This work has been funded by the National Biofilms Innovation Centre (NBIC) which is an Innovation and Knowledge Centre funded by the Biotechnology and Biological Sciences Research Council, Innovate UK and Hartree Centre [Awards BB/R012415/1 and BB/X002950/1] and by the UK CF Trust and USA CF Foundation Strategic Research Centre: &#39;An evidence-based preclinical framework for the development of antimicrobial therapeutics in cystic fibrosis&#39; (PIPE-CF) [Award SRC022].

1. Preparation of Synthetic Cystic Fibrosis media-2 (SCFM2) and plates
<ol>
	<li>Make SCFM2 according to the recipe described in Palmer et al.16 in a 500 mL borosilicate glass bottle at 2x concentration with a few modifications.
	<ol>
		<li>Weigh out and prepare stocks as indicated in Table 1. Prepare amino acid stocks according to Table 2.</li>
		<li>Weigh out 5 g mucin (from Porcine stomach (Type II)) and 600 mg eDNA (from salmon sperm) in 100 mL of dH2O in a 200 mL borosilicate glass bottle. Stir at 4 &#176;C overnight before sterilization by autoclaving.</li>
		<li>To a 500 mL sterilized bottle, add 0.54 g glucose, 6.06 g NaCl, and 2.228 g KCl. Add 10 mL each of the stocks prepared in step 1.1.1. Adjust pH to 6.9 using 1 M NaOH or HCl.</li>
		<li>Add 10 mL of dH2O followed by 10 mL of 0.175 M CaCl2 and 0.0606 M of MgCl2. Prepare and add 10 mL of 0.93 M L-lactic acid. Immediately before use, add 10 mL of 0.36 mM Fe(III)SO47H2O</li>
		<li>Filter sterilize the SCFM2 using a filter unit and vacuum pump, then add the autoclaved mucin and eDNA. 
		NOTE: The prepared SCFM2 can be stored at 4 &#176;C for up to 1 month.</li>
	</ol>
	</li>
	<li>Prepare SCFM2 agar plates as described below.
	<ol>
		<li>Preheat 25 mL of 2x SCFM2 in a 50 mL tube in a 55 &#176;C water bath before mixing with 25 mL of pre-melted 3% (w/v) technical agar, resulting in a mix of 1x SCFM2 and 1.5% (w/v) technical agar.</li>
		<li>Using a serological pipette, transfer 5 mL of the mixture into each well of a 6-well plate.</li>
		<li>Prepare the same number of 13 mm polycarbonate discs with a pore size of 0.2 &#181;m by placing them on the bottom of a Petri dish and applying UV-C for 600 s at a wavelength of 254 nm and a frequency of 60 Hz. Flip the discs over and repeat the UV-C procedure.</li>
		<li>Once the 1x SCFM2-1.5 % (w/v) technical agar has set, use sterile forceps to add a single polycarbonate disc to the surface of each well of the 6-well plate.</li>
	</ol>
	</li>
</ol>
2. Preparation of bacteria for infection
<ol>
	<li>Before setting up the model, prepare agar plate/s for the required bacteria and fungal&#160;strains according to the standard agar used in the lab for P. aeruginosa PAO1, S. aureus SH1000 and C. albicans SC5314 (e.g., Lysogeny broth with 1.2% (w/v) agar for bacteria and Sabouraud dextrose agar for C. albicans). 
	NOTE: To establish the model, specific laboratory reference strains have been used. However, this model has also been used with other strains, including clinical isolates, with some modifications, such as increasing the time for biofilm establishment before treatment for slower-growing strains.</li>
	<li>Use a 1 &#181;L inoculation loop to pick a single colony of each microorganism to inoculate 5 mL of standard laboratory liquid media (e.g., Lysogeny broth for P. aeruginosa, S. aureus and Yeast peptone dextrose (YPD) for C. albicans). Incubate these cultures until they reach the exponential growth phase at 200 rpm, 37 &#176;C for the bacteria, and 200 rpm, 30 &#176;C for C. albicans. 
	NOTE: Aeration will differ depending on the shaking incubator and radius of rotation</li>
</ol>
3. Setting up the solid air interface model
NOTE: An overall schematic representation of the model can be seen in Supplementary Figure 1.
<ol>
	<li>Transfer 1 mL of each liquid culture to a separate sterile 1.5 mL tube and centrifuge for 2 min at 8,000 x g to pellet the bacteria/fungi. Perform this and all the following centrifugation steps at room temperature.</li>
	<li>Aspirate the supernatant using a pipette and resuspend the pellets in 1 mL of PBS before centrifugation, once again, for 2 min at 8,000 x g.</li>
	<li>Remove the supernatant as before and resuspend the pellets in 1 mL of PBS. Dilute the resuspended P. aeruginosa and S. aureus cells 1:10 in PBS and measure optical density using a spectrophotometer at a wavelength of 600 nm.</li>
	<li>Adjust washed samples to 1 x 108 colony forming unit&#160;(CFU)/mL based on prior validation that an OD600 of 0.05 for P. aeruginosa and 0.1 for S. aureus is equivalent to 1 x 108 CFU/mL.</li>
	<li>For C. albicans, dilute washed samples 1:100 in PBS, add 10 &#181;L of each to two individual chambers of a hemocytometer, and use the fixed dimensions of the chamber and the dilution used to calculate CFU/ mL. Using the improved Neubauer hemocytometer the calculation will be as follow: 
	Number of cells = average cell count&#160;&#215; 10,000 / (dilution factor)</li>
	<li>The calculated CFUs represent C. albicans CFU/ mL in the pre-diluted sample. Adjust the C. albicans sample to 1 x 108 CFU/mL in PBS as with the bacterial cultures.</li>
	<li>To prepare the inoculum for addition to the biofilm, adjust the P. aeruginosa inoculum to 1 x 104 CFU/mL (2x 100-fold dilutions), C. albicans to 1 x 105 CFU/mL (100-fold dilution followed by a 10-fold dilution) and finally, S. aureus to 1 x 106 CFU/mL (100-fold dilution) using PBS.</li>
	<li>For mono-species biofilms, add 10 &#181;L of the diluted desired microbe to the center of the polycarbonate disc previously placed on the 1x SCFM2-1.5 % technical agar six-well plates and incubate statically at 37 &#176;C for 24 h before treatment or disruption.</li>
