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

bacteriophage-encoded enzymes for monitoring the presence of unwanted microorganisms in food and
foodstuff. Additional sausage processing and preservation approaches involve curing, fermentation,
ripening, heat treatment, high-pressure processing, marination, smoking, and drying [16,17] .

Sausage production conditions create an unfavorable environment for the microbiota. Therefore, only
microbes with specific metabolic adaptations may survive . Those microbes are members of the regional
[18]
microbiome, the development of which is influenced by environmental factors, including climate,
topography, and soil [19-21] . The distinctive microbiome of each sausage type influences the sensory
[22]
characteristics of the final product through well-orchestrated networks . Microbial interactions performed
among different taxonomic groups lead to the coexistence of specific, phylogenetically diverse microbiomes.
The development of co-occurring and co-exclusionary networks based on the presence/absence and relative
abundance of key microbes enables the understanding of the relationships among the members of the
community . Comparing interaction networks generated from microbial communities developed in
[23]
sausages produced using different manufacturing conditions and having different origins, may provide
insights into understanding the factors that influence the microbial diversity developed in different types of
sausages.

The present study aimed to characterize the microbial composition of Cypriot and Mytilini sausages using
metataxonomic sequencing, aspiring to enhance the existing knowledge of the microbial consortia that are
shaped in traditional and industrial sausages undergoing spontaneous fermentation. It is a follow-up study
[1]
of characterizing Cyprus sausages’ bacterial communities improved with the addition of more samples and
enriched with the characterization of the fungal diversity. Additionally, it provides a snapshot of Mytilini
sausages’ microbial communities. Using co-occurring and co-exclusionary networks, the outcome of the
study may increase our understanding of the microbial interactions developed in spontaneously fermented
sausages. Furthermore, the study provides insights into using the microbiome as an additional tool for
fingerprinting traditional sausages for authentication purposes.

METHODOLOGY
Sausages collection
Sausage samples were collected between December 2021 and January 2022. Samples included 22 pork
sausages from Cyprus and 8 from Mytilini [Table 1]. Sausages that were originated from the same region
were produced by different companies. All sausages were prepared without the addition of starter cultures
(spontaneous fermentation). Samples were put in cold storage boxes and transferred to University College
Cork, Ireland, after about 5 working days. All samples were stored at -20 °C until processing.

DNA isolation and DNA yield measurement
Sample homogenization was performed by mixing 5 grams of sausages with 45 mL of maximum recovery
diluent (MRD) in a Stomacher 400 Circulator (Seward, UK) at a shaking speed of 300 rpm for 2 min. Each
homogenized sample in a volume of 1.8 mL was transferred to a 2 mL tube and the cell-free supernatant was
separated from the cell pellet by centrifugation at 16,000 × g for 5 min at 4 °C. The T cell pellet was diluted
in 450 µL Solution MBL (MoBio Laboratories Inc., Carlsbad, CA, USA) and the microbial DNA was
extracted according to the protocol instructions of DNeasy® PowerFood® Microbial Kit (MoBio Laboratories
Inc., Carlsbad, CA, USA). The isolated DNA was kept at -20 °C until processing.

Extracted DNA yield measurement was performed using the Qubit 4.0 fluorometer (Invitrogen, Carlsbad,
CA) and Qubit dsDNA HS Assay Kit (Invitrogen). DNA quality was estimated by calculating the A260/
A280 and A260/A230 absorbance ratios in a spectrophotometer (NanoDrop Thermo Scientific, USA).