Iron chelation destabilizes bacterial biofilms and potentiates the antimicrobial activity of antibiotics against coagulase-negative Staphylococci

Iron chelation destabilizes bacterial biofilms and potentiates the antimicrobial activity of... Abstract OBJECTIVES The ability of certain bacteria to form biofilms underlies their capacity to cause medical device-associated infections. Most bacteria need the metal iron for their proliferation but also to form biofilms. The aim of this in vitro study was to investigate whether iron restriction upon application of the iron chelator deferiprone (DFP) impacts on bacterial biofilm formation and whether such an intervention can exert synergistic effects towards the antibacterial activity of three antibiotic compounds against coagulase-negative staphylococci (CNS) residing on titanium plates. METHODS Bacteria were seeded on titanium discs and cultured to obtain biofilms. Biofilms were then exposed to DFP and/or antibiotic treatment with clindamycin, gentamycin or vancomycin. Fluorescence microscopy and scanning electron microscopy (SEM) were used for morphological analysis of the biofilms before and after treatment. RESULTS Whereas DFP alone had only a moderate inhibitory effect on biofilm growth, the combination of DFP with the respective antibiotics resulted in a significant decline of bacterial numbers by two to three logs as compared to the effect of antibiotics alone. Fluorescence staining and SEM demonstrated severe damage to even complete destruction of biofilms after combined treatment with DFP and antibiotics that was not the case upon sole treatment with antibiotics. CONCLUSION Iron chelation is able to potentiate the antibacterial activity of conventional antibiotics by destroying bacterial biofilms that recommends this combination as a promising strategy for the treatment of chronic device infections with biofilm producing CNS. device-related infections, coagulase-negative staphylococci, biofilms, antibiotic resistance, iron chelation INTRODUCTION Coagulase-negative staphylococci (CNS) account for the majority of foreign body-related infections (Becker, Heilmann and Peters 2014) but often such bacteria have a reduced susceptibility or even resistance to conventional antibiotics (Hoyle and Costerton 1991; Giormezis et al.2014;Benito et al.2016). One additional mechanism contributing to this phenomenon and negatively affecting the antimicrobial susceptibility of CNS is the potential of these bacteria to produce biofilms when attached to foreign body surfaces (Giormezis et al.2014; Benito et al.2016). Biofilm formation enables bacteria in general to resists and survive under conditions with limited access to nutrients or in hostile environments (Hoyle and Costerton 1991). The ability to form biofilms equips certain bacteria with the capacity to cause chronic medical device-associated infections. Biofilm formation also explains why some normal flora organisms traditionally considered ‘a-pathogenic’ become virulent when they grow in the presence of foreign bodies (Tande and Patel 2014). The presence of biofilm results in tolerance of bacteria to antibiotics because many of them cannot sufficiently penetrate through biofilms (Singh et al.2010; Brauner et al.2016). Iron is an essential element for both eukaryotes and prokaryotes because it is involved in many central metabolic processes, and a sufficient supply of iron is central for cellular proliferation and growth. Thus, the control over iron homeostasis is considered to be a central battlefield in host-pathogen interaction and infection (Soares and Weiss 2015). Bacteria such as staphylococci have developed iron acquisition systems to guarantee a sufficient supply of iron needed for their growth and proliferation. These iron acquisition systems include cell-surface-associated heme-iron extraction and transfer mechanisms to obtain heme from hemoproteins, the production of high-affinity iron scavenging siderophores to capture iron from transferrin and lactoferrin, and ferric iron reductases and transporters for the acquisition of free inorganic iron (Sheldon and Heinrichs 2015). A sufficient availability of iron is not only linked to bacterial growth and pathogenicity, but