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Bacterial strains used in this work are listed in Table S1. Bacteria were routinely cultured for 18 h in Tryptic Soy Broth (TSB, Acumedia) with vigorous shaking. When required, tetracycline (Tc), gentamicin (Gm), and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal, Sigma) were added to the media. For A. baumannii, 50 μg/ml Tc, and 100 μg/ml Gm were used. For Escherichia coli 10 μg/ml Gm and 40 μg/ml X-Gal were used. When vitamins (Vit) were required, 19 μg /ml of nicotinic acid (Sigma-Aldrich), and 2 μg /ml of pyridoxal hydrochloride (MERCK) were added to the media. Bovine hemin chloride (Sigma-Aldrich) was freshly prepared in 10 mM NaOH. Bovine serum albumin (BSA, Sigma-Aldrich) was freshly prepared and added to the media at the final concentration of 5 mg/ml.

Three media have been used in this study: (i) BBL Mueller Hinton II (Cation-Adjusted) Broth (MHB, Becton Dickinson) was prepared according to the manufacturer's instructions; (ii) DMHB was prepared following the approved CLSI protocol for antimicrobial susceptibility testing (Hackel et al., 2018). Briefly, MHB was treated for 16 h at 4°C with 100 g/l of the metal-chelating Chelex® 100 resin (Bio-Rad) under moderate stirring, then filtered through Whatman no. 1 filter paper and pH adjusted to 7.3. After autoclaving, CaCl₂ and MgSO₄ were added to DMHB at the final concentrations of 22.5 and 11.25 μg/ml, respectively; and (iii) RPMI-1640 (Sigma-Aldrich) supplemented with 10% complement-free human serum (RPMI-HS). Human serum was collected from 140 healthy donors, pooled, filtered, and inactivated (30 min, 56°C), as previously described (Antunes et al., 2012). Bulk serum chemistry was: total serum proteins 80 mg/ml; total iron 0.70 μg/ml; ferritin 0.243 μg/ml; transferrin 2.63 mg/ml; total iron binding capacity 4.27 mg/ml (20% transferrin saturation).

Three Ga(III) compounds were used in this study: (i) GaN (Ga(NO₃)₃ × 6H₂O, Sigma-Aldrich; quality tested), freshly prepared as a 100 mM stock solution in water; (ii) GaM (NORAC Pharma, provided by Dr. Bernstein), freshly prepared as a 22 mM stock solution in water; and (iii) GaPPIX (Frontier Scientific), prepared as a 25 mM stock solution in dimethyl sulfoxide (DMSO), and stored at 4°C in the dark.

The iron concentration of MHB, DMHB, and RPMI-HS was measured by inductively coupled plasma optical emission spectrometry (ICP-OES) using an ICP-OES 710 Varian Spectrometer (Agilent Technologies). Briefly, the medium was mixed with 5% HNO₃, heated for 1 h at 90°C, and filtered through a Millipore membrane (pore size 0.45 μm) prior to ICP-OES analysis.

Siderophore production was determined by the chrome azurol S-Fe(III)-hexadecyltrimethylammonium bromide method (Schwyn and Neilands, 1987). Activity of the basA::lacZ reporter gene fusion carried by plasmid pMP220::PbasA (Antunes et al., 2012) was tested in the reference strain A. baumannii ATCC 17978, and β-galactosidase levels were expressed as Miller units (Miller, 1972).