	<li>For polymicrobial biofilms, add 10 &#181;L of each S. aureus and C. albicans on top of one another on the same disc. Incubate the inoculated disk statically for 24 h at 37 &#176;C. 
	NOTE: The order in which S. aureus and C. albicans inoculums are added does not affect the development of the model and generates the same results.</li>
	<li>Following this, transfer the polycarbonate discs to fresh 1x SCFM2-1.5% technical agar plates with sterile forceps and add 10 &#181;L of the 1 x 104 CFU/mL dilution of P. aeruginosa on top of the pre-established S. aureus - C. albicans biofilm. Incubate for another 24 h at 37 &#176;C. 
	NOTE: P. aeruginosa is added 24 h after S. aureus and C. albicans to reflect the order in which these organisms frequently colonize the CF lung in people with CF. This also prevents P. aeruginosa from outgrowing the other two microorganisms. Once P. aeruginosa has been added, the biofilm can be grown for longer than 24 h; however, the polycarbonate disk with the biofilm needs to be transferred to fresh media every 24 h. Before disruption, biofilms can be used for microscopy using strains that express fluorescent proteins, fluorescent dyes, or strain/microbe-specific antibodies. However, it is worth noting that some dyes stain the polycarbonate disc.</li>
</ol>
4. Biofilm disruption
<ol>
	<li>Add ceramic beads with a diameter of 2.8 mm to the plates and UV-crosslink the plates as described in step 1.3. Add 5 ceramic beads to a sterile 2 mL homogenizer tube using flame-sterilized forceps. Then, pipette 1 mL of phosphate-buffered saline (PBS) into the 2 mL homogenization tube.</li>
	<li>Vortex tubes for at least 10 s or until the biofilm has been removed from the polycarbonate disc, determined by visual inspection, at which point remove the polycarbonate disc.</li>
	<li>Place tubes with the beads in the homogenizer and beat for 2x for 10 s at 6 m/s with a 10-s interval between beats. 
	NOTE: This combination of beads, beating time, and homogenizer use results in sufficient disruption of the biofilm with minimal lysis as determined by microscopy and colony-forming units. This may vary depending on the specific bead beater used.</li>
	<li>Pour disrupted biofilm from the homogenizer tube into a 7 mL bijou containing 4 mL of PBS, resulting in a final volume of 5 mL.</li>
	<li>Ensure all liquid from the homogenizer tubes has been transferred to the bijou by centrifuging the tubes at 8,000 x g for 2 min and transferring the remaining liquid to the 7 mL bijou. 
	NOTE: From this 5 mL of disrupted biofilm, a range of assays can be conducted, but it is key to remember for any calculations the 1 mL biofilm PBS dilution into a final volume of 5 mL.</li>
</ol>
5. Metabolic activity testing
<ol>
	<li>Add 200 &#181;L of the disrupted biofilm from step 4 above to a clear-bottomed black 96 well plate. Then, add 10 &#181;L of 0.02% (w/v) resazurin solution.</li>
	<li>Incubate the resazurin-treated biofilm with a plate reader and read the metabolic activity. Incubate the plate at 37 &#176;C, applying orbital shaking every 30 min for 10 s at 200 rpm before taking any fluorescent readings.</li>
	<li>Measure the fluorescence using an excitation of 540 nm and an emission of 590 nm. Repeat this 30 min cycle for 4 h using a plate reader. 
	NOTE: The 4 h time point is used as it provides more distinct variations between treated versus untreated samples and between samples with varying antibiotic concentrations.</li>
</ol>
6. Antimicrobial sensitivity testing
<ol>
	<li>To test the antimicrobial tolerance of standard antibiotics, solubilize the antibiotics of choice in their relevant solvent and add this to the 1x SCFM2-1.5% (w/v) technical agar medium while still liquid before transferring 5 mL to each well of a six-well plate. Once set, transfer the biofilm grown on the polycarbonate disc to the top of the antibiotic-containing media using sterile forceps and add a 10 &#181;L drop of the corresponding antibiotic concentration onto the top of the biofilm. 
	NOTE: For this study, a 2-log fold serial dilution between 64 &#181;g/mL and 0.125 &#181;g/mL of meropenem and tobramycin was used to reflect clinically relevant concentrations. Administering antibiotics or other antimicrobial/anti-virulence treatments solely on top of the biofilm is sufficient, as similar responses have been observed when added to both the media and onto the biofilm.</li>
	<li>After 24 h of incubation at 37 &#176;C with the treatment, image biofilms and disrupt as described in step 4.</li>
	<li>From the 5 mL of disrupted culture, take an aliquot and carry out a 10-fold serial dilution using PBS. Plate out 10 &#181;L spots in triplicate on agar to determine colony-forming units using the Miles and Misra method30. 
	NOTE: The volume added to agar plates will impact the limit of detection, larger volumes will reduce the limit of detection but require more sample and agar plates.</li>
	<li>For mono-species biofilms streak P. aeruginosa and S. aureus onto LB agar plates and C. albicans onto SDA agar.</li>
	<li>For polymicrobial biofilms, place aliquots onto three different selective media containing antibiotics to isolate each of the three species and enable the determination of CFUs. This includes Pseudomonas isolation agar and mannitol salt agar containing nystatin at 10 &#181;g/mL to isolate P. aeruginosa and S. aureus, respectively, and Sabouraud dextrose agar with tetracycline at 125 &#181;g/mL to isolate C. albicans. Incubate S. aureus plates at 37 &#176;C overnight and P. aeruginosa and C. albicans plates at 30 &#176;C. 
	NOTE: Incubate P. aeruginosa plates at 30 &#176;C to minimize colony overgrowth and reduce swarming.</li>
</ol>

