LipNanoCar Technology - A Versatile and Scalable Technology for the Production of Lipid Nanoparticles.

The extensive knowledge in the miniemulsion technique used in biocatalysis applications by the authors allowed the development of drug delivery systems that constitutes the LipNanoCar technology core for the production of lipid nanoemulsions and solid lipid nanoparticles. The LipNanoCar technology, together with adequate formulations of different oils, fatty acids, surfactants, and temperature, allows the entrapment of several bioactive and therapeutic compounds in lipid nanoparticles for cosmetic, nutrition, and pharmaceutical applications.The LIpNanoCar technology allowed lipid nanoparticles production with average sizes ranging from 100 to 300 nm and Zeta Potentials between -55 and -20 mV. Concomitantly, high entrapment or encapsulation efficiencies (%EE) were achieved, as illustrated in this work for ß-carotene and vitamins derivatives (>85%) for cosmetic application, and for antibiotics currently used in chemotherapy, like rifampicin (69-85%) and pyrazinamide (14-29%) against Mycobacterium tuberculosis (TB), and ciprofloxacin (>65%) and tobramycin (~100%) in Cystic Fibrosis (CF) respiratory infections therapy. Ciprofloxacin presented, for example, a quick-release from the lipid nanoparticles using a dialysis tubing (96% in the first 7 h), but slower than the free antibiotic (95% in the first 3 h). This result suggests that ciprofloxacin is loaded near the external surface of the lipid nanoparticles.The toxicity and validation of entrapment of antibiotics in lipid nanoparticles for Cystic Fibrosis therapy were assessed using Caenorhabditis elegans as an animal model of bacterial infection. Fluorescence microscopy of an entrapped fluorescent dye (DiOC) confirmed the uptake of the lipid nanoparticles by ingestion, and their efficacy was successfully tested in C. elegans. Burkholderia contaminans IST408 and Burkholderia cenocepacia K56-2 infections were tested as model bacterial pathogens difficult to eradicate in Cystic Fibrosis respiratory diseases.

Broad Spectrum Functional Activity of Structurally Related Monoanionic Au(III) Bis(Dithiolene) Complexes.

The biological properties of sixteen structurally related monoanionic gold (III) bis(dithiolene/ diselenolene) complexes were evaluated. The complexes differ in the nature of the heteroatom connected to the gold atom (AuS for dithiolene, AuSe for diselenolene), the substituent on the nitrogen atom of the thiazoline ring (Me, Et, Pr, iPr and Bu), the nature of the exocyclic atom or group of atoms (O, S, Se, C(CN)2) and the counter-ion (Ph4P+ or Et4N+). The anticancer and antimicrobial activities of all the complexes were investigated, while the anti-HIV activity was evaluated only for selected complexes. Most complexes showed relevant anticancer activities against Cisplatin-sensitive and Cisplatin-resistant ovarian cancer cells A2780 and OVCAR8, respectively. After 48 h of incubation, the IC50 values ranged from 0.1-8 µM (A2780) and 0.8-29 µM (OVCAR8). The complexes with the Ph4P+ ([P]) counter-ion are in general more active than their Et4N+ ([N]) analogues, presenting IC50 values in the same order of magnitude or even lower than Auranofin. Studies in the zebrafish embryo model further showed that, despite their marked anticancer effect, the complexes with [P] counter-ion exhibited low in vivo toxicity. In general, the exocyclic exchange of sulfur by oxygen or ylidenemalononitrile (C(CN)2) enhanced the compounds toxicity. Most complexes containing the [P] counter ion exhibited exceptional antiplasmodial activity against the Plasmodium berghei parasite liver stages, with submicromolar IC50 values ranging from 400-700 nM. In contrast, antibacterial/fungi activities were highest for most complexes with the [N] counter-ion. Auranofin and two selected complexes [P][AuSBu(=S)] and [P][AuSEt(=S)] did not present anti-HIV activity in TZM-bl cells. Mechanistic studies for selected complexes support the idea that thioredoxin reductase, but not DNA, is a possible target for some of these complexes. The complexes [P] [AuSBu(=S)], [P] [AuSEt(=S)], [P] [AuSEt(=Se)] and [P] [AuSeiPr(=S)] displayed a strong quenching of the fluorescence intensity of human serum albumin (HSA), which indicates a strong interaction with this protein. Overall, the results highlight the promising biological activities of these complexes, warranting their further evaluation as future drug candidates with clinical applicability.

