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 strains (clade B, pp=1). The sequences isolated in the Milan metropolitan area tended to be interspersed in both clades, forming only small subclades of no more than 3 sequences. The most probable location of the tree-root was in Po Valley region. The mean tMRCA of the tree-root was estimated to be 69 YA (95%HPD: 16-165 YA), corresponding to a mean 1945. Phylogeographic reconstruction showed that S. Napoli spreads simultaneously towards the North-West and the South-East. Northwest diffusion followed the courses of the Ticino and Adda rivers, reaching the metropolitan area of Milan as soon as the early 1950s and expanding to the Northern area until reaching in 2000s the region of the great lakes (in Lombardy). The Southeast dispersal apparently followed the Po River, reaching Piacenza in 1960s-1970s. In the late 1980s-1990s it reached the main centers of the Emilia Romagna region (Parma, Reggio Emilia, Modena) up to Bologna in the 2000s. Most external branches reached the area near the Adriatic Sea (Forlì) only recently (2014). The comparative genomics analysis revealed that S. Napoli belongs to Typhi subclade in clade A, being Paratyphy A the most related serovar. As for typhoid serotypes, S. Napoli genome lacks the ß-glucuronidase gene and carries the invasive-determinant gene cdtB and the pathogenicity island SPI-18. Conclusions: S. Napoli is an emerging public health concern, and the enigma of the increase in infections in Italy remains unexplained. One difference between S. Napoli and the other Salmonella serovars concerns the proportion of cases in the different age groups, with a significantly greater involvement of young children, possibly due to different exposure patterns. 3 For young children, foodborne exposure may be less important than other routes. The three most affected provinces (Como, Lecco and Varese) are characterized by the presence of lakes, and analysis of data from the literature revealed that surface water contamination may be a direct (waterborne infection) or indirect (foodborne infection) vehicle for transmission of S. Napoli. Regarding molecular characterization, XbaI restriction enzyme profiles indicated genomic heterogeneity among strains. PFGE involves random screening of the entire genome, while MLST analysis is limited to nucleotides within the targeted genes. Therefore, if there is little or no variation in the nucleotide sequence of the genes targeted by MLST, this technique can provide little or no discrimination between strains tested. Our study demonstrates that MLST, using the genes tested, lacks the ability to discriminate between S. Napoli isolates, and that PFGE can still be considered the method of choice for the molecular typing of this serotype. However, there are several disadvantages of using this technique. The technological advancements of Whole Genome Sequencing (WGS) provides the opportunity to access the entire genome information. In the present study, we compared S. Napoli isolates from two different Italian regions. The results indicate that WGS coupled to SNP-based phylogeny seems an excellent approach to infer the genomic and geographic distribution of serovar Napoli. Phylogeographic analysis showed that S. Napoli diffusion followed the courses of the Po, Ticino and Adda rivers, supporting the hypothesis according to which the surface watersseems to be a risk factor for S. Napoli infection. Genomic comparison revealed that S. Napoli belongs to Typhi subclade in clade A. A combination of SPI-18 island and cdtB gene was previously reported only in S. Typhi, S. Paratyphi A and clade B serotypes, all of them associated to elevated rates of invasive disease. Thus, to the best of our knowledge, this is the first time that a clade A nontyphoidal serotype presents the same virulence-genes pattern of S. Typhi and S. Paratyphi A. This result suggestst that S. Napoli potential virulence deserves attention.