High-throughput sequencing of 16S rRNA amplicons reveals a high bacterial diversity in typical Italian dry fermented sausages

The final characteristics of fermented meats are strongly determined by the microbial species involved in ripening. Here we applied high-throughput sequencing Illumina technology (HTS) coupled to the PCR amplification of the 16s rRNA subunit to assess at fine scale level the bacterial diversity in batches of Salame Piacentino PDO (Protected Designation of Origin), a dry fermented sausage that is typical of a regional area of Northern Italy. Salami samples from 6 different local factories were analysed at 0, 21, 49 and 63 days of ripening; raw meat at time 0 and casing samples at 21 days of ripening where also analysed, and the effect of starter addition was included in the experimental set-up. Culture-based microbiological analyses and PCR-DGGE were also carried out in order to be compared with HTS results. A total of 722,196 high quality sequences targeting the two bacterial 16S hypervariable regions V3 and V4 were obtained; manual curation of 16S database allowed a correct taxonomical classification at the species level for 95 % of these reads. HTS results confirmed the presence of main bacterial species involved in the fermentation of salami, but the resolution and the number of identified species were much higher as compared to PCR-DGGE. Thirty-two different Staphylococcus and 33 Lactobacillus species were identified in the salami from different producers, while the whole data set obtained accounted for 13 main families and 98 rare ones, 23 of which were present in at least 10% of the investigated samples, with casings being the major sources of the diversity observed at DNA level. Multivariate analyses also showed that batches from 6 local producers tend to cluster altogether after 21 days of ripening, thus indicating a convergence in the overall diversity in different batches of the same local fermented dry sausage.