The inhibitory activity of Ga(III) compounds on ESKAPE pathogens was assessed by the microdilution method (Clinical Laboratory Standards Institute, 2015), with minor modifications. Bacteria were grown for 18 h in TSB, then washed in saline and diluted to obtain ca. 5 × 10⁵ CFU/ml in 200 μl of MHB, DMHB, or RPMI-HS, in the presence of increasing concentrations (0 to 128 μM) of each Ga(III) compound [GaN or GaM or GaPPIX], using 96-well microtiter plates. Plates were incubated for 24 h at 37°C with orbital shaking (110 rpm). The MIC of Ga(III) compounds was determined as the lowest concentration that completely inhibited bacterial growth as detected by the unaided eye (Clinical Laboratory Standards Institute, 2015). To test the effect of Fe(III) and hemin on GaPPIX antibacterial activity, freshly prepared FeCl₃ (Sigma-Aldrich) or bovine hemin chloride (Sigma-Aldrich) were added at the indicated final concentrations, into 200 μl of MHB inoculated with ca. 5 × 10⁵ CFU/ml, in the presence of GaPPIX supplied at the MIC. Microtiter plates were incubated for up to 24 h at 37°C and bacterial growth {optical density at 600 nm [OD₆₀₀]} was periodically measured using SPARK 10M TECAN reader.

The antibacterial activity of GaPPIX on A. baumannii strains was also assessed by disk diffusion assays. Briefly, 18 h cultures in TSB were washed and diluted in saline to OD₆₀₀ = 0.1, then seeded with a sterile swab on the surface of RPMI-HS supplemented with 15 g/l agar (Acumedia). Sterile 6-mm blank disks (ThermoFisher-Oxoid) soaked with 10 μl of a 15 mM solution of GaPPIX were deposited on the agar surface, and the growth inhibition halo was detected after 18 h incubation at 37°C.

Time-kill kinetic assays of Ga(III) compounds were performed on eleven ESKAPE pathogens according to a previously described procedure (Principe et al., 2009), with minor modifications. Briefly, tubes containing 1 ml of RPMI-HS supplemented with 28 μM of GaN, GaM or GaPPIX were inoculated with bacteria to a density ca. 5 × 10⁵ CFU/ml, and incubated at 37°C with gentle shaking (120 rpm). Aliquots were removed at time 0, 3, 6, and 24 h post-inoculation, and serially diluted in saline for determination of viable counts on Luria Bertani (LB) agar plates.

Previously described oligonucleotides and PCR conditions were used to check the presence of genes belonging to the heme iron-uptake gene cluster 2 (hereafter termed hemT cluster), and the heme iron-uptake gene cluster 3 (Antunes et al., 2011), which includes the hemO gene, hence named hemO cluster (Ou et al., 2015).

The 9,833 bp DNA fragment encompassing eight genes of the hemO cluster of ACICU (from ACICU_00873 to ACICU_00880 locus) was obtained by PCR amplification using primers HemO_FW (5′-CATTTGGTTTCCGAGTCTCG-3′) and HemO_RV (5′-CCATGATGCGTACCATGCA-3′). The PCR product was purified by the PCR Clean-Up System (Promega) and blunt-end ligated to the SmaI site of pVRL1 (Lucidi et al., 2018), yielding plasmid pVRL1hemO. The pVRL1hemO plasmid was introduced in A. baumannii ATCC 17978 by electroporation according to published procedures (Yildirim et al., 2016). Transformants were selected on LB agar plates supplemented with 100 μg/ml Gm.

