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.

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

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.

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 medium that mimics cystic fibrosis (CF) sputum 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 drug development pipelines, allowing the assessment of drug entry into biofilms, in in vivo-like conditions, prior to more detailed tests of biofilm killing.

Significance and Impact of the Study: The majority of studies of antibiotic efficacy in the treatment of chronic osteomyelitis are carried out in animals. We developed an in vitro model of Staphylococcus aureus infection of bone to evaluate the ability of antibiotics to eradicate mature biofilms on surfaces analogous to necrotic bone. The results demonstrated the difficulties which occur in osteomyelitis treatment, with only very high concentrations of antibiotic able to penetrate the bone sufficiently to reduce bacterial survival whilst still failing to eradicate biofilms. This model could be of use in initial screening of novel compounds intended for use in the treatment of osteomyelitis. AbstractChronic osteomyelitis is difficult to treat, with biofilm growth and the diffusion barrier to antibiotics presented by bone contributory factors. The aim of this study was to develop and evaluate an in vitro model of osteomyelitis. A bioluminescent strain of Staphylococcus aureus was grown in bone blocks made from bovine femur. Light output was insufficient for detection of bacterial cells within bone by 24 h and viable counting of crushed bone blocks was used to determine bacterial survival. Challenge of 72 h biofilms with gentamicin and daptomycin for 24 h demonstrated that only concentrations of 10 times the clinical peak serum target levels (100 mg l À1 gentamicin and 1000 mg l À1 daptomycin) resulted in significant reductions in cell viability compared to controls. Once daily dosing over 7 days resulted in ≥3 log reductions in cell numbers by 48 h. Thereafter no significant reduction was achieved, although emergence of resistance was suppressed. Determination of antibiotic concentration in bone blocks over 7 days indicated that neither agent was able to consistently reach levels in bone of >10% of the original dose. The model was, therefore, able to demonstrate the challenges posed by biofilm growth on and within bone.