Rafael Rotger | Universidad Complutense de Madrid (original) (raw)
Papers by Rafael Rotger
Anales De La Real Academia Nacional De Farmacia, 2011
Anales De La Real Academia Nacional De Farmacia, 2011
International Journal of Food Microbiology, Nov 1, 2009
Grape pomace is a natural product rich in dietary fiber and polyphenols. A major part of dietary ... more Grape pomace is a natural product rich in dietary fiber and polyphenols. A major part of dietary polyphenols is not absorbed in the small intestine and can interact with colonic microbiota. The influence of grape polyphenols on Lactobacillus acidophilus CECT 903 growth was investigated through agar diffusion assays and cultures in liquid media. Grape phenolic extracts and some standards of phenolic compounds (caffeic acid, gallic acid, tannic acid, catechin, epicatechin, and quercetin) were assayed. All phenolic compounds tested did not exert an inhibitory effect on L. acidophilus growth at a maximum concentration of 5000 μg/ disk in agar diffusion assays. In addition, a stimulatory trend in bacterial growth was observed in both grape phenolic extracts and tannic acid. The major finding was that grape pomace phenolic extract (1 mg/mL) induced a significant biomass increase of L. acidophilus grown in liquid culture media. Further research into the interaction between phenolic compounds and other intestinal bacteria, as well as healthy consequences, is required.
![Research paper thumbnail of [Change in resistance to quinolones and betalactams in different serogroups of Salmonella during the last decade in a Madrid hospital]](https://attachments.academia-assets.com/43448694/thumbnails/1.jpg)
](Revista espanola de quimioterapia: publicacion oficial de la Sociedad Espanola de Quimioterapia
The prevalence of antibiotic resistance was studied in 3230 strains of Salmonella enterica isolat... more The prevalence of antibiotic resistance was studied in 3230 strains of Salmonella enterica isolated in the Hospital Universitario La Paz in Madrid, Spain, from 1991 to 2001. Betalactam antibiotic resistance has been notorious in serogroup B4; the highest prevalence of ampicillin resistance (84%) was reached in 2000 and that of amoxicillin-clavulanic acid (45%) in 1996. Resistance to cephalosporins has been controlled, although in 2000 cefazolin resistance reached 37% in serogroup C2-8. An increase in first generation quinolone resistance was detected in every serogroup, especially in D9 and C2-8, which showed an increase from 6% and 15% in 1991, respectively, to 40% and 85% in 2001. Although important resistance to ciprofloxacin has not yet been detected, the activity of fluoroquinolones against Salmonella must be closely monitored.
Microbial Ecology in Health & Disease, 2005
The aim of this study was to follow the evolution of faecal bacteria over the first 34 weeks of l... more The aim of this study was to follow the evolution of faecal bacteria over the first 34 weeks of life using molecular methods in healthy infants, all of them initially breast-fed and then subjected to a similar succession of dietary changes. Faecal samples from six healthy, full-term, unrelated babies were subjected to PCR amplification with general primers for 16S bacterial rDNA. The PCR products were analysed by denaturing gradient gel electrophoresis (DGGE) to determine a pattern of predominant bacterial species in each sample. Detection of species of Bifidobacterium genus and Lactobacillus group was done using genus-or group-specific primers and sequencing DGGE bands. We detected an increment in the bacterial diversity in all infants, the more obvious changes being detected after introduction of a solid diet. Lactobacillus were detected from the third week and showed a frequent change of species. The presence of Bifidobacterium species was much more stable. Evolution of the microbiota in the six infants studied underwent significant changes until introduction of meat in the diet, but was unique for each infant despite the similarity of their feeding.
British Journal of Nutrition, 2013
Grape antioxidant dietary fibre (GADF) is a grape product rich in dietary fibre and natural antio... more Grape antioxidant dietary fibre (GADF) is a grape product rich in dietary fibre and natural antioxidants. We reported previously that GADF intake reduced apoptosis and induced a pro-reducing shift in the glutathione (GSH) redox status of the rat proximal colonic mucosa. The aim of the study was to elucidate the molecular mechanisms responsible for the anti-apoptotic effect of GADF and their association with the oxidative environment of the distal colonic mucosa. The ability of GADF to modify colonic crypt cell proliferation was also investigated. Male Wistar rats (n 20) were fed with diets containing either cellulose (control group) or GADF (GADF group) as fibre for 4 weeks. GADF did not modify cell proliferation but induced a significant reduction of colonic apoptosis. The anti-apoptotic proteins Bcl-2 (B-cell lymphoma-2) and Bcl-x L (B-cell lymphoma extra large) were up-regulated in the mitochondria and down-regulated in the cytosol of the GADF mucosa, whereas the opposite was found for the pro-apoptotic protein Bax (Bcl-2-associated X protein), leading to an anti-apoptotic shift in the pattern of expression of the Bcl-2 family. Cytosolic cytochrome c and cleaved caspase-3 levels and caspase-3 activity were reduced by GADF. The modulation of the antioxidant enzyme system and the increase of the cytosolic GSH:glutathione disulfide (GSSG) ratio elicited by GADF helped to reduce oxidative damage. The cytosolic GSH:GSSG ratio was negatively related to apoptosis. These results indicate that GADF acts on the expression of the pro-and anti-apoptotic Bcl-2 proteins, attenuating the mitochondrial apoptotic pathway in the distal colonic mucosa. This effect appears to be associated with the antioxidant properties of GADF.
