the One Health approach, linking animal, environmental, and human health within a sustainable food safety framework.

Clostridium perfringens and Clostridioides difficile are major pathogenic bacteria involved in human infections. C. perfringens is also one of the bacterial hazards identified in the Guides to Good Hygiene Practices and of application of HACCP principles in the slaughtering and cutting of bovine, pig and poultry sectors. As animals are healthy carriers of both species in their digestive tract, animal meats are privileged targets to explain the occurrence of toxic episodes involving these pathogens. Without questioning the adequacy and relevance of the control measures implemented, little or no data are currently available in France on the importance of these two hazards in slaughterhouses and cutting plants.

The ClostAbat project brings together academic partners and technical institutes with a wide range of expertise in food sciences and human clinical practice to develop knowledge and decision support tools to enable the sectors to have objective information on the importance of these hazards in slaughterhouses/cutting plants.

This project will allow:
i) to assess the contamination of the three meat sectors with regards to C. perfringens and C. difficile hazards and to consider the correlation between the occurrence of the digestive carriage at the slaughterhouse, contamination of carcasses, meat cuts and the production environment, as well as the bacterial profile within each slaughterhouse;
ii) to determine the pathogenic potential of the isolated strains for humans;
iii) to identify epidemiological markers allowing the isolates traceability according to the animal sectors;
iv) to define their persistence in slaughterhouse and the potential correlation between their presence and the abundance of other bacterial taxa in surface populations;
v) to determine the relative importance of the different transmission routes (animal or environmental) and their possible implication in infectious diseases in humans;
vi) to transfer the methodologies used for data processing to the technical institutes for the characterization and monitoring of the two pathogens in the different meat sectors;
vii) to set up recommendations by drafting guidelines describing strategies to be carried out by operators in order to efficiently reduce their involvement in contamination and their impact on Public Health;
viii) to propose an alternative exploratory strategy for the management of these hazards by LED light treatment and to assess its impact on meat quality.

The project has 5 Work Packages (WP): the WP1 concerns the organization of the sample collection in the slaughterhouses according to the specificities of each sector, the WP2 aims at determining the contamination frequencies of the meat sectors with regard to the hazards C. perfringens and C. difficile, the WP3 aims to determine the pathogenic potential of the isolated strains for humans, the WP4 aims to identify the relative importance of the sectors with respect to the two hazards and the WP5 aims to investigate the possibility of using LED combined with relative humidity/temperature to destroy the spores of C. perfringens and C. difficile.

Thus, the meat sectors’ operators will be better able to justify the adequacy and relevance of the control measures implemented with regard to the C. perfringens and C. difficile hazards, and, if necessary, to re-examine them and disseminate selected prevention practices.