Antibacterial activity of coffee extracts and selected coffee chemical compounds against enterobacteria

The in vitro antimicrobial activity of commercial coffee extracts and chemical compounds was investigated on nine strains of enterobacteria. The antimicrobial activity investigated by the disc diffusion method was observed in both the extracts and tested chemical compounds. Even though pH, color, and the contents of trigonelline, caffeine, and chlorogenic acids differed significantly among the coffee extracts, no significant differences were observed in their antimicrobial activity. Caffeic acid and trigonelline showed similar inhibitory effect against the growth of the microorganisms. Caffeine, chlorogenic acid, and protocatechuic acid showed particularly strong effect against Serratia marcescens and Enterobacter cloacae. The IC(50) and IC(90) for the compounds determined by the microtiter plate method indicated that trigonelline, caffeine, and protocatechuic acids are potential natural antimicrobial agents against Salmonella enterica. The concentrations of caffeine found in coffee extracts are enough to warrant 50% of the antimicrobial effect against S. enterica, which is relevant to human safety.     

Genetic Relationships and Variation among Biotypes of Dallisgrass (Paspalum dilatatum Poir.) and Related Species Using Random Amplified Polymorphic DNA Markers

The genus Paspalum L. consists of more than 400 species. Around twenty-five informal groups of species are recognized in Paspalum and the Dilatata group is of special interest because its members are excellent potential forage grasses. Seventy-five germplasm accessions, representing 15 taxa, were analyzed using randomly amplified polymorphic DNA (RAPD). Polymorphisms were observed with twenty-two primers in the Dilatata group and 16 of those were analyzed. Four hundred and four different RAPD fragments were generated, resulting in an average of 25.2 bands per primer. Among the 404 markers analyzed, 48 (11.88%) were exclusive for the P. dilatatum Poir. biotypes, 31 (7.67%) were exclusive to taxa belonging to other groups included in this study, 28 markers (6.93%) were diagnosed for other species of the Dilatata group and 16 (3.96%), for natural hybrids. Extensive RAPD variation was found among the species studied. Inter- and intra-taxonomic polymorphisms were detected. A dendrogram based on the RAPD data shows some clusters corresponding to the same taxa. However, the biotypes of P. dilatatum do not form a cluster. The present work confirms that the RAPD technique can be used to determine genetic relationships between the taxa belonging to the Dilatata group.     

Vitamin E composition of walnuts (Juglans regia L.): a 3-year comparative study of different cultivars

The tocopherol and tocotrienol composition of walnuts (Juglans regia L.) was determined for nine cultivars (cvs. Arco, Franquette, Hartley, Lara, Marbot, Mayette, Mellanaise, Parisienne, and Rego). Walnuts were harvested over three consecutive years from two different geographical origins (Bragan?a and Coimbra, Portugal), for a total of 26 samples. The methodology employed was a normal-phase high-performance liquid chromatography coupled to a series arrangement of a diode array detector followed by a fluorescence detector, allowing the simultaneous analysis of all tocopherols and tocotrienols. The analyses showed that all samples presented a similar qualitative profile composed of five compounds: alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, and gamma-tocotrienol. gamma-Tocopherol was the major compound in all samples, ranging from 172.6 to 262.0 mg/kg, followed by alpha- and delta-tocopherols, ranging from 8.7 to 16.6 mg/kg and from 8.2 to 16.9 mg/kg, respectively. Multivariate analysis of the data obtained showed the existence of significant differences in composition among cultivars. These differences were also significant when cultivars were grouped by year of production, showing that besides genetic factors, the vitamin E composition was influenced by environmental factors.     

Persistence and molecular evolution of Mycobacterium bovis population from cattle and wildlife in Doñana National Park revealed by genotype variation

The role of wildlife in tuberculosis epidemiology is being widely studied since it can affect the effectiveness of eradication campaigns in cattle. The health problem is enhanced when it concerns also wildlife welfare and biodiversity conservation. This study was performed to understand the epidemiology of Mycobacterium bovis population affecting livestock and wild animals in the Do?ana National Park using bacteriology and molecular characterisation techniques. Tuberculosis research was performed on 1209 cattle and wild animals (artiodactyla and carnivore) collected over 6 years in the Park. One hundred and sixty-three animals were found to be infected with M. bovis, comprising 7.96% of the cattle and 20.53% of the wild animals tested. Spoligotyping revealed nine patterns, being SB1232 and SB1230 the most prevalent (77.30% and 15.34% of infected animals, respectively). MIRU-VNTR analysis of a selected panel of 92 isolates showed eight different profiles, including several spoligotypes within the same MIRU-VNTR profile. The discriminatory capacity of both techniques in this panel was similar. The results obtained by combination of both techniques corroborate that wildlife species are infected with the M. bovis strains which are more prevalent in cattle and reveal their persistence. Genotype variation between isolates strongly suggests micro-evolutionary events in the M. bovis population in the same area. This study in the Do?ana National Park exposes the risk of introduction of domestic animals into wildlife areas when there is not a warranty of disease freedom, appropriate diagnostic techniques and control measures.     

