Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions

Background: Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results: We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (K-D) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a K-D of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions: We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

Sugarcane Growth Promotion by the Endophytic Bacterium Pantoea agglomerans 33.1

The promotion of sugarcane growth by the endophytic Pantoea agglomerans strain 33.1 was studied under gnotobiotic and greenhouse conditions. The green fluorescent protein (GFP)-tagged strain P. agglomerans 33.1: pNKGFP was monitored in vitro in sugarcane plants by microscopy, reisolation, and quantitative PCR (qPCR). Using qPCR and reisolation 4 and 15 days after inoculation, we observed that GFP-tagged strains reached similar density levels both in the rhizosphere and inside the roots and aerial plant tissues. Microscopic analysis was performed at 5, 10, and 18 days after inoculation. Under greenhouse conditions, P. agglomerans 33.1-inoculated sugarcane plants presented more dry mass 30 days after inoculation. Cross-colonization was confirmed by reisolation of the GFP-tagged strain. These data demonstrate that 33.1:pNKGFP is a superior colonizer of sugarcane due to its ability to colonize a number of different plant parts. The growth promotion observed in colonized plants may be related to the ability of P. agglomerans 33.1 to synthesize indoleacetic acid and solubilize phosphate. Additionally, this strain may trigger chitinase and cellulase production by plant roots, suggesting the induction of a plant defense system. However, levels of indigenous bacterial colonization did not vary between inoculated and noninoculated sugarcane plants under greenhouse conditions, suggesting that the presence of P. agglomerans 33.1 has no effect on these communities. In this study, different techniques were used to monitor 33.1:pNKGFP during sugarcane cross-colonization, and our results suggested that this plant growth promoter could be used with other crops. The interaction between sugarcane and P. agglomerans 33.1 has important benefits that promote the plant's growth and fitness.

Lactobacillus sakei 2a and its Concentrated Acid Extract on Inhibition of Food-Borne Salmonella strains

Lactic acid bacteria are used in food production to provide desirable organoleptic characteristics, and can also act as biopreservatives, controlling the growth of undesirable microorganisms. In this study, we examined the antimicrobial action of Lactobacillus sakei 2a and its concentrated acid extract against food-borne Salmonella spp. The extract was obtained by acid extraction from culture broth of L. sakei 2a and was designated extract 2a. We determined that extract 2a had significant activity (approximately 500 AU ml(-1)). We used different antimicrobial substances alone or in combination with extract 2a to evaluate the inhibitory activity of the various treatments on a pool of five Salmonella strains. The pathogen Listeria monocytogenes Scott A Cm-r Em(r) was used as an indicator strain of inhibitory activity. In summary, all antimicrobials substances that were tested showed an inhibitory effect against the growth of Salmonella, andthis action was enhanced in the presence of extract 2a. Moreover, among the treatments applied, the combination of extract 2a and 0.1% lactic acid exhibited the most potent inhibitory effect towards the pool of Salmonella strains. Our findings indicate that L. sakei 2a and extract 2a, especially in combination with other antimicrobials, present potential technological application in the control of salmonellae in foods.

Healthcare-associated infection in hematopoietic stem cell transplantation patients: risk factors and impact on outcome

Objective: The objective of this study was to analyze the incidence of and risk factors for healthcare-associated infections (HAI) among hematopoietic stem cell transplantation (HSCT) patients, and the impact of such infections on mortality during hospitalization. Methods: We conducted a 9-year (2001-2009) retrospective cohort study including patients submitted to HSCT at a reference center in Sao Paulo, Brazil. The incidence of HAI was calculated using days of neutropenia as the denominator. Data were analyzed using EpiInfo 3.5.1. Results: Over the 9-year period there were 429 neutropenic HSCT patients, with a total of 6816 days of neutropenia. Bloodstream infections (BSI) were the most frequent infection, presenting in 80 (18.6%) patients, with an incidence of 11.7 per 1000 days of neutropenia. Most bacteremia was due to Gram-negative bacteria: 43 (53.8%) cases were caused by Gram-negative species, while 33 (41.2%) were caused by Gram-positive species, and four (5%) by fungal species. Independent risk factors associated with HAI were prolonged neutropenia (odds ratio (OR) 1.07, 95% confidence interval (CI) 1.04-1.10) and duration of fever (OR 1.20, 95% CI 1.12-1.30). Risk factors associated with death in multivariate analyses were age (OR 1.02, 95% CI 1.01-1.43), being submitted to an allogeneic transplant (OR 3.08, 95% CI 1.68-5.56), a microbiologically documented infection (OR 2.96, 95% CI 1.87-4.6), invasive aspergillosis disease (OR 2.21, 95% CI 1.1-4.3), and acute leukemias (OR 2.24, 95% CI 1.3-3.6). Conclusions: BSI was the most frequent HAI, and there was a predominance of Gram-negative microorganisms. Independent risk factors associated with HAI were duration of neutropenia and fever, and the risk factors for a poor outcome were older age, type of transplant (allogeneic), the presence of a microbiologically documented infection, invasive aspergillosis, and acute leukemia. Further prospective studies with larger numbers of patients may confirm the role of these risk factors for a poor clinical outcome and death in this transplant population. (C) 2012 Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.