Differential effects of Th17 cytokines during the response of neutrophils to Burkholderia cenocepacia outer membrane protein A.

T helper 17 cells are involved in the immunopathology of cystic fibrosis. They play a key role in recruitment of neutrophils, which is the first line of defence against bacteria. Additionally, Burkholderia cenocepacia outer membrane protein A (OmpA) BCAL2958 is considered a potential protective epitope for vaccine development. The present study aimed to investigate the neutrophil response to OmpA in the presence of Th17 cytokines, IL-17 and IL-22 at different times of activation. Neutrophils were isolated from whole blood of healthy volunteers and activated with OmpA in the presence of IL-17, IL-22 or both cytokines together. Supernatant was collected after 1 h, 2 h, 4 h, 8 h, and 12 h. Neutrophil activation was assessed by measuring MPO, TNF-α, elastase, hydrogen peroxide, catalase and NO. The results revealed that the combination of IL-17 and IL-22 cytokines induced the release of NE, catalase, H2O2 and TNF-α from neutrophils activated with Burkholderia OmpA at late stages of activation. However, IL-22 alone or IL-17 alone decreased the myeloperoxidase (MPO), catalase and NE levels at early stages of neutrophil activation. The presence of IL-17 alone led to a significant increase in TNF-α level after 1 h and 12 h. However, the presence of IL-22 alone led to a significant increase in TNF-α level after only 1 h but a significant decrease after 8 h of activation was observed as compared to OmpA stimulated neutrophils. In conclusion, Th17 cytokines IL-17 and IL-22, have differential effects during the neutrophil response to Burkholderia OmpA.

Burkholderia puraquae sp. nov., a novel species of the Burkholderia cepacia complex isolated from hospital settings and agricultural soils.

Bacteria from the Burkholderia cepacia complex (Bcc) are capable of causing severe infections in patients with cystic fibrosis (CF). These opportunistic pathogens are also widely distributed in natural and man-made environments. After a 12-year epidemiological surveillance involving Bcc bacteria from respiratory secretions of Argentinean patients with CF and from hospital settings, we found six isolates of the Bcc with a concatenated species-specific allele sequence that differed by more than 3 % from those of the Bcc with validly published names. According to the multilocus sequence analysis (MLSA), these isolates clustered with the agricultural soil strain, Burkholderia sp. PBP 78, which was already deposited in the PubMLST database. The isolates were examined using a polyphasic approach, which included 16S rRNA, recA, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), DNA base composition, average nucleotide identities (ANIs), fatty acid profiles, and biochemical characterizations. The results of the present study demonstrate that the seven isolates represent a single novel species within the Bcc, for which the name Burkholderia puraquae sp. nov. is proposed. Burkholderia puraquae sp. nov. CAMPA 1040T (=LMG 29660T=DSM 103137T) was designated the type strain of the novel species, which can be differentiated from other species of the Bcc mainly from recA gene sequence analysis, MLSA, ANIb, MALDI-TOF MS analysis, and some biochemical tests, including the ability to grow at 42 °C, aesculin hydrolysis, and lysine decarboxylase and ß-galactosidase activities.

A Polyclonal Antibody against a Burkholderia cenocepacia OmpA-like Protein Strongly Impairs Pseudomonas aeruginosa and B. multivorans Virulence.

