0000000000015519
AUTHOR
Patricia Elizaquível
Recent developments in the use of viability dyes and quantitative PCR in the food microbiology field.
The increase in foodborne outbreaks highlights the need for rapid, sensitive and specific methods for food safety monitoring, enabling specific detection and quantification of viable foodborne pathogens. Real-time PCR (qPCR) combined with the use of viability dyes, recently introduced, fulfils all these requirements. The strategy relies on the use of DNA-binding molecules such as propidium monoazide (PMA) or ethidium monoazide (EMA) as sample pretreatment previous to the qPCR. These molecules permeate only membrane-compromised cells and have successfully been applied for different types of foodborne pathogens, including bacteria and viruses. Moreover, those dyes have been explored to monito…
A real-time PCR assay for detection and quantification of 2-branched (1,3)-β-D–glucan producing lactic acid bacteria in cider
28 p.-1 fig.-4 tab.
Potential microbial risk factors related to soil amendments and irrigation water of potato crops
Aims: This study assesses the potential microbial risk factors related to the use of soil amendments and irrigation water on potato crops, cultivated in one traditional and two intensive farms during two harvest seasons. Methods and Results: The natural microbiota and potentially pathogenic micro-organisms were evaluated in the soil amendment, irrigation water, soil and produce. Uncomposted amendments and residual and creek water samples showed the highest microbial counts. The microbial load of potatoes harvested in spring was similar among the tested farms despite the diverse microbial levels of Listeria spp. and faecal coliforms in the potential risk sources. However, differences in tota…
Characterization of lactic acid bacteria recovered from atole agrio, a traditional Mexican fermented beverage
Our aim was to identify and characterize the lactic acid bacteria (LAB) of atole agrio, a fermented Mexican maize-based beverage and to evaluate whether starters could be obtained to produce it under controlled conditions. Atole agrio fermentation process was variable with an abundant presence of Enterobacteriaceae throughout the fermentation. Based on RAPD-PCR, Weissella (29.2%), Pediococcus (24.0%), Lactococcus (17.8%) and Lactobacillus (16.4%) were the most abundant LAB genera. Out of 88 identified LAB strains, 87.5% produced folates, 71.6% degraded phytates, 38.6% produced exopolysaccharides (EPS) and 12.5% had amylolytic activity. The majority of the strains (81.8%) were resistant to a…
Lactic acid bacteria associated with vacuum-packed cooked meat product spoilage: population analysis by rDNA-based methods.
Aim: To determine the lactic acid bacteria (LAB) implicated in bloating spoilage of vacuum-packed and refrigerated meat products. Methods and Results: A total of 18 samples corresponding to four types of meat products, with and without spoilage symptoms, were studied. In all, 387 colonies growing on de Man, Rogosa and Sharpe, yeast glucose lactose peptone and trypticase soy yeast extract plates were identified by internal spacer region (ISR), ISR-restriction fragment length polymorphism and rapid amplified ribosomal DNA restriction analysis profiles as Lactobacillus (37%), Leuconostoc (43%), Carnobacterium (11%), Enterococcus (4%) and Lactococcus (2%). Leuconostoc mesenteroides dominated …
Simultaneous detection of the main black aspergilli responsible for ochratoxin A (OTA) contamination in grapes by multiplex real-time polymerase chain reaction
9 pages.
Comparative efficacy of Zataria multiflora Boiss., Origanum compactum and Eugenia caryophyllus essential oils against E. coli O157:H7, feline calicivirus and endogenous microbiota in commercial baby-leaf salads.
Abstract Ready-to-eat salads using baby-leaf and multi-leaf mixes are one of the most promising developments in the fresh-cut food industry. There is great interest in developing novel decontamination treatments, which are both safe for consumers and more efficient against foodborne pathogens. In this study, emulsions of essential oils (EOs) from Origanum compactum (oregano), Eugenia caryophyllus (clove), and Zataria multiflora Boiss (zataria) were applied by spray (0.8 ml) after the sanitizing washing step. The aim was to investigate their ability to control the growth of potentially cross-contaminating pathogens and endogenous microbiota in commercial baby leaves, processed in a fresh-cut…
Lactobacillus sicerae sp. nov., a lactic acid bacterium isolated from Spanish natural cider
Strains CUPV261T and CUPV262 were isolated from ropy natural ciders of the Basque Country, Spain, in 2007. Cells are Gram-stain positive, non-spore-forming, motile rods, facultative anaerobes and catalase-negative. The strains are obligately homofermentative (final product dl-lactate) and produce exopolysaccharides from sucrose. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the highest similarity to both isolates corresponded to the type strain of Lactobacillus vini (99.1 %), followed by Lactobacillus satsumensis (96.4 %), and Lactobacillus oeni (96.2 %), and for all other established species, 16S rRNA gene sequence similarities were below 96 %. The species delineatio…
Application of propidium monoazide quantitative PCR for selective detection of live Escherichia coli O157:H7 in vegetables after inactivation by essential oils.
