Selection versus transmission: Quantitative and organismic biology in antibiotic resistance.

We aimed to determine the importance of selection (mostly dependent on the anthropogenic use of antimicrobials) and transmission (mostly dependent on hygiene and sanitation) as drivers of the spread of antibiotic-resistant bacterial populations. The first obstacle to estimating the relative weight of both independent variables is the lack of detailed quantitative data concerning the number of bacterial cells, potentially either pathogenic or harmless, and bacterial species exposed to antimicrobial action in the microbiotas of specific environments. The second obstacle is the difficulty of considering the relative importance of the transmission and selection exerting their combined effects on antibiotic resistance across eco-biological levels. As a consequence, advances are urgently required in quantitative biology and organismic biology of antimicrobial resistance. The absolute number of humans exposed to antibiotics and the absolute number of potentially pathogenic and commensal bacteria in their microbiomes should influence both the selection and transmission of resistant bacterial populations. The "whole Earth" microbiome, with astonishingly high numbers of bacterial cells and species, which are also exposed to anthropogenic antimicrobials in various biogeographical spaces, shapes the antibiotic resistance landscape. These biogeographical spaces influence various intensities of selection and transmission of potentially pathogenic bacteria. While waiting for more precise data, biostatistics analysis and mathematical or computational modeling can provide proxies to compare the influence of selection and transmission in resistant bacteria. In European countries with lower sanitation levels, antibiotic consumption plays a major role in increasing antibiotic resistance; however, this is not the case in countries with high sanitation levels. Although both independent variables are linked, their relative influence on the level of antibiotic resistance varies according to the particular location. Therefore, interventions directed to decrease antibiotic resistance should be designed "a la carte" for specific locations with particular ecological conditions, including sanitation facilities.

Physicochemical and microbiological parameters during the manufacturing of a beer-type fermented beverage using selenized Saccharomycesboulardii.

Selenium is an essential trace element in human health. However, it has been considered a widespread selenium deficiency worldwide, although the recommended daily intake is very low (55 µg per day). Strategies have been implemented to comply with the recommended doses, for example, through bioavailable selenium such as selenoamino acids. Thus, this research aimed to elaborate on a beer-type fermented beverage produced with previously selenized Saccharomyces boulardii. For this, the yeast was selenized by adding a minimum inhibitory concentration of Na2SeO3 (74 ppm) to YPD media. Subsequently, barley must fermentations were carried out for 120 h. Kinetic parameters of the fermentation and physicochemical parameters and selenium content of the beverage were measured. The yeast accumulated up to 25.12 mg/g of dry cell. Furthermore, selenization affected the fermentation rate, but the beverage's physicochemical parameters were not different from those of the control. Due to the final concentration of selenium in the beverage (0.378 mg/kg), it is considered a process that confers advantages for the safe intake of selenium with bioavailable potential. In conclusion, fermented beverages enriched with organic selenium could be produced through cell selenization to produce functional beverages and food.

In vitro evaluation of the prebiotic effect of red and white grape polyphenolic extracts

Nowadays, the strong relationship between diet and health is well known. Although the primary role of diet is to provide nutrients to fulfill metabolic requirements, the use of foods to improve health and the state of well-being is an idea increasingly accepted by society in the last three decades. During the last years, an important number of scientific advances have been achieved in this field and, although in some situations, it is difficult to establish a distinction between "harmful" and "good" bacteria, experts agree in classifying the genera Bifidobacterium and Lactobacillus as beneficial bacteria. Thus, several strategies can be used to stimulate the proliferation of these beneficial intestinal bacteria, being one of them the consumption of prebiotics. The development of new prebiotics, with added functionality, is one of the most serious challenges shared not only by the scientific community but also by the food industry. The objective of this work was to evaluate the potential prebiotic effect of red and white grape residues, both obtained during the winemaking process. For such purpose, an in vitro study with pure cultures of Lactobacillus and Bifidobacterium was first conducted. Secondly, a study with mixed cultures using human fecal inocula was carried out in a simulator of the distal part of the colon. The obtained results showed an increase in the Lactobacillus and Bifidobacterium population, indicating that these ingredients are serious candidates to be considered as prebiotics

In vitro evaluation of the prebiotic effect of red and white grape polyphenolic extracts.

