PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Alessandra Dalmasso

Phd thesis

TITLE : Assessing Listeria monocytogenes Prevalence and Dynamics in Smoked Salmon Production

Scientific background/state of the art

 Listeria monocytogenes (Lm) is a ubiquitous, non-spore-forming bacterium responsible for listeriosis, one of the most reported human zoonosis in Europe. The invasive form of this infection can be fatal for vulnerable individuals and may lead to preterm birth or miscarriage in pregnant women [1]. In 2022, European Food safety Authority (EFSA) reported 35 listeriosis outbreaks, attributing the transmission of pathogen primarily to the consumption of contaminated ready-to-eat foods (RTE). Among RTE foods, vacuum-packed smoked salmon is highly vulnerable to Lm colonization. Potential origins of contamination in smoked salmon process include tools, equipment, and food handlers.

If Lm enters through raw material, the technological treatments used in this production (brining, smoking, cooling) are not able to completely eliminate the presence of pathogen. Once Lm has colonized a food processing plant, it may spread throughout the environment, leading to food contamination. Additionally Lm can form biofilm enhancing its stress resistance and persistence. Besides, the prolonged exposure to disinfectant may induce Lm to enter in a Viable But Not Culturable (VBNC) state, resulting in false negative during routine monitoring sampling.

The combination of an efficient food safety management system with a well-design environmental monitoring plan is crucial to mitigate the risk of Lm contamination in smoked salmon processing plant.

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:

1. to observe genetic relationship among strains.
2. to identify resident and transient Lm strains.
3. to test the ability to form biofilm
4. to assess the presence of genetic traits associated with sanitizer resistance
5. to evaluate the strains virulence potential
6. 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]and multi locus sequence typing (MLST) will be applied to analyze the genetic variability among strains.

Lm isolates will be subjected to further in-depth molecular characterization:

1. Whole Genome Sequencing (WGS) will be employed to investigate the genetic relationship among strains by Core Genome MLST (cgMLST) [8].
2. The presence of genes involved in sanitizer resistance will be determined by PCR reaction [9], [10].
3. Biofilm forming capacity will be tested using microtiter plate assay, staining biofilm layer with crystal violet
4. 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.

 

 

 

 

Research activities

Short summary of the results obtained during the 1st, and 2nd 

Sample collection was carried out in an Italian food facility specialized in smoked salmon production.
Environmental sampling was conducted across two distinct cutting lines, including food contact surfaces
(FCS) and non-food contact surfaces (NFCS). Food sampling was performed at different stages of production,
from raw material to finished product at the end of shelf life. To represent different productivity periods
(low, medium and high) both environmental and food sampling were conducted in triplicate in different
months. In total, 295 samples were collected: 85 from the processing environment and 210 from food
products. Samples were processed following the standard method EN ISO 11290-1:2017 for the detection of
Listeria monocytogenes. For each sample, a small aliquot was preserved for the analysis of total microbiota.
L.monocytogenes was confirmed in 13.22% (=39) samples, with 10.84% (=32) originating from environment
and 2.7% (=7) from food matrices. Preliminary molecular characterization performed using Multi Locus
Sequence Typing (MLST) as described by Ragon et al. (2008),grouped L.monocytogenes isolates into six
distinct Sequence Types (STs): ST5, ST6, ST7, ST9, ST14, ST155. Specifically:
• Subtypes ST9 (48.72%) and ST5(30.77%) were the most abundant and identified as “potentially
persistent”.
• Subtypes ST6(10.26%), ST7(5.13%), ST14 (2.56%), ST155 (2.56%) were considered as ‘‘sporadic’’.
• ST6, ST7, ST155 were identified as ‘’potentially virulent’’ .
In the following step, L.monocytogenes isolates were screened for genes associated with stress resistance.
In particular, the attention was focused on the five-gene Stress Survival Islet 1 (SSI-1), a genomic region
potentially involved in tolerance to high salt concentrations and acidic pH. All the isolates were screened
for SSI-1 according to the method described by Ryan et al. (2010). The results showed that most isolates
(84.62%) contained the complete SSI-1 gene set, while the remaining isolates carried only a subset of the
genes.

 

Project development

In parallel with molecular characterization, isolates are phenotypically characterized to assess their biofilm forming capacity and their tolerance to sanitizers commonly used in the smoked salmon industry. For a more in-depth genomic analyses, strains exhibiting interesting genotypic and phenotypic traits were selected as candidates for Whole Genome Sequencing (WGS) .
The analysis of total environmental and food microbiota is in progress and will be useful to evaluate: i) which other microbial genera populate the food processing environment, ii) how the microbial community evolves during food product transformation, and iii) how these microbes coexist with L. monocytogenes.
For this purpose, the total microbial DNA has been extracted and sent to an external company for 16S metabarcoding, library preparation and sequencing using the MiSeq System (Illumina Inc.).
I am currently undertaking my research period abroad at the Aristotle University of Thessaloniki, Greece.
During this period, microbiota data will be elaborated using QIIME 1.9.0 software and RStudio. Meanwhile, I am also working on drafting the first scientific paper focused on the presence of L.monocytogenes in the production environment of a smoked salmon facility, with particular emphasis on the use of the MLST method for strain characterization in combination with traditional culture-based techniques.

Next scheduled phases

During the period abroad, selected strains will be tested for their ability to adapt to acidic pH conditions. Moreover, strains identified as “acid-adapted” will undergo sanitizer tolerance assessment in both biofilm and planktonic states. These analyses will offer valuable insights into how L. monocytogenes may develop cross-adaptation mechanisms.
Regarding data from WGS, they will be elaborated according to the method of core genome multilocus sequence typing (cgMLST) in order to gain more information about strains diversity, stress resistance and virulence potential.