Michela Berta
- 502306
- Phd: 39th cycle
- Matriculation number: 1093679
Phd thesis
Aims
- Year 1
The environmental occurrence of Lm will be evaluated in an Italian smoked salmon plant.
The focus will be to identify the sites, niches, or machineries where Lm is present, constituting a risk for food contamination and cross-contamination.
Additionally, the complete environmental microbiota will be evaluated to determine which other bacteria coexist in the same environment.
- Year 2
The presence of Lm will be assessed even through the entire food production process, from raw materials until the final product at the end of shelf life.
This analysis will allow us to determine whether raw materials could be a possible source of Lm entrance in salmon facilities.
Besides, the microbiota of food materials will be analyzed. This assessment will provide information on the composition and dynamics of microbial communities throughout the production, offering valuable insights into the overall impact of the process on food hygiene.
- Years 1-3
Lm isolates from the previous described sampling steps will be molecularly characterized:
- to observe genetic relationship among strains.
- to identify resident and transient Lm strains.
- to test the ability to form biofilm.
- to assess the presence of genetic traits associated with sanitizer resistance.
- to evaluate the strains virulence potential.
- to verify the capability to shift in VBNC state.
Materials and methods
A total of 200 samples will be analyzed, 100 from food plant environment and 100 from food materials. Based on previous analysis performed on salmon processing plant, we expect to find at least 50 Lm strains.
Smoked swill be visited three times per year when there will be high, medium and low activity. Two producing lines will be included in environmental sampling, with at least 10 points sampled for each line. Raw fish, semi-finished, and finished product will be sampled.
Microbiological isolation will be performed following the standard protocols EN ISO 11290-1:2017. Suspected Lm will be confirmed by MALDI-TOF (Bruker Biotyper)
DNA will be extracted using Qiagen DNeasy Blood and Tissue Kit according to manufacturer instructions. Serotyping will be performed by multiplex PCR[6]
Multi locus sequence typing (MLST) will be applied to analyze the genetic variability among strains [7].
Lm isolates will be subjected to further in-depth molecular characterization:
- Whole Genome Sequencing (WGS) will be employed to investigate the genetic relationship among strains by Core Genome MLST (cgMLST) [8].
- The presence of genes involved in sanitizer resistance will be determined by PCR reaction [9], [10].
- Biofilm forming capacity will be tested using microtiter plate assay, staining biofilm layer with crystal violet
Total bacteria community present both in environment and food will be obtained by 16S metabarcoding [13].
Expected results
Assessing the prevalence of Lm in food plant environment and in food, together with microbiota analysis, will offer valuable insights to ensure the hygiene and safety of food production. (Reg.2073/2005)
Molecular characterization will provide additional information about differences in virulence and pathogenicity of Lm isolates. This data will be employed for a more accurate food plant risk assessment.
Moreover, information on biofilm-forming capacity and the presence of stress resistance genes will allow an improvement in food plant cleaning and disinfection procedures.