Phylogenetic and Evolutionary Genomic Analysis of Listeria monocytogenes Clinical Strains in the Framework of Foodborne Listeriosis Risk Assessment.

Listeria monocytogenes is one of the most important foodborne pathogens responsible for listeriosis, a severe disease with symptoms ranging from septicemia, meningoencephalitis, and abortion. Given the strong impact of listeriosis on human health and the difficulty of controlling L. monocytogenes along the food production chain, listeriosis has become a priority subjected to molecular surveillance in European Union/European Economic Area since 2007. From 2018, surveillance is based on whole-genome sequence using the core genome multilocus sequence type. The complete sequences of 132 clinical strains were used to define the evolutionary relatedness among subtypes of L. monocytogenes isolated in Italy from 2010 to 2016, allowing the identification of clades and/or clusters associated with outbreaks or sporadic cases of listeriosis. All the strains analyzed are clustered in lineages I and II, and the majority of the strains were classified as lineage II. A probable epidemic entrance in different years for every clade and cluster from each different region was defined. The persistence of the same specific clonal complexes of L. monocytogenes has been found over long periods; this may be related to the fact that some strains are able to survive better than others in a food production environment. Phylogenic studies, using whole-genome sequence data, are able to identify the emergence of highly persistent pathogenic variants, contributing to improving the hazard characterization of L. monocytogenes.

One-Day Molecular Detection of Salmonella and Campylobacter in Chicken Meat: A Pilot Study.

Salmonella and Campylobacter ssp. are bacterial pathogens responsible for most foodborne infections in EU countries. Poultry serves as a reservoir for these pathogens, and its important role in the meat industry makes it essential to develop a rapid detection assay able to provide results in one day. Indeed, the rapid identification of foodborne pathogens is an important instrument for the monitoring and prevention of epidemic outbreaks. To date, Salmonella and Campylobacter screening is mainly conducted through molecular methods (PCR or real-time PCR) performed after 18-24 h long enrichments. In this study, we evaluated short enrichments (0, 2, 4, and 6 h) combined with a colorimetric loop-mediated isothermal AMPlification (LAMP) or real-time PCR to detect Salmonella and Campylobacter in poultry meat contaminated at different concentration levels (101, 103, and 105 CFU/g). Our results show that real-time PCR allows the detection of Salmonella and Campylobacter, even after shorter enrichment times than prescribed by ISO references; particularly, it detected Salmonella down to 101 CFU/g since T0 and Campylobacter from 103 CFU/g since T0. Detection with LAMP was comparable to real-time PCR without the requirement of a thermal cycler and with shorter execution times. These characteristics make colorimetric LAMP a valid alternative when one-day results are needed, improving the timely identification of positive meat batches, even in the absence of specialized instrumentation.

Hepatitis E Virus Occurrence in Pigs Slaughtered in Italy.

In Europe, foodborne transmission has been clearly associated to sporadic cases and small clusters of hepatitis E in humans linked to the consumption of contaminated pig liver sausages, raw venison, or undercooked wild boar meat. In Europe, zoonotic HEV-genotype 3 strains are widespread in pig farms but little information is available on the prevalence of HEV positive pigs at slaughterhouse. In the present study, the prevalence of HEV-RNA positive pigs was assessed on 585 animals from 4 abattoirs located across Italy. Twenty-one pigs (3.6%) tested positive for HEV in either feces or liver by real-time RT-PCR. In these 21 pigs, eight diaphragm muscles resulted positive for HEV-RNA. Among animals collected in one abattoir, 4 out of 91 plasma tested positive for HEV-RNA. ELISA tests for the detection of total antibodies against HEV showed a high seroprevalence (76.8%), confirming the frequent exposure of pigs to the virus. The phylogenetic analyses conducted on sequences of both ORF1 and ORF2 fragments, shows the circulation of HEV-3c and of a novel unclassified subtype. This study provides information on HEV occurrence in pigs at the slaughterhouse, confirming that muscles are rarely contaminated by HEV-RNA compared to liver, which is the most frequently positive for HEV.

Hepatitis A Virus Strains Circulating in the Campania Region (2015-2018) Assessed through Bivalve Biomonitoring and Environmental Surveillance.

