Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples
A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of …
A comparison of strategies for the detection and recovery of Vibrio vulnificus from marine samples of the Western Mediterranean coast
Summary We have compared the effectiveness of culture-based methods and a DNA-based method for the detection, of Vibrio vulnificus from seawater and three types of shellfish collected from the coastal waters of Valencia, Spain. For culture-based method, we used two selective media, thiosulphate-citrate-bile salts-sucrose (TCBS), and cellobiose-polymyxin B-colistin (CPC) agars with and without previous enrichment in alkaline-saline-peptone-water (APWS). Presumptive colonies were confirmed as V. vulnificus by the polymerase chain reaction (PCR) using previously described 23S rRNA V. vulficus -specific sequences as primers (Dvu 9V and Dvu 45R). Direct detection was accomplished by a nested-PCR…
Lactobacillus tucceti sp. nov., a new lactic acid bacterium isolated from sausage
Abstract Following the application of several molecular techniques strain R 19c, isolated from sausage by Reuter in 1970 and deposited at the DSMZ as Lactobacillus sp., has been identified as pertaining to a new species. It showed singular ISR- Dde I and ISR- Hae III profiles that allowed its differentiation from 68 lactic acid bacteria reference strains analyzed. Phylogenetic analysis based on 16S rRNA gene sequences places this strain in the genus Lactobacillus within the Lactobacillus alimentarius group. Species L. versmoldensis is the closest phylogenetic neighbor with 96.3% sequence similarity. DNA–DNA hybridization experiments confirmed the independent status at species level of this …
Genomic Encyclopedia of Bacteria and Archaea (GEBA) VI: learning from type strains
Type strains of species are one of the most valuable resources in microbiology. During the last decade, the Genomic Encyclopedia of Bacteria and Archaea (GEBA) projects at the US Department of Energy Joint Genome Institute (JGI) and their collaborators have worked towards sequencing the genome of all the type strains of prokaryotic species. A new project GEBA VI extends these efforts to functional genomics, including pangenome and transcriptome sequencing and exometabolite analyses. As part of this project, investigators with interests in specific groups of prokaryotes are invited to submit samples for analysis at JGI.
A Polyphasic Approach to Study the Intraspecif ic Diversity Amongst Vibrio vulnificus Isolates
Summary A polyphasic taxonomic approach using phenotypic and molecular genetic techniques, was carried out on the species Vibrio vulnificus in order to study its intraspecific diversity. Seven techniques, including phenotypic (API 20E, BIOLOG, total protein profiles, serotyping, ELISA), and genotypic methods (ribotyping and AFLP), were employed on 80 V. vulnificus strains of biotypes 1 and 2, including 9 reference cultures. The isolates came from different geographic origins (USA, Spain, Belgium, Sweden, Norway, Japan, Thailand) and types of samples (clinical, health/diseased fish, seafood, water). Diversity indexes calculated for strains of both biotypes revealed a higher phenotypic and ge…
On the Specificity of PCR Detection of Listeria monocytogenes in Food: a Comparison of Published Primers
A total of nine pairs of primers, seven previously published and two newly developed, have been assayed for PCR detection of Listeria monocytogenes in food. They have been tested for specificity on a total of 72 strains including reference and food isolates belonging to L. monocytogenes and other species in the genus. First of all, a polyphasic approach has been carried out in order to establish a reference strain collection. They were biochemically and genetically characterized by API-Lis and randomly amplified polymorphic DNA PCR (RAPD-PCR), respectively. Random amplification of DNA was performed with M13, T7 and T3 universal primers and a data bank was created to compile the RAPD pattern…
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…
Antiviral properties of silver nanoparticles against norovirus surrogates and their efficacy in coated polyhydroxyalkanoates systems
Silver nanoparticles (AgNP) have strong broad-spectrum antimicrobial activity and gained increased attention for the development of AgNP based products, including medical and food applications. Initially, the efficacy of AgNP and silver nitrate (AgNO3) was evaluated for inactivating norovirus surrogates, the feline calicivirus (FCV) and the murine norovirus (MNV). These norovirus surrogates were exposed to AgNO3 and AgNP solutions for 24 h at 25 °C and then analyzed by cell-culture assays. Both AgNP and silver ions significantly decreased FCV and MNV infectivity in a dose-dependent manner between concentrations of 2.1 and 21 mg/L. Furthermore, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (…
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.
Comparative analysis of production and purification of homo- and hetero-polysaccharides produced by lactic acid bacteria
2 figuras, 2 tablas
Rapid and improved identification of drinking water bacteria using the Drinking Water Library, a dedicated MALDI-TOF MS database.