The use of propidium monoazide (PMA) is enjoying increased popularity among researchers in different fields of microbiology. Its use in combination with real-time PCR (qPCR) represents one of the most successful approaches to detect viable cells. PMA-qPCR has successfully been used to evaluate the efficacy of various disinfection technologies in different microorganisms. Initially, in this study the effect of four essential oils (EOs), cumin, clove, oregano and cinnamon, was evaluated on suspensions of the enterohemorrhagic Escherichia coli O157:H7 by PMA-qPCR, LIVE/DEAD BacLight flow cytometry analysis (LIVE/DEAD-FCM), and plate count. E. coli O157:H7 cells treated with EOs at killing conc…
Evaluation of Zataria multiflora Boiss. essential oil activity against Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes by propidium monoazide quantitative PCR in vegetables
Essential oils (EOs) have long been applied as flavoring agents in foods, and due to their content in antimicrobial compounds, they have potential as natural agents for food preservation. Recently, real-time PCR in combination with PMA has successfully been applied to discriminate between live Escherichia coli O157:H7 and dead bacteria killed by cumin, clove, oregano and cinnamon EOs. In this study, initial experiments were performed in order to elucidate the minimum bactericidal concentration of Zataria multiflora EOs on E. coli O157:H7, Salmonella enterica and Listeria monocytogenes. Thereafter PMA-qPCR was applied in order to selectively quantify life cells within a bacterial population …
A single method for recovery and concentration of enteric viruses and bacteria from fresh-cut vegetables
Fresh-cut vegetables are prone to be contaminated with foodborne pathogens during growth, harvest, transport and further processing and handling. As most of these products are generally eaten raw or mildly treated, there is an increase in the number of outbreaks caused by viruses and bacteria associated with fresh vegetables. Foodborne pathogens are usually present at very low levels and have to be concentrated (i.e. viruses) or enriched (i.e. bacteria) to enhance their detection. With this aim, a rapid concentration method has been developed for the simultaneous recovery of hepatitis A virus (HAV), norovirus (NV), murine norovirus (MNV) as a surrogate for NV, Escherichia coli O157:H7, List…
A multiplex RTi-PCR reaction for simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus on fresh, minimally processed vegetables.
In this work, a new multiplex single-tube real-time PCR approach is presented for the detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus, three of the more frequent food-borne bacterial pathogens that are usually investigated in a variety of food matrices. The study includes the design and specificity testing, of a new primer and probe specific for Salmonella spp. Reaction conditions were adjusted for the simultaneous amplification and detection of specific fragments in the beta-glucuronidase (uidA, E. coli) and Thermonulease (nuc, Sta. aureus) genes, and in the replication origin sequence (oriC, Salmonella spp.). Melting-curve analysis using a SYBR Green I RTi…
Molecular characterization of the black Aspergillus isolates responsible for ochratoxin A contamination in grapes and wine in relation to taxonomy of Aspergillus section Nigri.
This work examines ochratoxigenic mycobiota in grapes by ap-PCR analysis sequence analysis of the ITS and IGS regions and ability to produce OTA. A comparison was also made with many reference strains of Aspergillus section Nigri. Based on ap-PCR profiles, derived from two microsatellite primers, three main groups were obtained by UPGMA cluster analysis corresponding to A. carbonarius, A. niger and A. tubingensis. The cophenetic correlation values corresponding to ap-PCR UPGMA analysis revealed a higher genetic variability in A. niger and A. tubingensis than in A. carbonarius. In addition, no genotypical differences could be established between OTA producers and nonproducers in all species …
Disclosing diversity of exopolysaccharide-producing lactobacilli from Spanish natural ciders
24 p.-2 fig.-2 tab.-1 fig. supl.-1 tab.supl.
Evaluation of yogurt and various beverages as carriers of lactic acid bacteria producing 2-branched (1,3)-β-D-glucan
8 páginas, 2 figuras, 2 tablas -- PAGS nros. 3271-3278
The effect of essential oils on norovirus surrogates
Abstract Essential oils (EOs) have long been applied as flavoring agents in foods, and due to their content in antimicrobial compounds, they have potential as natural agents for food preservation. In this study the effect of three EOs, clove, oregano and zataria, was evaluated on the infectivity of norovirus surrogates, i.e. feline calicivirus (FCV) and murine norovirus (MNV). Different concentrations of EOs were individually mixed with each virus at titers of ca. 7–8 log TCID 50 /ml and incubated for 2 h at 4 °C and 37 °C. The infectivity of the recovered viruses after triplicate treatments was evaluated by cell-culture assays. 2% of oregano EO at 37 °C decreased the FCV titers by 3.75 log…
Application of propidium monoazide-qPCR to evaluate the ultrasonic inactivation of Escherichia coli O157:H7 in fresh-cut vegetable wash water.
