Evidence of viable Helicobacter pylori and other bacteria of public health interest associated with free-living amoeba in lettuce samples by next generation sequencing and other molecular techniques

[EN] Vegetables are one of the sources from which Helicobacter pylori can be acquired. This bacterium infects > 50% of the global population and is a recognized type I human carcinogen. H. pylori enters into the viable but nonculturable state when it is in the environment, and therefore the use of molecular techniques is much convenient for its detection. Free-living amoebae (FLA) are protozoans found in vegetables. They are transmission vehicles for amoeba-resistant bacteria, among which H. pylori is included. The aim of this study is to study the occurrence and viability of H. pylori from lettuce samples, H. pylori internalized into FLA and the microbiome of FLA isolated from these samples. Special focus was pointed to human pathogenic bacteria. H. pylori was not directly detected in any lettuce sample by means of molecular techniques and neither by culture. However, intra-amoebic H. pylori DNA was detected by means of PMA-qPCR in 55% of the samples and viable intra-amoebic H. pylori cells in 25% of the samples by means of DVC-FISH technique. When FLA microbiome was studied, 21 bacterial genera were part of FLA microbiome in all samples. Helicobacter genus was detected as part of the FLA microbiome in two samples. Other bacteria of public health interest such as Aeromonas sp., Arcobacter sp., Legionella sp., Mycobacterium sp., Pseudomonas sp. and Salmonella sp. were detected as part of FLA microbiome along the analysed samples. This study demonstrates for the first time that H. pylori is internalized as well as alive inside FLA isolated from vegetables. Moreover, this study shows that FLA promote H. pylori detection in environmental samples. In addition, as far as we are aware, this is the first study which studies the microbiome of FLA isolated from vegetables. Among the FLA microbiome, bacteria of public health interest were detected, pointing out that FLA are carriers of these pathogens which can reach humans and cause a public health concern.This study has been supported by the Conselleria de Educacion, Investigacion, Cultura y Deporte, of the Community of Valencia, Spain, within the program of support for research under project AICO/2018/273. The author Laura Moreno-Mesonero is the recipient of a technician contract funded by the Consellerfa de Educacion, Investigacion, Cultura y Deporte, of the Community of Valencia, Spain, within the program of support for research under project AICO/2018/273.Moreno-Mesonero, L.; Hortelano, I.; Moreno Trigos, MY.; Ferrús Pérez, MA. (2020). Evidence of viable Helicobacter pylori and other bacteria of public health interest associated with free-living amoeba in lettuce samples by next generation sequencing and other molecular techniques. 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Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrio parahaemolyticus in Oysters and Mussels

[EN] Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 mu g/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 degrees C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.García Hernández, J.; Hernández Pérez, M.; Moreno Trigos, MY. (2021). Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrio parahaemolyticus in Oysters and Mussels. Foods. 10(7):1-14. https://doi.org/10.3390/foods10071502S11410

Extraction and recovery of phenolic compounds from olive leaves

Póster presentado en: 9º congreso Internacional de Química de la ANQUE. Alimentos y bebidas, 17 a 20 de junio de 2018, MurciaThere is currently a great interest on the use of the residual biomass originated from the agricultural and food sectors as a bioresource instead as a waste, due to its high potential for the recovery of high added-value compounds. Olive leaves, an olive oil processing residue, contain high amounts of phenolic compounds with health-promoting properties, such as oleuropein, tyrosol and hydroxytyrosol. These compounds can be extracted from olive leaves to be added later in processed foods and thus increase their healthy properties. In this work, the recovery of phenolic compounds present in previously ground olive leaves (size less than 1 mm) was carried out by solid-liquid extraction using several solvents (hexane, ethanol, methanol and ethyl acetate). The olive leaves used were kindly provided by the cooperative "Pagos de Benaval" from olive trees of the variety "Serrana de Espadán", endemic of the Sierra de Espadán natural park (Castellón, Spain). The total phenolic content (TPC) in extracts was determined according to the Folin- Ciocalteau method. Olive leaf extract profile and quantitative determination of oleuropein and hydroxytyrosol were performed in an HPLC-DAD system, and also in a LC-MS system. The antioxidant activity of the extracts was determined by the DPPH and ABTS assays. The best results, with a high antioxidant activity and also a high oleuropein and hydroxytyrosol content in the extracts, were obtained when ethanol and methanol were used as solvents. Finally, a central composite design was performed to determine the effect of three factors (extraction temperature (25-40 ºC), solvent volumeleaf mass ratio (5-15 mL/g) and alcohol-to-water ratio (50-90 vol. %)) on TPC, antioxidant activity, and oleuropein and hydroxytyrosol content in the extracts, in order to optimize the extraction process.Junta de Castilla y León and the European Regional Development Fund through project BU055U1

