EPIDEMIOLOGIA DESCRITTIVA E SORVEGLIANZAMOLECOLARE DI SALMONELLA ENTERICA SUBSP.ENTERICA SIEROTIPO NAPOLI IN LOMBARDIA

Background: Salmonellosis is a major food-borne disease worldwide with an estimated 93.8 million cases occurring each year, resulting in 155,000 deaths. In Europe, infections caused by Salmonella enterica serovar Napoli (S. Napoli) have notably increased over the last few years, mainly affecting France, Switzerland and Italy. Information about its epidemiology, ecology and virulence is poor, and no foodborne or environmental factor has been identified so far. While foodborne transmission is the most common route for Salmonella infections, this does not appear to be the case for the increase of this serovar. Recent studies showed that exposure to surface water seems to be a risk factor for S. Napoli infection. This study aims at describing the epidemiology, the molecular characteristics and reconstructing the phylogeography of S. Napoli in Northern Italy. Methods: All S. Napoli cases in Lombardy Region between 2010 and 2016, reported from EnterNet Italia, a network of diagnostic laboratories, were included in this study. A random sample of human isolates (10%, N=104) collected by the Regional Reference Laboratory in the period 2010- 2016 were subtyped by Pulsed-Field Gel Electrophoresis (PFGE), according to standardized PulseNet protocol, with XbaI enzyme. Banding profile and clustering analysis was performed using the InfoQuest software. A total of 47 isolates were genotyped by Multi-Locus Sequence Typing (MLST), using previously described primers. Sequence types (STs) were assigned by comparison to the S. enterica MLST database. Forty-four S. Napoli strains isolated from human cases occurred in Lombardy and Emilia-Romagna between 2012-2014 were subjected to wholegenome sequencing (WGS), using the Illumina MiSeq platform. Bayesian SNP-based phylogeny was reconstructed using the kSNP software. The phylogenetic tree, model parameters, evolutionary rates demography models and phylogeography were co-estimated using a Bayesian Markov chain Monte Carlo (MCMC) method, implemented in the BEAST software. The significant migration rates were analysed and visualised using SPREAD. A comparative genomic analysis was performed to detect the differences between the S. Napoli genomes, in terms of nucleotide variations. Outbreak-related isolates were subjected to a genomic analysis based on sequence data for 93 core-genome loci, as described before, to define phylogenetic relationships of 15 Salmonella enterica subsp. enterica serotypes. Phylogenetic analysis was done using Maximum Likelihood method based on the Tamura-Nei model, using the Mega software. Results: A total of 885 (6,8%) isolates of S. Napoli out of 12,962 Salmonella spp. isolates were reported from 2010 to 2016, with the highest isolation rates among infants (0-5 years, 48,9%). The average annual incidence rate was 1.29 per 100,000 inhabitants, and the highest incidence rates were observed in the provinces of Como, Lecco and Varese. It was clear that there was a dramatic increase in this serovar from June to October, each year. S. Napoli isolates compared by PFGE exhibited high levels of diversity (67 XbaI pulsotypes, 8 clusters). MLST analyses showe that all the isolates belong to the same ST474, and to eBURST group (eBG) 60. Phylogenetic analysis revealed that S. Napoli isolates subjected to WGS are grouped in two main clades, that strongly correlate with their geographic origin. Clade A (n=16 isolates) included most of the isolates from Emilia-Romagna, and clade B (n=28 isolates) comprised most of the isolates from Lombardy. The analysis of the tree confirmed the existence of two highly significant clades: one mainly including the isolates sampled in the Po Valley area (clade A, pp=0.92) and the other encompassing the Western Prealpes stra