Nowadays, the strong relationship between diet and health is well known. Although the primary role of diet is to provide nutrients to fulfill metabolic requirements, the use of foods to improve health and the state of well-being is an idea increasingly accepted by society in the last three decades. During the last years, an important number of scientific advances have been achieved in this field and, although in some situations, it is difficult to establish a distinction between "harmful" and "good" bacteria, experts agree in classifying the genera Bifidobacterium and Lactobacillus as beneficial bacteria. Thus, several strategies can be used to stimulate the proliferation of these beneficial intestinal bacteria, being one of them the consumption of prebiotics. The development of new prebiotics, with added functionality, is one of the most serious challenges shared not only by the scientific community but also by the food industry. The objective of this work was to evaluate the potential prebiotic effect of red and white grape residues, both obtained during the winemaking process. For such purpose, an in vitro study with pure cultures of Lactobacillus and Bifidobacterium was first conducted. Secondly, a study with mixed cultures using human fecal inocula was carried out in a simulator of the distal part of the colon. The obtained results showed an increase in the Lactobacillus and Bifidobacterium population, indicating that these ingredients are serious candidates to be considered as prebiotics.

Topical application of probiotics in skin: adhesion, antimicrobial and antibiofilm in vitro assays.

AIMS: When skin dysbiosis occurs as a result of skin disorders, probiotics can act as modulators, restoring microbial balance. Several properties of selected probiotics were evaluated so that their topical application could be considered. METHODS AND RESULTS: Adhesion, antimicrobial, quorum sensing and antibiofilm assays were carried out with several probiotic strains and tested against selected skin pathogens. All tested strains displayed significant adhesion to keratin. All lactobacilli with the exception of Lactobacillus delbrueckii, showed antimicrobial activity against skin pathogens, mainly due to organic acid production. Most of them also prevented biofilm formation, but only Propioniferax innocua was able to break down mature biofilms. CONCLUSIONS: This study demonstrates that although all tested probiotics adhered to human keratin, they showed limited ability to prevent adhesion of some potential skin pathogens. Most of the tested probiotics successfully prevented biofilm formation, suggesting that they may be successfully used in the future as a complement to conventional therapies in the treatment of a range of skin disorders. SIGNIFICANCE AND IMPACT OF STUDY: The topically used probiotics may be a natural, targeted treatment approach to several skin disorders and a complement to conventional therapies which present many undesirable side effects.

Chitosan mouthwash: toxicity and in vivo validation.

A previous study showed that a chitosan mouthwash would be a valid alternative to current mouthwashes as it demonstrated, in vitro, significantly higher antibiofilm activity than two commercial mouthwashes. As such, the aim of this work was to verify the safety of the developed product and to validate, in vivo, the biological activity ascertained in vitro. Chitosan mouthwash safety was evaluated through Ames, MTT and V79 chromosomal aberration assay while antimicrobial activity was evaluated through in vivo assays. The results showed that the chitosan mouthwash was safe, presenting lower cytotoxicity than a commercial mouthwash, and that it effectively reduced viable counts of Streptococcus spp. and Enterococcus spp. by ca. 5.5 log of CFU. Furthermore, in direct comparison with a commercial mouthwash the chitosan mouthwash possessed significantly higher antimicrobial activity. The conjunction of these results proves that the chitosan mouthwash is a safe, effective, natural alternative to the existent chemical mouthwashes.

A comprehensive study into the impact of a chitosan mouthwash upon oral microorganism's biofilm formation in vitro.

Modern dentistry emphasizes the importance of dental plaque control to improve oral health. To that end the development of oral care formulations has been geared toward the incorporation of antiplaque agents that may play a crucial role in oral health maintenance. In later years the research into antiplaque agents has led to the discovery of compounds with significant capability to affect biofilm formation. Among these compounds was chitosan, a polysaccharide which showed great ability to interfere with Streptococcus mutans biofilm formation. As such the aim of this work was to incorporate chitosan into a mouthwash matrix and assess its effect upon biofilm formation of oral microorganisms. This assessment was performed via study of the impact the mouthwash upon microbial adherence, biofilm formation and mature biofilms. Additionally, the action of the chitosan mouthwash was compared with two commercially available mouthwashes. The results here obtained show that only the chitosan containing mouthwash was capable of interfering with all microorganisms' adherence, biofilm formation and mature biofilms while at the same time showing vastly superior activity than both commercial mouthwashes assayed. As such a chitosan mouthwash shows great potential as a natural and efficient alternative to traditional mouthwashes.

