Strains and plasmids used in this work are listed in Table 1. P. aeruginosa clinical isolates are listed in Table S1. P. aeruginosa strains from frozen cultures were maintained on Luria Bertani (LB) agar before being transferred to liquid culture media. Bacteria were cultured in iron-free Casamino Acids medium (DCAA, Visca et al., 1993) supplemented or not with 100 μM of FeCl₃ at 37°C, with vigorous shaking. When required, antibiotics were added to the media at the following concentrations for Escherichia coli, with the concentrations used for P. aeruginosa shown in parentheses: Ampicillin 100 μg/ml; carbenicillin (300 μg/ml in LB and 200 μg/ml in DCAA); and tetracycline 12.5 μg/ml (100 μg/ml). DCAA agar plates were prepared by the addition of 15 g/l bacteriological agar (Acumedia, Neogen corporation). When GaPPIX was required, a 50 mM of stock solution of GaPPIX (Frontier Scientific) was prepared in dimethyl sulfoxide (DMSO) and stored at 4°C in the dark. When Ga(NO₃)₃ was required, a 100 mM of stock solution of Ga(NO₃)₃ (Sigma-Aldrich), was prepared in double-distilled water and stored at −20°C.

The activity of GaPPIX, Ga(NO₃)₃ and Hemin (Hm) (Sigma-Aldrich) on P. aeruginosa was tested in 96-well microtiter plates (Falcon). Briefly, bacterial cells were grown over-night in DCAA supplemented with 100 μM FeCl₃ in order to obtain high cell densities, then washed in saline and diluted to an OD₆₀₀ of 0.01 in 200 μl of DCAA containing increasing concentrations (0–100 μM) of GaPPIX, Ga(NO₃)₃ or Hm. Microtiter plates were incubated for 24 h at 37°C with gentle shaking (120 rpm). Growth (OD₆₀₀) was measured in a Wallac 1420 Victor3 V multilabel plate reader (PerkinElmer). The minimum inhibitory concentration (MIC) of gallium compounds was visually determined as the lowest concentration that completely inhibited P. aeruginosa growth. As a control experiment the same procedure was performed, except that 100 μM FeCl₃ was added in the medium containing the highest concentration of gallium compounds tested (100 μM).

The antibacterial activity of gallium compounds was also assessed by disk diffusion assays. Briefly, cells from an over-night culture in DCAA supplemented with 100 μM FeCl₃ were washed and diluted in saline to OD₆₀₀ = 0.1, then seeded on the surface of DCAA agar plates supplemented or not with FeCl₃. Sterile 6-mm blank disks (ThermoFisher-Oxoid) soaked with 10 μl of a 15 mM solution of either GaPPIX or Ga(NO₃)₃ were deposited on the agar surface and the Zone Of growth Inhibition (ZOI) was measured (in mm) after 16 h of incubation at 37°C.

To observe the rescue effect of Hm and Hemoglobin (Hb), disks were soaked with 10 μl of a 7.5 mg/ml solution of bovine hemin chloride (Sigma-Aldrich) in 10 mM NaOH or bovine hemoglobin (Sigma-Aldrich) in phosphate buffered saline (PBS) and deposited on the plate surface nearby the disk soaked with GaPPIX. The appearance of a half-moon-shaped growth area around the disk soaked with Hm or Hb was detected after 16 h of incubation at 37°C.

Plasmid preparations and DNA cloning were performed according to standards methods (Sambrook et al., 1989). Restriction and DNA modifying enzymes were used following the instructions of the manufacturers. Oligonucleotide primers are listed in Table 1. To express hasR in the ΔhasRΔphuR mutant, a 2932 bp fragment containing the hasR gene with its own promoter region was amplified by PCR from the PAO1 genome using primers hasR compl FW and hasR compl RV (Table 1). The product was then digested with KpnI and BglII and directionally cloned into the corresponding sites of the shuttle vector pUCP18, giving plasmid pUCPhasR. To express phuR in ΔhasRΔphuR mutant, a 2575 bp fragment containing the phuR gene with its own promoter region was amplified by PCR from the PAO1 genome using primers phuR compl FW and phuR compl RV (Table 1). The product was then digested with EcoRI and KpnI and directionally cloned into the corresponding sites of the shuttle vector pUCP18, giving plasmid pUCPphuR. To express hasR and phuR in the ΔhasRΔphuR mutant strain, the pUCPhasRphuR plasmid previously described (Minandri et al., 2016) was used.

