Search

AIM To develop a defined multispecies root canal biofilm model ex vivo, and to perform viable compositional analysis following D,L-2-hydroxyisocaproic acid (HICA), alpha-mangostin, Calcicur® , and Odontopaste® exposure. METHODOLOGY Time-kill assays were conducted in vitro using HICA, alpha-mangostin, Calcicur® , Odontopaste® , and saline solution on the planktonic cultures of C. albicans, E. faecalis, L. rhamnosus, and S. gordonii. Human root dentine blocks were prepared (n = 100) ex vivo, and multispecies suspensions containing each of 1.5 × 108 CFU/mL C. albicans, E. faecalis, L. rhamnosus, and S. gordonii in brain heart infusion (BHI) were incubated within the root canals for 21 days. Canals (n = 20/group) were then exposed to medicaments for 7 days. Samples taken from the inner (first 0.1 mm) and deeper (second 0.1 mm) dentine by drilling with Ash Steel Burs No. 5 and No. 6, and residual roots were cultured in broth for 24 h. Cell growth was detected by spectrophotometry and confirmed by culture on agar. The other set of inner dentine, deeper dentine, and residual root samples were sonicated, and then exposed with 50 μM PMA before DNA was extracted using the QIAamp DNA mini kit. Real-time quantitative PCR was performed to determine the biofilm composition as well as the number of live and total cells remaining in the biofilm following each treatment. The OD data were analysed with Kruskal-Wallis and Friedman with Wilcoxon signed-rank test between and within groups, respectively, agar culture and qPCR data with Pearson chi-square with Mann-Whitney and Cochran with McNemar tests, respectively (p

Molecular fungal genotyping techniques developed and employed for epidemiological studies have understandably concentrated on establishing the genetic diversity of Aspergillus fumigatus in invasive aspergillosis due to its severity, the urgency for treatment, and the need to demonstrate possible sources. Some early studies suggested that these strains were phenotypically, if not genotypically, different from others. However, with improved discrimination and evaluations, incorporating environmental as well as clinical isolates from other Aspergillus conditions (e.g., chronic pulmonary aspergillosis and cystic fibrosis), this premise is no longer upheld. Moreover, with the onset of increased global triazole resistance, there has been a concerted effort to incorporate resistance profiling into genotyping studies and the realisation that the wider population of non-immunocompromised aspergillosis patients are at risk. This review summarises the developments in molecular genotyping studies that incorporate resistance profiling with attention to chronic pulmonary aspergillosis and an example of our UK experience.

Background Infections caused by triazole drug-resistant Aspergillus fumigatus are an increasing problem. The sensitivity of standard culture is poor, abrogating susceptibility testing. Early detection of resistance can improve patient outcomes, yet tools for this purpose are limited. Objectives To develop and validate a pyrosequencing technique to detect resistance-conferring cyp51A polymorphisms from clinical respiratory specimens and A. fumigatus isolates. Methods Method validation was performed by Sanger sequencing and pyrosequencing of 50 A. fumigatus isolates with a spectrum of triazole susceptibility patterns. Then, 326 Aspergillus quantitative PCR (qPCR)-positive respiratory samples collected over a 27 month period (January 2017–March 2019) from 160 patients at the UK National Aspergillosis Centre were assessed by cyp51A pyrosequencing. The Sanger sequencing and pyrosequencing results were compared with those from high-volume culture and standard susceptibility testing. Results The cyp51A genotypes of the 50 isolates analysed by pyrosequencing and Sanger sequencing matched. Of the 326 Aspergillus qPCR-positive respiratory specimens, 71.2% were reported with no A. fumigatus growth. Of these, 56.9% (132/232) demonstrated a WT cyp51A genotype and 31.5% (73/232) a resistant genotype by pyrosequencing. Pyrosequencing identified the environmental TR34/L98H mutation in 18.7% (61/326) of the samples in contrast to 6.4% (21/326) pan-azole resistance detected by culture. Importantly, pyrosequencing detected resistance earlier than culture in 23.3% of specimens. Conclusions The pyrosequencing assay described could detect a wide range of cyp51A polymorphisms associated with triazole resistance, including those not identified by commercial assays. This method allowed prompt recognition of resistance and the selection of appropriate antifungal treatment when culture was negative.