PROTEOMICS, 2004
A proteomic approach to study Salmonella typhi periplasmic proteins altered by a lack of the DsbA... more A proteomic approach to study Salmonella typhi periplasmic proteins altered by a lack of the DsbA thiol: Disulfide isomerase Two-dimensional electrophoresis (2-DE) was used to analyze the pleiotropic effects of a deficiency in DsbA, a periplasmic disulfide-bond oxidoreductase, in Salmonella typhi. With this aim, the dsbA gene was cloned and assayed for activity in a dsbA-null mutant of Escherichia coli. A dsbA/chloramphenicol acetylase construct was then used to disrupt the wild-type gene of S. typhi. The resultant dsbA-null mutant of S. typhi, like the E. coli mutant, exhibited a lack of flagellation and of glucose-1-phosphatase activity. Periplasmic extracts from the parental and mutant strains were analyzed by 2-DE using standard denaturing and nondenaturing conditions. Differences in protein expression were more marked in nondenaturing conditions. Ninety-nine protein spots were analyzed by peptide mass fingerprinting, and 65 spots were identified by searching a S. typhi database. Twenty-five spots were exclusively detected in the wild-type strain, 10 were found only in the mutant strain, and 21 were common to both strains. We observed a lack of DsbA, glucose-1-phosphatase and flagellin in the dsbA-null mutant, which explains two of the observed phenotypes. The AI-2 autoinducer-producing protein LuxS, which is involved in quorum-sensing signalling was also absent.
PROTEOMICS, 2009
Through acute enteric infection, Salmonella invades host enterocytes and reproduces intracellular... more Through acute enteric infection, Salmonella invades host enterocytes and reproduces intracellularly into specialized vacuolae. This involves changes in host cell signaling elicited by bacterial proteins delivered via type III secretion systems (TTSS). One of the two TTSSs of Salmonella enterica serovar Typhimurium encoded by the Salmonella pathogenicity island-1, triggers bacterial internalization. Among the effector proteins translocated by this TTSS, the GTPase modulator SopE/E2 and the phosphoinositide phosphatase SigD are known to play key roles in these processes. To better understand their contribution to re-programming host cell pathways, we used ZeptoMARK reverse-phase protein array technology, which allows printing 32-sample lysate arrays that can be analyzed with phospho-specific antibodies to evaluate the phosphorylation of signaling proteins. Lysates were obtained at different times after infection of HeLa cells with WT, TTSS-deficient, sopE/E2 and sigD single and double deletants, as well as different sigD Salmonella mutants. Our analysis detected activation of p38, JNK and ERK mitogen-activated protein kinases, mainly dependent on SopE/E2, as well as SigD-dependent phosphorylation of PKB/Akt and its targets GSK-3b and FKHR/FoxO. This is the first time that reverse-phase protein array technology is used in the cellular microbiology field, demonstrating its value to screen for host signaling events through bacterial infection.
Journal of Food Science, 2012
The digesta is a highly active biological system where epithelial cells, microbiota, nondigestibl... more The digesta is a highly active biological system where epithelial cells, microbiota, nondigestible dietary components, and a large number of metabolic products interact. The gut microbiota can be modulated by both endogenous and exogenous substrates. Undigested dietary residues are substrates for colonic microbiota and may influence gut microbial ecology. The objective of this work was to study the capacity of grape antioxidant dietary fiber (GADF), which is rich in polyphenols, to modify the bacterial profile in the cecum of rats. Male adult Wistar rats were fed for 4 wk with diets containing either cellulose or GADF as dietary fiber. The effect of GADF on bacterial growth was evaluated in vitro and on the cecal microbiota of rats using quantitative real time polymerase chain reaction (RT-PCR). The results showed that GADF intake stimulates proliferation of Lactobacillus and slightly affects the composition of Bifidobacterium species. GADF was also found to have a stimulative effect on Lactobacillus reuteri and Lactobacillus acidophilus in vitro. These findings suggest that the consumption of a diet rich in plant foods with high dietary fiber and polyphenol content may enhance the gastrointestinal health of the host through microbiota modulation.
