Molecular characterization of Yersinia enterocolitica strains to evaluate virulence associated genes.

INTRODUCTION: Yersinia enterocolitica (Ye) species is divided into 6 biotypes (BT), 1A, 1B, 2, 3, 4, 5 classified based on biochemical reactions and about 70 serotypes, classified based on the structure of the lipopolysaccharide O-antigen. The BT1A is considered non-pathogenic, while the BT 1B-5 are considered pathogenic. METHODS: Evaluate the distribution of eleven chromosomal and plasmid virulence genes, ail, ystA, ystB, myfA, hreP, fes, fepD, ymoA, sat, virF and yadA, in 87 Ye strains isolated from food, animals and humans, using two SYBR Green real-time PCR platforms. RESULTS: The main results showed the presence of the ail and ystA genes in all the pathogenic bioserotypes analyzed. The ystB, on the other hand, was identified in all non-pathogenic strains biotype 1A. The target fes, fepD, sat and hreP were found in both pathogenic biotypes and in BT1A strains. The myfA gene was found in all pathogenic biotype and in some Ye BT1A strains. The virF and yadA plasmid genes were mainly detected in bioserotype 4/O:3 and 2/O:9, while ymoA was identified in all strains. CONCLUSIONS: The two molecular platforms could be used to better define some specific molecular targets for the characterization and rapid detection of Ye in different sources which important implications for food safety and animal and human health.

A Cholera Case Imported from Bangladesh to Italy: Clinico-Epidemiological Management and Molecular Characterization in a Non-Endemic Country.

Despite the number of cholera outbreaks reported worldwide, only a few cases are recorded among returning European travellers. We describe the case of a 41-year-old male, returning to Italy after a stay in Bangladesh, his origin country, who presented with watery diarrhoea. Vibrio cholerae and norovirus were detected in the patient's stools via multiplex PCR methods. Direct microscopy, Gram staining, culture and antibiotic susceptibility tests were performed. The isolates were tested using end-point PCR for the detection of potentially enteropathogenic V. cholera. Serotype and cholera toxins identification were carried out. Whole genome sequencing and bioinformatics analysis were performed, and antimicrobial resistance genes identified. A phylogenetic tree with the most similar genomes of databases previously described was built. Sample of the food brought back by the patient were also collected and analysed. The patient was diagnosed with V. cholerae O1, serotype Inaba, norovirus and SARS-CoV-2 concomitant infection. The isolated V. cholerae strain was found to belong to ST69, encoding for cholera toxin, ctxB7 type and was phylogenetically related to the 2018 outbreak in Dhaka, Bangladesh. Adopting a multidisciplinary approach in a cholera non-endemic country ensured rapid and accurate diagnosis, timely clinical management, and epidemiological investigation at national and international level.

Foodborne Toxigenic Agents Investigated in Central Italy: An Overview of a Three-Year Experience (2018-2020).

Foodborne diseases (FBDs) represent a worldwide public health issue, given their spreadability and the difficulty of tracing the sources of contamination. This report summarises the incidence of foodborne pathogens and toxins found in food, environmental and clinical samples collected in relation to diagnosed or suspected FBD cases and submitted between 2018 and 2020 to the Food Microbiology Unit of the Istituto Zooprofilattico Sperimentale del Lazio e della Toscana (IZSLT). Data collected from 70 FBD investigations were analysed: 24.3% of them started with an FBD diagnosis, whereas a further 41.4% involved clinical diagnoses based on general symptomatology. In total, 5.6% of the 340 food samples analysed were positive for the presence of a bacterial pathogen, its toxins or both. Among the positive samples, more than half involved meat-derived products. Our data reveal the probable impact of the COVID-19 pandemic on the number of FBD investigations conducted. In spite of the serious impact of FBDs on human health and the economy, the investigation of many foodborne outbreaks fails to identify the source of infection. This indicates a need for the competent authorities to continue to develop and implement a more fully integrated health network.

Presence of Shiga Toxin-Producing Escherichia coli (STEC) in Fresh Beef Marketed in 13 Regions of ITALY (2017).