iron also plays an important role in the biofilm formation process (Lin et al.2012; Farrand et al.2015). Iron restriction strategies can limit the growth of bacteria in vitro and in vivo (Luo et al.2014; Lebeaux et al.2015; Liu et al.2017). Iron availability for bacteria can be restricted upon application of iron chelators that impair microbial proliferation (Neupane and Kim 2009; Thompson et al.2012; Nairz et al.2013). There are three classes of iron chelators currently available for clinical application. Deferiprone (DFP), as a bi-dentate chelator, and desferasirox that is a tri-dentate molecule, do not bind all six sites on the iron molecule capable of catalyzing free radical reactions. In contrast, deferoxamine is a hexa-dentate molecule capable of binding to all six iron coordination sites (Ma et al.2012; Horwitz and Horwitz 2014). DFP has a good tissue penetration (Thompson et al.2012), a certain antimicrobial activity (Kontoghiorghes et al.2010; Visca et al.2013; Ma, Gao and Maresso 2015; Gokarn and Pal 2018) and has also been shown to transport iron among cellular compartments (Sohn et al.2011). In addition to affecting the growth of bacteria, iron chelators may also impact on iron-dependent biofilm formation and thereby improve the therapeutic efficacy of antibiotics by promoting their penetration. To study this hypothesis we used a model of titanium discs colonized with biofilm forming CNS. We examined the effects of DFP, three antibiotic classes alone and combination thereof towards biofilm stability and antimicrobial efficacy. MATERIAL AND METHODS Bacterial strains Staphylococcus epidermidis (ATCC 12228) and a CNS (S. epidermidis) isolated from an implant-related infections patient were used in this study. The strains were suspended in Mueller–Hinton bouillon to a Mc Farland (McF, Mc Farland Densitometer, Den 1B, Grant Instruments (Cambridge) Ltd, Shepreth, GB) turbidity of 0.5 (1.5 × 108 CFU/mL). Antibiotics Gentamicin (gentamicin sulphate, dry powder, Heraeus Medical GmbH, Wehrheim, Germany), clindamycin (clindamycin phosphate, Dalacin-C® Phosphat (600 mg/4 mL), Pfizer Corporation Austria GmbH, Vienna, Austria) and vancomycin (vancomycin hydrochloride, Vancocin® (1g dry powder), AstroPharma GmbH, Vienna, Austria) were used. The concentration used for each antibiotic is described in Table 1. Table 1. Comparison of the MIC for S. epidermidis ATCC 12228 and a clinical CNS strain. Concentration (mg/L)  Antibiotic  S. epidermidis ATCC 12228  Clinical CNS  Gentamicin  0.064  0.064  Clindamycin  0.032  0.125  Vancomycin  2  2  Concentration (mg/L)  Antibiotic  S. epidermidis ATCC 12228  Clinical CNS  Gentamicin  0.064  0.064  Clindamycin  0.032  0.125  Vancomycin  2  2  View Large Table 1. Comparison of the MIC for S. epidermidis ATCC 12228 and a clinical CNS strain. Concentration (mg/L)  Antibiotic  S. epidermidis ATCC 12228  Clinical CNS  Gentamicin  0.064  0.064  Clindamycin  0.032  0.125  Vancomycin  2  2  Concentration (mg/L)  Antibiotic  S. epidermidis ATCC 12228  Clinical CNS  Gentamicin  0.064  0.064  Clindamycin  0.032  0.125  Vancomycin  2  2  View Large Iron chelator As iron chelator we used the commercially available and clinically used compound DFP (Sigma Aldrich, St. Louis, MI, USA), a 10 mM stock solution was stored at −20°C and a 25 μM working solution was prepared prior to the tests upon dilution with phosphate buffered saline (PBS, 9.55 g/l Dulbecco, Lonza). We chose to treat the biofilms first with DFP for 6 h and subsequently treat the biofilms with the antibiotics for 24 h. This treatment simulates an adequate clinical treatment where DFP would be applied locally by using, for example, a vacuum system direct in the wound and afterwards the patient would be systemically treated with the adequate antibiotic compound. Establishment of iron chelator doses To find an appropriate dose of DFP for subsequent experiments we exposed S. epidermidis ATCC 12228 and the CNS clinical isolate to different concentrations of DFP. For generation of biofilms 2 mL of bacterial suspensions (1.5 × 108 CFU/mL) of each strain were grown on multi-wells plates (VWR®, Radnor. Pennsylvania, USA). The plates were incubated on a shaker (Edmund Bühler GmbH, Hechingen, Germany) at 