Mast cells are upregulated in hidradenitis suppurativa tissue, associated with epithelialized tunnels and normalized by spleen tyrosine kinase antagonism.

Mast cells have traditionally been associated with allergic inflammatory responses; however, they play important roles in cutaneous innate immunity and wound healing. The Hidradenitis Suppurativa tissue transcriptome is associated with alterations in innate immunity and wound healing-associated pathways; however, the role of mast cells in the disease is unexplored. We demonstrate that mast cell-associated gene expression (using whole tissue RNAseq) is upregulated, and in-silico cellular deconvolution identifies activated mast cells upregulated and resting mast cells downregulated in lesional tissue. Tryptase/Chymase positive mast cells (identified using IHC) localize adjacent to epithelialized tunnels, fibrotic regions of the dermis and at perivascular sites associated with Neutrophil Extracellular Trap formation and TNF-alpha production. Treatment with Spleen Tyrosine Kinase antagonist (Fostamatinib) reduces the expression of mast cell-associated gene transcripts, associated biochemical pathways and the number of tryptase/chymase positive mast cells in lesional hidradenitis suppurativa tissue. This data indicates that although mast cells are not the most abundant cell type in Hidradenitis Suppurativa tissue, the dysregulation of mast cells is paralleled with B cell/plasma cell inflammation, inflammatory epithelialized tunnels and epithelial budding. This provides an explanation as to the mixed inflammatory activation signature seen in HS, the correlation with dysregulated wound healing and potential pathways involved in the development of epithelialized tunnels.

The efficacy of topical agents used in wounds for managing chronic biofilm infections: A systematic review.

OBJECTIVES: Clinicians have increasingly adopted the widespread use of topical agents to manage chronic wound infections, despite limited data on their effectiveness in vivo. This study sought to evaluate the evidence for commonly employed topical agents used in wounds for the purpose of treating chronic infections caused by biofilm. METHOD: We included in vitro, animal and human in vivo studies where topical agents were tested for their efficacy against biofilms, for use in wound care. For human studies, we only included those which utilised appropriate identification techniques for visualising and confirming the presence of biofilms. RESULT: A total of 640 articles were identified, with 43 included after meeting eligibility. In vitro testing accounted for 90% (n = 39) of all included studies, five studies using animal models and three human in vivo studies. Sixteen different laboratory models were utilised, with the most frequent being the minimum biofilm eradication concentration (MBEC™) / well plate assay (38%, n = 15 of 39). A total of 44 commercially available topical agents were grouped into twelve categories with the most commonly tested agents being silver, iodine and polyhexamethylene biguanide (PHMB). In vitro results on efficacy demonstrated iodine as having the highest mean log10 reductions of all agents (4.81, ±3.14). CONCLUSION: There is large disparity in the translation of laboratory studies to researchers undertaking human trials relating to the effectiveness of commercially available topical agents. There is insufficient human in vivo evidence to definitively recommend any commercially available topical agent over another for the treatment of chronic wound biofilms. The heterogeneity identified between study designs (in vitro to in vivo) further limits the generalisability of results.

Understanding the microbiome of diabetic foot osteomyelitis: insights from molecular and microscopic approaches.

OBJECTIVES: Rigorous visual evidence on whether or not biofilms are involved in diabetic foot osteomyelitis (DFO) is lacking. We employed a suite of molecular and microscopic approaches to investigate the microbiome, and phenotypic state of microorganisms involved in DFO. METHODS: In 20 consecutive subjects with suspected DFO, we collected intraoperative bone specimens. To explore the microbial diversity present in infected bone we performed next generation DNA sequencing. We used scanning electron microscopy (SEM) and peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) with confocal microscopy to visualize and confirm the presence of biofilms. RESULTS: In 19 of 20 (95%) studied patients presenting with DFO, it was associated with an infected diabetic foot ulcer. By DNA sequencing of infected bone, Corynebacterium sp. was the most commonly identified microorganism, followed by Finegoldia sp., Staphylococcus sp., Streptococcus sp., Porphyromonas sp., and Anaerococcus sp. Six of 20 bone samples (30%) contained only one or two pathogens, while the remaining 14 (70%) had polymicrobial communities. Using a combination of SEM and PNA-FISH, we identified microbial aggregates in biofilms in 16 (80%) bone specimens and found that they were typically coccoid or rod-shaped aggregates. CONCLUSIONS: The presence of biofilms in DFO may explain why non-surgical treatment of DFO, relying on systemic antibiotic therapy, may not resolve some chronic infections caused by biofilm-producing strains.

