Novel Same-Day method for viable Salmonella Enteritidis detection in chicken meat combining phage amplification and LAMP

Salmonella enterica is a major foodborne pathogen worldwide. Poultry products, especially eggs and meat, are the main responsible for human salmonellosis cases. Culture-based methods require at least 3 days to detect Salmonella positive samples. To facilitate food chain processes and provide a rapid response to food outbreaks, a simple and rapid detection method is necessary. For this purpose, nucleic acid amplification-based techniques are a potential solution. Loop-Mediated isothermal AMPlification (LAMP) has emerged as an alternative to qPCR due to the simple equipment necessary to perform the analysis while allowing the detection of living cells when combined with bacteriophages. The aim of this work was to develop a same-day protocol based in the combination of LAMP and a Salmonella phage (vB-SenS_PVP-SE2) to detect viable Salmonella Enteritidis cells in chicken meat. Specific LAMP primers were designed to target the capsid and endolysin genes of Salmonella phage vB-SenS_PVP-SE2. Two different detection strategies were developed: real-time fluorescence; and colorimetric (naked-eye detection). The LAMP method developed could detect down to 0.2 fg/L of pure phage DNA and concentrations of viral particles in buffered peptone water (BPW) of 10 pfu/mL. After optimization in spiked chicken samples, a 3 h sample pre-enrichment diluted 1/10 in BPW before phage addition to the samples followed by a co-incubation (with phage) of 4 h was established. The proposed method could determine the presence of S. Enteritidis in less than 8 h including sample processing, DNA isolation and LAMP analysis with a LOD of 1.5 cfu/25g and a LOD of 6.6 cfu/25g, both by fluorescence and naked-eye observation. The results were in close concordance with the reference method for Salmonella spp., the ISO 6579-1:2017. The described method represents a promising alternative for the rapid detection of Salmonella in the food chain.info:eu-repo/semantics/publishedVersio

Phage amplification coupled with loop-mediated isothermal amplification (PA-LAMP) for same-day detection of viable Salmonella Enteritidis in raw poultry meat

Salmonella Enteritidis is the main serotype responsible for human salmonellosis in the European Union. One of the main sources of Salmonella spp. in the food chain are poultry products, such as eggs or chicken meat. In recent years, molecular methods have become an alternative to culture dependent methods for the rapid screening of Salmonella spp. In this work, the strain S. Enteritidis S1400, and previously isolated and characterized bacteriophage PVP-SE2, were used to develop and evaluate a same-day detection method combining Phage Amplification and Loop-mediated isothermal amplification (PA-LAMP) to specifically detect viable S. Enteritidis in chicken breast. This method is based on the detection of the phage DNA rather than bacterial DNA. The virus is added to the sample during pre-enrichment in buffered peptone water, where it replicates in the presence of viable S. Enteritidis. The detection of phage DNA allows, on the one hand to detect viable bacteria, since viruses only replicate in them, and on the other hand to increase the sensitivity of the method since for each infected S. Enteritidis cell, hundreds of new viruses are produced. Two different PA-LAMP detection strategies were evaluated, a real time fluorescence and a naked-eye detection. The present method could down to 0.2 fg/L of pure phage DNA and a concentration of viral particles of 2.2 log PFU/mL. After a short Salmonella recovery step of 3 h and a co-culture of 4 h of the samples with phage particles, both real-time fluorescence and naked-eye method showed a LoD95 of 6.6 CFU/25 g and a LoD50 of 1.5/25 g in spiked chicken breast samples. The entire detection process, including DNA extraction and LAMP analysis, can be completed in around 8 h. In the current proof-of-concept, the novel PA-LAMP obtained comparable results to those of the reference method ISO 6579, to detect Salmonella Enteritidis in poultry meat.Dr. Alexandre Lamas was funded by a postdoctoral fellowship from Xunta de Galicia (Axudas de apoio a étapa de formación posdoutoral IN606B (Modalidade A)). Dr. Alejandro Garrido-Maestu acknowledges funding from the Fundação para a Ciência e Tecnologia through the Scientific Employment Stimulus Program (2021.02810.CEECIND).info:eu-repo/semantics/publishedVersio

Assessment of the presence of Acinetobacter spp. resistant to β-lactams in commercial ready-to-eat salad samples

