Draft genome sequence of Pseudomonas aeruginosa ATCC 9027, originally isolated from an outer ear infection

Pseudomonas aeruginosa ATCC 9027 was isolated in 1943 from a case of otitis externa and is commonly employed as a quality control strain for sterility, assessment of antibiofilm agents, and in vitro study of wound infection. Here, we present the 6.34-Mb draft genome sequence and highlight some pertinent genes that are associated with virulence

Alginate films augmented with chlorhexidine hexametaphosphate particles provide sustained antimicrobial properties for application in wound care

All chronic wounds are colonised by bacteria; for some, colonisation progresses to become infection. Alginate wound dressings are used for highly exuding chronic wounds as they are very absorbent, taking up large quantities of exudate while maintaining a moist wound bed to support healing. Some alginate dressings are doped with antimicrobials, most commonly silver, but evidence regarding the efficacy of these is largely inconclusive. This manuscript describes the development and in vitro assessment of alginate materials doped with chlorhexidine hexametaphosphate (CHX-HMP), a sparingly soluble salt which when exposed to aqueous environments provides sustained release of the common antiseptic chlorhexidine. Comparator materials were a commercial silver alginate dressing material and an alginate doped with chlorhexidine digluconate (CHXdg). CHX-HMP alginates provided a dose-dependent CHX release which was sustained for over 14 days, whereas CHXdg alginates released limited CHX and this ceased within 24 h. CHX-HMP and silver alginates were efficacious against 5 major wound pathogens (MRSA, E. coli, P. aeruginosa, K pneumoniae, A. baumannii) in a total viable count (TVC) and an agar diffusion zone of inhibition (ZOI) model. At baseline the silver alginate was more effective than the CHX-HMP alginate in the TVC assay but the CHX-HMP alginate was the more effective in the ZOI assay. After 7 days’ artificial aging the CHX-HMP alginate was more effective than the silver alginate for four of the five bacteria tested in both assays. These materials may ultimately find application in the development of wound dressings for chronic wounds that provide sustained antimicrobial protection

Manuka honey inhibits the development of Streptococcus pyogenes biofilms and causes reduced expression of two fibronectin binding proteins