Effect of disinfectant formulation and organic soil on the efficacy of oxidizing disinfectants against biofilms.

BACKGROUND: Biofilms that develop on dry surfaces in the healthcare environment have increased tolerance to disinfectants. This study compared the activity of formulated oxidizing disinfectants with products containing active ingredients against Staphylococcus aureus dry-surface biofilm (DSB) alone. METHODS: DSB was grown in the CDC bioreactor with alternating cycles of hydration and dehydration. Disinfectant efficacy was tested before and after treatment with neutral detergent for 30 s, and in the presence or absence of standardized soil. Biofilms were treated for 5 min with peracetic acid (Surfex and Proxitane), hydrogen peroxide (Oxivir and 6% H2O2 solution) and chlorine (Chlorclean and sodium dichloroisocyanurate tablets). Residual biofilm viability and mass were determined by plate culture and protein assay, respectively. FINDINGS: Biofilm viability was reduced by 2.8 log10 for the chlorine-based products and by 2 log10 for Proxitane, but these products failed to kill any biofilm in the presence of soil. In contrast, Surfex completely inactivated biofilm (6.3 log10 reduction in titre) in the presence of soil. H2O2 products had little effect against DSB. Biofilm mass removed in the presence and absence of soil was <30% by chlorine and approximately 65% by Surfex. Detergent treatment prior to disinfection had no effect. CONCLUSION: The additives in fully formulated disinfectants can act synergistically with active ingredients, and thus increase biofilm killing whilst decreasing the adverse effect of soil. It is suggested that purchasing officers should seek efficacy testing results, and consider whether efficacy testing has been conducted in the presence of biological soil and/or biofilm.

The validity of adenosine triphosphate measurement in detecting endoscope contamination.

BACKGROUND: Endoscopic procedures are vital to gastrointestinal disease diagnosis and management, but risk infection transmission. In Australia, endoscopes undergo monthly-to-quarterly microbiological testing, to prevent patient infection. Endoscopes are used more frequently, meaning contamination may not be detected by this surveillance before infection transmission occurs. AIM: To evaluate the use of adenosine triphosphate (ATP) measurement, alongside standard microbiological cultures, in detecting endoscope contamination before high-level disinfection. Using these results, we also aimed to confirm the efficacy of manual cleaning in reducing levels of ATP and cfu/mL. METHODS: Seventeen in-clinical-use gastroscopes and 24 in-clinical-use colonoscopes from the Liverpool Hospital Endoscopy unit were sampled across three separate cleaning stages before high-level disinfection. Colony counts and ATP measurements were then performed on these samples. FINDINGS: The correlation between the cfu/mL and RLU of samples collected from colonoscopes was 0.497 (95% confidence interval: 0.28-0.66; P < 0.0001). The correlation between cfu/mL and RLU for samples collected from gastroscopes was 0.377 (0.08-0.61; P = 0.0138). RLU and cfu/mL values were shown to fall significantly (P < 0.005) following precleaning and manual cleaning. CONCLUSION: There was a significant correlation between ATP and cfu/mL measured from samples collected before high-level disinfection. Precleaning and manual cleaning were shown to reduce ATP and microbiological load significantly. ATP measurement can be performed within minutes with little training and produces results that are easy to interpret. These findings warrant further research on the utility of ATP measurement as a screening tool for detecting endoscope contamination after high-level disinfection.

Evaluation of short exposure times of antimicrobial wound solutions against microbial biofilms: from in vitro to in vivo.

