Biology subjects

Harrison, F.

Publications and source records attributed to Harrison, F..

At least 19 recordsLinked to original sources

bioRxiv2026

Tetrasodium EDTA disrupts Pseudomonas aeruginosa membrane integrity, shows suppressed resistance evolution and reduced cytotoxicity compared to meropenem

Pseudomonas aeruginosa remains one of the most important clinical pathogens for which new drugs are needed, due to its resistance machinery. Consequently, there is an increasing effort to develop new and effective treatments against this pathogen. We recently showed that tetrasodium ethylenediaminetetraacetic acid (tEDTA) exhibits promising antibacterial and antibiofilm activity against P. aeruginosa in advanced biofilm models. tEDTA is known to chelate divalent cations, with predicted effects on the outer membrane; however, a full understanding of how this kills P. aeruginosa is lacking. Also, it is currently not clear how slowly or rapidly P. aeruginosa will evolve resistance to this treatment. Using membrane disruption assays and RNA-seq, we showed that tEDTA disrupts bacterial membrane potential and permeabilises P. aeruginosa membranes. RNA-seq revealed the significant upregulation of genes involved in the transport of iron, phosphate, potassium, and magnesium ion. The arnABCD operon which is involved in lipid A biosynthesis was also upregulated. Using a 7-day evolutionary ramp approach, we showed that P. aeruginosa could not evolve resistance to tEDTA under strong selection. Lastly, we carried out a cytotoxicity assay with Human Epithelial type 2 (HEp-2) cells and showed that there was reduced cytotoxicity of tEDTA compared to meropenem. This study provides good insight into the mechanism of action of tEDTA and further evidence of its potential as an alternative to antibiotics for P. aeruginosa infections.

microbiology↗

bioRxiv2026

Biofilm-deficient mutants of Pseudomonas aeruginosa have wild-type levels of antibiotic tolerance in a model of cystic fibrosis lung infection

1Opportunistic, biofilm-forming pathogens such as Pseudomonas aeruginosa can employ an array of strategies to reduce the impact of antibiotics on their survival. The biofilm matrix can prevent antibiotics from reaching bacteria embedded within it; general changes in metabolic activity alter susceptibility to specific drugs dependent on the target; changes in the membrane and the expression of channel or pump proteins embedded within it affect drug uptake and efflux; and production of antibiotic-degrading enzymes can remove the threat. In this study, we report that biofilm-deficient mutants of two well-studied lab strains of P. aeruginosa (PA14 and PAO1) have wild-type (WT) levels of tolerance to colistin and meropenem when allowed to establish mature populations in an ex vivo pig lung model of cystic fibrosis lung infection. This result was unexpected, given previous work in our lab that suggested physical protection of cells from colistin by the biofilm matrix was important for tolerance. The biofilm defects in the mutants were confirmed using electron and light microscopy, and cryo scanning electron microscopy was used to visualise the hydrated biofilm matrix in the WT. Using RNA sequencing of the PA14 WT and an isogenic mutant lacking the pel polysaccharide, we were able to identify a small number of differences in the responses of the two genotypes to the lung environment and to exposure to sub-bactericidal colistin in the lung model. Notably, there was differential upregulation of the MexXY-OprM and MexEF-OprN multidrug efflux pumps. Hence, mature biofilm structure is not essential for tolerance, as other mechanisms may be sufficient in this environment. However, the relative roles of biofilm matrix versus cellular changes in physiology in conferring antibiotic tolerance in this environment remain to be fully elucidated.