Streptococcus pyogenes (group A Streptococcus; GAS) is always of clinical significance in wounds where it can initiate infection, destroy skin grafts and persist as a biofilm. Manuka honey has broad spectrum antimicrobial activity and its use in the clinical setting is beginning to gain acceptance with the continuing emergence of antibiotic resistance and the inadequacy of established systemic therapies; novel inhibitors may affect clinical practice. In this study, the effect of manuka honey on S. pyogenes (M28) was investigated in vitro with planktonic and biofilm cultures using MIC, MBC, microscopy and aggregation efficiency. Bactericidal effects were found in both planktonic cultures and biofilms, although higher concentrations of manuka honey were needed to inhibit biofilms. Abrogation of adherence and intercellular aggregation was observed. Manuka honey permeated 24 h established biofilms of S. pyogenes, resulting in significant cell death and dissociation of cells from the biofilm. Sublethal concentrations of manuka honey effectively prevented the binding of S. pyogenes to the human tissue protein fibronectin, but did not inhibit binding to fibrinogen. The observed inhibition of fibronectin binding was confirmed by a reduction in the expression of genes encoding two major fibronectin-binding streptococcal surface proteins, Sof and SfbI. These findings indicate that manuka honey has potential in the topical treatment of wounds containing S. pyogenes. INTRODUCTION Streptococcus pyogenes (group A Streptococcus) colonizes the nasopharynx and skin of healthy individuals, forming part of the commensal microbiota. Under appropriate conditions, S. pyogenes can be transmitted to wounds and is especially problematic after surgery, following skin grafting and for military personal with traumatic or puncture wounds. Wounds provide a route of entry to the host and damaged tissues display a matrix of proteins including collagen, albumin, fibronectin and fibrinogen, which collectively provide a plethora of ligands to which opportunistic pathogens, including streptococci, adhere Biofilms have been associated with persistent or chronic wound infections and are a major obstacle to healing Honey has had a valued place in traditional medicine for many centuries and was reintroduced into modern medicine during the 21st century. Honey exhibits broad spectrum antibacterial activities and has been reported to inhibit more than 80 species of bacteria, including meticillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, Lancefield groups A, C and G streptococci, Pseudomonas aeruginosa and Actinomyces species METHODS Bacterial strains. S. pyogenes MGAS6180 (M28, invasive disease; Green et al., 2005) was grown in Todd-Hewitt (TH) broth (Oxoid) containing 0.5 % yeast extract; Bacto agar (Difco) was added to achieve a 1.4 % (w/v) final concentration in agar plates. Cultures were incubated at 37 uC in air supplemented with 5 % CO 2 . For analysis of aggregation properties, S. pyogenes was grown in C medium containing 0.2 % glucose (Lyon et al., 1998). Medical grade manuka honey. Sterile medical grade manuka honey (Medihoney) was kindly donated by Comvita. Medihoney was provided in sterile 50 g portions. Minimum inhibitory and bactericidal concentrations. The MIC for manuka honey against planktonically grown S. pyogenes MGAS6180 was determined by serial dilution (0-95 %; w/v) in a total volume of 5 ml iso-sensitest broth (Oxoid) (according to the British Society for Antimicrobial Chemotherapy methodology for determining MIC; Andrews, 2011). Cultures were incubated for 16 h at 37 uC in aerobic conditions with 5 % CO 2 . To establish the MBC, samples corresponding to the MIC and including three samples of a higher concentration were plated onto Todd-Hewitt agar and incubated for 16 h at 37 uC in aerobic conditions with 5 % CO 2 . Assays were done in triplicate on each of three separate occasions. Inhibition of bacterial growth. To determine the extent of growth inhibition by manuka honey, triplicate cultures of S. pyogenes MGAS6180 were grown for 8 h at 37 uC in 10 ml TH broth, in aerobic conditions with 5 % CO 2 , supplemented with manuka honey (0, 20 and 40 % w/v). Bacterial growth was monitored at OD 650 at hourly intervals. Growth experiments also utilized triplicate biological samples. Aggregation assays. S. pyogenes MGAS6180 was initially grown in C medium for 16 h (Lyon et al., 1998) and harvested by centrifugation at 5000 g for 10 min. Bacterial cell pellets were resuspended in either 1 ml C medium or an appropriate concentration of manuka honey (5 and 10 %; w/v) dissolved in C medium and the OD 600 of the culture was adjusted to 1.0 (±0.05) if necessary. In both cases, manuka honey at twice the desired concentration was dissolved in double strength TH and diluted to the required concentration using TH; cell pellets were resuspended in either 5 or 10 % (w/v) honey solutions. In an untreated control, manuka honey was replaced with PBS added to maintain the appropriate volume and concentration of TH media. Aggregation assays were carried out in triplicate as described previously Static biofilm model. S. pyogenes MGAS6180 was initially grown in C medium for 16 h and these stationary phase cultures were harvested by centrifugation and adjusted to OD 650 0.1. To determine whether manuka honey prevented biofilm formation, biofilms were established in 96-well microtitre plates (Greiner) in 50 ml TH, supplemented with manuka honey (0, 10, 20 and 40 % w/v), by inoculating each well with 5 ml harvested cells. Plates were incubated at 37 uC for 24 h, aerobically with 5 % CO 2 . To estimate biomass, unattached cells were gently aspirated and discarded, and adherent cells were washed twice with PBS and stained with crystal violet (0.25 %; w/v) for 10 min; following a further two washes with PBS, cell-bound crystal violet was resolubilized with 7 % acetic acid, and absorbance was measured at 595 nm (A 595 ) Live-Dead staining. Images of bacterial cells were collected for control cells (untreated) and for cells treated with 40 % (w/v) manuka honey for 2 h, to determine the effect of manuka honey on viability. Biofilms were grown in Petri dishes in 5 ml TH media, or 5 ml 40 % (w/v) honey dissolved in TH media, as previously described; liquid was aspirated from the plates and biofilms were washed with 1 ml PBS. Biofilms were scraped from the coverslips using a cell scraper, resuspended in 1 ml PBS and stained with Live-Dead BacLight bacterial viability kit (Invitrogen), following the manufacturer's instructions. Fluorescence microscopy images were obtained using a Nikon Eclipse 80i fluorescent microscope with oil immersion and 6100 lens. For detection of SYTO 9 (green channel) a 488 nm excitation and 520 nm emission filter was used. For propidium iodide detection (red channel) a 543 nm excitation and 572 nm emission filter was used. Image analysis used Volocity Software (PerkinElmer). Biofilm disruption. To determine whether manuka honey affected biofilm biomass by facilitating the dissociation of adherent cells