Abstract According to the European Directives (UE) 2020/2184 and 2009/54/EC, which establishes the sanitary criteria for water intended for human consumption in Europe, water suitable for human consumption must be free of the bacterial indicators Escherichia coli, Clostridium perfringens and Enterococcus spp. Drinking water is also monitored for heterotrophic bacteria, which are not a human health risk, but can serve as an index of bacteriological water quality. Therefore, a rapid, accurate, and cost-effective method for the identification of these colonies would improve our understanding of the culturable bacteria of drinking water and facilitate the task of water management by treatment f…
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…
Intraspecific diversity of Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus sakei, and Leuconostoc mesenteroides associated with vacuum-packed meat product spoilage analyzed by randomly amplified polymorphic DNA PCR
The intraspecific diversity of Leuconostoc mesenteroides, Lactobacillus curvatus, Lactobacillus sakei, and Lactobacillus plantarum was analyzed by randomly amplified polymorphic DNA (RAPD) PCR with universal primers M13 and T3. The study included 100 reference strains and 210 isolates recovered from two vacuum-packed Spanish meat products, fiambre de magro adobado and morcilla, previously identified by rDNA-restriction fragment length polymorphism profiles. The RAPD-M13 profiles identified isolates at species level in L. plantarum and L. mesenteroides, while RAPD-T3 provided profiles in L. sakei. The combination of RAPD-M13 and RAPD-T3 fingerprints revealed a total of 17 profiles in L. mese…
Iron-binding compounds and related outer membrane proteins in Vibrio cholerae non-O1 strains from aquatic environments
A total of 156 strains of Vibrio cholerae non-O1 from aquatic origins were examined for the presence of iron uptake mechanisms and compared with O1 strains and other Vibrio species. All non-O1 strains were able to grow in iron-limiting conditions, with MICs of ethylenediaminedi (O-hydroxyphenylacetic acid) ranging from 20 microM to 2 mM. The production of siderophores was demonstrated by growth in chrome azurol S agar and cross-feeding assays. All strains produced phenolate-type compounds, as assessed by the chemical tests and by bioassays with Salmonella typhimurium enb-7. Some of the strains also promoted the growth of S. typhimurium enb-1 (which can use only enterobactin as a siderophore…
Dextrans produced by lactic acid bacteria exhibit antiviral and immunomodulatory activity against salmonid viruses
36 p.-7 fig.-1 tab.-1 fig. supl.
In situ analysis of the bacterial communities associated to farmed eel by whole-cell hybridization.
Bacterial communities in water samples and eel slime were investigated by fluorescence in situ hybridization of whole bacterial cells in an eel intensive culture system over 1 year. A newly developed probe, matching 27 Vibrio spp., and a specific probe for Vibrio vulnificus were used. Phylogenetic probes complementary to selected regions of the 16S and 23S ribosomal RNA revealed that Proteobacteria of the alpha and beta subclass were predominant in water and eel slime. Members of the gamma subclass (e.g. vibrios and aeromonads) were more abundant in eel slime, although no V. vulnificus was detected.
Identification of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus by rDNA-based techniques
Summary Ribosomal DNA-based techniques including the analysis of profiles generated by ISR amplification, ISR restriction and ARDRA have been evaluated as molecular tools for identifying Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus. They have been applied for the molecular characterization of 91 strains with the following identities: eight Carnobacterium including the eight type species of the genus; 61 Lactobacillus including 40 type strains out of 45 species, 13 Leuconostoc, out of them 11 are type strains and three are subspecies of Lc. mesenteroides; and nine strains representing the six species of genus Pediococcus. The genetic relationship displayed between these species…
The effect of carvacrol on enteric viruses.
Carvacrol, a monoterpenic phenol, is said to have extensive antimicrobial activity in a wide range of food spoilage or pathogenic fungi, yeast and bacteria. The aim of this study was to assess its antiviral activity on norovirus surrogates, feline calicivirus (FCV), murine norovirus (MNV), and hepatitis A virus (HAV), as well as its potential in food applications. Initially, different concentrations of carvacrol (0.25, 0.5, 1%) were individually mixed with each virus at titers of ca. 6-7 log TCID50/ml and incubated 2h at 37°C. Carvacrol at 0.5% completely inactivated the two norovirus surrogates, whereas 1% concentration was required to achieve ca. 1 log reduction of HAV. In lettuce wash wa…
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…
Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids
The hepatitis E virus (HEV) is an emerging pathogen showing a considerable increase in the number of reported cases in Europe mainly related to the ingestion of contaminated food. As with other relevant viral foodborne pathogens, real-time reverse transcriptase polymerase chain reaction (RT-qPCR) is the gold standard for HEV detection in clinical, food, and environmental samples, but these procedures cannot discriminate between inactivated and potentially infectious viruses. Thus, the aim of this study was to develop a viability PCR method to discriminate between native, heat-, and high-pressure processing (HPP)-treated HEV using the hepatitis A virus (HAV) as a cultivable surrogate. To thi…
In situ riboflavin fortification of different kefir-like cereal-based beverages using selected Andean LAB strains.
