Six clinical CF Pseudomonas aeruginosa isolates were used in this study, selected from a set of 44 isolates from a single sputum sample as they showed a range of phenotypes in in vitro assays (Darch et al., 2015). PA14 was used as a control laboratory strain for comparison.

Minimum inhibitory concentrations were performed according to the EUCAST guidelines (EUCAST, 2019). Briefly, bacterial isolates were cultured on Luria-Bertani (LB) agar overnight at 37°C, resuspended in Mueller-Hinton broth (MHB) to OD₆₀₀ of 0.5 and diluted 1000 times. Meropenem (Sigma-Aldrich) and ciprofloxacin (Thermo Fisher) were two-fold serially diluted in MHB (256–0.0156 μg/mL), to a final volume of 50 μL in a 96-well plate (Corning). 50 μL of the diluted bacterial suspension (5 × 10⁵ cfu/mL final concentration) was added to all wells and incubated for 18 h at 37°C before the minimum inhibitory concentrations were determined.

Minimum biofilm eradication and inhibitory concentrations (MBEC and MBIC, respectively) were performed using Calgary device (peg lids biofilm assay) according to Moskowitz et al. (2004). Briefly, 100 μL of bacterial suspensions at 0.5 McFarland were aliquoted in U shaped 96-well plates (Corning), covered with peg lids (Thermo Electron) and incubated for 20 h at 37°C to form biofilms on the pegs. Peg lids were then washed three times in sterile phosphate-buffered saline (PBS), transferred to 96-well antibiotic challenge plates containing 100 μL of 2-fold serially diluted meropenem or ciprofloxacin (128–0.25 μg/mL) and incubated for 18 h at 37°C. Peg lids were washed three times in sterile PBS, transferred to 96-well recovery plates containing 100 μL MHB and sonicated for 5 min. Peg lids were replaced with standard plate lids, measured for absorbance at 600 nm and incubated for 6 h at 37°C before checked for turbidity and OD₆₀₀.

Dissection and infection of pig lungs were performed as described in Harrison et al. (Harrison and Diggle, 2016). Briefly, pig bronchioles were dissected, UV sterilized (using a Carlton germicidal cabinet with a G8T5-8 watt germicidal tube, generating shortwave ultra-violet radiation of 2537 å) and transferred to 24-well plates with 400 μL of 0.8% agarose/ASM [Artificial Sputum Medium (Palmer et al., 2007)] as a pad. Each bronchiole tissue was infected with the bacterial isolates using a sterile syringe then 500 μL of ASM were added to each well. Uninfected bronchiole tissues were used as negative controls. Plates were then covered with UV sterilized breathable membranes (Sigma-Aldrich) and incubated at 37°C for 7 days. Tissues were then washed in 500 μL of PBS, transferred to sterile bead tubes containing 1 gm of metal beads (2.4 mm, Fisher Scientific) and 1 mL of PBS, and homogenized using a FastPrep-24^TM 5G homogenizer (MP Biomedicals) for 40 s at 4.0 m/sec. Biofilm homogenate was transferred to 96-well plates, serially diluted 10-fold and plated on LB agar plates for calculating the bacterial load.

For assessing the effect of different media, replicate infected tissues were treated with antibiotics by transferring the washed infected tissues to 48-well plates containing 300 μL of either MHB or ASM containing ciprofloxacin (Thermo Fisher), meropenem (Sigma), tobramycin (Thermo Fisher) or combination therapy at the specified concentrations and incubated at 37°C for 24 h. Antibiotic-treated tissues were then washed, homogenized and plated as previously described.

Total proteases were quantified according to Harrison et al. (2014). Briefly, 100 μL of tissue homogenate or surrounding ASM were added to 900 μL of azocasein solution (final concentration of 5 mg/mL dissolved in 100 mM Tris–HCl, 1 mM CaCl₂) in a 2 mL tube and incubated for 15 min at 37°C with shaking at 170 rpm. Then, 500 μL of 10% trichloroacetic acid were added as a stopping solution, and tubes were centrifuged at 13,000 rpm for 1 min at room temperature. 200 μL of the supernatant were transferred into a clean 96-well plate and the absorbance was measured at 400 nm. PBS was used as a negative control and a standard curve using proteinase K (Supplementary Figure S1) was used to estimate the total amount of proteases.

