Nomorfilm papers

Cyanobacterial diversity held in microbial biological resource centers as a biotechnological asset: the case study of the newly established LEGE culture collection

Cyanobacteria are a well-known source of bioproducts which renders culturable strains a valuable resource for biotechnology. We describe here the establishment of a cyanobacterial culture collection (CC) and present the first version of the strainand its online database (http://lege.ciimar.up.pt/). The LEGE CC holds 386 strains, mainly collected in coastal (48%),(11%), and fresh (34%) water bodies, for the most part from Portugal (84%). By following the most recent taxonomic classification, LEGE CC strains were classified into at least 46 genera from six orders (41% belong to the Synechococcales), several of them are unique among the phylogenetic diversity of the cyanobacteria. For all strains, primary data were obtained anddata were surveyed and reviewed, which can be reached through the strain sheets either in the catalog or in the online. An overview on the notable biodiversity of LEGE CC strains is showcased, including a searchable phylogenetic tree and images for all strains. With this work, 80% of the LEGE CC strains have now their 16S rRNA gene sequences deposited in GenBank. Also, based in primary data, it is demonstrated that several LEGE CC strains are a promising source of extracellular polymeric substances (EPS). Through a review of previously published data, it is exposed that LEGE CC strains have the potential or actual capacity to produce a variety of biotechnologically interesting compounds, including common cyanotoxins or unprecedented bioactive molecules. Phylogenetic diversity of LEGE CC strains does not entirely reflect chemodiversity. Further bioprospecting should, therefore, account for strain specificity of the valuable cyanobacterial holdings of LEGE CC.

Paper Cyanobacterial diversity held in microbial biological resource centers as a biotechnological asset: the case study of the newly established LEGE culture collection

The Sea as a Source of Antibiotics

Over the past few decades, there has been a decline in antibiotics development, resulting in fewer new antimicrobial agents approvals. Thus, the era of antibiotics began in the 1920s and 30s with the discovery of penicillin by Alexander Fleming. There are now over 13 classes of antibiotics, some into their fifth generation, consisting on synthetic derivatives from older antibiotics and natural compounds. In the first decades, about 15-20 new antibiotics were developed each decade, but in the last ten years only 6 new drugs have come on the market. In addition to the scarce development of antibiotics, the emergence and increase of multiresistant bacteria to the existent antibiotics used in clinical practice are limiting treatment options for patients.

Paper The Sea as a Source of Antibiotics

Expanding the potential of NAI-107 for treating serious ESKAPE pathogens: synergistic combinations against Gram-negatives and bactericidal activity against non-dividing cells

Objectives: To characterize NAI-107 and related lantibiotics for their in vitro activity against Gram-negative pathogens, alone or in combination with polymyxin, and against non-dividing cells or biofilms of Staphylococcus aureus. NAI-107 was also evaluated for its propensity to select or induce self-resistance in Gram-positive bacteria.

Paper Expanding the potential of NAI-107 for treating serious ESKAPE pathogens: synergistic combinations against Gram-negatives and bactericidal activity against non-dividing cells

Porcine Models of Biofilm Infections with Focus on Pathomorphology

Bacterial biofilm formation is one of the main reasons for a negative treatment outcome and a high recurrence rate for many chronic infections in humans. The optimal way to study both the biofilm forming bacteria and the host response simultaneously is by using discriminative, reliable, and reproducible animal models of the infections. In this review, the advantages of in vivo studies are compared to in vitro studies of biofilm formation in infectious diseases. The pig is the animal of choice when developing and applying large animal models of infectious diseases due to its similarity of anatomy, physiology, and immune system to humans. Furthermore, conventional pigs spontaneously develop many of the same chronic bacterial infections as seen in humans. Therefore, in this review porcine models of five different infectious diseases all associated with biofilm formation and chronicity in humans are described. The infectious diseases are: chronic wounds, endocarditis, pyelonephritis, hematogenous osteomyelitis, and implant-associated osteomyelitis (IAO).

