The outer membranes of Brucella spp. are not barriers to hydrophobic permeants

The patterns of susceptibility to hydrophobic and hydrophilic drugs and the uptake of the fluorescent probe N-phenyl-naphthylamine in Brucella spp., Haemophilus influenzae, Escherichia coli, and deep rough Salmonella minnesota mutants were compared. The results show that the outer membranes of smooth and naturally rough Brucella spp. do not represent barriers to hydrophobic permeants and that this absence of a barrier relates at least in part to the properties of Brucella lipopolysaccharide

The outer membranes of Brucella spp. are resistant to bactericidal cationic peptides

The actions of polymyxin B, rabbit polymorphonuclear lysosome extracts, 14 polycationic peptides (including defensin NP-2, cecropin P1, lactoferricin B, and active peptides from cationic protein 18 and bactenecin), EDTA, and Tris on Brucella spp. were studied, with other gram-negative bacteria as controls. Brucella spp. were comparatively resistant to all of the agents listed above and bound less polymyxin B, and their outer membranes (OMs) were neither morphologically altered nor permeabilized to lysozyme by polymyxin B concentrations, although both effects were observed for controls. EDTA and peptides increased or accelerated the partition of the hydrophobic probe N-phenyl-naphthylamine into Escherichia coli and Haemophilus influenzae OMs but had no effect on Brucella OMs. Since Brucella and H. influenzae OMs are permeable to hydrophobic compounds (G. Martínez de Tejada and I. Moriyón, J. Bacteriol. 175:5273-5275, 1993), the results show that such unusual permeability is not necessarily related to resistance to polycations. Although rough (R) B. abortus and B. ovis were more resistant than the controls were, there were qualitative and quantitative differences with smooth (S) brucellae; this may explain known host range and virulence differences. Brucella S-lipopolysaccharides (LPSs) had reduced affinities for polycations, and insertion of Brucella and Salmonella montevideo S-LPSs into the OM of a Brucella R-LPS mutant increased and decreased, respectively, its resistance to cationic peptides. The results show that the core lipid A of Brucella LPS plays a major role in polycation resistance and that O-chain density also contributes significantly. It is proposed that the features described above contribute to Brucella resistance to the oxygen-independent systems of phagocytes

A permeability-increasing drug synergizes with bacterial efux pump inhibitors and restores susceptibility to antibiotics in multi-drug resistant Pseudomonas aeruginosa strains

Resistance to antibiotics poses a major global threat according to the World Health Organization. Restoring the activity of existing drugs is an attractive alternative to address this challenge. One of the most efficient mechanisms of bacterial resistance involves the expression of efflux pump systems capable of expelling antibiotics from the cell. Although there are efflux pump inhibitors (EPIs) available, these molecules are toxic for humans. We hypothesized that permeability-increasing antimicrobial peptides (AMPs) could lower the amount of EPI necessary to sensitize bacteria to antibiotics that are efflux substrates. To test this hypothesis, we measured the ability of polymyxin B nonapeptide (PMBN), to synergize with antibiotics in the presence of EPIs. Assays were performed using planktonic and biofilm-forming cells of Pseudomonas aeruginosa strains overexpressing the MexAB-OprM efflux system. Synergy between PMBN and EPIs boosted azithromycin activity by a factor of 2,133 and sensitized P. aeruginosa to all tested antibiotics. This reduced several orders of magnitude the amount of inhibitor needed for antibiotic sensitization. The selected antibiotic-EPI-PMBN combination caused a 10 million-fold reduction in the viability of biofilm forming cells. We proved that AMPs can synergize with EPIs and that this phenomenon can be exploited to sensitize bacteria to antibiotics

Evaluation of the role of the Bvg intermediate phase in Bordetella pertussis during experimental respiratory infection

The BvgAS system of Bordetella pertussis was traditionally considered to mediate a transition between two phenotypic phases (Bvg(+) and Bvg(-)) in response to environmental signals. We characterized a third state, the intermediate (Bvg(i)) phase, which can be induced by introducing a 1-bp substitution into bvgS (the bvgS-I1 mutation) or by growing B. pertussis under conditions intermediate between those leading to the Bvg(+) and Bvg(-) phases. Like B. bronchiseptica, B. pertussis displays in its Bvg(i) phase a characteristic colony morphology and hemolytic activity and expresses a Bvg(i)-phase-specific polypeptide called BipA, whose synthesis is regulated by bvgAS at the transcriptional level. Based on our results, we hypothesize that the Bvg(i) phase of B. pertussis may be involved in facilitating transmission between hosts. Thus, a B. pertussis mutant carrying the bvgS-I1 mutation (GMT1i) persisted at wild-type levels only in the upper murine respiratory tract. Interestingly, a bipA deletion derivative of GMT1i displayed a reduced ability to colonize the nasal cavity of mice compared with GMT1i. However, in experimental mixed infections GMT1i expressing the Bvg(i) phase could establish an initial colonization in the nose and trachea of mice as efficiently as GMT1, but the wild-type strain outcompeted GMT1i at a later time point at all sites of the respiratory tract, suggesting that the Bvg(i) phase does not serve as a phenotypic phase specialized in colonization. Finally, even though B. pertussis expresses in vitro the Bvg(i) phase at the human nasal temperature, anti-BipA antibodies were undetectable in a large collection of sera from pertussis patients