Total pyocyanin was quantified according to Saha et al. (2008) with minor modifications. Briefly, pyocyanin was extracted using chloroform in a ratio of 5:3. The chloroform mixture was vortexed for 2 min, then centrifuged at room temperature at 10,000 rpm for 5 min. The bottom layer was transferred to a new 2 mL tube and an equal volume of 0.2 M HCl was added. Tubes were vortexed for 2 min, centrifuged at room temperature at 10,000 rpm for 5 min and 200 μL of the top phase was transferred to a black 96-well plate and the absorbance was measured at 520 nm (Saha et al., 2008). The concentration of pyocyanin (μg/mL) was calculated by multiplying the OD₅₂₀ by 17.072 (Essar et al., 1990).

All data were analyzed by ANOVA to test for the main effect and interactions of different lung, strains and antibiotic treatments using RStudio v1.1.463 (2009-2018 RStudio, Inc.). Unpaired t-tests were performed for pairwise statistical analysis using GraphPad Prism (v8.0.1) and the familywise error rate correction for multiple comparisons.

Clinical P. aeruginosa strains were resistant to ciprofloxacin (MIC 2 μg/mL) and sensitive to meropenem (MIC ≤ 0.25 μg/mL) by standard antibiotic susceptibility testing using the broth dilution method; PA14 was sensitive to both antibiotics (Table 1). Determination of MBECs for clinical isolates using the Calgary device demonstrated an increase of 2–4-fold and 64–128-fold MIC for ciprofloxacin and meropenem, respectively (Table 1). Determination of MBICs for clinical isolates demonstrated an increase of 4–8-fold and 64–512-fold MIC for ciprofloxacin and meropenem, respectively (Table 1). Additionally, the MBEC recovery plates showed no visible production of pyocyanin, pyochelin or pyoverdine (Supplementary Figure S2).

Figure 1 represents a schematic diagram of the work flow as previously described. Supplementary Figures S3, S4 show pieces of tissues infected with P. aeruginosa strains after 7 days of biofilm formation and the mucoid phenotype of the strains in the tissues, respectively, to represent chronic CF infection (Harrington et al., 2020). Our previous work with the EVPL model confirms that P. aeruginosa forms structured biofilms in this model [Alcian blue staining confirms presence of exopolysaccharide matrix, and mutant studies showed formation of structured aggregates on tissue required the gacS/gacA pathway and pel polysaccharide (Harrington et al., 2020)].

The effect of antibiotics on the bacterial load was first tested by transferring bronchiole sections containing developed biofilms to MHB (standard medium for microdilution assays) containing antibiotics. This allowed to directly compare the inhibitory effect of these antibiotics in EVPL versus in standard diagnostics assays, without the effect of the medium or any physiological differences induced by CF lung mucus. Treatment with ciprofloxacin at 32-fold MIC (8–16-fold MBEC) resulted in a 1–2 log decrease in bacterial load across all tested clinical strains (Figure 2A). However, exposure to meropenem at 256–1024-fold MIC (4–8-fold MBEC) resulted in only about 1 log decrease of the bacterial load across all strains except SED 34, which showed less than a log decrease in the bacterial count (Figure 2B). ANOVA showed a significant effect of ciprofloxacin [F(1,69) = 749.5, p < 0.001] and meropenem treatment [F(1,69) = 115.5, p < 0.001] on bacterial load, the magnitude of which was strain dependent [strain × treatment interaction F(6,69) = 3.1, p < 0.01] for ciprofloxacin but strain independent for meropenem [strain × treatment interaction F(6,69) = 0.71, p = 0.64] despite the differences in MICs and MBECs.

To investigate the effect of the environment on antibiotic tolerance, the bacterial load of the clinical isolate SED 43 was compared with and without ciprofloxacin or meropenem in MHB and ASM: a chemically defined medium which mimics the chemistry of chronically infected CF sputum (Palmer et al., 2007; Figure 3). As shown in Figure 3, there was a statistically significant increase in tolerance to both ciprofloxacin and meropenem treatment in ASM (0.84 and 0.54 log decrease) compared with MHB (1.68 and 1.02 log decrease). ANOVA showed significant effects of changing of medium [F(1,20) = 9.04, p < 0.01], antibiotic treatment [F(1,20) = 44.66, p < 0.001], and a medium × antibiotic interaction F(1,20) = 5.56, p < 0.05.

