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Biomedical subjects

M Manzano

Publications and source records attributed to M Manzano.

At least 19 recordsLinked to original sources
PubMed2006

Classical and molecular analyses to characterize commercial dry yeasts used in wine fermentations.

AIMS: The aim of the work was to apply PCR-temperature gradient gel electrophoresis (PCR-TGGE) and restriction enzyme analysis (RE) assays to identify commercially available starters of Saccharomyces cerevisiae sensu stricto complex. METHODS AND RESULTS: To characterize an analysed pool of 62 active dry yeasts of different brands used in wine fermentation practices, classical microbiological tests were also performed as well as evaluation of contamination with lactic acid bacteria and non-Saccharomyces yeasts. PCR-TGGE and RE were used in order to provide fast and reliable methods to identify and differentiate enological yeasts. Proposed molecular methods enabled to identify particular strains within 36 h after colony isolation and directly from dry yeast suspension. CONCLUSIONS: The methods are highly recommended to obtain reliable results on yeast strain differentiation in a significantly shorter time if compared to classical fermentation tests. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtaining of yeast strain differentiation in a short time and without plating is a good tool for a rapid discrimination among enological strains used as starters in enological practices.

Colony Count, Microbial↗
PubMed2006

Simazine treatment history determines a significant herbicide degradation potential in soils that is not improved by bioaugmentation with Pseudomonas sp. ADP.

AIMS: To study biological removal of the herbicide simazine in soils with different history of herbicide treatment and to test bioaugmentation with a simazine-degrading bacterial strain. METHODS AND RESULTS: Simazine removal was studied in microcosms prepared with soils that had been differentially exposed to this herbicide. Simazine removal was much higher in previously exposed soils than in unexposed ones. Terminal restriction fragment length polymorphism analysis and multivariate analysis showed that soils previously exposed to simazine contained bacterial communities that were significantly impacted by simazine but also had an increased resilience. The biodegradation potential was also related to the presence of high levels of the atz-like gene sequences involved in simazine degradation. Bioaugmentation with Pseudomonas sp. ADP resulted in an increased initial rate of simazine removal, but this strain scarcely survived. After 28 days, residual simazine removals were the same in bioaugmented and not bioaugmented microcosms. CONCLUSIONS: In soils with a history of simazine treatment bacterial communities were able to overcome subsequent impacts with the herbicide. The success of bioaugmentation was limited by the low survival of the introduced strain. SIGNIFICANCE AND IMPACT OF THE STUDY: Conclusions from this work provided insights on simazine biodegradation potential of soils and the convenience of bioaugmentation.

Biodegradation, Environmental↗
PubMed2004

Genetic organization of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 (pJP4) reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways.

Ralstonia eutropha JMP134 (pJP4) is a useful model for the study of bacterial degradation of substituted aromatic pollutants. Several key degrading capabilities, encoded by tfd genes, are located in the 88 kb, self-transmissible, IncP-1 beta plasmid pJP4. The complete sequence of the 87,688 nucleotides of pJP4, encoding 83 open reading frames (ORFs), is reported. Most of the coding sequence corresponds to a well-conserved IncP-1 beta backbone and the previously reported tfd genes. In addition, we found hypothetical proteins putatively involved in the transport of aromatic compounds and short-chain fatty acid oxidation. ORFs related to mobile elements, including the Tn501-encoded mercury resistance determinants, an IS1071-based composite transposon and a cryptic class II transposon, are also present in pJP4. These mobile elements are inefficient in transposition and are located in two regions of pJP4 that are rich in remnants of lateral gene transfer events. pJP4 plasmid was able to capture chromosomal genes and form hybrid plasmids with the IncP-1 alpha plasmid RP4. These observations are integrated into a model for the evolution of pJP4, which reveals mechanisms of bacterial adaptation to degrade pollutants.

Adaptation, Physiological↗
PubMed2003

A molecular method to detect Bacillus cereus from a coffee concentrate sample used in industrial preparations.