Acinetobacter baumannii is a well-known nosocomial infection causing agent. However, other Acinetobacter spp. have also been implicated in cases of human infection. Additionally, these bacteria are known for the development of antibiotic resistance thus making the treatment of the infections they cause, challenging. Due to their relevance in clinical setups less attention has been paid to their presence in foods, and its relation with infection/ dissemination routes. In the current study commercial Ready-To-Eat (RTE) salads were analyzed seeking for antibiotic resistant Acinetobacter spp. A preliminary screening allowed us to recover Gram-negative bacteria resistant to β – lactams using cefotaxime, third generation cephalosporins, as the selective agent, and this was followed by identification with CHROMagar™ Acinetobacter and 16S rDNA sequencing. Finally, the isolates identified as Acinetobacter spp. were reanalyzed by PCR to determine the presence of nine potential Extended Spectrum β Lactamases (ESBL). Two commercial RTE salad brands were included in the study (2 batches per brand and 8 samples of each batch making a total of 32 independent samples), and compared against an organic lettuce. High concentrations of β – lactam, resistant bacteria were found in all the samples tested (5 log CFU/g). Additionally, 209 isolates were phenotypically characterized on CHROMagar Acinetobacter. Finally, PCR analysis identified the presence of different ESBL genes, being positive for blaACC, blaSHV, blaDHA and blaVEB; out of these, blaACC was the most prevalent. None of the isolates screened were positive for more than one gene. To conclude, it is important to highlight the fact that pathogenic species within the genus Acinetobacter spp., other than A. baumannii, have been identified bearing resistance genes not typically associated to these microorganisms highlight the importance of continuous surveillance.info:eu-repo/semantics/publishedVersio

Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples

Serovar Enteritidis represents 45.7% of all Salmonella reported human cases identified in Europe. Additionally, minced meat and meat preparations from poultry have a high level of non-compliance, regarding Salmonella regulation. In the current study, a novel method based on the amplification of the Salmonella bacteriophage vB_SenS_PVP-SE2, coupled with real-time PCR (qPCR), was developed and evaluated, for the rapid detection of viable Salmonella Enteritidis in chicken samples. The results obtained indicated that the qPCR method could detect down to 0.22 fg/L of pure virus DNA and a concentration of viral particles of 103pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8cfu/25g could be detected within 10h, including the time for DNA extraction and qPCR analysis. Additionally, the evaluation of the performance parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values, and index kappa of concordance, obtained values higher than 92%, and the acceptability limit values were within the limits. All these results demonstrate that the proposed methodology is a powerful tool for the rapid detection of viable Salmonella Enteritidis.This work was supported by the project Nanotechnology Based Functional Solutions (NORTE-01-0145-FEDER-000019), supported by NortePortugal Regional Operational Programme(NORTE2020), andby the “NanoBioSensor: Desenvolvimento de nanosensores para avaliação da qualidade microbiológica de produtos à base de fruta” (POCI-010247-FEDER-033925), supported bytheOperational ThematicProgram for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio

Whole genome sequencing in the palm of your hand: how to implement a MinION Galaxy-based workflow in a food safety laboratory for rapid Salmonella spp. serotyping, virulence, and antimicrobial resistance gene identification

IntroductionWhole Genome Sequencing (WGS) implementation in food safety laboratories is a significant advancement in food pathogen control and outbreak tracking. However, the initial investment for acquiring next-generation sequencing platforms and the need for bioinformatic skills represented an obstacle for the widespread use of WGS. Long-reading technologies, such as the one developed by Oxford Nanopore Technologies, can be easily implemented with a minor initial investment and with simple protocols that can be performed with basic laboratory equipment.MethodsHerein, we report a simple MinION Galaxy-based workflow with analysis parameters that allow its implementation in food safety laboratories with limited computer resources and without previous knowledge in bioinformatics for rapid Salmonella serotyping, virulence, and identification of antimicrobial resistance genes. For that purpose, the single use Flongle flow cells, along with the MinION Mk1B for WGS, and the community-driven web-based analysis platform Galaxy for bioinformatic analysis was used. Three strains belonging to three different serotypes, monophasic S. Typhimurium, S. Grancanaria, and S. Senftenberg, were sequenced.ResultsAfter 24 h of sequencing, enough coverage was achieved in order to perform de novo assembly in all three strains. After evaluating different tools, Flye de novo assemblies with medaka polishing were shown to be optimal for in silico Salmonella spp. serotyping with SISRT tool followed by antimicrobial and virulence gene identification with ABRicate.DiscussionThe implementation of the present workflow in food safety laboratories with limited computer resources allows a rapid characterization of Salmonella spp. isolates