For a comparative assessment of the antibacterial effect of the three Ga(III) compounds, a representative collection of ESKAPE species was used, including reference strains and multidrug-resistant (MDR) clinical isolates (Table S1). Since Ga(III) is an Fe(III)-mimetic acting as a metabolic competitor of Fe(III), its antibacterial activity depends on the iron concentration in the test medium, being enhanced by conditions of relative iron scarcity (Minandri et al., 2014). Therefore, both chemical analyses and functional assays were performed to probe iron content and availability in MHB, DMHB, and RPMI-HS media, prior to Ga(III)-susceptibility testing. ICP-OES measurements (Figure S1) showed that the iron concentrations in DMHB (0.43 μM) and RPMI-HS (1.95 μM) were lower than in MHB (3.38 μM). The relatively high iron concentration of RPMI-HS can be ascribed to partially (ca. 20%) iron-saturated transferrin in human serum, since only iron traces (0.11 μM) are present in serum-free RPMI-1640 (data not shown). To evaluate whether DMHB and RPMI-HS are perceived by bacteria as iron-poor media, both siderophore production and iron-repressible gene expression were investigated by using A. baumannii ATCC 17978 as a biosensor organism. Notably, high siderophore levels were produced in both DMHB and RPMI-HS, as opposed to MHB (Figure S1). Moreover, a transcriptional fusion between the promoter of the iron-repressible basA gene and the reporter lacZ gene (Antunes et al., 2012) was expressed by A. baumannii ATCC 17978 at higher levels in DMHB and RPMI-HS than in MHB (Figure S1). These data indicate that DMHB and RPMI-HS are low-iron media that induce an iron-starvation response during bacterial growth. ESKAPE pathogens share similar iron-mediated regulatory mechanisms of gene expression, all possessing the Ferric uptake regulator protein Fur, which drives the expression of iron-repressible genes, including those for siderophore-biosynthesis (i.e., basA). Therefore, it can be assumed that the basA::lacZ transcriptional fusion provides an indirect estimate of the intracellular iron levels of ESKAPE bacteria grown in different media (Ochsner and Vasil, 1996; Achenbach and Yang, 1997; Haley and Skaar, 2012; Mortensen and Skaar, 2013; Carpenter and Payne, 2014; Latorre et al., 2018). The ability of ESKAPE pathogens to grow in DMHB and RPMI-HS was then tested for the reference strains of each species (Figure S2). For all strains tested, evident growth reduction (12–60%) was observed in DMHB compared with MHB. Addition of 3 μM FeCl₃ to DMHB (i.e., restoring the iron concentration of MHB before Chelex® 100 treatment) rescued the growth of all strains, except E. faecium ATCC 19434 and S. aureus ATCC 25923 (Figure S2A). For these two species, the residual growth reduction observed in iron-replete DMHB is likely due to the removal of other metabolically relevant metals, besides iron. Moreover, all but one strain grew in RPMI-HS, although at different rates (Figure S2B). The only exception was E. faecium ATCC 19434, whose growth was rescued by the addition of two vitamins, namely nicotinic acid and pyridoxal hydrochloride. These cofactors were added to RPMI-HS to allow Ga-susceptibility testing of E. faecium (Figure S2C). These preliminary experiments allowed us to establish iron-poor culture conditions in conventional media that support the growth of all ESKAPE strains tested, thus being suitable for comparative testing of the antibacterial activity of Ga(III) compounds.

The activity of the three Ga(III) compounds was tested on a total of 24 ESKAPE strains in three selected media (Table 1). Arbitrarily assuming the resistance breakpoint at MIC > 32 μM, which roughly corresponds to the peak serum concentration of Ga(III) achievable during human therapy (Bernstein, 1998; Collery et al., 2002), all strains were resistant to the three Ga(III) compounds tested in MHB and DMHB, except S. aureus and A. baumannii, which were susceptible to GaPPIX (MIC ≤ 32 μM). Notably, the MIC of GaPPIX for S. aureus was extremely low in both MHB and DMHB (0.06–0.12 μM). Moreover, no differences in MIC values for S. aureus and A. baumannii were observed between MHB and DMHB, in spite of their different iron content, and thus of the different iron starvation status of bacteria (Figure S1). This suggests a mechanism of action of GaPPIX that is not responsive to iron. In fact, addition to MHB of 100 μM FeCl₃, i.e., in excess over GaPPIX, did not abrogate the growth inhibitory effect of GaPPIX for all S. aureus and A. baumannii strains tested (Figure 2), while 100 μM FeCl₃ alone, used as control in MHB, did not influence bacterial growth (data not shown). Since GaPPIX is likely to exert its antibacterial effect by acting as a heme analog (Stojiljkovic et al., 1999; Hijazi et al., 2017), we wondered whether hemin might counteract bacterial GaPPIX susceptibility. To verify this hypothesis, several concentrations of hemin (from 5 to 400 μM) were added to MHB together with GaPPIX, provided at the MIC (Table 1). The addition of 5 μM hemin was sufficient to completely abrogate the activity of GaPPIX against all S. aureus strains, except S. aureus UD95, for which a higher hemin concentration (50 μM) was required (Figure 2A). Conversely, not even the highest hemin concentration tested (400 μM) was able to fully reverse the growth-inhibitory effect of GaPPIX in A. baumannii (Figure 2B). Of note, also FeCl₃ provided at 400 μM (i.e., equimolar with the highest hemin concentration tested) did not rescue the growth of all A. baumannii strains tested.