The emergence of azole-resistant Aspergillus fumigatus (ARAf) complicates the treatment of aspergillosis and can nearly double the mortality from invasive aspergillosis (IA). ARAf has been isolated from many different environmental sites and indoor environments and thus presents a significant risk for susceptible patients. Local surveillance of environmental ARAf can guide antifungal prescribing and improve patient outcomes. In this study, seventy-four soils samples collected from the surface of a variety of root vegetables from farm shops and private gardens covering a wide geographical area of the UK, were cultured to assess the presence of A. fumigatus, and the prevalence and nature of any resistance mechanisms. A high-throughput in-house antifungal susceptibility screening method was developed and validated using the EUCAST MIC reference method, E.DEF 9.3.1. A total of 146 isolates were recovered and analysed. Even though the study premise was that soil-covered root vegetables and other fresh produce could represent a conduit for ARAf exposure in vulnerable patients, no ARAf were found in the soil samples despite 55% of samples harbouring A. fumigatus. The sample type and screening method used could be suitable for more extensive monitoring of the soil to detect trends in the prevalence of ARAf.

Management of biofilm infections relies on time-consuming laboratory techniques and monitoring treatment by subjective clinical evaluations. Due to these limitations, there is a need to explore alternative strategies. The aims of this study were to assess the feasibility of using volatile organic compound (VOC) biomarkers to monitor treatment response and measure anti-biofilm efficacy of electrical stimulation (ES) in vitro and in human cutaneous wound biofilm models. Staphylococcus aureus (MSSA) and Pseudomonas aeruginosa (PA) biofilms were exposed to ES, ciprofloxacin, or both, with efficacy assessed and quantified by fluorescence staining, enumeration, metabolic assays, and biomass quantification; VOCs were measured by gas chromatography-mass spectrometry. In vitro MSSA and PA and ex vivo PA biofilms exposed to ES showed significantly reduced bacterial viability, metabolic activity, and biomass compared to controls (p < 0.05). There was significant variation in the relative abundance of VOCs in in vitro MSSA and PA and in ex vivo PA biofilms exposed to ES and antibiotic (p < 0.05). 2-methyl-1-propanol was associated with MSSA viability (R = 0.93, p < 0.05), biomass (R = 0.97, p < 0.05), and metabolic activity (R = 0.93, p < 0.05) and 3-methyl-1-butanol was associated with PA biomass (R = 0.93, p < 0.05). We showed that ES and VOC biomarkers are possible options for alternative nonpharmacological antimicrobial management of biofilms and noninvasive monitoring of wound infection treatment responses, respectively.

Guidelines on the diagnosis and management of Aspergillus disease recommend a multi-test approach including CT scans, culture, fungal biomarker tests, microscopy and fungal PCR. The first-line treatment of confirmed invasive aspergillosis (IA) consists of drugs in the azole family; however, the emergence of azole-resistant isolates has negatively impacted the management of IA. Failure to detect azole-resistance dramatically increases the mortality rates of azole-treated patients. Despite drug susceptibility tests not being routinely performed currently, we suggest including resistance testing whilst diagnosing Aspergillus disease. Multiple tools, including DNA sequencing, are available to screen for drug-resistant Aspergillus in clinical samples. This is particularly beneficial as a large proportion of IA samples are culture negative, consequently impeding susceptibility testing through conventional methods. Pyrosequencing is a promising in-house DNA sequencing method that can rapidly screen for genetic hotspots associated with antifungal resistance. Pyrosequencing outperforms other susceptibility testing methods due to its fast turnaround time, accurate detection of polymorphisms within critical genes, including simultaneous detection of wild type and mutated sequences, and—most importantly—it is not limited to specific genes nor fungal species. Here we review current diagnostic methods and highlight the potential of pyrosequencing to aid in a diagnosis complete with a resistance profile to improve clinical outcomes.