37°C (30 cycles/min) for 72 h for the obtainment of biofilms. The growth medium was not changed during this period. Thereafter, the media containing the bacteria were removed and the wells were washed with PBS for the removal of planktonic cells. All the wells containing biofilm were incubated with different concentrations of DFP (2.5, 25 and 250 μM) dissolved in 1 mL of medium at 37°C for 6 h. After incubation the wells were washed in PBS once, fresh PBS was added and the plates were sonicated for 1 min at high intensity (Bactosonic, Bandelin electronic GmbH & Co. KG, Berlin, Germany). After sonication the fluid was transferred to a Mueller–Hinton agar plate (10 μL) and incubated for 24 h at 36°C. After incubation the number of colony forming units (CFU) was defined (Fig. 1). The control wells were sonicated after the incubation of the biofilms with DFP and the CFU was counted. Figure 1. View largeDownload slide Effects of increasing DFP dosages on two isolates of S. epidermidis in vitro (average/SD). The CFU for the control group is significantly higher than all DFP treated groups. Staphylococcus epidermidis ATCC 12228 (P < 0.0001); CNS patient isolate (P < 0.005). The experiment was carried out in technical triplicate. Figure 1. View largeDownload slide Effects of increasing DFP dosages on two isolates of S. epidermidis in vitro (average/SD). The CFU for the control group is significantly higher than all DFP treated groups. Staphylococcus epidermidis ATCC 12228 (P < 0.0001); CNS patient isolate (P < 0.005). The experiment was carried out in technical triplicate. Antibiotic susceptibility tests Standard susceptibility tests for the estimation of minimal inhibitory concentration (MIC) of the antibiotics were carried out using E-test stripes (bioMérieux Austria GmbH, Vienna, Austria). The bacterial strains were plated on Mueller–Hinton agar plates (10 μL of 1.5 × 108 CFU/mL solution) with a sterile loop and afterwards the E-test stripes for each antibiotic (Lannacher Heilmittel GmbH, Lannach, Austria) were placed on the plates. The plates were incubated at 37°C for 24 h. After incubation the zones of inhibition were read from each stripe for the obtainment of MIC. The estimation of MIC for each antibiotic helped us establish the concentration used for subsequent experiments. Biofilm growth The bacterial suspensions were incubated on sterile titanium alloy discs (TiMo12Zr6Fe2, diameter 7 mm; Stryker GmbH & Co KG, Duisburg, Germany) to allow for biofilm formation. Specifically, 2 mL of bacterial suspension (1.5 × 108 CFU/mL) of each strain was added to different multi-well plates (VWR®, Radnor. Pennsylvania, USA) containing sterile titanium discs. The plates containing the inoculated discs were incubated under humidity control on a shaker (Edmund Bühler GmbH, Hechingen, Germany) at 37°C (30 cycles/min) for 72 h for the obtainment of biofilms. The growth medium was not changed during this period. CFU quantification After the growth of the biofilms, the discs containing bacteria with biofilms were removed from the media and washed in PBS for the removal of planktonic cells. The discs were then placed in a new multi-well plate (VWR®, Radnor. Pennsylvania, USA) containing fresh Mueller–Hinton medium. The discs containing biofilm were incubated in 1 mL of 25 μM of DFP for 6 h at 37°C or medium serving as control. As control the discs containing biofilms were also treated with 1 mL of 25 μM of ferric chloride or concomitantly with 25 μM of ferric chloride and 25 μM of DFP. After the incubation with DFP or control the discs were exposed to antibiotic treatment. For the antibiotic treatment the discs were incubated with gentamicin, clindamycin and vancomycin at the indicated dosages (section 2.2) at 37°C for 24 h. After that the discs were washed in PBS once and sonicated in fresh PBS for 1 min at high intensity (Bactosonic, Bandelin electronic GmbH & Co. KG, Berlin, Germany). The sonication fluid was transferred to a Mueller–Hinton agar plate (10 μL) and incubated for 24 h at 37°C. After incubation the number ofCFU was determined. Fluorescence microscopy A Live/Dead Double Staining Kit (EMD Millipore Corporation, San Diego, USA) for determination of bacterial