Assessment of the Microbiological Safety and Hygiene of Raw and Thermally Treated Milk Cheeses Marketed in Central Italy between 2013 and 2020.

A profile of the microbial safety and hygiene of cheese in central Italy was defined based on an analysis of 1373 cheeses sampled under the Italian National Control Plan for Food Safety spanning the years 2013 to 2020 and tested according to Commission Regulation (EC) No. 2073/2005 (as amended). A total of 97.4% of cheese samples were assessed as being satisfactory for food safety criteria and 80.5% for process hygiene criteria. Staphylococcal enterotoxin was found in 2/414 samples, while Salmonella spp. and Listeria monocytogenes were detected in 15 samples out of 373 and 437, respectively. Escherichia coli and coagulase-positive staphylococci counts were found unsatisfactory in 12/61 and 17/88 cheese samples, respectively. The impact of milking species, milk thermal treatment, and cheese hardness category was considered. A statistically significant association (p < 0.05) was found between milk thermal treatment and the prevalence of coagulase-positive staphylococci and Listeria monocytogenes and between hardness and unsatisfactory levels of Escherichia coli. The data depict a contained public health risk associated with these products and confirm, at the same time, the importance of strict compliance with good hygiene practices during milk and cheese production. These results can assist in bolstering risk analysis and providing insights for food safety decision making.

Monophasic Variant of Salmonella Typhimurium 4,[5],12:i:- (ACSSuGmTmpSxt Type) Outbreak in Central Italy Linked to the Consumption of a Roasted Pork Product (Porchetta).

The monophasic variant of S. Typhimurium 4,[5],12:i:- (MVST) is the third most commonly reported Salmonella serovar involved in human infections (8.8%) in the EU and ranks after S. Enteritidis (54.6%) and S. Typhimurium (11.4%). In Italy, in contrast, the MVST has achieved peculiar epidemiological and ecological success which has allowed it to be, since 2011, the serovar most frequently isolated from humans. In the summer of 2022, a foodborne outbreak of the MVST involving 63 people occurred in the Marche Region (Central Italy). A common food exposure source among some human cases was a roasted, ready-to-eat (RTE) pork product, porchetta, which is a typical product of Central Italy. This paper describes the results of investigations conducted to clarify this outbreak. The porchetta was produced by a local manufacturing plant and distributed to at least two local retail stores, one of which was the retail outlet for the manufacturing plant. The MVST was isolated from surface samples collected at the porchetta manufacturing plant and at both local retail stores via bacterial analysis, and the porchetta sampled at one store contained the MVST. These data confirm this type of RTE pork product can be a source of Salmonella infection in humans.

The Emerging Nosocomial Pathogen Klebsiella michiganensis: Genetic Analysis of a KPC-3 Producing Strain Isolated from Venus Clam.

The recovery and characterization of a multidrug-resistant, KPC-3-producing Klebsiella michiganensis that was obtained from Venus clam samples is reported in this study. A whole-genome sequencing (WGS) analysis using Illumina and Nanopore technologies of the K. michiganensis 23999A2 isolate revealed that the strain belonged to the new sequence type 382 (ST382) and carried seven plasmid replicon sequences, including four IncF type plasmids (FII, FIIY, FIIk, and FIB), one IncHI1 plasmid, and two Col plasmids. The FIB and FIIk plasmids showed high homology to each other and to multireplicon pKpQIL-like plasmids that are found in epidemic KPC-K. pneumoniae clones worldwide. The strain carried multiple ß-lactamase genes on the IncF plasmids: blaOXA-9 and blaTEM-1A on FIB, blaKPC-3 inserted in a Tn4401a on FIIK, and blaSHV-12 on FIIY. The IncHI1-ST11 harbored no resistance gene. The curing of the strain caused the loss of all of the bla genes and a rearrangement of the IncF plasmids. Conjugal transfer of the blaOXA-9, blaTEM-1A and blaKPC-3 genes occurred at a frequency of 5 × 10-7, using K. quasipneumoniae as a recipient, and all of the bla genes were transferred through a pKpQIL that originated from the recombination of the FIB and FIIk plasmids of the donor. A comparison with 31 K. michiganensis genomes that are available in the NCBI database showed that the closest phylogenetic relatives of K. michiganensis 23999A2 are an environmental isolate from soil in South Korea and a clinical isolate from human sputum in Japan. Finally, a pan-genome analysis showed a large accessory genome of the strain as well as the great genomic plasticity of the K. michiganensis species. IMPORTANCE Klebsiella michiganensis is an emerging nosocomial pathogen, and, so far, few studies describe isolates of clinical origin in the environment. This study contributes to the understanding of how the dissemination of carbapenem-resistance outside the hospital setting may be related to the circulation of pKpQIL-like plasmids that are derived from epidemic Klebsiella pneumoniae strains. The recovery of a carbapenem-resistant isolate in clams is of great concern, as bivalves could represent vehicles of transmission of pathogens and resistance genes to humans via the food chain. The study demonstrates the plasticity of K. michiganensis genome, which is probably useful to multiple environment adaptation and to the evolution of the species.

