Recreating lung microbiota in vitro to determine antimicrobial susceptibility

Introduction Bacterial aggregates are one of the most relevant causes of persistent infections. At least 65% of all bacterial infections are associated with it. Mucus is a human-designed barrier that in the lungs protect us against the constant exposure to inhaled pathogens, particles and toxic molecules, as well as physical insults. Yet, in some airway disorders, as cystic fibrosis (CF), mucus hypersecretion and accumulation provide a suitable site for bacterial infections to thrive resulting on chronic infections that lead to morbidity and mortality of patients. Infections of CF patients are characterised by the presence of different bacteria, which microbial complexity and microenvironment are difficult to recreate in current bacterial culture substrates and in animal models. Therefore, novel models are needed to either screen already available antimicrobial agents in a patient-specific manner or design new molecules to tackle airway mucus infections [1]. Antimicrobial treatment of airway CF infections has been of main importance to increase the survival rates in patients with CF. However, bacteria in CF airways evolve in the form of aggregates, which makes it difficult to eradicate. Experimental Methods Three-dimensional mucus models were adopted, CF-Muc3Gel, as models of CF mucus to recreate lung microbiota in vitro. CF-Muc3Gel is mostly composed of 2.5 %(w/v) commercial mucins (porcine stomach type III that contains Muc5AC - one of the most important mucins found in airway mucus) and 0.71 %(w/v) NaCl that fall within the ranges determined on CF sputum. Extensive rheological analyses were carried out to characterize the viscoelastic properties of CF-Muc3Gel. In vitro infections were induced within CF-Muc3Gel by culturing Pseudomonas aeruginosa and/or Staphylococcus aureus, the prevalent bacteria colonizing the airway CF mucus. Bacterial organization was characterized through confocal microscopy. Vertical microprofiles of oxygen tension throughout the CF-Muc3Gel structure were determined through O2- microsensor measurements of chemical gradients with or without cultured bacteria. The ability of CF-Muc3Gel to act as a platform to determine antimicrobial susceptibility was further assessed by treating the different generated in vitro infections with three antimicrobial agents routinely employed in the clinics to manage chronic infections by these bacteria. Their effectiveness was further compared to standard bacteria cultures. Results and Discussion CF-Muc3Gel exhibits similar viscoelastic properties alterations to those reported for CF mucus with the presence of structural gradients. CF-Muc3Gel successfully sustained the growth of P. aeruginosa and S. aureus either in monoculture or co-culture with a bacterial number representative of CF patients after 24 h of infection. Bacteria cultured within CF-Muc3Gel exhibited many pathophysiological features, as these not only were able to colonise all its structure, but also generate microcolony aggregates, which size and shape resembled those observed in the mucus of CF patients. CF-Muc3Gel was also able to sustain the growth of P. aeruginosa and S. aureus derived from clinical isolates. Measurements of O2- microprofiles in CF-Muc3Gel without bacteria, revealed a gradual decrease of O2 tension. Similar to what was previously reported in CF sputum with chronic P. aeruginosa airway infections, CF-Muc3Gel infected with either P. aeruginosa or S. aureus exhibited dual O2 distribution with an upper aerobic region and a lower region that was completely anoxic after 48 hours of infection. The interplay of all these features resulted in similar barrier to antimicrobial treatment. Bacteria resulted more susceptible to antimicrobial treatment under planktonic conditions than when cultured within CF-Muc3Gel, where these instead displayed increased antibiotic tolerances even at high concentrations of antimicrobial drugs (10 MIC). The sensibility difference between CF-Muc3Gel and planktonic cultures confirmed the well-reported mismatch between planktonically cultured bacteria and and clinical outcomes. CF-Muc3Gel is a very versatile bacteria culture substrate that can be further complicated with other components, such as proteins, can integrate different culture media and be exploited for different culture applications to model the mucus of different body districts. In addition, from the antimicrobial studies, rheological data and microstructural inspections, it is possible to propose CF-Muc3Gel as a new, valid tool for the screening of antimicrobial agents for the research and development of new antimicrobial agents. CF-Muc3Gel is ready-to-use without requiring any technical skills and offers superior reproducibility.