Objectives: Test the performance of topical antimicrobial wound solutions against microbial biofilms using in vitro, ex vivo and in vivo model systems at clinically relevant exposure times. Methods: Topical antimicrobial wound solutions were tested under three different conditions: (in vitro) 4% w/v Melaleuca oil, polyhexamethylene biguanide, chlorhexidine, povidone iodine and hypochlorous acid were tested at short duration exposure times for 15 min against 3 day mature biofilms of Staphylococcus aureus and Pseudomonas aeruginosa; (ex vivo) hypochlorous acid was tested in a porcine skin explant model with 12 cycles of 10 min exposure, over 24 h, against 3 day mature P. aeruginosa biofilms; and (in vivo) 4% w/v Melaleuca oil was applied for 15 min exposure, daily, for 7 days, in 10 patients with chronic non-healing diabetic foot ulcers complicated by biofilm. Results: In vitro assessment demonstrated variable efficacy in reducing biofilms ranging from 0.5 log10 reductions to full eradication. Repeated instillation of hypochlorous acid in a porcine model achieved <1 log10 reduction (0.77 log10, P = 0.1). Application of 4% w/v Melaleuca oil in vivo resulted in no change to the total microbial load of diabetic foot ulcers complicated by biofilm (median log10 microbial load pre-treatment = 4.9 log10 versus 4.8 log10, P = 0.43). Conclusions: Short durations of exposure to topical antimicrobial wound solutions commonly utilized by clinicians are ineffective against microbial biofilms, particularly when used in vivo. Wound solutions should not be used as a sole therapy and clinicians should consider multifaceted strategies that include sharp debridement as the gold standard.

Staphylococcus aureus dry-surface biofilms are more resistant to heat treatment than traditional hydrated biofilms.

BACKGROUND: The importance of biofilms to clinical practice is being increasingly realized. Biofilm tolerance to antibiotics is well described but limited work has been conducted on the efficacy of heat disinfection and sterilization against biofilms. AIM: To test the susceptibility of planktonic, hydrated biofilm and dry-surface biofilm forms of Staphylococcus aureus, to dry-heat and wet-heat treatments. METHODS: S. aureus was grown as both hydrated biofilm and dry-surface biofilm in the CDC biofilm generator. Biofilm was subjected to a range of temperatures in a hot-air oven (dry heat), water bath or autoclave (wet heat). FINDINGS: Dry-surface biofilms remained culture positive even when treated with the harshest dry-heat condition of 100°C for 60min. Following autoclaving samples were culture negative but 62-74% of bacteria in dry-surface biofilms remained alive as demonstrated by live/dead staining and confocal microscopy. Dry-surface biofilms subjected to autoclaving at 121°C for up to 30min recovered and released planktonic cells. Recovery did not occur following autoclaving for longer or at 134°C, at least during the time-period tested. Hydrated biofilm recovered following dry-heat treatment up to 100°C for 10min but failed to recover following autoclaving despite the presence of 43-60% live cells as demonstrated by live/dead staining. CONCLUSION: S. aureus dry-surface biofilms are less susceptible to killing by dry heat and steam autoclaving than hydrated biofilms, which are less susceptible to heat treatment than planktonic suspensions.

Next Generation DNA Sequencing of Tissues from Infected Diabetic Foot Ulcers.

We used next generation DNA sequencing to profile the microbiome of infected Diabetic Foot Ulcers (DFUs). The microbiota was correlated to clinical parameters and treatment outcomes to determine if directed antimicrobial therapy based on conventional microbiological cultures are relevant based on genomic analysis. Patients≥18years presenting with a new Diabetic Foot Infection (DFI) who had not received topical or oral antimicrobials in the two weeks prior to presentation, were eligible for enrolment. Tissue punch biopsies were obtained from infected DFUs for analysis. Demographics, clinical and laboratory data were collected and correlated against microbiota data. Thirty-nine patients with infected DFUs were recruited over twelve-months. Shorter duration DFUs (

Effect of cadexomer iodine on the microbial load and diversity of chronic non-healing diabetic foot ulcers complicated by biofilm in vivo.

Objectives: The performance of cadexomer iodine was determined against microbial populations from chronic non-healing diabetic foot ulcers (DFUs) complicated by biofilm in vivo , using molecular, microscopy and zymography methods. Methods: Chronic non-healing DFUs due to suspected biofilm involvement were eligible for enrolment. DNA sequencing and real-time quantitative PCR was used to determine the microbial load and diversity of tissue punch biopsies obtained pre- and post-treatment. Scanning electron microscopy and/or fluorescence in situ hybridization confirmed the presence or absence of biofilm. Zymography was used to determine levels of wound proteases. Results: Seventeen participants were recruited over a 6 month period. Scanning electron microscopy and or fluorescence in situ hybridization confirmed the presence of biofilm in all samples. Eleven participants exhibited log 10 reductions in microbial load after treatment (range 1-2 log 10 ) in comparison with six patients who experienced <1 log 10 reduction ( P = 0.04). Samples were tested for levels of wound proteases pre- and post-treatment. Reductions in the microbial load correlated to reductions in wound proteases pre- and post-treatment ( P = 0.03). Conclusions: To the best of our knowledge, this study represents the first in vivo evidence, employing a range of molecular and microscopy techniques, of the ability of cadexomer iodine to reduce the microbial load of chronic non-healing DFUs complicated by biofilm. Further analyses correlating log reductions to optimal duration of therapy and improvements in clinical parameters of wound healing in a larger cohort are required.

Can molecular DNA-based techniques unravel the truth about diabetic foot infections?

Diabetes foot infections are a common condition and a major causal pathway to lower extremity amputation. Identification of causative pathogens is vital in directing antimicrobial therapy. Historically, clinicians have relied upon culture-dependent techniques that are now acknowledged as both being selective for microorganisms that thrive under the physiological and nutritional constraints of the microbiology laboratory and that grossly underestimate the microbial diversity of a sample. The amplification and sequence analysis of the 16S rRNA gene has revealed a diversity of microorganisms in diabetes foot infections, extending the view of the diabetic foot microbiome. The interpretation of these findings and their relevance to clinical care remains largely unexplored. The advent of molecular methods that are culture-independent and employ massively parallel DNA sequencing technology represents a potential 'game changer'. Metagenomics and its shotgun approach to surveying all DNA within a sample (whole genome sequencing) affords the possibility to characterize not only the microbial diversity within a diabetes foot infection (i.e. 'which microorganisms are present') but the biological functions of the community such as virulence and pathogenicity (i.e. 'what are the microorganisms capable of doing'), moving the focus from single species as pathogens to groups of species. This review will examine the new molecular techniques for exploration of the microbiome of infected and uninfected diabetic foot ulcers, exploring the potential of these new technologies and postulating how they could translate to improved clinical care. Copyright © 2016 John Wiley & Sons, Ltd.

MALDI-TOF MS meets WGS in a VRE outbreak investigation.

The use of MALDI-TOF MS (matrix-assisted laser desorption/ ionization-time of flight mass spectrometry) and WGS (whole genome sequencing) has been described for identification and strain relatedness determination. We describe the complementary use of MALDI-TOF MS and WGS in a VRE (vancomycin-resistant enterococci) outbreak investigation, and discuss some of the challenges with defining strain similarity across these two platforms. Although both assays indicated multiple clusters involved in the outbreak of vancomycin resistant Enterococcus faecium isolates from positive blood cultures of four haematology-oncology patients, the small cohort and discrepancies between findings indicate the limitations of MALDI-TOF MS and the cautious interpretation of MALDI-TOF MS dendrograms during outbreaks. For definitive determination of the evolutionary distance between isolates, WGS can be used.

Staphylococcus aureus dry-surface biofilms are not killed by sodium hypochlorite: implications for infection control.

BACKGROUND: Dry hospital environments are contaminated with pathogenic bacteria in biofilms, which suggests that current cleaning practices and disinfectants are failing. AIM: To test the efficacy of sodium hypochlorite solution against Staphylococcus aureus dry-surface biofilms. METHODS: The Centers for Disease Control and Prevention Biofilm Reactor was adapted to create a dry-surface biofilm, containing 1.36 × 10(7)S. aureus/coupon, by alternating cycles of growth and dehydration over 12 days. Biofilm was detected qualitatively using live/dead stain confocal laser scanning microscopy (CLSM), and quantitatively with sonicated viable plate counts and crystal violet assay. Sodium hypochlorite (1000-20,000parts per million) was applied to the dry-surface biofilm for 10min, coupons were rinsed three times, and residual biofilm viability was determined by CLSM, plate counts and prolonged culture up to 16 days. Isolates before and after exposure underwent minimum inhibitory concentration (MIC) and minimum eradication concentration (MEC) testing, and one pair underwent whole-genome sequencing. FINDINGS: Hypochlorite exposure reduced plate counts by a factor of 7 log10, and reduced biofilm biomass by a factor of 100; however, staining of residual biofilm showed that live S. aureus cells remained. On prolonged incubation, S. aureus regrew and formed biofilms. Post-exposure S. aureus isolates had MICs and MECs that were not significantly different from the parent strains. Whole-genome sequencing of one pre- and post-exposure pair found that they were virtually identical. CONCLUSIONS: Hypochlorite exposure led to a 7-log kill but the organisms regrew. No resistance mutations occurred, implying that hypochlorite resistance is an intrinsic property of S. aureus biofilms. The clinical significance of this warrants further study.

