DVC-FISH to identify potentially pathogenic Legionella inside free-living amoebae from water sources

[EN] Despite all safety efforts, drinking and wastewater can still be contaminated by Legionella and free-living amoebae (FLA) since these microorganisms are capable of resisting disinfection treatments. An amoebae cyst harboring pathogenic Legionella spp. can be a transporter of this organism, protecting it and enhancing its infection abilities. Therefore, the aim of this work is to identify by DVC-FISH viable Legionella spp and Legionella pneumophila cells inside FLA from water sources in a specific and rapid way with the aim of assessing the real risk of these waters. A total of 55 water samples were processed, 30 reclaimed wastewater and 25 drinking water. FLA presence was detected in 52.7% of the total processed water samples. When DVC-FISH technique was applied, the presence of viable internalized Legionella spp. cells was identified in 69.0% of the total FLA-positive samples, concretely in 70.0% and 66.7% of wastewater and drinking water samples, respectively. L. pneumophila was simultaneously identified in 48.3% of the total FLA-positive samples, specifically in 50.0% and 44.4% of wastewater and drinking water samples, respectively. By culture, potentially pathogenic Legionella cells were recovered in 27.6% of the total FLA-positive bacteria, particularly in 35.0% and 11.1% of wastewater and drinking water samples, respectively. These findings demonstrate that FLA may promote resistance of bacteria to the performed disinfection treatments for drinking as well as for wastewater. So, in addition to the risk for the presence of pathogenic FLA in water it is necessary to take into account that these can be transporters of the pathogenic bacteria Legionella, which are able to survive inside them. The DVC-FISH method described here has been proved to be a rapid and specific tool to identify pathogenic Legionella spp. and L. penumophila viable cells harboured by FLA in these water sources, posing particular public health concern.This study has been supported by First research projects funding (PAID-06-18), Vice-Rectorate for Research, Innovation and Transfer of Universitat Politècnica de València" (UPV), València, Spain.Moreno Trigos, MY.; Moreno-Mesonero, L.; García Hernández, J. (2019). DVC-FISH to identify potentially pathogenic Legionella inside free-living amoebae from water sources. Environmental Research. 176:1-7. https://doi.org/10.1016/j.envres.2019.06.002S1717

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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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

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

In vitro antimicrobial activity of immobilised essential oil components against Helicobacter pylori

[EN] Available disinfection methods and therapies against Helicobacter pylori have multiple disadvantages, such as increased prevalence of antibiotic-resistant strains, which requires the search for novel effective antimicrobial agents against H. pylori. Among them, naturally-occurring antimicrobial compounds, like essential oil components (EOCs), have been reported as substances with anti-H. pylori potential. To avoid the disadvantages associated with using EOCs in their free form, including volatility, low water solubility and intense sensory properties, their immobilisation in inert supports has recently been developed. This study sought to evaluate the inhibitory properties of EOCs immobilised on silica microparticles against H. pylori and to elucidate the mechanism of action of the immobilised antimicrobials. After the preparation and characterisation of the antimicrobial supports, the susceptibility of H. pylori in the presence of the immobilised compounds was assessed by plate count, fluorescent viability staining and direct viable count-fluorescent in situ hybridisation analyses. The antimicrobial supports were found to inhibit H. pylori growth, and to induce morphological and metabolic alterations to the H. pylori membrane, with a minimum bactericidal concentration value between 25 and 50 mu g/ml according to the tested EOC. These findings indicate that immobilised EOCs can be used as potential antimicrobial agents for H. pylori clearance and treatment.Authors gratefully acknowledge the financial support from the Ministerio de Ciencia, Innovacion y Universidades, the Agencia Estatal de Investigacion and FEDER-EU (Project RTI2018-101599-B-C21). M.R.R. acknowledges the Generalitat Valenciana for their Postdoctoral Fellowship (APOSTD/2019/118).Ruiz Rico, M.; Moreno Trigos, MY.; Barat Baviera, JM. (2020). In vitro antimicrobial activity of immobilised essential oil components against Helicobacter pylori. World Journal of Microbiology and Biotechnology. 36(1):1-9. https://doi.org/10.1007/s11274-019-2782-yS19361Adams BL, Bates TC, Oliver JD (2003) Survival of Helicobacter pylori in a natural freshwater environment. Appl Environ Microbiol 69:7462–7466. https://doi.org/10.1128/AEM.69.12.7462-7466.2003Ali S, Khan A, Ahmed I et al (2005) Antimicrobial activities of eugenol and cinnamaldehyde against the human gastric pathogen Helicobacter pylori. Ann Clin Microbiol Antimicrob 4:20. https://doi.org/10.1186/1476-0711-4-20Altiok D, Altiok E, Tihminlioglu F (2010) Physical, antibacterial and antioxidant properties of chitosan films incorporated with thyme oil for potential wound healing applications. 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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

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

Detection and enumeration of viable Listeria monocytogenes cells from ready-to-eat and processed vegetable foods by culture and DVC-FISH

Listeria monocytogenes is an important agent of foodborne disease, showing low prevalence but high mortality. There is evidence that vegetables are important vehicles of transmission, especially those minimally processed (fresh, fresh-cut vegetables under modified atmosphere packaging (MAP) or frozen) and directly exposed to consumers. The aim of this work was to detect and enumerate viable . L. . monocytogenes cells by culture and molecular methods from vegetable foods.A total of 191 vegetable samples (fresh, frozen and fresh-cut under modified atmosphere packaging) were studied. . L. monocytogenes was detected and identified by selective plating, PCR and DVC-FISH.An isolation rate of 4.19% was obtained by culture, with a higher incidence in frozen vegetables. Six isolates belonged to serotype 1/2a and 2 to 4b. Counts were below <100 cfu/g for the eight positive samples, according to the food safety criteria established for the RTE by Commission Regulation EC No 1441/2007.Multiplex PCR method yielded a greater number of positive samples (10.47%). DVC-FISH technique determined that viable cells of L. monocytogenes were present in 32.98% of the samples, containing up to 4.97 log 10 viable cells/g. © 2012 Elsevier Ltd.This work was supported by the grant AGL2008-05275-C03-02 (national and FEDER funds) from Ministerio de Ciencia e Innovacion, Spain.Moreno Trigos, MY.; Sánchez Contreras, J.; Montes Estellés, RM.; García Hernández, J.; Ballesteros, L.; Ferrús Pérez, MA. (2012). Detection and enumeration of viable Listeria monocytogenes cells from ready-to-eat and processed vegetable foods by culture and DVC-FISH. Food Control. 27(2):374-379. https://doi.org/10.1016/j.foodcont.2012.04.01737437927

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

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