microbiology↗

bioRxiv2026

Uncovering the multifaceted mechanism of action of a historical antimicrobial

Natural products have provided most of our modern pharmacopoeia, serving as active molecules or scaffolds for active molecules. Their use in drug development is often inspired by their traditional or historical medical use. For many decades, this discovery pipeline has focused on identifying a single molecule responsible for much of the biological activity of a raw natural product preparation (e.g. a whole-plant extract) and scoping this molecule for clinical potential. However, it is increasingly realised that historical/traditional remedies with significant biological activity may owe this activity to the combined action of multiple molecules. Concomitantly, microbiologists increasingly argue that effectively fighting antimicrobial-resistant infections will rely on combination therapies that combine multiple antimicrobials and/or adjuvant molecules. We previously reconstructed a complex historical remedy, Bald's eyesalve. Our reconstruction of this remedy had strong antibiofilm activity, which relied on the presence of multiple ingredients. Here, we report that Bald's eyesalve has multiple antibacterial effects on exemplar Gram-positive (Staphylococcus aureus) and Gram-negative (Acinetobacter baumannii) pathogens. Bald's eyesalve disrupts bacterial membrane integrity; inhibits expression of genes associated with bacterial adhesins, virulence factors and efflux pumps in both S. aureus and A. baumannii; inhibits quorum sensing in S. aureus; and causes downregulation of genes involved in de novo nucleotide biosynthesis in S. aureus. Lastly, we show that this multifaceted mechanism of action makes it difficult for S. aureus, A. baumannii, and Pseudomonas aeruginosa to evolve resistance against Bald's eyesalve. Bald's eyesalve could be used to identify a defined cocktail of natural products suitable for preclinical testing as a multi-target antibacterial preparation to which resistance may arise more slowly than current single-molecule antibiotics.

microbiology↗

bioRxiv2026

Tracking eco-evolutionary dynamics among lung pathobiome members from children with cystic fibrosis

Polymicrobial lung infections are common in individuals with cystic fibrosis (CF). Pathogen communities typically follow an ecological succession in which early colonizers such as Haemophilus influenzae and Staphylococcus aureus are later joined by Pseudomonas aeruginosa. Although adaptation to the lung environment is well described for single pathogens in adult people with CF, much less is known about the role of pathogen interactions and how changes in individual pathogens influence community dynamics at the early stages of disease. To address these questions, we combined genome sequencing with phenotypic screens and pathogen interaction assays using longitudinal clinical isolates (19 P. aeruginosa, 44 S. aureus, and 21 H. influenzae) collected from 23 children with CF (0-7.9 years of age) enrolled in the SCILD (Swiss CF Infant Lung Development) cohort. Our analysis revealed that early pathogen communities are characterized by a combination of strain turnover and persistence of isolates undergoing first steps of within-host evolution. Most notably, quorum-sensing-deficient P. aeruginosa variants repeatedly emerged, showing reduced protease production and diminished inhibition of S. aureus and H. influenzae. These changes indicate that P. aeruginosa becomes less antagonistic towards co-occurring pathogens, possibly promoting community stability. Together, our results show that ecological and evolutionary dynamics between pathobiome members may play an underappreciated role in shaping CF lung disease during early childhood.

evolutionary biology↗

bioRxiv2025

A Novel Stress Response Pathway Mediates Antibiotic Tolerance and Architecture in Pseudomonas aeruginosa Biofilms

Pseudomonas aeruginosa is a multidrug-resistant opportunistic pathogen, with chronic infections often associated with biofilm formation. Here, we investigate the previously uncharacterized gene PA3049, which is upregulated under biofilm conditions, to determine its role in infection, biofilm formation, and antimicrobial tolerance. We show that the small uncharacterised protein PA3049, renamed as Biofilm antibiotic tolerance Regulator (BatR), promotes biofilm establishment and enhances bacterial survival in sub-inhibitory concentrations of antibiotics. Proteomic analysis revealed that BatR influences the R2/F2 pyocin cluster, which drives explosive cell lysis and extracellular DNA (eDNA) release during biofilm development. We further identify a specific interaction between BatR and PA0486 (SrkA), an uncharacterised Ser/Thr protein kinase. We show that SrkA controls biofilm and pyocyanin production, and lysis-mediated eDNA release through regulation of the R2/F2 pyocin cluster and activation of bacteriophage Pf1. Our findings support a model in which SrkA directly regulates key biofilm-associated phenotypes, while BatR acts as a modulatory partner that tunes SrkA activity under specific conditions. Finally, BatR function was tested in high- validity infection models, including the ex vivo pig lung model of cystic fibrosis infection and a synthetic chronic-wound model. In these models, BatR contributes to biofilm architecture and antibiotic tolerance, and modulates pyocyanin production. Our study implicates the BatR/SrkA system in the response of P. aeruginosa biofilms to antibiotic challenge in lung infections.