from S. E. Maddocks and others 782 Microbiology 158 the biofilm, assays were conducted to monitor the numbers of viable planktonic cells that were released into the liquid phase, from established biofilms following treatment with manuka honey. Biofilms were grown for 24 h as described above. The liquid was aspirated from each well, biofilms were washed twice with PBS to remove any planktonic or loosely adherent cells, and manuka honey over a range of concentrations [0, 10, 20 and 40 % (w/v), respectively] was added to the 24 h established biofilms. Following the application of honey, biofilms were incubated for a further 2 h at 37 uC as above and samples of the liquid above the biofilm were collected at 30 min intervals. Bacterial cells were enumerated using the total viable cell (TVC) counting method described by 21 . Fibronectin-and fibrinogen-binding assays. To determine the effect of manuka honey on adherence of S. pyogenes cells to immobilized fibronectin and fibrinogen, a crystal violet assay was conducted as described previously, using 1 % BSA to block wells prior to assaying cell binding RNA extraction from S. pyogenes biofilms. Large scale, static biofilms of S. pyogenes were grown in duplicate in 5 ml C medium (with 20 % honey for the 'treated' biofilms) in sterile Petri dishes for 24 h at 37 uC, as for the small scale biofilms. The liquid was aspirated and the biofilm was scraped from the surface of the Petri dish using a sterile cell scraper. Biofilms were resuspended in 500 ml PBS and vortexed for 1 min to break up cell aggregates. Honey-treated and untreated cell suspensions were equilibrated (to approximately 2.5610 9 c.f.u.) prior to treatment with mutanolysin (100 mg) and lysozyme (100 mg) for 20 min at 37 uC. RNA extraction was carried out using the SV Wizard total RNA extraction kit (Promega) according the manufacturer's instructions. RNA quantification was performed by spectrophotometric measurement using a NanoDrop ND-1000 (NanoDrop Technologies) and each RNA sample was adjusted to give a final concentration of 10 ng ml 21 . End point RT-PCR to determine the relative expression of sof and sfbI. PCR primers were designed to amplify a 1100 bp fragment of the sof gene (sof-fwd: 59-ACTTAGAAAGTTATCTGTAGGG; sofrev: 59-TCTCTCGAGCTTTATGGATAG) and 1200 bp fragment of the sfbI gene (sbfI-fwd: 59-AACTGCTTTAGGAACAGCTTC; sbfI-rev: 59-CCACCATAGCCACAATGCT). The complete genome sequence for S. pyogenes MGAS6180 was obtained from the NCBI database (www.ncbi.nlm.nih.gov) and used as a basis to design the primers used in this study. Internal control primers were designed to amplify a 900 bp internal fragment of the glr (murI; glutamate racemase) gene (glr-fwd: 59-ATGGATACAAGACCAATTGGG; glr-rev: 59-TCATAA-GGTGACATGCTCCAC), a known housekeeping gene in S. pyogenes that is commonly used for MLST (http://spyogenes.mlst.net/misc/ info.asp) RESULTS Inhibition of planktonic S. pyogenes by manuka honey The MIC of manuka honey against S. pyogenes MGAS6180 was found to be 20 % (w/v) and the MBC was found to be 45 % (w/v). Growth curves with 20 % (w/v) manuka honey resulted in a reduced growth rate and reduction in overall cell number ( S. pyogenes aggregation and biofilm development are inhibited by sublethal concentrations of manuka honey To test the capacity for manuka honey to inhibit intercellular aggregation, suspensions of S. pyogenes MGAS6180 were mixed with 10 % (w/v) manuka honey. In the absence of manuka honey, cells strongly aggregated but in the presence of 10 % (w/v) honey, aggregation was completely inhibited (P,0.05) When biofilms of S. pyogenes were initiated in the presence of 10, 20 and 40 % (w/v) manuka honey, a statistically significant reduction in biomass was observed in each case. A reduction of 75 % (P50.03) was observed with 10 % (w/ v) manuka honey; 20 and 40 % (w/v) manuka honey resulted in between 90 % (P50.02) and 96 % (P50.01) reduction in biomass, respectively Manuka honey facilitates cell death and dissociation of bacterial cells from established S. pyogenes biofilms To determine the effect of manuka honey against established biofilms of S. pyogenes MGAS6180, biofilms were grown for 24 h prior to honey treatment. Following 2 h treatment with 10 and 20 % manuka honey (w/v), a 72 % (P50.32) and 77 % (P50.08) reduction in biomass was observed, respectively. Following treatment for 2 h with 40 % (w/v) manuka honey, the observed reduction in biofilm biomass was even greater, and statistically significant at 85 % (P50.01) Live-Dead viability staining (Invitrogen) of untreated biofilms grown for 24 h at 37 uC, honey-treated (40 % w/ v) biofilms grown for 24 h at 37 uC, and biofilms that were grown for 24 h at 37 uC, then treated for 2 h with 40 % (w/ v) manuka honey, showed that in both cases, the honey treatment resulted in cell death With time, bacterial cells may dissociate from a biofilm. To determine whether manuka honey facilitated the dissociation of S. pyogenes from biofilms, the levels of planktonic cells in the liquid phase were monitored for 2 h following the application of manuka honey (the biofilm data described above showed that the biomass was reduced following 2 h treatment, so this time period was deemed appropriate for these experiments). Immediately after application of the manuka honey (0 min) the c.f.u. was approximately the same for each condition. After 30 min and throughout the duration of the experiment (120 min), higher c.f.u. values were recovered from biofilms treated with 10 and 20 % (w/v) manuka honey (subinhibitory concentrations) compared with untreated biofilms, with a maximum recovery of 1.2610 6 c.f.u. S. E. Maddocks and others 784 Microbiology To establish whether manuka honey affected binding of S. pyogenes MGAS6180 to the wound-associated proteins fibronectin and fibrinogen, 1 mg of each of these two protein ligands was immobilized to the surface of a microtitre plate. The extent of binding inhibition using a sublethal dose (20 % w/v) of manuka honey was compared with adherence observed in the absence of manuka honey. Binding of S. pyogenes to fibronectin in the presence of 20 % (w/v) manuka honey was reduced by 74 %, which was found to be statistically significant (P50.01); conversely no such reduction in binding was observed for fibrinogen (P50.38) Genes encoding the surface adhesins Sof and SbfI are differentially expressed in response to exposure to a sublethal concentration of manuka honey To determine whether the decreased binding to fibronectin was the result of differential expression of two of the major fibronectin-binding proteins (Sof and SfbI) of S. pyogenes MGAS6180 in response to sublethal concentrations of honey, end point RT-PCR was employed. The PCR products were analysed by densitometry and quantified relative to the New England Biolabs 1 kb quantitative molecular mass Streptococcus pyogenes biofilms and honey http://mic.sgmjournals.org 785 marker (thresholds for detection ¢0.15 nmol). The results confirmed that both sfbI and sof were expressed to a lesser extent in the presence of 20 % (w/v) manuka hone

