Evidence of bacterial biofilms within acute wounds: a systematic review.

OBJECTIVE: The prevalence and role of biofilm formation in acute wounds has seldom been investigated. Understanding the presence of biofilm in acute wounds would allow earlier, biofilm-targeted management, thus decreasing the morbidity and mortality associated with wound infection, improving patient experience and potentially reducing healthcare costs. The purpose of this study was to summarise the evidence for biofilm formation within acute wounds. METHOD: We conducted a systematic literature review for studies which reported evidence of bacterial biofilm formation in acute wounds. An electronic search of four databases was carried out, without restrictions on date. The search terms included 'bacteria', 'biofilm', 'acute' and 'wound'. RESULTS: A total of 13 studies met the inclusion criteria. Of the studies, 69.2% showed evidence of biofilm formation within 14 days of acute wound formation, with 38.5% showing evidence of biofilm 48 hours after wound formed. CONCLUSION: The evidence from this review suggests that biofilm formation plays a greater role within acute wounds than previously considered.

Assessment of mutations induced by cold atmospheric plasma jet treatment relative to known mutagens in Escherichia coli.

The main bactericidal components of cold atmospheric plasma (CAP) are thought to be reactive oxygen and nitrogen species (RONS) and UV-radiation, both of which have the capacity to cause DNA damage and mutations. Here, the mutagenic effects of CAP on Escherichia coli were assessed in comparison to X- and UV-irradiation. DNA damage and mutagenesis were screened for using a diffusion-based DNA fragmentation assay and modified Ames test, respectively. Mutant colonies obtained from the latter were quantitated and sequenced. CAP was found to elicit a similar mutation spectrum to X-irradiation, which did not resemble that for UV implying that CAP-produced RONS are more likely the mutagenic component of CAP. CAP treatment was also shown to promote resistance to the antibiotic ciprofloxacin. Our data suggest that CAP treatment has mutagenic effects that may have important phenotypic consequences.

An ESIPT Probe for the Ratiometric Imaging of Peroxynitrite Facilitated by Binding to Aß-Aggregates.

A series of 3-hydroxyflavone (3-HF) ESIPT (excited-state intramolecular proton transfer) boronate-based fluorescent probes have been developed for the detection of peroxynitrite (ONOO-). The dyes are environmentally sensitive, and each probe exhibited a ratiometric response toward ONOO- in a micellar environment. The probes were used to image different aggregation states of amyloid-ß (Aß) in the presence of ONOO-. The 3-HF-OMe probe was found to produce a ratiometric response toward ONOO- when bound to Aß aggregates, resulting in a novel host-guest ensemble, which adds insight into the development of other ESIPT-based probes for the simultaneous sensing of fibrous proteins/peptides and environmental ROS/RNS.

Investigating the lytic activity and structural properties of Staphylococcus aureus phenol soluble modulin (PSM) peptide toxins.

The ubiquitous bacterial pathogen, Staphylococcus aureus, expresses a large arsenal of virulence factors essential for pathogenesis. The phenol-soluble modulins (PSMs) are a family of cytolytic peptide toxins which have multiple roles in staphylococcal virulence. To gain an insight into which specific factors are important in PSM-mediated cell membrane disruption, the lytic activity of individual PSM peptides against phospholipid vesicles and T cells was investigated. Vesicles were most susceptible to lysis by the PSMα subclass of peptides (α1-3 in particular), when containing between 10 and 30mol% cholesterol, which for these vesicles is the mixed solid ordered (so)-liquid ordered (lo) phase. Our results show that the PSMß class of peptides has little effect on vesicles at concentrations comparable to that of the PSMα class and exhibited no cytotoxicity. Furthermore, within the PSMα class, differences emerged with PSMα4 showing decreased vesicle and cytotoxic activity in comparison to its counterparts, in contrast to previous studies. In order to understand this, peptides were studied using helical wheel projections and circular dichroism measurements. The degree of amphipathicity, alpha-helicity and properties such as charge and hydrophobicity were calculated, allowing a structure-function relationship to be inferred. The degree of alpha-helicity of the peptides was the single most important property of the seven peptides studied in predicting their lytic activity. These results help to redefine this class of peptide toxins and also highlight certain membrane parameters required for efficient lysis.

