Rafael Rotger

Stimulatory role of grape pomace polyphenols on Lactobacillus acidophilus growth.

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 microg/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 (1mg/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.

Analysis of the leaching and toxicity of new amine activators for the curing of acrylic bone cements and composites

The comparative reactivity of new tertiary amine activators with the basic chemical structure of N,N-dimethyl-4-toluidine, but reduced toxicity, is analysed. The leaching of the amine compounds from cured cements was studied by analysis of the concentration of the corresponding amine in a physiological saline solution after 3 months of immersion, giving lower values for the new amine compounds as compared to N,N-dimethyl-4-toluidine. The acute toxicity was determined by intravenous injection of saline solutions of the corresponding chlorhydrates in mice and the cytotoxicity by the evolution of specific culture media. The results obtained demonstrate a lower acute toxicity and cytotoxicity of the new activators, even with a noticeable antiseptic action, which makes these materials very interesting from a practical point of view as activators of the curing process of acrylic bone cements for orthopaedic surgery and dentistry.

Chronic colonization by Pseudomonas aeruginosa of patients with obstructive lung diseases: cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary disease

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.

Grape Antioxidant Dietary Fiber Stimulates Lactobacillus Growth in Rat Cecum

UNLABELLED 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. PRACTICAL APPLICATION Grape antioxidant fiber combines nutritional and physiological properties of dietary fiber and natural antioxidants from grapes. Grape antioxidant fiber could be used as an ingredient for functional foods and as a dietary supplement to increase the intake of dietary fiber and bioactive compounds.

Homologous regions of the Salmonella enteritidis virulence plasmid and the chromosome of Salmonella typhi encode thiol: disulphide oxidoreductases belonging to the DsbA thioredoxin family

The nucleotide sequence relatedness between the chromosome of Salmonella typhi and the virulence plasmid of Salmonella enteritidis was investigated using short DNA probes of < 2 kb covering the whole virulence plasmid sequence. Only one homologous region was detected. This region was subsequently cloned and partially sequenced. Sequences closely related to the pefl gene and the ORFs orf7, orf8 and orf9, which are located downstream of the fimbrial pef operon of the Salmonella typhimurium virulence plasmid, were detected. Sequencing of the cloned S. typhi DNA fragment also revealed identity with genes of the fimbrial sef operon characterized in the chromosome of S. enteritidis. These nucleotide sequences mapped upstream of the S. typhi chromosomal region homologous to the S. enteritidis virulence plasmid. The general organization of the cloned S. typhi chromosomal fragment was similar to the fimbriae-encoding region of the S. typhimurium virulence plasmid. The deduced product of orf8 in the S. typhimurium virulence plasmid, as well as those of the corresponding ORFs in the homologous region of the S. typhi chromosome and in the S. enteritidis virulence plasmid (designated dlt and dlp, respectively), appeared to be related to the thioredoxin family of thiol: disulphide oxidoreductases. The dlp gene was able to complement the DTT-sensitive phenotype, the inability to metabolize glucose 1-phosphate and the low alkaline phosphatase activity of a dsbA mutant of Escherichia coli. The dlt gene partially complemented the lack of alkaline phosphatase activity, but not the other mutant phenotypes. The products of both genes could be detected using the T7 RNA polymerase promoter expression system. The estimated molecular masses of the products of the dlt and dlp genes by SDS-PAGE were 26 and 23 kDa, respectively, the first being in agreement with the deduced amino acid sequence and the latter, somewhat smaller. The processing of a possible leader peptide in the Dlp protein, but not in the Dlt protein, could be responsible for this difference. The Dlp protein appeared as a doublet band on SDS-PAGE, which is characteristic of the oxidized and reduced states of this kind of protein.

A multi-resistance plasmid isolated from commensal Neisseria species is closely related to the enterobacterial plasmid RSF1010

pFM739, an R plasmid from Neisseria sicca that encodes penicillin, streptomycin and sulphonamide resistance, and the enterobacterial IncQ(P-4) plasmid RSF1010, which encodes streptomycin and sulphonamide resistance, were incompatible, and were mobilized by the same conjugative plasmids. Restriction mapping confirmed a high degree of similarity between both R plasmids; pFM739 carried DNA fragments corresponding to the known replication and resistance regions of RSF1010. pFM739 also carried an extra segment with the same restriction map as that described for the beta-lactamase-coding region of transposon Tn3. It is suggested that the R plasmids isolated from commensal Neisseria sp. could have resulted from transposition of a Tn3-like genetic element to an RSF1010-like plasmid, and that they contain deletion derivatives of transposon Tn3.