A Strong Evidence Outbreak of Salmonella Enteritidis in Central Italy Linked to the Consumption of Contaminated Raw Sheep Milk Cheese.

Salmonellosis is the second most commonly reported gastrointestinal infection in humans after campylobacteriosis, and an important cause of foodborne outbreaks in the EU/EEA. The vast majority (72.4%) of the salmonellosis foodborne outbreaks reported in EU in 2019 were caused by Salmonella Enteritidis, even if their total number due to this serovar decreased. In spring 2020, a foodborne outbreak of S. Enteritidis occurred in the Marche region (Central Italy), involving 85 people. The common exposure source was a cheese, pecorino "primo sale", produced with raw sheep milk. The cheese batches were produced by two local dairies, with a livestock production facility, also including a sheep farm, being part of one dairy. Bacteriological analysis of samples collected allowed the detection of S. Enteritidis in animal faeces, environmental samples, raw-milk bulk tanks and milk taken from single animals. These data confirm that, despite the scarce scientific evidence, S. Enteritidis can infect sheep and be shed into the animals' milk. Hence, this is a real risk for public health when unpasteurized milk is used in production of such cheese. The present paper describes the results of the investigations conducted to clarify this outbreak.

Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria monocytogenes Food and Human Isolates and In Vitro Adhesion/Invasion Assessment.

Listeria monocytogenes (Lm) is the causative agent of human listeriosis. Lm strains have different virulence potential. For this reason, we preliminarily characterised via Whole-Genome Sequencing (WGS) some Lm strains for their key genomic features and virulence-associated determinants, assigning the clonal complex (CC). Moreover, the ability of the same strains to adhere to and invade human colon carcinoma cell line Caco-2, evaluating the possible correspondence with their genetic virulence profile, was also assessed. The clinical strains typed belonged to clonal complex (CC)1, CC31, and CC101 and showed a very low invasiveness. The Lm strains isolated from food were assigned to CC1, CC7, CC9, and CC121. All CC1 carried the hypervirulence pathogenicity island LIPI-3 in addition to LIPI-1. Premature stop codons in the inlA gene were found only in Lm of food origin belonging to CC9 and CC121. The presence of LIPI2_inlII was observed in all the CCs except CC1. The CC7 strain, belonging to an epidemic cluster, also carried the internalin genes inlG and inlL and showed the highest level of invasion. In contrast, the human CC31 strain lacked the lapB and vip genes and presented the lowest level of invasiveness. In Lm, the genetic determinants of hypo- or hypervirulence are not necessarily predictive of a cell adhesion and/or invasion ability in vitro. Moreover, since listeriosis results from the interplay between host and virulence features of the pathogen, even hypovirulent clones are able to cause infection in immunocompromised people.

Heavy Metals Contamination in Shellfish: Benefit-Risk Evaluation in Central Italy.

Seafood is a source of nutrients in human diet but also of environmental contaminants and its consumption could pose a risk to consumers' health. A survey regarding the exposure to cadmium, lead and mercury through the consumption of bivalve mollusks, gastropods and sea urchins collected on Italian coasts was carried out among central Italian population over a period of three years. A limited number of samples exceeds the threshold set by legislation (6 samples) and the average level of contamination was low in all the species considered. The contribution Acceptable Daily Intake (ADI) was higher for cadmium (9.17%) than lead (1.44%) and mercury (0.20%). The benefit-risk evaluation suggests that the bivalve mollusks and sea urchins consumption (Benefit Risk Quotient < 1) could be increased without health detrimental effects.

Vibrio parahaemolyticus control in mussels by a Halobacteriovorax isolated from the Adriatic sea, Italy.