The genetic diversity of Hepatitis A Virus (HAV) circulating in the Campania Region in years 2015-2018 was investigated through the monitoring of sentinel bivalve shellfish and water matrices. Overall, 463 water samples (71 sewage samples, 353 coastal discharge waters, and 39 seawaters samples), and 746 bivalve shellfish samples were analyzed. Positivity for HAV was detected in 20/71 sewage samples, 14/353 coastal discharge waters, 5/39 seawaters, and 102/746 bivalve shellfish. Sixty-one of the positive samples were successfully sequenced and were characterized as genotype IA (n = 50) and IB (n = 11). The prevalent strain circulating in 2015 in both bivalves and waters was the IA strain responsible for the outbreak occurring around the same time in the Naples area. This variant was no longer identified in subsequent years (2017-2018) when, instead, appeared two of the IA variants of the multistate outbreak affecting men who have sex with men (MSM), VRD_521_2016, and RIVM-HAV16-090, with the former prevailing in both shellfish and water environments. HAV IB isolates were detected over the years in shellfish and in water matrices, but not in clinical samples, suggesting that this genotype had been circulating silently. An integrated surveillance system (environment/food/clinical cases) can be a useful tool to monitor changes in viral variants in the population, as well as an early warning system.

Phylogenetic analysis and epidemiological history of Hepatitis E virus 3f and 3c in swine and wild boar, Italy.

BACKGROUND: Hepatitis E virus (HEV) genotype 3 has a worldwide distribution. The food-borne transmission of HEV associated with the consumption of products derived from domestic pig, wild boar has been reported in various countries. In this study the genetic diversity, evolutionary rates of HEV 3f, 3c among swine and wild boar in Italy were estimated. METHODS: Sampling was performed on a wild boar population living in an area located in Abruzzo region. The HEV RNA amplification was performed by real-time RT-PCR. Nested RT-PCR and sequencing of the ORF2 region were carried out by the Super Script III First-Strand Synthesis System. Sequencing of purified PCR products was carried out by the Genome Lab Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit. The maximum likelihood trees were generated by using Phyml. The mean evolutionary rates and the dated trees were co-estimated by BEAST. RESULTS: The phylogenetic analysis showed that the HEV ORF2 isolates from Abruzzo region belonged to 3f subtype. The prevalent subtypes in Italy were those belonging to 3f and 3c. The estimated mean values of the HEV ORF2 capsid gene evolutionary rates were 1.915 × 10-2 substitutions/site/year (95% HPD: 1.64 × 10-3 - 3.97 × 10-2) and 2.81 × 10-2 substitutions/site/year (1.83 × 10-2 - 3.8 × 10-2) for 3f and 3c subtype datasets, respectively.The HEV 3f dated back to 1985 (1960-2000), whereas the 3c subtype entered in Italy during the year 2006 (2005-2006). The majority of the HEV 3f sequences collected from swine didn't appear intermixed, except in two cases. The HEV 3c population circulating in Italy remained segregated without significant transfer to swine. CONCLUSION: Our study provide insight into the evolution, circulation of HEV 3f and 3c in Italy.Continued genomic surveillance of HEV in animal reservoir, as well as improving sanitary control measures are required.

Detection and quantification of Campylobacter in foods: New analytic approaches to detect and quantify Campylobacter spp. in food samples.

The aim of the present study was to develop rapid qualitative and quantitative methods based on the use of Real-Time PCR and Droplet Digital PCR (ddPCR), in order to have reliable techniques to detect and quantify Campylobacter spp. in food samples. The gene 16S-rRNA was used as specific target for Campylobacter spp. Real- Time PCR evaluation assay and a not competitive internal control was ushered in it. To investigate the selectivity of the method, 26 Campylobacter strains and 40 non-Campylobacter strains were tested and in order to verify the application of Real- Time PCR method, 5 pork meat samples were experimentally inoculated with a Campylobacter jejuni strain. Subsequently, dilutions with a bacterial load of Campylobacter jejuni within 10-106 CFU/mL were chosen for the optimization of the ddPCR assay. Lastly, a total of 54 naturally contaminated foods samples were analyzed through molecular (Real-Time PCR and ddPCR) and traditional methods. The Real-Time PCR protocol demonstrated to amplify only the Campylobacter spp. strains and when Campylobacter jejuni was experimentally inoculated in meat samples the pathogen was always detected. The ddPCRs assay allowed to quantify a level of contamination of 10 CFU/mL, but it was unable to quantify levels of 105 - 106 CFU/mL. Lastly, Campylobacter spp. was never detected in the 54 samples tested. In conclusion, the novel analytic approach proposed, based on an initial screening of the samples with Real-Time PCR and then on quantification of Campylobacter spp. with a ddPCR on those positive, represents a quick monitoring tool and, if used correctly, it would allow the implementation of food safety.

