Validation of ELISA-based detection of L. monocytogenes and E. coli O157:H7 in fresh cut vegetables

Innovative diagnostic methods were developed for the detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed fresh cut fruits and vegetables. The aim of the present study was to validate the technical efficiency of these methods and evaluate their efficacy and viability for routine analysis. To this purpose, ready-to-eat fresh fruits and vegetables were collected throughout the production chain. A multidisciplinary approach, including a newly developed ELISA method compared to ISO procedures, was applied to detect the pathogenic bacteria after harvesting, processing and shelf-life. Results obtained exhibited the technical efficiency of the developed methods showing similar sensitivity, specificity, negative predictive values and negative likelihood ratios

Development and performance characteristics evaluation of a new Bioelectric Recognition Assay (BERA) method for rapid Sars-CoV-2 detection in clinical samples

Introduction: As the second wave of COVID-19 pandemic is in progress the development of fast and cost-effective approaches for diagnosis is essential. The aim of the present study was to develop and evaluate the performance characteristics of a new Bioelectric Recognition Assay (BERA) regarding Sars-CoV-2 detection in clinical samples and its potential to be used as a point of care test. Materials and methods: All tests were performed using a custom portable hardware device developed by EMBIO DIAGNOSTICS (EMBIO DIAGNOSTICS Ltd, Cyprus). 110 positive and 136 negative samples tested by RT-PCR were used in order to define the lower limit of detection (L.O.D.) of the system, as well as the sensitivity and the specificity of the method. Results: The system was able to detect a viral concentration of 4 genome copies/μL. The method displayed total sensitivity of 92.7 % (95 %CI: 86.2–96.8) and 97.8 % specificity (95 %CI: 93.7–99.5). When samples were grouped according to the recorded Ct values the BERA biosensor displayed 100.00 % sensitivity (95 %CI: 84.6–100.0) for Ct values <20−30. For the aforementioned Ct values the Positive Predictive Value (PPV) of the method was estimated at 31.4 % for COVID-19 prevalence of 1% and at 70.5 % for 5% prevalence. At the same time the Negative Predictive Value (NPV) of the BERA biosensor was at 100.0 % for both prevalence rates. Conclusions: EMBIO DIAGNOSTICS BERA for the detection of SARS-CoV-2 infection has the potential to allow rapid and cost-effective detection and subsequent isolation of confirmed cases, and therefore reduce household and community transmissions. © 2021 Elsevier B.V

Antimicrobial activity of lactic acid bacteria on biofilm formed by Listeria monocytogenes

Lactic acid bacteria are proposed as an innovative eco-friendly strategy to control relevant food-borne pathogens including Listeria monocytogenes and L. monocytogenes represent a safety concern for the food industry due to its ability to survive and grow under several harsh conditions associated with food processing and preservation. In addition, L. monocytogenes may persist in food plants and equipment since able to form biofilms. In the present work, we screened 152 lactic acid bacteria for their ability to inhibit the growth of L. monocytogenes serovar ½a and 4b from animal and vegetable origin. The antagonistic effect was investigated by analysing the halo of inhibition on agar plates co-inoculated with LAB strains or in presence of the correspondent cell-free supernatant. The same approach was employed to evaluate the reduction of biofilm formation on glass, polystirene, and stainless steel. L. monocytogenes biofilms were quantified spectrophotometrically and the viability of the pathogen assessed by qPCR. LAB were clustered in four categories according to their inhibitor effect. The strains with the stronger antagonistic activity suggest a potential employment to control L. monocytogenes proliferation and the corresponding biofilm formation in food processing and plants

Estimation of listeria monocytogenes and escherichia coli O157:H7 prevalence and levels in naturally contaminated rocket and cucumber samples by deterministic and stochastic approaches