The effects of a video intervention on posthospitalization pulmonary rehabilitation uptake

Rationale: Pulmonary rehabilitation (PR) after hospitalizations for exacerbations of chronic obstructive pulmonary disease (COPD) improves exercise capacity and health-related quality of life and reduces readmissions. However, posthospitalization PR uptake is low. To date, no trials of interventions to increase uptake have been conducted.Objectives: To study the effect of a codesigned education video as an adjunct to usual care on posthospitalization PR uptake.Methods: The present study was an assessor- and statistician-blinded randomized controlled trial with nested, qualitative interviews of participants in the intervention group. Participants hospitalized with COPD exacerbations were assigned 1:1 to receive either usual care (COPD discharge bundle including PR information leaflet) or usual care plus the codesigned education video delivered via a handheld tablet device at discharge. Randomization used minimization to balance age, sex, FEV1 % predicted, frailty, transport availability, and previous PR experience.Measurements and Main Results: The primary outcome was PR uptake within 28 days of hospital discharge. A total of 200 patients were recruited, and 196 were randomized (51% female, median FEV1% predicted, 36 [interquartile range, 27-48]). PR uptake was 41% and 34% in the usual care and intervention groups, respectively (P = 0.37), with no differences in secondary (PR referral and completion) or safety (readmissions and death) endpoints. A total of 6 of the 15 participants interviewed could not recall receiving the video.Conclusions: A codesigned education video delivered at hospital discharge did not improve posthospitalization PR uptake, referral, or completion

Chlorhexidine hexametaphosphate as a wound care material coating: antimicrobial efficacy, toxicity and effect on healing.