Cavitary pneumonia in an AIDS patient caused by an unusual Bordetella bronchiseptica variant producing reduced amounts of pertactin and other major antigens

Although Bordetella bronchiseptica can infect and colonize immunocompromised humans, its role as a primary pathogen in pneumonia and other respiratory processes affecting those patients remains controversial. A case of cavitary pneumonia caused by B. bronchiseptica in an AIDS patient is presented, and the basis of the seemingly enhanced pathogenic potential of this isolate (designated 814) is investigated. B. bronchiseptica was the only microorganism recovered from sputum, bronchoalveolar lavage fluid, and samples taken through the protected brush catheter. Unlike previous work reporting the involvement of B. bronchiseptica in cases of pneumonia, antibiotic treatment selected on the basis of in vitro antibacterial activity resulted in clearance of the infection and resolution of the pulmonary infiltrate. Although isolate 814 produced reduced amounts of several major antigens including at least one Bvg-activated factor (pertactin), the molecular basis of this deficiency was found to be BvgAS independent since the defect persisted after the bvgAS locus of isolate 814 was replaced with a wild-type bvgAS allele. Despite its prominent phenotype, isolate 814 displayed only a modest yet a significant deficiency in its ability to colonize the respiratory tracts of immunocompetent rats at an early time point. Interestingly, the antibody response elicited by isolate 814 in these animals was almost undetectable. We propose that isolate 814 may be more virulent in immunocompromised patients due, at least in part, to its innate ability to produce low amounts of immunogenic factors which may be required at only normal levels for the interaction of this pathogen with its immunocompetent natural hosts

Lime-based rendering mortars with photocatalytic and hydrophobic agents: assessment of the water repellency and biocide effect

Different rendering mortars were prepared by mixing air lime and air lime-pozzolanic nanosilica with TiO2 and sodium oleate as, respectively, photocatalytic and water repellent agents, added in bulk. The aim of the work was to design and obtain new rendering mortars with improved durability focusing in the reduction of the water absorption of these materials and in their self-cleaning and biocide effect. To achieve a better distribution of the TiO2 particles, which was expected to enhance their efficiency, different dispersing agents were also incorporated to the fresh mixtures. Four diverse polycarboxylate ethers superplasticizers and a poly-naphthalene-sulfonate were tested. Workability and fluidity of the fresh rendering mortars were determined to guarantee the applicability of the final products. Water contact angle was monitored with the aim of assessing the hydrophobicity of the mortars lent by the water repeller. The biocide effect was studied by means of the culture of a strain of Pseudomonas fluorescens. The colonization of the mortars’ surface was analyzed by determining the number of colonies forming units (CFU) after several days subjecting the samples to suitable T and RH conditions. At the same time, the surface of the mortars was irradiated with solar light to activate the photocatalyst. Results showed the efficiency of the sodium oleate in reducing the water uptake of the rendering mortars. Good compatibility between the water repellent agent, the pozzolanic additive and some of the polycarboxylate superplasticizers was observed. The presence of the photocatalyst was found to be very effective in preventing microbiological colonization

Structural features governing the activity of lactoferricin-derived peptides that act in synergy with antibiotics against Pseudomonas aeruginosa in vitro and in vivo