Nine-Year Nationwide Environmental Surveillance of Hepatitis E Virus in Urban Wastewaters in Italy (2011-2019).

Hepatitis E virus (HEV) is an emerging causative agent of acute hepatitis worldwide. To provide insights into the epidemiology of HEV in Italy, a large-scale investigation was conducted into urban sewage over nine years (2011-2019), collecting 1374 sewage samples from 48 wastewater treatment plants located in all the 20 regions of Italy. Broadly reactive primers targeting the ORF1 and ORF2 regions were used for the detection and typing of HEV, followed by Sanger and next generation sequencing (NGS). Real-time RT-qPCR was also used to attempt quantification of positive samples. HEV RNA detection occurred in 74 urban sewage samples (5.4%), with a statistically significant higher frequency (7.1%) in central Italy. Fifty-six samples were characterized as G3 strains and 18 as G1. While the detection of G3 strains occurred in all the surveillance period, G1 strains were mainly detected in 2011-2012, and never in 2017-2019. Typing was achieved in 2 samples (3f subtype). Viral concentrations in quantifiable samples ranged from 1.2 × 103 g.c./L to 2.8 × 104 g.c./L. Our results suggest the considerable circulation of the virus in the Italian population, despite a relatively small number of notified cases, a higher occurrence in central Italy, and a noteworthy predominance of G3 strains.

Quantification and genetic diversity of Hepatitis E virus in wild boar (Sus scrofa) hunted for domestic consumption in Central Italy.

Hepatitis E is an emerging disease in industrialized countries. The food-borne transmission of hepatitis E virus (HEV) is associated principally with products derived from the domestic pig, the wild boar, and deer; however, few quantitative data are available on HEV loads in animals used in food production. This study assessed HEV occurrence, viral load and genetic variability in wild boar hunted for domestic consumption in the district of Viterbo (Central Italy) where high anti-HEV IgG seroprevalence values are reported in humans. A total of 332 liver and 69 intestine samples were obtained from wild boar hunted between 2011 and 2014. The liver tissue in 54 of the animals (16.3%) was HEV-positive. Viral loads in quantifiable liver samples (n = 29) ranged between 3.2 × 102 and 3.8 × 105 genome copies (g.c.)/g with a mean value of 1.85 × 104 g.c./g. A statistically significant positive correlation was found between viral concentration in liver and intestinal tissue, though mean viral load in the intestines was lower (3.13 × 103 g.c./g). Twenty-six samples were characterized molecularly as genotype 3 (G3) and four subtypes (a, c, f and l) were detected. Finally, twelve samples with near identical sequences were identified as G3 but could not be assigned to any of the known subtypes, and could therefore represent a potentially new subtype.

Foodborne botulism: an evolving public health challenge.

Foodborne botulism is a life-threatening disease caused by the ingestion of food containing preformed botulinum neurotoxins, the most potent natural poisons known to humans. On the basis of the new challenges in management of the diseases as well as considering the potential use of botulinum toxins as biological weapons, foodborne botulism is still considered a public health emergency. Each suspected case should be immediately notified to public health authorities with the aim of preparing a prompt response. With the aim of improving botulism surveillance systems, health authorities as well as governmental organizations should enhance national and international cooperation. Education and training activities devoted to operators involved in the disease management, and to general population, may significantly contribute to strengthen the system.

Fresh produce and microbial contamination: persistence during the shelf life and efficacy of domestic washing methods.

The transmission of enteric pathogens by fresh produce depends on the survival of the bacteria organisms during the product shelf-life. The removal of any potentially hazardous microorganism from the vegetables is therefore dependent on the washing and sanitizing techniques employed by individual households. For this purpose, in this work we investigated the persistence of enteric bacteria, using as model Salmonella enterica serovar Napoli (S. Napoli) and Yersinia enterocolitica, in vegetables stored at refrigeration temperature (4 °C). The efficiency of tap water and different chlorine solutions for cleaning vegetables experimentally contaminated with Y. enterocolitica were tested. The results showed that in lettuce spiked with different concentrations of S. Napoli and Y. enterocolitica, both microorganisms were still detected after seven days of storage at 4 °C. Lettuce contaminated with low concentrations of Y. enterocolitica was not decontaminated by washing with tap water or with water added with 60 ppm of chlorine. The presence of Y. enterocolitica in lettuce was reduced of about 1-2 logs after washing with water added with 220 ppm of chlorine. The addition of low concentration of chlorine in post harvest washing processes represents a useful tool to reduce the contamination of the vegetables, with consequent reduction of the risks. However, since complete decontamination was not achieved, foodborne infections linked to fresh produce can still be possible, although contamination is avoided during primary production.