Paper Porcine Models of Biofilm Infections with Focus on Pathomorphology

Molecular Interactions of Human Plasminogen with Fibronectin-binding Protein B (FnBPB), a Fibrinogen/ Fibronectin-binding Protein from Staphylococcus aureus*

Staphylococcus aureus is a commensal bacterium that has the ability to cause superficial and deep-seated infections. Like several other invasive pathogens, S. aureus can capture plasminogen from the human host where it can be converted to plasmin by host plasminogen activators or by endogenously expressed staphylokinase. This study demonstrates that sortase-anchored cell wall-associated proteins are responsible for capturing the bulk of bound plasminogen. Two cell wall-associated proteins, the fibrinogen- and fibronectin-binding proteins A and B, were found to bind plasminogen, and one of them, FnBPB, was studied in detail. Plasminogen captured on the surface of S. aureusor Lactococcus lactis-expressing FnBPB could be activated to the potent serine protease plasmin by staphylokinase and tissue plasminogen activator. Plasminogen bound to recombinant FnBPB with a KD of 0.532 M as determined by surface plasmon resonance. Plasminogen binding did not to occur by the same mechanism through which FnBPB binds to fibrinogen. Indeed, FnBPB could bind both ligands simultaneously indicating that their binding sites do not overlap. The N3 subdomain of FnBPB contains the full plasminogen-binding site, and this includes, at least in part, two conserved patches of surface-located lysine residues that were recognized by kringle 4 of the host protein

Paper Molecular Interactions of Human Plasminogen with Fibronectin-binding Protein B (FnBPB), a Fibrinogen/ Fibronectin-binding Protein from Staphylococcus aureus*

Combined Staining Techniques for Demonstration of Staphylococcus aureus Biofilm in Routine Histopathology

Aim: Visualization of Staphylococcus aureus biofilm using histochemical staining and combined histochemistry (HC) and immunohistochemistry (IHC).

Paper Combined Staining Techniques for Demonstration of Staphylococcus aureus Biofilm in Routine Histopathology

Novel porcine model of implant‐associated osteomyelitis: A comprehensive analysis of local, regional, and systemic response

Pigs are favorable experimental animals for infectious diseases in humans. However, implant‐associated osteomyelitis (IAO) models in pigs have only been evaluated using high‐inoculum infection (>108 CFU) models in 1975 and 1993. Therefore, the aim of this paper was to present a new low inoculum porcine model of human IAO based on 42 experimental pigs. The model was created by drilling an implant cavity in the tibial bone followed by insertion of a small steel implant and simultaneous inoculation of Staphylococcus aureus bacteria (n = 32) or saline (n = 10). The infected pigs were either inoculated with 104 CFU (n = 26) or 102 and 103 CFU (n = 6). All animals were euthanized 5 days after insertion of implants. Pigs receiving the high‐inoculum infections showed a significantly higher volume of bone lesion, number of neutrophils around the implant, concentrations of acute phase proteins in serum, and enlargement of regional lymph nodes. A positive correlation was present between a high number of surrounding neutrophils and high values of all other parameters. Furthermore, a threshold of 40 neutrophils per 10 high power fields for the histopathological diagnosis of high grade IAO was defined. In conclusion: This paper describes a novel low‐inoculum S. aureus porcine model of IAO which was demonstrated to be reliable, reproducible and discriminative to human IAO, and represents a requested and valuable tool in orthopedic research. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2211–2221, 2017.

Paper Novel porcine model of implant‐associated osteomyelitis: A comprehensive analysis of local, regional, and systemic response

BIOPELICULAS Y SU RELACIŁN CON LAS INFECCIONES PROTÉSICAS

La formación de biopelículas es especialmente importante en infecciones relacionadas con implantes y catéteres. Aunque algunos de estos microorganismos que colonizan no causan infección, pueden promover una reacción inmune, que da lugar a la inflamación en el tejido subyacente. Esto, finalmente, provoca una liberación del implante, que debe ser eliminado y reemplazado por uno nuevo. Las bacterias que forman biopelículas son difíciles de erradicar, debido a la resistencia a los antimicrobianos conferida por la matriz y a las células persistentes, lo cual hace necesario buscar nuevas herramientas terapéuticas, como se revisan en el presente trabajo. Página 36.