viability was used. The staining solution was prepared in a 2 mL Eppendorf tube. The 1 mL PBS was mixed with 1 μL solution A (1 mM Cyto-Dye-calcein AM) and 1 μL solution B (2.5 mg/mL propidium iodide). The solution was then covered with aluminium foil to protect from light. The biofilms were grown in a microtiter plate containing glass discs (cover slips: 12 mm) with 2 mL of an inoculum of S. epidermidis ATCC 12228 and the CNS in Mueller–Hinton medium (McF turbidity of 0.5 each). Here cover slips were used instead of metal discs to allow the microscopy of the biofilms by fluorescence light microscopy. The discs were incubated for 72 h at 37°C in a moist chamber in an incubator shaker. After incubation, the discs containing biofilm were incubated in 1 mL of 25 μM of DFP for 6 h at 37°C or medium serving as control. After the incubation with DFP or control the discs were exposed to antibiotic treatment. For the antibiotic treatment the discs were incubated with gentamicin, clindamycin and vancomycin at the indicated dosages at 37°C for 24 h. After the treatment with antibiotics, the discs were washed with PBS for the removal of planktonic cells and were covered with the staining solution and placed upside down on glass slides. The slides were incubated in darkness for 15 min at 37°C. The fluorescence microscopy was done with an Axio Scope A1 (Carl Zeiss Microscopy GmbH, Jena, Germany) and images were captured by an AxioCamERc 5s (Carl Zeiss Microscopy GmbH, Jena, Germany). Fluorescence from propidium iodide was detected using a filter with excitation wavelength of 540–580 nm and an emission filter of 600–660 nm. Fluorescence from Cyto was detected using a filter with excitation wavelength of 465–495 nm and an emission filter of 515–555 nm. Scanning electron microscopy After biofilm growth and subsequent treatments, the discs were removed from media, washed for removal of planktonic cells and treated in 1 mL glutaraldehyde 2.5% for fixation. After fixating for 24 h at 4°C, the discs were dehydrated with an ascending alcohol series (50%–70%–80%–99.9% ethanol). Each step lasted for 5 min. After the last step the discs were placed in an incubator at 37°C for 1 h. The dried discs were placed on aluminum pins and fixed with conductive carbon cement (Leit-C; Göcke, Plano GmbH, Wetzlar, Germany). The pins were sputtered with gold (Agar Sputter Coater, Agar Scientific Ltd, Stansted, GB) for 1 min, thickness 10 nm approximately and analyzed by scanning electron microscopy (SEM, JSM-6010LV, JEOL GmbH, Freising, Germany). Data analysis The results were evaluated by using GraphPad Prism 7.00 (GraphPad Software, Inc., La Jolla, CA, USA). The results obtained for the establishment of the iron chelator dosis were statistically analysed with two-way Analysis of Variance (ANOVA) and Bonferroni's multiple comparisons test. For the difference between CFU counting within each group of strains one-way ANOVA with Turkey's multiple comparison test was carried out. Multiple t tests were carried out to compare the different treatments for the different bacterial strains. RESULTS AND DISCUSSION Susceptibility tests The MICs for gentamicin, clindamycin and vancomycin for S. epidermidis ATCC 12228 strains and a clinical isolate of CNS are shown in Table 1. These MICs were then used for the subsequent combination experiments with DFP. Establishment of iron chelator doses We then investigated the antibacterial effects of increasing dosages of DFP towards biofilm producing bacterial strains. Whereas exposure of S. epidermidis ATCC 12228 to DFP resulted in a moderate, dose-dependent reduction of biofilm formation already at a concentration of 2.5 μM, this effect was only weak with the clinical isolate of CNS and not increased by escalating dosages of DFP (Fig. 1). We thus chose the dosage of 25 μM DFP for subsequent in vitro experiments (range 10–400 μM). The results were statistically analysed with two-way ANOVA and Bonferroni's multiple comparisons test. Combined effects of iron chelation and antibiotics As control the discs containing biofilms were also treated with ferric chloride and concomitantly with ferric chloride and DFP. This treatment showed that the effect of DFP can be over-come by