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The respiratory tract is a compartmentalised and heterogenous environment. The nasopharynx and sinuses of the upper airways have distinct properties from the lungs and these differences may shape bacterial adaptation and evolution. Upper airway niches act as early colonisation sites for respiratory bacterial pathogens, including those, such as Pseu...
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Context 1
... P. aeruginosa isolates used in this work are shown in Table 1. Bacteria were routinely streaked onto Tryptone Soy Agar (TSA) (Sigma-Aldrich) from bead stocks and restreaked every other day to maintain a fresh stock or from bead stocks, as required. ...Context 2
... we describe, step-by step, the procedure used to prepare 1L of each of the four developed media. Reagents and equipment used in this study are listed in Table 16. ...Context 3
... Under constant stirring with magnetic stirrer, from the stocks prepared, add the amounts of each chemical to the base media as given in Table 10. ...Context 4
... Prepare stocks of lysozyme, lactoferrin, spermidine, spermine, putrescine, CaCl 2 , MgCl 2 , FeCl 2 , CuCl 2 , ZnCl 2 , sialic acid and galactose in the concentrations given in Table 11 for healthy lungs by dissolving each powder in deionised water. ...Context 5
... Under constant stirring with magnetic stirrer, from the stocks prepared, add the amounts of each chemical to the base media as given in Table 12. 4. Add 26.8 μl of neat spermidine solution (9.296 g/l) to the base media. 5. Add amino acids in the same way of preparation of the CF media, see Table 3 for concentrations. ...Context 6
... conditions differ between lung-and sinus media. The incubation conditions for each media are given in Table 13. Conditions of 5% CO 2 can be achieved by incubating the media in GasPak Jars with candles, using Campygen packs or microaerobic incubators. ...Context 7
... wrinkly structures have been suggested to be the result of redox-driven adaptation that maximizes oxygen accessibility in biofilms, to increase their surface area in oxidant limitation. 17 Colony morphotypes in Figure 7D and E were visible only after transfer 10 and were unique to CF media (Table 14). ...Context 8
... envisage these media can help reduce the need for use of animals for the purposes of experimental evolution, investigation of host-pathogen interactions and for virulence screening. (Table 14). ...Context 9
... developed media were designed to be cost effective for the purposes of long-term experiments (Table 15). The media should also prove readily accessible, as they can be prepared with standard laboratory equipment and without the need for any specialist training. ...Context 10
... was higher than the M9 control for at least one tested concentration of albumin, amino acids, mucin, spermine, zinc, lysozyme and lactoferrin. Relative to PAO1, the airway adapted isolate LESB65 appeared better able to Table 16. Materials and Equipment used in the study and supplier information. ...Context 11
... Table 14, I believe the title is somewhat of a misnomer. The table shows when each morphotype appears and whether it is maintained in the populations. ...Context 12
... Figure 7 and Table 14: Do you have quantitative data for colony morphotype frequencies during the experiment? Data is now quite descriptive. ...Context 13
... agree that algU is clearly different from the other genes and have acknowledged this in the manuscript text. Figure 7 and Table 14: Do you have quantitative data for colony morphotype frequencies during the experiment? Data is now quite descriptive. ...Context 14
... P. aeruginosa isolates used in this work are shown in Table 1. Bacteria were routinely streaked onto Tryptone Soy Agar (TSA) (Sigma-Aldrich) from bead stocks and restreaked every other day to maintain a fresh stock or from bead stocks, as required. ...Context 15
... we describe, step-by step, the procedure used to prepare 1L of each of the four developed media. Reagents and equipment used in this study are listed in Table 16. ...Context 16
... Under constant stirring with magnetic stirrer, from the stocks prepared, add the amounts of each chemical to the base media as given in Table 10. ...Context 17
... Prepare stocks of lysozyme, lactoferrin, spermidine, spermine, putrescine, CaCl 2 , MgCl 2 , FeCl 2 , CuCl 2 , ZnCl 2 , sialic acid and galactose in the concentrations given in Table 11 for healthy lungs by dissolving each powder in deionised water. ...Context 18
... Under constant stirring with magnetic stirrer, from the stocks prepared, add the amounts of each chemical to the base media as given in Table 12. 4. Add 26.8 μl of neat spermidine solution (9.296 g/l) to the base media. 5. Add amino acids in the same way of preparation of the CF media, see Table 3 for concentrations. ...Context 19
... conditions differ between lung-and sinus media. The incubation conditions for each media are given in Table 13. Conditions of 5% CO 2 can be achieved by incubating the media in GasPak Jars with candles, using Campygen packs or microaerobic incubators. ...Context 20