AIMS: The aim of this work was to develop specific primers which are able to detect Bacillus cereus in a coffee concentrate sample. METHODS AND RESULTS: A pre-PCR step to clean the DNA, used for PCR, was developed to avoid PCR inhibition by Maillard products. The combination of centrifugation and washing the pellet, employing EDTA and water, before DNA extraction improved the detection of low numbers of B. cereus cells (10 cells ml-1). The development of specific primers enabled to detect low numbers of B. cereus without the need of a pre-enrichment step. CONCLUSIONS: The data obtained demonstrated the specificity and the sensitivity of the primers that could be used to check the presence of B. cereus in different food products, avoiding the need for labourious and time-consuming culture-based techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: The method could help food microbiologists to check food samples quickly for the presence of B. cereus.

Bacillus cereus↗
PubMed2001

Denaturing gradient gel electrophoresis analysis of the 16S rRNA gene V1 region to monitor dynamic changes in the bacterial population during fermentation of Italian sausages.

In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) protocol was used to monitor the dynamic changes in the microbial population during ripening of natural fermented sausages. The method was first optimized by using control strains from international collections, and a natural sausage fermentation was studied by PCR-DGGE and traditional methods. Total microbial DNA and RNA were extracted directly from the sausages and subjected to PCR and reverse transcription-PCR, and the amplicons obtained were analyzed by DGGE. Lactic acid bacteria (LAB) were present together with other organisms, mainly members of the family Micrococcaceae and meat contaminants, such as Brochothrix thermosphacta and Enterococcus sp., during the first 3 days of fermentation. After 3 days, LAB represented the main population, which was responsible for the acidification and proteolysis that determined the characteristic organoleptic profile of the Friuli Venezia Giulia fermented sausages. The PCR-DGGE protocol for studying sausage fermentation proved to be a good tool for monitoring the process in real time, and it makes technological adjustments possible when they are required.

Animals↗
PubMed2000

Mitochondrial DNA restriction enzyme analysis and evaluation of the enological characteristics of Saccharomyces cerevisiae strains isolated from grapes of the wine-producing area of Collio (Italy).

A total of 70 strains of Saccharomyces cerevisiae were isolated from different grapes from the Collio Region. Chemical parameters and mitochondrial DNA (mtDNA) restriction patterns were determined. Higher alcohols were the main parameter useful for differentiating between strains, whereas the mtDNA analysis demonstrated a high genetic variability between strains. A weak correlation was observed when the dendrograms obtained from the chemical and genetic results were compared.

DNA, Mitochondrial↗
PubMed2000

Development and evaluation of a PCR-microplate capture hybridization method for direct detection of verotoxigenic Escherichia coli strains in artificially contaminated food samples.

For the purpose of detecting, directly in food, verotoxigenic Escherichia coli, a microplate hybridization method for the detection of PCR products from the SLT I and SLT II genes, was developed and evaluated. Two pairs of primers and two probes, specific for the SLT I gene and for the SLT II gene, were designed and tested. For the strains containing both genes, two PCR products of different molecular weights were obtained, whereas when only one gene was present only one fragment resulted from PCR. The use of the biotin-labeled probes allowed the immobilization of the PCR products in the microtiter plate wells and by this means their detection was possible using an ELISA-based technique. Forty artificially contaminated and fifty naturally contaminated food samples were analyzed by using the PCR-microplate hybridization technique developed in this study. All the artificially contaminated food samples were positive, independently of the number of cells inoculated before the enrichment step, whereas the naturally contaminated food samples were all negative.

Bacterial Toxins↗
PubMed2000

Biochemical responses in a Candida famata strain adapted to high copper concentrations.

A strain of Candida famata adapted to high copper concentration (1.26 mM) and a number of biochemical parameters have been tested, in order to get information on the mechanisms of metal toxicity and detoxification as well as on the metabolic responses to the treatment. The cytoplasmic levels of superoxide dismutase, peroxidase and glutathione were found significantly increased with respect to control cells, in contrast to catalase which is not affected. The activities of enolase and of triosephosphate isomerase are found to decrease as a consequence of the exposure to copper. Statistically significant differences in the content of some aminoacids are found between copper-treated and control cells.