A novel direct contact method for the assessment of the antimicrobial activity of dental cements.

Dental cements are a crucial part of the odontological treatment, however, due to the hazardous nature and reduced biological efficiency of some of the used materials, newer and safer alternatives are needed, particularly so those possessing higher antimicrobial activity than their traditional counterparts. The evaluation of the antimicrobial properties of solid and semi-solid antimicrobials, such as dental cements and gels, is challenging, particularly due to the low sensitivity of the current methods. Thus, the main aim of this study was the evaluation of the antimicrobial activity of a novel chitosan containing dental cement while simultaneous assessing/validating a new, more efficient, method for the evaluation of the antimicrobial activity of solid and gel like materials. The results obtained showed that the proposed method exhibited a higher sensitivity than the standard 96 well microtiter assay and allowed the determination of bactericidal activity. Additionally, it is interesting to note that the chitosan containing cement, which presented higher antimicrobial activity than the traditional zinc oxide/eugenol mix, was capable of inducing a viable count reduction above 5 log of CFU for all of the studied microorganisms.

Metagenomics of human microbiome: beyond 16s rDNA.

The gut microbiota presents a symbiotic relationship with the human host playing a beneficial role in human health. Since its establishment, the bacterial community is subjected to the influence of many different factors that shape its composition within each individual. However, an important convergence is observed at functional level in the gut microbiota. A metatranscriptomic study of healthy individuals showed homogeneity in the composition of the active microbiota that increased further at functional level.

Immunization of mice with Neisseria meningitidis serogroup B genomic expression libraries elicits functional antibodies and reduces the level of bacteremia in an infant rat infection model.

The feasibility of expression library immunization against the pathogenic bacterium Neisseria meningitidis was studied. A genomic library of N. meningitidis serogroup B strain CU385, containing 6000 individual clones, was constructed and divided into 10 sublibraries. Immunization of BALB/c mice with plasmid DNA from six sublibraries induced a humoral response, with recognition of several meningococcal proteins by Western blot. Three of these sublibraries elicited bactericidal antibodies against the homologous strain, and sera from mice immunized with one of these sublibraries reduced significantly the number of viable bacteria in blood of infant rats challenged with N. meningitidis. In addition, after DNA immunization, mice were boosted intraperitoneally with 5 x 10(2) colony forming units of strain CU385. Mice immunized with nine of the 10 libraries developed bactericidal antibodies 1 week after the boost and controls did not, demonstrating the priming capacity and specificity of our immunization strategy. Our study demonstrates, for the first time, that genomic immunization offers a novel approach for screening possible vaccine candidates against N. meningitidis.

Fluorinated analogues of tert-butyl alcohol as novel protecting groups for use in fluorous synthesis.

[reaction--see text] A series of fluorous derivatives of tert-butyl alcohol were prepared and evaluated as reagents for the protection of carboxylic acids for use in fluorous synthesis. Alcohol 3b can be employed efficiently to protect and immobilize medium-size nonpolar carboxylic acids in a fluorous phase.

Kinetic control in the palladium-catalyzed synthesis of C(2)-symmetric hexabenzotriphenylene. A conformational study

The hitherto unisolated, thermodynamically unstable C(2)-symmetric conformer of hexabenzotriphenylene (1) has been efficiently synthesized by palladium-catalyzed cyclotrimerization of 9, 10-didehydrophenanthrene (4). The barriers to conformational interconversion of 1 are examined experimentally and by computational studies.

[Evaluation of the effectiveness of a new vaccine against human leptospirosis in groups at risk]. / Evaluación de la efectividad de una nueva vacuna contra la leptospirosis humana en grupos en riesgo.

In order to evaluate the effectiveness of a new vaccine against human leptospirosis, a prospective cohort study was done with persons in the Cuban province of Holguín who were at risk of becoming ill with leptospirosis. The study included 118,018 persons from 15 to 65 years old who were considered to face either permanent or temporary risk. The vaccinated cohort consisted of 101,137 persons. They received two vaccine doses, 6 weeks apart, of 0.50 mL via deep intramuscular injection into the deltoid muscle of the nondominant arm. The unvaccinated cohort consisted of 16,881 persons. Epidemiological surveillance began 21 days after the application of the second vaccine dose and continued for 1 year. The same criteria for suspected and confirmed cases were maintained throughout the study period. At the end of the surveillance period effectiveness was calculated as being higher than 97%. It is estimated that the vaccination program prevented eight out of ten cases that would have otherwise occurred. Vaccine reactogenicity was also measured in a subsample of 1,500 persons between 15 and 65 years old. The observed symptomatology was low. Slight pain at the injection site was the most frequent symptom (25%). The results of the study indicate the usefulness of the vaccine for disease prevention among people at risk, and its use is thus recommended.