For mutant construction, E. coli and P. aeruginosa strains were grown in LB, with or without antibiotics, at 37 and 42°C, respectively, with vigorous aeration. Previously described suicide plasmids (Table 1) were used according to procedures detailed elsewhere (Milton et al., 1996; Frangipani et al., 2008).

Cytochrome c oxidase activity was assayed by using the artificial electron donor N,N,N',N'tetramethyl-p-phenylene diamine (TMPD) (Fluka). Briefly, bacteria were grown over-night in DCAA supplemented with 100 μM FeCl₃, then washed in saline and inoculated in DCAA to a final OD₆₀₀ = 0.05. When the mid-exponential growth phase was reached (≈6 h post inoculum), cells were washed once in saline and adjusted to an OD₆₀₀ = 1 (corresponding to ≈10⁹ CFU/ml).

Then, 10⁸ bacterial cells (100 μl) were suspended in 1.4 ml of 33 mM potassium phosphate buffer (KPi, pH 7.0). The reaction was started by the addition of 5 μl of a 0.54 M TMPD solution to the sample cuvette. The rate of TMPD oxidation was recorded spectrophotometrically at 520 nm for 8 min at 25°C. Results were expressed as μmol TMPD oxidized/min⁻¹/10⁸cells using 6.1 as the millimolar extinction coefficient of TMPD (Matsushita et al., 1982).

OMPs were isolated following the sarcosyl solubilization method (Filip et al., 1973), with some modifications. Briefly, bacteria from over-night cultures in DCAA supplemented with 100 μM FeCl₃ and 200 μg/ml Cb were washed in saline, then diluted to OD₆₀₀ = 0.05 in 60 ml DCAA supplemented with 200 μg/ml Cb, and incubated over-night at 37°C. Cells were collected by centrifugation (2500 × g, 20 min), washed with 5 ml of 30 mM Tris HCl (pH 8, Sigma-Aldrich) and suspended in 1 ml of the same buffer. Bacteria were lysed by sonication in an ice bath (8 × 20 s cycles in a Sonics Vibra-Cell™ VCX 130 sonicator), punctuated by 20 s intervals (50% power). Phenyl methyl sulfonyl fluoride (PMSF, Sigma-Aldrich) was added to cell lysate at 1 mM final concentration. Unbroken cells were removed by centrifugation at 2400 × g for 20 min, and supernatants were transferred to fresh tubes. Sarcosyl (N-laurylsarcosinate sodium salt, Sigma) was added to the supernatant to a final concentration of 2%. After 1 h incubation at room temperature with gentle shaking, the mixture was centrifuged for 2 h at 55,000 × g at 4°C. OMP pellets were suspended in 40 μl 2 x SDS-PAGE loading dye (Sambrook et al., 1989), boiled for 10 min, then separated by 8% SDS-PAGE and visualized by Coomassie brilliant blue staining.

Statistical analysis was performed with the software GraphPad Instat (GraphPad Software, Inc., La Jolla, CA), using One-Way Analysis of Variance (ANOVA), followed by Tukey-Kramer Multiple Comparisons Test.

It has been previously reported that GaPPIX has no effect on P. aeruginosa (Stojiljkovic et al., 1999). This results is quite surprising given that P. aeruginosa is able to utilize heme as an iron source, by expressing two heme-uptake systems, i.e., has and phu (Ochsner et al., 2000). However, since the effect of GaPPIX has previously been investigated in iron-rich media (Stojiljkovic et al., 1999), we sought that under these conditions iron availability would have impaired Ga(III) activity. To verify this hypothesis, we preliminary tested the effect of GaPPIX on P. aeruginosa PAO1 growth using the iron-poor medium DCAA (Visca et al., 1993), supplemented with increasing concentrations of GaPPIX, the iron-binding porphyrin Hemin, or Ga(NO₃)₃, the latter resulting very active on P. aeruginosa in this medium (Bonchi et al., 2015). Ga(NO₃)₃ completely inhibited P. aeruginosa growth at 12.5 μM, and its activity was abrogated by the addition of FeCl₃ (Figure 2A) consistent with previous findings (Kaneko et al., 2007; Frangipani et al., 2014). Although the minimal inhibitory concentration (MIC) could not be determined for up to 100 μM GaPPIX (Figure 2A), exposure of PAO1 to GaPPIX reduced bacterial growth by 50% (IC₅₀) at 12.5 μM (Figure 2A). Also in the case of GaPPIX, growth inhibition was completely reversed by the addition of FeCl₃ (Figure 2A). As expected, exposure P. aeruginosa PAO1 to Hemin promoted bacterial growth at concentrations ranging between 1.55 and 25 μM, in line with the ability of P. aeruginosa to use Hemin as an iron source (Ochsner et al., 2000).