Bacteriophage Can Prevent Encrustation and Blockage of Urinary Catheters by Proteus mirabilis.

Proteus mirabilis forms dense crystalline biofilms on catheter surfaces that occlude urine flow, leading to serious clinical complications in long-term catheterized patients, but there are presently no truly effective approaches to control catheter blockage by this organism. This study evaluated the potential for bacteriophage therapy to control P. mirabilis infection and prevent catheter blockage. Representative in vitro models of the catheterized urinary tract, simulating a complete closed drainage system as used in clinical practice, were employed to evaluate the performance of phage therapy in preventing blockage. Models mimicking either an established infection or early colonization of the catheterized urinary tract were treated with a single dose of a 3-phage cocktail, and the impact on time taken for catheters to block, as well as levels of crystalline biofilm formation, was measured. In models of established infection, phage treatment significantly increased time taken for catheters to block (∼ 3-fold) compared to untreated controls. However, in models simulating early-stage infection, phage treatment eradicated P. mirabilis and prevented blockage entirely. Analysis of catheters from models of established infection 10 h after phage application demonstrated that phage significantly reduced crystalline biofilm formation but did not significantly reduce the level of planktonic cells in the residual bladder urine. Taken together, these results show that bacteriophage constitute a promising strategy for the prevention of catheter blockage but that methods to deliver phage in sufficient numbers and within a key therapeutic window (early infection) will also be important to the successful application of phage to this problem.

Exploiting the reversible covalent bonding of boronic acids: recognition, sensing, and assembly.

Boronic acids can interact with Lewis bases to generate boronate anions, and they can also bind with diol units to form cyclic boronate esters. Boronic acid based receptor designs originated when Lorand and Edwards used the pH drop observed upon the addition of saccharides to boronic acids to determine their association constants. The inherent acidity of the boronic acid is enhanced when 1,2-, 1,3-, or 1,4-diols react with boronic acids to form cyclic boronic esters (5, 6, or 7 membered rings) in aqueous media, and these interactions form the cornerstone of diol-based receptors used in the construction of sensors and separation systems. In addition, the recognition of saccharides through boronic acid complex (or boronic ester) formation often relies on an interaction between a Lewis acidic boronic acid and a Lewis base (proximal tertiary amine or anion). These properties of boronic acids have led to them being exploited in sensing and separation systems for anions (Lewis bases) and saccharides (diols). The fast and stable bond formation between boronic acids and diols to form boronate esters can serve as the basis for forming reversible molecular assemblies. In spite of the stability of the boronate esters' covalent B-O bonds, their formation is reversible under certain conditions or under the action of certain external stimuli. The reversibility of boronate ester formation and Lewis acid-base interactions has also resulted in the development and use of boronic acids within multicomponent systems. The dynamic covalent functionality of boronic acids with structure-directing potential has led researchers to develop a variety of self-organizing systems including macrocycles, cages, capsules, and polymers. This Account gives an overview of research published about boronic acids over the last 5 years. We hope that this Account will inspire others to continue the work on boronic acids and reversible covalent chemistry.

Development and early testing of a simple, low cost, fast sensor for maternal and neonatal group B Streptococcus.

Streptococcus agalactiae, (Group B Streptococcus (GBS)), is a common colonizer of the female vagina. In women giving birth it can be transmitted to the baby and cause serious illness and even death to the child. We have developed a biosensor comprising of phospholipids and fatty acids vesicles encapsulating high concentration, self-quenched carboxyfluorescein, which is released by the lysis of the vesicle by virulence factors expressed by GBS, becoming diluted and fluorescent. The microbial specificity of the sensor was tested against a number of GBS strains and other microbes including Candida albicans, Enterococcus faecalis and Staphylococcus epidermidis and a statistically significant response to GBS measured over these other microbes. To test the invivo efficacy of the biosensor, a pilot study using donated lower vaginal swabs from non-pregnant women was conducted, where 58 female adults were recruited. Participants donated two swabs, one which was used for the vesicle test and one for the 'gold standard', enriched culture media (ECM) test. An overall GBS carriage rate of 17.2% was measured using the ECM test. The vesicle biosensor test took 45 min to obtain a result, and showed a sensitivity of 83.3%, specificity of 85.7% and accuracy of 85.3%. The test accuracy is in line with current novel GBS identification tests, with the advantage of being rapid, easy to use, low-cost and able to be conducted by bedside during start of labour.