Adaptation, Physiological↗
PubMed2000

Development of a rapid method for the identification of Lactobacillus spp. isolated from naturally fermented italian sausages using a polymerase chain reaction-temperature gradient gel electrophoresis.

A rapid method for the identification of Lactobacillus spp. isolated from naturally fermented Italian sausages was developed. It is based on the amplification of a small fragment from the 16S rRNA gene followed by temperature gradient gel electrophoresis (TGGE). Lactobacillus sakei, L. curvatus, L. alimentarius, L. casei, L. plantarum and L. brevis, obtained from International Collections, were used to optimize the method. Thiry-nine strains of Lactobacillus spp. were isolated from naturally fermented sausages and, after traditional identification, were tested by the PCR-TGGE protocol developed. No differences were observed comparing the results obtained, apart from five strains identified as L. curvatus that showed a PCR-TGGE profile identical to L. sakei.

Bacterial Typing Techniques↗
PubMed2000

A multiplex-PCR method to detect enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli in artificially contaminated foods.

In view of the considerable public health risk of the enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli, a multiplex-PCR based method was used for the amplification of the slt genes and of the eaeA gene. Three pairs of primers, different from the oligonucleotides previously used by other authors, were exploited for the amplification. Different E. coli serotypes were tested with the optimized protocol. Fifty three artificially contaminated samples and sixty naturally contaminated samples were processed with the multiplex-PCR. All the artificially contaminated samples gave positive results independently of the number of cells inoculated. On the contrary, the naturally contaminated samples were all negative. The results obtained from this experiment demonstrated that this protocol could be used for monitoring the spread of these organisms in food.

DNA Primers↗
PubMed2000

Role of tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II) gene modules in catabolism of 3-chlorobenzoate by Ralstonia eutropha JMP134(pJP4).

The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4, tfdC(I)D(I)E(I)F(I) (module I) and tfdD(II)C(II)E(II)F(II) (module II), putatively encoding these enzymes. To assess the role of both tfd modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene. These constructs were introduced into R. eutropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,2-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I. The R. eutropha JMP222 derivative containing tfdR-tfdC(I)D(I)E(I)F(I) grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR-tfdD(II)C(II)E(II)F(II). In the case of P. putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB. These results indicate that efficient degradation of 3-CB by R. eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II.

Bacterial Proteins↗
PubMed2000

A lexical database for Spanish-speaking children.

This article presents a computerized database of words for use in experimental research in cognitive psychology and psycholinguistics. The data are based on the oral vocabulary of 200 Spanish-speaking children aged from 11.16 to 49.16 months. The database includes 15,428 Spanish words (tokens) and comprises 1,259 different words (types). It provides information about age of acquisition, orthography, grammar, semantics, and frequency.

Child↗
PubMed2000

Temperature gradient gel electrophoresis of the amplified product of a small 16S rRNA gene fragment for the identification of Listeria species isolated from food.

The development of a rapid method for the identification of Listeria spp. is described. It is based on the polymerase chain reaction amplification of a small fragment from the 16S rRNA gene followed by temperature gradient gel electrophoresis. Forty-five strains of Listeria spp. (Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri) were used for the optimization of the protocol. No differences were observed between the results of the identification of the strains tested using traditional methods and those obtained by polymerase chain reaction-temperature gradient gel electrophoresis analysis.

Electrophoresis, Polyacrylamide Gel↗
PubMed1999

The impact of the Aznalcóllar mine tailing spill on groundwater.

