antibiotic-efficacy-testing-an-ex-vivo-model-pseudomonas-aeruginosa

Antibiotic Efficacy Testing in an Ex vivo Model of <em>Pseudomonas aeruginosa</em> and <em>Staphylococcus aureus</em> Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

University of Warwick

A key aim in microbiology is to determine the genetic and phenotypic bases of bacterial virulence, persistence and antimicrobial resistance in chronic biofilm infections. This requires tractable, high-throughput models that reflect the physical and chemical environment encountered in specific infection contexts. Such models will increase the predictive power of microbiological experiments and provide platforms for enhanced testing of novel antibacterial or antivirulence therapies. We present an optimized ex vivo model of cystic fibrosis lung infection: ex vivo culture of pig bronchiolar tissue in artificial cystic fibrosis mucus. We focus on the formation of biofilms by Pseudomonas aeruginosa. We show highly repeatable and specific formation of biofilms that resemble clinical biofilms by a commonly studied laboratory strain and ten cystic fibrosis isolates of this key opportunistic pathogen.

An ex vivo lung model to study bronchioles infected with Pseudomonas aeruginosa biofilms.

Bacterial traits that contribute to disease are termed "virulence factors" and there is much interest in therapeutic approaches that disrupt such traits. What remains less clear is whether a virulence factor identified as such in a particular context is also important in infections involving different host and pathogen types. Here, we address this question using a meta-analytic approach. We statistically analyzed the infection outcomes of 81 experiments associated with one well-studied virulence factor-pyoverdine, an iron-scavenging compound secreted by the opportunistic pathogen . We found that this factor is consistently involved with virulence across different infection contexts. However, the magnitude of the effect of pyoverdine on virulence varied considerably. Moreover, its effect on virulence was relatively minor in many cases, suggesting that pyoverdine is not indispensable in infections. Our works supports theoretical models from ecology predicting that disease severity is multifactorial and context dependent, a fact that might complicate our efforts to identify the most important virulence factors. More generally, our study highlights how comparative approaches can be used to quantify the magnitude and general importance of virulence factors, key knowledge informing future anti-virulence treatment strategies.

Do Bacterial "Virulence Factors" Always Increase Virulence? A Meta-Analysis of Pyoverdine Production in  As a Test Case.

Bacteria produce a wide variety of exoproducts that favourably modify their environment and increase their fitness. These are often termed 'public goods' because they are costly for individuals to produce and can be exploited by non-producers (cheats). The outcome of conflict over public goods is dependent upon the prevailing environment and the phenotype of the individuals in competition. Many bacterial species use quorum sensing (QS) signalling molecules to regulate the production of public goods. QS, therefore, determines the cooperative phenotype of individuals, and influences conflict over public goods. In addition to their regulatory functions, many QS molecules have additional properties that directly modify the prevailing environment. This leads to the possibility that QS molecules could influence conflict over public goods indirectly through non-signalling effects, and the impact of this on social competition has not previously been explored. The  QS signal molecule PQS is a powerful chelator of iron which can cause an iron starvation response. Here, we show that PQS stimulates a concentration-dependent increase in the cooperative production of iron scavenging siderophores, resulting in an increase in the relative fitness of non-producing siderophore cheats. This is likely due to an increased cost of siderophore output by producing cells and a concurrent increase in the shared benefits, which accrue to both producers and cheats. Although PQS can be a beneficial signalling molecule for , our data suggest that it can also render a siderophore-producing population vulnerable to competition from cheating strains. More generally, our results indicate that the production of one social trait can indirectly affect the costs and benefits of another social trait.

Environmental modification via a quorum sensing molecule influences the social landscape of siderophore production.

Experiments examining the social dynamics of bacterial quorum sensing (QS) have focused on mutants which do not respond to signals and the role of QS-regulated exoproducts as public goods. The potential for QS signal molecules to themselves be social public goods has received much less attention. Here, we analyze how signal-deficient () mutants of the opportunistic pathogen  interact with wild-type cells in an environment where QS is required for growth. We show that when growth requires a "private" intracellular metabolic mechanism activated by the presence of QS signal,  mutants act as social cheats and outcompete signal-producing wild-type bacteria in mixed cultures, because they can exploit the signals produced by wild-type cells. However, reducing the ability of signal molecules to diffuse through the growth medium results in signal molecules becoming less accessible to mutants, leading to reduced cheating. Our results indicate that QS signal molecules can be considered social public goods in a way that has been previously described for other exoproducts but that spatial structuring of populations reduces exploitation by noncooperative signal cheats. Bacteria communicate via signaling molecules to regulate the expression of a whole range of genes. This process, termed quorum sensing (QS), moderates bacterial metabolism under many environmental conditions, from soil and water (where QS-regulated genes influence nutrient cycling) to animal hosts (where QS-regulated genes determine pathogen virulence). Understanding the ecology of QS could therefore yield vital clues to how we might modify bacterial behavior for environmental or clinical gains. Here, we demonstrate that QS signals act as shareable public goods. This means that their evolution, and therefore population-level responses to interference with QS, will be constrained by population structure. Further, we show that environmental structure (constraints on signal diffusion) alters the accessibility of QS signals and demonstrates that we need to consider population and environmental structure to help us further our understanding of QS signaling systems.