Effect of lipid and fatty acid composition of phospholipid vesicles on long-term stability and their response to Staphylococcus aureus and Pseudomonas aeruginosa supernatants.

Phospholipid vesicles have been the focus of attention as potential vehicles for drug delivery, as they are biomimetic, easy to produce, and contain an aqueous compartment which can be used to carry hydrophilic material, such as drugs or dyes. Lipid vesicles used for this purpose present a particular challenge, as they are not especially stable and can rapidly break down and release their contents away from the target area, especially at physiological temperatures/environments. This study aims to investigate optimum methods for vesicle stabilization where the vesicles are employed as part of a system or technology that signals the presence of pathogenic bacteria via the effect of secreted cytolytic virulence factors on a sensor interface. A number of approaches have been investigated and are presented here as a systematic study of the long-term (14 day) stability at 37 °C, and at various pHs. The response of vesicles, both in suspension and within hydrogels, to Staphylococcus aureus (RN 4282) and Pseudomonas aeruginosa (PAO1) whole bacteria, and supernatants from overnight cultures of both (containing secreted proteins but free of cells), was measured via a sensitive encapsulated carboxyfluorescein release assay. The results showed that lipid chain length, cholesterol concentration, and stabilization via photopolymer stable components were critical in achieving stability. Finally, dispersion of the optimum vesicle formulation in hydrogel matrixes was investigated, culminating in the in vivo demonstration of a simple prototype wound dressing.

Using electrocardiogram electrodes to monitor skin impedance spectroscopic response when skin is subjected to sustained static pressure.

Background: Impedance spectroscopy is a non-invasive technique which can be used to monitor skin barrier function, with potential applications in early-stage pressure ulcer detection. This paper describes how changes in skin impedance, due to mechanical damage of the stratum corneum by tape stripping or applied pressure, can be straightforwardly measured using commercial electrocardiogram electrodes and a relatively low-cost impedance analyser. Two models of pressure injury were studied, an ex vivo porcine and in vivo human skin model. Objectives: Determine whether impedance spectroscopy may have potential utility in measuring the effect on skin of applied pressure on early-stage pressure injury. Methods: Two models were utilized to measure the effect of pressure. Porcine model: 0, 7.5, 15 or 22.5 mmHg of pressure was applied for up to 24 h (N = 4) and monitored at various time intervals. Human Model: 88 mmHg of pressure was applied for four sets of three-minute intervals (N = 13) and post-pressure recovery was monitored for 4 h. For each model, skin impedance was monitored at 0.1 Hz-50 kHz using disposable Ag/AgCl electrodes. The data was analysed using Ordinary One-Way Analysis of Variance. Results: Porcine model: after 24 h, the impedance of pressure-loaded skin was significantly reduced compared to the non-loaded control group (p ≤ 0.0001); this reduction in impedance was proportional to the degree of mechanical loading. Histology images of skin cross-sections provided qualitative evidence that the epidermis was structurally compromised by pressure. Human Model: the response of healthy skin to applied pressure displayed inter-variation. Participants with a significant change in skin impedance (p ≤ 0.01) also demonstrated signs of erythema. Conclusions: This study suggests that using impedance spectroscopy to measure skin (stratum corneum) resistance may have utility in giving early warning of skin pressure injury prior to clinical symptoms, with a good correlation between observed erythema and reduction in skin resistance. Further work should be initiated on patients at risk of pressure injury to improve intervention strategies, including in darker skin tones where early-stage pressure injuries may not be visually distinct.

Detecting and monitoring incontinence associated dermatitis: Does impedance spectroscopy have a part to play?