Adaptable CF Model for Antimicrobial Testing and Microbial Dynamics Analysis

<ol>
	<li>. 2021 Cf foundation patient registry highlights report Available from: <a target="_blank" href="https://cysticfibrosis.msu.edu/index.php/welcome/news-and-events/39-2021-cf-foundation-patient-registry-highlights-report">https://cysticfibrosis.msu.edu/index.php/welcome/news-and-events/39-2021-cf-foundation-patient-registry-highlights-report</a> (2022)</li><li>Guo, J., Garratt, A., Hill, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worldwide+rates+of+diagnosis+and+effective+treatment+for+cystic+fibrosis.">Worldwide rates of diagnosis and effective treatment for cystic fibrosis.</a> J Cyst Fibros. 21 (3), 456-462 (2022).</li><li>Mandal, V., Ghosh, N. N., Mitra, P. K., Mandal, S., Mandal, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+a+broad-spectrum+antimicrobial+5-butyl-2-pyridine+carboxylic+acid+from+Aspergillus+fumigatus+nhf-01.">Production and characterization of a broad-spectrum antimicrobial 5-butyl-2-pyridine carboxylic acid from Aspergillus fumigatus nhf-01.</a> Sci Rep. 12 (1), 6006 (2022).</li><li>Fran&#231;oise, A., H&#233;ry-Arnaud, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microbiome+in+cystic+fibrosis+pulmonary+disease.">The microbiome in cystic fibrosis pulmonary disease.</a> Genes. 11 (5), 536 (2020).</li><li>Grasemann, H., Ratjen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis.">Cystic fibrosis.</a> New Engl J Med. 389 (18), 1693-1707 (2023).</li><li>Camus, L., Vandenesch, F., Moreau, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+genotype+to+phenotype:+Adaptations+of+pseudomonas+aeruginosa+to+the+cystic+fibrosis+environment.">From genotype to phenotype: Adaptations of pseudomonas aeruginosa to the cystic fibrosis environment.</a> Microb Genom. 7 (3), mgen000513 (2021).</li><li>Rossi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+adaptation+and+evolution+in+patients+with+cystic+fibrosis.">Pseudomonas aeruginosa adaptation and evolution in patients with cystic fibrosis.</a> Nat Rev Microbiol. 19 (5), 331-342 (2021).</li><li>Flemming, H. -. C., Wingender, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm+matrix.">The biofilm matrix.</a> Nat Rev Microbiol. 8 (9), 623-633 (2010).</li><li>Grainha, T., Jorge, P., Alves, D., Lopes, S. P., Pereira, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+pseudomonas+aeruginosa+and+candida+albicans+communication+in+coinfection+scenarios:+Insights+through+network+analysis.">Unraveling pseudomonas aeruginosa and candida albicans communication in coinfection scenarios: Insights through network analysis.</a> Front Cell Infect Microbiol. 10, 550505 (2020).</li><li>Flemming, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+An+emergent+form+of+bacterial+life.">Biofilms: An emergent form of bacterial life.</a> Nat Rev Microbiol. 14 (9), 563-575 (2016).</li><li>H&#248;iby, N., Bjarnsholt, T., Givskov, M., Molin, S., Ciofu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+of+bacterial+biofilms.">Antibiotic resistance of bacterial biofilms.</a> Int J Antimicrob Agents. 35 (4), 322-332 (2010).</li><li>Lorusso, A. B., Carrara, J. A., Barroso, C. D. N., Tuon, F. F., Faoro, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+efflux+pumps+on+antimicrobial+resistance+in+Pseudomonas+aeruginosa.">Role of efflux pumps on antimicrobial resistance in Pseudomonas aeruginosa.</a> Int J Mol Sci. 23 (24), 15779 (2022).</li><li>Michaelis, C., Grohmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+gene+transfer+of+antibiotic+resistance+genes+in+biofilms.">Horizontal gene transfer of antibiotic resistance genes in biofilms.</a> Antibiotics .(Basel). 12 (2), 328 (2023).</li><li>Worlitzsch, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+reduced+mucus+oxygen+concentration+in+airway+pseudomonas+infections+of+cystic+fibrosis+patients.">Effects of reduced mucus oxygen concentration in airway pseudomonas infections of cystic fibrosis patients.</a> J Clin Invest. 109 (3), 317-325 (2002).</li><li>K&#246;nig, J., Schreiber, R., Voelcker, T., Mall, M., Kunzelmann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cystic+fibrosis+transmembrane+conductance+regulator+(CFTR)+inhibits+ENaC+through+an+increase+in+the+intracellular+Cl-+concentration.">The cystic fibrosis transmembrane conductance regulator (CFTR) inhibits ENaC through an increase in the intracellular Cl- concentration.</a> EMBO Reps. 2 (11), 1047-1051 (2001).</li><li>Palmer, K. L., Mashburn, L. M., Singh, P. K., Whiteley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+sputum+supports+growth+and+cues+key+aspects+of+Pseudomonas+aeruginosa+physiology.">Cystic fibrosis sputum supports growth and cues key aspects of Pseudomonas aeruginosa physiology.</a> J Bacteriol. 187 (15), 5267-5277 (2005).</li><li>Valenza, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+antimicrobial+susceptibility+of+microorganisms+isolated+from+sputa+of+patients+with+cystic+fibrosis.">Prevalence and antimicrobial susceptibility of microorganisms isolated from sputa of patients with cystic fibrosis.</a> J Cyst Fibros. 7 (2), 123-127 (2008).</li><li>Alam, F., Catlow, D., Di Maio, A., Blair, J. M. A., Hall, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+enhances+meropenem+tolerance+of+Pseudomonas+aeruginosa+in+a+dual-species+biofilm.">Candida albicans enhances meropenem tolerance of Pseudomonas aeruginosa in a dual-species biofilm.</a> J Antimicrob Chemother. 75 (4), 925-935 (2020).</li><li>Burm&#248;lle, M., Ren, D., Bjarnsholt, T., S&#248;rensen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+in+multispecies+biofilms:+Do+they+actually+matter.">Interactions in multispecies biofilms: Do they actually matter.