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The aims of our study are firstly to investigate the diagnostic and triage performance of symptom checkers, secondly to assess their potential impact on healthcare utilisation and thirdly to investigate for variation in performance between systems. Publicly available symptom checkers for patient use. Publicly available symptom-checkers were identified. A standardised set of 50 clinical vignettes were developed and systematically run through each system by a non-clinical researcher. System accuracy was assessed by measuring the percentage of times the correct diagnosis was a) listed first, b) within the top five diagnoses listed and c) listed at all. The safety of the disposition advice was assessed by comparing it with national guidelines for each vignette. Twelve tools were identified and included. Mean diagnostic accuracy of the systems was poor, with the correct diagnosis being present in the top five diagnoses on 51.0% (Range 22.2 to 84.0%). Safety of disposition advice decreased with condition urgency (being 71.8% for emergency cases vs 87.3% for non-urgent cases). 51.0% of systems suggested additional resource utilisation above that recommended by national guidelines (range 18.0% to 61.2%). Both diagnostic accuracy and appropriate resource recommendation varied substantially between systems. There is wide variation in performance between available symptom checkers and overall performance is significantly below what would be accepted in any other medical field, though some do achieve a good level of accuracy and safety of disposition. External validation and regulation are urgently required to ensure these public facing tools are safe.

A novel alternative to synthetic preservatives is the use of natural products such as essential oil (EO) as a natural food-grade preservative. EOs are Generally Recognized as Safe (GRAS), so they could be considered an alternative way to increase the shelf-life of highly perishable food products by impeding the proliferation of food-borne pathogens. The mounting interest within the food industry and consumer preference for “natural” and “safe” products means that scientific evidence on plant-derived essential oils (EOs) needs to be examined in-depth, including the underlying mechanisms of action. Understanding the mechanism of action that individual components of EO exert on the cell is imperative to design strategies to eradicate food-borne pathogens. Results from published works showed that most EOs are more active against Gram-positive bacteria than Gram-negative bacteria due to the difference in the cell wall structure. In addition, the application of EOs at a commercial scale has been minimal, as their flavour and odour could be imparted to food. This review provides a comprehensive summary of the research carried out on EOs, emphasizing the antibacterial activity of fruit peel EOs, and the antibacterial mechanism of action of the individual components of EOs. A brief outline of recent contributions of EOs in the food matrix is highlighted. The findings from the literature have been encouraging, and further research is recommended to develop strategies for the application of EO at an industrial scale.

A rapid and easy method that takes advantage of an inexpensive and portable fibre-based spectroscopic system (optrode) to determine the ratio of live to dead bacteria is proposed. Mixtures of live and dead Escherichia coli with proportions of live:dead cells varying from 0 to 100% were stained using SYTO 9 and propidium iodide (PI) and measured using the optrode. We demonstrated several approaches to obtaining the proportions of live:dead E. coli in a mixture of both live and dead, from analyses of the fluorescence spectra collected by the optrode. To find a suitable technique for predicting the percentage of live bacteria in a sample, four analysis methods were assessed and compared: SYTO 9:PI fluorescence intensity ratio, an adjusted fluorescence intensity ratio, single-spectrum support vector regression (SVR) and multi-spectra SVR. Of the four analysis methods, multi-spectra SVR obtained the most reliable results and was able to predict the percentage of live bacteria in 108 bacteria/mL samples between c. 7 and 100% live, and in 107 bacteria/mL samples between c. 7 and 73% live. By demonstrating the use of multi-spectra SVR and the optrode to monitor E. coli viability, we raise points of consideration for spectroscopic analysis of SYTO 9 and PI and aim to lay the foundation for future work that uses similar methods for different bacterial species.

Bacteria secrete RNAs, some of which have effects on other cells and on other species as signalling RNAs. Prokaryotic membrane vesicles (MVs) contain a range of RNA types. The MV structure offers protection from degradation as well as receptors to facilitate delivery to target cells. Microscopic imaging and molecular biology analyses have provided evidence to demonstrate that bacterial MVs deliver their RNA into eukaryotic cells. Moreover, in some cases the RNA cargo is demonstrably functional and phenotypic changes can be identified in MV-RNA treated target cells. The challenge now is to dissect the effect of MV-RNA on target cells away from the effects of non-RNA components of the MV such as lipopolysaccharide that can co-purify with RNA.