Cereal-based functional beverages represent social, economic, and environmental sustainable opportunities to cope with emerging trends in food consumption and global nutrition. Here we report, for the first time, the polyphasic characterization of three cereal-based kefir-like riboflavin-enriched beverages, obtained from oat, maize and barley flours, and their comparison with classical milk-based kefir. The four matrices were successfully fermented with commercial starters: i) milk-kefir and ii) water-kefir, proving the potential of cereal ingredients in the formulation of dairy-like fermented beverages with milk-kefir starter behavior better in these matrices. In the light of their potenti…
Genomic Insights Into Five Strains of Lactobacillus plantarum With Biotechnological Potential Isolated From chicha, a Traditional Maize-Based Fermented Beverage From Northwestern Argentina
Lactic acid bacteria (LAB) are indigenous microorganisms that have been involved in food fermentations throughout history to preserve food and also to give special characteristics to them. The traditional fermented foods that are still being elaborated in indigenous populations around the world are a potential source of LAB with important biotechnological properties and/or beneficial to health. In a previous work, LAB biodiversity associated with chicha, a traditional maize-based fermented beverage from Northwestern Argentina, was studied, both by culture dependent and independent methods. From that study, 392 isolates were recovered, mostly members of Lactobacillus and Leuconostoc. Biotech…
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 …
DNA-DNA hybridization and ribotyping of Acidovorax delafieldii isolates from eels and aquatic environments
Eleven strains of Acidovorax isolated from a freshwater lake and from healthy and diseased European eels in a hatchery were investigated by genotypic methods. DNA-DNA slot blot hybridization confirmed the identity of all strains as A. delafieldii. Isolates were heterogeneous in EcoR1 ribopatterns with four types recognizable. Most freshwater and healthy eel strains had the same ribotype but differed from the disease-associated strains. Typical ribopatterns of A. delafieldii contained 1·3, 1·5, and 3·2 kb fragments which provided useful additional taxonomic markers for species identification.
Nested PCR method for rapid and sensitive detection of Vibrio vulnificus in fish, sediments, and water
A nested PCR for the detection of Vibrio vulnificus in fish farms was developed as an alternative to cultural methods by using universal primers flanking the V. vulnificus-specific sequences directed against 23S rRNA genes. This specific assay detected 10 fg of DNA or 12 to 120 cells in artificially inoculated samples without enrichment and within 24 h.
Exploring diversity and biotechnological potential of lactic acid bacteria from tocosh - traditional Peruvian fermented potatoes - by high throughput sequencing (HTS) and culturing
Lactic acid bacteria (LAB) diversity associated with tocosh, Peruvian traditional fermented potatoes, was for the first time investigated by culturing and high throughput sequencing (HTS) approaches. They were applied on three samples i.e. freshly harvested potatoes, one-month and eight-months production. While by culture-dependent approach a few Lactobacillus (Lb) species (Lb. sakei, Lb. casei, Lb. farciminis, Lb. brevis, Lb. fermentum) and Leuconostoc (Ln) mesenteroides were identified, twenty-four OTUs belonging to six LAB genera were considered in tocosh samples by HTS, being Lactobacillus dominant in all three samples. LAB predominated on fresh potatoes, while Clostridium, Zymophilus a…
Evaluation of viability PCR performance for assessing norovirus infectivity in fresh-cut vegetables and irrigation water
Norovirus (NoV) detection in food and water is mainly carried out by quantitative RT-PCR (RT-qPCR). The inability to differentiate between infectious and inactivated viruses and the resulting overestimation of viral targets is considered a major disadvantage of RT-qPCR. Initially, conventional photoactivatable dyes (i.e. propidium monoazide, PMA and ethidium monoazide, EMA) and newly developed ones (i.e. PMAxx and PEMAX) were evaluated for the discrimination between infectious and thermally inactivated NoV genogroup I (GI) and II (GII) suspensions. Results showed that PMAxx was the best photoactivatable dye to assess NoV infectivity. This procedure was further optimized in artificially inoc…
Simultaneous detection of the main black aspergilli responsible for ochratoxin A (OTA) contamination in grapes by multiplex real-time polymerase chain reaction
9 pages.
Dextransucrase Expression Is Concomitant with that of Replication and Maintenance Functions of the pMN1 Plasmid in Lactobacillus sakei MN1
The exopolysaccharide synthesized by Lactobacillus sakei MN1 is a dextran with antiviral and immunomodulatory properties of potential utility in aquaculture. In this work we have investigated the genetic basis of dextran production by this bacterium. Southern blot hybridization experiments demonstrated the plasmidic location of the dsrLS gene, which encodes the dextransucrase involved in dextran synthesis. DNA sequencing of the 11,126 kbp plasmid (pMN1) revealed that it belongs to a family which replicates by the theta mechanism, whose prototype is pUCL287. The plasmid comprises the origin of replication, repA, repB, and dsrLS genes, as well as seven open reading frames of uncharacterized f…
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…
PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food.