</a> Trends Microbiol. 22 (2), 84-91 (2014).</li><li>Lories, B., Belpaire, T. E. R., Smeets, B., Steenackers, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competition+quenching+strategies+reduce+antibiotic+tolerance+in+polymicrobial+biofilms.">Competition quenching strategies reduce antibiotic tolerance in polymicrobial biofilms.</a> NPJ Biofilms Microbiomes. 10 (1), 23 (2024).</li><li>Nabb, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymicrobial+interactions+induce+multidrug+tolerance+in+Staphylococcus+aureus+through+energy+depletion.">Polymicrobial interactions induce multidrug tolerance in Staphylococcus aureus through energy depletion.</a> Front Microbiol. 10, 2803 (2019).</li><li>Cornforth, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+transcriptome+during+human+infection.">Pseudomonas aeruginosa transcriptome during human infection.</a> Proc Natl Acad Sci U S A. 115 (22), E5125-E5134 (2018).</li><li>Wolter, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+small-colony+variants+are+independently+associated+with+worse+lung+disease+in+children+with+cystic+fibrosis.">Staphylococcus aureus small-colony variants are independently associated with worse lung disease in children with cystic fibrosis.</a> Clin Infect Dis. 57 (3), 384-391 (2013).</li><li>Takagi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucin+o-glycans+are+natural+inhibitors+of+Candida+albicans+pathogenicity.">Mucin o-glycans are natural inhibitors of Candida albicans pathogenicity.</a> Nat Chem Biol. 18 (7), 762-773 (2022).</li><li>Cinical and Laboratory Standard Institute. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods for dilution susceptibility tests for bacteria that grow aerobically. 35 (2), (2015).</li><li>Roberts, A. E., Kragh, K. N., Bjarnsholt, T., Diggle, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+limitations+of+in+vitro+experimentation+in+understanding+biofilms+and+chronic+infection.">The limitations of in vitro experimentation in understanding biofilms and chronic infection.</a> J Mol Biol. 427 (23), 3646-3661 (2015).</li><li>Macia, M. D., Rojo-Molinero, E., Oliver, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+susceptibility+testing+in+biofilm-growing+bacteria.">Antimicrobial susceptibility testing in biofilm-growing bacteria.</a> Clin Microbiol Infect. 20 (10), 981-990 (2014).</li><li>Coenye, T., Goeres, D., Van Bambeke, F., Bjarnsholt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+standardized+susceptibility+testing+for+microbial+biofilms+be+introduced+in+clinical+practice.">Should standardized susceptibility testing for microbial biofilms be introduced in clinical practice.</a> Clin Microbiol Infect. 24 (6), 570-572 (2018).</li><li>Turner, K. H., Wessel, A. K., Palmer, G. C., Murray, J. L., Whiteley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+genome+of+Pseudomonas+aeruginosa+in+cystic+fibrosis+sputum.">Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum.</a> Proc Natl Acad Sci U S A. 112 (13), 4110-4115 (2015).</li><li>Miles, A. A., Misra, S. S., Irwin, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+estimation+of+the+bactericidal+power+of+the+blood.">The estimation of the bactericidal power of the blood.</a> J Hyg. 38 (6), 732-749 (1938).</li><li>Robertson, S. N., Romero, M., Fenn, S., Kohler Riedi, P. L., C&#225;mara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+characterization,+and+evaluation+of+a+simple+polymicrobial+colony+biofilm+model+for+testing+of+antimicrobial+wound+dressings.">Development, characterization, and evaluation of a simple polymicrobial colony biofilm model for testing of antimicrobial wound dressings.</a> J Appl Microbiol. 135 (3), lxae042 (2024).</li><li>Rumpf, C., Lange, J., Schwartbeck, B., Kahl, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+and+cystic+fibrosis-a+close+relationship.+What+can+we+learn+from+sequencing+studies.">Staphylococcus aureus and cystic fibrosis-a close relationship. What can we learn from sequencing studies.</a> Pathogens. 10 (9), 1177 (2021).</li><li>Tay, W. H., Chong, K. K. L., Kline, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymicrobial-host+interactions+during+infection.">Polymicrobial-host interactions during infection.</a> J Mol Biol. 428 (17), 3355-3371 (2016).</li><li>Orazi, G., O'toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&amp;#34;It+takes+a+village&amp;#34;:+Mechanisms+underlying+antimicrobial+recalcitrance+of+polymicrobial+biofilms.">&amp;#34;It takes a village&amp;#34;: Mechanisms underlying antimicrobial recalcitrance of polymicrobial biofilms.</a> J Bacteriol. 202 (1), e00530-e00519 (2019).</li><li>Arjes, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+biofilm+colony+growth+supports+a+mutualism+involving+matrix+and+nutrient+sharing.">Three-dimensional biofilm colony growth supports a mutualism involving matrix and nutrient sharing.</a> eLife. 10, e64145 (2021).</li><li>Kasza, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciprofloxacin+poly(&#946;-amino+ester)+conjugates+enhance+antibiofilm+activity+and+slow+the+development+of+resistance.">Ciprofloxacin poly(&#946;-amino ester) conjugates enhance antibiofilm activity and slow the development of resistance.</a> ACS Appl Mater Interf. 16 (5), 5412-5425 (2024).</li><li>Martin, I., Waters, V., Grasemann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+to+targeting+bacterial+biofilms+in+cystic+fibrosis+airways.">Approaches to targeting bacterial biofilms in cystic fibrosis airways.</a> Int J Mol Sci. 22 (4), 2155 (2021).</li></ol>

No conflict of interest or competing financial interest is declared for this work.