The GaPPIX susceptibility of P. aeruginosa PAO1 was also tested using the disk diffusion assays in DCAA agar plates supplemented or not with an excess of FeCl₃ (600 μM) (Figure 2B). In FeCl₃-supplemented DCAA, both GaPPIX and Ga(NO₃)₃ caused no inhibition of PAO1 growth. Conversely, in DCAA a clear ZOI was observed around the GaPPIX and Ga(NO₃)₃ disks (Figure 2B). Different from the ZOI formed by Ga(NO₃)₃, the ZOI formed by GaPPIX was less transparent (Figure 2B), consistent with the evidence that no MIC (full inhibition) could be determined for GaPPIX in liquid DCAA (Figure 2A). Although more transparent, the ZOI caused by Ga(NO₃)₃ was smaller than that of GaPPIX (Figure 2B). These preliminary data indicate that iron-deplete conditions render P. aeruginosa PAO1 susceptible to GaPPIX-mediated growth inhibition.

The above results prompted us to investigate the effect of the intracellular iron content on GaPPIX-dependent growth inhibition. To this aim, the effect of GaPPIX was compared between P. aeruginosa PAO1 cells that had been pre-cultured in either DCAA containing 100 μM FeCl₃ (to increase the intracellular iron content) or DCAA without FeCl₃ (to lower the intracellular iron content). Iron-starved bacterial cells were significantly more susceptible to GaPPIX (P < 0.001) compared with those pre-cultured with FeCl₃ (Figure 3A). In particular, upon the addition of 0.38 μM GaPPIX, the growth of iron-starved PAO1 cells was reduced by 40% compared with cells pre-cultured in the presence of 100 μM FeCl₃ (Figure 3A).

To further investigate the correlation between the intracellular iron content and GaPPIX-dependent growth inhibition, GaPPIX susceptibility was evaluated on P. aeruginosa mutants impaired in Fe(III)-siderophore uptake systems, i.e., mutants unable to synthesize pyoverdine (ΔpvdA), pyochelin (ΔpchD), or both siderophores (ΔpvdAΔpchD) (Figure 3B). While GaPPIX-dependent growth inhibition was similar in the wild type and the ΔpchD mutant, both ΔpvdA and ΔpvdAΔpchD mutants were extremely sensitive to GaPPIX (Figure 3B). In particular, 0.38 μM GaPPIX inhibited the growth of the ΔpvdA and ΔpvdAΔpchD mutant strains by 75 and 78%, respectively, compared with the untreated cultures, while it reduced the growth of the wild-type strain and of the ΔpchD mutant by only 40 and 30%, respectively (Figure 3B). Altogether, these data indicate that the response of P. aeruginosa PAO1 to GaPPIX also depends on the carryover of intracellular iron.

To investigate the hypothesis that GaPPIX may enter P. aeruginosa cells by exploiting the same routes as heme, P. aeruginosa mutants carrying a deletion of either of the known heme receptors (ΔhasR and ΔphuR mutants; Table 1) were generated. The effect of GaPPIX on these mutants, as well as on a ΔhasRΔphuR double mutant lacking both heme receptors (Minandri et al., 2016), was investigated in DCAA in the presence of 12.5 μM GaPPIX (IC₅₀; Figure 4A). While all strains showed the same growth profiles in the untreated medium, both ΔphuR and ΔhasRΔphuR mutants grew better than the wild type or the ΔhasR mutant in the presence of 12.5 μM GaPPIX, displaying ≈50% higher growth levels relative to the wild type or the ΔhasR mutant (Figure 4A). These data suggest that, among the P. aeruginosa heme-uptake systems, phu has a more prominent role than has in the uptake of GaPPIX. Then, the effect of GaPPIX on heme-receptor mutants was evaluated in DCAA agar plates, by performing the disk diffusion assays (Figure 4B). Results showed a similar ZOI (27.6 ± 2.0 mm) for both the wild-type strain and the ΔhasR mutant, while a smaller ZOI (24.5 ± 0.7 mm) was observed for the ΔphuR mutant, indicating a less susceptible phenotype (Figure 4B, Table S2). In addition, no ZOI was observed for the ΔhasRΔphuR double mutant, indicating a fully resistant phenotype (Figure 4B). These observations indicate that both has and phu systems are implicated in GaPPIX transport, although the phu system appears to be the preferential route for the entrance of GaPPIX in P. aeruginosa cells (Figure 4B).