In this review, current understanding of the prevention and treatment of Incontinence Associated Dermatitis (IAD) is discussed. The need for preventative measures which target specific faecal/urinary irritants is highlighted, including the role of urease inhibitors. There is no existing internationally and clinically accepted method to diagnose and categorise the severity of IAD. Diagnosis currently relies on visual inspection; non-invasive techniques to assess skin barrier function could remove subjectiveness, particularly in darker skin tones. Impedance spectroscopy is a non-invasive technique which can be used to monitor skin barrier function, supporting visual assessments. Six studies (2003-2021) which used impedance to assess dermatitis were reviewed; inflamed skin was distinguishable from healthy skin in each case. This suggests that impedance spectroscopy could be useful in diagnosis early-stage IAD, potentially enabling earlier intervention. Finally, the authors present their initial findings on the role of urease in skin breakdown in an in vivo IAD model, using impedance spectroscopy.

A TCF-based fluorescent probe to determine nitroreductase (NTR) activity for a broad-spectrum of bacterial species.

A nitroreductase (NTR) responsive fluorescent probe with long wavelength fluorescence emission was used to determine the NTR activity of a selection of bacterial species under a range of different bacterial growth conditions ensuring applicability under multiple complex clinical environments, where sensitivity, reaction time, and the detection accuracy were suitable for planktonic cultures and biofilms.

The development of boronic acids as sensors and separation tools.

Synthetic receptors for diols that incorporate boronic acid motifs have been developed as new sensors and separation tools. Utilizing the reversible interactions of diols with boronic acids to form boronic esters under new binding regimes has provided new hydrogel constructs that have found use as dye-displacement sensors and electrophoretic separation tools; similarly, molecular boronic-acid-containing chemosensors were constructed that offer applications in the sensing of diols. This review provides a somewhat-personal perspective of developments in boronic-acid-mediated sensing and separation, placed in the context of the seminal works of others in the area, as well as offering a concise summary of the contributions of the co-authors in the area.

Aß42 oligomers, but not fibrils, simultaneously bind to and cause damage to ganglioside-containing lipid membranes.

Aß (amyloid-ß peptide) assembles to form amyloid fibres that accumulate in senile plaques associated with AD (Alzheimer's disease). The major constituent, a 42-residue Aß, has the propensity to assemble and form soluble and potentially cytotoxic oligomers, as well as ordered stable amyloid fibres. It is widely believed that the cytotoxicity is a result of the formation of transient soluble oligomers. This observed toxicity may be associated with the ability of oligomers to associate with and cause permeation of lipid membranes. In the present study, we have investigated the ability of oligomeric and fibrillar Aß42 to simultaneously associate with and affect the integrity of biomimetic membranes in vitro. Surface plasmon field-enhanced fluorescence spectroscopy reveals that the binding of the freshly dissolved oligomeric 42-residue peptide binds with a two-step association with the lipid bilayer, and causes disruption of the membrane resulting in leakage from vesicles. In contrast, fibrils bind with a 2-fold reduced avidity, and their addition results in approximately 2-fold less fluorophore leakage compared with oligomeric Aß. Binding of the oligomers may be, in part, mediated by the GM1 ganglioside receptors as there is a 1.8-fold increase in oligomeric Aß binding and a 2-fold increase in permeation compared with when GM1 is not present. Atomic force microscopy reveals the formation of defects and holes in response to oligomeric Aß, but not preformed fibrillar Aß. The results of the present study indicate that significant membrane disruption arises from association of low-molecular-mass Aß and this may be mediated by mechanical damage to the membranes by Aß aggregation. This membrane disruption may play a key role in the mechanism of Aß-related cell toxicity in AD.

Variation in definitions of burn wound infection limits the validity of systematic review findings in burn care: A systematic review of systematic reviews.