The biofilm model described here enables us to mimic some aspects of the CF lung infection environment by incorporating a range of common polymicrobial interactions in a more accessible way than currently used in vitro antimicrobial testing methods such as the CDC reactor and the Lubbock biofilm model31. This is important when a range of factors, including the physical properties of the biofilm, nutrient availability, and molecular interactions between the species within the biofilm, can affect both the activity of the antimicrobial agent and the response of the microbes to the antimicrobial21. By incorporating these factors, we have been able to develop a highly versatile model that may more accurately enable the prediction of antimicrobial treatment outcomes than current models that do not consider the aforementioned factors.
P. aeruginosa, S. aureus, and C. albicans were chosen as representative Gram-positive, Gram-negative, and fungi commonly isolated in CF32. However, we have also been able to show that this model can be used for a range of other CF-relevant microbes, including Burkholderia cenocepacia, Burkholderia multivorans, Aspergillus fumigatus, and is currently being adapted for use with anaerobic bacteria such as Prevotella melaninogenica. This demonstrates the versatility of the model as a wide range of microbes could be incorporated, highlighting this model&#39;s adaptability for antimicrobial testing against a wide range of microbial pathogens. The model can also be easily adapted to a range of environmental conditions for more personalized antimicrobial sensitivity testing, including the replacement of SCFM2 with sputum from the patient.
The consideration of polymicrobial biofilm models for the testing of new antimicrobials is also paramount, as they can cover an existing gap in the antimicrobial development pipeline prior to their use in animal or human studies33,34. Alongside this, the model maintains a relatively high throughput and allows for a range of outputs relevant to both industry and research.
Although we have only exemplified CFUs and metabolic activity in this methods paper, we have also demonstrated the utility of the model in the analysis of the biofilm exo-metabolome using liquid extraction surface analysis mass spectrometry (LESA). This allows for probing some of the molecular mechanisms underpinning antimicrobial tolerance35. In addition to these other outputs, we have also demonstrated the use of this model for testing novel antimicrobial delivery systems. These include polymer-ciprofloxacin conjugates to enhance the penetration of this antibiotic within the biofilm while increasing its activity and reducing the development of resistance36.
Critical steps and considerations 
It is important to maintain sterility, especially when producing SCFM2 and media, as contamination can result in the introduction of non-naturally occurring species, which can interfere with the results. Different strains of the same microbe and different microbes may require different environmental conditions for growth or may have varying growth characteristics. Therefore, the model must be optimized before carrying out any antibiotic sensitivity testing. We recommend carrying out a small pilot study of 24 h and 48 h for mono species and polymicrobial biofilm to assess growth under standard model conditions. We have found that different P. aeruginosa strains and certain antibiotic concentrations can induce swarming off the polycarbonate disc. This can be easily overcome by using larger-sized discs which are commercially available. Biofilm disruption may also require optimization depending on the bead beater and the microbes used. We optimized the disruption method by determining the CFU/mL of a known sample before and after bead beating. We also used microscopy to visualize the bead beat samples to visualize how well the biofilm was disrupted.
One of the limitations of this CF-mimicking biofilm model is the attachment to a solid surface containing polycarbonate, which is not found in the CF lung, rather than being free-floating aggregates within the CF sputum37 . However, the solid agar model presented here precludes the need to wash the biofilms to remove planktonic microbes and ensures that the results only include microbes derived from the biofilm as opposed to a mixed planktonic biofilm culture. We have also found that the penetration of some antibiotics to the deeper areas of this biofilm model can be reduced, and the use of DNase may be required to alter the rheology of the extracellular matrix and decrease the electrostatic interactions with eDNA.
We believe that the model is highly suitable for antimicrobial testing due to its versatility, adaptability, and inclusion of environmental factors that many existing models fail to take into account. Its use in fundamental research and pre-clinical antimicrobial sensitivity testing can provide a more clinically relevant approach for AST of clinical samples and the development of novel therapeutics.