Helicobacter pylori growth pattern in reference media and extracts from selected minimally processed vegetables

[EN] Helicobacter pylori is an emergent foodborne pathogen of concern, the entrance of which into the food chain has been recently related with the possible contamination of raw or minimally processed vegetables. The present study documented the growth kinetics of the bacterium at 5, 20 and 37 degrees C, in reference media and vegetable substrates, to be fitted to the Gompertz equation. H. pylori was able to grow at 37 degrees C and 20 degrees C, but not at refrigeration temperature. Incubation temperature decrease significantly (p-value < 0.05) affected growth kinetic parameters, with the elongation of lag phase duration (lambda) and the reduction of the maximum specific growth rate (mu(max)) (0.10 log(10)(CFU/ml)/h at 37 degrees C; 0.04 log(10)(CFU/ml)/h at 20 degrees C). In vegetable extracts, the microorganism remained in a viable culturable (VC) form for a maximum of 5 days (20 degrees C), being unable to grow significantly in chard, spinach and in kale. In lettuce, H. pylori achieved growth of close to 1 log(10) cycle (after 5 days at 20 degrees C) (mu(max) 0.79 log(10)(CFU/ml)/d). The present study is the first reporting kinetic parameter values describing the growth behavior of H. pylori at its optimum growth temperature and, also studying the most relevant handling temperatures for minimally processed vegetables: commercial distribution (room temperature 20 degrees C), and refrigeration temperature. (C) 2017 Elsevier Ltd. All rights reserved.The present research work has been supported by the funds provided by the Spanish Ministry of Economy and Competitiveness (MINECO) as HELICOFOOD project with reference AGL2014-53875-R. Postdoctoral contract of M.C. Pina-Perez as Juan de la Cierva-Incorporacion granted by the MINECO is also acknowledged.Pina Pérez, MC.; González Pellicer, A.; Moreno Trigos, MY.; Ferrús Pérez, MA. (2017). Helicobacter pylori growth pattern in reference media and extracts from selected minimally processed vegetables. Food Control. 86:389-396. doi:10.1016/j.foodcont.2017.11.044S3893968

Optimization of pre- treatments with Propidium Monoazide and PEMAX¿ before real-time quantitative PCR for detection and quantification of viable Helicobacter pylori cells

[EN] Accurate detection of H. pylori in different environmental and clinical samples is essential for public health studies. Now, a big effort is being made to design PCR methodologies that allow for the detection of viable and viable but non-culturable (VBNC) H. pylori cells, by achieving complete exclusion of dead cells amplification signals. The use of DNA intercalating dyes has been proposed. However, its efficacy is still not well determined. In this study, we aimed to test the suitability of PMA and PEMAXTM dyes used prior to qPCR for only detecting viable cells of H. pylori. Their efficiency was evaluated with cells submitted to different disinfection treatments and confirmed by the absence of growth on culture media and by LIVE/DEAD counts. Our results indicated that an incubation period of 5 min for both, PMA and PEMAXTM, did not affect viable cells. Our study also demonstrated that results obtained by using intercalating dyes may vary depending on the cell stress conditions. In all dead cell¿s samples, both PMA and PEMAXTM pre-qPCR treatments decreased the amplification signal (>103 Genomic Units (GU)), although none of them allowed for its disappearance confirming that intercalating dyes, although useful for screening purposes, cannot be considered as universal viability markers. To investigate the applicability of the method specifically to detect H. pylori cells in environmental samples, PMA-qPCR was performed on samples containing the different morphological and viability states that H. pylori can acquire in environment. The optimized PMA-qPCR methodology showed to be useful to detect mostly (but not only) viable forms, regardless the morphological state of the cell.This work was supported by the Conselleria de Educacion, Investigacion, Cultura y Deporte, of the Community of Valencia, Spain, under project AICO/2018/273, and by the Spanish Ministerio de Ciencia e Innovacion PID2019-105691RB-I00 Grant and by the Spanish Ministry of Economy and Competitiveness AGL2014/53875-R Grant.Hortelano, I.; Moreno Trigos, MY.; García Hernández, J.; Ferrús Pérez, MA. (2021). Optimization of pre- treatments with Propidium Monoazide and PEMAX¿ before real-time quantitative PCR for detection and quantification of viable Helicobacter pylori cells. Journal of Microbiological Methods. 185:1-9. https://doi.org/10.1016/j.mimet.2021.1062231918