The Fitness of  Quorum Sensing Signal Cheats Is Influenced by the Diffusivity of the Environment.

Erratum for Mund et al., "The Fitness of  Quorum Sensing Signal Cheats Is Influenced by the Diffusivity of the Environment".

The potential for siderophore mutants of Pseudomonas aeruginosa to attenuate virulence during infection, and the possibility of exploiting this for clinical ends, have attracted much discussion. This has largely been based on the results of in vitro experiments conducted in iron-limited growth medium, in which siderophore mutants act as social 'cheats:' increasing in frequency at the expense of the wild type to result in low-productivity, low-virulence populations dominated by mutants. We show that insights from in vitro experiments cannot necessarily be transferred to infection contexts. First, most published experiments use an undefined siderophore mutant. Whole-genome sequencing of this strain revealed a range of mutations affecting phenotypes other than siderophore production. Second, iron-limited medium provides a very different environment from that encountered in chronic infections. We conducted cheating assays using defined siderophore deletion mutants, in conditions designed to model infected fluids and tissue in cystic fibrosis lung infection and non-healing wounds. Depending on the environment, siderophore loss led to cheating, simple fitness defects, or no fitness effect at all. Our results show that it is crucial to develop defined in vitro models in order to predict whether siderophores are social, cheatable and suitable for clinical exploitation in specific infection contexts.

Optimised chronic infection models demonstrate that siderophore 'cheating' in Pseudomonas aeruginosa is context specific.

Assessing evolutionary risks of resistance for new antimicrobial therapies.

The pharmacopeia used by physicians and laypeople in medieval Europe has largely been dismissed as placebo or superstition. While we now recognize that some of the  used by medieval physicians could have had useful biological properties, research in this area is limited by the labor-intensive process of searching and interpreting historical medical texts. Here, we demonstrate the potential power of turning medieval medical texts into contextualized electronic databases amenable to exploration by the use of an algorithm. We used established methodologies from network science to reveal patterns in ingredient selection and usage in a key text, the 15th-century , focusing on remedies to treat symptoms of microbial infection. In providing a worked example of data-driven textual analysis, we demonstrate the potential of this approach to encourage interdisciplinary collaboration and to shine a new light on the ethnopharmacology of historical medical texts. We used established methodologies from network science to identify patterns in medicinal ingredient combinations in a key medieval text, the 15th-century , focusing on recipes for topical treatments for symptoms of microbial infection. We conducted experiments screening the antimicrobial activity of selected ingredients. These experiments revealed interesting examples of ingredients that potentiated or interfered with each other's activity and that would be useful bases for future, more detailed experiments. Our results highlight (i) the potential to use methodologies from network science to analyze medieval data sets and detect patterns of ingredient combination, (ii) the potential of interdisciplinary collaboration to reveal different aspects of the ethnopharmacology of historical medical texts, and (iii) the potential development of novel therapeutics inspired by premodern remedies in a time of increased need for new antibiotics.

Data Mining a Medieval Medical Text Reveals Patterns in Ingredient Choice That Reflect Biological Activity against Infectious Agents.

Novel antimicrobials are urgently needed to combat drug-resistant bacteria and to overcome the inherent difficulties in treating biofilm-associated infections. Studying plants and other natural materials used in historical infection remedies may enable further discoveries to help fill the antibiotic discovery gap. We previously reconstructed a 1,000-year-old remedy containing onion, garlic, wine, and bile salts, known as 'Bald's eyesalve', and showed it had promising antibacterial activity. In this current paper, we have found this bactericidal activity extends to 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 soft-tissue wound biofilm model. While the presence of garlic in the mixture can explain the activity against planktonic cultures, garlic has no activity against biofilms. We have found the potent anti-biofilm activity of Bald's 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 and underlines the importance of working with biofilm models when exploring natural products for the anti-biofilm pipeline.

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

Bacterial biofilms are known to have high antibiotic tolerance which directly affects clearance of bacterial infections in people with cystic fibrosis (CF). Current antibiotic susceptibility testing methods are either based on planktonic cells or do not reflect the complexity of biofilms . Consequently, inaccurate diagnostics affect treatment choice, preventing bacterial clearance and potentially selecting for antibiotic resistance. This leads to prolonged, ineffective treatment.

Predicting Antibiotic-Associated Virulence of  Using an  Lung Biofilm Model.