This study evaluated the application of a Halobacteriovorax isolated from water of the Adriatic Sea (Italy) in controlling V. parahaemolyticus in mussels (Mytilus galloprovincialis). Two 72 h laboratory-scale V. parahaemolyticus decontamination experiments of mussels were performed. The test microcosm of experiment 1 was prepared using predator/prey free mussels experimentally contaminated with Halobacteriovorax/V. parahaemolyticus at a ratio of 103 PFU/105 CFU per ml, while that of experiment 2 using mussels naturally harbouring Halobacteriovorax that were experimentally contaminated with 105 CFU per ml of V. parahaemolyticus. For experiment 1, was also tested a control microcosm only contaminated with 105 CFU per ml of V. parahaemolyticus.. Double layer agar plating and pour plate techniques were used to enumerate Halobacteriovorax and V. parahaemolyticus, respectively. 16 S rRNA analysis was used to identify Halobacteriovorax. For both experiments in the test microcosm the concentration of prey remained at the same level as that experimentally added, i.e. 5 log for the entire analysis period. In experiment 1, V. parahaemolyticus counts in mussels were significantly lower in the test microcosm than the control with the maximum difference of 2.2 log at 24 h. Results demonstrate that Halobacteriovorax can modulate V. parahaemolyticus level in the mussels. The public impact of V. parahaemolyticus in bivalves is relevant and current decontamination processes are not always effective. Halobacteriovorax is a suitable candidate in the development of a biological approach to the purification of V. parahaemolyticus in mussels.

Vibrio parahaemolyticus-specific Halobacteriovorax From Seawater of a Mussel Harvesting Area in the Adriatic Sea: Abundance, Diversity, Efficiency and Relationship With the Prey Natural Level.

This research aimed to study the abundance and molecular diversity of Vibrio parahaemolyticus-specific Halobacteriovorax strains isolated from seawater of the Adriatic Sea and the relationship between predator and prey abundances. Moreover, predator efficiency of the Halobacteriovorax isolates toward V. parahaemolyticus and Vibrio cholerae non-O1/O139 strains was tested. V. parahaemolyticus NCTC 10885 was used as primary host for the isolation of Halobacteriovorax from seawater by the plaque assay. Molecular identification was performed by PCR detection of a fragment of the 16S rRNA gene of the Halobacteriovoraceae family members. Moreover, 700 bp PCR products were sequenced and compared between them and to clones described for other sampling sites. Vibrio counts were performed on TCBS agar from 100 ml of filtered water samples and presumptive colonies were confirmed by standard methods. Predatory efficiency of Halobacteriovorax isolates was tested by monitoring abilities of 3-day enrichments to form clear lytic halos on a lawn of Vibrio preys, by the plaque assay. Out of 12 seawater samples monthly collected from June 2017 to May 2018, 10 were positive for V. parahaemolyticus specific Halobacteriovorax with counts ranging from 4 to 1.4 × 103 PFU per 7.5 ml. No significant relationship was found between Halobacteriovorax and Vibrio abundances. The 16SrRNA sequences of our Halobacteriovorax strains, one for each positive sample, were divided into three lineages. Within the lineages, some sequences had 100% similarity. Sequence similarity between lineages was always <94.5% suggesting that they may therefore well belong to three different species. All Halobacteriovorax isolates had the ability to prey all tested Vibrio strains. Additional research is necessary to assess whether stable strains of Halobacteriovorax are present in the Adriatic Sea and to understand the mechanisms by which Halobacteriovorax may modulate the abundance of V. parahaemolyticus and other vibrios in a complex marine ecosystem.

Occurrence of Arcobacter spp. and correlation with the bacterial indicator of faecal contamination Escherichia coli in bivalve molluscs from the Central Adriatic, Italy.

A total of 162 samples of bivalve molluscs (45 mussels and 117 clams) collected between December 2012 and 2014 from harvesting areas of the Central Adriatic were analysed by a culturing method for the presence of Arcobacter spp. Species identification was performed by PCR and sequencing analysis of a fragment of the rpoB gene. Overall, Arcobacter species were detected in 30% of samples, specifically 33% clams and 22% mussels. A. butzleri was the most common species (20% of the samples), followed by A. cryaerophilus (9%) and A. skirrowii (1%). A seasonal association of A. butzleri contamination was detected. A. butzleri was significantly more commonly recovered from samples collected during the winter-spring period (29%) than from those of the summer-autumn (8%). A. cryaerophilus was cultured from 6% to 11% of the samples collected in summer-autumn and winter-spring, respectively, but these differences were not statistically significant. A. skirrowii was recovered from a sample of mussels harvested in May 2014. To identify associations between the occurrence of Arcobacter spp. and E. coli levels, samples were divided into groups generating results with E. coli at >230MPN/100g and E. coli at ≤230MPN/100g, the latter corresponding to EU microbiological criteria allowed for live bivalve molluscs at retail level. A. butzleri was significantly more commonly detected in samples with higher E. coli levels (48%) than in those with lower levels of E. coli (10%), providing evidence for considering E. coli as an index organism for A. butzleri contamination in bivalve molluscs.