Despite advances in therapies, bacterial chronic respiratory infections persist as life-threatening to patients suffering from cystic fibrosis (CF). Pseudomonas aeruginosa and bacteria of the Burkholderia cepacia complex are among the most difficult of these infections to treat, due to factors like their resistance to multiple antibiotics and ability to form biofilms. The lack of effective antimicrobial strategies prompted our search for alternative immunotherapies that can effectively control and reduce those infections among CF patients. Previous work from our group showed that the anti-BCAL2645 goat polyclonal antibody strongly inhibited Burkholderia cenocepacia to adhere and invade cultured epithelial cells. In this work, we showed that the polyclonal antibody anti-BCAL2645 also strongly inhibited the ability of P. aeruginosa to form biofilms, and to adhere and invade the human bronchial epithelial cell line CFBE41o-. The polyclonal antibody also inhibited, to a lesser extent, the ability of B. multivorans to adhere and invade the human bronchial epithelial cell line CFBE41o. We also show that the ability of B. cenocepacia, P. aeruginosa and B. multivorans to kill larvae of the Galleria mellonella model of infection was impaired when bacteria were incubated with the anti-BCAL2645 antibody prior to the infection. Our findings show that an antibody against BCAL2645 possesses a significant potential for the development of new immunotherapies against these three important bacterial species capable of causing devastating and often lethal infections among CF patients.

Surface-Exposed Protein Moieties of Burkholderia cenocepacia J2315 in Microaerophilic and Aerobic Conditions.

Burkholderia cepacia complex infections remain life-threatening to cystic fibrosis patients, and due to the limited eradication efficiency of current treatments, novel antimicrobial therapies are urgently needed. Surface proteins are among the best targets to develop new therapeutic strategies since they are exposed to the host's immune system. A surface-shaving approach was performed using Burkholderia cenocepacia J2315 to quantitatively compare the relative abundance of surface-exposed proteins (SEPs) expressed by the bacterium when grown under aerobic and microaerophilic conditions. After trypsin incubation of live bacteria and identification of resulting peptides by liquid chromatography coupled with mass spectrometry, a total of 461 proteins with ≥2 unique peptides were identified. Bioinformatics analyses revealed a total of 53 proteins predicted as localized at the outer membrane (OM) or extracellularly (E). Additionally, 37 proteins were predicted as moonlight proteins with OM or E secondary localization. B-cell linear epitope bioinformatics analysis of the proteins predicted to be OM and E-localized revealed 71 SEP moieties with predicted immunogenic epitopes. The protegenicity higher scores of proteins BCAM2761, BCAS0104, BCAL0151, and BCAL0849 point out these proteins as the best antigens for vaccine development. Additionally, 10 of the OM proteins also presented a high probability of playing important roles in adhesion to host cells, making them potential targets for passive immunotherapeutic approaches. The immunoreactivity of three of the OM proteins identified was experimentally demonstrated using serum samples from cystic fibrosis patients, validating our strategy for identifying immunoreactive moieties from surface-exposed proteins of potential interest for future immunotherapies development.

Antitumoral and Antimicrobial Activities of Block Copolymer Micelles Containing Gold Bisdithiolate Complexes.

Gold(III) bisdithiolate complexes have been reported as potential antimicrobial and antitumoral agents. The complex [Au(cdc)2]- (cdc=cyanodithioimido carbonate) displayed antimicrobial and outstanding antitumor activity against the ovarian cancer cells A2780 and A2780cisR, which are sensitive and resistant to cisplatin, respectively. However, poor water solubility may hamper its clinical use. Block copolymer micelles (BCMs) may solubilize hydrophobic drugs, improving their bioavailability and circulation time in blood. Aiming to provide water solubility, prolonged availability, and enhanced therapeutic indexes, BCMs loaded with [Au(cdc)2]- were synthesized and characterized. The BCM-[Au(cdc)2] micelles were prepared with a loading efficiency of 64.6% and a loading content of 35.3 mg [Au(cdc)2]-/gBCM. A hydrodynamic diameter of 77.31 ± 27.00 nm and a low polydispersity index of 0.18 indicated that the micelles were homogenous and good candidates for drug delivery. Cytotoxic activity studies against A2780/A2780cisR cells showed that BCM-[Au(cdc)2] maintained relevant cytotoxic activity comparable to the cytotoxicity observed for the same concentration of gold complexes. The Au uptake in A2780 cells, determined by PIXE, was ca. 17% higher for BCMs-[Au(cdc)2] compared to [Au(cdc)2]-. The BCMs-[Au(cdc)2] presented antimicrobial activity against S. aureus Newman and C. glabrata CBS138. These results evidenced the potential of BCM-[Au(cdc)2] for drug delivery and its promising anticancer and antimicrobial activities.

