Occurrence of Clostridium spp. in ewe’s milk: enumeration and identification of isolates

Abstract

The presence of gas-producing clostridia in ewe’s milk can lead to the occurrence of late-blowing defects in cheeses. However, data on this aspect are limited. In the present study, using the most probable number (MPN) method, clostridial spores were enumerated in 527 ewe’s milk samples collected in the Grosseto Province (Tuscany, Italy) from autumn 2014 to summer 2015. In addition, using polymerase chain reaction (PCR), we identified the species most frequently involved in late-blowing defects in cheese (Clostridium tyrobutyricum, Clostridium butyricum, Clostridium beijerinckii, and Clostridium sporogenes), and of Clostridium perfringens. Gas-producing clostridial spores were detected in 99% of samples. Spore concentrations ranged from 360 to more than 110,000 spores∙L−1. We observed that 86% of samples had a spore load higher than 1000 spores∙L−1. During autumn and summer, spore concentrations were significantly higher than in winter and spring (P < 0.001). A total of 222 isolates obtained from 77 MPN positive tubes from different milk samples were subjected to PCR. Colonies from 63/77 (82%) MPN positive tubes were taxonomically identified. Among the 63 PCR-positive samples, C. perfringens was the most frequently detected species (56%), followed by C. sporogenes (44%), C. tyrobutyricum (7.9%), C. butyricum (1.6%), and C. beijerinkii (1.6%). In addition, in 11% of the MPN positive tubes, at least two clostridial species were found to be present simultaneously. This work highlights the presence of clostridial spores in ovine milk from central Italy (Tuscany) and led to the identification of some of the clostridia species involved in such high spore loads.