microbiology↗

bioRxiv2025

Low concentrations of tetrasodium EDTA cause significant killing of biofilm-associated P. aeruginosa in high validity models of chronic wound and CF lung infections but not in a model of endotracheal tube colonisation

Pseudomonas aeruginosa is a pathogen notorious for its antimicrobial resistance and is currently classified as a high-priority pathogen for which new drugs are needed. Tetrasodium EDTA (tEDTA) is one of the new antimicrobial compounds that have been shown to have good antibacterial and antibiofilm efficacy against P. aeruginosa. Due to the diversity and highly drug-tolerant nature of P. aeruginosa biofilms in different infection environments, it is important to carry out pre-clinical testing of new antibiofilm agents against this pathogen in media and models that accurately mimic diverse infection environments. In this study, we used different high validity media and biofilm models that mimic chronic wounds, endotracheal tubes, and cystic fibrosis lung infections to assess the efficacy of tEDTA against P. aeruginosa biofilms. We report that different infection environments influence the susceptibility of both planktonic and biofilm forms of P. aeruginosa to tEDTA. The highest tolerance to tEDTA was observed in the media and biofilm model that mimics the endotracheal tube environment. In conclusion, we show that although different infection environments influence the efficacy of tEDTA against P. aeruginosa biofilms, it has good potential for use as an alternative antimicrobial in treating P. aeruginosa-associated biofilm infections.

microbiology↗

bioRxiv2025

Optimising synthetic cystic fibrosis sputum media for growth of non-typeable Haemophilus influenzae

Non-typeable Haemophilus influenzae (NTHi) is an early pathogen isolated from the lungs of children with cystic fibrosis (CF). However, its role in the progression of CF lung infection is poorly understood. Additionally, whether it forms biofilms in the lungs of people with CF is an open question. The development of synthetic cystic fibrosis sputum media has given key insights into the microbiology of later CF pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, through replicating the chemical composition of CF sputum. However, growth of NTHi in these media has not previously been reported. We show that NTHi grows poorly in three variants of synthetic cystic fibrosis sputum media commonly used to induce in vivo -like growth of P. aeruginosa and S. aureus (SCFM1, SCFM2 and SCFM3). The addition of NAD and hemin to SCFM1 and SCFM2 promoted the planktonic growth and biofilm formation of both laboratory and clinical NTHi isolates, and we were able to develop a modified variant of SCFM2 that allows culture of NTHis. We show that NTHi cannot be identified in an established ex-vivo model of CF infection, which uses SCFM and porcine bronchiolar tissue. This may in part be due to the presence of endogenous bacteria on the pig lung tissue which outcompete NTHi, but the lack of selective agar to isolate NTHi from endogenous bacteria, and the fact that NTHi is an exclusively human pathogen, make it hard to conclude that this is the case. Through spiking modified SCFM2 with filter sterilized lung homogenate, biofilm growth of clinical NTHi isolates was enhanced. Our results highlight that there are crucial components present in the lung tissue which NTHi require for growth, that are not present in any published variant of SCFM from the Palmer et al. 2007 lineage. Our results may inform future modifications to SCFM recipes to truly mimic the environment of CF lung sputum, and thus, to facilitate study of a wide range of CF pathogens. Data SummaryThe authors confirm that all supporting data, code and protocols have been provided within the article or through supplementary data files. All raw data has been uploaded to FigShare (https://doi.org/10.6084/m9.figshare.28175300.v1).