Paper BIOPELICULAS Y SU RELACIŁN CON LAS INFECCIONES PROTÉSICAS

Comparative study of structure and properties of thermal spray coatings using conventional and nanostructured hydroxyapatite powder, for applications in medical implants

The objective of this paper is to study and compare the structure and properties of thermal spray coatings prepared with conventional and nanostructured hydroxyapatite powder, in order to assess their suitability for future use in medical implants. Four coatings were prepared via Atmospheric Plasma Spray process (APS). Two kinds of feedstock material were employed for the spray process, namely, commercial XPT-D-703 hydroxyapatite powder for half the samples and novel nanostructured PYRO 4 hydroxyapatite powder, which had previously been mechanically treated, for the remaining samples. The substrate of all the samples was stainless steel 304. Finally, the plasma spray parameters were altered for two out of the four coatings, each produced with a different type of powder, in order for a sufficient amount of porosity to be achieved for future incorporation of biomolecules. The coatings were also examined in terms of bioactivity in vitro. It was concluded that the coating produced with the nanostructured powder, under lower plasma energy and at greater spraying distance presented the best results regarding its roughness, bioactive response, crystallinity, adherence and porosity content.

Paper Comparative study of structure and properties of thermal spray coatings using conventional and nanostructured hydroxyapatite powder, for applications in medical implants

Microstructural investigation of porous titanium coatings, produced by thermal spraying techniques, using plasma atomization and hydride-dehydride powders, for orthopedic implants

The present study focuses on porous titanium (commercially pure Ti) coatings for future use on medical implants, manufactured by specific thermal spraymethod. Its objective is to investigate the possibility of Ti porosity to offer room for medication storage in addition to bone growth promotion. Three specimens for each different type of coating were produced by different sets of thermal spray process/feedstock powder, namely, (1) Flame Spray (FS) with spherical Plasma Atomization (PA) powder, (2) Atmospheric Plasma Spray (APS) with spherical PA powder and (3) APS with angular Hydride – Dehydride (HDH) powder. The specific types of specimens along with the two powders produced by the PA and HDH processes were characterized using various techniques, namely (a) Roughness measurements of the coated surfaces and thickness measurements of the coatings, which disclosed the highest values for the FS-PA coating, Ra = 11.19 ± 0.21 μm and average thickness of 281 μm, (b) Optical microscopy and SEM-EDS to study the surface morphology and microstructure of the coatings and discover any possible oxidation or other phases. The porosity percentage was measured and the pore size distribution was also estimated. The porosity percentages of the coatings ranged from 12% on the APS-PA coating to 34% on the FS-PA coating, (c) XRD analyses which showed varying titanium oxide formations for each type of specimen. These oxides may prove to be beneficial for the development of osseous tissue, (d) assessment of biological response of the coatings in vitro that resulted in satisfactory primary response in a physiological environment and the formation of calcium phosphate phases with Ca/P ratio of 1.61 and (e) Vickers microhardnessmeasurements, which resulted in values that fluctuated from 786.6 for the FS-PA coating to 967.6 for the APS-HDH coating. Overall, the produced FS-PA coating is characterized by high porosity content and satisfactory bioactivity, in order to fulfill the objective of the project for biomolecule incorporation and drug storage.

Paper Microstructural investigation of porous titanium coatings, produced by thermal spraying techniques, using plasma atomization and hydride-dehydride powders, for orthopedic implants

In Vivo Gentamicin Susceptibility Test for Prevention of Bacterial Biofilms in Bone Tissue and on Implants

The objective of this study was to set up an in vivo gentamicin susceptibility test for biofilm prevention in bone tissue and on implants. In vivo, the antimicrobial tolerance of the inoculated planktonic bacteria was increased by in vivo-specific factors of acute inflammation. This resulted in bacterial aggregation and biofilm formation, which further increased the gentamicin tolerance. Thus, susceptibility patterns in vitro might not reflect the actual in vivo susceptibility locally within a developing infectious area..