The amino-terminal non-catalytic region of Salmonella typhimurium SigD affects actin organization in yeast and mammalian cells.

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‐bisphosphate 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.

Interaction of the Salmonella Typhimurium effector protein SopB with host cell Cdc42 is involved in intracellular replication

The phosphoinositide phosphatase SopB/SigD is a type III secretion system effector that plays multiple roles in Salmonella internalization and intracellular survival. We previously reported that SopB complexed with and inhibited the small GTPase Cdc42 when expressed in a yeast model system, independently of its phosphatase activity. Here we show that human Cdc42, but not Rac1, interacts with catalytically inactive SopB when coexpressed in Saccharomyces cerevisiae. This interaction occurs with both constitutively active and non‐activatable Cdc42, suggesting that SopB binds Cdc42 independently of its activation state. By mutational analysis we have narrowed the Cdc42‐interacting region of SopB to the first 142 amino acids, and isolated a collection of point mutations in this region, mainly affecting leucine residues conserved in the related Shigella IpgD protein. Such mutations yielded SopB unable to interact with Cdc42 but maintained phosphatase activity. SopB mutant proteins defective for binding Cdc42 were ubiquitinated upon translocation in mammalian cells, but their localization to the Salmonella‐containing vacuole was reduced compared with wild‐type SopB. Whereas invasion of mammalian cells by Salmonella bearing these sopB mutations was not affected, intracellular replication was less efficient, suggesting that SopB–Cdc42 interaction contributes to the adaptation of Salmonella to the intracellular environment.

A ColE1-type plasmid from Salmonella enteritidis encodes a DNA cytosine methyltransferase

The multicopy plasmid pFM366 was isolated from a virulent Salmonella enteritidis strain and was found to code for DNA methylase activity (Ibáñez and Rotger, 1993). The present work was aimed at characterizing the genetic organization and functional features of this 5.6 kb plasmid. We found pFM366 almost identical to the plasmid P4 isolated from Shigella sonnei, that encodes the SsoII restriction-modification system (Karyagina et al., 1993), and related to other ColE1-type plasmids. Examination of these plasmids revealed a common organization which suggests they were the result of similar recombinational events. The cytosine methylase of pFM366 is nearly identical to M. SsoII, whereas the gene encoding the restrictase homologous to R. SsoII is truncated and its product is inactive. The expression of the cytosine methylase encoded by pFM366 is strongly affected by deletion of regions located upstream and downstream of its ORF, and is negatively controlled by the rpoS gene in Escherichia coli. The methylase activity encoded by pFM366 induces the SOS response, which could be responsible for the observed delay in the growth of E. coli.

Multiresistance plasmid from commensal Neisseria strains.

Antibiotic-resistant commensal strains of Neisseria spp. and Branhamella catarrhalis were isolated from throat cultures, on the basis of their capacity to grow in the presence of penicillin, streptomycin, or sulfamethoxazole-trimethoprim. Several strains, which belonged to different species of Neisseria, were resistant to beta-lactams, streptomycin, sulfamethoxazole, and trimethoprim, harbored a 6.0-megadalton plasmid with identical HinfI restriction patterns, and produced beta-lactamase and streptomycin phosphotransferase. The resistance determinants for beta-lactams, streptomycin, and sulfamethoxazole, but not for trimethoprim, were transferred from all these strains to Escherichia coli by conjugation or transformation. The resulting transconjugants or transformants acquired the plasmid and the capacity to produce beta-lactamase and streptomycin phosphotransferase. The 6.0-megadalton plasmid complemented a mutation which determines production of thermosensitive dihydropteroate synthetase in E. coli. We conclude that an R plasmid coding for beta-lactamase, streptomycin phosphotransferase, and a sulfonamide-resistant dihydropteroate synthetase is common to these strains. Images