Identification of Feed-Borne Microbial Spoilers and Pathogens by MALDI-TOF MS in the Dairy Environment

Spore forming bacteria are observed as the common spoilage-causing microflora in dairy sector due to their spore-forming ability and production of spoilage-causing enzymes which can influence quality and functionality of the end products. Despite their significance, reliable, practical, and rapid identification methods are few. Furthermore, several commercial identification systems for anaerobic bacteria have failed to accurately identify these bacteria at species level. Matrix-assisted laser desorption/ionization time-off light mass spectrometry (MALDI-TOF MS) is an increasingly used technique enabling rapid identification of isolates to the species level. It is a valuable alternative to 16S rDNA gene sequencing which is an expensive and time-consuming technique. A total of 157 anaerobic isolated colonies and 40 aerobic isolated colonies of from the Grana Padano PDO production chain, plus 6 reference strains were used to study the population of spore forming bacteria in the dairy production chain. The aim of this study was the optimization of spore forming bacteria identification by using MALDI TOF MS Biotyper and the comparison of the results with those obtained by 16S rDNA gene sequencing. A hypervariable region amplification protocol (16S rDNA) was used to differentiate bacterial species after specific fragment amplification and sequencing of generated amplicons. The sequences were compared with those in the GenBank database using the BLAST. 99% of homology was considered the cutoff for the identification. Then, all samples were identified by using MALDI-TOF MS Biotyper by Extended Direct Transfer sample preparation procedure (eDT). A total of 113 out of 157 anaerobic isolated colonies were identified to the species level by 16S rDNA gene sequencing. Altogether 99 anaerobic isolates identification were used for the final comparison with the 16S rDNA sequencing; Identical identification species level by both methods were obtained in isolates 71.72% (71/99). Identification results (113 sorted by ≥ 99%) by 16S rDNA sequencing were assigned to the genus clostridium 63.72%, genus Paenibacillus 10.62%, genus Lacrimispora 15.04%, genus Anaerocolumna 2.65%, genus Paraclostridium 3.54%, and genus Bacillus 4.42%. These strains were identified to their genus level or species level by MALDI TOF MS Biotyper analysis which included a total of 8 different types at species level such as 77% of Clostridium sporogenes, 13% of Clostridium beijerincki, 2% of Clostridium celerecrescens, 1% of Clostridium aerotolerans, 1% of Paenibacillus polymyxa, 1% of Clostridium tyrobutyricum, 4% of Paraclostridium bifermentans, and 1% of Clostridium butyricum. Aerobic isolates were identified as Bacillus subtilis 72.5%, Bacillus cereus 25%, and Bacillus mojavensis 2.5%. The reliability of the identification results obtained by MALDI TOF MS were comparable to 16S rDNA sequencing at species level. It can be concluded that MALDI-TOF MS has been successfully proved to be a useful tool for the identification of spore forming bacteria in the dairy industry. The best results have been achieved by the Extended Direct Transfer sample preparation procedure of isolates at 24h of incubation, to prevent sporulation.