Paper In Vivo Gentamicin Susceptibility Test

JAGN1 is required for fungal killing in neutrophil extracellular traps: Implications for severe congenital neutropenia

Mutations in the gene JAGN1 were recently discovered in patients with severe congenital neutropenia (SCN). Neutrophils release neutrophil extracellular traps (NETs) consisting of decondensed chromatin decorated with various granular proteins such as neutrophil elastase and myeloperoxidase (MPO) to combat microbial infections. However, whether JAGN1 is required for the formation or function of NETs is not known. Here, we analyzed primary neutrophils from a patient with homozygous JAGN1 mutations with respect to phorbol myristate acetate (PMA)-induced NET formation. NET release was observed, but there appeared to be a reduced level of expression of MPO in the NETs. To study this further, we differentiated HL-60 cells into neutrophil-like cells and silenced JAGN1 expression by transfection with siRNA. These cells remained capable of producing NETs, but MPO expression was severely affected, and NETs released by JAGN1-silenced cells were ineffective in killing Candida albicans. The candidacidal function was restored upon treatment with GM-CSF or addition of MPO. GM-CSF also up-regulated the expression of calprotectin in NETs. Notably, JAGN1 did not impact on N-glycosylation of MPO in neutrophil-like HL-60 cells. These studies shed light on the susceptibility of SCN patients to fungal infections and the role of JAGN1 for the antimicrobial function of neutrophils exerted by NETs.

Paper JAGN1 is required for fungal killing

Biofilms in the Food Industry: Health Aspects and Control Methods

Diverse microorganisms are able to grow on food matrixes and along food industry infrastructures. This growth may give rise to biofilms. This review summarizes, on the one hand, the current knowledge regarding the main bacterial species responsible for initial colonization, maturation and dispersal of food industry biofilms, as well as their associated health issues in dairy products, ready-to-eat foods and other food matrixes. These human pathogens include Bacillus cereus (which secretes toxins that can cause diarrhea and vomiting symptoms), Escherichia coli (which may include enterotoxigenic and even enterohemorrhagic strains), Listeria monocytogenes (a ubiquitous species in soil and water that can lead to abortion in pregnant women and other serious complications in children and the elderly), Salmonella enterica (which, when contaminating a food pipeline biofilm, may induce massive outbreaks and even death in children and elderly), and Staphylococcus aureus (known for its numerous enteric toxins). On the other hand, this review describes the currently available biofilm prevention and disruption methods in food factories, including steel surface modifications (such as nanoparticles with different metal oxides, nanocomposites, antimicrobial polymers, hydrogels or liposomes), cell-signaling inhibition strategies (such as lactic and citric acids), chemical treatments (such as ozone, quaternary ammonium compounds, NaOCl and other sanitizers), enzymatic disruption strategies (such as cellulases, proteases, glycosidases and DNAses), non-thermal plasma treatments, the use of bacteriophages (such as P100), bacteriocins (such us nisin), biosurfactants (such as lichenysin or surfactin) and plant essential oils (such as citral- or carvacrol-containing oils).

Paper Biofilms in the Food Industry: Health Aspects and Control Methods

Pigs are useful for the molecular study of bone inflammation and regeneration in humans

Pigs are used with increased frequency to model different kinds of orthopedic surgical conditions. In order to show the full potential of porcine models in orthopedic research, it is therefore required to examine the expression of bone regulatory genes in pigs affected by orthopedic surgery and compare it to the expression in humans and mice as mice, are one of the most applied animal species in orthopedics today. In the present study, the local molecular response to drilling of a tibial implant cavity, and the subsequent insertion of a steel implant was examined in a porcine model. Pigs were euthanized five days after drilling of the bone. The molecular response of 73 different genes was analyzed using a high-throughput quantitative polymerase chain reaction platform and compared to histopathology. Histologically, it was found that bone remodeling was initiated on day 5 after surgery and was associated with upregulation of several genes involved in bone degradation and formation (CTSK, ACP5, IBSP, RANK, RANKL and COL1A1). Interleukin-6 and several acutephase proteins (C3, SAA and ITIH4) were significantly upregulated, indicating their importance in the initial process of healing and osseointegration. All tested bone morphogenic proteins (BMP2, -4 and -7) including their inhibitor noggin were also significantly upregulated. Surprisingly, vascular endothelial growth factor A was not found to be regulated five days after surgery while several other vascular growth factors (ANGPT1, ANGPT2 and PTN) were upregulated. The pig was found to be a useful model for elucidation of bone regulatory genes in humans.

Paper Pigs are useful for the molecular study of bone inflammation and regeneration in humans

Inhibition of Bacterial and Fungal Biofilm Formation by 675 Extracts from Microalgae and Cyanobacteria

Paper Inhibition of Bacterial and Fungal Biofilm Formation by 675 Extracts from Microalgae and Cyanobacteria

NMR characterization and evaluation of antibacterial and antiobiofilm activity of organic extracts from stationary phase batch cultures of five marine microalgae (Dunaliella sp., D. salina, Chaetoceros calcitrans, C. gracilis and Tisochrysis lutea)

Paper NMR characterization and evaluation of antibacterial and antiobiofilm activity of organic extracts from stationary phase batch cultures of five marine microalgae

Impact of manganese on biofilm formation and cell morphology of Candida parapsilosis clinical isolates with different biofilm forming abilities

The commensal species Candida parapsilosis is an emerging human pathogen that has the ability to form biofilms. In this study, we explored the impact of the divalent cations cobalt (Co2+), copper (Cu2+), iron (Fe3+), manganese (Mn2+), nickel (Ni2+) and zinc (Zn2+) on biofilm formation of clinical isolates of C. parapsilosis with no, low and high biofilm forming abilities at 30 and 37◦C. All strains besides one isolate showed a concentration-dependent enhancement of biofilm formation at 30◦C in the presence of Mn2+ with a maximum at 2 mM. The biofilm forming ability of no and low biofilm forming isolates was >2-fold enhanced in the presence of 2 mM Mn2+, while the effect in high biofilm forming isolate was significantly less pronounced. Of note, cells in the biofilms of no and low biofilm forming strains differentiated into yeast and pseudohyphal cells similar in morphology to high biofilm formers. The biofilm transcriptional activator BCR1 has a dual developmental role in the absence and presence of 2 mM Mn2+ as it promoted biofilm formation of no biofilm forming strains, and, surprisingly, suppressed cells of no biofilm forming strains to develop into pseudohyphae and/or hyphae. Thus, environmental conditions can significantly affect the amount of biofilm formation and cell morphology of C. parapsilosis with Mn2+ to overcome developmental blocks to trigger biofilm formation and to partially relieve BCR1 suppressed cell differentiation

Paper Impact of manganese on biofilm formation and cell morphology of Candida parapsilosis clinical isolates with different biofilm forming abilities

Fibronectin-binding protein B (FnBPB) from Staphylococcus aureus protects against the antimicrobial activity of histones

Staphylococcus aureus is a Gram-positive bacterium that can cause both superficial and deep-seated infections. Histones released by neutrophils kill bacteria by binding to the bacterial cell surface and causing membrane damage. We postulated that cell wall–anchored proteins protect S. aureus from the bactericidal effects of histones by binding to and sequestering histones away from the cell envelope. Here, we focused on S. aureus strain LAC and by using an array of FnBPB variants defective in fibrinogen binding also did not bind H3. An FnBPB-deletion mutant of S. aureus LAC bound less H3 and was more susceptible to its bactericidal activity and to neutrophil extracellular traps, whereas an FnBPB-overexpressing mutant bound more H3 and was more resistant than the wild type. FnBPB bound simultaneously to H3 and plasminogen, which after activation by tissue plasminogen activator cleaved the bound histone. We conclude that FnBPB provides a dual immune-evasion function that captures histones and prevents them from reaching the bacterial membrane and simultaneously binds plasminogen, thereby promoting its conversion to plasmin to destroy the bound histone.

Paper Fibronectin-binding protein B (FnBPB) from Staphylococcus aureus protects against the antimicrobial activity of histones

Guideline for Preclinical Studies of Bone Infections in Large Animals Based on a Systematic Review of 316 Non-Rodent Models

In recent years, animal models of bone infections have been used with increased frequency in order to evaluate novel diagnostic and anti-infective technologies, like antibacterial coating of bone implants or local antibiotic carrier products. Therefore, it is highly relevant to evaluate the scientific quality of existing bone infection models.

Paper Guideline for Preclinical Studies of Bone Infections in Large Animals Based on a Systematic Review of 316 Non-Rodent Models

The usefulness of Microal-gae Compounds for pre-venting Biofilm Infections

Paper The usefulness of Microal-gae Compounds for pre-venting Biofilm Infections

Antibacterial Aromatic Polyketides Incorporating the Unusual Amino Acid Enduracididine

The increasing incidence of infections caused by drug-resistant pathogens requires new efforts for the discovery of novel antibiotics. By screening microbial extracts in an assay aimed at identifying compounds interfering with cell wall biosynthesis, based on differential activity against a Staphylococcus aureus strain and its isogenic L-form, the potent enduracyclinones (1, 2), containing the uncommon amino acid enduracididine linked to a six-ring aromatic skeleton, were discovered from different Nonomuraea strains. The structures of 1 and 2 were established through a combination of derivatizations, oxidative cleavages, and NMR analyses of natural and 13C−15N-labeled compounds. Analysis of the biosynthetic cluster provides the combination of genes for the synthesis of enduracididine and type II polyketide synthases. Enduracyclinones are active against Gram-positive pathogens (especially Staphylococcus spp.), including multi-drug-resistant strains, with minimal inhibitory concentrations in the range of 0.0005 to 4 μg mL−1 and with limited toxicity toward eukaryotic cells. The combined results from assays and macromolecular syntheses suggest a possible dual mechanism of action in which both peptidoglycan and DNA syntheses are inhibited by these molecules.

Paper Antibacterial Aromatic Polyketides Incorporating the Unusual Amino Acid Enduracididine

The inflammatory response to bone infection – a review based on animal models and human patients

Bone infections are difficult to diagnose and treat, especially when a prosthetic joint replacement or implant is involved. Bone loss is a major complication of osteomyelitis, but the mechanism behind has mainly been investigated in cell cul- tures and has not been confirmed in human settings. Inflammation is important in initiating an appropriate immune response to invading pathogens. However, many of the signaling molecules used by the immune system can also modu- late bone remodeling and contribute to bone resorption during osteomyelitis. Our current knowledge of the inflamma- tory response relies heavily on animal models as research based on human samples is scarce. Staphylococcus aureus is one of the most common causes of bone infections and is the pathogen of choice in animal models. The regulation of inflammatory genes during prosthetic joint infections and implant-associated osteomyelitis has only been studied in rodent models. It is important to consider the validity of an animal model when results are extrapolated to humans, and both bone composition and the immune system of pigs has been shown to be more similar to humans, than to rodents. Here in vivo studies on the inflammatory response to prosthetic joint infections and implant-associated osteomyelitis are reviewed.

Paper The inflammatory response to bone infection – a review based on animal models and human patients

The host response to bacterial bone infection involves a local upregulation of several acute phase proteins

Bone infections often become chronic and can be difficult to diagnose. In the present study, the osseous gene expression of several acute phase proteins (APPs) during osteomyelitis was investigated in a porcine model of implant associated osteomyelitis (IAO) (sampled 5, 10 and 15 days after infection) and in slaughter pigs with spontaneous hematogenous osteomyelitis, and compared to gene expression in liver tissue. Furthermore, im- munohistochemical (IHC) staining of the APP complement component C3 (C3) was performed on the porcine osteomyelitis lesions together with material from human patients with chronic osteomyelitis. In the porcine bone samples a local upregulation of the expression of several APP genes, including serum amyloid A (SAA) and C3, was observed during infection. In the liver, only C-reactive protein (CRP) and Inter-Alpha-Trypsin Inhibitor Heavy Chain 4 were significantly upregulated. Serum concentrations of CRP, SAA and haptoglobin were only upregulated at day 5 in infected animals of the IAO model. This indicates a limited systemic response to os- teomyelitis. Similar numbers of positive IHC stained C3 leukocytes were found in human and porcine bone samples with chronic osteomyelitis, indicating a high transcriptional value of porcine models of osteomyelitis. The local upregulation of APPs could potentially be used for diagnosing osteomyelitis.

Paper The host response to bacterial bone infection involves a local upregulation of several acute phase proteins