To further investigate the contribution of the HasR and PhuR receptors to GaPPIX-uptake, we individually expressed multicopy hasR, phuR, or both hasR and phuR in the ΔhasRΔphuR mutant strain (using plasmids pUCPhasR, pUCPphuR, or pUCPhasRphuR, respectively) (Figure 5A). The effect of GaPPIX on these strains was initially tested by the disk diffusion assays (Figure 5A). While, the empty pUCP18 vector did not alter the susceptibility of ΔhasRΔphuR to GaPPIX (cfr Figures 5A, 4B), the expression of hasR from the multicopy plasmid pUCPhasR made the ΔhasRΔphuR mutant more susceptible to GaPPIX (ZOI = 27.6 ± 2.0 mm) (Figure 5A, Table S2). The effect of GaPPIX was even more pronounced in the ΔhasΔphuR mutant overexpressing either phuR (ΔhasΔphuR carrying the multicopy plasmid pUCPphuR; ZOI = 34.0 ± 1.0 mm) or both hasR and phuR (ΔhasΔphuR carrying the multicopy plasmid pUCPhasRphuR; ZOI = 33.3 ± 0.5 mm) (Figure 5A, Table S2). GaPPIX sensitivity of the ΔhasΔphuR strain expressing hasR, phuR, or both genes, was also evaluated in DCAA liquid medium, in the presence of different concentrations of GaPPIX (Figure 5B). All strains grew equally in the untreated medium, and GaPPIX did not affect the growth of ΔhasRΔphuR/pUCP18 up to 25 μM (Figure 5B). Conversely, strains ΔhasRΔphuR/pUCPhasR, ΔhasRΔphuR/pUCPphuR, and ΔhasRΔphuR/pUCPhasRphuR were very sensitive to GaPPIX. In particular, 0.38 μM GaPPIX reduced the growth of the ΔhasRΔphuR/pUCPhasR strain by 56%, and by >80% in both ΔhasRΔphuR/pUCPphuR and ΔhasRΔphuR/pUCPhasRphuR strains (Figure 5B). This effect was much more pronounced than that observed for the parental strain PAO1 (Figure 2A). Of note, no further growth reduction was observed for both the ΔhasRΔphuR/pUCPphuR and ΔhasRΔphuR/pUCPhasRphuR mutant strains at > 0.38 μM GaPPIX. The increased sensitivity of the ΔhasRΔphuR strain expressing either hasR or phuR, relative to the wild type, can be explained by the overexpression of heme receptors from the multicopy plasmid pUCP18 (Figure 5A). To confirm this hypothesis, HasR and PhuR protein levels were visualized by SDS-PAGE analysis of OMPs purified from the different P. aeruginosa strains cultured in DCAA (Figure 5C). By comparing P. aeruginosa outer-membrane-proteins profiles of the wild type, the ΔphuR or the ΔhasRΔphuR mutant strains, the lack of a ca. 75 kDa protein in the ΔphuR or the ΔhasRΔphuR mutants, was observed. This was in good agreement with a predicted molecular mass of 82 kDa for the mature PhuR receptor. Moreover, a protein band at that position was evident in SDS-PAGE electropherograms of the ΔhasRΔphuR/pUCPphuR and the ΔhasRΔphuR/pUCPhasRphuR complemented mutants (Figure 5C). Similarly, a protein band corresponding to ca. 94 kDa, consistent with the HasR receptor mass, was absent in the ΔhasR and ΔhasRΔphuR mutants, while it was clearly detectable in the ΔhasRΔphuR/pUCPhasR and ΔhasRΔphuR/pUCPhasRphuR complemented mutants (Figure 5C). In line with previous results (Ochsner et al., 2000), protein levels greatly differed between PhuR and HasR, the latter being poorly expressed in wild-type PAO1. These results confirm that both HasR and PhuR direct GaPPIX entrance in P. aeruginosa cells, and argue for a prominent role of PhuR as a consequence of its higher expression levels, compared with HasR.