concomitant addition of iron to the medium (Fig. 2A and B). We then studied the effect of antibiotics with/without DFP on bacterial proliferation in vitro. The addition of antibiotics at MIC as well as DFP application when used alone resulted in a significant reduction of both bacterial isolates by several logs, however, no complete killing was observed. After exposure of biofilm producing bacteria to 25 μM of DFP for 6 h prior to the treatment with the antibiotics, a complete elimination of bacteria was observed for the S. epidermidis ATCC 12228 (Fig. 2A). We also found a significant reduction in bacterial numbers for the clinical CNS isolate (from 107 to 103 CFU/μL) upon combined treatment with DFP and antibiotics as compared to sole treatment with the respective compounds (Fig. 2B). This indicated that the addition of the iron chelator DFP amplified the antibacterial activity of conventional antibiotics against S. epidermidis. Our results are in good agreement with observations made with biofilm producing Pseudomonas aeruginosas using the metal chelator ethylenediamine tetraacetic acid (EDTA) [14]. Similarly, EDTA enhanced the antimicrobial activity of selected antibiotics against strains isolated from infected intravascular catheters (Lebeaux et al.2015). While our studies in addition highlight the importance of the specific chelation of iron we provide further details on the underlying mechanisms of this synergistic effect by disrupting biofilm integrity. Gokarn and Pal (2018) hypothesized that iron deprivation caused by iron chelators in bacteria may result in inhibition or inactivation of proteins and enzymes involved in vital functions. Siderophores may increase the biofilm permeability by scavenging iron thereby increasing the bacteria's antibiotic susceptibility (Gokarn and Pal 2018). Hypothesizing that altering bacterial iron homeostasis could help developing new antimicrobial strategies, Ma, Gao and Maresso (2015) found that E. coli is eliminated by sub-lethal doses of antibiotics when combined with the iron chelator DFP. This process is associated with increased permeability of the outer bacterial membrane, with an iron starvation response of bacteria, as measured by upregulation of the ferric uptake regulator Fur, and a rise in the level of intracellular oxygen radicals (Ma, Gao and Maresso 2015). Figure 2. View largeDownload slide Effect of combined antibiotic and DFP treatment on bacterial growth. Staphylococcus epidermidis ATCC 12228 (A) and clinical isolated CNS (B) were treated with 25 μM of DFP pure, 25 μM ferric chloride, 25 μM ferric chloride + 25 μM DFP, 0.064 mg/L gentamicin, 0032 mg/L clindamycin and 2 mg/L vancomycin pure, or combination of DFP and antibiotics for 6 h and CFU were then quantified. Within the group of S. epidermidis ATCC 12228, the CFU counting for 25 μM of DFP pure, 0.064 mg/L gentamicin, 0032 mg/L clindamycin and 2 mg/L vancomycin pure, or combination of both were significantly lower in comparison with the control group (P < 0.0001). Significant higher bacteria killing could be observed on strains treated with 25 μM DFP + antibiotics in comparison with antibiotics alone (P < 0.0001). The same could be observed within the group of CNS patient isolate (P < 0.0001). Comparing the treatments between the strains, 25 μM of DFP alone*, 25 μM DFP + 0.064 mg/L gentamicin**, and 25 μM DFP + 0032 mg/L clindamycin* showed significantly less CFUs for S. epidermidis ATCC 12228 in comparison with CNS patient isolate (** = P < 0.05; * = P < 0.0001). The combined results (average/SD) of three independent experiments performed in technical triplicates are shown. Figure 2. View largeDownload slide Effect of combined antibiotic and DFP treatment on bacterial growth. Staphylococcus epidermidis ATCC 12228 (A) and clinical isolated CNS (B) were treated with 25 μM of DFP pure, 25 μM ferric chloride, 25 μM ferric chloride + 25 μM DFP, 0.064 mg/L gentamicin, 0032 mg/L clindamycin and 2 mg/L vancomycin pure, or combination of DFP and antibiotics for 6 h and CFU were then quantified. Within the group of S. epidermidis ATCC 12228, the CFU counting for 25 μM of DFP pure, 0.064 mg/L gentamicin, 0032 mg/L clindamycin and 2 mg/L vancomycin pure, or combination