Cystic fibrosis (CF) is a genetic condition that affects over 11,000 people in the UK and 162,000 people globally1,2. Although CF is a multi-organ disease, a key symptom experienced by people with cystic fibrosis (pwCF) is the formation of abnormally thick, dehydrated mucus within their respiratory tract3. This, along with reduced cilia beating, can enhance the colonization of the lungs by a wide range of bacteria, fungi, viruses, and archaea4,5. Despite the CF lung providing conditions and selective pressures to limit the growth and survival of microorganisms, bacteria such as Pseudomonas aeruginosa are highly adapted to these harsh environments6. This allows for both colonization and survival, resulting in the formation of persistent chronic infections7.
Many of the microbes that cause these chronic infections do so using a phenotypic shift from an initial planktonic to a biofilm style of growth, either surface attached or in aggregates7. These biofilms are characterized by tightly packed bacterial communities encased by an exopolysaccharide (EPS) matrix consisting of a variety of components, including polysaccharides, proteins, lipids, and environmental DNA (eDNA)8. This matrix is a common feature between microorganisms; however, its composition can differ. For example, the main polysaccharide components of the bacterial P. aeruginosa matrix are Psl, Pel, and alginate, unlike in the fungi Candida albicans, whose main polysaccharide matrix components are mannans and glucans9. In mono-species biofilms, this matrix can greatly affect antibiotic tolerance compared to planktonic by reducing the penetration of antibiotics into the biofilm, decreasing their effectiveness10. The biofilm style of growth also induces the formation of persister cells with reduced metabolic activity compared to their planktonic counterparts and, therefore, further decreased susceptibility to antibiotics11. This style of growth is also characterized by the upregulation of some antibiotic resistance mechanisms, such as efflux pumps and those required for horizontal gene transfer, enabling the exchange of resistance genes11,12,13. In addition to these, the disease-related environment of the host very much influences the microbial physiology and the way they respond to antibiotics. This includes the increased micro-aerobiosis within the thick mucus as well as the availability of non-standard carbon sources such as amino acids and eDNA, either host-derived or from microbial degradation of lung products14,15,16.
These specific interactions are further complicated by the polymicrobial nature of biofilms, which introduces an additional layer of complexity with complex interactions not just between bacteria but also between bacteria and fungi. Compared to bacterial biofilms, there is less known regarding some of the interactions between bacterial and fungal biofilms, despite C. albicans being isolated from over 75% of people with CF15. Generally, the interactions between C. albicans and bacteria like P. aeruginosa are antagonistic but can result in more chronic and severe infections18. The combination of microbial interactions, both pathogenic and commensal, alongside a range of CF-related environmental factors can ultimately result in increased antibiotic tolerance19,20. Many of these factors are not often considered in preclinical antibiotic testing despite being attributed to increased antibiotic tolerance in existing models21.
These conditions are also difficult to recapitulate in vitro, and as a result, many models lack the presence of specific tolerance-inducing factors present in pwCF, such as those increasing beta-lactamase production in P. aeruginosa22, the induction of small colony variants in Staphylococcus aureus and the inhibition of hyphenation in C. albicans; all of which have been shown to occur in CF sputum23,24.
There is, therefore, a large disparity between the conditions used in current antibiotic sensitivity testing methods, based on planktonic or agar-plate grown cultures in standardized media such as Mueller-Hinton with disk diffusion, Etest, and CLSI broth microdilution assays, and the conditions encountered in the host environment25. This frequently fails to accurately determine the antibiotic sensitivity26. This problem is further complicated by a lack of standardization in antimicrobial biofilm testing, making it hard to accurately translate antimicrobial efficacy from the laboratory to the clinic27,28.
The polymicrobial model we have developed here demonstrates enhanced tolerance of Pseudomonas aeruginosa to a range of antimicrobials, including meropenem and tobramycin. This illustrates the large variation between current antimicrobial testing using mono-species biofilms and the polymicrobial biofilm model developed in this study to determine minimum inhibitory concentrations. This model also maintains a relatively high throughput and low-cost desirable for antimicrobial testing. The model can also be used to study the impact of antimicrobial therapy on polymicrobial dynamics and establish whether a particular treatment may lead to a particular pathogen becoming dominant, enabling the prediction of further complications. Although this model permits the build-up of complex biofilms, its set-up does not require sophisticated laboratory equipment and provides a platform for a wide range of clinical and research outputs.