INTRODUCTION: Systematic reviews (SR) of high-quality randomised controlled trials can identify effective treatments for burn wound infections (BWIs). Clinical heterogeneity in outcome definitions can prevent valid evidence synthesis, which may limit the reliability of the findings of SRs affected by this heterogeneity. This SR aimed to investigate whether there is variation BWI definitions across studies in SRs of burn care interventions and its impact on identification of effective treatments for patients with burn injuries. METHODS: A systematic search of five databases was conducted. Included SRs were: in English, published from January 2010 to October 2018, assessed intervention effects for patients with a burn injury, and reported data about BWI. RESULTS: Twenty-nine SRs, which included 248 studies reporting BWI outcomes, were included in our final dataset. Three SRs used a definition of BWI to select studies for inclusion. Fourteen reported BWI definitions from included studies in the review results. There was heterogeneity of BWI definition in their included studies; across 29 SRs, 32 different BWI indicators were used, with the median across SRs ranging from 1 to 7 (range 1-14). Fourteen SRs accounted for BWI definition heterogeneity in their conclusions, indicating that the issue impacted whether a conclusion could be drawn, and limited the validity of the SR findings. CONCLUSIONS: There is variation in BWI definition across SRs and within the studies included in SRs of interventions assessing BWI outcomes. This heterogeneity has prevented conclusions about intervention effects being drawn, and only half of the SR authors have accounted for it in their findings. Reviews that have collated this data without reference to the heterogeneity should be viewed with caution, since it may limit the validity of evidence for the identification of effective treatments for BWI. The use of a newly developed core indicator set to support consistent reporting of indicators and standardisation of BWI outcome reporting will enable effective evidence synthesis.

TCF-based fluorescent probe for monitoring superoxide anion produced in bacteria under chloramphenicol- and heat-induced stress.

We report on a superoxide anion (O2˙-) responsive fluorescent probe called TCF-OTf. TCF-OTf is able to monitor O2˙- production when the bacterial species Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Enterococcus faecalis are exposed to chloramphenicol and heat shock at 50 and 58 °C.

Optimisation of a lozenge-based sensor for detecting impending blockage of urinary catheters.

Catheter-associated urinary tract infections resulting from urease-positive microorganisms are more likely to cause a urinary catheter blockage owing to the urease activity of the microbes. Catheter blockage can be dangerous and increases the risk of severe infections, such as sepsis. Ureases, a virulence factor in Proteus mirabilis, cause an increase in urine pH - leading to blockage. An optimised biosensor "lozenge" is presented here, which is able to detect impending catheter blockage. This lozenge has been optimised to allow easy manufacture and commercialisation. It functions as a sensor in a physiologically representative model of a catheterised urinary tract, providing 6.7 h warning prior to catheter blockage. The lozenge is stable in healthy human urine and can be sterilized for clinical use by ethylene oxide. Clinically, the lozenge will provide a visible indication of impending catheter blockage, enabling quicker clinical intervention and thus reducing the morbidity and mortality associated with blockage.

TCF-ALP: a fluorescent probe for the selective detection of Staphylococcus bacteria and application in "smart" wound dressings.

Alkaline phosphatase (ALP) is an important enzyme-based biomarker present in several bacterial species; however, it is currently undervalued as a strategy to detect pathogenic bacteria. Here, we explore our ALP-responsive colorimetric and fluorescent probe (TCF-ALP) for such applications. TCF-ALP displayed a colorimetric and fluorescence response towards Staphylococcus aureus (S. aureus), with a limit of detection of 3.7 × 106 CFU mL-1 after 24 h incubation. To our surprise, TCF-ALP proved selective towards Staphylococcus bacteria when compared with Enterococcus faecalis (E. faecalis), and Gram-negative P. aeruginosa and E. coli. Selectivity was also seen in clinically relevant S. aureus biofilms. Owing to the high prevalence and surface location of S. aureus in chronic wounds, TCF-ALP was subsequently encapsulated in polyvinyl alcohol (PVA)-based hydrogels as a proof-of-concept "smart" wound dressing. TCF-ALP hydrogels were capable of detecting S. aureus in planktonic and biofilm assays, and displayed a clear colour change from yellow to purple after 24 h incubation using ex vivo porcine skin models. Overall, TCF-ALP is a simple tool that requires no prior knowledge, training, or specialist equipment, and has the potential to overcome issues related to invasive swabbing and tissue biopsy methods. Thus, TCF-ALP could be used as a tool to monitor the early development of infection in a wound and allow for the rapid provision of appropriate treatment for Staphylococcal bacterial infections.

