Ecological and evolutionary mechanisms driving within-patient emergence of antimicrobial resistance.

The ecological and evolutionary mechanisms of antimicrobial resistance (AMR) emergence within patients and how these vary across bacterial infections are poorly understood. Increasingly widespread use of pathogen genome sequencing in the clinic enables a deeper understanding of these processes. In this Review, we explore the clinical evidence to support four major mechanisms of within-patient AMR emergence in bacteria: spontaneous resistance mutations; in situ horizontal gene transfer of resistance genes; selection of pre-existing resistance; and immigration of resistant lineages. Within-patient AMR emergence occurs across a wide range of host niches and bacterial species, but the importance of each mechanism varies between bacterial species and infection sites within the body. We identify potential drivers of such differences and discuss how ecological and evolutionary analysis could be embedded within clinical trials of antimicrobials, which are powerful but underused tools for understanding why these mechanisms vary between pathogens, infections and individuals. Ultimately, improving understanding of how host niche, bacterial species and antibiotic mode of action combine to govern the ecological and evolutionary mechanism of AMR emergence in patients will enable more predictive and personalized diagnosis and antimicrobial therapies.

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 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 cuing 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 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. It provides a more accurate P. aeruginosa growth environment for determining antimicrobial resistance that quickly drives P. aeruginosa into a chronic-like infection phenotype. IMPORTANCE Pseudomonas aeruginosa lung infections that affect people with cystic fibrosis are resistant to most available 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.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus 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 poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo pig lung, EVPL). Several in vitro assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays. The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

An ex vivo cystic fibrosis model recapitulates key clinical aspects of chronic Staphylococcus aureus infection.

Staphylococcus aureus 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 ex vivo model of CF infection comprising porcine bronchiolar tissue and synthetic mucus, S. aureus 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 S. aureus in people with CF.

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 work and CF lung infection.

Predicting Antibiotic-Associated Virulence of Pseudomonas aeruginosa Using an ex vivo Lung Biofilm Model.

BACKGROUND: 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 in vivo. Consequently, inaccurate diagnostics affect treatment choice, preventing bacterial clearance and potentially selecting for antibiotic resistance. This leads to prolonged, ineffective treatment. METHODS: In this study, we use an ex vivo lung biofilm model to study antibiotic tolerance and virulence of Pseudomonas aeruginosa. Sections of pig bronchiole were dissected, prepared and infected with clinical isolates of P. aeruginosa and incubated in artificial sputum media to form biofilms, as previously described. Then, lung-associated biofilms were challenged with antibiotics, at therapeutically relevant concentrations, before their bacterial load and virulence were quantified and detected, respectively. RESULTS: The results demonstrated minimal effect on the bacterial load with therapeutically relevant concentrations of ciprofloxacin and meropenem, with the latter causing an increased production of proteases and pyocyanin. A combination of meropenem and tobramycin did not show any additional decrease in bacterial load but demonstrated a slight decrease in total proteases and pyocyanin production. CONCLUSION: In this initial study of six clinical isolates of P. aeruginosa showed high levels of antibiotic tolerance, with minimal effect on bacterial load and increased proteases production, which could negatively affect lung function. Thus, the ex vivo lung model has the potential to be effectively used in larger studies of antibiotic tolerance in in vivo-like biofilms, and show how sub optimal antibiotic treatment of biofilms may potentially contribute to exacerbations and eventual lung failure. We demonstrate a realistic model for understanding antibiotic resistance and tolerance in biofilms clinically and for molecules screening in anti-biofilm drug development.