Faster monitoring of the invasive alien species (IAS) Dreissena polymorpha in river basins through isothermal amplification

Zebra mussel (Dreissena polymorpha) is considered as one of the 100 most harmful IAS in the world. Traditional detection methods have limitations, and PCR based environmental DNA detection has provided interesting results for early warning. However, in the last years, the development of isothermal amplification methods has received increasing attention. Among them, loop-mediated isothermal amplification (LAMP) has several advantages, including its higher tolerance to the presence of inhibitors and the possibility of naked-eye detection, which enables and simplifies its potential use in decentralized settings. In the current study, a real-time LAMP (qLAMP) method for the detection of Dreissena polymorpha was developed and tested with samples from the Guadalquivir River basin, together with two real-time PCR (qPCR) methods using different detection chemistries, targeting a specific region of the mitochondrial gene cytochrome C oxidase subunit I. All three developed approaches were evaluated regarding specificity, sensitivity and time required for detection. Regarding sensitivity, both qPCR approaches were more sensitive than qLAMP by one order of magnitude, however the qLAMP method proved to be as specific and much faster being performed in just 9 min versus 23 and 29 min for the qPCR methods based on hydrolysis probe and intercalating dye respectivelyThis work was supported by a partnership agreement project between the Confederación Hidrográfica del Guadalquivir and INL for the development of a system for early detection of zebra mussels through analysis of environmental DNA, and by project Nanotechnology Based Functional Solutions (NORTE-01-0145-FEDER-000019), supported by Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund. S. A. acknowledges the Portuguese funding institution FCT – Fundação para Ciência e Tecnologia for Ph.D. scholarship SFRH/BD/140396/2018S

Green synthesis of lignin nano- and micro-particles: Physicochemical characterization, bioactive properties and cytotoxicity assessment

Lignin particles (LPs) have gained prominence due to their biodegradability and bioactive properties. LP production at nano and micro scale produced from organosolv lignin and the understanding of size's effect on their properties is unexplored. This work aimed to produce and characterize lignin nanoparticles and microparticles using a green synthesis process, based on ethanol-solubilized lignin and water. Spherical shape LPs, with a mean size of 75 nm and 215 nm and with a low polydispersity were produced, as confirmed by transmission electron microscopy and dynamic light scattering. LPs thermal stability improved over raw lignin, and the chemical structure of lignin was not affected by the production method. The antimicrobial tests proved that LPs presented a bacteriostatic effect on Escherichiacoli and Salmonella enterica. Regarding the antioxidant potential, LPs had a good antioxidant activity that increased with the reaction time and LPs concentration. LPs also presented an antioxidant effect against intracellular ROS, reducing the intracellular ROS levels significantly. Furthermore, the LPs showed a low cytotoxic effect in Caco-2 cell line. These results showed that LPs at different scales (nano and micro) present biological properties and are safe to be used in different high value industrial sectors, such as biomedical, pharmaceutical and food.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit, BioTecNorte operation (NORTE-01-0145-FEDER000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, and MICRODIGEST project (grant agreement 037716) co-funded by FCT and ERDF through COMPETE2020.info:eu-repo/semantics/publishedVersio

Interlaboratory validation of a multiplex qPCR method for the detection of listeria monocytogenes in a ready-to-eat seafood product

Listeria monocytogenes is a major foodborne pathogen which mainly infects susceptible individuals through the consumption of contaminated foods. To this end, ready-to-eat (RTE) food products are of particular concern as this microorganism is widely distributed, can survive, and even grow, under adverse conditions, and thus must be carefully controlled. In the present study, an interlaboratory ring trial was organized to evaluate an open formula qPCR-based method for the detection of L. monocytogenes. The molecular method was evaluated on a novel RTE seafood product, developed in the framework of a European project, the SEAFOODAGE (EAPA_758/2018). Six laboratories located in Spain and Portugal participated in the study, and the results obtained indicated that this new method presented high diagnostic sensitivity (100%) reaching a low limit of detection (<10CFU/25 g) with an overall agreement with the reference method, attending to the Cohen's k, of 0.97 that is interpreted as almost complete agreement.info:eu-repo/semantics/publishedVersio