Trh (tdh-/trh+) gene analysis of clinical, environmental and food isolates of Vibrio parahaemolyticus as a tool for investigating pathogenicity.

Sequencing analysis of the trh gene encoding the TDH-related haemolysin of tdh-/trh+ Vibrio parahaemolyticus isolated in Italy between 2002 and 2011 from clinical, environmental, and food samples revealed the presence of the trh2 variant in all isolates. The trh2 of the clinical isolate was 100% identical to other clinical tdh-/trh2 V. parahaemolyticus from Europe. Nucleotide and amino acid differences in the trh2 sequences of clinical isolates from Italy and other countries allowed a differentiation of the clinical strains from the majority of environmental or food strains isolated in Italy. Aspartic acid and isoleucine at positions 113 and 115, encoded by nucleotide triplets GAT and ATT at positions 337-339 and 343-345 of the complete trh gene sequence, were present in clinical strains from Europe (Italy, Norway and Germany), Asia and the United States. Only 35.5% of the tdh-/trh2 V. parahaemolyticus of environmental or food origin from Italy shared the same triplets/amino acid detected in clinical isolates, while 64.5% of isolates from the marine environment were different from those of clinical origins, demonstrating that differences occur amongst the trh2 sequences of strains from the environment and these polymorphisms may differentiate potentially pathogenic from less or non-pathogenic cultures found in the environment and seafood. In addition the distribution of T3SS2 genes was investigated in this group of tdh-/trh+ V. parahaemolyticus from different sources and in three clinical tdh+/trh- V. parahaemolyticus isolates. All tdh-/trh+ V. parahaemolyticus of environmental or food source, independent of year of isolation or geographical origin, amplified all the screened T3SS2ß genes and tested negative to PCR assays for all five T3SS2α genes, as the tdh-/trh+ clinical V. parahaemolyticus isolate. The vopC genes, encoding for one of the effector proteins of T3SS2, were partially sequenced and compared to clinical tdh-/trh+ and tdh+/trh+ V. parahaemolyticus isolates from other countries. Analysis of T3SS2ß vopC sequences revealed variation in tdh-/trh2 isolates from Italy, which were separated from a group of vopC sequences derived from trh2 V. parahaemolyticus from the USA.

Bioaccumulation experiments in mussels contaminated with the food-borne pathogen Arcobacter butzleri: preliminary data for risk assessment.

The aim of this study was to evaluate, at a laboratory scale, the ability of this microorganism to grow in seawater and bioaccumulate in mussels (Mytilus galloprovincialis) maintained in constantly aerated tanks, containing twenty litres of artificial seawater. Three concentrations of A. butzleri LMG 10828(T) were tested (about 5 × 106 CFU/mL, 5 × 104 CFU/mL, and 5 × 10² CFU/mL). Following contamination, enumeration of A. butzleri was performed from water and mussels each day, for up to 96 h. Three contamination experiments with artificial seawater in absence of mussels were also performed in the same manner. In the experiments with mussels, A. butzleri declined in water of approximately 1 log every 24 h from the contamination. In artificial seawater without mussels the concentration of A. butzleri remained on the same logarithmic level in the first 48 h and then decreased of about 1 log every 24 hours. In mussels, the concentration was approximately 2 log lower than the exposition level after 24 h from the contamination, and then it decreased exponentially of 1 log every 24 h. Our findings suggest that in the experimental conditions tested A. butzleri is neither able to effectively grow in seawater nor bioaccumulate in mussels, at least in the free and cultivable form.

A severe case of Aeromonas veronii biovar sobria travellers' diarrhoea characterized by Vibrio parahaemolyticus co-isolation.

We report a severe case of travellers' diarrhoea in a patient returning from Ecuador to Italy with the concomitant presence of Aeromonas veronii biovar sobria and Vibrio parahaemolyticus in their faeces. Based on diagnostic results, epidemiological information and the clinical outcome, we conclude that the real aetiological agent was A. veronii biovar sobria, while V. parahaemolyticus was only transient in the intestine of the patient.

Nontoxigenic Vibrio parahaemolyticus strains causing acute gastroenteritis.