Antibody-Based Immunotherapies as a Tool for Tackling Multidrug-Resistant Bacterial Infections.

The discovery of antimicrobials is an outstanding achievement of mankind that led to the development of modern medicine. However, increasing antimicrobial resistance observed worldwide is rendering commercially available antimicrobials ineffective. This problem results from the bacterial ability to adapt to selective pressure, leading to the development or acquisition of multiple types of resistance mechanisms that can severely affect the efficacy of antimicrobials. The misuse, over-prescription, and poor treatment adherence by patients are factors strongly aggravating this issue, with an epidemic of infections untreatable by first-line therapies occurring over decades. Alternatives are required to tackle this problem, and immunotherapies are emerging as pathogen-specific and nonresistance-generating alternatives to antimicrobials. In this work, four types of antibody formats and their potential for the development of antibody-based immunotherapies against bacteria are discussed. These antibody isotypes include conventional mammalian polyclonal antibodies that are used for the neutralization of toxins; conventional mammalian monoclonal antibodies that currently have 100 IgG mAbs approved for therapeutic use; immunoglobulin Y found in birds and an excellent source of high-quality polyclonal antibodies able to be purified noninvasively from egg yolks; and single domain antibodies (also known as nanobodies), a recently discovered antibody format (found in camelids and nurse sharks) that allows for a low-cost synthesis in microbial systems, access to hidden or hard-to-reach epitopes, and exhibits a high modularity for the development of complex structures.

The second RNA chaperone, Hfq2, is also required for survival under stress and full virulence of Burkholderia cenocepacia J2315.

Burkholderia cenocepacia J2315 is a highly virulent and epidemic clinical isolate of the B. cepacia complex (Bcc), a group of bacteria that have emerged as important pathogens to cystic fibrosis patients. This bacterium, together with all Bcc strains and a few other prokaryotes, is unusual for encoding in its genome two distinct and functional Hfq-like proteins. In this work, we show results indicating that the 188-amino-acid Hfq2 protein is required for the full virulence and stress resistance of B. cenocepacia J2315, despite the presence on its genome of the functional 79-amino-acid Hfq protein encoded by the hfq gene. Similar to other Hfq proteins, Hfq2 is able to bind RNA. However, Hfq2 is unique in its ability to apparently form trimers in vitro. Maximal transcription of hfq was observed in B. cenocepacia J2315 cells in the early exponential phase of growth. In contrast, hfq2 transcription reached maximal levels in cells in the stationary phase, depending on the CepR quorum-sensing regulator. These results suggest that tight regulation of the expression of these two RNA chaperones is required to maximize the fitness and virulence of this bacterium. In addition, the ability of Hfq2 to bind DNA, not observed for Hfq, suggests that Hfq2 might play additional roles besides acting as an RNA chaperone.

Immunization and Immunotherapy Approaches against Pseudomonas aeruginosa and Burkholderia cepacia Complex Infections.

Human infections caused by the opportunist pathogens Burkholderia cepacia complex and Pseudomonas aeruginosa are of particular concern due to their severity, their multiple antibiotic resistance, and the limited eradication efficiency of the current available treatments. New therapeutic options have been pursued, being vaccination strategies to prevent or limit these infections as a rational approach to tackle these infections. In this review, immunization and immunotherapy approaches currently available and under study against these bacterial pathogens is reviewed. Ongoing active and passive immunization clinical trials against P. aeruginosa infections is also reviewed. Novel identified bacterial targets and their possible exploitation for the development of immunization and immunotherapy strategies against P. aeruginosa and B. cepacia complex and infections are also presented and discussed.