A Photonic Label-Free Biosensor to Detect Salmonella spp. in Fresh Vegetables and Meat

[EN] This paper presents a method that can be used to detect and identify Salmonella spp. in fresh meat and vegetable samples using a photonic biosensor with specialized bioreceptors. Detection was based on photon transduction. Silicon-nitride-based resonant cavities were used to capture the change in light response when there is specific binding of the immobilized antibody to the sensor surface against the target antigen. A control immobilization experiment was conducted to validate the immobilization process on the biosensor surface prior to biofunctionalization for Salmonella spp. detection. This experiment involved immobilization of pre-selected antibodies on silicon nitride surfaces. Two types of antibodies were suitable. The first was a specific polyclonal antibody with superior antigen-binding capacity across a wide range of concentrations. The second was a monoclonal antibody designed for effective binding at lower concentrations. Rigorous validation was performed. The outcomes were compared with those of the habitual method used to detect Salmonella spp. (reference method). Replicates from different batches of contaminated meat and vegetable samples were analyzed. This comprehensive approach provides a methodologically robust, highly sensitive, and accurate way of rapidly detecting Salmonella spp. in food samples. It has potential implications for improved food safety and quality controlThis research was financially supported by the AVI (Valencian Innovation Agency) and received funding from the European Union through FEDER funds. The project, identified as INNEST/2021/338, is known as the BACTERIO project , which focuses on Integrated Photonics for integral microbiological control in the agri-food sector .Fernández Blanco, A.; Hernández Pérez, M.; Moreno Trigos, MY.; García Hernández, J. (2023). A Photonic Label-Free Biosensor to Detect Salmonella spp. in Fresh Vegetables and Meat. Applied Sciences. 13(24). https://doi.org/10.3390/app132413103132

Rapid Identification of Viable H. pylori Cells in Feces by DVCFISH

[EN] Helicobacter pylori isolation in fecal samples is a less invasive and more comfortable practice than those that require patient endoscopy, particularly in children. However, culture of this pathogen from stools is usually unsuccessful. Other techniques such as PCR or H. pylori Stool Antigen (HpSA) are used to detect the presence of H. pylori in feces; nevertheless, a positive result by using these techniques does not involve viability of the pathogen. Direct Viable Count combined with Fluorescent in situ Hybridization (DVC-FISH) technique has been successfully applied to detect viable H. pylori cells in highly contaminated environmental samples. To assess the suitability of DVC-FISH technique to identify viable H. pylori cells in stools, experimentally inoculated feces and fecal samples from infected patients were analyzed. qPCR and culture techniques were also used. Viable H. pylori cells were detected by DVC-FISH in all inoculated samples with a specific DNA/LNA probe. H. pylori colonies were also identified on agar. DVC-FISH gave positive results in all the patients' fecal samples, while qPCR only detected H. pylori in two patients. DVC-FISH technique with LNA/DNA probes has the potential to be used as a specific and effective non-invasive method for the detection of viable H. pylori in stools samples. Moreover, our results evidence the presence of viable H. pylori cells in fecal samples from infected patients, supporting the evidence that H. pylori is transmitted via the fecal route.The work has been funded by AGL2014-53875-R grant (Ministerio de Economía y Competitividad, Spain).Moreno Trigos, MY.; Pérez-Santonja, R.; Ramirez, M.; Calvet, X.; Santiago Cuellar, P.; Ferrús Pérez, MA. (2015). Rapid Identification of Viable H. pylori Cells in Feces by DVCFISH. JSM Gastroenterology and Hepatology. 3(3). http://hdl.handle.net/10251/97911S3

Bodegas, lagares y almazaras en el territorio de Kelin (siglos v-iii a. C.): el caso de la Rambla de la Alcantarilla (Requena, Valencia)

Las evidencias arqueológicas datan la expansión del cultivo de la vid en el territorio de Kelin durante los siglos v-iii a. C., aunque los primeros indicios se fechan ya en el siglo vii a. C. Una de las singularidades de este territorio es la existencia de estructuras de piedra al aire libre para la producción de vino y aceite. Todas ellas se localizan en una zona concreta del territorio (Ramblas de la Alcantarilla y de los Morenos) (Requena, Valencia) y aparecen asociadas a materiales arqueológicos que permiten datarlas como ibéricas. En este artículo abordamos cómo se organiza el poblamiento en la rambla de la Alcantarilla y presentamos en detalle los yacimientos de la Rambla de la Alcantarilla y Solana de Cantos 2.Archaeological evidences date the expansion of grape vineyards in the territory of the Iberian Iron Age city of Kelin from the 5th to 3rd centuries BC; although the origins are documented in the 7th BC. One of the singularities of this territory is the existence of outdoors stone structures for the production of wine and oil. All are located in a specific area of the territory (la Alcantarilla and Los Morenos ravines) (Requena, Valencia), and are associated with archaeological materials that allow date them as Iberian. In this paper we approach the rural settlement pattern in la Alcantarilla ravine and we present in detail the esarchaeological sites of Rambla de la Alcantarilla and Solana de Cantos 2

