Droplet Digital PCR (ddPCR) Analysis for Detecting Shiga-Toxin-Producing Escherichia coli (STEC)

Verocytotoxin-producing Escherichia coli, also referred to as Shiga-toxin-producing Escherichia coli (STEC), can be transmitted to humans through person-to-person contact, consumption of contaminated food or water, or by direct contact with animals. Its clinical and economic consequences have prompted the development of alternative approaches to the official method of analysis “UNI CEN ISO/TS 13136: 2012”, which describes the identification of STEC through the detection of its main virulence genes. Recently, droplet digital PCR (ddPCR) has been proposed as a technique for the sequence-specific detection and direct quantification of nucleic acids. The present study aimed to investigate if ddPCR could be able to detect STEC in less time than that required by the official method. This study consisted of the ddPCR of slices of beef contaminated with STEC and of the sponges used for beef official control at the slaughter stage. The results showed the ability of ddPCR to detect STEC in slices of beef already after sample incubation for 7 h at 37 °C while, in the case of sponges used for official controls, 9 h at 37 °C was needed. In this way, the ddPCR could represent an efficient method for detecting STEC and providing results in less time than the official method

Droplet Digital PCR (ddPCR) Analysis for Detecting Shiga-Toxin-Producing Escherichia coli (STEC)

Verocytotoxin-producing Escherichia coli, also referred to as Shiga-toxin-producing Escherichia coli (STEC), can be transmitted to humans through person-to-person contact, consumption of contaminated food or water, or by direct contact with animals. Its clinical and economic consequences have prompted the development of alternative approaches to the official method of analysis “UNI CEN ISO/TS 13136: 2012”, which describes the identification of STEC through the detection of its main virulence genes. Recently, droplet digital PCR (ddPCR) has been proposed as a technique for the sequence-specific detection and direct quantification of nucleic acids. The present study aimed to investigate if ddPCR could be able to detect STEC in less time than that required by the official method. This study consisted of the ddPCR of slices of beef contaminated with STEC and of the sponges used for beef official control at the slaughter stage. The results showed the ability of ddPCR to detect STEC in slices of beef already after sample incubation for 7 h at 37 °C while, in the case of sponges used for official controls, 9 h at 37 °C was needed. In this way, the ddPCR could represent an efficient method for detecting STEC and providing results in less time than the official method

Exposure to Bacillus cereus in Water Buffalo Mozzarella Cheese

Bacillus cereus is a spoilage bacterium and is recognized as an agent of food poisoning. Two food-borne illnesses are caused by B. cereus: a diarrheal disease, associated with cytotoxin K, hemolysin BL, non-hemolytic enterotoxin and enterotoxin FM, and an emetic syndrome, associated with the cereulide toxin. Owing to the heat resistance of B. cereus and its ability to grow in milk, this organism should be considered potentially hazardous in dairy products. The present study assessed the risk of B. cereus poisoning due to the consumption of water buffalo mozzarella cheese. A total of 340 samples were analyzed to determine B. cereus counts (ISO 7932:2005); isolates underwent molecular characterization to detect the presence of genes encoding toxins. Eighty-nine (26.1%) samples harbored B. cereus strains, with values ranging from 2.2 × 102 to 2.6 × 106 CFU/g. Isolates showed eight different molecular profiles, and some displayed virulence characteristics. Bacterial counts and the toxin profiles of isolates were evaluated both separately and jointly to assess the risk of enteritis due to B. cereus following the consumption of buffalo mozzarella cheese. In conclusion, the results of the present study showed that the risk of poisoning by B. cereus following the consumption of this cheese was moderate