Effects on cognitive and clinical insight with the use of Guided Self-Determination in outpatients with schizophrenia: A randomized open trial.

Poor insight has a negative impact on the outcome in schizophrenia; consequently, poor insight is a logical target for treatment. However, neither medication nor psychosocial interventions have been demonstrated to improve poor insight. A method originally designed for diabetes patients to improve their illness management, Guided Self-Determination (GSD), has been adapted for use in patients with schizophrenia (GSD-SZ). The purpose of this study was to investigate the effect on insight of GSD-SZ as a supplement to treatment as usual (TAU) as compared to TAU alone in outpatients diagnosed with schizophrenia. The design was an open randomized trial. The primary hypothesis was cognitive insight would improve in those patients who received GSD-SZ+TAU as assessed by the BCIS. We additionally explored whether the intervention led to changes in clinical insight, self-perceived recovery, self-esteem, social functioning and symptom severity. Assessments were conducted at baseline, and at 3-, 6- and 12-month follow-up. Analysis was based on the principles of intention to treat and potential confounders were taken into account through applying a multivariate approach. A total of 101 participants were randomized to GSD-SZ+TAU (n=50) or to TAU alone (n=51). No statistically significant differences were found on the cognitive insight. However, at 12-month follow-up, clinical insight (measured by G12 from the Positive and Negative Syndrome Scale), symptom severity, and social functioning had statistically significantly improved in the intervention group as compared to the control group. "Improving insight in patients diagnosed with schizophrenia", NCT01282307, http://clinicaltrials.gov/.

Second-generation antipsychotic effect on cognition in patients with schizophrenia--a meta-analysis of randomized clinical trials.

OBJECTIVE: To investigate the effect of second-generation antipsychotics on cognitive function in patients diagnosed with schizophrenia or schizoaffective disorder. METHOD: Multiple-treatments meta-analysis model. RESULTS: On cognitive composite score, sertindole was superior to clozapine, effect size (ES) 0.87; 95% CI: 0.12-1.63, quetiapine, ES 0.75; 95% CI: 0.00-1.49, and first-generation antipsychotics (FGAs), ES 0.89; 95% CI: 0.14-1.64. Analyses on each cognitive domain showed clozapine, ES 0.37; 95% CI: 0.00-0.74, olanzapine, ES 0.31; 95%CI: 0.02-0.59, quetiapine, ES 0.34; 95% CI: 0.03-0.64, and FGAs, ES 0.51; 95% CI: 0.18-0.83 performing poorer on verbal working memory than ziprasidone, as well as FGAs performing poorer than risperidone, ES 0.31; 95% CI: 0.04-0.58. On executive function, sertindole performed better than clozapine, ES 0.82; 95% CI: 0.06-1.58, olanzapine, ES 0.81; 95% CI: 0.07-1.55, quetiapine, ES 0.76; 95% CI: 0.02-1.51, ziprasidone, ES 0.90; 95% CI: 0.14-1.67, and FGAs, ES 0.83; 95% CI: 0.08-1.58. On processing speed, FGAs performed poorer than sertindole, ES 0.97; 95% CI: 0.02-1.91, and quetiapine, ES 0.36; 95% CI: 0.01-0.72. On long-term verbal working memory, clozapine performed poorer than olanzapine, ES 0.41; 95% CI: 0.06-0.76. On verbal fluency, FGAs performed poorer than olanzapine, ES 0.26; 95% CI: 0.01-0.50, and clozapine, ES 0.44; 95% CI: 0.06-0.81. Lastly, FGAs, ES 0.41; 95% CI: 0.04-0.78, and clozapine, ES 0.44; 95% CI: 0.05-0.83, performed poorer on visuospatial skill compared to olanzapine. CONCLUSION: The meta-analysis was able to detect some trends in the data analyzed, but did not show any drug having a uniform positive cognitive profile.