International Journal of Food Microbiology, 2009
Grape pomace is a natural product rich in dietary fiber and polyphenols. A major part of dietary ... more Grape pomace is a natural product rich in dietary fiber and polyphenols. A major part of dietary polyphenols is not absorbed in the small intestine and can interact with colonic microbiota. The influence of grape polyphenols on Lactobacillus acidophilus CECT 903 growth was investigated through agar diffusion assays and cultures in liquid media. Grape phenolic extracts and some standards of phenolic compounds (caffeic acid, gallic acid, tannic acid, catechin, epicatechin, and quercetin) were assayed. All phenolic compounds tested did not exert an inhibitory effect on L. acidophilus growth at a maximum concentration of 5000 μg/ disk in agar diffusion assays. In addition, a stimulatory trend in bacterial growth was observed in both grape phenolic extracts and tannic acid. The major finding was that grape pomace phenolic extract (1 mg/mL) induced a significant biomass increase of L. acidophilus grown in liquid culture media. Further research into the interaction between phenolic compounds and other intestinal bacteria, as well as healthy consequences, is required.
International Journal of Food Microbiology, 2005
The potential health-improving effects of both a prebiotic and a probiotic infant formula have be... more The potential health-improving effects of both a prebiotic and a probiotic infant formula have been evaluated in a rat model. Two groups of 10 rats were fed with either prebiotics containing fructo-oligosaccharides or probiotics containing viable Bifidobacterium lactis and Streptococcus thermophilus. The composition of their caecal microbiota was analyzed both by classical plate count of the main bacterial groups and by PCR amplification of a V3 fragment of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE). Both diets induced a significant reduction of clostridia and Bacteroides spp. compared to a control diet, whereas prebiotics were also able to reduce the number of coliforms and to increase the presence of bifidobacteria. DGGE analysis showed a significant increase of 16S rRNA gene fragments in rats fed with either probotics or prebiotics. Nineteen bands were sequenced and most of them showed similarity to cultured bacteria. Detection of Bifidobacterium spp. by this technique using genus-specific primers only permitted these bacteria to be detected in prebiotics-fed rats, whereas the use of Lactobacillus group-specific primers gave similar results in rats fed with any diet, in agreement with the plate count results. D
International Journal of Antimicrobial Agents, 2004
We characterized 29 antimicrobial-resistant Salmonella enterica serovar Typhimurium strains, incl... more We characterized 29 antimicrobial-resistant Salmonella enterica serovar Typhimurium strains, including four belonging to the monophasic variant 4,5,12:i:-, mostly isolated from infants. They were selected from 3230 strains isolated in the years 1990-2001 on the basis of resistance to ampicillin and variable susceptibility to the amoxicillin-clavulanate combination. Twenty-three strains were resistant to more than four antibiotics. All the strains carried the bla TEM gene and most were able to transfer this gene by conjugation. Sequencing of the gene from one of the amoxicillin-clavulanate-resistant strains allowed identification of the encoded -lactamase as TEM-1; all of these strains carried a second gene encoding -lactamase production, either pse-1 or oxa1. However, the association of bla TEM plus pse-1 genes did not always confer resistance to amoxicillin-clavulanate. The pse-1 gene, found in 17 strains, was located in the Salmonella Genomic Island-1 (SGI1), which carries two integrons and encodes multiple drug-resistance. None of the oxa1-bearing strains had the SGI1, yet this gene was found as part of an integron that also carried the aadA1 gene and was not plasmid-associated. Thirteen of the strains harbouring SGI1 belonged to the definitive phage type (DT) 104, and most of those remaining to DT104b and U302; particularly, strains carrying the oxa1-aadA1 integron belonged to the last two phage types. Pulsed field electrophoresis confirmed the clonal organization of DT104 strains, whereas U302 strains fell into different groups, depending on their resistance determinants.
Food Control, 2011
The presence of pathogenic bacteria in spices represents a public health risk as a possible cause... more The presence of pathogenic bacteria in spices represents a public health risk as a possible cause of food contamination. Salmonella has been found in several spices and it has been involved in food-borne outbreaks, but this bacterium has not been reported as a contaminant of saffron (Crocus sativus L.). We examined a possible antibacterial effect of saffron using samples from Iran, Greece and Spain which were artificially contaminated with clinical isolates belonging to five different serovars of Salmonella. We detected a loss of viability during the room-temperature storage of the saffron samples, with bacteria being undetectable at day 16 except in the case of the DT104 strain of the Typhimurium serovar, in all of the samples, and of the Hadar serovar in the Iranian sample, both of which gave negative culture at day 32. The laboratory strain LT2 of the Typhimurium serovar was undetectable at day 4. To gain an insight into the basis for this bactericidal effect, we measured the inhibitory and bactericidal concentrations of safranal and crocin, the main compounds responsible for the flavouring and colouring capabilities of saffron. They were in the order of 8e16 mg/mL and 64e128 mg/mL for safranal and crocin, respectively. These data suggest that these compounds, and probably their chemical relatives, are involved in the antibacterial activity of saffron, and that this effect can significantly reduce the risk of food contamination with Salmonella by this spice.