The bactericidal effect of human secreted group IID phospholipase A(2) results from both hydrolytic and non-hydrolytic activities

The Human Secreted Group IID Phospholipase A(2) (hsPLA2GIID) may be involved in the human acute immune response. Here we have demonstrated that the hsPLA2GIID presents bactericidal and Ca2+-independent liposome membrane-damaging activities and we have compared these effects with the catalytic activity of active-site mutants of the protein. All mutants showed reduced hydrolytic activity against DOPC:DOPG liposome membranes, however bactericidal effects against Escherichia coli and Micrococcus luteus were less affected, with the D49K mutant retaining 30% killing of the Gram-negative bacteria at a concentration of 10 mu g/mL despite the absence of catalytic activity. The H48Q mutant maintained Ca2+-independent membrane-damaging activity whereas the G30S and D49K mutants were approximately 50% of the wild-type protein, demonstrating that phospholipid bilayer permeabilization by the hsPLA2GIID is independent of catalytic activity. We suggest that this Ca2+-independent damaging activity may play a role in the bactericidal function of the protein. (C) 2012 Elsevier Masson SAS. All rights reserved.

Mast cells act as phagocytes against the periodontopathogen Aggregatibacter Actinomycetemcomitans

Background: Evidence to date shows that mast cells play a critical role in immune defenses against infectious agents, but there have been no reports about involvement of these cells in eliminating periodontopathogens. In this study, the phagocytic ability of mast cells against Aggregatibacter actinomycetemcomitans compared with macrophages is evaluated. Methods: In vitro phagocytic assays were conducted using murine mast cells and macrophages, incubated with A. actinomycetemcomitans, either opsonized or not, with different bacterial load ratios. After 1 hour, cells were stained with acridine orange and assessed by confocal laser-scanning electronmicroscopy. Results: Phagocytic ability of murine mast cells against A. actinomycetemcomitans was confirmed. In addition, the percentage of mast cells with internalized bacteria was higher in the absence of opsonization than in the presence of opsonization. Both cell types showed significant phagocytic activity against A. actinomycetemcomitans. However, the percentage of mast cells with non-opsonized bacteria was higher than that of macrophages with opsonized bacteria in one of the ratios (1:10). Conclusions: This is the first report about the participation of murine mast cells as phagocytes against A. actinomycetemcomitans, mainly in the absence of opsonization with human serum. Our results may indicate that mast cells act as professional phagocytes in the pathogenesis of biofilmassociated periodontal disease

Proinflammatory profile of in vitro monocytes in the ageing is affected by lymphocytes presence

Background: Aging is associated with complex and constant remodeling of the immune function, resulting in an increasing susceptibility to infection and others diseases. The infections caused by Gram-negative microorganisms, present in nursing homes and hospitals, constitute one of the most common infections in the elderly, and are mainly combated by innate immune cells. Although the functions of innate immunity seem more preserved during aging than of adaptive immune mechanisms, two systems operate in an integrated way in the body, so that injury in one part of the immune system inevitably affects the other as they are part of a defensive network. The aim of this study was to investigate the in vitro production of proinflammatory (TNF-α, IL-6, IL-1α, CXCL-8 and MCP-1) and antiinflammatory (TGF-α and IL-10) cytokines by monocytes, stimulated or not (basal) with lipopolysaccharide, from healthy young and elderly subjects. By means of PBMCs, we also studied if cytokine profile is altered in these different patient groups, in the presence of lymphocytes, under the same experimental conditions. Results: The monocytes from elderly presented higher basal production of TNF-α, MCP-1 and lower of TGF-α than young monocytes. PBMC showed similar cytokines production, irrespective age or stimulation presence. In the presence of lymphocytes, the spontaneous production of IL-10 was higher and of TGF-α was lower than monocytes, regardless of age. After LPS-stimulation, the presence of lymphocytes resulted in increased IL-6, IL-1α, MCP-1 and IL-10 and decreased CXCL-8 and TGF-α in comparison to pure culture of monocytes from young patients. With age, the same differences were observed, except for CXCL-8 and TGF-α which production was the same between monocytes and PBMC stimulated with LPS.