Aims: To evaluate the specificity of nuc targeted primers for PCR detection of Staphylococcus aureus in different food matrices and to establish a RTQ-PCR procedure suitable for the routine detection and quantification of this pathogen in food. Methods and Results: Specificity of nuc targeted primers (Pri1–Pri2 and the newly designed RTQ-PCR primers) was tested on a total of 157 strains of genetically confirmed identity, including reference and food isolates. PCR detection on artificially inoculated beef samples by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) showed a sensitivity value around 103 CFU g−1. The two RTQ-PCR systems, incorporating SYBR-Green I and T…
Genome Sequence of Bifidobacterium breve INIA P734 (CECT 8178), a Strain Isolated from Human Breast Milk
Departamento de Tecnología de Alimentos (INIA)
Fostering the antiviral activity of green tea extract for sanitizing purposes through controlled storage conditions
Food-contact surfaces is considered an important vehicle for the indirect transmission of foodborne viral diseases with enteric viruses, especially human norovirus (HuNoV) and hepatitis A virus (HAV). The aim of the present study was to evaluate the antiviral activity of green tea extract (GTE) at room temperature as a function of pH and storage time and to relate it with changes in composition as a consequence of degradation and epimerization reactions in the storage conditions. The obtained results revealed that freshly prepared GTE was very effective in inactivating murine norovirus (MNV) and HAV at neutral and alkaline pH but was ineffective at pH 5.5. Additionally, storage of the solut…
Real-time detection of riboflavin production by Lactobacillus plantarum strains and tracking of their gastrointestinal survival and functionality in vitro and in vivo using mCherry labeling
Some strains of lactic acid bacteria (LAB) produce riboflavin, a water-soluble vitamin of the B complex, essential for human beings. Here, we have evaluated riboflavin (B2 vitamin) production by five Lactobacillus plantarum strains isolated from chicha, a traditional maize-based fermented alcoholic beverage from north-western Argentina and their isogenic riboflavin-overproducing derivatives previously selected using roseoflavin. A direct fluorescence spectroscopic detection method to quantify riboflavin production in bacterial culture supernatants has been tested. Comparison of the efficiency for riboflavin fluorescence quantification with and without prior HPLC fractionation showed that th…
Sanitizing food contact surfaces by the use of essential oils
Chemical sanitizers continue to be widely used by the food industry to disinfect food contact surfaces. However, as some chemical disinfectants have been reported to produce unhealthy by-products, alternative and natural compounds need to be investigated. To this end, nine essential oils (EOs) were screened to develop a natural sanitizing solution (SAN) for disinfecting food contact surfaces. Once extracted, their antimicrobial activity and chemical composition were determined. An exploratory multivariate approach was used to investigate the relationships between the chemical and microbiological data sets. Among the tested EOs, Thymbra capitata EO, containing up to 93.31% oxygenated monoter…
Mesonia oceanica sp. Nov., isolated from oceans during the tara oceans expedition, with a preference for mesopelagic waters
10 pages, 4 figures, 3 tables
Genetic relatedness among environmental, clinical, and diseased-eel Vibrio vulnificus isolates from different geographic regions by ribotyping and randomly amplified polymorphic DNA PCR.
ABSTRACT Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with Kpn I and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high l…
Physico-chemical and bacteriological parameters in a hypereutrophic lagoon (Albufera Lake, Valencia, Spain).
Several physico-chemical parameters related to water quality, as well as saprophytic and public health-related heterotrophic bacterial groups were studied in a hypereutrophic lake (Albufera, Valencia) at different seasons. Total microscopic and viable counts were compared, and, together with faecal indicators, were determined in water samples from different sites. Heterotrophic bacteria grown on nonselective medium were identified to genus level and a diversity index was calculated. Pseudomonas-Alcaligenes was the most frequently isolated group from all sampling sites along the study, following by Moraxella, Acinetobacter, Vibrio and Aeromonas. The relationships between all parameters were …
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…
Effect of (−)-epigallocatechin gallate at different pH conditions on enteric viruses.
Epigallocatechin gallate (EGCG), a flavonoid from green tea, is said to have extensive antimicrobial activity in a wide range of food spoilage or pathogenic fungi, yeast and bacteria. In this work, the antiviral activity of EGCG was assessed against hepatitis A virus (HAV) and murine norovirus (MNV), a human norovirus surrogate, at different temperatures, contact times and pH conditions by cell-culture methods. EGCG was effective in reducing the titers of HAV and MNV in a dose-dependent manner at neutral pH and 25 and 37 °C, while no effect was reported at 4 °C. HAV and MNV infectivity was completely removed after overnight treatment with EGCG at 2.5 mg/mL at 37 °C. Furthermore, results als…
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…
Surface and virulence properties of environmental Vibrio cholerae non-O1 from Albufera Lake (Valencia, Spain).
A total of 140 environmental Vibrio cholerae non-O1 isolates, together with several culture collection strains from both environmental and clinical sources, were studied in relation to hemagglutination, surface hydrophobicity, and the enzymatic, hemolytic, cytotoxic, and enterotoxic activities of their extracellular products. A total of 78 and 62% of the strains produced hemagglutinins and exohemagglutinins, respectively. Four different hemagglutinating and two exohemagglutinating activities were found by using eight sugars in the inhibition assays. Cell-bound mannose-sensitive hemagglutination was detected mainly in chicken blood, whereas fucose-sensitive hemagglutination was recorded only…
Low incidence of Vibrio vulnificus among Vibrio isolates from sea water and shellfish of the western Mediterranean coast.