A severe outbreak of listeriosis in central Italy with a rare pulsotype associated with processed pork products.

PURPOSE: From May 2015 to March 2016, an outbreak due to Listeria monocytogenes serotype 1/2a and clinical pulsotype never previously isolated in Europe occurred in central Italy, involving 24 confirmed clinical cases. The article provides a description of the outbreak and the investigation carried out by a multidisciplinary network. METHODOLOGY: Epidemiological and microbiological surveillance was conducted to confirm the outbreak and to detect the food vehicle of infection. The origin and destination of the implicated food and its ingredients were investigated by tracing-back and -forward investigation. RESULTS: Next-generation sequencing confirmed the unique outbreak strain. On 4 January 2016, a L. monocytogenes strain with pulsotype indistinguishable from that isolated from clinical cases in the outbreak was detected in a sample of hog head cheese purchased from a retail supermarket by one of the patients. The hog head cheese was produced by a small meat processing plant in the Marche region, where microbiological investigation confirmed environmental and food contamination by the outbreak strain. Plant production was suspended and all contaminated batches of the hog head cheese were withdrawn from the market by 19 February by local health authority. We subsequently observed a sharp decline in clinical cases, the last being reported on 11 March 2016. CONCLUSION: The key factor in the timely conclusion of this investigation was intersectoral collaboration among epidemiologists, microbiologists, veterinarians, statisticians and health and food safety authorities at national, regional and local levels.

Botulism in Italy, 1986 to 2015.

Botulism is a rare but severe neuroparalytic disease caused by botulinum toxins. Because of its high potential impact on public health, botulism is a closely monitored communicable disease in Europe. In Italy, which has one of the highest incidence rates in Europe (0.03 cases per 100,000 population), botulism is monitored through a case-based passive surveillance system: the front-line physician who diagnoses a suspected case must notify the Local Health Units immediately, and the Ministry of Health's office within 12 hours. From 1986 to 2015, 466 confirmed cases of botulism were recorded in Italy (of 1,257 suspected cases). Of these, 421 were food-borne (the most frequently seen form of botulism due to the consumption of improperly home-canned foods), 36 were infant botulism, which accounts for ca 50% of all these types of cases registered in Europe, six were wound-related and three were due to adult intestinal colonisation. This scenario suggests that stronger efforts should be made towards raising public awareness of the risk of food-borne botulism, especially with respect to home-preserved foods, as well as improving the training of front-line medical personnel, to ensure that a quick and accurate diagnosis of botulism can be made.

Occurrence and Trend of Hepatitis A Virus in Bivalve Molluscs Production Areas Following a Contamination Event.

The aim of this study was to assess the trend of hepatitis A virus (HAV) in a coastal zone impacted by a contamination event, providing data for the development of management strategies. A total of 352 samples, including four bivalve mollusc species (Mytilus galloprovincialis, Solen vagina, Venus gallina and Donax trunculus), were taken over a period of 6 months from 27 production areas of the coast and analysis were performed according to ISO/TS 15216-1:2013. HAV presence was detected in 77 samples from 11 production areas and all positive results were related to samples collected in the first 3 months of the surveillance, during which HAV prevalence was 39.9% and values as high as 5096 genome copies/g were detected. A progressive reduction of viral contamination was evident during the first trimester of the monitoring, with prevalence decreasing from 78.8% in the first month, to 37.8% in the second and 3.9% in the third and quantitative levels reduced from an average value of 672 genome copies/g to 255 genome copies/g over a period of 4 weeks (virus half-life: 21.5 days). A regression analysis showed that, during the decreasing phase of the contamination, the data fitted a reciprocal quadratic model (Ra2 = 0.921) and, based on the model, a residual presence of HAV could be estimated after negativization of the production areas. The statistical analysis of the results per shellfish species and per production area showed that there were limited differences in contamination prevalence and levels among diverse bivalve species, while a statistically significant difference was present in quantitative levels of one production area. These data could be useful for the development of both risk assessment models and code of practice for the management of viral contamination in primary production.