The efficacy of sanitizing technologies in produce or in vegetable wash water is generally evaluated by plate count in selective media. This procedure is time consuming and can lead to misinterpretations because environmental conditions and sanitizing processes may affect bacterial growth or culturable capability. Thus, the aim of this study was to determine the applicability of a propidium monoazide real-time PCR (PMA-qPCR) method to monitor the inactivation by ultrasound treatment of foodborne bacteria in fresh-cut vegetable wash water. To this aim, lettuce wash water was artificially inoculated with Escherichia coli O157:H7 (10⁶ CFU/mL) and treated by means of a continuous ultrasonic irr…
Quantification of Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 in non-spiked food products and evaluation of real-time PCR as a diagnostic tool in routine food analysis
Abstract Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp. are foodborne pathogens frequently associated with foods such as poultry, ready-to-eat products, fruits and vegetables. PCR-based procedures are rapid, sensitive and accurate; in particular, real-time PCR (qPCR), which besides being an automated high-throughput technique, allows quantification of foodborne pathogens. In the present work, qPCR-based methods were applied for the quantitative detection of E. coli O157:H7, Salmonella spp. and L. monocytogenes in a total of 306 non-spiked food samples in a study carried out in two laboratories simultaneously. qPCR allowed the detection of the three pathogens in around …
Virucidal effect of high power ultrasound combined with a chemical sanitizer containing peroxyacetic acid for water reconditioning in the fresh-cut industry
The use of chlorine-based sanitizers is widespread throughout the fresh produce industry in the process water to maintain microbial safety of produce, avoid cross-contamination and recycle water. In this study, alternative disinfection technologies were investigated due to the negative reports showing chlorine instability in the presence of organic matter and the undesirable by-product residues. Tsunami ® (15.2% peroxyacetic acid and 11.2% hydrogen peroxide) and high-power ultrasound (HPU, 0.56 kW/l, 20 kHz), alone or in combination, were evaluated in lettuce process water analyzing their ability to reduce MNV during either produce washing or water recycling. PAA concentrations of 6.4, 12.8…
Pyrosequencing vs. culture-dependent approaches to analyze lactic acid bacteria associated to chicha, a traditional maize-based fermented beverage from Northwestern Argentina.
The diversity of lactic acid bacteria (LAB) associated with chicha, a traditional maize-based fermented alcoholicbeverage from Northwestern Argentina, was analyzed using culture-dependent and culture-independent approaches.Samples corresponding to 10 production steps were obtained from two local producers at Maimará(chicha M) and Tumbaya (chicha T). Whereas by culture-dependent approach a few number of species(Lactobacillus plantarum and Weissella viridescens in chicha M, and Enterococcus faecium and Leuconostocmesenteroides in chicha T) were identified, a higher quantitative distribution of taxa was found in both beveragesby pyrosequencing. The relative abundance of OTUs was higher in chic…
Quantitative detection of viable foodborne E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables combining propidium monoazide and real-time PCR
Abstract The increase of foodborne outbreaks associated with fresh vegetables has highlighted the importance of developing rapid and specific methods for the detection and quantification of foodborne pathogens. In this sense, real-time PCR (qPCR) fulfills these requirements although it may detect dead cells. Recently, a potential strategy to specifically detect viable cells has been proposed relying on the use of DNA binding molecules as sample pretreatment previous to the qPCR. In this study propidium monoazide (PMA) and reagent D, combined with qPCR, were evaluated for the detection and quantification of viable Escherichia coli O157:H7, Salmonella and Listeria monocytogenes. Initially, th…
Comparison of Four Commercial DNA Extraction Kits for PCR Detection of Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, and Staphylococcus aureus in Fresh, Minimally Processed Vegetables
Four commercial DNA extraction methods, PrepMan Ultra (Applied Biosystems), InstaGene Matrix (BioRad), DNeasy Tissue kit (Qiagen), and UltraClean (MoBio), were tested for PCR detection of Listeria monocytogenes, Escherichia coli O157: H7, Salmonella, and Staphylococcus aureus in fresh, minimally processed vegetables. For comparative purposes, sensitivity assays with specific PCRs were carried out after DNA extraction with the four methods in green pepper, broccoli, and onion artificially inoculated with the four pathogens separately. As confirmed by statistical analysis, the DNeasy Tissue kit rendered the highest sensitivity values in the three matrices assayed for Salmonella, L. monocytoge…
A TaqMan-based real-time PCR assay for the specific detection and quantification ofLeuconostoc mesenteroidesin meat products
A new real-time PCR procedure was developed for the specific detection and quantification of Leuconostoc mesenteroides in meat products. It is a TaqMan assay based on 23S rRNA gene targeted primers and probe. Specificity was evaluated using purified DNA from 132 strains: 102 lactic acid bacteria (LAB), including 57 reference strains and 46 food isolates, belonging to genus Leuconostoc and related genera, and 30 non-LAB strains. Quantification was linear over at least 5 log units using both serial dilutions of purified DNA and calibrated cell suspensions from Leuconostoc mesenteroides ssp. dextranicum CECT 912T. This assay was able to detect at least five genomic equivalents, using purified …