A specific search for Vibrio vulnificus in natural marine samples from the Spanish Mediterranean Sea was carried out by nested PCR and cultural approaches using thiosulphate-citrate-bile salts-sucrose agar (TCBS) and cellobiose-polymixin B-colistin agar (CPC), incubated at 40 degrees C, as selective media. Presumptive colonies were identified by PCR using specific primers against 23S rRNA sequences. This species was isolated from sea water and edible bivalves, mainly after preenrichment in alkaline peptone water (APW) at 40 degrees C followed by CPC agar. None of the V. vulnificus isolates identified corresponded to serovar E. Dominant Vibrio species on directly inoculated TCBS plates incub…
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 …
Different Modes of Regulation of the Expression of Dextransucrase in Leuconostoc lactis AV1n and Lactobacillus sakei MN1
Leuconostoc lactis AV1 strain isolated from a Tunisian avocado was characterized as a dextran producer. The promoter PdsrLL and the dsrLL gene encoding the DsrLL dextransucrase responsible for the dextran synthesis were transcriptionally fused to the mCherry coding gene generating the pRCR20 plasmid. Upon plasmid transfer, both AV1n and the dextran non-producing Leuconostoc mesenteroides CM70 became red due to expression of the mCherry from the PdsrLL-dsr-mrfp transcriptional fusion. Characterization of the polymers present in cultures supernatants revealed that the DsrLL encoded from pRCR20 in the recombinant bacteria was able to synthesize dextran. The production of dextran by the DsrLL i…
Siderophore production by environmental strains ofSalmonellaspecies
Iron uptake mechanisms were investigated in different species of Salmonella isolated from environmental waters. All strains examined were able to grow in the presence of high concentrations (10 mM) of the iron chelator EDDA. All strains excreted phenolate and hydroxamate siderophores, as assessed by bioassays and chemical tests. Bioassays with different indicator strains showed that all Salmonella strains can cross-feed other Enterobacteria, as well as mutants of Salmonella typhimurium deficient in the Enterobactin system, suggesting that this siderophore may be produced by the environmental Salmonella strains. The siderophore aerobactin may also be produced by one of the strains, according…
Dextran production by Lactobacillus sakei MN1 coincides with reduced autoagglutination, biofilm formation and epithelial cell adhesion
40 p.-7 fig.-4 fig.supl.
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…
Antilisterial peptides from Spanish dry-cured hams: Purification and identification.
The typical Spanish dry-cured ham has a particular sensory quality that makes it a distinctive food, highly appreciated for consumers worldwide. Its particular physicochemical properties, such as high salt content and reduced water activity contribute to their shelf-stability. However, post-processing actions carried out for the commercialization of these products such as slicing may increase the risk of development of pathogenic microorganisms as Listeria monocytogenes. During ripening, muscle proteins are hydrolyzed by muscle peptidases releasing peptides and free amino acids. Some of these peptides have been described to exert biological activities such as antioxidant and ACE-inhibition.…
Functional and Nutritious Beverages Produced by Lactic Acid Bacteria
Abstract Currently, in the developed countries, there is a concern about leading a healthy lifestyle including eating “organic” and “functional” food. Moreover, in recent years, the percentage of the population with food allergies or intolerances has increased. Within this framework, lactic acid bacteria (LAB), which are able to ferment a variety of foodstuffs and also colonize the human digestive tract, can play a beneficial role. Many LAB strains have the generally recognized as safe (GRAS) status and are therefore being used in the food industry, especially to produce different types of fermented beverages, because they confer “functionality” to drinks due to the production of bioactive …
Biopreservation potential of lactic acid bacteria from Andean fermented food of vegetal origin
Abstract Microbial fermentations have long represented a way of natural biopreservation of raw materials, which frequently originated new food products. Among them, traditionally fermented products still manufactured by native populations all around the world are source of lactic acid bacteria (LAB) strains with high biotechnological potential. LAB are food grade microorganisms and therefore a good alternative to chemicals to be applied in food preservation. A total of 130 LAB isolates recovered from “chicha” and “tocosh”, traditional fermented Andean products of vegetal origin, were screened for antimicrobial activities against spoiler fungi Meyerozyma guilliermondii CECT 1021 (synonym Pic…
Shimia thalassica sp. nov., and reclassification of Pseudopelagicola gijangensis as Shimia gijangensis comb. nov., and Thalassobius activus as Cognatishimia activa comb. nov.