microbiology↗

bioRxiv2024

BatR: A novel regulator of antibiotic tolerance in Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa is a multidrug-resistant opportunistic human pathogen. Chronic infections are associated with biofilms, conferring resistance to antibiotics and complicating treatment strategies. This study focuses on understanding the role of the uncharacterized gene PA3049, upregulated under biofilm conditions. In the context of P. aeruginosa biofilms, PA3049 plays a role in withstanding antimicrobial challenges both in vitro and in clinically validated infection models. Under sub-inhibitory concentrations of antibiotic, the deletion of PA3049 resulted in reduced pyocyanin production and altered abundance of enzymes controlling denitrification, pyoverdine, and hydrogen cyanide biosynthesis. Notably, PA3049 directly interacts with two kinases implicated in stress response, inactivating their active sites. Renamed as the Biofilm antibiotic tolerance Regulator (BatR), PA3049 is a key player in P. aeruginosa biofilm maintenance and antimicrobial tolerance. These findings contribute to understanding the complex bacterial lifestyle in biofilms, shedding light on a previously uncharacterized gene with significant implications for combating multidrug-resistant infections. IMPORTANCEP. aeruginosa is a multidrug-resistant ESKAPE pathogen that causes chronic biofilm-based infections and is a leading cause of mortality in cystic fibrosis (CF) patients. Understanding the molecular mechanisms underlying P. aeruginosa biofilm resilience and antimicrobial resistance is crucial for developing effective therapeutic interventions. This study focuses on characterizing the gene PA3049, now known as the biofilm antibiotic tolerance Regulator (batR). BatR plays a central role within P. aeruginosa biofilms, orchestrating adaptive responses to antimicrobial challenges. Our work sheds light on the contribution of batR to biofilm biology and its relevance in lung infections, where subinhibitory antibiotic concentrations make BatR pivotal for bacterial survival. By advancing our understanding of P. aeruginosa biofilm regulation, this study holds significant promise for the development of innovative approaches against biofilm-associated infections to mitigate the growing threat of antimicrobial resistance.

microbiology↗

bioRxiv2024

A new model of endotracheal tube biofilm identifies combinations of matrix-degrading enzymes and antimicrobials able to eradicate biofilms of pathogens that cause ventilator-associated pneumonia

Defined as a pneumonia occurring after more than 48 hours of mechanical ventilation via an endotracheal tube, ventilator-associated pneumonia results from biofilm formation on the indwelling tube, seeding the patients lower airways with pathogenic microbes such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Candida albicans. Currently there is a lack of accurate in vitro models of ventilator-associated pneumonia development. This greatly limits our understanding of how the in-host environment alters pathogen physiology and the efficacy of ventilator-associated pneumonia prevention or treatment strategies. Here, we showcase a reproducible model that simulates biofilm formation of these pathogens in a host-mimicking environment, and demonstrate that the biofilm matrix produced differs from that observed in standard laboratory growth medium. In our model, pathogens are grown on endotracheal tube segments in the presence of a novel synthetic ventilator airway mucus (SVAM) medium that simulates the in-host environment. Matrix-degrading enzymes and cryo-SEM were employed to characterise the system in terms of biofilm matrix composition and structure, as compared to standard laboratory growth medium. As seen in patients, the biofilms of ventilator-associated pneumonia pathogens in our model either required very high concentrations of antimicrobials for eradication, or could not be eradicated. However, combining matrix-degrading enzymes with antimicrobials greatly improved biofilm eradication of all pathogens. Our in vitro endotracheal tube (IVETT) model informs on fundamental microbiology in the ventilator-associated pneumonia context, and has broad applicability as a screening platform for antibiofilm measures including the use of matrix-degrading enzymes as antimicrobial adjuvants. ImportanceThe incidence of ventilator-associated pneumonia in mechanically ventilated patients is between 5-40%, increasing to 50-80% in patients suffering from coronavirus disease 2019 (COVID-19). The mortality rate of ventilator-associated pneumonia patients can reach 45%. Treatment of the endotracheal tube biofilms that cause ventilator-associated pneumonia is extremely challenging, with causative organisms able to persist in endotracheal tube biofilm despite appropriate antimicrobial treatment in 56% of ventilator-associated pneumonia patients. Flawed antimicrobial susceptibility testing often means that ventilator-associated pneumonia pathogens are insufficiently treated, resulting in patients experiencing ventilator-associated pneumonia recurrence. Here we present an in vitro endotracheal tube biofilm model that recapitulates key aspects of endotracheal tube biofilms, including dense biofilm growth and elevated antimicrobial tolerance. Thus our biofilm model can be used as a ventilated airway simulating environment, aiding the development of anti-ventilator-associated pneumonia therapies and antimicrobial endotracheal tubes that can one day improve the clinical outcomes of mechanically ventilated patients.

