Chiesa, F.

Civera, T.

Di Ciccio, P.

Rubiola, S.

English

Institutional Research Information System University of Turin

19/11/20191Genetic Determinants Associated with Biofilm Formation of Listeria Monocytogenes from Food and Food Processing EnvironmentP. Di Ciccio, F. Chiesa, S. Rubiola and T. CiveraDepartment of Veterinary Science, University of Turin, Largo P. Braccini 2, Grugliasco, TO 10095, Italypierluigialdo.diciccio@unito.itScanning electron microscopy (SEM) images of Listeria monocytogenes in (A) planktonic and (B) biofilm form on stainless steel(A.Ianieri, 2008)Microbial Biofilm structured communities of bacterial cells attached to a surface (biotic or abiotic) embedded in a self-produced matrix of polymeric substances (EPS) exhibiting an altered phenotype and gene expression Introduction19/11/20192Biofilm in the Food processing environmentserious concern: can develop on various surfaces: worksurfaces, conveyors, pipes… more resistant to antimicrobials source of contamination of food productsIntroductionbiofilmattached monolayer microcolonypersistent cellsstress responseListeria monocytogenes (L.m) is a psychrotolerant, gram-positive, ubiquitous bacteriumcan grow both in planktonic and sessile form, organizing in Biofilmsurvival and persistence under various stressful environmental conditionsin food processing environmentsIntroduction19/11/20193In the EU in 2017, a total of 2,480 confirmed invasive human listeriosis were reported by 28 member states, corresponding to an EU notification rate of 0.48 cases per 100.000 population and a fatality rate of 13.8% (EFSA and ECDC, 2018)Different types of cheeses (especially fresh-soft and semi-hard varieties) and meat products were often found to be vehicles in listeriosis outbreaks L.m is of interest because infection is often associated with a high mortality rate, particularly among the elderly (CDC, 2018)IntroductionL.m Biofilm in food-processing environment is considered to be one of the main sources of repeated food contaminationsBiofilm production of L.m is a phenotypic character influenced by many genetic and environmental factors such as temperature, surface type, lineage and serotype etc.In the past, attempts to correlate Biofilm phenotype to serotype, origin or lineage gaveconflicting results between studiessuch comparisons can be, now, addressed at the level of the genome by using Whole Genome Sequencing (WGS).Introduction19/11/20194The purposes of this study were: • to assess the ability to produce Biofilm of L.m isolates from food and food environment • to compare the levels of Biofilm formation with the presence/absence or truncations of Biofilm associated genesAim of the study• The biofilm formation of 57 L.m isolates (previously, whole genome sequenced) selectedfor their genetic traits (Clonal Complex) and their origins (28 from meat origin and 29from dairy origin) was evaluated according to a previously described method(Stepanovic et al., 2007)Materials and MethodsCrystal Violet staining• DO ≤ DOc = no biofilm producers; • DOc < DO ≤ 2×DOc = weak biofilm producers; • 2×DOc < DO ≤ 4×DOc = moderate biofilm producers; • 4×DOc < DO= strong biofilm producers.19/11/20195• A nucleotide BLAST of each gene against each genome assemblywas performed to determine the presence or absence or truncation of apreviously described set of Biofilm associated genespresence or absence (SSI-1; intL): significant hits were defined as those with coverage of at least 80% and a percent identity greater than or equal to 80%. (BLAST analysis) (Pirone-Davies et al., 2018)truncations (intA; actA: premature stop codons: PMSCs) were defined as presentif a sequence was missing at least ten amino acids from the end of the sequenceas compared to the EGD-e reference sequence.Sequences were translated to amino acids, aligned with MUSCLE, and manuallyinspected for truncations.Materials and Methods• 58% weak Biofilm producers• 38.5% moderate Biofilm producers• 3.5% strong Biofilm producersResultsBiofilm formation• 2/57 isolates from meat origin were strong Biofilm producer58%38.5% 3.5%• All L.m isolates were classified as Biofilm producer19/11/20196ResultsWGS• A higher Biofilm formation was observed in L.m isolates belonging to CC9 and CC31• No difference in biofilm production was observed between food and environment isolates• The percentage of isolates from meat products (16%), classified as moderate/strong biofilm producers, was higher than the percentage obtained for isolates from dairy products (7%) - (p < 0,05)ResultsBiofilm formation19/11/20197ResultsWGS• The presence of the five genescluster lmo0444-lmo0448 (StressSurvival Islet 1) and atruncated inlA protein, was significantlyassociated with increased levels ofBiofilm• The presence of the inlL and atruncated actA protein, was not significantlyassociated with increased levels of Biofilm19/11/20198Conclusions• To date, the most WGS studies are carried out on L.m clinical isolatesL.m isolates from Food sector was evaluated in our study• In accordance with the literature, the presence of Stress Survival Islet 1 and a truncated inlA protein was associated in the Biofilm‐forming capacity in L.m(Franciosa et al., 2009; Ryan et al., 2010; Keeney at al., 2018)• In literature the inlL and a truncated actA protein was associated in the Biofilm‐forming capacity in L.m (Travier et al., 2013; Popowska et al., 2017). Our finding did not show this evidence• Associating WGS-genotypes and specific Biofilm phenotypes could improve prediction of microbial (L.m) behaviors• The implementation of this information in Hazard Identification and Exposure Assessment processes will open new possibilities to feed quantitative Microbial Risk Assessment modelsConclusions19/11/20199THANK YOU FOR YOUR ATTENTIONpierluigialdo.diccio@unito.it

Genetic Determinants Associated with Biofilm Formation of Listeria Monocytogenes from Food and Food Processing Environment

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Genetic Determinants Associated with Biofilm Formation of Listeria Monocytogenes from Food and Food Processing Environment

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