<table><tbody><tr><td>1 &amp;micro;L inoculation loops&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>13 mm 0.2 &amp;micro;m pore size polycarbonate discs&amp;nbsp;</td><td>Isopore&amp;nbsp;</td><td>GTTP01300</td><td>Larger discs are also available&amp;nbsp;</td></tr><tr><td>2 mL reinforced tubes&amp;nbsp;</td><td>Thermofisher&amp;nbsp;</td><td>15545809</td><td></td></tr><tr><td>2.5 mL ceramic beads&amp;nbsp;</td><td>Qiagen</td><td>13114-325</td><td></td></tr><tr><td>500 mL borosilicate glass Duran bottle</td><td>Sigma Aldrich&amp;nbsp;</td><td>Z305197</td><td>larger bottles available in larger volumes are desired</td></tr><tr><td>6-well culture plates</td><td>Greiner</td><td>657165</td><td></td></tr><tr><td>7 mL Bijou</td><td>Thermofisher&amp;nbsp;</td><td>129B</td><td></td></tr><tr><td>96-well plates</td><td>Thermofisher&amp;nbsp;</td><td>167008</td><td>for serial dilutions in CFU assay</td></tr><tr><td>Agar plates for preparing plates of &lt;em&gt;P. aerugnisa, S. aureus, &lt;/em&gt;and &lt;em&gt;C. albicans&lt;/em&gt;</td><td></td><td></td><td>&amp;nbsp;LB miller for &lt;em&gt;P. aeruginosa &lt;/em&gt;and &lt;em&gt;S. aureus &lt;/em&gt;and Sabouraud dextrose agar&amp;nbsp; for &lt;em&gt;C. albicans&amp;nbsp;&lt;/em&gt;</td></tr><tr><td>Bead beater - suitable for 2 mL tubes</td><td>Fisherbrand&amp;nbsp;</td><td>15515799</td><td>Thermofisher bead mill 24</td></tr><tr><td>bench top centrifuge&amp;nbsp;</td><td></td><td></td><td>must be capable of at least 8000 x g</td></tr><tr><td>Black clear bottom 96 well plates</td><td>Costar&amp;nbsp;</td><td>3603</td><td></td></tr><tr><td>Bunsen Burner&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Containers for disposing of contaminated equipment and material according to the institutes health and safety regulations.&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>deionised water&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>eDNA&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>31149</td><td></td></tr><tr><td>Filter unit&amp;nbsp;</td><td>Fisherbrand&amp;nbsp;</td><td>FB12566504</td><td>Interchangeable depending on the vacuum pump used but must have a pore size of 0.2 &amp;micro;m</td></tr><tr><td>Haemocytometer and cover slip</td><td>Hawksley&amp;nbsp;</td><td>HC001</td><td>Haemocytometers may differ in size and volume. Double check and adjust CFU calculations accordingly&amp;nbsp;</td></tr><tr><td>LB broth&amp;nbsp;</td><td>oxoid&amp;nbsp;</td><td>1.46813</td><td></td></tr><tr><td>Mannitol salt agar&amp;nbsp;</td><td>Oxoid&amp;nbsp;</td><td>CM0085B</td><td></td></tr><tr><td>meropenem</td><td>abcr</td><td>Ab243429</td><td></td></tr><tr><td>Mucin from porcine stomach Type II</td><td>Sigma Aldrich&amp;nbsp;</td><td>M2378</td><td></td></tr><tr><td>Nystatin</td><td>Millipore&amp;nbsp;</td><td>1003352658</td><td></td></tr><tr><td>petri dishes&amp;nbsp;</td><td>SLS</td><td>SLS2000</td><td></td></tr><tr><td>Phosphate buffered saline&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Pseudomonas isolation agar</td><td>Millipore&amp;nbsp;</td><td>17208</td><td></td></tr><tr><td>Resazurin sodium salt&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>199303</td><td></td></tr><tr><td>Sabouraud dextrose agar&amp;nbsp;</td><td>Oxoid&amp;nbsp;</td><td>CM0041</td><td></td></tr><tr><td>selection of forceps&amp;nbsp;</td><td></td><td></td><td>fine tipped and tissue forceps with teeth for transferring ceramic beads</td></tr><tr><td>serological pipette</td><td></td><td></td><td></td></tr><tr><td>shaking and static incubators&amp;nbsp;</td><td></td><td></td><td>must be temperature controlled</td></tr><tr><td>Sparks microtitre plate reader</td><td>Tecan&amp;nbsp;</td><td></td><td>For Resazurin assay the microtitre plate reader must have the appropriate filters or be a monochromator for detecting flourescence.&amp;nbsp;</td></tr><tr><td>spectrophotometer</td><td></td><td></td><td></td></tr><tr><td>Technical agar (Agar Technical No.2 )</td><td>Oxoid&amp;nbsp;</td><td>LP0012B&amp;nbsp;</td><td></td></tr><tr><td>tetracycline</td><td>Sigma Aldrich&amp;nbsp;</td><td>T7660</td><td></td></tr><tr><td>UV crosslinker&amp;nbsp;</td><td>Spectroline&amp;nbsp;</td><td>11-992-89</td><td></td></tr><tr><td>vacuum pump/ flask</td><td>Fisherbrand&amp;nbsp;</td><td>FB12566504</td><td></td></tr><tr><td>water bath&amp;nbsp;</td><td></td><td></td><td>must be capable of maintaining 55 &amp;deg;C</td></tr><tr><td>YPD broth&amp;nbsp;</td><td>Millipore&amp;nbsp;</td><td>Y1375&amp;nbsp;</td><td>Can be bought pre-made or made using the base ingredients</td></tr></tbody></table>

development of a polymicrobial colony biofilm model to test antimicrobials in cystic fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

