Genetic Diversity of Clostridium spp. Isolated from Spoiled Hard-cooked and Semi-hard Types of Cheese

Introduction: Butyric acid fermentation, the late-blowing defect in cheese, caused by the outgrowth of Clostridia spores present in raw milk, can lead to considerable loss of product, especially in the production of semi-hard and Gruyère cheeses. Although Clostridium tyrobutyricum is the most frequently isolated strain from late-blown cheeses, spores of other clostridia, particularly C. sporogenes, C. beijerinckii, and C. butyricum, have also been isolated from natural and processed cheeses and raw milk. Conventional methods for the isolation of Clostridium spp. from cheeses with late-blowing defects are tedious and the identification of isolates is often complicated.

Purpose: The aim of this work was to develop and evaluate the use of molecular typing tools to detect and differentiate major species involved in late-blown cheeses.

Methods: A collection of over 300 Clostridia isolates was analysed using various molecular typing methods to perform clusters (REP-PCR) and assess genetic relatedness with a multi-locus sequence typing (MSLT) method targeting 5 different genes (recA, groEL, tpi, rpoB, 16S rDNA). Specific PCR methods were developed i) to detect the major species involved in late-blown cheeses and ii) differentiate the major targeted species in particular distinguish closely related C. sporogenes and C. botulinum.  

Results: 274 isolates were analysed using the REP-PCR method combined to the 16S rDNA sequencing (>600bp), providing more than 60 clearly differentiated groups. Several primer sets were designed to specifically detect C. sporogenes, C. butyricum and differentiate C. botulinum from other dairy-related Clostridia. Primers pairs were designed in the recA gene and the tpi gene for specific PCR detection of C. sporogenes and C. butyricum, respectively. A different primer pair was designed in the recA gene to specifically detect group A C. botulinum. The optimised protocols can distinguish the three target Clostridia species and no specific amplifications were obtained among other Clostridium spp. or non-target species (Streptococcus thermophilus and thermophilic Lactobacilli). The MLST of 16 C. sporogenes isolated revealed important intra-species diversity and locus frequencies that ranged from 4 to 8 alleles per locus. 11 unique profile patterns or STs were identified. The 16-23S rDNA PCR method yielded discriminative inter-species genomic fingerprint.  

Significance: Clostridia strains isolated from raw milks and spoiled dairy products have been analyzed, leading to the set up of a characterized collection. Moreover, several methods have been developed to type and identify the isolates, respectively a REP-PCR fingerprinting method, as well as a MLST and species specific PCR methods.