Synthesis and Characterization of Camphorimine Au(I) Complexes with a Remarkably High Antibacterial Activity towards B. contaminans and P. aeruginosa.

Fourteen new camphorimine Au(I) complexes were synthesized and characterized by spectroscopic (NMR, FTIR) and elemental analysis. The structural arrangement of three selected examples were computed by Density Functional Theory (DFT) showing that the complexes essentially keep the {AuI-CN} unit. The Minimum Inhibition Concentrations (MIC) were assessed for all complexes showing that they are active towards the Gram-negative strains E. coli ATCC25922, P. aeruginosa 477, and B. contaminans IST408 and the Gram-positive strain S. aureus Newman. The complexes display very high activity towards P. aeruginosa 477 and B. contaminans IST408 with selectivity towards B. contaminans. An inverse correlation between the MIC values and the gold content was found for B. contaminans and P. aeruginosa. However, plots of MIC values and Au content for P. aeruginosa 477 and B. contaminans IST408 follow distinct trends. No clear relationship could be established between the MIC values and the redox potentials of the complexes measured by cyclic voltammetry. The MIC values are essentially independent of the redox potentials either cathodic or anodic. The complexes K3[{Au(CN)2}3(A4L)] (8, Y = m-OHC6H4) and K3[{Au(CN)2}3(B2L)]·3H2O (14, Z = p-C6H4) display the lower MIC values for the two strains. In normal fibroblast cells, the IC50 values for the complexes are ca. one order of magnitude lower than their MIC values, although higher than that of the precursor KAu(CN)2.

Bacterial Nosocomial Infections: Multidrug Resistance as a Trigger for the Development of Novel Antimicrobials.

Nosocomial bacterial infections are associated with high morbidity and mortality, posing a huge burden to healthcare systems worldwide. The ongoing COVID-19 pandemic, with the raised hospitalization of patients and the increased use of antimicrobial agents, boosted the emergence of difficult-to-treat multidrug-resistant (MDR) bacteria in hospital settings. Therefore, current available antibiotic treatments often have limited or no efficacy against nosocomial bacterial infections, and novel therapeutic approaches need to be considered. In this review, we analyze current antibacterial alternatives under investigation, focusing on metal-based complexes, antimicrobial peptides, and antisense antimicrobial therapeutics. The association of new compounds with older, commercially available antibiotics and the repurposing of existing drugs are also revised in this work.

A Polyclonal Antibody Raised against the Burkholderia cenocepacia OmpA-like Protein BCAL2645 Impairs the Bacterium Adhesion and Invasion of Human Epithelial Cells In Vitro.

Respiratory infections by bacteria of the Burkholderia cepacia complex (Bcc) remain a life threat to cystic fibrosis (CF) patients, due to the faster lung function decline and the absence of effective eradication strategies. Immunotherapies are regarded as an attractive alternative to control and reduce the damages caused by these infections. In this work, we report the cloning and functional characterization of the OmpA-like BCAL2645 protein, previously identified and found to be immunoreactive against sera from CF patients with a record of Bcc infections. The BCAL2645 protein is shown to play a role in biofilm formation, adherence to mucins and invasion of human lung epithelial cells. The expression of the BCAL2645 protein was found to be increased in culture medium, mimicking the lungs of CF patients and microaerophilic conditions characteristic of the CF lung. Moreover, a polyclonal antibody raised against BCAL2645 was found to inhibit, by about 75 and 85%, the ability of B. cenocepacia K56-2 to bind and invade in vitro CFBE41o- human bronchial epithelial cells. These results highlight the potential of anti-BCAL2645 antibodies for the development of passive immunization therapies to protect CF patients against Bcc infections.

Key Parameters on the Antibacterial Activity of Silver Camphor Complexes.