microbiology↗

bioRxiv2023

Differential development of antibiotic resistance and virulence between Acinetobacter species

The two species that account for most cases of Acinetobacter-associated bacteraemia in the UK are Acinetobacter lwoffii, often a commensal but also an emerging pathogen, and A. baumannii, a well-known antibiotic-resistant species. While these species both cause similar types of human infection and occupy the same niche, A. lwoffii (unlike A. baumannii) has thus far remained susceptible to antibiotics. Comparatively little is known about the biology of A. lwoffii and this is the largest study on it conducted to date, providing valuable insights into its behaviour and potential threat to human health. This study aimed to explain the antibiotic susceptibility, virulence, and fundamental biological differences between these two species. The relative susceptibility of A. lwoffii, was explained as it encoded fewer antibiotic resistance and efflux pump genes than A. baumannii (9 and 30 respectively). While both species had markers of horizontal gene transfer, A. lwoffii encoded more DNA defence systems and harboured a far more restricted range of plasmids. Furthermore, A. lwoffii displayed a reduced ability to select for antibiotic resistance mutations, form biofilm and infect both in vivo and in vitro models of infection. This study suggests that the emerging pathogen A. lwoffii has remained susceptible to antibiotics because mechanisms exist to make it highly selective about the DNA it acquires, and we hypothesise that the fact that it only harbours a single RND system restricts the ability to select for resistance mutations. This provides valuable insights into how development of resistance can be constrained in Gram negative bacteria. ImportanceAcinetobacter lwoffii is often a harmless commensal but is also an emerging pathogen and is the most common cause of Acinetobacter-derived blood stream infections in England and Wales. In contrast to the well-studied, and often highly drug resistant A. baumannii, A. lwoffii has remained susceptible to antibiotics. This study explains why this organism has not evolved resistance to antibiotics. These new insights are important to understand why and how some species develop antibiotic resistance, while others do not and could inform future novel treatment strategies.

microbiology↗

bioRxiv2023

Sweet and sour synergy: exploring the antibacterial and antibiofilm activity of acetic acid and vinegar combined with medical-grade honeys

Oxymel, a combination of honey and vinegar, has been used as a remedy for wounds and infections from antiquity to the present day. While honey is now clinically used to treat infected wounds, this use of a complex, raw natural product (NP) mixture is unusual in modern western medicine. Research into the antimicrobial activity of NPs more usually focusses on finding a single active compound. The acetic acid in vinegar is known to have antibacterial activity at low concentrations and is in clinical use to treat burn wound infections. Here, we investigated the potential for synergistic activity of different compounds present in a complex ingredient used in historical medicine (vinegar) and in an ingredient mixture (oxymel). We conducted a systematic review to investigate published evidence for antimicrobial effects of vinegars against human pathogenic bacteria and fungi. No published studies explicitly compared the activity of vinegar with that of a comparable concentration of acetic acid. We then characterised selected vinegars by high-performance liquid chromatography (HPLC) and assessed the antibacterial and antibiofilm activity of the vinegars and acetic acid, alone and in combination with medical-grade honeys, against P. aeruginosa and S. aureus. We found that some vinegars have antibacterial activity that exceeds that predicted by their acetic acid content alone, but that this depends on the bacterial species being investigated and the growth conditions (media type, planktonic vs. biofilm). Pomegranate vinegars may be particularly interesting candidates for further study. We also conclude that there is potential for acetic acid, and some vinegars, to show synergistic antibiofilm activity with manuka honey. Data SummaryRaw quantitative data and R code for analyses are provided in the supplementary data (Document S1).

microbiology↗

bioRxiv2022

Pseudomonas aeruginosa transcriptome analysis in a cystic fibrosis lung model reveals metabolic changes accompanying biofilm maturation and increased antibiotic tolerance over time.