Pseudomonas aeruginosa is naturally resistant to many antibiotics, and infections caused by this organism are a serious threat, especially to hospitalized patients. The intrinsic low permeability of P. aeruginosa to antibiotics results from the coordinated action of several mechanisms, such as the presence of restrictive porins and the expression of multidrug efflux pump systems. Our goal was to develop antimicrobial peptides with an improved bacterial membrane-permeabilizing ability, so that they enhance the antibacterial activity of antibiotics. We carried out a structure activity relationship analysis to investigate the parameters that govern the permeabilizing activity of short (8- to 12-amino-acid) lactoferricin-derived peptides. We used a new class of constitutional and sequence-dependent descriptors called PEDES (peptide descriptors from sequence) that allowed us to predict (Spearman's ρ = 0.74; P < 0.001) the permeabilizing activity of a new peptide generation. To study if peptide-mediated permeabilization could neutralize antibiotic resistance mechanisms, the most potent peptides were combined with antibiotics, and the antimicrobial activities of the combinations were determined on P. aeruginosa strains whose mechanisms of resistance to those antibiotics had been previously characterized. A subinhibitory concentration of compound P2-15 or P2-27 sensitized P. aeruginosa to most classes of antibiotics tested and counteracted several mechanisms of antibiotic resistance, including loss of the OprD porin and overexpression of several multidrug efflux pump systems. Using a mouse model of lethal infection, we demonstrated that whereas P2-15 and erythromycin were unable to protect mice when administered separately, concomitant administration of the compounds afforded long-lasting protection to one-third of the animals

Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides

Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS

Diseño racional de péptidos derivados de la lactoferricina humana con actividad permeabilizante mejorada y capacidad de producir Permeabilidad Asociada al Efecto Postantibiótico

Para la realización de este estudio, se establecieron los siguientes objetivos: 1. Desarrollar un modelo cuantitativo que relacione la estructura química con la actividad permeabilizante de los péptidos disponibles actualmente y diseñar una nueva generación de péptidos con capacidad permeabilizante mejorada. 2. Determinar la actividad permeabilizante y antimicrobiana de los nuevos péptidos frente a P. aeruginosa y caracterizar su espectro de acción frente a otras especies bacterianas. 3. Caracterizar la capacidad neutralizante de endotoxina de los nuevos péptidos y estudiar posibles asociaciones entre parámetros con interés terapéutico. 4. Determinar la toxicidad y actividad hemolítica de los nuevos péptidos. 5. Estudiar las relaciones estructura-actividad y establecer las bases estructurales que determinan la actividad permeabilizante de péptidos catiónicos sobre P. aeruginosa. 6. Seleccionar los mejores péptidos permeabilizantes y evaluar su capacidad para potenciar antibióticos in vitro y en un modelo animal de sepsis por P. aeruginosa. 7. Determinar si los péptidos pueden producir efecto postantibiótico y si la presencia simultánea de antibióticos en las terapias combinadas puede potenciar el efecto post-antibiótico de los péptidos

Caracterización de los mecanismos de resistencia de aislamientos clínicos de Pseudomonas aeruginosa no sensibles a carbapenems y factores de riesgo asociados a su adquisición

The epidemiology-related features of antibiotic resistant Pseudomonas aeruginosa strains (Pa) isolated in the University Hospital of Navarra from January 2002 to February 2009 were analyzed. An increase of Pa isolates not susceptible to carbapenems (Pa-nsc) was detected and the profile of antibiotic resistance of these organisms was markedly different from that of carbapenem susceptible Pa (Pasc). Both Pa-nsc and the overall Pa isolates increased their resistance to several antibiotic classes, whereas Pa-sc maintained their level of susceptibility. Phenotypic methods most commonly used for detection of metallo-beta-lactamase producing strains (MBL) including E-test MBL, simple microdilution test, double disk synergy test and disks combined test were compared and found to render 100% sensitivity. The combined disk test using imipenem and EDTA provided the greatest specificity. The susceptibility to doripenem in Pa isolates not susceptible to imipenem and meropenem was analyzed. Depending on the cut-off points established by CLSI, EUCAST and FDA, it was found that 28.6% to 11.9% of the strains not susceptible to imipenem were susceptible to doripenem, while 26.2% to 10.7% of strains not susceptible to meropenem were susceptible to doripenem. The mechanisms of resistance to carbapenems in clinical isolates of Pa-nsc were characterized. The altered expression of the porin OprD was the most common resistance mechanism (87.5%), followed by the overproduction of AmpC (33.3%). Four MBL-producing strains of the VIM-2 family were detected including the first MBL-producing strain sensitive to doripenem (MIC 1 ì/ml). The use of urinary catheter and the consumption of broad-spectrum antibiotics (piperacillin-tazobactam, carbapenems, fluoroquinolones and aminoglycosides) were established as risk factors for the acquisition of Pa-nsc. Exposure to hospital environment and the use of mechanical ventilation were identified as risk factors when the groups under comparison were Pa-nsc isolates versus isolates different from Pa. Finally, a prolonged hospital admission, the isolation of multiresistant Pa, the administration of chemotherapy and the use of immunosuppressive drugs were predictors of mortality attributable to Pa-nsc