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Kamilari et al. Microbiome Res Rep 2025;4:3 https://dx.doi.org/10.20517/mrr.2024.47 Page 5 of 15

[37]
[35]
Cytoscape 3.2.1 , as described by Kamilari et al. .
The raw sequence data were archived in the NCBI sequence read archive (SRA) with BioProject PRJNA
1010532.

RESULTS
Microbial population and diversity metrics in sausages
Sausages originating from Cyprus (Nicosia, Lemesos, Pitsilia) and Mytilini were separately examined for
their microbial diversity. The 16S rRNA amplicon sequencing results showed that the 30 specimens
generated an average of 37,851.47 sequencing reads (range = 19,570-107,301, STD = 15,275.43,
Supplementary Table 1), and 155.97 OTUs (range = 32-696, SD = 137.37; Supplementary Table 1) per
sample. The ITS loci amplicon sequencing results from 23 samples produced an average of 47,687
sequencing reads (range = 23,571-81,687, STD = 13,842; Supplementary Table 2), and 202 OTUs (range =
37-327, SD = 87; Supplementary Table 2) per specimen. Seven samples were excluded from the analysis
owing to a reduced number of reads (< 8,000).

Initially, the Shannon, Simpson, and Chao1 indices were analyzed to estimate the alpha diversity of the
bacterial and fungal communities [Supplementary Tables 1 and 2]. The results indicated that there was no
significant difference among the areas of sausage production, or the applied manufacturing conditions,
using the Kruskal–Wallis test [Supplementary Table 3, Shannon index].

Regional microbial beta diversity
To assess the existence of unique microbial signatures among sausages produced in different areas or
between traditionally and industrially produced Cypriot sausages, we calculated the beta diversity based on
the weighted and unweighted UniFrac distances [Supplementary Table 4]. No notable difference was
observed in microbial diversity among sausages produced in different areas, or between traditionally and
industrially produced sausages, based on the PERMANOVA test.

Microbial community taxonomic profile
The predominant bacterial genus in Cypriot and Mytilini sausages was Lactobacillus [Figure 1A and B].
Some Cypriot sausages were defined by a higher relative abundance of Leuconostoc (7%-27%). Additionally,
Pseudomonas and Brochothrix were detected in lower relative abundances in some sausages (0%-7% and 0%-
8%, respectively). Industrially produced sausages from Mytilini were dominated by the presence of
Lactobacillales (52%-89%), whereas traditionally produced were dominated by Lactobacillus delbrueckii
(15%-28%), Lactococcus (9%-16%), and Streptococcus (15%-26%). The genera Leuconostoc (0%-16%),
Salinivibrio (0%-4%), and Pseudomonas (0%-17%) were detected in lower relative abundances in
traditionally produced Mytilini sausages. Regarding the fungal diversity, Cypriot sausages exhibited an
enhanced representation of the species Debaryomyces hansenii (0.1%-83%), Candida zeylanoides (0.2%-
52%), and Candida sake (0%-32%) [Figure 1C]. Reduced relative abundances were also observed for
members of the genera Saccharomyces (0%-19%) and Alternaria (0%-4%), the species Aspergillus
penicillioides (0%-5%), and Wallemia sebi (0%-9%). Most Mytilini sausages were excluded from subsequent
analyses due to insufficient sequencing output (< 10,000 reads cutoff after filtering). One Mytilini sausage
was dominated by the species C. zeylanoides (96%) [Figure 1D]. The other two showed an enhanced
representation of the species Xeromyces bisporus (10%-14%) and members of the genus Alternaria (8%-
12%). D. hansenii was among the dominant species of one sausage (16%). Other species that were detected
in lower relative abundances included Wallemia sebi (0.2%-8%), Botrytis sp. (0%-4%), Vishniacozyma
tephrensis (0%-2%), Vishniacozyma carnescens (0%-2%), Pichia cephalocereana (0%-3%), and Cladosporium
tenuissimum (0%-3%).