Identification of Genes Involved in Polysaccharide-Independent Staphylococcus aureus Biofilm Formation

Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor α2-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Flow cell biofilm formation of <i>graS</i> and <i>imp</i> mutants in different strain backgrounds.

<p><i>S. aureus</i> strains SH1000 (A) and LAC* (B), and <i>graS</i> and <i>imp</i> mutations in each strain, were grown in a flow cell apparatus for two days. A <i>z</i> series of images was obtained with CLSM, reconstructed with Volocity software, and each side of a grid square is 20 µm in the image reconstruction. The addition of a complementing plasmid containing either the <i>graRS</i> or <i>imp</i> genes is shown in the last column.</p

Assessment of biofilm formation in transposon mutants.

<p>Microtiter biofilm assays were performed in the absence (A) or presence (B) of the protease inhibitor α₂-macroglobulin in triplicate, and the percentages of biofilm formation for each mutant relative to that of the wild type are shown. Error bars show standard deviation. Color coding is as follows: black bars indicate mutants with normal extracellular protease activity, blue bars indicate high protease activity and biofilm recovery with α₂-macroglobulin, and red bars indicate the <i>graS</i> (37 C9) and <i>imp</i> (60 G7, 64 G10) mutations that did not recover with α₂-macroglobulin.</p

Extracellular protease activity of biofilm mutants.

<p>Protease activity in cell-free supernatants from cultures grown in TSB for 12 hours was measured with Azocoll reagent as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010146#s4" target="_blank">Materials and Methods</a>. Supernatant protease activity of the wild-type strain (SH1000) was set to 1 as a reference. The graph shows the mean of three samples; error bars show standard deviation. Blue bars indicate mutants that show consistently higher levels of protease activity.</p

Biofilm defective mutants.

a<p>Order of mutants listed matches the ordering in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010146#pone-0010146-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010146#pone-0010146-g002" target="_blank">2</a>.</p>b<p>ORF numbers, gene names, and descriptions are from the National Microbial Pathogen Data Resource web site (<a href="http://www.nmpdr.org" target="_blank">www.nmpdr.org</a>).</p>c<p>Percentages of biofilm formation for each mutant relative to wild type represent the means (+/− standard deviations) from four independent experiments each performed in triplicate.</p

Extracellular nuclease activity of selected biofilm defective mutants.

<p>Nuclease activity was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010146#s4" target="_blank">Materials and Methods</a> and plotted relative to SH1000 levels. For the <i>imp</i> mutant, only the results of transposon mutant 60 G7 are shown. Results with <i>imp</i> mutant 64 G10 were indistinguishable (data not shown). Underneath the plotted nuclease activity results are representative images of colonies grown on methyl green DNase agar plates. Clearing zones around the colonies is indicative of increased nuclease activity. Error bars show standard deviation.</p

Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis

<p>Anti-neutrophil cytoplasmic antibody-associated (ANCA-associated) small vessel necrotizing vasculitis is caused by immune-mediated inflammation of the vessel wall and is diagnosed in some cases by the presence of myeloperoxidase-specific antibodies (MPO-ANCA). This multicenter study sought to determine whether differences in ANCA epitope specificity explain why, in some cases, conventional serologic assays do not correlate with disease activity, why naturally occurring anti-MPO autoantibodies can exist in disease-free individuals, and why ANCA are undetected in patients with ANCA-negative disease. Autoantibodies from human and murine samples were epitope mapped using a highly sensitive epitope excision/mass spectrometry approach. Data indicated that MPO autoantibodies from healthy individuals had epitope specificities different from those present in ANCA disease. Importantly, this methodology led to the discovery of MPO-ANCA in ANCA-negative disease that reacted against a sole linear sequence. Autoantibodies against this epitope had pathogenic properties, as demonstrated by their capacity to activate neutrophils in vitro and to induce nephritis in mice. The confounder for serological detection of these autoantibodies was the presence of a fragment of ceruloplasmin in serum, which was eliminated in purified IgG, allowing detection. These findings implicate immunodominant epitopes in the pathology of ANCA-associated vasculitis and suggest that autoantibody diversity may be common to other autoimmune diseases.</p>

Autoimmunity to the alpha 3 chain of type IV collagen in glomerulonephritis is triggered by 'autoantigen complementarity'

'Autoantigen complementarity' is a theory proposing that the initiator of an autoimmune response is not necessarily the autoantigen or its molecular mimic, but may instead be a peptide that is 'antisense/complementary' to the autoantigen. We investigated whether such complementary proteins play a role in the immunopathogenesis of autoimmune glomerulonephritis. Experimental autoimmune glomerulonephritis, a model of anti-glomerular basement membrane (GBM) disease, can be induced in Wistar Kyoto (WKY) rats by immunization with the α3 chain of type IV collagen. In this study, WKY rats were immunized with a complementary α3 peptide (c-α3-Gly) comprised of amino acids that 'complement' the well characterized epitope on α3(IV)NC1, pCol(24-38). Within 8 weeks post-immunization, these animals developed cresentic glomerulonephritis, similar to pCol(24-38)-immunized rats, while animals immunized with scrambled peptide were normal. Anti-idiotypic antibodies to epitopes from c-α3-Gly-immunized animals were shown to be specific for α3 protein, binding in a region containing sense pCol(24-38) sequence. Interestingly, anti-complementary α3 antibodies were identified in sera from patients with anti-GBM disease, suggesting a role for 'autoantigen complementarity' in immunopathogenesis of the human disease. This work supports the idea that autoimmune glomerulonephritis can be initiated through an immune response against a peptide that is anti-sense or complementary to the autoantigen. The implications of this discovery may be far reaching, and other autoimmune diseases could be due to responses to these once unsuspected 'complementary' antigens