Antigens from Rhipicephalus sanguineus ticks elicit potent cell-mediated immune responses in resistant but not in susceptible animals

In the present study we compared the immunological reactions between Rhipicephalus sanguineus tick-infested susceptible (dogs and mice) and tick-resistant hosts (guinea pigs), elucidating some of the components of efficient protective responses against ticks. We found that T-cells from guinea pigs infested with adult ticks proliferate vigorously in the presence of concanavalin A (ConA), whereas ConA-induced cell proliferation of tick-infested mice and dogs was significantly decreased at 43.1 and 94.0%, respectively, compared to non-infested controls. Moreover, cells from mice and dogs submitted to one or three successive infestations did not exhibit a T-cell proliferative response to tick antigens, whilst cells from thrice tick-infested guinea pigs, when cultured with either a tick extract or tick saliva, displayed a significant increase in cell proliferation. Also, we evaluated the response of tick-infested mice to a cutaneous hypersensitivity test induced by a tick extract. Tick-infested mice developed a significant immediate reaction, whereby a 29.9% increase in the footpad thickness was observed. No delayed-type hypersensitivity (DTH) reaction was detected. Finally, the differential cell count at the tick attachment site in repeatedly infested mice exhibited a 6.6- and 4.1-fold increase in the percentage of eosinophils and neutrophils, respectively, compared to non-infested animals, while a decrease of 77.0-40.9 in the percentage of mononuclear cells was observed. The results of the cutaneous hypersensitivity test and the cellular counts at the tick feeding site for mice support the view that tick-infested mice develop an immune response to R. sanguineus ticks very similar to dogs, the natural host of this species of tick, but very different from guinea pigs (resistant host), which develop a DTH reaction in addition to a basophil and mononuclear cell infiltration at the tick-attachment site. In conclusion, saliva introduced during tick infestations reduces the ability of a susceptible animal host to respond to tick antigens that could stimulate a protective immune response. As a consequence, the animals present a lack of DTH response and disturbed cellular migration to tick feeding site, which can represent a deficient response against ticks.     

Routes of transmission and consequences of small ruminant lentiviruses (SRLVs) infection and eradication schemes

Small ruminant lentiviruses (SRLV = maedi-visna in sheep and caprine arthritis encephalitis in goats) are distributed throughout most countries of the world, particularly Europe. Laboratories from 16 European countries established collaborations within the framework of a COST (CO-operation in the field of Scientific and Technical Research) action sponsored by the European Union in order to (i) better organize their research programmes on SRLVs and (ii) to coordinate efforts to combat these two diseases. After five years, a consensus conference--the first one in the veterinary medicine field--concluded the work of this network of laboratories by reviewing the present position and discussing three important questions in the field of SRLVs: routes of transmission, consequences of infection and potential role of eradication programmes at either a European or local level, according to the situation in each country or region. This paper brings together existing information regarding these questions and identifies areas for future research.     

Genetic variability of rhizobacteria from wild populations of four Lupinus species based on PCR-RAPDs§

The metabolic capacities of rhizosphere bacteria can depend on intraspecific genetic variability at strain level. We sampled bacteria from the rhizosphere of three populations of four different Lupinus species at two growth stages (flowering: GS1 and fruiting: GS2). Isolates were identified to the genus level by classical biochemical tests. The most abundant genera found were Bacillus, Aureobacterium, Cellulomonas, Pseudomonas, and Arthrobacter. Genetic divergence of rhizobacteria was tested by PCR-RAPDs. The genetic distances were low, with mean values of 37 % for Bacillus, 25 % for Aureobacterium, 46 % for Cellulomonas, 16 % for Pseudomonas, and 23 % for Arthrobacter. Aureobacterium, the most abundant genus, predominated in the rhizosphere of all populations and at both growth stages (GS1 and GS2) of L. angustifolius. The Aureobacterium strains consisted of 11 groups with 90 % similarity indexes. The cluster analysis of these groups shows that strains isolated from different lupin species and sampling times have extraordinary low diversity indexes, or are even identical. This fact, together with the low genetic distance detected in the rhizosphere, reveals a clear specificity in the plant-bacteria interaction. This specificity could be related with several aspects of plant physiology.     

