Detection, quantification and genomic characterization of Clostridioides difficile in pasteurized poultry products

Abstract Pasteurized products made from poultry meat or mechanically separated meat (MSM) are understudied and poorly characterized regarding Clostridioides difficile contamination. In this study, both qualitative and quantitative methods were employed to assess contamination rates. Various types of pasteurized sausages produced from a single batch of chicken MSM, as well as retail samples containing either poultry MSM or non-MSM meat along with additional ingredients, were analyzed. Overall, C. difficile was detected in 60.6% (20/33) of the samples. In positive samples, enumeration using the most probable number (MPN) method revealed C. difficile levels ranging from < 0.090 to 1.600 MPN/g. Whole-genome sequencing of the isolates identified 10 different sequence types (STs), with ST1255 as a novel ST. Virulence gene analysis revealed that 21 isolates carried tcdA , 28 carried tcdB and two had an intact CDT locus. Most isolates (19/32; 59.4%) belonged to ST3 and formed a common wgMLST cluster; of these, 18 isolates were from the same batch of MSM. Another wgMLST cluster comprised four ST58 isolates, which were associated with three different food business operators. Although antimicrobial resistance (AMR) genes or AMR-associated mutations were detected at relatively low frequencies, they included determinants associated with resistance to several antimicrobial classes. The high C. difficile contamination rate observed in pasteurized poultry sausages, despite relatively low pathogen counts, highlights the need for a comprehensive risk assessment to evaluate potential public health implications.