Surprisingly, a dramatic loss of GaPPIX activity was observed in RPMI-HS for S. aureus and A. baumannii (MICs ≥ 128 μM for 4/4 and 2/4 strains, respectively), as opposed to the activity of GaN and GaM which was enhanced for almost all species tested in RPMI-HS, compared with MHB or DMHB (Table 1). Non-fermenting Gram-negative species, P. aeruginosa and A. baumannii, showed low MIC values for both GaN and GaM (0.5 μM < MIC < 16 μM) with some intra-species variability. Intriguingly, the hypervirulent P. aeruginosa LesB58 (Liverpool strain) showed the lowest MIC values for all Ga(III) compounds, and was the only GaPPIX-sensitive P. aeruginosa strain (Table 1). Interestingly, half of K. pneumoniae strains and one isolate of E. cloacae were also susceptible to GaN and GaM in RPMI-HS (MIC ≤ 16 μM). Moreover, no GaPPIX MIC could be determined in RPMI-HS for all Enterobacteriaceae tested (MIC > 128 μM) (Table 1). Of note, Ga(III) susceptibility was not limited to antibiotic-sensitive ESKAPE strains, but also exerted on MDR clinical isolates (Table 1).

A bactericidal activity has previously been reported for GaN and GaPPIX on P. aeruginosa and A. baumannii, respectively. However, previous killing assays were conducted in rich laboratory media, which do not mimic the condition encountered in vivo by infecting pathogens (Kaneko et al., 2007; Arivett et al., 2015). For this reason, we devised to assess the bactericidal activity of the three Ga(III) compounds in RPMI-HS, i.e., under conditions that trend to mimic biological fluids. Only susceptible strains showing MIC values < 32 μM (Table 1) were selected for bactericidal activity testing of GaN, GaM and GaPPIX, all provided at 28 μM, which corresponds to the peak serum concentration of Ga(III) achievable during human therapy (Bernstein, 1998; Collery et al., 2002). Interestingly, GaN and GaM showed only a bacteriostatic effect (Figure S3, Figure 3), whereas the response to GaPPIX varied among the susceptible strains (Figure 3). The presence of 28 μM GaPPIX caused 2–3 log reduction of viable cells (CFU counts) of A. baumannii ACICU, A. baumannii C13-373, and P. aeruginosa LesB58 after 6 h of incubation at 37°C (Figure 3), indicating a bactericidal effect of GaPPIX in RPMI-HS.