Bacterium-to-host signalling during infection is a complex process involving proteins, lipids and other diffusible signals that manipulate host cell biology for pathogen survival. Bacteria also release membrane vesicles (MV) that can carry a cargo of effector molecules directly into host cells. Supported by recent publications, we hypothesised that these MVs also associate with RNA, which may be directly involved in the modulation of the host response to infection. Using the uropathogenic Escherichia coli (UPEC) strain 536, we have isolated MVs and found they carry a range of RNA species. Density gradient centrifugation further fractionated and characterised the MV preparation and confirmed that the isolated RNA was associated with the highest particle and protein containing fractions. Using a new approach, RNA-sequencing of libraries derived from three different 'size' RNA populations (<50nt, 50-200nt and 200nt+) isolated from MVs has enabled us to now report the first example of a complete bacterial MV-RNA profile. These data show that MVs carry rRNA, tRNAs, other small RNAs as well as full-length protein coding mRNAs. Confocal microscopy visualised the delivery of lipid labelled MVs into cultured bladder epithelial cells and showed their RNA cargo labelled with 5-EU (5-ethynyl uridine), was transported into the host cell cytoplasm and nucleus. MV RNA uptake by the cells was confirmed by droplet digital RT-PCR of csrC. It was estimated that 1% of MV RNA cargo is delivered into cultured cells. These data add to the growing evidence of pathogenic bacterial MV being associated a wide range of RNAs. It further raises the plausibility for MV-RNA-mediated cross-kingdom communication whereby they influence host cell function during the infection process.

We have isolated a filamentous fungus that actively secretes a pigmented exudate when growing on agar plates. The fungus was identified as being a strain of Epicoccum nigrum. The fungal exudate presented strong antifungal activity against both yeasts and filamentous fungi, and inhibited the germination of fungal spores. The chemical characterization of the exudate showed that the pigmented molecule presenting antifungal activity is the disalt of epipyrone A—a water-soluble polyene metabolite with a molecular mass of 612.29 and maximal UV–Vis absorbance at 428 nm. This antifungal compound showed excellent stability to different temperatures and neutral to alkaline pH.

Prokaryotes release membrane vesicles (MVs) with direct roles in disease pathogenesis. MVs are heterogeneous when isolated from bacterial cultures so Density Gradient Centrifugation (DGC) is valuable for separation of MV subgroups from contaminating material. Here we report the technical variability and natural biological heterogeneity seen between DGC preparations of MVs for Mycobacterium smegmatis and Escherichia coli and compare these DGC data with size exclusion chromatography (SEC) columns. Crude preparations of MVs, isolated from cultures by ultrafiltration and ultracentrifugation were separated by DGC with fractions manually collected as guided by visible bands. Yields of protein, RNA and endotoxin, protein banding and particle counts were analysed in these. DGC and SEC methods enabled separation of molecularly distinct MV populations from crude MVs. DGC banding profiles were unique for each of the two species of bacteria tested and further altered by changing culture conditions, for example with iron supplementation. SEC is time efficient, reproducible and cost effective method that may also allow partial LPS removal from Gram-negative bacterial MVs. In summary, both DGC and SEC are suitable for the separation of mixed populations of MVs and we advise trials of both, coupled with complete molecular and single vesicle characterisation prior to downstream experimentation.

Polyaniline (PANI) and functionalised polyanilines (fPANI) are novel antimicrobial agents whose mechanism of action was investigated. Escherichia coli single gene deletion mutants revealed that the antimicrobial mechanism of PANI likely involves production of hydrogen peroxide while homopolymer poly(3-aminobenzoic acid), P3ABA, used as an example of a fPANI, disrupts metabolic and respiratory machinery, by targeting ATP synthase and causes acid stress. PANI was more active against E. coli in aerobic, compared to anaerobic, conditions, while this was apparent for P3ABA only in rich media. Greater activity in aerobic conditions suggests involvement of reactive oxygen species. P3ABA treatment causes an increase in intracellular free iron, which is linked to perturbation of metabolic enzymes and could promote reactive oxygen species production. Addition of exogenous catalase protected E. coli from PANI antimicrobial action; however, this was not apparent for P3ABA treated cells. The results presented suggest that PANI induces production of hydrogen peroxide, which can promote formation of hydroxyl radicals causing biomolecule damage and potentially cell death. P3ABA is thought to act as an uncoupler by targeting ATP synthase resulting in a futile cycle, which precipitates dysregulation of iron homeostasis, oxidative stress, acid stress, and potentially the fatal loss of proton motive force.