of both were significantly lower in comparison with the control group (P < 0.0001). Significant higher bacteria killing could be observed on strains treated with 25 μM DFP + antibiotics in comparison with antibiotics alone (P < 0.0001). The same could be observed within the group of CNS patient isolate (P < 0.0001). Comparing the treatments between the strains, 25 μM of DFP alone*, 25 μM DFP + 0.064 mg/L gentamicin**, and 25 μM DFP + 0032 mg/L clindamycin* showed significantly less CFUs for S. epidermidis ATCC 12228 in comparison with CNS patient isolate (** = P < 0.05; * = P < 0.0001). The combined results (average/SD) of three independent experiments performed in technical triplicates are shown. Effect of combination therapy on biofilm integrity We then performed fluorescence staining for investigating the effects of single and combined treatments on biofilm integrity and the presence of live/dead bacteria. Biofilm producing bacteria residing on titanium discs treated with DFP prior to antibiotic exposure showed more dead bacteria (red areas) as compared to biofilms treated with antibiotics alone. Staphylococcus epidermidis ATCC 12228 cultivated under control conditions showed formation of biofilms, which cover the complete surface of cover slips (Fig. 3A). All live bacteria were stained in green to show viability. The same could be observed for biofilms formed by the patient isolate; here the biofilms were formed as clumps of live bacteria (Fig. 3B). The biofilms treated with 25 μM of DFP resulted in impaired growth and more dead bacteria for both strains (Fig. 3C and D). Staphylococcus epidermidis ATCC 12228 biofilms treated with gentamicin showed less live bacteria (Fig. 3E) in contrast to biofilms cultivated from the patient CNS strain. Here the biofilms were massive and no dead bacteria were observed (Fig. 3F). Clindamycin was effective against S. epidermidis ATCC 12228 (Fig. 3G) resulting in reduced biofilm growth and partial bacterial killing as reflected by the red areas. The same could be observed on the image of the CNS patient isolate (Fig. 3H). Vancomycin exposure caused killing of S. epidermidis ATCC 12228 biofilms (Fig. 3I), but only to a limited extend with the patient isolate of CNS (Fig. 3J). The combination of DFP and antibiotics showed more profound effects on the killing of bacteria residing within biofilms. Gentamicin treatment combined with DFP showed more dead S. epidermidis (Fig. 3K) and CNS (Fig. 3L) in comparison to gentamicin alone (Fig. 3E and F). The same could be observed for the combination of clindamycin and DFP (Fig. 3M and N) as well as for vancomycin plus DFP (Fig. 3O and P). Figure 3. View largeDownload slide Live and dead fluorescence staining of S. epidermidis ATCC 12228; 10x (A) and patient isolated CNS; 40x (B) biofilms. Live and dead fluorescence staining of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with 25 μM of DFP; 10x and 10x (C and D), with gentamicin; 10x and 40x (E and F), with clindamycin; 40x and 40x (G and H), with vancomycin; 40x and 40x (I and J), with the combination of gentamicin and 25 μM of DFP; 40x and 40x (K and L), with clindamycin and 25 μM of DFP; 40x and 40x (M and N) and with vancomycin and 25 μM of DFP; 40x and 40x (O and P). Live/dead staining using green-fluorescent nucleic acid stain and propidium iodide solution; green staining = living cells; yellow staining = damaged cells; red staining = dead cells. One out of three independent experiments performed in technical triplicate is shown. Figure 3. View largeDownload slide Live and dead fluorescence staining of S. epidermidis ATCC 12228; 10x (A) and patient isolated CNS; 40x (B) biofilms. Live and dead fluorescence staining of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with 25 μM of DFP; 10x and 10x (C and D), with gentamicin; 10x and 40x (E and F), with clindamycin; 40x and 40x (G and H), with vancomycin; 40x and 40x (I and J), with the combination of gentamicin and 25 μM of DFP; 40x and 40x (K and L), with clindamycin and 25 μM of DFP; 40x and 40x (M and N) and with vancomycin and 25 μM of DFP; 40x and 40x (O and P). Live/dead staining using green-fluorescent nucleic acid stain and propidium iodide solution; green staining = living