Multiple-locus variable number of tandem repeat analysis as a tool for molecular epidemiology of botulism: The Italian experience.

Clostridium botulinum is the bacterial agent of botulism, a rare but severe neuro-paralytic disease. Because of its high impact, in Italy botulism is monitored by an ad hoc surveillance system. The National Reference Centre for Botulism, as part of this system, collects and analyzes all demographic, epidemiologic, microbiological, and molecular data recovered during cases and/or outbreaks occurred in Italy. A panel of 312 C. botulinum strains belonging to group I were submitted to MLVA sub-typing. Strains, isolated from clinical specimens, food and environmental samples collected during the surveillance activities, were representative of all forms of botulism from all Italian regions. Through clustering analysis isolates were grouped into 12 main clusters. No regional or temporal clustering was detected, demonstrating the high heterogeneity of strains circulating in Italy. This study confirmed that MLVA is capable of sub-typing C. botulinum strains. Moreover, MLVA is effective at tracing and tracking the source of contamination and is helpful for the surveillance system in terms of planning and upgrading of procedures, activities and data collection forms.

Key Role of Sequencing to Trace Hepatitis A Viruses Circulating in Italy During a Large Multi-Country European Foodborne Outbreak in 2013.

BACKGROUND: Foodborne Hepatitis A Virus (HAV) outbreaks are being recognized as an emerging public health problem in industrialized countries. In 2013 three foodborne HAV outbreaks occurred in Europe and one in USA. During the largest of the three European outbreaks, most cases occurred in Italy (>1,200 cases as of March 31, 2014). A national Task Force was established at the beginning of the outbreak by the Ministry of Health. Mixed frozen berries were early demonstrated to be the source of infection by the identity of viral sequences in patients and in food. In the present study the molecular characterization of HAV isolates from 355 Italian cases is reported. METHODS: Molecular characterization was carried out by PCR/sequencing (VP1/2A region), comparison with reference strains and phylogenetic analysis. RESULTS: A unique strain was responsible for most characterized cases (235/355, 66.1%). Molecular data had a key role in tracing this outbreak, allowing 110 out of the 235 outbreak cases (46.8%) to be recognized in absence of any other link. The data also showed background circulation of further unrelated strains, both autochthonous and travel related, whose sequence comparison highlighted minor outbreaks and small clusters, most of them unrecognized on the basis of epidemiological data. Phylogenetic analysis showed most isolates from travel related cases clustering with reference strains originating from the same geographical area of travel. CONCLUSIONS: In conclusion, the study documents, in a real outbreak context, the crucial role of molecular analysis in investigating an old but re-emerging pathogen. Improving the molecular knowledge of HAV strains, both autochthonous and circulating in countries from which potentially contaminated foods are imported, will become increasingly important to control outbreaks by supporting trace back activities, aiming to identify the geographical source(s) of contaminated food, as well as public health interventions.

Genomic characterization of Italian Clostridium botulinum group I strains.

Clostridium botulinum is a gram-positive bacterium capable of producing the botulinum neurotoxin, a powerful poison that causes botulism, a severe neuroparalytic disease. Its genome has been sequenced entirely and its gene content has been analyzed. To date, 19 full genomes and 64 draft genomes are available. The geographical origin of these genomes is predominantly from the US. In the present study, 10 Italian genomes of C. botulinum group I were analyzed and compared with previously sequenced group I genomes, in order to genetically characterize the Italian population of C. botulinum group I and to investigate the phylogenetic relationships among different lineages. Using the suites of software ClonalFrame and ClonalOrigin to perform genomic analysis, we demonstrated that Italian C. botulinum group I population is phylogenetically heterogeneous encompassing different and distant lineages including overseas strains, too. Moreover, a high recombination rate was demonstrated in the evolution of C. botulinum group I species. Finally, genome sequencing of the strain 357 led us to identify a novel botulinum neurotoxin subtype, F8.

Foodborne botulism associated with home-preserved turnip tops in Italy.

In Italy, foodborne botulism is a rare disease mainly due to home-preserved food. In the case reported here, clinical diagnosis was performed on the basis of clinical signs and referred consumption of home-preserved turnip tops in oil. Definitive diagnosis was performed by detection of botulinum toxin in sera and neuro-toxigenic organisms in stools and leftover food. This case report highlights the need of a high medical awareness, prompt clinical diagnosis, and synergic collaboration among the health authorities for a correct management of botulism as well as disease containment.