MR findings in hereditary spherocytosis.

The magnetic resonance findings of intrathoracic extramedullary hematopoiesis associated with bone marrow reconversion, and reticuloendothelial hemosiderosis in a 50-years-old are reported. Radiologic finding of extramedullary hematopoiesis was the first step to the diagnosis of previously unknown hereditary spherocytosis.

Novel anti-inflammatory undecapeptides that contain anisolyated glutamic acid derivatives.

In various animal models of tissue injury, corticotropin-releasing factor (CRF) and related peptides inhibit swelling, edema and loss of protein from the vascular compartment. To search for smaller peptide segments of CRF that might retain anti-inflammatory activity, the authors tested peptides similar to the carboxy terminals of ovine (o) and human/rat (h/r) CRF. Also, because h/rCRF(35-39), -Arg-Lys-Leu-Met-Glu-, resembles Arg-Lys-Leu-Leu-Glu-, a sequence found in many intermediate filament proteins, analogous peptides were evaluated. Ovine CRF(21-41), -Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys- Leu-Asp-Ile-Ala-NH2, its carboxy terminal carboxyl derivative, oCRF(21-41)-OH, and the fragments, oCRF(26-41) and oCRF(30-41), were inactive when assayed at 5 mg/kg i.v. on edema induced in the pentobarbital-anesthetized rat's hindpaw after immersion in 58 degrees C water for 1 min. Crude peptides, D-Leu-Ala-Thr-D-Tyr-Arg-Lys-Leu-Leu-Glu-Ile-D-Leu-NH2 and D-Ala-His-Ser-D-Asn-Arg-Lys-Leu-Leu-Glu-Ile-D-Leu-NH2, were found to have activity in this bioassay. Characterization of the structures within the crude mixture revealed that substitution of the glutamic acid residue with an anisolyated glutamic acid (2-amino-5-(methoxyphenyl)-5-oxopentanoic acid) derivative, designated as (A*), increased the overall potency. The glutamyl-anisole derivative was a by-product of the temperature-dependent Friedel-Crafts acylation reaction that occurs during hydrogen fluoride cleavage of glutamyl-containing peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Prey-catching and predator-avoidance in frog and toad: defining the schemas.

The present model postulates the construction of motor actions through the interaction of different motor schemas via a process of competition and co-operation wherein there is no need for a unique schema to win the competition (although that might well be the result) since two or more schemas may simultaneously be active and co-operate to yield a more complicated motor pattern. Based on lesion data, our model is structured on the principles of segregation of co-ordinate systems and participation of maps intermediate between sensory and motor schemas. The motor schemas are driven by specific internal maps which between them constitute a distributed internal representation of the world. These maps collectively provide the transition from topographically-coded sensory information to frequency-coded inputs to the diverse motor schemas that drive muscle activity. We stimulate data on approach and avoidance behavior of the frog or toad under normal conditions and under lesion of different brain centers. For example, the model generates different motor zones for prey-catching behavior which match those observed experimentally in normal conditions and in the medullary hemifield deficit, and offers predictions for new experiments on both approach and avoidance behaviors.

Prenatal development of the intrinsic neurons of the rat neocortex: a comparative study of the distribution of GABA-immunoreactive cells and the GABAA receptor.