Guidelines on the diagnosis and management of Aspergillus disease recommend a multi-test approach including CT scans, culture, fungal biomarker tests, microscopy and fungal PCR. The first-line treatment of confirmed invasive aspergillosis (IA) consists of drugs in the azole family; however, the emergence of azole-resistant isolates has negatively impacted the management of IA. Failure to detect azole-resistance dramatically increases the mortality rates of azole-treated patients. Despite drug susceptibility tests not being routinely performed currently, we suggest including resistance testing whilst diagnosing Aspergillus disease. Multiple tools, including DNA sequencing, are available to screen for drug-resistant Aspergillus in clinical samples. This is particularly beneficial as a large proportion of IA samples are culture negative, consequently impeding susceptibility testing through conventional methods. Pyrosequencing is a promising in-house DNA sequencing method that can rapidly screen for genetic hotspots associated with antifungal resistance. Pyrosequencing outperforms other susceptibility testing methods due to its fast turnaround time, accurate detection of polymorphisms within critical genes, including simultaneous detection of wild type and mutated sequences, and—most importantly—it is not limited to specific genes nor fungal species. Here we review current diagnostic methods and highlight the potential of pyrosequencing to aid in a diagnosis complete with a resistance profile to improve clinical outcomes.

The emergence of azole-resistant Aspergillus fumigatus (ARAf) complicates the treatment of aspergillosis and can nearly double the mortality from invasive aspergillosis (IA). ARAf has been isolated from many different environmental sites and indoor environments and thus presents a significant risk for susceptible patients. Local surveillance of environmental ARAf can guide antifungal prescribing and improve patient outcomes. In this study, seventy-four soils samples collected from the surface of a variety of root vegetables from farm shops and private gardens covering a wide geographical area of the UK, were cultured to assess the presence of A. fumigatus, and the prevalence and nature of any resistance mechanisms. A high-throughput in-house antifungal susceptibility screening method was developed and validated using the EUCAST MIC reference method, E.DEF 9.3.1. A total of 146 isolates were recovered and analysed. Even though the study premise was that soil-covered root vegetables and other fresh produce could represent a conduit for ARAf exposure in vulnerable patients, no ARAf were found in the soil samples despite 55% of samples harbouring A. fumigatus. The sample type and screening method used could be suitable for more extensive monitoring of the soil to detect trends in the prevalence of ARAf.

Aspergillus fumigatus is a critical pathogen of humans. Exposure to A. fumigatus conidia occurs frequently but is normally cleared from the respiratory airways. In contrast, individuals with respiratory diseases are often highly colonized by fungi. Here, we use genome-edited epithelial cells to show that the genetic variant rs35699176 in ZNF77 causes loss of integrity of the bronchial epithelium and increases levels of extracellular matrix proteins. These changes promote A. fumigatus conidial adhesion, germination and growth. RNA-seq and LC/MS-MS analysis reveal rs35699176 upregulates vesicle trafficking leading to an increment of adhesion proteins. These changes make cells carrying rs35699176 more receptive to A. fumigatus in the early stages of infection. Moreover, patients with fungal asthma carrying rs35699176+/− have higher A. fumigatus loads in their respiratory airway. Our results indicate ZNF77 as a key controller of Aspergillus colonization and suggest its utility as a risk-marker for patient stratification. Aspergillus fumigatus regularly colonises the lungs but in the context of respiratory disease can be associated with increased pathology. Here the authors show mutations in ZNF77 result in altered bronchial epithelial characteristics and enhanced fungal burden.