The rise in antibiotic resistance has stimulated research into adjuvants that can improve the efficacy of broad-spectrum antibiotics. Lactoferrin is a candidate adjuvant; it is a multifunctional iron-binding protein with antimicrobial properties. It is known to show dose-dependent antimicrobial activity against Staphylococcus aureus through iron sequestration and repression of β –lactamase expression. However, S. aureus can extract iron from lactoferrin through siderophores for their growth, which confounds the resolution of lactoferrin’s method of action. We measured the minimum inhibitory concentration (MIC) for a range of lactoferrin/ β –lactam antibiotic dose combinations and observed that at low doses (< 0.39 μM ), lactoferrin contributes to increased S. aureus growth, but at higher doses (> 6.25 μM ), iron-depleted native lactoferrin reduced bacterial growth and reduced the MIC of the β -lactam-antibiotic cefazolin. This differential behaviour points to a bacterial population response to the lactoferrin/ β –lactam dose combination. Here, with the aid of a mathematical model, we show that lactoferrin stratifies the bacterial population, and the resulting population heterogeneity is at the basis of the dose dependent response seen. Further, lactoferrin disables a sub-population from β -lactam-induced production of β -lactamase, which when sufficiently large reduces the population’s ability to recover after being treated by an antibiotic. Our analysis shows that an optimal dose of lactoferrin acts as a suitable adjuvant to eliminate S. aureus colonies using β -lactams, but sub-inhibitory doses of lactoferrin reduces the efficacy of β -lactams.

Objective: Fungal corneal infections are challenging to treat due to delayed diagnostic procedures, bacterial co-infections, and limited antifungal efficacy. This study investigates the therapeutic potential of ultraviolet C (UVC) light alone and combined with antifungal drugs. Methods: A subsurface infection model was developed in semi-solid agar droplets, with Candida albicans cells or Aspergillus brasiliensis spores inoculated into 0.75% w/v yeast peptone dextrose (YPD) agar in a 96-well microplate (5 µL per well). Two treatment groups were tested: (1) UVC exposure (265 nm, 1.93 mW/cm2) for durations of 0 s, 5 s, 10 s, 15 s, 30 s, 60 s, or 120 s, and (2) UVC combined with antifungal drugs (Amphotericin B and Natamycin) at their minimum inhibitory concentrations (MICs), determined in YPD broth. After treatment, agar droplets were homogenized, diluted, and plated for microbial enumeration. The most effective UVC doses were further tested in an ex vivo C. albicans porcine keratitis model, where the corneal epithelium was debrided, infected with C. albicans, and exposed to UVC. Corneas were then homogenized and plated to evaluate treatment efficacy. Results: UVC exposure of ≥15 s inhibited C. albicans, and ≥10 s inhibited A. brasiliensis (all p < 0.05). The broth MICs were 0.1875 µg/mL for Amphotericin B against C. albicans, 6.25 µg/mL against A. brasiliensis, and 0.78125 µg/mL for Natamycin against C. albicans, 7.8125 µg/mL against A. brasiliensis. The broth MIC did not eradicate fungi in the subsurface model. Combined treatments enhanced inhibition (all p < 0.05), with 30 s UVC + amphotericin B for C. albicans (p = 0.0218) and 30 s UVC + natamycin for A. brasiliensis (p = 0.0017). Ex vivo, 15 s and 30 s UVC inhibited growth (p = 0.0476), but no differences were seen between groups (all p > 0.05). Conclusion: UVC demonstrated strong antifungal efficacy, with supplementary benefits from combining UVC with low doses of antifungal drugs.