Strain CECT 7735T, a marine Gram-reaction negative, aerobic, non-motile bacterium, was isolated from coastal seawater in Valencia, Spain. Strain CECT 7735T is chemoorganotrophic, mesophilic, slightly halophilic, grows at 15–28 °C but not at 4 or 37 °C, requires seawater for growth and grows up to 6 % salinity. The major cellular fatty acid is summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The G+C content of the genome is 55.7 mol%. Comparative analysis of the 16S rRNA gene sequence shows the strain is affiliated to the family Rhodobacteraceae , in the class Alphaproteobacteria , with highest similarities to Phaeobacter species (97.0–97.5 %), Shimia species (96.5–97.3 %) and Pseudopelagico…
Thalassocella blandensis gen. nov., sp. nov., a novel member of the family Cellvibrionaceae
9 pages, 1 figure, 3 tables
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 …
Ribotyping of Vibrio Populations Associated with Cultured Oysters (Ostrea edulis)
The intraspecific variability of Vibrio splendidus, V. harveyi and V. tubiashii recovered from oysters (Ostrea edulis) collected at the Mediterranean coast near Valencia, Spain, was analyzed by ribotyping. The two former species represented the most abundant ones, and the third one was the only species described as pathogenic for oysters. A total of 115 environmental strains were studied, 84 of V. splendidus, 23 of V. harveyi and 8 of V. tubiashii. Chromosomal DNA was digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Ribotyping among natural populations of the three species rendered 5 to 9 bands, and showed a h…
Disclosing diversity of exopolysaccharide-producing lactobacilli from Spanish natural ciders
24 p.-2 fig.-2 tab.-1 fig. supl.-1 tab.supl.
Characterization of R-plasmids in environmental isolates ofsalmonella: Host range and stability
Four environmental isolates ofSalmonella, resistant to several drugs, were examined for plasmid carriage with four different plasmid DNA isolation procedures. The method of Birnboim and Doly gave the best results. Three of the strains possessed a single plasmid with molecular weights of 60 (kanamycin resistant), 44.5 (kanamcin resistant), and 23.4 Md (ampicillin and amoxicillin resistant); the other strain (resistant to tetracycline) harbored two plasmids of 69.8 and 2.2 Md. The 69.8 Md was the one responsible for resistance. All plasmids were fi−, and the 44.5 Md Kcr plasmid synthesized a sex pilus type F. Some properties related to the dissemination of R-plasmids, such as host range, tran…
Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection
Johne´s disease is a chronic granulomatous enteritis of ruminants caused by the intracellular bacterium Mycobacterium avium subsp. paratuberculosis (Map). We previously demonstrated that Map isolates from sheep persisted within host macrophages in lower CFUs than cattle isolates after 7 days of infection. In the current study, we hypothesize that these phenotypic differences between Map isolates may be driven be the fatty acids (FAs) present on the phosphadidyl-1-myo-inositol mannosides of the Map cell wall that mediate recognition by the mannose receptors of host macrophages. FAs modifications may influence Map´s envelope fluidity ultimately affecting pathogenicity. To test this hypothesis…
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 viability PCR to discriminate the infectivity of hepatitis A virus in food samples.
Abstract Transmitted through the fecal–oral route, the hepatitis A virus (HAV) is acquired primarily through close personal contact and foodborne transmission. HAV detection in food is mainly carried out by quantitative RT-PCR (RT-qPCR). The discrimination of infectious and inactivated viruses remains a key obstacle when using RT-qPCR to quantify enteric viruses in food samples. Initially, viability dyes, propidium monoazide (PMA) and ethidium monoazide (EMA), were evaluated for the detection and quantification of infectious HAV in lettuce wash water. Results showed that PMA combined with 0.5% Triton X-100 (Triton) was the best pretreatment to assess HAV infectivity and completely eliminate…
Hepatitis E virus in lettuce and water samples: A method-comparison study
The hepatitis E virus (HEV), which is an increasing cause of acute viral hepatitis in Europe, is a zoonotic virus that is mainly transmitted through contaminated water, consumption of raw or undercooked meat from pigs or wild boar, blood transfusion, and organ transplantation. Although the role of HEV transmission through contaminated produce has not been confirmed, the presence of HEV has been reported in irrigation waters and in vegetables. The present study used a World Health Organization (WHO) international standard and clinical samples to evaluate the performance characteristics of three RT-qPCR assays for detection and quantification of HEV. Two of the evaluated assays provided good …
Effect of green tea extract on enteric viruses and its application as natural sanitizer
In this work, the effect of green tea extract (GTE) was assessed against murine norovirus (MNV) and hepatitis A virus (HAV) at different temperatures, exposure times and pH conditions. Initially, GTE at 0.5 and 5 mg/ml were individually mixed with each virus at 5 log TCID50/ml and incubated 2 h at 37 °C at different pHs (from 5.5 to 8.5). GTE affected both viruses depending on pH with higher reductions observed in alkaline conditions. Secondly, different concentrations of GTE (0.5 and 5 mg/ml) were mixed with viral suspensions and incubated for 2 or 16 h at 4, 25 and 37 °C at pH 7.2. A concentration-, temperature- and exposure time-dependent response was showed by GTE in suspension tests, w…
Simultaneous detection of Carnobacterium and Leuconostoc in meat products by multiplex PCR.
M.C. MACIAN, E. CHENOLL AND R. AZNAR. 2004. Aims: To develop a multiplex PCR approach for simultaneous detection of Leuconostoc and Carnobacterium and its validation in meat products. Methods and Results: Two multiplex PCR assays were developed using newly designed 16S rDNA-directed primers adapted to the current taxonomic situation of genera Leuconostoc and Carnobacterium that allow: (i) simultaneous detection of both genera, and members of the nonmotile species of genus Carnobacterium and (ii) identification in a single assay of the nonmotile species C. divergens, C. maltaromicum and C. gallinarum. Sensitivity values of 10 3 and 10 4 CFU g )1 were determined for multiplex PCR detection of…
Siderophores and related outer membrane proteins produced by pseudomonads isolated from eels and freshwater.