AIM: In this study, chlorhexidine hexametaphosphate (CHX-HMP) is investigated as a persistent antimicrobial coating for wound care materials. MATERIALS & METHODS: CHX-HMP was used as a wound care material coating and compared with chlorhexidine digluconate materials with respect to antimicrobial efficacy, toxicity and wound closure. RESULTS: Antimicrobial efficacy at day 1, 3 and 7 was observed with experimental and commercial materials. CHX-HMP coated materials had less toxic effect on human placental cells than commercial chlorhexidine dressings. CHX-HMP in pluronic gel did not delay healing but reduced wound colonization by E. faecalis. CONCLUSION: CHX-HMP could become a useful component of wound care materials with sustained antimicrobial efficacy, lower toxicity than chlorhexidine digluconate materials, and reduction in wound colonization without affecting closure

Residential Moving and Preventable Hospitalizations

OBJECTIVES: To investigate the association between moving home in the first year of life and subsequent emergency admissions for potentially preventable hospitalizations. METHODS: We undertook a cohort analysis of linked anonymized data on 237 842 children in the Welsh Electronic Cohort for Children. We included children born in Wales between April 1, 1999 and December 31, 2008. The exposure was the number of residential moves from birth up to 1 year. The main outcome was emergency admissions for potentially preventable hospitalizations (PPH) between the age of 1 and 5 years. RESULTS: After adjustment for confounders, we identified that moving home frequently in the first year of life was associated with an increased risk of emergency PPH between the ages of 1 and 5 when compared with not moving. We found significant differences associated with ≥2 moves for the following: ear, nose, and throat infections (incidence risk ratio [IRR], 1.44; 95% confidence interval [CI], 1.29–1.61); convulsions/epilepsy (IRR, 1.58; 95% CI, 1.23–2.04); injuries (IRR, 1.33; 95% CI, 1.18–1.51); dehydration/gastroenteritis (IRR, 1.51; 95% CI, 1.21–1.88); asthma (IRR, 1.61; 95% CI, 1.19–2.16); influenza/pneumonia (IRR, 1.15; 95% CI, 1.00–1.32); and dental conditions (IRR, 1.30; 95% CI, 1.03–1.64) for ≥1 moves. CONCLUSIONS: Children who move home in the first year of life are at substantially increased risk of emergency admissions for PPH in early childhood. Additional research that focuses on enhancing health and social support services for highly mobile families, educating parents about safety risks, and improving housing quality is warranted

BORA ON THE NORTHERN ADRIATIC, 12-18 APRIL 1982

Analysis is presented of a bora case on 12-18 April 1982, characterized by the longest bora duration in Senj (138 hours) during the ALPEX-SOP. The bora was observed only on the northern Adriatic. The vertical wind and stability profiles indicate that the part of the upstream inversion layer decoupled and descended toward the sea which is confirmed in the aircraft data analysis (Smith, 1987). The application of the generalized hydraulic theory on the continuously stratified atmosphere showed that the theory can successfully explain the bora phenomenon in the postfrontal bora situation

Does frequent residential mobility in early years affect the uptake and timeliness of routine immunisations? An anonymised cohort study

Background: There are conflicting findings regarding the impact of residential mobility on immunisationstatus. Our aim was to determine whether there was any association between residential mobility andtake up of immunisations and whether they were delayed in administration. Methods: We carried out a cohort analysis of children born in Wales, UK. Uptake and time of immunisationwere collected electronically. We defined frequent movers as those who had moved: 2 or more times inthe period prior to the final scheduled on-time date (4 months) for 5 in 1 vaccinations; and 3 or moretimes in the period prior to the final scheduled on-time date (12 months) for MMR, pneumococcal andmeningitis C vaccinations. We defined immunisations due at 2–4 months delayed if they had not beengiven by age 1; and those due at 12–13 months as delayed if they had not been given by age 2. Results: Uptake rates of routine immunisations and whether they were given within the specified time-frame were high for both groups. There was no increased risk (odds ratios (95% confidence intervals)between frequent movers compared to non-movers for the uptake of: primary MMR 1.08 (0.88–1.32);booster Meningitis C 1.65 (0.93–2.92); booster pneumococcal 1.60 (0.59–4.31); primary 5 in 1 1.28(0.92–1.78); and timeliness: primary MMR 0.92 (0.79–1.07); booster Meningitis C 1.26 (0.77–2.07);booster pneumococcal 1.69 (0.23–12.14); and primary 5 in 1 1.04 (0.88–1.23). Discussion: Findings suggest that children who move home frequently are not adversely affected in termsof the uptake of immunisations and whether they were given within a specified timeframe. Both werehigh and may reflect proactive behaviour in the primary healthcare setting to meet Government coveragerates for immunisation