Two RND proteins involved in heavy metal efflux in Caulobacter crescentus belong to separate clusters within proteobacteria

Abstract Background Heavy metal Resistance-Nodulation-Division (HME-RND) efflux systems help Gram-negative bacteria to keep the intracellular homeostasis under high metal concentrations. These proteins constitute the cytoplasmic membrane channel of the tripartite RND transport systems. Caulobacter crescentus NA1000 possess two HME-RND proteins, and the aim of this work was to determine their involvement in the response to cadmium, zinc, cobalt and nickel, and to analyze the phylogenetic distribution and characteristic signatures of orthologs of these two proteins. Results Expression assays of the czrCBA operon showed significant induction in the presence of cadmium and zinc, and moderate induction by cobalt and nickel. The nczCBA operon is highly induced in the presence of nickel and cobalt, moderately induced by zinc and not induced by cadmium. Analysis of the resistance phenotype of mutant strains showed that the ΔczrA strain is highly sensitive to cadmium, zinc and cobalt, but resistant to nickel. The ΔnczA strain and the double mutant strain showed reduced growth in the presence of all metals tested. Phylogenetic analysis of the C. crescentus HME-RND proteins showed that CzrA-like proteins, in contrast to those similar to NczA, are almost exclusively found in the Alphaproteobacteria group, and the characteristic protein signatures of each group were highlighted. Conclusions The czrCBA efflux system is involved mainly in response to cadmium and zinc with a secondary role in response to cobalt. The nczCBA efflux system is involved mainly in response to nickel and cobalt, with a secondary role in response to cadmium and zinc. CzrA belongs to the HME2 subfamily, which is almost exclusively found in the Alphaproteobacteria group, as shown by phylogenetic analysis. NczA belongs to the HME1 subfamily which is more widespread among diverse Proteobacteria groups. Each of these subfamilies present distinctive amino acid signatures.

Antimicrobial effectiveness of silver nanoparticles co-stabilized by the bioactive copolymer pluronic F68

Abstract Background Silver nanoparticles (AgNps) have attracted much interest in biomedical engineering, since they have excellent antimicrobial properties. Therefore, AgNps have often been considered for incorporation into medical products for skin pathologies to reduce the risk of contamination. This study aims at evaluating the antimicrobial effectiveness of AgNps stabilized by pluronic™ F68 associated with other polymers such as polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP). Methods AgNps antimicrobial activity was evaluated using the minimum inhibitory concentration (MIC) method. The action spectrum was evaluated for different polymers associated with pluronic™ F68 against the gram negative bacteria P. aeuroginosa and E. coli and the gram positive bacteria S. Aureus. Results AgNps stabilized with PVP or PVA and co-stabilized with pluronic™ F68 are effective against E. coli and P. aeruginosa microorganisms, with MIC values as low as 0.78% of the concentration of the original AgNps dispersion. The antimicrobial action against S. aureus is poor, with MIC values not lower than 25%. Conclusions AgNps stabilized by different polymeric systems have shown improved antimicrobial activity against gram-negative microorganisms in comparison to unstabilized AgNps. Co-stabilization with the bioactive copolymer pluronic™ F68 has further enhanced the antimicrobial effectiveness against both microorganisms. A poor effectiveness has been found against the gram-positive S. aureus microorganism. Future assays are being delineated targeting possible therapeutic applications.

Molecular and functional characterization of the chemotactic genes in the PCBs-degrader Pseudomonas pseudoalcaligenes KF707

Although bacteria represent the simplest form of life on Earth, they have a great impact on all living beings. For example the degrader bacterium Pseudomonas pseudoalcaligenes KF707 is used in bioremediation procedures for the recovery of polluted sites. Indeed, KF707 strain is know for its ability to degrade biphenyl and polychlorinated biphenyls - to which is chemotactically attracted - and to tolerate the oxydative stress due to toxic metal oxyanions such as tellurite and selenite. Moreover, in bioremediation processes, target compounds can be easily accessible to KF707 through biofilm formation. All these considerations suggest that KF707 is such a unique microorganism and this Thesis work has been focused on determining the molecular nature of some of the peculiar physiological traits of this strain. The genome project provided a large set of informations: putative genes involved in the degradation of aromatic and toxic compounds and associated to stress response were identified. Notably, multiple chemotactic operons and cheA genes were also found. Deleted mutants in the cheA genes were constructed and their role in motility, chemotaxis and biofilm formation were assessed and compared to those previously attributed to a cheA1 gene in a KF707 mutant constructed by a mini-Tn5 transposon insertion and which was impaired in motility and biofilm development. The results of this present Thesis work, taken together, were interpreted to suggest that in Pseudomonas pseudoalcaligenes KF707 strain, multiple factors are involved in these networks and they might play different roles depending on the environmental conditions. The ability of KF707 strain to produce signal molecules possibly involved in cell-to-cell communication, was also investigated: lack of a lux-like QS system - which is conversely widely present in Gram negative bacteria – keeps open the question about the actual molecular nature of KF707 quorum sensing mechanism.

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Eosinophils in the gastrointestinal tract: friends or foes?