Deletion of the GI-2 integrase and the wbkA flanking transposase improves the stability of Brucella melitensis Rev 1 vaccine

Brucella melitensis Rev 1 is the best vaccine available for the prophylaxis of small ruminant brucellosis and, indirectly, for reducing human brucellosis. However, Rev 1 shows anomalously high rates of spontaneous dissociation from smooth (S) to rough (R) bacteria, the latter being inefficacious as vaccines. This S-R instability results from the loss of the O-polysaccharide. To overcome this problem, we investigated whether some recently described mechanisms promoting mutations in O-polysaccharide genes were involved in Rev 1 S-R dissociation. We found that a proportion of Rev 1 R mutants result from genome rearrangements affecting the wbo O-polysaccharide loci of genomic island GI-2 and the wbkA O-polysaccharide glycosyltransferase gene of the wbk region. Accordingly, we mutated the GI-2 int gene and the wbk IS transposase involved in those arrangements, and found that these Rev 1 mutants maintained the S phenotype and showed lower dissociation levels. Combining these two mutations resulted in a strain (Rev 2) displaying a 95% decrease in dissociation with respect to parental Rev 1 under conditions promoting dissociation. Rev 2 did not differ from Rev 1 in the characteristics used in Rev 1 typing (growth rate, colonial size, reactivity with O-polysaccharide antibodies, phage, dye and antibiotic susceptibility). Moreover, Rev 2 and Rev 1 showed similar attenuation and afforded similar protection in the mouse model of brucellosis vaccines. We conclude that mutations targeting genes and DNA sequences involved in spontaneous O-polysaccharide loss enhance the stability of a critical vaccine phenotype and complement the empirical stabilization precautions taken during S Brucella vaccine production.     

pH-Dependent Solution Structure and Activity of a Reduced Form of the Host-Defense Peptide Myticin C (Myt C) from the Mussel Mytilus galloprovincialis

Myticin C (Myt C) is a highly variable host-defense peptide (HDP) associated to the immune response in the mediterranean mussel (Mytilus galloprovincialis), which has shown to be active across species due to its strong antiviral activity against a fish rhabdovirus found in fish cells overexpressing this HDP. However, the potential antimicrobial properties of any synthetic analogue of Myt C has not yet been analysed. Thus, in this work we have synthesised the sequence of the mature peptide of Myt C variant c and analysed the structure activity relationships of its reduced (non-oxidized) form (red-MytCc). In contrast to results previously reported for oxidized isoforms of mussel myticins, red-MytCc was not active against bacteria at physiological pH and showed a moderate antiviral activity against the viral haemorrhagic septicaemia (VHS) rhabdovirus. However, its chemotactic properties remained active. Structure/function studies in neutral and acid environments by means of infrared spectroscopy indicated that the structure of red-MytCc is pH dependent, with acid media increasing its alpha-helical content. Furthermore, red-MytCc was able to efficiently aggregate artificial phospholipid membranes at low pH, as well as to inhibit the Escherichia coli growth, suggesting that this activity is attributable to its more structured form in an acidic environment. All together, these results highlight the dynamic and environmentally sensitive behavior of red-Myt C in solution, and provide important insights into Myt C structure/activity relationships and the requirements to exert its antimicrobial/immunomodulatory activities. On the other hand, the pH-dependent direct antimicrobial activity of Myt C suggests that this HDP may be a suitable template for the development of antimicrobial agents that would function selectively in specific pH environments, which are sorely needed in this “antibiotic-resistance era”.     

Asymmetric segregation of polarized antigen on B cell division shapes presentation capacity

During the activation of humoral immune responses, B cells acquire antigen for subsequent presentation to cognate T cells. Here we show that after mouse B cells accumulate antigen, it is maintained in a polarized distribution for extended periods in vivo. Using high-throughput imaging flow cytometry, we observed that this polarization is preserved during B cell division, promoting asymmetric antigen segregation among progeny. Antigen inheritance correlates with the ability of progeny to activate T cells: Daughter cells receiving larger antigen stores exhibit a prolonged capacity to present antigen, which renders them more effective in competing for T cell help. The generation of progeny with differential capacities for antigen presentation may have implications for somatic hypermutation and class switching during affinity maturation and as B cells commit to effector cell fates.