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 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.

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This method allows you to grow bacteria in an environment that triggers similar cell physiology and biofilm morphology to that which you see in cystic fibrosis lung infections. The lungs are a waste products in the meat industry, so there are no ethical concerns. The model offers a platform to mimic human infection without the need for a live animal model.

This technique gives researchers a new way to screen candidate drugs for the potential to enter and destroy the biofilms that form in cystic fibrosis lungs. With further development and validation, we believe that the EVPR model could have potential use for specialized and individualized antibiotic susceptibility testing. Obtain lungs immediately after slaughter and transport them to the laboratory in a domestic cool box.

Work on a sterilized surface and under a flame. Place the lungs on a clean plastic chopping board covered with autoclaved aluminum foil, and check that the bronchioles remain intact. The lungs are not suitable for use if there has been any damage at the abattoir or during transport.

Heat a pallet knife under a flame and very briefly touch the knife to the area of the lung surrounding the bronchiole to sterilize the surface of the tissue. Cut away the surface tissue surrounding the bronchiole using a sterile-mounted razor blade, making incisions parallel to the bronchiole to prevent any damage. Once the bronchiole has been exposed, make a cross-sectional incision through the bronchiole at the highest point visible.

Using sterile forceps, lightly hold the free end of the bronchiole and cut away any remaining unwanted tissue using a sterile-mounted razor blade. Make a final cross-sectional incision across the bronchiole before any branching is visible to remove the bronchiole from the lungs. Place the bronchiole in the first DMEM RPMI 1640 wash.

Leave it in the wash and harvest additional sections of bronchiole from the same lung to yield sufficient tissue sections for the planned experiment. Place any additional bronchiolar sections from the same lung into the wash and leave them in the wash for at least two minutes. Remove the bronchioles from the first wash, and place the samples in a sterile Petri dish.

Lightly hold each bronchiole with sterile forceps, taking care not to damage the tissue. Remove as much remaining soft tissue as possible, and cut the tissue into five-millimeter-wide strips using sterile dissection scissors. Place all of the bronchiolar tissue strips into the second DMEM RPMI 1640 wash.

Leave them in the wash for least two minutes, then remove the tissue strips from the second wash using sterile forceps and place them in a clean, sterile Petri dish. Remove any remaining soft tissue attached to the bronchiole, and cut the strips into squares using sterile dissection scissors. Add the third DMEM RPMI 1640 wash into the Petri dish and lightly mix the tissue pieces in the wash by swirling the dish.

Pour the third wash out of the Petri dish without removing the tissue pieces. Then add the final SCFM wash, ensuring that all of the tissue pieces are covered. Sterilize the tissue in SCFM under UV light for five minutes.

Use sterile forceps to transfer each sterilized bronchiolar tissue piece into individual wells of a 24-well plate containing SCFM agarose pads. To infect each tissue piece with the desired bacterial strain, touch a colony grown on an agar plate with the tip of a 29-gauge needle attached to a sterile 0.5 milliliter insulin syringe, then touch the colony onto the tissue piece, gently pricking the surface. For the uninfected controls, gently prick the surface of the tissue piece with the tip of the needle, then use a pipette to add 500 microliters of SCFM to each well.

Sterilize a breathable sealing membrane for each 24-well plate under ultraviolet light for 10 minutes. Remove the lid from the 24-well plate and replace it with the breathable membrane, then incubate the plates at 37 degrees Celsius without shaking. Set up replicate sets of lung pieces from at least two independent lungs, using one set for a negative control, and one set per each concentration of antibiotic to be tested.

After 48 hours of incubation, visually inspect the uninfected tissue pieces for growth of endogenous bacteria, which would cause the medium around these sections to be turbid. If growth typical of the selected study species is observed, restart the experiment with fresh lungs. If the uninfected tissue sections show none or minimal bacterial growth, prepare one 24-well wash plate and one 24-well treatment plate.

Each well of the treatment plate should contain 500 microliters of fresh SCFM without antibiotics, or with the antibiotic of interest. Remove each infected tissue piece from the incubation plate with flame-sterilized forceps. Swirl it briefly in a fresh well of the wash plate to remove any non-biofilm-associated bacterial cells and transfer it to the appropriate well of the treatment plate.

Seal the treatment plate with a fresh breathable membrane, then incubate it at 37 degrees Celsius without shaking for 18 to 24 hours. Use flame-sterilized forceps to remove each lung piece from the 24-well plate, and put it in a sterile two-milliliter homogenization tube containing one milliliter of PBS and one gram of metal beads. Bead beat for 40 seconds at four meters per second.

Serially dilute the lung homogenate using PBS, and plate it on LB agar to determine the colony forming units in individual, untreated, and antibiotic-treated tissue pieces. When grown in the ex-vivo pig lung or EVPL, biofilms of P.aeruginosa and S.aureus demonstrate increased tolerance to antibiotics compared to susceptibility in standard, industry-approved broth MIC and disk assays using standard media. The various effects of different antibiotics on an EVPL-established biofilm are distinguishable.

For example, P.aeruginosa killing is achieved in EVPL with 4 to 16X MIC ciprofloxacin, but not with 4 to 8X MIC chloramphenicol. S.aureus populations that are susceptible to linezolid in the disk assay are able to survive target-serum concentrations and higher in EVPL. Even an optimized in-vitro assay cannot accurately predict P.aeruginosa susceptibility to colistin in the EVPL.

The amount of antibiotic required to achieve three log 10 killing of EVPL-grown bacteria is often significantly higher than the MIC or the MBEC calculated from standard in-vitro assays. In addition to distinguishing differences between anti-microbial agents, this model can highlight changes in susceptibility at different bacterial growth stages and for different antibiotic dosing intervals. The increasing tolerance of P.aeruginosa biofilms to meropenem as they mature is shown here.

S.aureus survival was measured at 4 and 24 hours post-exposure to flucloxacillin, making it possible to observe differences in the reduction of bacterial cell counts across time and between isolates. Variations in bacterial load often increase with extended culture times. This can be seen in the untreated control following 48-hour biofilm development and a further 24-hour exposure to account for antibiotic dosing interval.

It's crucial to maintain excellent sterile technique throughout the dissection, so we recommend doing the dissection in a lab or in an area of your lab that you never use for microbiology work. We recently used the model to explore the entry of colistin into biofilm, and the model has good potential for use in research to improve drug delivery into biofilms.

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Journal (Immunology and Infection)

Journal (Immunology and Infection)

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

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