Exposure to Bacillus cereus in Water Buffalo Mozzarella Cheese

: Bacillus cereus is a spoilage bacterium and is recognized as an agent of food poisoning. Two food-borne illnesses are caused by B. cereus: a diarrheal disease, associated with cytotoxin K, hemolysin BL, non-hemolytic enterotoxin and enterotoxin FM, and an emetic syndrome, associated with the cereulide toxin. Owing to the heat resistance of B. cereus and its ability to grow in milk, this organism should be considered potentially hazardous in dairy products. The present study assessed the risk of B. cereus poisoning due to the consumption of water buffalo mozzarella cheese. A total of 340 samples were analyzed to determine B. cereus counts (ISO 7932:2005); isolates underwent molecular characterization to detect the presence of genes encoding toxins. Eighty-nine (26.1%) samples harbored B. cereus strains, with values ranging from 2.2 × 102 to 2.6 × 106 CFU/g. Isolates showed eight different molecular profiles, and some displayed virulence characteristics. Bacterial counts and the toxin profiles of isolates were evaluated both separately and jointly to assess the risk of enteritis due to B. cereus following the consumption of buffalo mozzarella cheese. In conclusion, the results of the present study showed that the risk of poisoning by B. cereus following the consumption of this cheese was moderate

Detection of pathogenic <i>Vibrio</i> spp. in foods: polymerase chain reaction-based screening strategy to rapidly detect pathogenic <i>Vibrio parahaemolyticus</i>, <i>Vibrio cholerae</i>, and <i>Vibrio vulnificus</i> in bivalve mollusks and preliminary results

The majority of human diseases attributed to seafood are caused by Vibrio spp. and the most commonly reported species are Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae. The conventional methods for the detection of Vibrio species involve the use of selective media, which are inexpensive and simple but time-consuming. The present work aimed to develop a rapid method based on the use of multiplex real-time polymerase chain reaction (PCR) to detect V. parahaemolyticus, V. vulnificus, and V. cholerae in bivalve mollusks. 30 aliquots of bivalve mollusks (Mytilus galloprovincialis) were experimentally inoculated with two levels of V. parahaemolyticus, V. vulnificus, and V. cholerae. ISO 21872-1:2017 was used in parallel for qualitative analysis. The limit of detection of 50% was 7.67 CFU/g for V. cholerae, 0.024 CFU/g for V. vulnificus, and 1.36 CFU/g for V. parahaemolyticus. For V. vulnificus and V. cholerae, the real-time PCR protocol was demonstrated to amplify the pathogens in samples seeded with the lowest and highest levels. The molecular method evaluated showed a concordance rate of 100% with the reference microbiological method. V. parahaemolyticus was never detected in samples contaminated with the lowest level, and it was detected in 14 samples (93.33%) seeded with the highest concentration. In conclusion, the multiplex real-time PCR developed proved to be reliable for V. vulnificus and V. cholerae. Results for V. parahaemolyticus are promising, but further analysis is needed. The proposed method could represent a quick monitoring tool and, if used, would allow the implementation of food safety

Detection of SARS-CoV-2 RNA in Bivalve Mollusks by Droplet Digital RT-PCR (dd RT-PCR)

Bivalve shellfish are readily contaminated by human pathogens present in waters impacted by municipal sewage, and the detection of SARS-CoV-2 in feces of infected patients and in wastewater has drawn attention to the possible presence of the virus in bivalves. The aim of this study was to collect data on SARS-CoV-2 prevalence in bivalve mollusks from harvesting areas of Campania region. A total of 179 samples were collected between September 2019 and April 2021 and were tested using droplet digital RT-PCR (dd RT-PCR) and real-time RT-PCR. Combining results obtained with different assays, SARS-CoV-2 presence was detected in 27/179 (15.1%) of samples. A median viral concentration of 1.1 &times; 102 and 1.4 &times; 102 g.c./g was obtained using either Orf1b nsp14 or RdRp/gene E, respectively. Positive results were unevenly distributed among harvesting areas and over time, positive samples being more frequent after January 2021. Partial sequencing of the spike region was achieved for five samples, one of which displaying mutations characteristic of the Alpha variant (lineage B.1.1.7). This study confirms that bivalve mollusks may bioaccumulate SARS-CoV-2 to detectable levels and that they may represent a valuable approach to track SARS-CoV-2 in water bodies and to monitor outbreak trends and viral diversity

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&ndash;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&ndash;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&ndash;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