Delivery of a Chlamydial Adhesin N-PmpC Subunit Vaccine to the Ocular Mucosa Using Particulate Carriers

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), remains the world's leading preventable infectious cause of blindness. Recent attempts to develop effective vaccines rely on modified chlamydial antigen delivery platforms. As the mechanisms engaged in the pathology of the disease are not fully understood, designing a subunit vaccine specific to chlamydial antigens could improve safety for human use. We propose the delivery of chlamydia-specific antigens to the ocular mucosa using particulate carriers, bacterial ghosts (BGs). We therefore characterized humoral and cellular immune responses after conjunctival and subcutaneous immunization with a N-terminal portion (amino acid 1-893) of the chlamydial polymorphic membrane protein C (PmpC) of Ct serovar B, expressed in probiotic Escherichia coli Nissle 1917 bacterial ghosts (EcN BGs) in BALB/cmice. Three immunizations were performed at two-week intervals, and the immune responses were evaluated two weeks after the final immunization in mice. In a guinea pig model of ocular infection animals were immunized in the same manner as the mice, and protection against challenge was assessed two weeks after the last immunization. N-PmpC was successfully expressed within BGs and delivery to the ocularmucosa was well tolerated without signs of inflammation. N-PmpC- specific mucosal IgA levels in tears yielded significantly increased levels in the group immunized via the conjunctiva compared with the subcutaneously immunized mice. Immunization with N-PmpC EcN BGs via both immunization routes prompted the establishment of an N-PmpC-specific IFN gamma immune response. Immunization via the conjunctiva resulted in a decrease in intensity of the transitional inflammatory reaction in conjunctiva of challenged guinea pigs compared with subcutaneously and non-immunized animals. The delivery of the chlamydial subunit vaccine to the ocular mucosa using a particulate carrier, such as BGs, induced both humoral and cellular immune responses. Further investigations are needed to improve the immunization scheme and dosage

Estudio comparativo de comunidades bacterianas en monumentos mediante técnicas moleculares y microbiológicas

23 páginas, 3 figuras, 4 tablas, 37 referencias.La aplicación de métodos moleculares, especialmente la amplificación del ADN que codifica para el ARN ribosómico 16S, permite el estudio de las comunidades bacterianas no cultivables de los monumentos y su comparación con las cultivables.Esta investigación forma parte de las actividades desarrolladas dentro del Proyecto Europeo "Novel molecular tools for the analysis of unknown microbial communities of mural paintings and their implementation into the conservation/restoration practice" (ENV4-CT98-0705). El trabajo de G. Piñar ha sido financiado por una beca de la Comunidad Europea "Marie Curie" (BI04-CT98-5057).Peer reviewe

Culture-independent analyses of bacterial communities on paleolithic paintings and surrounding rock walls in karstic caves. Altamira, Tito Bustillo, La Garma and Llonín

3 páginas, 1 tabla, 1 figura. Trabajo publicado en la revista digital editada por el IRNASE. CSIC Thematic Network on Cultural Heritage. Electronic Newsletter - CSIC - Ministerio de Educación y Ciencia. Special issue: COALITION Workshop "Molecular approaches and standarization on Cultural Heritage".Paleolithic painting microbiology deserves attention, since it has been reported that microorganisms can affect painting pigments. Caves represent nutrient-poor ecosystems subjected to stable and low temperatures and although providing extreme environments for life, they are inhabited by a variety of microorganisms. However, few studies on cultivated bacteria in caves have been conducted so far. While these cultivation-based studies most probably revealed just a very minor and not representative part of cave population, culture-independent identification methods allowed the detection of unexpected and unknown bacteria and gave insight into a greater bacterial taxonomic diversity in caves (Engel et al. 2001; Holmes et al. 2001; Northup et al. 2000; Vlasceanu et al. 2000). Recently, the combination of PCR amplification of bacterial 16S rRNA genes (16S rDNA), phylogenetic sequence analyses, and genetic community fingerprinting by denaturing gradient gel electrophoresis (DGGE) was shown to be a proper method to study the bacterial communities on the valuable Paleolithic paintings and on the rock surfaces near the paintings in four Spanish caves of Altamira, La Garma, Llonín, and Tito Bustillo (Schabereiter-Gurtner et al. 2001, 2002a, b, c).This research was supported by the European Comission, contract ENV4-CT95- 0104 and the Austrian-Spanish Cooperation Agreement HU 1997-0035. The facilities provided by the archaeologists responsible of the caves sampled are acknowledged.Peer reviewe