... wrinkly structures have been suggested to be the result of redox-driven adaptation that maximizes oxygen accessibility in biofilms, to increase their surface area in oxidant limitation. 17 Colony morphotypes in Figure 7D and E were visible only after transfer 10 and were unique to CF media (Table 14). ...Context 21
... envisage these media can help reduce the need for use of animals for the purposes of experimental evolution, investigation of host-pathogen interactions and for virulence screening. (Table 14). ...Context 22
... developed media were designed to be cost effective for the purposes of long-term experiments (Table 15). The media should also prove readily accessible, as they can be prepared with standard laboratory equipment and without the need for any specialist training. ...Context 23
... was higher than the M9 control for at least one tested concentration of albumin, amino acids, mucin, spermine, zinc, lysozyme and lactoferrin. Relative to PAO1, the airway adapted isolate LESB65 appeared better able to Table 16. Materials and Equipment used in the study and supplier information. ...Context 24
... Table 14, I believe the title is somewhat of a misnomer. The table shows when each morphotype appears and whether it is maintained in the populations. ...Citations
... SCFM2 was found to be able to reproduce similar gene expression levels to those identified in sputum samples for P. aeruginosa, including when compared to a murine lung infection model [25]. Recent media have been formulated to mimic conditions found in both the lungs and sinuses of pwCF [26]. These media contain elevated glucose concentrations to replicate CF-associated diabetes, and bile salts, which are a comorbidity of CF-associated gastrointestinal reflux disease. ...
A workshop was held by the PIPE-CF strategic research centre to consider preclinical testing of antimicrobials for cystic fibrosis (CF). The workshop brought together groups of people from the CF community to discuss current challenges and identify priorities when developing CF therapeutics. This paper summarizes the key points from the workshop from the different sessions, including talks given by presenters on the day and round table discussions. Currently, it is felt that there is a large disconnect throughout the community, with communication between patients, clinicians and researchers being the main issue. This leads to little consideration being given to factors such as treatment regimes, routes of administration and side effects when developing new therapies, that could alter the day-to-day lifestyles of people living with CF. Translation of numerical data that are obtained in the laboratory to successful outcomes of clinical trials is also a key challenge facing researchers today. Laboratory assays in preclinical testing involve basing results on bacterial clearance and decrease in viable cells, when these are not factors that are considered when determining the success of a treatment in the clinic. However, there are several models currently in development that seek to tackle some of these issues, such as the organ-on-a-chip technology and adaptation of a hollow-fibre model, as well as the development of media that aim to mimic the niche environments of a CF respiratory tract. It is hoped that by summarizing these opinions and discussing current research, the communication gap between groups can begin to close.
Pseudomonas aeruginosa is a cause of chronic respiratory tract infections in people with cystic fibrosis (CF), non-CF bronchiectasis and chronic obstructive pulmonary disease. Prolonged infection allows accumulation of mutations and horizontal gene transfer, increasing the likelihood of adaptive phenotypic traits. Adaptation is proposed to arise first in bacterial populations colonising upper airway environments. Here, we model this process using an experimental evolution approach. P. aeruginosa PAO1, which is not airway adapted, was serially passaged, separately, in media chemically reflective of upper or lower airway environments. To explore whether the CF environment selects for unique traits, we separately passaged PAO1 in airway-mimicking media with or without CF-specific factors. Our findings demonstrated that all airway environments – sinus and lungs, under CF and non-CF conditions – selected for loss of twitching motility, increased resistance to multiple antibiotic classes and a hyper-biofilm phenotype. These traits conferred increased airway colonisation potential in an in vivo model. CF-like conditions exerted stronger selective pressures, leading to emergence of more pronounced phenotypes. Loss of twitching was associated with mutations in type IV pili genes. Type IV pili mediate surface attachment, twitching and induction of cAMP signalling. We additionally identified multiple evolutionary routes to increased biofilm formation involving regulation of cyclic-di-GMP signalling. These included loss of function mutations in bifA and dipA phosphodiesterase genes and activating mutations in the siaA phosphatase. These data highlight that airway environments select for traits associated with sessile lifestyles and suggest upper airway niches support emergence of phenotypes that promote establishment of lung infection.