Changes in the diagnosed incidence of early onset schizophrenia over four decades.

OBJECTIVE: To explore changes in the diagnosed incidence of early onset schizophrenia (EOS) from 1971 to 2010. METHOD: Examination of incidence rates of schizophrenia in patients under 18 years of age, using a nationwide, population-based, mental health register. RESULTS: The age-standardized incidence rate (IR) of EOS in the period 1971-2010 was 3.17 (95% CI: 3.16, 3.18) per 100 000 person years in the age group 0-18 years, and 9.10 (95% CI: 9.00, 9.21) in the age group 12-18 years. In the period 1971-1993, the age-standardized IR of EOS was 1.80 (95% CI: 1.79, 1.82) per 100 000 person years in the age group 0-18 years, and 5.02 (95% CI: 4.92, 5.11) in the age group 12-18 years. In the period 1994-2010, the age-standardized IR of EOS was 5.15 (95% CI: 5.10, 5.20) per 100 000 person years in the age group 0-18 years, and 15.73 (95% CI: 15.22, 16.22) in the age group 12-18 years. The IR was higher for males than females in the periods 1971-1993 and 1971-2010, but in the period 1994-2010 the IR was higher for females than males. CONCLUSION: In recent years, the diagnosed incidence of EOS has increased and the usual male excess has disappeared. The changes in IR could be a result of changes in the diagnostic system, increased awareness of early psychosis or a reflection of actual underlying incidence of the disorder.

Carriage of an ACME II variant may have contributed to methicillin-resistant Staphylococcus aureus sequence type 239-like strain replacement in Liverpool Hospital, Sydney, Australia.

Approximately 39% of methicillin-resistant Staphylococcus aureus (MRSA) sequence type 239 (ST239)-like bloodstream isolates from Liverpool Hospital (obtained between 1997 and 2008) carry an arginine catabolic mobile element (ACME). Whole-genome sequencing revealed that an ACME II variant is located between orfX and SCCmec III, and based on pulsed-field gel electrophoresis patterns and temporal relationships of all ST239-like isolates (n = 360), ACME carriage may have contributed to subpulsotype strain replacement.

Cell division in Bacillus subtilis: FtsZ and FtsA association is Z-ring independent, and FtsA is required for efficient midcell Z-Ring assembly.

The earliest stage in cell division in bacteria is the assembly of a Z ring at the division site at midcell. Other division proteins are also recruited to this site to orchestrate the septation process. FtsA is a cytosolic division protein that interacts directly with FtsZ. Its function remains unknown. It is generally believed that FtsA localization to the division site occurs immediately after Z-ring formation or concomitantly with it and that FtsA is responsible for recruiting the later-assembling membrane-bound division proteins to the division site. Here, we report the development of an in vivo chemical cross-linking assay to examine the association between FtsZ and FtsA in Bacillus subtilis cells. We subsequently use this assay in a synchronous cell cycle to show that these two proteins can interact prior to Z-ring formation. We further show that in a B. subtilis strain containing an ftsA deletion, FtsZ localized at regular intervals along the filament but the majority of Z rings were abnormal. FtsA in this organism is therefore critical for the efficient formation of functional Z rings. This is the first report of abnormal Z-ring formation resulting from the loss of a single septation protein. These results suggest that in this organism, and perhaps others, FtsA ensures recruitment of the membrane-bound division proteins by ensuring correct formation of the Z ring.

Domain organisation in phosphomannose isomerases (types I and II).

Phosphomannose isomerase (PMI) types I and II were found to possess a conserved protein motif. This motif coincides with the catalytic site of the Candida albicans type I PMI, indicating a common catalytic process for both PMI types. The type II PMI are bifunctional enzymes possessing PMI and guanosine diphospho-D-mannose pyrophosphorylase (GMP) activity in separate catalytic domains, which in some species may function as separate proteins.