In many bacterial pathogens, severe iron limitation induces the expression of heme-uptake systems, therefore increasing the susceptibility to GaPPIX (Stojiljkovic et al., 1999; Hijazi et al., 2017). Intriguingly, S. aureus was very sensitive to GaPPIX in both MHB and DMHB, while no MIC was determined for GaPPIX in RPMI-HS (Table 1), even though RPMI-HS is iron-poor and induces an iron-starvation response in bacteria (Figure S1). Likewise, A. baumannii ATCC 17978 and AYE were much more susceptible to GaPPIX in MHB and DMHB than in RPMI-HS (Table 1). However, while all S. aureus strains were resistant to GaPPIX in RPMI-HS (MIC > 128 μM, Table 1), they became extremely sensitive to GaPPIX in RPMI-1640 without HS (MIC ≤ 0.25 μM, Table 2). Likewise, A. baumannii ATCC 17978 and AYE became sensitive to GaPPIX in RPMI-1640 without HS (MIC = 8 μM, Table 2), although showing high GaPPIX MIC in RPMI-HS (128 μM, Table 1). These results argue for the presence of HS compound(s) capable of counteracting the antibacterial activity of GaPPIX. Since human serum albumin (HSA), the most abundant plasma protein, binds a variety of ligands including heme (Adams and Berman, 1980), we hypothesized that HSA could bind GaPPIX, due to its chemical similarity with heme, thus impairing its translocation across the cell membrane via heme-uptake systems. To test this hypothesis, the susceptibility of S. aureus and A. baumannii to GaPPIX was determined in MHB and RPMI-1640 supplemented or not with BSA, a protein sharing 76% sequence identity and the same heme-binding properties as HSA (Goncharova et al., 2013). The amount of added BSA was 5 mg/ml, equaling the final concentration of HSA in RPMI-HS. Consistent with our hypothesis, addition of 5 mg/ml BSA to RPMI-1640 dramatically increased (67 to 533 fold) the MICs of GaPPIX on both S. aureus and A. baumannii (Table 2). A similar effect was also observed upon addition of BSA to MHB (Table 2). Taken together, these results suggest that the poor susceptibility to GaPPIX observed for S. aureus and A. baumannii in RPMI-HS is due to the presence of HSA which binds GaPPIX and neutralizes its inhibitory effect.

The wide range of GaPPIX susceptibility observed among A. baumannii isolates led us to hypothesize that the presence of different GaPPIX-uptake capabilities could be the source of this variability. GaPPIX is known to exploit heme-uptake routes to enter bacterial cells (Stojiljkovic et al., 1999; Hijazi et al., 2017), and all A. baumannii strains sequenced so far, including ATCC 17978, AYE and ACICU, possess the hemT heme-uptake cluster (homolog of the iron-uptake gene cluster 2 in Antunes et al., 2011). Interestingly, A. baumannii ACICU possesses an additional heme-uptake cluster, named hemO (iron-uptake gene cluster 3 in Antunes et al., 2011) (Figure 4A). Since A. baumannii ACICU showed an extremely low GaPPIX MIC (1 μM), we hypothesized that the presence of hemO could be responsible for the increased GaPPIX susceptibility, likely providing a more efficient GaPPIX uptake route. Indeed, PCR analysis revealed the presence of both hemO and hemT clusters in A. baumannii C13-373 (data not shown), another strain showing very low GaPPIX MIC (0.25 μM), similar to A. baumannii ACICU (Figure 4B). To shed more light on the relationship between heme uptake and the antibacterial activity of GaPPIX in A. baumannii, the whole hemO cluster of strain ACICU (9,833 bp), was cloned and expressed in trans in A. baumannii ATCC 17978. GaPPIX susceptibility of A. baumannii ATCC 17978 expressing the whole hemO cluster from plasmid pVRL1hemO or harboring the empty vector (pVRL1) was then assessed in both solid and liquid RPMI-HS (Figure 4B). In RPMI-HS agar plates, a much larger growth inhibition halo was observed around the GaPPIX-soaked disk for A. baumannii ATCC 17978 (pVRL1hemO), compared with A. baumannii ATCC 17978 (pVRL1), indicating that expression of the hemO cluster from a multicopy plasmid in trans greatly increases A. baumannii ATCC 17978 susceptibility to GaPPIX (Figure 4B). As expected, the presence of the empty vector pVRL1 did not affect the susceptibility of A. baumannii ATCC 17978 to GaPPIX (Figure 4B). In line with the results of the disk diffusion assay, MIC data confirmed that A. baumannii ATCC 17978 (pVRL1hemO) is more susceptible to GaPPIX than A. baumannii ATCC 17978 (pVRL1), the former showing a MIC of 0.25 μM in RPMI-HS (Figure 4B). Altogether, these data indicate that the susceptibility of A. baumannii to GaPPIX is increased by the presence of the hemO gene cluster.

LB holds several patents for possible applications of GaM in human and veterinary medicine and is affiliated with a company (Gallixa LLC) that would like to obtain regulatory approval for topical gallium maltolate as a therapeutic agent. LB did not participate in data collection for this study. The remaining 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.