To confirm the specificity of GaPPIX for both heme-uptake systems, we investigated whether the growth inhibitory effect of GaPPIX could be rescued by the presence of Hemin (Hm) or Hemoglobin (Hb), which are known to deliver iron via heme-uptake receptors (Ochsner et al., 2000). To this aim, the heme-uptake mutant ΔhasRΔphuR overexpressing either PhuR or HasR was tested in the GaPPIX disk diffusion assays in the presence of Hm and Hb (Figure 5D). Both Hm and Hb partly rescued the growth of the ΔhasRΔphuR mutant overexpressing either PhuR (from pUCPphuR) or HasR (from pUCPhasR) thus confirming that (i) Hm, Hb and GaPPIX compete with heme receptors and (ii) GaPPIX enters P. aeruginosa cells through PhuR and HasR (Figure 5D).

It has been observed that P. aeruginosa isolates evolving during chronic lung infection in CF patients tend to accumulate mutations in siderophore loci, concomitant with preferential utilization of heme iron (Cornelis and Dingemans, 2013; Marvig et al., 2014; Andersen et al., 2015). To simulate this situation, we tested GaPPIX susceptibility of a siderphore-defective P. aeruginosa mutant overexpressing both PhuR and HasR receptors (ΔpvdAΔpchD/pUCPhasRphuR). Whereas, exposure of the ΔpvdAΔpchD mutant to GaPPIX reduced bacterial growth by 82% at 0.38 μM, expression of both hasR and phuR from multicopy plasmid pUCPhasRphuR made the ΔpvdAΔpchD mutant extremely susceptible to GaPPIX, displaying 90% growth reduction (IC₉₀) at 0.38 μM (Figure 5E). Notably, full inhibition of the ΔpvdAΔpchD/pUCPhasRphuR strain was observed upon challenge with 50 μM GaPPIX.

GaPPIX has been proven effective against a wide range of pathogenic bacteria by targeting metabolic pathways that require heme as an enzymatic cofactor, such as cellular respiration (Stojiljkovic et al., 1999). Thus, we investigated whether GaPPIX could interfere with the activity of terminal oxidases implicated in P. aeruginosa aerobic respiration. In particular, we focused on Cco-1, Cco-2, and Cio, which have been shown to sustain P. aeruginosa growth under low oxygen conditions, as those encountered in the lung of CF patients (Alvarez-Ortega and Harwood, 2007; Kawakami et al., 2010). To this aim, we initially tested the sensitivity of cytochrome c oxidases (i.e., Cox, Cco-1, and Cco-2) to GaPPIX. Strains deleted of the whole operon encoding the terminal oxidase Cox (Δcox) or both the Cco-1 and Cco-2 terminal oxidases (Δcco) were generated in the same parental strain used to generate the heme-receptor mutants (Table 1). The effect of GaPPIX was then assayed on these cytochrome-defective mutants using the TMPD redox indicator, which is an artificial electron donor to the cytochrome c (Matsushita et al., 1982). Oxidation of TMPD to a blue indophenol compound indicates electron flow to the cytochrome c terminal oxidases. Thus, cytochrome c oxidase activity was measured on P. aeruginosa PAO1 and in the Δcox and Δcco mutants grown in DCAA supplemented or not with a sub-inhibitory concentration of GaPPIX (4 μM). In whole cells cultured in the untreated medium, no cytochrome c oxidase activity could be measured in the Δcco strain (Figure 6A), confirming that in our conditions the TMPD test mainly measures the activity of Cco. Indeed, the Δcox mutation does not affect the TMPD oxidase activity (Figure 6A), as previously reported (Frangipani and Haas, 2009). This is because Cox is known to be poorly expressed during P. aeruginosa exponential growth (Kawakami et al., 2010). Interestingly, 4 μM GaPPIX reduced the respiratory activity by more than 50% in the wild-type strain PAO1 and the Δcox mutant, compared with the untreated condition (Figure 6A). These observations suggest that Cco-1 and Cco-2 terminal oxidases are sensitive to GaPPIX. To confirm these preliminary results, the effect of GaPPIX was tested on a mutant expressing only Cco-1 and Cco-2. To this aim, a ΔcyoΔcioΔcox triple mutant strain was generated. Disk diffusion assays showed that the ΔcyoΔcioΔcox mutant was more sensitive to GaPPIX than the wild type (ZOI = 34.6 ± 1.24 vs 27.6 ± 2.0 mm, respectively) (Figure 6B, Table S2). Similar results were obtained in DCAA liquid cultures. PAO1 wild type and the ΔcyoΔcioΔcox mutant showed a similar growth profile in the untreated medium (Figure 6C), whereas exposure to 0.38 μM GaPPIX reduced bacterial growth by 40% and 68%, respectively, relative to the untreated cultures (Figure 6C). Interestingly, it was possible to determine an IC₉₀ at 82 μM for the ΔcyoΔcioΔcox mutant strain (Figure 6C). These results confirm that Cco-1 and Cco-2 are targeted by GaPPIX.