The ontogenesis of cells showing GABA-like immunoreactivity, and the distribution of the immunoreactivity for the GABAA receptor were studied immunocytochemically in the prenatal rat brain. By embryonic day 14, a few GABA-like immunoreactive (GABA-positive) cells scattered at the subpial limit of the marginal zone (primordial plexiform layer) in the lateral part of the developing cortex. GABA-positive cells appeared progressively within the dorsal and medial sectors of the primordial plexiform layer, occupying deeper positions within the layer. The immunoreactivity for the GABAA receptor covered the whole thickness of the primordial plexiform layer. By embryonic day 16, most GABA-positive cells populated three distinct laminar compartments of the developing cortex: the prospective lamina I, the subplate, and the lower part of the intermediate zone. The GABA-positive cells of the lower intermediate zone appeared to be typical of the developing cerebral cortex of the rat: their neuronal nature was assessed immunocytochemically, using monoclonal antibodies against microtubule-associated protein 2, mainly expressed in neuronal somata and dendrites, and against intermediate filament protein vimentin, expressed in glia. The lower intermediate zone contained cells immunoreactive for microtubule-associated protein 2, although the immunostaining was less intense than in the prospective lamina I and the subplate. Preliminary results showed no vimentin-positive cells in the lower intermediate zone. At embryonic day 16, immunoreactivity for the GABAA receptor was present within the prospective lamina 1 and the subplate. Preliminary results showed no vimentin-positive cell in the lower intermediate zone. At embryonic day 16, immunoreactivity for the GABAA receptor was present within the prospective lamina 1 and the subplate, but not in the lower intermediate zone. From embryonic day 18 onwards, the immunostaining for the GABAA receptor labelled, unambiguously, the subplate as a lamina clearly separated from the suprajacent cortical plate. At embryonic day 18, the GABAA receptor started to be expressed within the lower, differentiating part of the cortical plate. Within the cortical plate, the expression of GABA in neural cell perikarya, and the immunostaining for the GABAA receptor, followed a similar spatio-temporal ("inside-out") gradient during pre- and early postnatal stages. Most GABA-positive cells of the lower intermediate zone started to disappear (or stopped the expression of GABA) by embryonic day 20, but some remained until adulthood. A similar time-course was observed for the microtubule-associated protein 2-immunoreactive cell population located at the same level.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurogenesis of glutamic acid decarboxylase immunoreactive cells in the hippocampus of the mouse. I: Regio superior and regio inferior.

The neurogenetic gradients of neurons showing glutamic acid decarboxylase (GAD) immunoreactivity were determined in the regio superior and in the regio inferior of the mouse hippocampus. Pregnant C57Bl mice received pulse injections of (3H)thymidine from E11 through E17 (E0 being the day of mating). Distributions of (3H)thymidine-labeled, GAD-positive neurons in the different strata of the hippocampus proper were recorded in adult animals. GAD-positive neurons in this region are generated prenatally. Radial gradients of neurogenesis of GAD-positive cells are characterized by two main features: 1) with the exception of the stratum lacunosum-moleculare and its interface with the stratum radiatum, GAD-positive neurons of the plexiform strata are generated before those destined for the pyramidal layer; 2) within the pyramidal layer, GAD-positive cells are positioned according to an inside-out sequence. In the transverse axis, neurogenesis of GAD-positive cells follows a regio inferior to regio superior gradient. This gradient is due to prolonged neurogenesis of GAD-positive cells for the pyramidal layer in the regio superior. Given the selective laminar disposition of the GABAergic interneurons in the hippocampus, the present authors explored whether or not the diverse types of these interneurons could have specific birth dates and concluded that no relationship exists between birth dates and adult phenotypes of GAD-immunoreactive cells in the mouse hippocampus proper.

Neurogenesis of glutamic acid decarboxylase immunoreactive cells in the hippocampus of the mouse. II: Area dentata.

The temporal patterns of neurogenesis of cells showing glutamic acid decarboxylase (GAD) immunoreactivity were determined in the area dentata of the mouse. Pregnant C57Bl mice received pulse injections of (3H)thymidine from E11 through E17 (E0 being the day of mating). The distribution of (3H)thymidine-labeled, GAD-positive neurons in the hilus and in the different strata of the fascia dentata (stratum infragranulosum, stratum granulosum, stratum moleculare) were recorded in adult animals. A radial gradient of neurogenesis of GAD-positive cells in the area dentata was not apparent. In the transverse axis, neurogenesis of GAD-positive cells seemed to follow a faint suprapyramidal to infrapyramidal gradient, which was due to differential timing of neurogenesis of GAD-positive cells destined for the stratum infragranulosum of the suprapyramidal and infrapyramidal blades of the fascia dentata. GABAergic neurons in the fascia dentata comprise a limited number of well-defined cell types. All of the different morphologic types of GAD-positive neurons present in the area dentata were generated prenatally. These diverse forms did not have specific times of neurogenesis. These results support the concept that the adult morphology of GAD-positive cells in the area dentata of the mouse do not bear any relationship to their times of origin.