FEMS Microbiology Letters, 2009
Salmonella uses type III secretion systems (TTSS) to deliver pathogenic proteins into the host ce... more Salmonella uses type III secretion systems (TTSS) to deliver pathogenic proteins into the host cells. These translocated effectors induce bacterial internalization and intracellular proliferation by targeting important cellular processes that are conserved among eukaryotes. Here, we assessed the feasibility of performing a genetic screen in yeast to identify novel Salmonella effectors, by searching for genes that produce toxicity when expressed in this model system. We identified several known TTSS-translocated effectors and found that two of them, SteC and SseF, from Salmonella enterica serovar Typhimurium, interfere with cytoskeletal dynamics as they do in mammalian cells. We also identified 11 genes of unknown function (seven from S. Typhi and four from S. Typhimurium) that display features commonly showed by effector proteins, such as a (G1C) content lower than the average for the chromosome, suggesting their acquisition by horizontal transfer processes. Five of these proteins are highly conserved only among Salmonella serovars, whereas the other six are also conserved in other pathogenic or opportunistic enterobacteria. Moreover, we identified other proteins that share specific activity domains with either translocated or bacterial-confined proteins known to be involved in pathogenesis, which might also act as virulence proteins.
FEMS Microbiology Letters, 1986
Enfermedades Infecciosas y Microbiología Clínica, 2011
The digesta is a highly active biological system where epithelial cells, microbiota, nondigestibl... more The digesta is a highly active biological system where epithelial cells, microbiota, nondigestible dietary components, and a large number of metabolic products interact. The gut microbiota can be modulated by both endogenous and exogenous substrates. Undigested dietary residues are substrates for colonic microbiota and may influence gut microbial ecology. The objective of this work was to study the capacity of grape antioxidant dietary fiber (GADF), which is rich in polyphenols, to modify the bacterial profile in the cecum of rats. Male adult Wistar rats were fed for 4 wk with diets containing either cellulose or GADF as dietary fiber. The effect of GADF on bacterial growth was evaluated in vitro and on the cecal microbiota of rats using quantitative real time polymerase chain reaction (RT-PCR). The results showed that GADF intake stimulates proliferation of Lactobacillus and slightly affects the composition of Bifidobacterium species. GADF was also found to have a stimulative effect on Lactobacillus reuteri and Lactobacillus acidophilus in vitro. These findings suggest that the consumption of a diet rich in plant foods with high dietary fiber and polyphenol content may enhance the gastrointestinal health of the host through microbiota modulation.
Diagnostic Microbiology and Infectious Disease, 2010
Pseudomonas aeruginosa is isolated in sputum cultures from cystic fibrosis (CF) patients and adul... more Pseudomonas aeruginosa is isolated in sputum cultures from cystic fibrosis (CF) patients and adults with bronchiectasis (BS) and chronic obstructive pulmonary disease, but it is not well known if the characteristics of colonization in these latter patients are similar to those with CF. We examined 125 P. aeruginosa isolates obtained from 31 patients suffering from these diseases by pulsed field gel electrophoresis and genotyping of mucA and fpvA genes. The pattern of colonization, with dominance of a clonal strain and incidence of mucoid phenotypes, was similar in every group of patients; however, in some CF and BS patients, we detected the replacement or coexistence of 2 main clones. The main differences were found in the nucleotide position of less common mucA mutations, other than mucA22, and in the predominance of the different types of the pyoverdine receptor. Our results support a similar colonization pattern by P. aeruginosa in the different obstructive pulmonary diseases.
Cellular Microbiology, 2005
The internalization of Salmonella into epithelial cells relies on the function of bacterial prote... more The internalization of Salmonella into epithelial cells relies on the function of bacterial proteins which are injected into the cell by a specialized type III secretion system. Such bacterial effectors interfere with host cell signalling and induce local cytoskeletal rearrangements. One of such effectors is SigD/SopB, which shares homology with mammalian inositol phosphatases. We made use of the Saccharomyces cerevisiae model for elucidating new aspects of SigD function. Endogenous expression of SigD in yeast caused severe growth inhibition. Surprisingly, sigD alleles mutated in the catalytic site or even deleted for the whole C-terminal phosphatase domain still inhibited yeast growth by inducing loss of actin polarization and precluding the budding process. Accordingly, when expressed in HeLa cells, the same sigD alleles lost the ability of depleting phosphatidylinositol 4,5-bis phosphate from the plasma membrane, but still caused disappearance of actin fibres and loss of adherence. We delineate a region of 25 amino acids (residues 118-142) that is necessary for the effect of SigD on actin in HeLa cells. Our data indicate that SigD exerts a toxic effect linked to its N-terminal region and independent of its phosphatase activity.