To understand the effect of antibiotics on the virulence of P. aeruginosa strains, PA14 and three clinical isolates (SED 20, SED 41, and SED 43) were assessed for the production of two readily quantifiable and well-studied virulence factors, proteases and pyocyanin in the lung tissues homogenate and surrounding ASM, separately, in the presence and absence of antibiotics. In this work, we compared our results to P. aeruginosa infected untreated tissues and all data were normalized to uninfected untreated tissues to show the effects that are due to antibiotics’ exposure. The ASM surrounding tissue sections at the end of antibiotic exposure remained visibly clear, suggesting that bacterial cells did not detach from the biofilm or grow in the surrounding ASM at appreciable rates. Total proteases and pyocyanin were shown to be mainly released from the biofilm into the ASM, with a similar pattern in the tissues (Figures 4A,B). The control strain, PA14, showed the highest protease production (63.95–76.22 μg/mL in ASM), while clinical isolates SED 20 and SED 41 showed increased total proteases from 38.87 to 50.86 and 27.24 to 43.15 μg/mL, respectively, with meropenem treatment (Figure 4A). Interestingly, pyocyanin production varied between clinical strains with meropenem treatment. SED 20 and SED 43 infected tissues demonstrated decreased production by 68% in comparison to untreated, while SED 41 showed increased pyocyanin secretion by 162% with exposure to meropenem (Figure 4B and Supplementary Tables S1, S2). Surprisingly, ciprofloxacin treatment also showed a significantly higher pyocyanin by 116% (Figure 4B and Supplementary Table S2). Proteases and pyocyanin concentrations in tissue and surrounding ASM, and total fold increases associated with antibiotic treatments are summarized in Supplementary Tables S1, S2, respectively.

ANOVA showed a significant effect of strain [F(4,20) = 6.798, p < 0.01] and meropenem treatment [F(1,20) = 6.519, p < 0.05] on proteases production in the tissues, and the effect of meropenem did not differ between strains [strain × treatment interaction F(4,20) = 0.825, p = 0.52]. In the surrounding ASM, similar effects were observed with strain [F(4,20) = 20.92, p < 0.001] and meropenem treatment [F(1,20) = 3.193, p < 0.1]. Total pyocyanin produced in the tissues and ASM was also significantly different between strains [F(4,20) = 5.03, p < 0.01 and F(4,20) = 6.40, p < 0.01, respectively], and the effect of meropenem significantly differed between strains [strain × treatment interaction F(4,20) = 3.31, p < 0.05].

To further assess the potential effects on virulence of different clinically relevant antibiotics, we exposed EVPL biofilms of strain SED 43 to ciprofloxacin, tobramycin and a combination of meropenem and tobramycin, in ASM. Isolates are resistant to tobramycin (Darch et al., 2015). Interestingly, SED 43 showed a greater total production of proteases, in ASM, compared with the other clinical isolates (50.91 μg/mL), and slightly increased total proteases with meropenem (54.85 μg/mL, 109%) and tobramycin at low concentration (67.24 μg/mL, 133%). Both treatments led to comparable decreases in bacterial load (Supplementary Figure S5). The increased bacterial death with ciprofloxacin and tobramycin, at high concentration, (Supplementary Figure S5) correlated with decreased total proteases of 37.55 μg/mL (74%) and 39.7 μg/mL (77%), respectively. Combination treatment of tobramycin at 4 or 200 μg/mL with meropenem (64 μg/mL) showed a total protease of 33.83 (62%) and 31.07 μg/mL (59%) (Figure 4A and Supplementary Table S2), respectively.

As the total concentrations of proteases and pyocyanin discussed above are a function of both altered cellular production levels and altered cell numbers, we then normalized total proteases and pyocyanin concentrations by bacterial counts, to determine how antibiotic exposure affected per-cell production. The amount of proteases/cfu and pyocyanin/cfu measured in the tissues were slightly lower or equal to that of the surrounding ASM, respectively (Figures 4C,D). Exposure to meropenem (64 μg/mL), ciprofloxacin (64 μg/mL), and tobramycin (4 μg/mL) slightly increased the production of proteases and pyocyanin by bacterial cfu, while a significant increase was found with tobramycin (200 μg/mL) and a combination of meropenem/tobramycin (64/200 μg/mL) (Figures 4C,D). The latter treatments have a greater effect on bacterial load (Supplementary Figure S5). The total amount of tissue and ASM proteases/cfu and pyocyanin/cfu is shown in Supplementary Figure S6.

ANOVA analysis showed a significant effect of antibiotic treatment on the amount of protease/cfu [tissue F(6,13) = 11.27, p < 0.001] and [surrounding ASM F(6,13) = 37.09, p < 0.001], and production of pyocyanin [tissue F(6,13) = 25.61, p < 0.001] and [surrounding ASM F(6,13) = 17.88, p < 0.001].