Biofilms are major contributors to delayed wound healing and there is a need for clinically relevant experimental models to assess theranostics. Microorganisms release volatile organic compounds (VOCs) and the ability to identify these in infected cutaneous wounds could lead to efficient non-invasive diagnosis. The aims here were to develop and assess bacterial biofilm formation and identify their VOC profiles in an in vitro model and validate in human ex vivo incisional and excisional cutaneous wound models. Biofilm development was assessed using multiple microscopy techniques with biofilm-forming deficient controls and quantified using metabolic and biomass assays; and VOC production measured by gas chromatography-mass spectrometry. The production of most VOCs was affected by biofilm development and model used. Some VOCs were specific either for planktonic or biofilm growth. The relative abundance of some VOCs was significantly increased or decreased by biofilm growth phase (P Staphylococcus aureus and Pseudomonas aeruginosa VOCs correlated with biofilm metabolic activity and biomass (R ≤ −0.5; ≥0.5). We present for the first time bacterial biofilm formation in human ex vivo cutaneous wound models and their specific VOC profiles. These models provide a vehicle for human skin-relevant biofilm studies and VOC detection has potential clinical translatability in efficient non-invasive diagnosis of wound infection.

Management of biofilm infections relies on time-consuming laboratory techniques and monitoring treatment by subjective clinical evaluations. Due to these limitations, there is a need to explore alternative strategies. The aims of this study were to assess the feasibility of using volatile organic compound (VOC) biomarkers to monitor treatment response and measure anti-biofilm efficacy of electrical stimulation (ES) in vitro and in human cutaneous wound biofilm models. Staphylococcus aureus (MSSA) and Pseudomonas aeruginosa (PA) biofilms were exposed to ES, ciprofloxacin, or both, with efficacy assessed and quantified by fluorescence staining, enumeration, metabolic assays, and biomass quantification; VOCs were measured by gas chromatography-mass spectrometry. In vitro MSSA and PA and ex vivo PA biofilms exposed to ES showed significantly reduced bacterial viability, metabolic activity, and biomass compared to controls (p0.05). There was significant variation in the relative abundance of VOCs in in vitro MSSA and PA and in ex vivo PA biofilms exposed to ES and antibiotic (p0.05). 2-methyl-1-propanol was associated with MSSA viability (R = 0.93, p0.05), biomass (R = 0.97, p0.05), and metabolic activity (R = 0.93, p0.05) and 3-methyl-1-butanol was associated with PA biomass (R = 0.93, p0.05). We showed that ES and VOC biomarkers are possible options for alternative nonpharmacological antimicrobial management of biofilms and noninvasive monitoring of wound infection treatment responses, respectively.

Chronic biofilm infections are often accompanied by a chronic inflammatory response, leading to impaired healing and increased, irreversible damage to host tissues. Biofilm formation is a major virulence factor forCandida albicansand a challenge for treatment. Most current antifungals have proved ineffective in eradicating infections attributed to biofilms. The biofilm structure protectsCandidaspecies against antifungals and provides a way for them to evade host immune systems. This leads to a very distinct inflammatory response compared to that seen in planktonic infections. Previously, we showed the superior efficacy ofdl-2-hydroxyisocaproic acid (HICA) against various bacteria and fungi. However, the immunomodulatory properties of HICA have not been studied. Our aim was to investigate the potential anti-inflammatory response to HICAin vivo. We hypothesized that HICA reduces the levels of immune mediators and attenuates the inflammatory response. In a murine model, a robust biofilm was formed for 5 days in a diffusion chamber implanted underneath mouse skin. The biofilm was treated for 12 h with HICA, while caspofungin and phosphate-buffered saline (PBS) were used as controls. The pathophysiology and immunoexpression in the tissues surrounding the chamber were determined by immunohistochemistry. Histopathological examination showed an attenuated inflammatory response together with reduced expression of matrix metalloproteinase 9 (MMP-9) and myeloperoxidase (MPO) compared to those of chambers containing caspofungin and PBS. Interestingly, the expression of developmental endothelial locus 1 (Del-1), an antagonist of neutrophil extravasation, increased after treatment with HICA. Considering its anti-inflammatory and antimicrobial activity, HICA may have enormous therapeutic potential in the treatment of chronic biofilm infections and inflammation, such as those seen with chronic wounds.