Anales De La Real Academia Nacional De Farmacia, 2011
Anales De La Real Academia Nacional De Farmacia, 2011
International Journal of Food Microbiology, Nov 1, 2009
Grape pomace is a natural product rich in dietary fiber and polyphenols. A major part of dietary ... more Grape pomace is a natural product rich in dietary fiber and polyphenols. A major part of dietary polyphenols is not absorbed in the small intestine and can interact with colonic microbiota. The influence of grape polyphenols on Lactobacillus acidophilus CECT 903 growth was investigated through agar diffusion assays and cultures in liquid media. Grape phenolic extracts and some standards of phenolic compounds (caffeic acid, gallic acid, tannic acid, catechin, epicatechin, and quercetin) were assayed. All phenolic compounds tested did not exert an inhibitory effect on L. acidophilus growth at a maximum concentration of 5000 μg/ disk in agar diffusion assays. In addition, a stimulatory trend in bacterial growth was observed in both grape phenolic extracts and tannic acid. The major finding was that grape pomace phenolic extract (1 mg/mL) induced a significant biomass increase of L. acidophilus grown in liquid culture media. Further research into the interaction between phenolic compounds and other intestinal bacteria, as well as healthy consequences, is required.
Revista espanola de quimioterapia: publicacion oficial de la Sociedad Espanola de Quimioterapia
The prevalence of antibiotic resistance was studied in 3230 strains of Salmonella enterica isolat... more The prevalence of antibiotic resistance was studied in 3230 strains of Salmonella enterica isolated in the Hospital Universitario La Paz in Madrid, Spain, from 1991 to 2001. Betalactam antibiotic resistance has been notorious in serogroup B4; the highest prevalence of ampicillin resistance (84%) was reached in 2000 and that of amoxicillin-clavulanic acid (45%) in 1996. Resistance to cephalosporins has been controlled, although in 2000 cefazolin resistance reached 37% in serogroup C2-8. An increase in first generation quinolone resistance was detected in every serogroup, especially in D9 and C2-8, which showed an increase from 6% and 15% in 1991, respectively, to 40% and 85% in 2001. Although important resistance to ciprofloxacin has not yet been detected, the activity of fluoroquinolones against Salmonella must be closely monitored.
Microbial Ecology in Health & Disease, 2005
The aim of this study was to follow the evolution of faecal bacteria over the first 34 weeks of l... more The aim of this study was to follow the evolution of faecal bacteria over the first 34 weeks of life using molecular methods in healthy infants, all of them initially breast-fed and then subjected to a similar succession of dietary changes. Faecal samples from six healthy, full-term, unrelated babies were subjected to PCR amplification with general primers for 16S bacterial rDNA. The PCR products were analysed by denaturing gradient gel electrophoresis (DGGE) to determine a pattern of predominant bacterial species in each sample. Detection of species of Bifidobacterium genus and Lactobacillus group was done using genus-or group-specific primers and sequencing DGGE bands. We detected an increment in the bacterial diversity in all infants, the more obvious changes being detected after introduction of a solid diet. Lactobacillus were detected from the third week and showed a frequent change of species. The presence of Bifidobacterium species was much more stable. Evolution of the microbiota in the six infants studied underwent significant changes until introduction of meat in the diet, but was unique for each infant despite the similarity of their feeding.
British Journal of Nutrition, 2013
Grape antioxidant dietary fibre (GADF) is a grape product rich in dietary fibre and natural antio... more Grape antioxidant dietary fibre (GADF) is a grape product rich in dietary fibre and natural antioxidants. We reported previously that GADF intake reduced apoptosis and induced a pro-reducing shift in the glutathione (GSH) redox status of the rat proximal colonic mucosa. The aim of the study was to elucidate the molecular mechanisms responsible for the anti-apoptotic effect of GADF and their association with the oxidative environment of the distal colonic mucosa. The ability of GADF to modify colonic crypt cell proliferation was also investigated. Male Wistar rats (n 20) were fed with diets containing either cellulose (control group) or GADF (GADF group) as fibre for 4 weeks. GADF did not modify cell proliferation but induced a significant reduction of colonic apoptosis. The anti-apoptotic proteins Bcl-2 (B-cell lymphoma-2) and Bcl-x L (B-cell lymphoma extra large) were up-regulated in the mitochondria and down-regulated in the cytosol of the GADF mucosa, whereas the opposite was found for the pro-apoptotic protein Bax (Bcl-2-associated X protein), leading to an anti-apoptotic shift in the pattern of expression of the Bcl-2 family. Cytosolic cytochrome c and cleaved caspase-3 levels and caspase-3 activity were reduced by GADF. The modulation of the antioxidant enzyme system and the increase of the cytosolic GSH:glutathione disulfide (GSSG) ratio elicited by GADF helped to reduce oxidative damage. The cytosolic GSH:GSSG ratio was negatively related to apoptosis. These results indicate that GADF acts on the expression of the pro-and anti-apoptotic Bcl-2 proteins, attenuating the mitochondrial apoptotic pathway in the distal colonic mucosa. This effect appears to be associated with the antioxidant properties of GADF.