The opportunistic pathogen Pseudomonas aeruginosa forms biofilm infections in the lungs of people with the genetic condition cystic fibrosis (CF) that can persist for decades. There are numerous P. aeruginosa lifestyle changes associated with chronic biofilm infection cued by the CF lung environment. These include a loss of virulence, metabolic changes and increased antimicrobial tolerance. We have investigated P. aeruginosa PA14 biofilm infection over 7 d in an ex vivo pig lung (EVPL) model for CF, previously shown to facilitate formation of a clinically relevant P. aeruginosa biofilm structure with expression of key genes comparable to human infection. We have compared P. aeruginosa gene expression between sequential time points: 24 h, 48 h and 7 d post infection, and investigated tolerance to polymyxins. Our results demonstrate that the EVPL model can maintain a P. aeruginosa biofilm population, which exhibits increased antibiotic tolerance, for at least 7 d. Differential expression of antimicrobial resistance-associated genes was not observed, however there was significant upregulation of sulfur metabolism and maintenance of a structured biofilm. Our findings provide further insight into the increased P. aeruginosa antibiotic tolerance during chronic infection of the CF lung, and suggest we can cue aspects of chronic infection in 7 d under the right lab conditions.

microbiology↗

bioRxiv2021

An assessment of the evidence for antibacterial activity of stinging nettle (Urtica dioica) extracts

Stinging nettles (Urtica spp.) have been used in a diverse range of traditional and historical medicines from around the world for the treatment of skin diseases, wounds, urinary disorders, respiratory diseases, bone and joint pain, anaemia and other circulatory problems, as well as in cosmetic preparations for skin and haircare. As part of an interdisciplinary exploration of nettle-based remedies, we performed a systematic review of published evidence for the antimicrobial activity of Urtica spp. extracts against bacteria and fungi that commonly cause skin, soft tissue and respiratory infections. We focussed on studies in which minimum inhibitory concentration (MIC) assays of U. dioica were conducted on the common bacterial opportunistic pathogens Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus. No studies used fresh leaves (all were dried prior to use), and no studies prepared nettles in weak acid (corresponding to vinegar) or in fats/oils, which are common combinations in historical and traditional preparations. We addressed this gap by conducting new antibacterial tests of extracts of fresh U. dioica leaves prepared in vinegar, butter or olive oil against P. aeruginosa and S. aureus. Our systematic review and additional experimental data leads us to conclude that there is no strong evidence for nettles containing molecules with clinically useful antimicrobial activity. It seems most likely that the utility of nettles in traditional topical preparations for wounds may simply be as a "safe" absorbent medium for keeping antibacterial (vinegar) or emollient (oils) ingredients at the treatment site. Data SummaryAll data relating to the systematic review are supplied in the Datasets and Supplementary tables contained in the Supporting Data file.

microbiology↗

bioRxiv2021

Transcriptome analysis of Pseudomonas aeruginosa biofilm infection in an ex vivo pig model of the cystic fibrosis lung