A combination of direct viable count and fluorescence in situ hibridization for specific enumeration of viable Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus

[EN] Aims: We have developed a direct viable count (DVC)-FISH procedure for quickly and easily discriminating between viable and nonviable cells of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains, the traditional yogurt bacteria. Methods and Results: direct viable count method has been modified and adapted for Lact. delbrueckii subsp, bulgaricus and Strep. thermophilus analysis by testing different times of incubation and concentrations of DNA-gyrase inhibitors. DVC procedure has been combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of both bacteria with specific rRNA oligonucleotide probes (DVC-FISH). Of the four antibiotics tested (novobiocin, nalidixic acid, pipemidic acid and ciprofloxacin), novobiocin was the most effective for DVC method and the optimum incubation time was 7 h for both bacteria. The number of viable cells was obtained by the enumeration of specific hybridized cells that were elongated at least twice their original length for Lactobacillus and twice their original size for Streptococcus. Conclusions: This technique was successfully applied to detect viable cells in inoculated faeces. Significance and Impact of the Study: Results showed that this DVC-FISH procedure is a quick and culture-independent useful method to specifically detect viable Lact, delbrueckii subsp, bulgaricus and Strep thermophilus in different samples, being applied for the first time to lactic acid bacteria.García Hernández, J.; Moreno Trigos, MY.; Amorocho Cruz, CM.; Hernández Pérez, M. (2011). A combination of direct viable count and fluorescence in situ hibridization for specific enumeration of viable Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. Letters in Applied Microbiology. 54(3):247-254. doi:10.1111/j.1472-765X.2011.03201.x24725454

Detection of Helicobacter pylori in drinking water treatment plants in Bogotá, Colombia, using cultural and molecular techniques

[EN] Helicobacter pylori is one of the most common causes of chronic bacterial infection in humans, and a predisposing factor for peptic ulcer and gastric cancer. The infection has been consistently associated with lack of access to clean water and proper sanitation. H. pylori has been detected in surface water, wastewater and drinking water. However, its ability to survive in an infectious state in the environment is hindered because it rapidly loses its cultivability. The aim of this study was to determine the presence of cultivable and therefore viable H. pylori in influent and effluent water from drinking water treatment plants (DWTP). A total of 310 influent and effluent water samples were collected from three drinking water treatment plants located at Bogota city, Colombia. Specific detection of H. pylori was achieved by culture, qPCR and FISH techniques. Fifty-six positive H. pylori cultures were obtained from the water samples. Characteristic colonies were covered by the growth of a large number of other bacteria present In the water samples, making isolation difficult to perform. Thus, the mixed cultures were submitted to Fluorescent in situ Hybridization (FISH) and qPCR analysis, followed by sequencing of the amplicons for confirmation. By qPCR, 77 water samples, both from the influent and the effluent, were positive for the presence of H. pylori. The results of our study demonstrate that viable H. pylori cells were present In both, influent and effluent water samples obtained from drinking water treatment plants in Bogota and provide further evidence that contaminated water may act as a transmission vehicle for H. pylori. Moreover, FISH and qPCR methods result rapid and specific techniques to identify H. pylori from complex environmental samples such as influent water.This work was supported by COLCIENCIAS, Project 120356933870, CTO 642-2013, Colombia and the Spanish Ministry of Economy and Competitiveness AGL2014/53875-R Project, Spain. We greatly acknowledge the collaboration of the Aqueduct and Sewage Company of Bogota (EAB), Colombia.Vesga-Pérez, FJ.; Moreno Trigos, MY.; Ferrús Pérez, MA.; Campos Pinilla, C.; Trespalacios Rangel, AA. (2018). Detection of Helicobacter pylori in drinking water treatment plants in Bogotá, Colombia, using cultural and molecular techniques. International Journal of Hygiene and Environmental Health. 221(4):595-601. https://doi.org/10.1016/j.ijheh.2018.04.010S595601221