Nine new complexes with camphor imine or camphor sulfonimine ligands were synthesized and analytically and spectroscopically characterized, aiming to identify the key parameters that drive the antibacterial activity of the complexes with metal cores and imine substituents with distinct electronic and steric characteristics. The antimicrobial activity of all complexes was evaluated by determining their minimum inhibitory concentrations (MIC) against the Gram-negative Escherichia coli ATCC25922, Pseudomonas aeruginosa 477, and Burkholderia contaminans IST408, and the Gram-positive Staphylococcus aureus Newman. Camphor imine complexes based on the hydroxyl silver center ({Ag(OH)}) typically perform better than those based on the nitrate silver center ({Ag(NO3)}), while ligands prone to establish hydrogen bonding facilitate interactions with the bacterial cell surface structures. A different trend is observed for the silver camphor sulfonimine complexes that are almost non-sensitive to the nature of the metal cores {Ag(OH)} or {Ag(NO3)} and display low sensitivity to the Y substituent. The antibacterial activities of the Ag(I) camphor sulfonimine complexes are higher than those of the camphor imine analogues. All the complexes display higher activity towards Gram-negative strains than towards the Gram-positive strain.

The hfq gene is required for stress resistance and full virulence of Burkholderia cepacia to the nematode Caenorhabditis elegans.

The Burkholderia cepacia complex (Bcc) emerged as problematic opportunistic pathogens to cystic fibrosis (CF) patients. Although several virulence factors have been identified in Bcc, the knowledge of their relative contribution to Bcc pathogenicity remains scarce. In this work, we describe the identification and characterization of a B. cepacia IST408 mutant containing a disruption in the hfq gene. In other bacteria, Hfq is a global regulator of metabolism, acting as an RNA chaperone involved in the riboregulation of target mRNAs by small regulatory non-coding RNAs (sRNAs). The B. cepacia Hfq protein was overproduced as a histidine-tagged derivative, and we show evidence that the protein forms hexamers and binds sRNAs. When provided in trans, the B. cepacia IST408 hfq gene complemented the Escherichia coli hfq mutant strain GS081. Our results also show that the B. cepacia hfq mutant is more susceptible to stress conditions mimicking those faced by Bcc bacteria when infecting the CF host. In addition, the B. cepacia hfq mutant and two hfq mutants derived from B. dolosa and B. ambifaria clinical isolates also exhibited a reduced ability to colonize and kill the nematode Caenorhabditis elegans, used as an infection model. These data, together with the conservation of Hfq orthologues among Bcc, strongly suggest that Hfq plays a major role in the survival of Bcc under stress conditions, contributing to the success of Bcc as CF pathogens.

Distribution of cepacian biosynthesis genes among environmental and clinical Burkholderia strains and role of cepacian exopolysaccharide in resistance to stress conditions.

The genus Burkholderia includes strains pathogenic to animals and plants, bioremediators, or plant growth promoters. Genome sequence analyses of representative Burkholderia cepacia complex (Bcc) and non-Bcc strains for the presence of the bce-I gene cluster, directing the biosynthesis of the exopolysaccharide (EPS) cepacian, further extended this previously described cluster by another 9 genes. The genes in the bce-II cluster were named bceM to bceU and encode products putatively involved in nucleotide sugar precursor biosynthesis and repeat unit assembly, modification, and translocation across the cytoplasmic membrane. Disruption of the B. cepacia IST408 bceQ and bceR genes, encoding a putative repeat unit flippase and a glycosyltransferase, respectively, resulted in the abolishment of cepacian biosynthesis. A mutation in the bceS gene, encoding a putative acyltransferase, did not affect EPS production yield significantly but decreased its acetylation content by approximately 20%. Quantitative real-time reverse transcription-PCR experiments confirmed the induction of genes in the bce-I and bce-II clusters in a Burkholderia multivorans EPS producer clinical isolate in comparison to the level for its isogenic EPS-defective strain. Fourier Transform infrared spectroscopy analysis confirmed that the exopolysaccharide produced by 10 Burkholderia isolates tested was cepacian. The ability of Burkholderia strains to withstand desiccation and metal ion stress was higher when bacteria were incubated in the presence of 2.5 g/liter of cepacian, suggesting that this EPS plays a role in the survival of these bacteria by contributing to their ability to thrive in different environments.