Pseudomonas aeruginosa is the predominant cause of chronic biofilm infections that form in the lungs of people with cystic fibrosis (CF). These infections are highly resistant to antibiotics and persist for years in the respiratory tract. One of the main research challenges is that current laboratory models do not accurately replicate key aspects of a chronic P. aeruginosa biofilm infection, highlighted by previous RNA-sequencing studies. We compared the P. aeruginosa PA14 transcriptome in an ex vivo pig lung (EVPL) model of CF and a well-studied synthetic cystic fibrosis sputum medium (SCFM). P. aeruginosa was grown in the EVPL model for 1, 2 and 7 days, and in vitro in SCFM for 1 and 2 days. The RNA was extracted and sequenced at each time point. Our findings demonstrate that expression of antimicrobial resistance genes was cued by growth in the EVPL model, highlighting the importance of growth environment in determining accurate resistance profiles. The EVPL model created two distinct growth environments: tissue-associated biofilm and the SCFM surrounding tissue, each of which cued a transcriptome distinct from that seen in SCFM in vitro. The expression of quorum sensing associated genes in the EVPL tissue-associated biofilm at 48 h relative to in vitro SCFM was found to be similar to CF sputum versus in vitro conditions. Hence, the EVPL model can replicate key aspects of in vivo biofilm infection that are missing from other current models and provides a more accurate P. aeruginosa growth environment for determining antimicrobial resistance. Significance StatementPseudomonas aeruginosa lung infections that affect people with cystic fibrosis are a well-researched area, yet they continue to evade antimicrobial treatments. The lack of a laboratory model that captures all key aspects of these infections hinders not only research progression but also clinical diagnostics. We used transcriptome analysis to demonstrate how a model using pig lungs can more accurately replicate key characteristics of P. aeruginosa lung infection, including mechanisms of antibiotic resistance and infection establishment. Therefore, this model may be used in the future to further understand infection dynamics to develop novel treatments and more accurate treatment plans. This could improve clinical outcomes as well as quality of life for individuals affected by these infections.

microbiology↗

bioRxiv2020

Effect of host-mimicking medium and biofilm growth on the ability of colistin to kill Pseudomonas aeruginosa.

In vivo biofilms cause recalcitrant infections with extensive and unpredictable antibiotic tolerance. Here, we demonstrate increased tolerance of colistin by Pseudomonas aeruginosa when grown in cystic fibrosis-mimicking medium versus standard medium in in vitro biofilm assays, and drastically increased tolerance when grown in an ex vivo CF model versus the in vitro assay. We used colistin conjugated to the fluorescent dye BODIPY to assess the penetration of the antibiotic into ex vivo biofilms and showed that poor penetration partly explains the high doses of drug necessary to kill bacteria in these biofilms. The ability of antibiotics to penetrate the biofilm matrix is key to their clinical success, but hard to measure. Our results demonstrate both the importance of reduced entry into the matrix in in vivo-like biofilm, and the tractability of using a fluorescent tag and benchtop fluorimeter to assess antibiotic entry into biofilms. This method could be a relatively quick, cheap and useful addition to diagnostic and R&D pipelines, allowing the assessment of drug entry into biofilms, in in vivo-like conditions, prior to more detailed tests of biofilm killing.

microbiology↗

bioRxiv2020

The safety profile of Bald's eyesalve for the treatment of bacterial infections

The rise in antimicrobial resistance has prompted the development of alternatives, such as plant-derived compounds, to combat bacterial infections. Balds eyesalve, a remedy used in the Early Medieval period, has previously been shown to have efficacy against Staphylococcus aureus grown in an in vitro model of soft tissue infection. This remedy also had bactericidal activity against methicillin-resistant S. aureus (MRSA) in a chronic mouse wound. However, the safety profile of Balds eyesalve has not yet been demonstrated, and this is vital before testing in humans. Here, we determined the safety potential of Balds eyesalve using in vitro, ex vivo, and in vivo models representative of skin or eye infections. We also confirmed that Balds eyesalve is active against an important eye pathogen, Neisseria gonorrhoeae. Low levels of cytotoxicity were observed in eyesalve-treated cell lines representative of skin and immune cells. Results from a bovine corneal opacity and permeability test demonstrated slight irritation to the cornea that resolved within 10 minutes. The slug mucosal irritation assay revealed that a low level of mucus was secreted by slugs exposed to eyesalve, indicating mild mucosal irritation. We obtained promising results from mouse wound closure experiments; no visible signs of irritation or inflammation were observed. Our results suggest that Balds eyesalve could be tested further on human volunteers to assess safety for topical application against bacterial infections. ImportanceAlternative treatment for bacterial infections are needed to combat the ever increasing repertoire of bacteria resistant to antibiotics. A medieval plant-based remedy, Balds eyesalve, shows promise as a substitute for the treatment of these infections. For any substance to be effective in the treatment of bacterial infections in humans, it is important to consider the safety profile. This is a key consideration in order to have the necessary regulatory approval. We demonstrate the safety profile of Balds eyesalve using a variety of models, including whole-organ and whole-animal models. Our results show that Balds eyesalve is mildly toxic to cultured human cells, but potentially suitable for patch tests on healthy human volunteers to assess safety for later clinical trials. Our work has the potential to transform the management of diseases caused by bacterial infections, such as diabetic foot ulcers, through topical application of a natural product cocktail based on Balds eyesalve.