A total of 46 environmental pseudomonads, together with six type strains, were examined for their siderophore-producing activity. All strains were able to grow under iron-limiting conditions, gave orange halos in the CAS agar assay, and produced hydroxamates, and some of them also produced phenolate-type compounds. Bioassays showed that all strains, except Pseudomonas aeruginosa, promoted growth of mutant strain Arthrobacter flavescens JG-9, deficient in hydroxamate production, and some of them promoted growth of Salmonella typhimurium enb-1, which requires enterobactin for growth. The presence of iron-regulated outer membrane proteins was observed, the molecular size of the main induced pr…
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…
Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques.
Abstract The possibility of using PCR for rapid identification of food-borne Staphylococcus aureus isolates was evaluated as an alternative to the API-Staph system. A total of 158 strains, 15 S. aureus , 12 other staphylococcal species, and 131 isolates recovered from 164 food samples were studied. They were phenotypically characterized by API-Staph profiles and tested for PCR amplification with specific primers directed to thermonuclease ( nuc ) and enterotoxin ( sea to see ) genes. Disagreement between the PCR results and API-Staph identification was further assessed by the analysis of randomly amplified polymorphic DNA (RAPD) profiles obtained with three universal primers (M13, T3, and T…
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…
The Ability of Riboflavin-Overproducing Lactiplantibacillus plantarum Strains to Survive Under Gastrointestinal Conditions
17 p.-5 fig.-4 tab.+mat. supl.:5 p.-3 fig.-1 tab.
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…
Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples
Shellfish contamination by human noroviruses (HuNoVs) is a serious health and economic problem. Recently an ISO procedure based on RT-qPCR for the quantitative detection of HuNoVs in shellfish has been issued, but these procedures cannot discriminate between inactivated and potentially infectious viruses. The aim of the present study was to optimize a pretreatment using PMAxx to better discriminate between intact and heat-treated HuNoVs in shellfish and sewage. To this end, the optimal conditions (30 min incubation with 100 μM of PMAxx and 0.5% of Triton, and double photoactivation) were applied to mussels, oysters and cockles artificially inoculated with thermally-inactivated (99 °C for 5 …
Antiviral activity of aged green tea extract in model food systems and under gastric conditions.
Aged-green tea extract (GTE) is known to reduce the infectivity of hepatitis A virus (HAV) and murine norovirus (MNV), a human norovirus surrogate, in vitro and in washing solutions. Initially, the effect of aged-GTE was evaluated on virus like particles (VLPs) of human norovirus (HuNoV) genogroup I (GI) by a porcine gastric mucine (PGM)-enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM), and on HuNoV GI suspensions by an in situ capture-RT-qPCR method, suggesting that HuNoVs are very sensitive to aged-GTE treatment at 37 °C. Moreover, the potential application of aged-GTE was evaluated using model foods and simulated gastric conditions. Then, aged-GTE samp…
Discrimination of non-infectious SARS-CoV-2 particles from fomites by viability RT-qPCR.
The ongoing coronavirus 2019 (COVID-19) pandemic constitutes a concerning global threat to public health and economy. In the midst of this pandemic scenario, the role of environment-to-human COVID-19 spread is still a matter of debate because mixed results have been reported concerning SARS-CoV-2 stability on high-touch surfaces in real-life scenarios. Up to now, no alternative and accessible procedures for cell culture have been applied to evaluate SARS-CoV-2 infectivity on fomites. Several strategies based on viral capsid integrity have latterly been developed using viability markers to selectively remove false-positive qPCR signals resulting from free nucleic acids and damaged viruses. T…
Real-time PCR based procedures for detection and quantification of Aspergillus carbonarius in wine grapes
9 pages, 4 tables, 1 figure.
Lactobacillus rennini sp. nov., isolated from rennin and associated with cheese spoilage.
Two bacterial strains, DSM 20253T and DSM 20254, isolated from rennin and regarded as causing cheese spoilage, were deposited in the DSMZ as Lactobacillus sp. by J. Stadhouders. The strains show 99·9 % 16S rRNA gene sequence similarity and have less than 94·3 % similarity with any other species of the genus. Lactobacillus coryniformis is their closest phylogenetic neighbour. DNA–DNA hybridization experiments confirmed that the two strains are members of the same species with separate status within the genus Lactobacillus. The strains are homofermentative lactic acid bacteria and can be phenotypically and genotypically distinguished from their closest relatives. 16S rRNA gene-targeted specif…
Multiplex Detection of Aspergillus Species
Multiplex real-time polymerase chain reaction (PCR) provides a fast and accurate DNA-based tool for the simultaneous amplification of more than one target sequence in a single reaction. Here a duplex real-time PCR assay is described for the simultaneous detection of Aspergillus carbonarius and members of the Aspergillus niger aggregate, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the beta-ketosynthase and the acyl transferase domains of the polyketide synthase of A. carbonarius and the A. niger aggregate, respectively.Besides, a rapid and efficient fungi DNA extraction procedure is described suitable to be applied …
Lactobacillus vini sp. nov., a wine lactic acid bacterium homofermentative for pentoses.