The aim of this study was to determine the prevalence of Shiga toxin-producing Escherichia coli in fresh beef marketed in 2017 in 13 regions of Italy, to evaluate the potential risk to human health. According to the ISO/TS 13136:2012 standard, 239 samples were analysed and nine were STEC positive, from which 20 strains were isolated. The STEC-positive samples were obtained from Calabria (n = 1), Campania (n = 1), Lazio (n = 2), Liguria (n = 1), Lombardia (n = 1) and Veneto (n = 3). All STEC strains were analysed for serogroups O26, O45, O55, O91, O103, O104, O111, O113, O121, O128, O145, O146 and O157, using Real-Time PCR. Three serogroups were identified amongst the 20 strains: O91 (n = 5), O113 (n = 2), and O157 (n = 1); the O-group for each of the 12 remaining STEC strains was not identified. Six stx subtypes were detected: stx1a, stx1c, stx2a, stx2b, stx2c and stx2d. Subtype stx2c was the most common, followed by stx2d and stx2b. Subtype stx2a was identified in only one eae-negative strain and occurred in combination with stx1a, stx1c and stx2b. The presence in meat of STEC strains being potentially harmful to human health shows the importance, during harvest, of implementing additional measures to reduce contamination risk.

Prevalence and Molecular Characterisation of Shiga Toxin-Producing Escherichia Coli in Raw Milk Cheeses from Lazio Region, Italy.

In recent years, the incidence of foodborne diseases caused by Shiga toxin-producing Escherichia coli (STEC) has increased globally. For this reason, within the specific regional control plan for the detection of STEC in food products in Italy, the presence of STEC in unpasteurised milk cheeses was investigated. In total, 203 samples obtained from March 2011 to December 2013 were analysed, with two standard methods (ISO 16654:2001 and ISO 13136:2012). Two strains of E. coli O157 were isolated (2/161, 1.2%) but did not carry any virulence-associated genes and 22 stx-positive samples (22/146, 15.1%) were detected in enrichment cultures, mostly from ovine cheeses. Only two strains isolated from different ovine cheeses carried stx gene and none of these was eae-positive. This study confirms the presence of stx-positive E. coli and suggests that this type of food cannot be excluded as a potential vehicle of STEC.

European validation of Real-Time PCR method for detection of Salmonella spp. in pork meat.

The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories.

European validation of a real-time PCR-based method for detection of Listeria monocytogenes in soft cheese.

The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories.

Extended-spectrum beta-lactamases in Escherichia coli isolated from dogs and cats in Rome, Italy, from 2001 to 2003.

We report expanded-spectrum cephalosporin resistance in Escherichia coli from dogs and cats in Rome, Italy. Three major beta-lactamases (CMY-2, SHV-12, and CTX-M-1) are reported for the first time in E. coli from sick and healthy dogs and cats. Molecular characterization suggests the presence of several combinations of beta-lactamase genes in E. coli from companion animals.

Chronic hyperglycemia impairs insulin secretion by affecting insulin receptor expression, splicing, and signaling in RIN beta cell line and human islets of Langerhans.

Recent evidence suggests that insulin signaling through the insulin receptor A type (Ex11-), regulates insulin gene transcription. Because chronic hyperglycemia negatively affects insulin receptor function and regulates alternative splicing of the insulin receptor, we inquired whether chronic exposure of pancreatic beta-cells to high glucose results in alterations in insulin signaling due to changes in insulin receptor expression and relative abundance of its spliced isoforms. Our results demonstrate that the insulin receptor is localized in insulin secretory vescicles in human pancreatic beta-cells. Furthermore, we find that alterations in insulin expression and secretion caused by chronic exposure to high glucose are paralleled by decreased insulin receptor expression and increased relative abundance of the Ex11+ isoform in both human islets and RIN beta-cells. PDX-1 and HMGI(Y) transcription factors are down-regulated by high glucose. These changes are associated with defects in insulin signaling involving insulin receptor-associated PI 3-kinase/Akt/PHAS-I pathway in RIN beta-cells. Re-expression in RIN beta-cells chronically exposed to high glucose of the Ex11-, but not the Ex11+, isoform restored insulin mRNA expression. These data suggest that changes in early steps of insulin receptor signaling may play a role in determining beta-cell dysfunction caused by chronic hyperglycemia.