PROTEOMICS, 2004
A proteomic approach to study Salmonella typhi periplasmic proteins altered by a lack of the DsbA... more A proteomic approach to study Salmonella typhi periplasmic proteins altered by a lack of the DsbA thiol: Disulfide isomerase Two-dimensional electrophoresis (2-DE) was used to analyze the pleiotropic effects of a deficiency in DsbA, a periplasmic disulfide-bond oxidoreductase, in Salmonella typhi. With this aim, the dsbA gene was cloned and assayed for activity in a dsbA-null mutant of Escherichia coli. A dsbA/chloramphenicol acetylase construct was then used to disrupt the wild-type gene of S. typhi. The resultant dsbA-null mutant of S. typhi, like the E. coli mutant, exhibited a lack of flagellation and of glucose-1-phosphatase activity. Periplasmic extracts from the parental and mutant strains were analyzed by 2-DE using standard denaturing and nondenaturing conditions. Differences in protein expression were more marked in nondenaturing conditions. Ninety-nine protein spots were analyzed by peptide mass fingerprinting, and 65 spots were identified by searching a S. typhi database. Twenty-five spots were exclusively detected in the wild-type strain, 10 were found only in the mutant strain, and 21 were common to both strains. We observed a lack of DsbA, glucose-1-phosphatase and flagellin in the dsbA-null mutant, which explains two of the observed phenotypes. The AI-2 autoinducer-producing protein LuxS, which is involved in quorum-sensing signalling was also absent.
PROTEOMICS, 2009
Through acute enteric infection, Salmonella invades host enterocytes and reproduces intracellular... more Through acute enteric infection, Salmonella invades host enterocytes and reproduces intracellularly into specialized vacuolae. This involves changes in host cell signaling elicited by bacterial proteins delivered via type III secretion systems (TTSS). One of the two TTSSs of Salmonella enterica serovar Typhimurium encoded by the Salmonella pathogenicity island-1, triggers bacterial internalization. Among the effector proteins translocated by this TTSS, the GTPase modulator SopE/E2 and the phosphoinositide phosphatase SigD are known to play key roles in these processes. To better understand their contribution to re-programming host cell pathways, we used ZeptoMARK reverse-phase protein array technology, which allows printing 32-sample lysate arrays that can be analyzed with phospho-specific antibodies to evaluate the phosphorylation of signaling proteins. Lysates were obtained at different times after infection of HeLa cells with WT, TTSS-deficient, sopE/E2 and sigD single and double deletants, as well as different sigD Salmonella mutants. Our analysis detected activation of p38, JNK and ERK mitogen-activated protein kinases, mainly dependent on SopE/E2, as well as SigD-dependent phosphorylation of PKB/Akt and its targets GSK-3b and FKHR/FoxO. This is the first time that reverse-phase protein array technology is used in the cellular microbiology field, demonstrating its value to screen for host signaling events through bacterial infection.
Journal of Food Science, 2012
The digesta is a highly active biological system where epithelial cells, microbiota, nondigestibl... more The digesta is a highly active biological system where epithelial cells, microbiota, nondigestible dietary components, and a large number of metabolic products interact. The gut microbiota can be modulated by both endogenous and exogenous substrates. Undigested dietary residues are substrates for colonic microbiota and may influence gut microbial ecology. The objective of this work was to study the capacity of grape antioxidant dietary fiber (GADF), which is rich in polyphenols, to modify the bacterial profile in the cecum of rats. Male adult Wistar rats were fed for 4 wk with diets containing either cellulose or GADF as dietary fiber. The effect of GADF on bacterial growth was evaluated in vitro and on the cecal microbiota of rats using quantitative real time polymerase chain reaction (RT-PCR). The results showed that GADF intake stimulates proliferation of Lactobacillus and slightly affects the composition of Bifidobacterium species. GADF was also found to have a stimulative effect on Lactobacillus reuteri and Lactobacillus acidophilus in vitro. These findings suggest that the consumption of a diet rich in plant foods with high dietary fiber and polyphenol content may enhance the gastrointestinal health of the host through microbiota modulation.