Six strains with more than 99·5 % 16S rRNA gene sequence similarity, identical internal spacer region profiles and restriction analysis of the amplified 16S rRNA gene patterns were isolated from fermenting grape musts during independent studies carried out in France and Spain many years apart. Strains are Gram-positive, motile, facultatively anaerobic rods that do not exhibit catalase activity and have the ability to utilize pentose sugars (ribose and/or l-arabinose), although they are homofermentative bacteria. Strains ferment pentoses exclusively yielding lactic acid as the end product. A broad set of molecular techniques has been applied to characterize these strains and the results show…
Numerical Taxonomy of Pseudomonads Isolated from Water, Sediment and Eels
Summary A numerical taxonomic study was performed on 129 pseudomonad isolates from aquatic environments. 67 out of 98 field strains were isolated from water and sediment samples from Albufera lake, a hypereutrophic lake near Valencia, and 31 strains came from an eel hatchery. 10 clusters were formed at 84% similarity level when the Sokal-Michener similarity coefficient was used. Five clusters were designated as Pseudomonas putida (16 strains), P. fluorescens biotype I (9 strains), P. pseudoalcaligenes (11 strains), P. stutzen (4 strains) and Shewanella putrefaciens (3 strains). A subcluster of 7 strains (out of 15) was ascribed to the species Xanthomonas maltophilia , while the other strain…
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 …
Improving efficiency of viability-qPCR for selective detection of infectious HAV in food and water samples.
Aim: To improve the efficacy of intercalating dyes to distinguishing between infectious and inactivated hepatitis A virus (HAV) in food. Methods and Results: Different intercalating dyes were evaluated for the discrimination between infectious and thermally inactivated HAV suspensions combining with the RT‐qPCR proposed in the ISO 15216. Among them, PMAxx was the best dye in removing the RT‐qPCR signal from inactivated HAV. Applied to lettuce and spinach, PMAxx–Triton pretreatment resulted in complete removal of the RT‐qPCR signal from inactivated HAV. Likewise, this study demonstrates that this pretreatment is suitable for the discrimination of inactivated HAV in shellfish without further …
Evaluation of a Real-Time PCR Assay for the Detection and Quantification of Group Spores in Food
A procedure based on quantitative real-time PCR was evaluated for the detection and quantification of Bacillus cereus spores. Several methods for DNA isolation, such as various heat treatments and germination solutions, were evaluated on spore suspensions of representative strains of the B. cereus group. Overall, the commercially available DNeasy tissue kit yielded the maximum amount of DNA. The procedure also was used to construct calibration curves for different food matrices, with a wide spore quantification range of 5 log units using serial dilutions of spore suspensions of B. cereus CECT 148T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted i…
Occurrence of enteric viruses in reclaimed and surface irrigation water: relationship with microbiological and physicochemical indicators.
Aims To assess the prevalence of enteric viruses in different irrigation water sources and in the irrigated produce, and the possible links with microbiological and physicochemical water characteristics. Methods and results The prevalence and levels of Escherichia coli, Norovirus (NoV) genogroup I (GI) and II (GII), as well as Hepatitis A virus were assessed in three types of water: surface water (surface-W), reclaimed water subjected to secondary treatment (secondary-W) and reclaimed water subjected to tertiary treatment (tertiary-W), as well as in zucchini irrigated with these irrigation water sources. Chemical oxygen demand (COD), turbidity, total suspended solids, alkalinity, and maximu…
R plasmids in environmental Vibrio cholerae non-O1 strains.
The occurrence of drug resistance and its plasmid-mediated transferability was investigated in 140 environmental strains of Vibrio cholerae non-O1 and 6 strains of Vibrio cholerae, both O1 and non-O1, of clinical origin. Of the 146 strains tested, 93% were resistant to at least one drug and 74% were resistant to two or more antibiotics. The O1 strains were susceptible to all antibiotics used. A total of 26 of 28 selected resistant wild strains carried R plasmids that were transferable by intraspecific and intergeneric matings. The most common transmissible R factor determined resistance to ampicillin, amoxicillin, and sulfanilamide (30%), followed by resistance to ampicillin and amoxicillin…
Chryseobacterium potabilaquae sp. nov., Chryseobacterium aquaeductus sp. nov. and Chryseobacterium fistulae sp. nov., from drinking water systems
A polyphasic taxonomic study was conducted on three strains isolated from drinking water systems that had previously been deposited as Chryseobacterium species at the Spanish Type Culture Collection in order to complete their classification. Strains CECT 9293T, CECT 9390T and CECT 9393T were isolated from sites in Barcelona, Spain, in the framework of a project aimed at generating the first MALDI-TOF database specific for bacteria present in water for human consumption. Their partial 16S rRNA sequences showed that their closest relatives among the type strains of Chryseobacterium exhibited 98 % similarity or less, supporting their taxonomic novelty. At the same time, comparison between them…