Then, the effect of GaPPIX on the Cio terminal oxidase was assessed. To this purpose, sodium azide (NaN₃) was used as a specific inhibitor of copper-dependent oxidases, i.e., all terminal oxidases except Cio (Cunningham and Williams, 1995). Preliminarily, we determined the minimal NaN₃ concentration inhibiting all terminal oxidases except Cio in DCAA, by comparing the growth of wild-type PAO1 and the Δcio mutant in the presence of increasing NaN₃ concentrations (250–1000 μM). We observed that 350 μM of NaN₃ completely inhibited the Δcio mutant without affecting PAO1 growth (data not shown). Then, the sensitivity of Cio to GaPPIX was tested by performing a GaPPIX disk diffusion assays with wild-type PAO1 in DCAA supplemented or not with 350 μM NaN₃. It was observed that PAO1 remains sensitive to GaPPIX in the presence of 350 μM NaN₃, displaying a ZOI even greater than that obtained for PAO1 without NaN₃ (36.6 ± 3.0 vs 27.6 ± 2.0 mm, respectively) (Figure 7A, Table S2). This result provides evidence that Cio is a target for GaPPIX. To strengthen this evidence, a P. aeruginosa ΔcyoΔccoΔcox triple mutant, which expresses only Cio (Table 1) was constructed and assayed for GaPPIX susceptibility. Disk diffusion assay results showed that the ΔcyoΔccoΔcox mutant was more sensitive to GaPPIX than the wild-type PAO1 (ZOI = 30.0 ± 0.7 vs 27.6 ± 2.0 mm, respectively) (Figure 7A, Table S2). Similar results were also obtained in DCAA liquid medium, showing that GaPPIX significantly reduced (P < 0.001) the growth of the ΔcyoΔccoΔcox mutant relative to the wild type, at concentrations ranging between 0.38 and 6.25 μM (Figure 7B). Altogether, the above results indicate that P. aeruginosa Cco-1, Cco-2, and Cio terminal oxidases are targets for GaPPIX.

The expression of P. aeruginosa genes encoding heme-uptake systems has recently been detected in sputum samples collected from CF patients (Konings et al., 2013), and an evolution toward preferential heme utilization has been documented in P. aeruginosa during the course of chronic lung infection in CF patients (Marvig et al., 2014; Nguyen et al., 2014). Given the importance of heme in sustaining P. aeruginosa growth during infection, we have comparatively assessed the response to Ga(NO₃)₃ and GaPPIX in a collection of P. aeruginosa clinical isolates from CF and non-CF patients (Figure 8, Table S1). Although GaPPIX (up to 100 μM) never abolished P. aeruginosa growth, the majority of clinical isolates (>70%) was sensitive to GaPPIX, displaying an IC₅₀ values in the range 0.1–15.2 μM (Table S1). Moreover, all but one P. aeruginosa clinical isolates were significantly more susceptible than the reference PAO1 strain (Figure 8). In line with previous reports (Bonchi et al., 2015), all clinical isolates except one (FM1, Table S1) were very sensitive to Ga(NO₃)₃, showing IC₅₀ values ranging from 0.2 to 9 μM (Table S1).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.