The amino acid derivative 2-hydroxyisocaproic acid (HICA) is a nutritional additive used to increase muscle mass. Low levels can be detected in human plasma as a result of leucine metabolism. It has broad antibacterial activity but its efficacy against pathogenic fungi is not known. The aim was to test the efficacy of HICA against Candida and Aspergillus species. Efficacy of HICA against 19 clinical and reference isolates representing five Candida and three Aspergillus species with variable azole antifungal sensitivity profiles was tested using a microdilution method. The concentrations were 18, 36 and 72�mg�ml(-1) . Growth was determined spectrophotometrically for Candida isolates and by visual inspection for Aspergillus isolates, viability was tested by culture and impact on morphology by microscopy. HICA of 72�mg�ml(-1) was fungicidal against all Candida and Aspergillus fumigatus and Aspergillus terreus isolates. Lower concentrations were fungistatic. Aspergillus flavus was not inhibited by HICA. HICA inhibited hyphal formation in susceptible Candida albicans and A. fumigatus isolates and affected cell wall integrity. In conclusion, HICA has broad antifungal activity against Candida and Aspergillus at concentrations relevant for topical therapy. As a fungicidal agent with broad-spectrum bactericidal activity, it may be useful in the topical treatment of multispecies superficial infections.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. Reactive oxygen species (ROS) is a novel therapeutic strategy for topical or local application to wounds, mucosa or internal structures where there may be heavy bacterial bioburden with biofilm and chronic inflammation. Bacterial biofilms are a significant problem in clinical settings owing to their increased tolerance towards conventionally prescribed antibiotics and their propensity for selection of further antibacterial resistance. There is therefore a pressing need for the development of alternative therapeutic strategies that can improve antibiotic efficacy towards biofilms. ROS has been successful in treating chronic wounds and in clearing multidrug-resistant organisms, including methicillin-resistant Staphylococcus aureus (MRSA), and carbapenemase-producing isolates from wounds and vascular line sites. There is significant antifungal activity of ROS against planktonic and biofilm forms. Nebulised ROS has been evaluated in limited subjects to assess reductions in bioburden in chronically colonised respiratory tracts. The antibiofilm activity of ROS could have great implications for the treatment of a variety of persistent respiratory conditions. Use of ROS on internal prosthetic devices shows promise. Avariety of novel delivery mechanisms are being developed to apply ROS activity to different anatomical sites.

Chronic pulmonary aspergillosis (CPA) is estimated to affect 3 million people worldwide making it an under recognised, but significant health problem across the globe, conferring significant morbidity and mortality. With variable disease forms, high levels of associated respiratory co-morbidity, limited therapeutic options and prolonged treatment strategies, CPA is a challenging disease for both patients and healthcare professionals. CPA can mimic smear-negativetuberculosis (TB), pulmonary histoplasmosis or coccidioidomycosis. Cultures for Aspergillus are usually negative, however, the detection of Aspergillus IgG is a simple and sensitive test widely used in diagnosis. When a fungal ball/aspergilloma is visible radiologically, the diagnosis has been madelate. Sometimes weight loss and fatigue are predominant symptoms; pyrexia is rare. Despite the efforts of the mycology community, and significant strides being taken in optimising the care of these patients, much remains to be learnt about this patient population, the disease itself and the best useof available therapies, with the development of new therapies being a key priority. Here, current knowledge and practices are reviewed, and areas of research priority highlighted.

This listing covers the period May 1, 1997 through to June 30, 1997. Citations are arranged in groups which roughly correspond with the British Mycological Society's Special Interest Committees. All correspondence about this item should be addressed to the Executive Editor. Reprints of this feature will not be available.