The rise in antimicrobial resistance has prompted the development of alternatives to combat bacterial infections. Bald's eyesalve, a remedy used in the Early Medieval period, has previously been shown to have efficacy against Staphylococcus aureus in in vitro and in vivo models of chronic wounds. However, the safety profile of Bald's eyesalve has not yet been demonstrated, and this is vital before testing in humans. Here, we determined the safety potential of Bald's eyesalve using in vitro, ex vivo, and in vivo models representative of skin or eye infections. We also confirmed that Bald's 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 min. The slug mucosal irritation assay revealed that a low level of mucus was secreted by slugs indicating moderate mucosal irritation. We obtained promising results from mouse wound closure experiments; no visible signs of irritation or inflammation were observed. Our results suggest that Bald's eyesalve could be tested further on human volunteers to assess safety for topical application against bacterial infections.

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

 is the most prevalent organism isolated from the airways of people with cystic fibrosis (CF), predominantly early in life. Yet its role in the pathology of lung disease is poorly understood. In mice, and many experiments using cell lines, the bacterium invades cells or interstitium, and forms abscesses. This is at odds with the limited available clinical data: interstitial bacteria are rare in CF biopsies and abscesses are highly unusual. Bacteria instead appear to localize in mucus plugs in the lumens of bronchioles. We show that, in an established  model of CF infection comprising porcine bronchiolar tissue and synthetic mucus,  demonstrates clinically significant characteristics including colonization of the airway lumen, with preferential localization as multicellular aggregates in mucus, initiation of a small colony variant phenotype and increased antibiotic tolerance of tissue-associated aggregates. Tissue invasion and abscesses were not observed. Our results may inform ongoing debates relating to clinical responses to  in people with CF.

An  cystic fibrosis model recapitulates key clinical aspects of chronic  infection.

 biofilms cause recalcitrant infections with extensive and unpredictable antibiotic tolerance. Here, we demonstrate increased tolerance of colistin by  when grown in medium that mimics cystic fibrosis (CF) sputum versus standard medium in  biofilm assays, and drastically increased tolerance when grown in an  CF model versus the  assay. We used colistin conjugated to the fluorescent dye BODIPY to assess the penetration of the antibiotic into  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 -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 drug development pipelines, allowing the assessment of drug entry into biofilms, in -like conditions, prior to more detailed tests of biofilm killing.

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

 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  pig lung (EVPL) model for  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  PA14 for key genes: ,  and . Our results demonstrate that EVPL recapitulates a key aspect of  infection metabolism, and that the pathogen forms a biofilm with a clinically realistic structure not seen in other  studies. Two pathways known to be required for  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  work and CF lung infection.

Article	Year
An ex vivo lung model to study bronchioles infected with Pseudomonas aeruginosa biofilms. Microbiology (Reading, England)\| PubMed ID: 27520088	2016
Do Bacterial "Virulence Factors" Always Increase Virulence? A Meta-Analysis of Pyoverdine Production in As a Test Case. Frontiers in microbiology\| PubMed ID: 28018298	2016
Environmental modification via a quorum sensing molecule influences the social landscape of siderophore production. Proceedings. Biological sciences\| PubMed ID: 28404780	2017
The Fitness of Quorum Sensing Signal Cheats Is Influenced by the Diffusivity of the Environment. mBio\| PubMed ID: 28465424	2017
Erratum for Mund et al., "The Fitness of Quorum Sensing Signal Cheats Is Influenced by the Diffusivity of the Environment". mBio\| PubMed ID: 28611251	2017
Optimised chronic infection models demonstrate that siderophore 'cheating' in Pseudomonas aeruginosa is context specific. The ISME journal\| PubMed ID: 28696423	2017
Assessing evolutionary risks of resistance for new antimicrobial therapies. Nature ecology & evolution\| PubMed ID: 30886376	2019
Data Mining a Medieval Medical Text Reveals Patterns in Ingredient Choice That Reflect Biological Activity against Infectious Agents. mBio\| PubMed ID: 32047130	2020
Anti-biofilm efficacy of a medieval treatment for bacterial infection requires the combination of multiple ingredients. Scientific reports\| PubMed ID: 32724094	2020
Predicting Antibiotic-Associated Virulence of Using an Lung Biofilm Model. Frontiers in microbiology\| PubMed ID: 32983077	2020
The safety profile of Bald's eyesalve for the treatment of bacterial infections. Scientific reports\| PubMed ID: 33060666	2020
An cystic fibrosis model recapitulates key clinical aspects of chronic infection. Microbiology (Reading, England)\| PubMed ID: 33186093	2020
Effect of host-mimicking medium and biofilm growth on the ability of colistin to kill . Microbiology (Reading, England)\| PubMed ID: 33253080	2020
Building a better biofilm - Formation of -like biofilm structures by in a porcine model of cystic fibrosis lung infection. Biofilm\| PubMed ID: 33381751	2020
Editorial: Polymicrobial Biofilms in Chronic Infectious Disease. Frontiers in cellular and infection microbiology\| PubMed ID: 33425788	2020
Professor Pangloss and the Pangenome: Does Have the Best of All Possible Worlds? American journal of respiratory and critical care medicine\| PubMed ID: 33428861	2021

Freya Harrison

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