The colonisation of building materials by microorganisms as revealed by culturing and molecular methods

6 pages, 4 figures, 1 table, 21 references.Microbial commumties colonising monuments can be analysed by culture-dependent and -independent techniques. However, the results obtained from these studies varied considerably. This apparent disparity was investigated using an experimental system composed of common building materials inoculated wíth a consortium of 14 bacteria. The bacterial population present after 6 months incubation was determined using 16S rDNA amplification and DGGE, construction of 16S rDNA clone libraries and sequence analyses, and by culture on appropriate growth media. Culture-independent techniques revealed the presence of a diversity of microorganisms including halophilic bacteria and Archaea, while culture-dependent techniques showed the presence of spore-forming bacteria. These differences are explained in terms of population dynamic and influence of the environmental conditions during incubation.Laiz and Piñar's works were supported by CSIC-13P programme, social European Fund, and European Community Marie Curie Grant (contract BIO4-CT98-5057)respectively. This work was carried out in the framework of the EC projects: CATS (EVK4-CT2000-00028) and COALITION (EVK4-CT-999-20001).Peer reviewe

Monitoring the colonization of monuments by bacteria: cultivation versus molecular methods

Building materials commonly used in wall paintings and monuments (mortar, limestone and sandstone) were inoculated with an artificial consortium composed of 14 microorganisms and incubated for 6 months at 28°C. The colonization of the different materials by the consortium was investigated. Culture-independent techniques revealed the presence of a diversity of bacteria, whereas culture-dependent techniques yielded mainly spore-forming bacteria. The data suggest that plating leads to an overestimation of the number of spore-forming bacteria with respect to quiescent vegetative forms; the latter are less easily cultured, but are readily detected by culture-independent techniques.L. Laiz’s and G. Piñar’s work was supported by CSIC-I3P programme, Social European Fund, and European Community Marie Curie Grant (contract BIO4-CT98-5057) respectively. This work was carried out in the framework of the EC projects: CATS (EVK4-CT2000-00028) and COALITION (EVK4-CT-1999-20001).Peer Reviewe

Fed-Batch Production of Bacterial Ghosts Using Dielectric Spectroscopy for Dynamic Process Control

The Bacterial Ghost (BG) platform technology evolved from a microbiological expression system incorporating the ϕX174 lysis gene E. E-lysis generates empty but structurally intact cell envelopes (BGs) from Gram-negative bacteria which have been suggested as candidate vaccines, immunotherapeutic agents or drug delivery vehicles. E-lysis is a highly dynamic and complex biological process that puts exceptional demands towards process understanding and control. The development of a both economic and robust fed-batch production process for BGs required a toolset capable of dealing with rapidly changing concentrations of viable biomass during the E-lysis phase. This challenge was addressed using a transfer function combining dielectric spectroscopy and soft-sensor based biomass estimation for monitoring the rapid decline of viable biomass during the E-lysis phase. The transfer function was implemented to a feed-controller, which followed the permittivity signal closely and was capable of maintaining a constant specific substrate uptake rate during lysis phase. With the described toolset, we were able to increase the yield of BG production processes by a factor of 8–10 when compared to currently used batch procedures reaching lysis efficiencies >98%. This provides elevated potentials for commercial application of the Bacterial Ghost platform technology

Acidobacteria in Paleolithic painting caves

The Acidobacterium division represents a practically unknown but apparently widely distributed group within the Bacteria. Recently, the use of molecular techniques has opened the possibility for their detection in a large variety of natural ecosystems.This research was supported by the European Commission (ENV4-CT-95-0104 and ENK4-CT-1999-20001-COALITION).Peer reviewe