Growth environment greatly alters many facets of pathogen physiology, including pathogenesis and antimicrobial tolerance. The importance of host-mimicking environments for attaining an accurate picture of pathogen behaviour is widely recognised. Whilst this recognition has translated into the extensive development of artificial cystic fibrosis (CF) sputum medium, attempts to mimic the growth environment in other respiratory disease states have been completely neglected. The composition of the airway surface liquid (ASL) in different pulmonary diseases is far less well characterised than CF sputum, making it very difficult for researchers to model these infection environments. In this review, we discuss the components of human ASL, how different lung pathologies affect ASL composition, and how different pathogens interact with these components. This will provide researchers interested in mimicking different respiratory environments with the information necessary to design a host-mimicking medium, allowing for better understanding of how to treat pathogens causing infection in these environments.
When cultured together under standard laboratory conditions Pseudomonas aeruginosa has been shown to be an effective inhibitor of Staphylococcus aureus . However, P. aeruginosa and S. aureus are commonly observed in coinfections of individuals with cystic fibrosis (CF) and in chronic wounds. Previous work from our group revealed that S. aureus isolates from CF infections are able to persist in the presence of P. aeruginosa strain PAO1 with a range of tolerances with some isolates being eliminated entirely and others maintaining large populations. In this study, we designed a serial transfer, evolution experiment to identify mutations that allow S. aureus to survive in the presence of P. aeruginosa . Using S. aureus USA300 JE2 as our ancestral strain, populations of S. aureus were repeatedly cocultured with fresh P. aeruginosa PAO1. After eight coculture periods, S. aureus populations that survived better in the presence of PAO1 were observed. We found two independent mutations in the highly conserved S. aureus aspartate transporter, gltT , that were unique to evolved P. aeruginosa -tolerant isolates. Subsequent phenotypic testing demonstrated that gltT mutants have reduced uptake of glutamate and outcompeted wild-type S. aureus when glutamate was absent from chemically defined media. These findings together demonstrate that the presence of P. aeruginosa exerts selective pressure on S. aureus to alter its uptake and metabolism of key amino acids when the two are cultured together.
Pseudomonas aeruginosa is a major nosocomial pathogen that causes severe disease including sepsis. Carbapenem-resistant P. aeruginosa is recognised by the World Health Organisation as a priority 1 pathogen, with urgent need for new therapeutics. As such, there is renewed interest in using bacteriophages as a therapeutic. However, the dynamics of treating pan-resistant P. aeruginosa with phage in vivo are poorly understood. Using a pan-resistant P. aeruginosa in vivo infection model, phage therapy displays strong therapeutic potential, clearing infection from the blood, kidneys, and spleen. Remaining bacteria in the lungs and liver displays phage resistance due to limiting phage adsorption. Yet, resistance to phage results in re-sensitisation to a wide range of antibiotics. In this work, we use phage steering in vivo, pre-exposing a pan resistant P. aeruginosa infection with a phage cocktail to re-sensitise bacteria to antibiotics, clearing the infection from all organs.
Despite enabling Streptococcus pneumoniae to acquire antibiotic resistance and evade vaccine-induced immunity, transformation occurs at variable rates across pneumococci. Phase variants of isolate RMV7, distinguished by altered methylation patterns driven by the translocating variable restriction-modification (tvr) locus, differed significantly in their transformation efficiencies and biofilm thicknesses. These differences were replicated when the corresponding tvr alleles were introduced into an RMV7 derivative lacking the locus. RNA-seq identified differential expression of the type 1 pilus, causing the variation in biofilm formation, and inhibition of competence induction in the less transformable variant, RMV7domi. This was partly attributable to RMV7domi’s lower expression of ManLMN, which promoted competence induction through importing N-acetylglucosamine. This effect was potentiated by analogues of some proteobacterial competence regulatory machinery. Additionally, one of RMV7domi’s phage-related chromosomal island was relatively active, which inhibited transformation by increasing expression of the stress response proteins ClpP and HrcA. However, HrcA increased competence induction in the other variant, with its effects depending on Ca2+ supplementation and heat shock. Hence the heterogeneity in transformation efficiency likely reflects the diverse signalling pathways by which it is affected. This regulatory complexity will modulate population-wide responses to synchronising quorum sensing signals to produce co-ordinated yet stochastic bet hedging behaviour.