microbiology↗

bioRxiv2020

Anti-biofilm efficacy of a medieval treatment for bacterial infection requires the combination of multiple ingredients.

Novel antimicrobials are urgently needed to combat the increasing occurrence of drug-resistant bacteria and to overcome the inherent difficulties in treating biofilm-associated infections. Research into natural antimicrobials could provide candidates to fill the antibiotic discovery gap, and the study of plants and other natural materials used in historical infection remedies may enable further discoveries of natural products with useful antimicrobial activity. We previously reconstructed a 1,000-year-old remedy containing onion, garlic, wine, and bile salts, which is known as Balds eyesalve, and showed it to have promising antibacterial activity. In this paper, we have found this remedy has bactericidal activity against a range of Gram-negative and Gram-positive wound pathogens in planktonic culture and, crucially, that this activity is maintained against Acinetobacter baumannii, Stenotrophomonas maltophilia, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes in a model of soft-tissue wound biofilm. While the presence of garlic in the mixture is sufficient to explain activity against planktonic cultures, garlic alone has no activity against biofilms. We have found the potent anti-biofilm activity of Balds eyesalve cannot be attributed to a single ingredient and requires the combination of all ingredients to achieve full activity. Our work highlights the need to explore not only single compounds but also mixtures of natural products for treating biofilm infections. These results also underline the importance of working with biofilm models when exploring natural products for the anti-biofilm pipeline. ImportanceBacteria can live in two ways, as individual planktonic cells or as a multicellular biofilm. Biofilm helps protect bacteria from antibiotics and makes them much harder to treat. Both the biofilm lifestyle and the evolution of antibiotic resistance mean we urgently need new drugs to treat infections. Here, we show that a medieval remedy made from onion, garlic, wine, and bile can kill a range of problematic bacteria grown both planktonically and as biofilms. A single component of the remedy - allicin, derived from garlic - is sufficient to kill planktonic bacteria. However, garlic or allicin alone do nothing against bacteria when they form a biofilm. All four ingredients are needed to fully kill bacterial biofilm communities, hinting that these ingredients work together to kill the bacteria. This suggests that future discovery of antibiotics from natural products could be enhanced by studying combinations of ingredients, rather than single plants or compounds.

microbiology↗

bioRxiv2019

Building a better biofilm - formation of in vivo-like biofilm structures by Pseudomonas aeruginosa in a porcine model of cystic fibrosis lung infection

Pseudomonas aeruginosa biofilm infections in the cystic fibrosis (CF) lung are highly resistant to current antimicrobial treatments and are associated with increased mortality rates. The existing models for such infections are not able to reliably mimic the clinical biofilms observed. We aimed to further optimise an ex vivo pig lung (EVPL) model for P. aeruginosa CF lung infection that can be used to increase understanding of chronic CF biofilm infection. The EVPL model will facilitate discovery of novel infection prevention methods and treatments, and enhanced exploration of biofilm architecture. We investigated purine metabolism and biofilm formation in the model using transposon insertion mutants in P. aeruginosa PA14 for key genes: purD, gacA and pelA. Our results demonstrate that EVPL recapitulates a key aspect of in vivo P. aeruginosa infection metabolism, and that the pathogen forms a biofilm with a clinically realistic structure not seen in other in vitro studies. Two pathways known to be required for in vivo biofilm infection - the Gac regulatory pathway and production of the Pel exopolysaccharide - are essential to the formation of this mature, structured biofilm on EVPL tissue. We propose the high-throughput EVPL model as a validated biofilm platform to bridge the gap between in vitro and CF lung infection.

microbiology↗