International Journal of Food Microbiology, 2009
Grape pomace is a natural product rich in dietary fiber and polyphenols. A major part of dietary ... more Grape pomace is a natural product rich in dietary fiber and polyphenols. A major part of dietary polyphenols is not absorbed in the small intestine and can interact with colonic microbiota. The influence of grape polyphenols on Lactobacillus acidophilus CECT 903 growth was investigated through agar diffusion assays and cultures in liquid media. Grape phenolic extracts and some standards of phenolic compounds (caffeic acid, gallic acid, tannic acid, catechin, epicatechin, and quercetin) were assayed. All phenolic compounds tested did not exert an inhibitory effect on L. acidophilus growth at a maximum concentration of 5000 μg/ disk in agar diffusion assays. In addition, a stimulatory trend in bacterial growth was observed in both grape phenolic extracts and tannic acid. The major finding was that grape pomace phenolic extract (1 mg/mL) induced a significant biomass increase of L. acidophilus grown in liquid culture media. Further research into the interaction between phenolic compounds and other intestinal bacteria, as well as healthy consequences, is required.
International Journal of Food Microbiology, 2005
The potential health-improving effects of both a prebiotic and a probiotic infant formula have be... more The potential health-improving effects of both a prebiotic and a probiotic infant formula have been evaluated in a rat model. Two groups of 10 rats were fed with either prebiotics containing fructo-oligosaccharides or probiotics containing viable Bifidobacterium lactis and Streptococcus thermophilus. The composition of their caecal microbiota was analyzed both by classical plate count of the main bacterial groups and by PCR amplification of a V3 fragment of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE). Both diets induced a significant reduction of clostridia and Bacteroides spp. compared to a control diet, whereas prebiotics were also able to reduce the number of coliforms and to increase the presence of bifidobacteria. DGGE analysis showed a significant increase of 16S rRNA gene fragments in rats fed with either probotics or prebiotics. Nineteen bands were sequenced and most of them showed similarity to cultured bacteria. Detection of Bifidobacterium spp. by this technique using genus-specific primers only permitted these bacteria to be detected in prebiotics-fed rats, whereas the use of Lactobacillus group-specific primers gave similar results in rats fed with any diet, in agreement with the plate count results. D
International Journal of Antimicrobial Agents, 2004
We characterized 29 antimicrobial-resistant Salmonella enterica serovar Typhimurium strains, incl... more We characterized 29 antimicrobial-resistant Salmonella enterica serovar Typhimurium strains, including four belonging to the monophasic variant 4,5,12:i:-, mostly isolated from infants. They were selected from 3230 strains isolated in the years 1990-2001 on the basis of resistance to ampicillin and variable susceptibility to the amoxicillin-clavulanate combination. Twenty-three strains were resistant to more than four antibiotics. All the strains carried the bla TEM gene and most were able to transfer this gene by conjugation. Sequencing of the gene from one of the amoxicillin-clavulanate-resistant strains allowed identification of the encoded -lactamase as TEM-1; all of these strains carried a second gene encoding -lactamase production, either pse-1 or oxa1. However, the association of bla TEM plus pse-1 genes did not always confer resistance to amoxicillin-clavulanate. The pse-1 gene, found in 17 strains, was located in the Salmonella Genomic Island-1 (SGI1), which carries two integrons and encodes multiple drug-resistance. None of the oxa1-bearing strains had the SGI1, yet this gene was found as part of an integron that also carried the aadA1 gene and was not plasmid-associated. Thirteen of the strains harbouring SGI1 belonged to the definitive phage type (DT) 104, and most of those remaining to DT104b and U302; particularly, strains carrying the oxa1-aadA1 integron belonged to the last two phage types. Pulsed field electrophoresis confirmed the clonal organization of DT104 strains, whereas U302 strains fell into different groups, depending on their resistance determinants.
Food Control, 2011
The presence of pathogenic bacteria in spices represents a public health risk as a possible cause... more The presence of pathogenic bacteria in spices represents a public health risk as a possible cause of food contamination. Salmonella has been found in several spices and it has been involved in food-borne outbreaks, but this bacterium has not been reported as a contaminant of saffron (Crocus sativus L.). We examined a possible antibacterial effect of saffron using samples from Iran, Greece and Spain which were artificially contaminated with clinical isolates belonging to five different serovars of Salmonella. We detected a loss of viability during the room-temperature storage of the saffron samples, with bacteria being undetectable at day 16 except in the case of the DT104 strain of the Typhimurium serovar, in all of the samples, and of the Hadar serovar in the Iranian sample, both of which gave negative culture at day 32. The laboratory strain LT2 of the Typhimurium serovar was undetectable at day 4. To gain an insight into the basis for this bactericidal effect, we measured the inhibitory and bactericidal concentrations of safranal and crocin, the main compounds responsible for the flavouring and colouring capabilities of saffron. They were in the order of 8e16 mg/mL and 64e128 mg/mL for safranal and crocin, respectively. These data suggest that these compounds, and probably their chemical relatives, are involved in the antibacterial activity of saffron, and that this effect can significantly reduce the risk of food contamination with Salmonella by this spice.