Eosinophils play an important role in the mucosal immune system of the gastrointestinal tract under resting and under inflammatory conditions. Under steady-state conditions, the mucosa of the digestive tract is the only organ harboring a substantial number of eosinophils, which, if need be, get activated and exert several effector and immunoregulatory functions. The precise function of these late-phase inflammatory cells is not yet completely understood. Nevertheless, it has recently been demonstrated that lipopolysaccharides from gram-negative bacteria activate eosinophils to rapidly release mitochondrial DNA in the extracellular space. Released mitochondrial DNA and eosinophil granule proteins form extracellular structures able to bind and inactivate bacteria. These findings suggest a novel mechanism of eosinophil-mediated innate immune responses that might be important in maintaining the intestinal barrier function. Moreover, eosinophils also play a crucial role in several inflammatory conditions, such as intestinal infections, immune-mediated inflammations and hypersensitivity reactions. Under chronic inflammatory conditions, the ability of the eosinophils to induce repair can lead to pathological sequelae in the tissue, such as esophageal remodeling in eosinophilic esophagitis. It is established that the uncontrolled eosinophilic inflammation induces fibrosis, esophageal wall thickening and strictures leading to damage that results in a loss of esophageal function. One potential mechanism of this remodeling is so-called 'epithelial mesenchymal transition', which is triggered by eosinophils and is potentially reversible under successful anti-eosinophil treatment. Therefore, eosinophils may act either as friends or as foes, depending on the microenvironment.

Bacterial decontamination of surgical wounds treated with Lavasept

In a prospective randomized controlled double-blind study in 50 acutely injured patients, bacterially contaminated type 2-4 soft tissue wounds were treated with moist dressings of 0.2% Lavasept (fractionated polyhexamethylenbiguanide and macrogolum 4000) solution (n=28) in comparison with Ringer solution (n=22). Standardized swabs were taken on days 0, 2, 8 and 15 and investigated for microorganisms. For a quantitative evaluation, the number of colony forming units (CFU) was determined by a serial dilution technique. The tissue compatibility and anti-inflammatory effect were rated on a scale of 0 (=bad) to 3 (=very good). The most frequently found microorganism was Staphylococcus aureus, which was isolated from 13 wounds. Use of Lavasept led to a faster and significant reduction in microorganisms on the wound surfaces. The number of CFU per wound remained constant or decreased, in contrast to the wounds treated with Ringer solution. This was true for both Gram-positive and Gram-negative bacteria. There was no evidence of impaired wound healing in either group. The anti-inflammatory effect and the tissue compatibility of Lavasept were rated significantly better than that of Ringer solution. It is concluded that Lavasept combines antiseptic action with good tissue compatibility.

A case of necrotizing fasciitis with septic shock in a cat caused by Acinetobacter baumannii

A 4-year-old, neutered female, domestic shorthair cat admitted to the animal hospital for recurrent constipation presumed to be due to post-traumatic injuries, went into shock with signs including fever and ataxia followed by stupor. On the fifth day of hospitalization, the cat developed severe, diffuse oedema of the ventral abdomen with multifocal to coalescing erythematous areas and small vesicle formation. The results of bacteriological cultures of liver, spleen and kidney specimens led to the diagnosis of Acinetobacter baumannii sepsis. Histopathological findings of skin samples taken during necropsy showed an extensive epidermal and dermal necrosis with septic vasculitis and numerous intralesional gram-negative bacteria. Detection of the bla(OXA-51-like) gene specific for A. baumannii by PCR, performed retrospectively on samples of the deep layers of the skin, confirmed the presence of A. baumannii also in the cutaneous lesions. To our knowledge this is the first report of a necrotizing fasciitis with septic shock in a cat caused by A. baumannii.

Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense

Although eosinophils are considered useful in defense mechanisms against parasites, their exact function in innate immunity remains unclear. The aim of this study is to better understand the role of eosinophils within the gastrointestinal immune system. We show here that lipopolysaccharide from Gram-negative bacteria activates interleukin-5 (IL-5)- or interferon-gamma-primed eosinophils to release mitochondrial DNA in a reactive oxygen species-dependent manner, but independent of eosinophil death. Notably, the process of DNA release occurs rapidly in a catapult-like manner--in less than one second. In the extracellular space, the mitochondrial DNA and the granule proteins form extracellular structures able to bind and kill bacteria both in vitro and under inflammatory conditions in vivo. Moreover, after cecal ligation and puncture, Il5-transgenic but not wild-type mice show intestinal eosinophil infiltration and extracellular DNA deposition in association with protection against microbial sepsis. These data suggest a previously undescribed mechanism of eosinophil-mediated innate immune responses that might be crucial for maintaining the intestinal barrier function after inflammation-associated epithelial cell damage, preventing the host from uncontrolled invasion of bacteria.