The Burkholderia cenocepacia K56-2 pleiotropic regulator Pbr, is required for stress resistance and virulence.

Burkholderia cenocepacia is one of the most virulent species of the Burkholderia cepacia complex, a group of bacteria that emerged as important pathogens, especially to cystic fibrosis (CF) patients. In this study, we report the identification and characterization of a mutant strain derived form the CF isolate Burkholderia cenocepacia K56-2, carrying a plasposon insertion in a gene, located in a 3516 bp chromosomal region with an atypical G+C content, encoding a 80 amino acid putative regulatory protein named Pbr. Besides its inability to produce phenazines, the B. cenocepacia K56-2 pbr mutant exhibited a pleiotropic phenotype, including impaired survival to oxidative and osmotic stress, aromatic amino acid and prolonged nutrient starvation periods. In addition, the pbr mutant exhibited decreased virulence the nematode Caenorhabditis elegans. Altogether, our results demonstrate the involvement of Pbr on the regulation of phenazine biosynthesis, and an important role for this regulatory protein on several cellular processes related to stress resistance and virulence.

A new methodology combining PCR, cloning, and sequencing of clones discriminated by RFLP for the study of microbial populations: application to an UASB reactor sample.

This work describes a methodology combining DNA extraction, polymerase chain reaction amplification with primers targeting 16S ribosomal RNA genes, cloning, and sequencing of clones previously analyzed by restriction fragment length polymorphism (RFLP), which can be applied to study the microbial diversity in a given habitat. The methodology allows the minimization of the sequencing effort, which is particularly relevant when analyzing large numbers of clones. The methodology does not require particularly skilled personnel and can easily be adaptable to the molecular characterization of virtually any particular microbial population, provided that both adequate primers and suitable restriction enzymes for RFLP analysis of the clone library have been chosen. An example of application is presented, in which a sample taken from a continuously operating upflow anaerobic sludge blanket reactor was analyzed. RFLP analysis of the initial 162 clones with HaeIII allowed the identification of only 28 distinct profiles. As expected, identical RFLP profiles corresponded to identical nucleotide sequences.

Pathogenicity, virulence factors, and strategies to fight against Burkholderia cepacia complex pathogens and related species.

The Burkholderia cepacia complex (Bcc) is a group of 17 closely related species of the beta-proteobacteria subdivision that emerged in the 1980s as important human pathogens, especially to patients suffering from cystic fibrosis. Since then, a remarkable progress has been achieved on the taxonomy and molecular identification of these bacteria. Although some progress have been achieved on the knowledge of the pathogenesis traits and virulence factors used by these bacteria, further work envisaging the identification of potential targets for the scientifically based design of new therapeutic strategies is urgently needed, due to the very difficult eradication of these bacteria with available therapies. An overview of these aspects of Bcc pathogenesis and opportunities for the design of future therapies is presented and discussed in this work.

Characterization of the Burkholderia cenocepacia J2315 Surface-Exposed Immunoproteome.

Infections by the Burkholderia cepacia complex (Bcc) remain seriously life threatening to cystic fibrosis (CF) patients, and no effective eradication is available. A vaccine to protect patients against Bcc infections is a highly attractive therapeutic option, but none is available. A strategy combining the bioinformatics identification of putative surface-exposed proteins with an experimental approach encompassing the "shaving" of surface-exposed proteins with trypsin followed by peptide identification by liquid chromatography and mass spectrometry is here reported. The methodology allowed the bioinformatics identification of 263 potentially surface-exposed proteins, 16 of them also experimentally identified by the "shaving" approach. Of the proteins identified, 143 have a high probability of containing B-cell epitopes that are surface-exposed. The immunogenicity of three of these proteins was demonstrated using serum samples from Bcc-infected CF patients and Western blotting, validating the usefulness of this methodology in identifying potentially immunogenic surface-exposed proteins that might be used for the development of Bcc-protective vaccines.