Enhanced PFGE protocol to study the genomic diversity of Clostridium spp. isolated from Manchego cheeses with late blowing defect

Late blowing defect (LBD) is caused by the outgrowth in cheese of strains of the genus Clostridium capable of fermenting lactic acid with production of butyric acid, acetic acid, carbon dioxide and hydrogen, resulting in the appearance of texture and flavour defects. In the present work, 223 Clostridium isolates (C. beijerinckii, C. butyricum, C. sporogenes and C. tyrobutyricum) from 45 Manchego cheeses with LBD were typed by a modified short pulsed field gel electrophoresis (PFGE) protocol that solved several problems of the original protocol such as DNA degradation, incomplete DNA digestion and reduced DNA release. Our modified protocol allowed the typification of all isolates and the study of their genetic diversity.Out of the 84 Clostridium pulsotypes found in Manchego cheeses with LBD, 54 pulsotypes were represented by more than one isolate. Isolates of the same pulsotype were found in the same cheese for 36 of those 54 pulsotypes, in different cheeses from the same factory for 5 pulsotypes, and in cheeses from different factories for 13 pulsotypes. Dates of manufacture of the cheeses from the same factory containing the same pulsotype differed by less than 40 days. Thirty-six out of the 45 cheeses with LBD contained more than one Clostridium pulsotype.Clostridium isolates from Manchego cheeses with LBD showed a high genetic diversity and could be grouped by their pulsotype according to the factory and the date of cheese manufacture.

► Clostridium isolates from Manchego cheeses with late blowing defect showed high genetic diversity. ► Clostridium pulsotypes could be grouped according to the factory and date of cheese manufacture. ► The PFGE protocol used allowed typing and characterization of all Clostridium isolates.