The aims of the present study were to determine the prevalence and levels of Listeria monocytogenes and Escherichia coli O157:H7 in rocket and cucumber samples by deterministic (estimation of a single value) and stochastic (estimation of a range of values) approaches. In parallel, the chromogenic media commonly used for the recovery of these microorganisms were evaluated and compared, and the efficiency of an enzyme-linked immunosorbent assay (ELISA)-based protocol was validated. L. monocytogenes and E. coli O157:H7 were detected and enumerated using agar Listeria according to Ottaviani and Agosti plus RAPID'L.mono medium and Fluorocult plus sorbitol MacConkey medium with cefixime and tellurite in parallel, respectively. Identity was confirmed with biochemical and molecular tests and the ELISA. Performance indices of the media and the prevalence of both pathogens were estimated using Bayesian inference. In rocket, prevalence of both L. monocytogenes and E. coli O157:H7 was estimated at 7% (7 of 100 samples). In cucumber, prevalence was 6% (6 of 100 samples) and 3% (3 of 100 samples) for L. monocytogenes and E. coli O157:H7, respectively. The levels derived from the presence-absence data using Bayesian modeling were estimated at 0.12 CFU/25 g (0.06 to 0.20) and 0.09 CFU/25 g (0.04 to 0.170) for L. monocytogenes in rocket and cucumber samples, respectively. The corresponding values for E. coli O157:H7 were 0.59 CFU/25 g (0.43 to 0.78) and 1.78 CFU/25 g (1.38 to 2.24), respectively. The sensitivity and specificity of the culture media differed for rocket and cucumber samples. The ELISA technique had a high level of cross-reactivity. Parallel testing with at least two culture media was required to achieve a reliable result for L. monocytogenes or E. coli O157:H7 prevalence in rocket and cucumber samples

In Vitro Gene Transcription of Listeria monocytogenes after Exposure to Human Gastric and Duodenal Aspirates

The aim of the present study was to assess, for the first time to our knowledge, Listeria monocytogenes CFU changes, as well as to determine the transcription of key virulence genes, namely, sigB, prfA, hly, plcA, plcB, inlA, inlB, inlC, inlJ, inlP, and lmo2672 after in vitro exposure to human gastric and duodenal aspirates. Furthermore, investigations of the potential correlation between CFU changes and gene regulation with factors influencing gastric (proton pump inhibitor intake and presence of gastric atrophy) and duodenal pH were the secondary study aims. Gastric and duodenal fluids that were collected from 25 individuals undergoing upper gastrointestinal endoscopy were inoculated with L. monocytogenes serotype 4b strain LQC 15257 at 9 log CFU•mL-1 and incubated at 378C for 100 min and 2 h, respectively, with the time corresponding to the actual exposure time to gastric and duodenal fluids in the human gastrointestinal tract. Sampling was performed upon gastric fluid inoculation, after incubation of the inoculated gastric fluids, upon pathogen resuspension in duodenal fluids and after incubation of the inoculated duodenal fluids. L. monocytogenes CFU changes were assessed by colony counting, as well as reverse transcription quantitative PCR by using inlB as a target. Gene transcription was assessed by reverse transcription quantitative PCR. In 56% of the cases, reduction of the pathogen CFU occurred immediately after exposure to gastric aspirate. Upregulation of hly and inlC was observed in 52 and 58% of the cases, respectively. On the contrary, no upregulation or downregulation was noticed regarding sigB, prfA, plcA, plcB, inlA, inlB, inlJ, inlP, and lmo2672. In addition, sigB and plcA transcription was positively and negatively associated, respectively, with an increase of the pH value, and inlA transcription was negatively associated with the presence of gastric atrophy. Finally, a positive correlation between the transcriptomic responses of plcB, inlA, inlB, inlC, inlJ, inlP, and lmo2672 was detected. This study revealed that the CFU of the pathogen was negatively affected after exposure to human gastroduodenal aspirates, as well as significant correlations between the characteristics of the aspirates with the virulence potential of the pathogen. © 2020 International Association for Food Protection. All rights reserved

Validation of innovative methods for human pathogen bacteria detection in fresh cut vegetables

In the framework of the QUAFETY FP7 \u2013 EU project innovative diagnostic methods have been developed for the detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed fresh cut fruits and vegetables. The aim of the present study was to validate the technical efficiency of these methods and evaluate their efficacy and viability for routine analysis. For this purpose, ready-to-eat fresh fruits and vegetables, have been collected throughout the production chain. More accurately, a total of 48 samples of rocket, mixed salad and piel de sapo melon have been provided by Italian, Portuguese and Greek SMEs. A multidisciplinary approach, including newly developed ELISA and MPN-qPCR methods as well as ISO procedures have been used to detect the pathogenic bacteria after harvesting, processing, packaging and shelf-life. Results obtained exhibited the technical efficiency of the developed methods. More accurately, both methods had similar sensitivity, specificity, negative predictive values and negative likelihood ratios. False positive results obtained by the ELISA method resulted in the reduction of positive predictive values. Regarding their efficacy and viability for routine analysis it is mostly dependent upon available equipment and technical expertise