FEMS Microbiology Letters, 2009
Salmonella uses type III secretion systems (TTSS) to deliver pathogenic proteins into the host ce... more Salmonella uses type III secretion systems (TTSS) to deliver pathogenic proteins into the host cells. These translocated effectors induce bacterial internalization and intracellular proliferation by targeting important cellular processes that are conserved among eukaryotes. Here, we assessed the feasibility of performing a genetic screen in yeast to identify novel Salmonella effectors, by searching for genes that produce toxicity when expressed in this model system. We identified several known TTSS-translocated effectors and found that two of them, SteC and SseF, from Salmonella enterica serovar Typhimurium, interfere with cytoskeletal dynamics as they do in mammalian cells. We also identified 11 genes of unknown function (seven from S. Typhi and four from S. Typhimurium) that display features commonly showed by effector proteins, such as a (G1C) content lower than the average for the chromosome, suggesting their acquisition by horizontal transfer processes. Five of these proteins are highly conserved only among Salmonella serovars, whereas the other six are also conserved in other pathogenic or opportunistic enterobacteria. Moreover, we identified other proteins that share specific activity domains with either translocated or bacterial-confined proteins known to be involved in pathogenesis, which might also act as virulence proteins.
FEMS Microbiology Letters, 1986
Enfermedades Infecciosas y Microbiología Clínica, 2011
The digesta is a highly active biological system where epithelial cells, microbiota, nondigestibl... more The digesta is a highly active biological system where epithelial cells, microbiota, nondigestible dietary components, and a large number of metabolic products interact. The gut microbiota can be modulated by both endogenous and exogenous substrates. Undigested dietary residues are substrates for colonic microbiota and may influence gut microbial ecology. The objective of this work was to study the capacity of grape antioxidant dietary fiber (GADF), which is rich in polyphenols, to modify the bacterial profile in the cecum of rats. Male adult Wistar rats were fed for 4 wk with diets containing either cellulose or GADF as dietary fiber. The effect of GADF on bacterial growth was evaluated in vitro and on the cecal microbiota of rats using quantitative real time polymerase chain reaction (RT-PCR). The results showed that GADF intake stimulates proliferation of Lactobacillus and slightly affects the composition of Bifidobacterium species. GADF was also found to have a stimulative effect on Lactobacillus reuteri and Lactobacillus acidophilus in vitro. These findings suggest that the consumption of a diet rich in plant foods with high dietary fiber and polyphenol content may enhance the gastrointestinal health of the host through microbiota modulation.
Diagnostic Microbiology and Infectious Disease, 2010
Pseudomonas aeruginosa is isolated in sputum cultures from cystic fibrosis (CF) patients and adul... more Pseudomonas aeruginosa is isolated in sputum cultures from cystic fibrosis (CF) patients and adults with bronchiectasis (BS) and chronic obstructive pulmonary disease, but it is not well known if the characteristics of colonization in these latter patients are similar to those with CF. We examined 125 P. aeruginosa isolates obtained from 31 patients suffering from these diseases by pulsed field gel electrophoresis and genotyping of mucA and fpvA genes. The pattern of colonization, with dominance of a clonal strain and incidence of mucoid phenotypes, was similar in every group of patients; however, in some CF and BS patients, we detected the replacement or coexistence of 2 main clones. The main differences were found in the nucleotide position of less common mucA mutations, other than mucA22, and in the predominance of the different types of the pyoverdine receptor. Our results support a similar colonization pattern by P. aeruginosa in the different obstructive pulmonary diseases.
Cellular Microbiology, 2005
The internalization of Salmonella into epithelial cells relies on the function of bacterial prote... more The internalization of Salmonella into epithelial cells relies on the function of bacterial proteins which are injected into the cell by a specialized type III secretion system. Such bacterial effectors interfere with host cell signalling and induce local cytoskeletal rearrangements. One of such effectors is SigD/SopB, which shares homology with mammalian inositol phosphatases. We made use of the Saccharomyces cerevisiae model for elucidating new aspects of SigD function. Endogenous expression of SigD in yeast caused severe growth inhibition. Surprisingly, sigD alleles mutated in the catalytic site or even deleted for the whole C-terminal phosphatase domain still inhibited yeast growth by inducing loss of actin polarization and precluding the budding process. Accordingly, when expressed in HeLa cells, the same sigD alleles lost the ability of depleting phosphatidylinositol 4,5-bis phosphate from the plasma membrane, but still caused disappearance of actin fibres and loss of adherence. We delineate a region of 25 amino acids (residues 118-142) that is necessary for the effect of SigD on actin in HeLa cells. Our data indicate that SigD exerts a toxic effect linked to its N-terminal region and independent of its phosphatase activity.