A small-molecular inhibitor against Proteus mirabilis urease to treat catheter-associated urinary tract infections.

Infection and blockage of indwelling urinary catheters is significant owing to its high incidence rate and severe medical consequences. Bacterial enzymes are employed as targets for small molecular intervention in human bacterial infections. Urease is a metalloenzyme known to play a crucial role in the pathogenesis and virulence of catheter-associated Proteus mirabilis infection. Targeting urease as a therapeutic candidate facilitates the disarming of bacterial virulence without affecting bacterial fitness, thereby limiting the selective pressure placed on the invading population and lowering the rate at which it will acquire resistance. We describe the design, synthesis, and in vitro evaluation of the small molecular enzyme inhibitor 2-mercaptoacetamide (2-MA), which can prevent encrustation and blockage of urinary catheters in a physiologically representative in vitro model of the catheterized urinary tract. 2-MA is a structural analogue of urea, showing promising competitive activity against urease. In silico docking experiments demonstrated 2-MA's competitive inhibition, whilst further quantum level modelling suggests two possible binding mechanisms.

A thin film detection/response system for pathogenic bacteria.

This paper describes the modification of nonwoven fabric such that it responds by releasing an encapsulated antimicrobial from within an attached vesicle in response to two species of pathogenic bacteria (Staphylococcus aureus MSSA 476 and Pseudomonas aeruginosa PAO1), but does not respond to nonpathogenic Escherichia coli DH5alpha. This concept is based on the generalization that a majority of pathogenic bacteria secrete virulence factors such as toxins and lipases that actively damage cell membranes, typically observed as tissue damage around infected wounds, while nonpathogenic bacteria do not (or not at high concentration). The eventual aim of this work is to produce responsive dressings which release antimicrobials and change color only on infected wounds. This paper details preliminary approaches to achieving this goal, including vesicle-bacteria studies in aqueous suspension, and fluorescence imaging of fluorescein containing vesicles lysed by S. aureus and P. aeruginosa, but not by E. coli.

Photorhabdus adhesion modification protein (Pam) binds extracellular polysaccharide and alters bacterial attachment.

BACKGROUND: Photorhabdus are Gram-negative nematode-symbiotic and insect-pathogenic bacteria. The species Photorhabdus asymbiotica is able to infect humans as well as insects. We investigated the secreted proteome of a clinical isolate of P. asymbiotica at different temperatures in order to identify proteins relevant to the infection of the two different hosts. RESULTS: A comparison of the proteins secreted by a clinical isolate of P. asymbiotica at simulated insect (28 degrees C) and human (37 degrees C) temperatures led to the identification of a small and highly abundant protein, designated Pam, that is only secreted at the lower temperature. The pam gene is present in all Photorhabdus strains tested and shows a high level of conservation across the whole genus, suggesting it is both ancestral to the genus and probably important to the biology of the bacterium. The Pam protein shows limited sequence similarity to the 13.6 kDa component of a binary toxin of Bacillus thuringiensis. Nevertheless, injection or feeding of heterologously produced Pam showed no insecticidal activity to either Galleria mellonella or Manduca sexta larvae. In bacterial colonies, Pam is associated with an extracellular polysaccharide (EPS)-like matrix, and modifies the ability of wild-type cells to attach to an artificial surface. Interestingly, Surface Plasmon Resonance (SPR) binding studies revealed that the Pam protein itself has adhesive properties. Although Pam is produced throughout insect infection, genetic knockout does not affect either insect virulence or the ability of P. luminescens to form a symbiotic association with its host nematode, Heterorhabditis bacteriophora. CONCLUSIONS: We studied a highly abundant protein, Pam, which is secreted in a temperature-dependent manner in P. asymbiotica. Our findings indicate that Pam plays an important role in enhancing surface attachment in insect blood. Its association with exopolysaccharide suggests it may exert its effect through mediation of EPS properties. Despite its abundance and conservation in the genus, we find no evidence for a role of Pam in either virulence or symbiosis.