Molecular Characterization of a Novel Staphylococcus Aureus Surface Protein (SasC) Involved in Cell Aggregation and Biofilm Accumulation

BACKGROUND:Staphylococci belong to the most important pathogens causing implant-associated infections. Colonization of the implanted medical devices by the formation of a three-dimensional structure made of bacteria and host material called biofilm is considered the most critical factor in these infections. To form a biofilm, bacteria first attach to the surface of the medical device, and then proliferate and accumulate into multilayered cell clusters. Biofilm accumulation may be mediated by polysaccharide and protein factors. METHODOLOGY/PRINCIPAL FINDINGS:The information on Staphylococcus aureus protein factors involved in biofilm accumulation is limited, therefore, we searched the S. aureus Col genome for LPXTG-motif containing potential surface proteins and chose the so far uncharacterized S. aureus surface protein C (SasC) for further investigation. The deduced SasC sequence consists of 2186 amino acids with a molecular mass of 238 kDa and has features typical of gram-positive surface proteins, such as an N-terminal signal peptide, a C-terminal LPXTG cell wall anchorage motif, and a repeat region consisting of 17 repeats similar to the domain of unknown function 1542 (DUF1542). We heterologously expressed sasC in Staphylococcus carnosus, which led to the formation of huge cell aggregates indicative of intercellular adhesion and biofilm accumulation. To localize the domain conferring cell aggregation, we expressed two subclones of sasC encoding either the N-terminal domain including a motif that is found in various architectures (FIVAR) or 8 of the DUF1542 repeats. SasC or its N-terminal domain, but not the DUF1542 repeat region conferred production of huge cell aggregates, higher attachment to polystyrene, and enhanced biofilm formation to S. carnosus and S. aureus. SasC does not mediate binding to fibrinogen, thrombospondin-1, von Willebrand factor, or platelets as determined by flow cytometry. CONCLUSIONS/SIGNIFICANCE:Thus, SasC represents a novel S. aureus protein factor involved in cell aggregation and biofilm formation, which may play an important role in colonization during infection with this important pathogen

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

Spatio-temporal population modelling for enhanced assessment of urban exposure to flood risk

There is a growing need for high resolution spatio-temporal population estimates which allow accurate assessment of population exposure to natural hazards. Current approaches to population estimation are usually limited either by the use of arbitrary administrative boundaries or insufficient resolution in the temporal dimension. The innovative approach proposed here combines the use of a spatio-temporal gridded population model with flood inundation data to estimate time-specific variations in population exposed to natural hazards. The approach is exemplified through an application centred on Southampton (UK) using Environment Agency flood map inundation data. Results demonstrate that large fluctuations occur over time in the population distribution within flood risk zones. Variations in the spatio-temporal distribution of population subgroups are explored. Analysis using GIS indicates a diurnal shift in exposure between fluvial and tidal flooding, particularly attributable to the movement of the working age population. This illustrates the improvements achievable to flood risk management as well as potential application to other natural hazard scenarios both within the UK and globally

SasC-mediated cell aggregation of <i>S. carnosus</i>.

<p>Cell aggregation is visible macroscopically (A, B) and microscopically (C) and is protease-sensitive (B, C). A: Visible cell cluster formation in liquid medium. Strains were incubated overnight in TSB with (B) or without (A) 1% xylose with shaking. The cells were harvested by centrifugation, resuspended in PBS, and observed in the wells of 24-well microtiter plates. 1: <i>S. carnosus</i> (pCX19); 2, <i>S. carnosus</i> (pSasCCol); 3, <i>S. carnosus</i> (pSasC4074). B: After overnight growth in TSB with 1% xylose, <i>S. carnosus</i> cells were treated for 3 h at 37°C with PBS (A), 2.5 mg/ml trypsin (B), or 2 mg/ml proteinase K (C). 1: <i>S. carnosus</i> (pCX19); 2, <i>S. carnosus</i> (pSasCCol); 3, <i>S. carnosus</i> (pSasC4074). C: Phase-contrast micrographs. Cells were treated overnight at 37°C with different concentrations of trypsin (0.25 mg/ml, 3; 0.5 mg/ml, 4; 1.25 mg/ml, 5; 2.5 mg/ml, 6) or left untreated (1, 2). 1: <i>S. carnosus</i> (pCX19); 2–6: <i>S. carnosus</i> (pSasC4074).</p

<i>S. aureus</i> SH1000 <i>sasC</i> shows reduced biofilm formation.

<p>Quantitative assay of biofilm formation. Lanes: 1, <i>S. aureus</i> SH1000; 2, <i>S. aureus</i> SH1000 <i>sasC</i>. A, 1% xylose; B, 0.25% glucose; C, no additional carbohydrate source.</p

Expression of <i>sasC</i> or <i>sasC</i> subfragments.

<p>A: SDS-PAGE (10% separation gel) of cell lysates of <i>S. carnosus</i> or <i>S. aureus</i> expressing <i>sasC</i> or <i>sasC</i> subfragments. The lanes contained: 1, 10, 15, marker proteins; 2, <i>S. carnosus</i> (pCX19); 3, <i>S. carnosus</i> (pSasC4074); 4, <i>S. carnosus</i> (pSasCsub1); 5, <i>S. carnosus</i> (pSasCsub2); 6, <i>S. aureus</i> SH1000; 7, <i>S. aureus</i> SH1000 (pSasC4074); 8, <i>S. aureus</i> SH1000 (pSasCsub1); 9, <i>S. aureus</i> SH1000 (pSasCsub2); 11, <i>S. aureus</i> 4074; 12, <i>S. aureus</i> 4074 (pSasC4074); 13, <i>S. aureus</i> 4074 (pSasCsub1); 14, <i>S. aureus</i> 4074 (pSasCsub2). The sizes of marker proteins are shown on the left (prestained protein ladder; Fermentas, Leon-Rot, Germany); in the right margin the sizes (kilodaltons) of SasC and truncated SasC proteins are indicated. B: Expression and purification of 6 x His-DUF1542. SDS-PAGE (10% separation gel) of crude cell lysates from non-induced <i>E. coli</i> (pHis-DUF1542) (lane 2), IPTG-induced <i>E. coli</i> (pHis-DUF1542) (lane 3), and purified 6 x His-DUF1542 (1.5 µg) (lane 4). The size of one marker protein is shown on the left (lane 1; prestained protein ladder, Fermentas).</p

Oligonucleotide primers used in this study.

<p>Oligonucleotide primers used in this study.</p

SasC does not bind to extracellular matrix proteins Fg, vWf, and TSP-1 or to platelets.

<p>Bars: black, <i>S. aureus</i> 4074; dark grey, <i>S. carnosus</i> (pCX19); light grey, <i>S. carnosus</i> (pSasC4074); white, <i>S. carnosus</i> (pSasCsub2). Solid black line: <i>S. aureus</i> 4074; small dashed line: <i>S. carnosus</i> (pCX19); dashed black line, <i>S. carnosus</i> (pSasC4074); dashed grey line, <i>S. carnosus</i> (pSasCsub2). The results represent the mean of three independent experiments. Standard deviations are indicated.</p

SasC-mediated initial attachment of <i>S. carnosus</i> or <i>S. aureus</i> strains.

<p>Phase-contrast micrographs of attached cells of <i>S. carnosus</i> (TM300) or <i>S. aureus</i> (SH1000) strains on polystyrene Petri dishes (A) or on glass slides (B).</p