The simplicity of the solid-air interface biofilm model enables the screening of a large number of antimicrobials under different clinically relevant conditions at one time. This model allows the evaluation of the effectiveness of antibiotics using CFU counts and metabolic assays in both mono and polymicrobial biofilms to be carried out in a week. Due to the nature of the model, it also allows easy manipulation of the environmental conditions, such as changing the media composition and placing the biofilms under reduced oxygen and anaerobic conditions. Using this model, we have shown changes in tolerance to two antibiotics commonly used in pwCF between P. aeruginosa grown in mono-species and polymicrobial biofilms (Figure 1). Alongside this, we have been able to determine how antimicrobial treatment can impact the population dynamics within polymicrobial biofilms (Figure 2).
To carry out these experiments, the initial inoculum was prepared as specified in the above protocol, leading to the formation of stable mono species and polymicrobial biofilms with reproducible CFU/mL and small standard deviations as determined by one or two-way ANOVA. This model also highlighted consistent growth and CFU recovery, as well as allowing for the possibility of combining these classical microbiology techniques with other techniques not performed in this study without the need for sophisticated processing. These include imaging such as live/dead, matrix visualization, and molecular analysis, which other three-dimensional models are unable to offer in such an accessible manner (Figure 1)31. It should be noted that the standard deviation expected for CFU/mL increases when data is displayed as the percentage of survival compared to CFU/mL (Figure 1). When comparing the effectiveness of antibiotic treatment between mono species and polymicrobial biofilms, there was a significant increase in all the antibiotic concentrations required to achieve 50% killing. A 2-log increase in antibiotic concentration was needed to achieve this level of killing for meropenem and tobramycin (Figure 1). There was also an overall increase in P. aeruginosa survival in the presence of S. aureus and C. albicans in the polymicrobial biofilm when treated with 64 &#181;g/mL tobramycin, whereas the opposite was true for meropenem.
The method used to determine the metabolic activity measures the overall activity of the whole polymicrobial biofilm without being able to distinguish the individual contribution from each species. For this reason, only mono-species metabolic changes are shown to demonstrate the use of metabolic activity assays for this model. For mono species biofilms there was a strong relationship between P. aeruginosa survival and metabolic activity for both meropenem and tobramycin (Figure 2). The utilization of both metabolic activity and CFU counts from the same samples enables the easy identification of both bacteriostatic and bactericidal effects. For polymicrobial biofilms, the determination of metabolic activity may indicate how the targeting of one species may induce an overall increase in biofilm metabolic activity and, therefore, potentially increase the microbial growth of other species.
Although CFUs are the gold standard for antimicrobial sensitivity testing, in polymicrobial biofilms they also allow the assessment of species composition within the biofilm. By treating P. aeruginosa in polymicrobial biofilms, we are not only able to show the increased MBEC50 for this organism but also establish the effect this has on the other co-isolated species (Figure 3). This is, for instance, seen with meropenem (antipseudomonal), where the reduction of P. aeruginosa is accompanied by an increase in S. aureus and C. albicans CFU, resulting in S. aureus becoming the most prevalent organism upon exposure to certain antibiotic concentrations (Figure 3A). This highlights the importance of considering the polymicrobial nature of the biofilm due to the potential increase in the population size of other disease-causing species when treating a particular pathogen.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67213/67213fig01.jpg" /> 
Figure 1: Variation in antimicrobial tolerance of P. aeruginosa to meropenem and tobramycin between mono and polymicrobial biofilms grown in the solid-air interface model. P. aeruginosa PAO1 grown in mono or poly microbial biofilms with S. aureus and C. albicans was established using the solid-air interface models for 24 h on SCFM2. Biofilms were treated with a range of (A) meropenem or (B) tobramycin concentrations from 0.125 &#181;g/mL to 64 &#181;g/mL along with a no antibiotic control. The CFU/mL of each biofilm was determined. P. aeruginosa CFUs were determined in PIA and converted into percentage survival by using the negative control as the 100% survival. Error bars denote standard deviation, and each data point is derived from 3 biological repeats, each with three technical repeats. <a href="https://www.jove.com/files/ftp_upload/67213/67213fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67213/67213fig02.jpg" /> 
Figure 2: Variation in metabolic activity of P. aeruginosa biofilms in the presence of meropenem or tobramycin grown in the solid-air interface model. P. aeruginosa PAO1 biofilms were grown on SCFM2 in the solid-air interface model, and a range of (A) meropenem or (B) tobramycin concentrations from 0.125 &#181;g/mL to 64 &#181;g/mL were added along with a no antibiotic control. The plate was read fluorescently at an excitation of 540 nm and emission of 590 nm every 30 min. Percentage metabolic activity was calculated by determining the percentage of this activity on each antibiotic-treated sample compared to the no antibiotic control. Error bars denote standard deviation, and each data point is derived from 3 biological repeats, each with three technical repeats. <a href="https://www.jove.com/files/ftp_upload/67213/67213fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67213/67213fig03.jpg" /> 
Figure 3: Impact on total CFU counts of P. aeruginosa, S. aureus, and C. albicans recovered from polymicrobial solid-air interface model following antimicrobial treatment.&#160;S. aureus and C. albicans were grown on SCFM2 on the solid-air interface model. P. aeruginosa was added, and a range of 0.125 &#181;g/mL to 64 &#181;g/mL concentrations of (A) meropenem or (B) tobramycin were added to the biofilms alongside a no-antibiotic control. The CFU/mL of each biofilm was determined. Error bars denote standard deviation, and each data point was derived from 3 biological repeats, each with three technical repeats. <a href="https://www.jove.com/files/ftp_upload/67213/67213fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Establishment and workflow of the solid-air interface model. Graphical representation of the solid-air interface model establishment, biofilm disruption, and outputs. <a href="https://www.jove.com/files/ftp_upload/67213/Supplementary figure 1.zip" target="_blank">Please click here to download this File.</a>

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

development-polymicrobial-colony-biofilm-model-to-test-antimicrobials

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

796 Views.  University of Nottingham. This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

Video: Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for establishing new therapeutic approaches in this field. Therefore, animal models are required that are able to closely mimic the human disease and also sufficiently deal with scientific questions. The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. In this model, a small stent is surgically inserted into the ascending colon of mice or rats leading to a continuous leakage of intestinal bacteria into the peritoneal cavity. The procedure results in peritonitis, systemic bacteraemia, organ infection by gut bacteria, and systemic but also local release of several pro- and anti-inflammatory cytokines. The lethality of CASP can be controlled by the diameter of the inserted stent. A variant of this model, the so-called CASP with intervention (CASPI), raises opportunity to remove the septic focus by a second operation according to common procedures in clinical practice. CASP is an easily learnable and highly reproducible model that closely mimics the clinical course of abdominal sepsis. It leads way to study on questions in several scientific fields e.g. immunology, infectiology, or surgery.

Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. This article describes the surgical procedure of CASP. The CASP model and its variants allow the systematic investigation of various problems concerning the subject of sepsis.

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for ...

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

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&#160;pig lung, EVPL).
Several in vitro&#160;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.

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

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

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

In Vitro Polymicrobial Interactions in Cystic Fibrosis Lung Infections

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This is particularly true in the context of hard-to-treat, chronic, and polymicrobial biofilm-based infections detected in the airways of individuals living with cystic fibrosis (CF), a multiorgan genetic disease. While multiple microbiome studies have defined the microbial compositions detected in the airway of people with CF (pwCF), no in vitro models thus far have fully integrated critical CF-relevant lung features. Therefore, a significant knowledge gap exists in the capacity to investigate the mechanisms driving the pathogenesis of mixed species CF lung infections. Here, we describe a recently developed four-species microbial community model, including Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in CF-like conditions. Through the utilization of this system, clinically relevant phenotypes such as antimicrobial&#160;recalcitrance of several pathogens were observed and explored at the molecular level. The usefulness of this in vitro model resides in its standardized workflow that can facilitate the study of interspecies interactions in the context of chronic CF lung infections.

This protocol describes a cystic fibrosis (CF) lung-relevant four-species polymicrobial biofilm model that can be used to explore the impact of bacterial interspecies interactions.

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This ...

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

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Chapters in this video

Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

Microtiter Dish Biofilm Formation Assay

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

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

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes