Bioinspired Nanofeatured Substrates: Suitable Environment for Bone Regeneration.

Bone mimicking coatings provide a complex microenvironment in which material, through its inherent properties (such as nanostructure and composition), affects the commitment of stem cells into bone lineage and the production of bone tissue regulating factors required for bone healing and regeneration. Herein, a bioactive mineral/biopolymer composite made of calcium phosphate/chitosan and hyaluronic acid (CaP-CHI-HA) was elaborated using a versatile simultaneous spray coating of interacting species. The resulting CaP-CHI-HA coating was mainly constituted of bioactive, carbonated and crystalline hydroxyapatite with 277 ± 98 nm of roughness, 1 μm of thickness, and 2.3 ± 1 GPa of stiffness. After five days of culture, CaP-CHI-HA suggested a synergistic effect of intrinsic biophysical features and biopolymers on stem cell mechanobiology and nuclear organization, leading to the expression of an early osteoblast-like phenotype and the production of bone tissue regulating factors such as osteoprotegerin and vascular endothelial growth factor. More interestingly, amalgamation with biopolymers conferred to the mineral a bacterial antiadhesive property. These significant data shed light on the potential regenerative application of CaP-CHI-HA bioinspired coating in providing a suitable environment for stem cell bone regeneration and an ideal strategy to prevent implant-associated infections.journal article2017 Apr 122017 03 30importe

Bone Environment Influences Irreversible Adhesion of a Methicillin-Susceptible Staphylococcus aureus Strain

Prosthesis and joint infections are an important threat in public health, especially due to the development of bacterial biofilms and their high resistance to antimicrobials. Biofilm-associated infections increase mortality and morbidity rates as well as hospitalization costs. Prevention is the best strategy for this serious issue, so there is an urgent need to understand the signals that could induce irreversible bacterial adhesion on a prosthesis. In this context, we investigated the influence of the bone environment on surface adhesion by a methicillin-susceptible Staphylococcus aureus strain. Using static and dynamic biofilm models, we tested various bone environment factors and showed that the presence of Mg2+, lack of oxygen, and starvation each increased bacterial adhesion. It was observed that human osteoblast-like cell culture supernatants, which contain secreted components that would be found in the bone environment, increased bacterial adhesion capacity by 2-fold (p = 0.015) compared to the medium control. Moreover, supernatants from osteoblast-like cells stimulated with TNF-α to mimic inflammatory conditions increased bacterial adhesion by almost 5-fold (p = 0.003) without impacting on the overall biomass. Interestingly, the effect of osteoblast-like cell supernatants on bacterial adhesion could be counteracted by the activity of synthetic antibiofilm peptides. Overall, the results of this study demonstrate that factors within the bone environment and products of osteoblast-like cells directly influence S. aureus adhesion and could contribute to biofilm initiation on bone and/or prosthetics implants

Survival in amoeba: a major selection pressure on the presence of bacterial copper and zinc resistance determinants?: identification of a "copper pathogenicity island"

The presence of metal resistance determinants in bacteria usually is attributed to geological or anthropogenic metal contamination in different environments or associated with the use of antimicrobial metals in human healthcare or in agriculture. While this is certainly true, we hypothesize that protozoan predation and macrophage killing are also responsible for selection of copper/zinc resistance genes in bacteria. In this review, we outline evidence supporting this hypothesis, as well as highlight the correlation between metal resistance and pathogenicity in bacteria. In addition, we introduce and characterize the "copper pathogenicity island" identified in Escherichia coli and Salmonella strains isolated from copper- and zinc-fed Danish pigs

Nitric oxide (NO) elicits aminoglycoside tolerance in Escherichia coli but antibiotic resistance gene carriage and NO sensitivity have not co-evolved

The spread of multidrug-resistance in Gram-negative bacterial pathogens presents a major clinical challenge, and new approaches are required to combat these organisms. Nitric oxide (NO) is a well-known antimicrobial that is produced by the immune system in response to infection, and numerous studies have demonstrated that NO is a respiratory inhibitor with both bacteriostatic and bactericidal properties. However, given that loss of aerobic respiratory complexes is known to diminish antibiotic efficacy, it was hypothesised that the potent respiratory inhibitor NO would elicit similar effects. Indeed, the current work demonstrates that pre-exposure to NO-releasers elicits a >10-fold increase in IC50 for gentamicin against pathogenic E. coli (i.e. a huge decrease in lethality). It was therefore hypothesised that hyper-sensitivity to NO may have arisen in bacterial pathogens, and that this trait could promote the acquisition of antibiotic-resistance mechanisms through enabling cells to persist in the presence of toxic levels of antibiotic. To test this hypothesis, genomics and microbiological approaches were used to screen a collection of E. coli clinical isolates for antibiotic susceptibility and NO tolerance, although the data did not support a correlation between increased carriage of antibiotic resistance genes and NO tolerance. However, the current work has important implications for how antibiotic susceptibility might be measured in future (i.e. +/- NO), and underlines the evolutionary advantage for bacterial pathogens to maintain tolerance to toxic levels of NO

Specific antibacterial activity of copper alloy touch surfaces in five long-term care facilities for older adults

International audienc

SlyA Is a Transcriptional Regulator Involved in the Virulence of Enterococcus faecalis

Phylogenetic analysis of the crystal structure of the Enterococcus faecalis SlyA (EF_3002) transcriptional factor places it between the SlyA and MarR regulator subfamilies. Proteins of these families are often involved in the regulation of genes important for bacterial virulence and stress response. To gather evidence for the role of this putative regulator in E. faecalis biology, we dissected the genetic organization of the slyA-EF_3001 locus and constructed a slyA deletion mutant as well as complemented strains. Interestingly, compared to the wild-type parent, the ΔslyA mutant is more virulent in an insect infection model (Galleria mellonella), exhibits increased persistence in mouse kidneys and liver, and survives better inside peritoneal macrophages. In order to identify a possible SlyA regulon, global microarray transcriptional analysis was performed. This study revealed that the slyA-EF_3001 locus appears to be autoregulated and that 117 genes were differentially regulated in the ΔslyA mutant. In the mutant strain, 111 were underexpressed and 6 overexpressed, indicating that SlyA functions mainly as an activator of transcription

Broad-Spectrum Anti-biofilm Peptide That Targets a Cellular Stress Response

<div><p>Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate a 10 to 1000 fold increase in adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. Here we identified a potent anti-biofilm peptide 1018 that worked by blocking (p)ppGpp, an important signal in biofilm development. At concentrations that did not affect planktonic growth, peptide treatment completely prevented biofilm formation and led to the eradication of mature biofilms in representative strains of both Gram-negative and Gram-positive bacterial pathogens including <i>Pseudomonas aeruginosa</i>, <i>Escherichia coli</i>, <i>Acinetobacter baumannii</i>, <i>Klebsiella pneumoniae</i>, methicillin resistant <i>Staphylococcus aureus</i>, <i>Salmonella</i> Typhimurium and <i>Burkholderia cenocepacia</i>. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. We hypothesized that the peptide acted to inhibit a common stress response in target species, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this, increasing (p)ppGpp synthesis by addition of serine hydroxamate or over-expression of <i>relA</i> led to reduced susceptibility to the peptide. Furthermore, <i>relA</i> and <i>spoT</i> mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to a reduction of biofilm formation in the four tested target species. Also, eliminating (p)ppGpp expression after two days of biofilm growth by removal of arabinose from a strain expressing <i>relA</i> behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp within 30 minutes, and <i>in vitro</i> directly interacted with ppGpp. We thus propose that 1018 targets (p)ppGpp and marks it for degradation in cells. Targeting (p)ppGpp represents a new approach against biofilm-related drug resistance.</p></div

Genetic complementation of (p)ppGpp synthetase enzymes restored the ability to form biofilms.

<p>The biofilm deficiency of <i>Pseudomonas aeruginosa</i> PAO1 and <i>Staphylococcus aureus</i> HG001 (p)ppGpp mutants (Δ<i>relAspoT</i> and <i>rsh_Syn</i> respectively) was rescued by genetic complementation [Δ<i>relAspoT</i> + <i>relAspoT⁺</i> (+SR) as described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004152#ppat.1004152-Nguyen1" target="_blank">[12]</a> and <i>rsh⁺</i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004152#ppat.1004152-Geiger1" target="_blank">[30]</a>, respectively] leading to biofilm formation equivalent to WT shown in the left-most panels. After 3 days, bacteria were stained green with the all bacteria stain Syto-9 and red with the dead-bacteria stain propidium iodide (merge shows as yellow to red) prior to confocal imaging. Each panel shows reconstructions from the top in the large panel and sides in the right and bottom panels (xy, yz and xz dimensions).</p

Peptide 1018 exhibited potent broad-spectrum direct anti-biofilm activity but weak antibacterial activity for planktonic cells.

<p>Comparison of planktonic cell MIC to MBIC₅₀ and MBIC₁₀₀, which are the minimal biofilm inhibitory concentrations leading to 50% and 100% decrease in biofilm growth, respectively.</p

Peptide 1018 prevented (p)ppGpp accumulation <i>in vivo</i> as revealed by thin layer chromatography separation of guanine nucleotides extracted from intact cells.

<p>(<b>A</b>) Anti-biofilm peptide 1018 at 5 µg/ml directly prevented (p)ppGpp accumulation. (<b>B</b>) Treatment with peptide 1018 led to (p)ppGpp elimination within 30 min in <i>P. aeruginosa</i> PAO1 cells containing pre-accumulated (p)ppGpp due to SHX treatment. In panel <b>A</b>, bacteria were grown overnight in modified MOPS minimal medium containing 0.4% glucose, 2 mM phosphate (KH₂PO₄), and 0.2% CAA. For experiments evaluating the ability of the peptide to directly degrade (p)ppGpp in panel <b>B</b>, the cells were grown as described previously, induced with SHX and allowed to synthesize (p)ppGpp for 3 h prior to peptide treatment. After growth for both A and B, the cells were then diluted 1∶20 in the same MOPS minimal medium except containing 0.4 mM phosphate (KH₂PO₄) and 500 µM serine hydroxamate (SHX) to induce (p)ppGpp synthesis, in the presence or absence of peptide 1018 and cells were labelled with 10 µCi/ml ³²P for 3 h. Samples were then extracted with frozen 13 M formic acid by three cycles of freeze-thaw. Aliquots of the supernatants were applied to 20×20 cm PEI cellulose TLC plates, resolved with 1.5 M KH₂PO₄, pH 3.4 for 4 h. After chromatography, nucleotides were visualized by autoradiography and quantified with a MolecularImager FX PhosphorImager and Quantity One software (Bio-Rad). Controls were performed to demonstrate that the Δ<i>relAspoT</i> mutation also prevented (p)ppGpp formation.</p