We investigated the virulence properties of four Vibrio parahaemolyticus strains causing acute gastroenteritis following consumption of indigenous mussels in Italy. The isolated strains were cytotoxic and adhesive but, surprisingly, lacked tdh, trh, and type three secretion system 2 (T3SS2) genes. We emphasize that nontoxigenic V. parahaemolyticus can induce acute gastroenteritis, highlighting the need for more investigation of the pathogenicity of this microorganism.

Unusual case of necrotizing fasciitis caused by Vibrio cholerae O137.

We report a case of necrotizing fasciitis caused by Vibrio cholerae O137 in an immunocompromised 49-year-old man. The infection was acquired following a minor traumatic injury and exposure to seawater during the summer of 2009 in Italy. Although highly immunocompromised, the patient survived. The strain was cytotoxic, invasive, and adhesive and contained a fragment of the El Tor-like hemolysin (El Tor hlyA) gene.

Vibrio parahaemolyticus-associated gastroenteritis in Italy: persistent occurrence of O3:K6 pandemic clone and emergence of O1:KUT serotype.

We report 2 cases of O3:K6 and O1:KUT Vibrio parahaemolyticus gastroenteritis associated with consumption of local mussels in Italy in 2008. Serotypic, antibiogram, toxigenic, and pulsed-field gel electrophoresis patterns of these strains were compared to those of other isolates collected from local clinical and seafood samples in 2007 to 2008. We underline the recurrent presence of O3:K6 pandemic clone and the emergence of trh-positive O1:KUT serotype in Italy.

Prevalence, serotyping and molecular characterization of Vibrio parahaemolyticus in mussels from Italian growing areas, Adriatic Sea.

The aims of this study were to investigate the prevalence of total and pathogenic Vibrio parahaemolyticus strains in Italian mussels from different geographical areas of the Adriatic Sea and to determine their serotypes, toxigenic profiles and pandemic potential. Out of 559 samples analysed during 2007, 65 (11.6%) were positive for V. parahaemolyticus. None of the isolates had the genes for thermostable direct haemolysin (tdh) and pandemic marker (toxRS), while five strains (7.7%) had that for TDH-related haemolysin (trh). Regarding geographical location of the toxigenic strains, three were from the Adriatic coast of Puglia, one from Veneto, and one from the Marches. The trh-positive V. parahaemolyticus isolates from Puglia belonged to O1:KUT (2/3) and O1:K37(1/3) serotypes, the trh-positive isolate from the Marches to OUT(O untypeable):KUT serotype, and that from Veneto to O3:KUT. The prevalence of trh-positive V. parahaemolyticus obtained from mussels in this study was higher respect to that reported in previous studies from other European and Extraeuropean countries. The Health Authorities should be more aware of the epidemiological role of environmental V. parahaemolyticus in local food-borne diseases, and increase the microbial surveillance on these microorganisms isolated from shellfish.

Prevalence and virulence properties of non-O1 non-O139 Vibrio cholerae strains from seafood and clinical samples collected in Italy.

Seafood and clinical samples collected in Italy during 2006 were analyzed to evaluate prevalence, serological and virulence properties of non-O1 non-O139 Vibrio cholerae (NCV) isolates. Biochemical and serological characterization of the strains was performed by standardized procedures while virulence properties of NCVs were assayed by molecular, in vivo and in vitro toxicological methods. Of the 300 seafood samples examined, including mussel, cod, mackerel, anchovy, clam, prawn and cuttlefish, 5.6% were positive for NCVs: 4.7% and 8.5% from local and imported seafood, respectively. The prevalence of NCVs was highest in prawn (16.6%) and mussel (7.7%). Of 58 hospitalized patients that presented acute diarrhea, 3.4% eliminated NCVs in stools 24-48 h after consumption of seafood. All NCVs had ToxR and hlyAET genes but lacked ctxA, zot, and tcpA genes. One isolate from prawn had stn/sto gene. All strains were hemolytic, cytotoxic, and able to induce intestinal and extraintestinal effects on the suckling mouse model. Our results confirm that non-toxigenic NCVs that express the gene encoding El Tor-like hemolysin can be isolated from patients suffering a cholera-like syndrome after consumption of seafood. This evidence along with the virulence and enteropathogenicity of all the ctxA(-) tcpA(-) zot(-) stn/sto(-) hlyAET(+) NCV isolates in the experimental model, suggest that El Tor-like hemolysin may play an important role in human pathogenesis. Moreover, the isolates from seafood showed molecular, biological and enzymatic patterns similar to those isolated from clinical samples, underlining that environmental NCVs are potentially able to induce human infections and confirming the important role of seafood as a vehicle of V. cholerae diseases.