As a consequence of a mine tailing dam collapse on the 25th April 1998, more than 4000 ha of the Guadiamar riverflat and farmlands were flooded by 4 hm3 of sulphide slurry. A number of open wells (12 of the 47 analyzed) were also flooded and the water was contaminated. Before the spill, the groundwater in the aquifers was of calcium-carbonate and calcium-sulphate type, with moderate mineralisation and near neutral pH. With the exception of some of the wells close to the mine, this groundwater had a low concentration of the metals associated with the Aznalcóllar mine. After the flood the following metals had anomalous concentrations in well water: Zn, Mn, Pb, Co, Cd and Tl. Of these, Zn seems to be the best tracer of the contamination, owing to its high concentrations. During the 5 months following the spill, water from the unflooded wells did not show an increase in metal concentration. Apart from some exceptions in August, the metal concentration in the affected wells showed a progressive decrease reaching levels closer to those in the wells free from contamination. Nevertheless, in the following dry seasons the draw-down of the water level may lead to exposure and weathering of sulphides in the wells, which could cause an increase in pollution. Therefore, thorough cleaning of all highly contaminated wells is strongly recommended.

Accidents, Occupational↗
PubMed1999

Induction of the multispecific organic anion transporter (cMoat/mrp2) gene and biliary glutathione secretion by the herbicide 2,4,5-trichlorophenoxyacetic acid in the mouse liver.

The canalicular multispecific organic anion transporter, cMoat, is an ATP-binding-cassette protein expressed in the canalicular domain of hepatocytes. In addition to the transport of endo- and xenobiotics, cMoat has also been proposed to transport GSH into bile, the major driving force of bile-acid-independent bile flow. We have shown previously that the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a peroxisome-proliferator agent, significantly increases bile-acid-independent bile flow in mice. On this basis, the effect of the herbicide on cMoat gene expression was studied. A 3.6-fold increase in cMoat mRNA levels and a 2.5-fold increase in cMoat protein content were observed in the liver of mice fed on a diet supplemented with 0.125% 2,4,5-T. These effects were due to an increased rate of gene transcription (3.9-fold) and were not associated with peroxisome proliferation. Significant increases in bile flow (2.23+/-0.39 versus 1.13+/-0.15 microl/min per g of liver; P<0.05) and biliary GSH output (7.40+/-3.30 versus 2.65+/-0.34 nmol/min per g of liver; P<0.05) were observed in treated animals. The hepatocellular concentration of total glutathione also increased in hepatocytes of treated mice (10.95+/-0.84 versus 5.12+/-0.47 mM; P<0.05), because of the induction (2.4-fold) of the heavy subunit of the gamma-glutamylcysteine synthetase (GCS-HS) gene. This is the first model of co-induction of cMoat and GCS-HS genes in vivo in the mouse liver, associated with increased glutathione synthesis and biliary glutathione output. Our observations are consistent with the hypothesis that the cMoat transporter plays a crucial role in the secretion of biliary GSH.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1998

A rapid method for the identification and partial serotyping of Listeria monocytogenes in food by PCR and restriction enzyme analysis.

Two highly specific primers for Listeria monocytogenes were used to yield from foods such as milk, soft cheese and meat, PCR products that were cleaved with the restriction enzyme HindIII. The fragments generated allowed a distinction between two groups of L. monocytogenes serovars: serovars 1/2a and 1/2c cluster in one group and serovars 1/2b, 3b and 4b in the other subgroup. Since this procedure can be completed in 24 h, an epidemiological association between human disease and suspected sources can be rapidly confirmed at the subgroup level in the laboratory.

Animals↗
PubMed1998

Development of a PCR microplate-capture hybridization method for simple, fast and sensitive detection of Salmonella serovars in food.

The authors have developed an easy and rapid detection and identification system for Salmonella spp. in food. The gene inv A was selected as the target sequence. Oligonucleotides derived from conserved regions of this gene were able to exclusively prime the amplification of a 389 bp fragment when Salmonella spp. DNA was used as the template. An internal Salmonella spp. specific DNA probe was used for confirmation of the amplified polymerase chain reaction(PCR)product, by Southern blot or microplate-capture hybridization assay. In this fashion the sensitivity of the method was increased 100-fold (4.5 fg total DNA). To validate the method, a total of 75 food samples were tested. The PCR-microplate capture hybridization assay is easy to perform and much faster than traditional detection methods for Salmonella spp. in food. Hybridization in microtitre plates is more readily observed than in Southern blot and is more sensitive than conventional agarose gel electrophoresis.

Biotin↗