cells; yellow staining = damaged cells; red staining = dead cells. One out of three independent experiments performed in technical triplicate is shown. To further verify the effects of single and combined therapies observed upon CFU quantification and fluorescence microscopy, we next performed SEM of bacterial biofilms placed on titanium cover slips. The control groups showed biofilm growth for both strains. Massive biofilms showing channels and slime formation were observed for both strains. Staphylococcus epidermidis ATCC 12228 showed larger and more structured biofilms (Fig. 4A) in comparison to the patient isolate that presented with small slime-rich islands containing the biofilms (Fig. 4B). The treatment with DFP did not allow the growth of massive biofilms. In this case only small biofilms were observed in S. epidermidis ATCC 12228 cultures (Fig. 4C) and the CNS patient isolate (Fig. 4D). In contrast to the samples treated with DFP alone, the samples treated with iron or iron plus DFP showed more biofilm formation. In these cases cluster of cells was observed distributed on the surface of the discs (Fig. 4E and H). Little biofilm formation was observed on the strains treated with gentamicin (Fig. 5A and B) in comparison to the control groups. In the groups treated with clindamycin alone, small biofilms could be observed for both strains (Fig. 5C and D), whereas only minimum biofilm formation could be observed in the groups treated with vancomycin. In this latter case, still some small biofilms or free cells could be observed on the surface of the discs (Fig. 5E and F). In comparison to the strains treated with gentamicin alone, the strains treated with the combination of gentamicin and DFP hardly any biofilm formation was evident (Fig. 6A and B). Complete killing of bacteria and biofilm disruption was achieved by combined treatment of DFP with either clindamycin (Fig. 6C and D) or vancomycin (Fig. 6E and F). These results, as well as the results of fluorescence staining (above) on biofilm integrity, strongly suggest that iron chelator treatment enhances the susceptibly of biofilm forming staphylococci to conventional antibiotics that can be largely explained by disruption of biofilm formation by the iron chelator which then enables a better penetration of antibiotics into bacteria with subsequent promotion of bacterial killing. In addition, a direct, albeit smaller antibacterial effect of iron chelators due to limitation of bacterial iron availability may also play a role in this setting and may further contribute to the synergistic action of DFP and antibiotics (Cassat and Skaar 2013; Weiss and Carver 2018). Figure 4. View largeDownload slide SEM of S. epidermidis ATCC 12228 (A) and patient isolated CNS (B) biofilms. SEM of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with 25 μM of DFP (C and D—magnification X 1.600), with 25 μM of ferric chloride (E and F); and with 25 μM of ferric chlodire and 25 μM of DFP (G and H). Magnification: (A) ×3.000; (B) ×5.500; (C) ×4.000; (D) ×1.600; (E) ×8.000; (F) ×4.500; (G) ×8.500; (H) ×3.500. Specimens were analyzed by SEM (JSM-6010LV, JEOL GmbH, Freising, Germany). One out of three independent experiments perforemd in technical triplicates is shown. Figure 4. View largeDownload slide SEM of S. epidermidis ATCC 12228 (A) and patient isolated CNS (B) biofilms. SEM of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with 25 μM of DFP (C and D—magnification X 1.600), with 25 μM of ferric chloride (E and F); and with 25 μM of ferric chlodire and 25 μM of DFP (G and H). Magnification: (A) ×3.000; (B) ×5.500; (C) ×4.000; (D) ×1.600; (E) ×8.000; (F) ×4.500; (G) ×8.500; (H) ×3.500. Specimens were analyzed by SEM (JSM-6010LV, JEOL GmbH, Freising, Germany). One out of three independent experiments perforemd in technical triplicates is shown. Figure 5. View largeDownload slide SEM of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with gentamicin (A and B), with clindamycin (C and D) and with vancomycin (E and F). Magnification: (A) ×3.700; (B) ×3.500; (C) ×1.900; (D) ×6.000; (E) ×4.300; (F) ×2.500. Specimens were analyzed by SEM (JSM-6010LV, JEOL GmbH, Freising, Germany). The experiment was carried out in technical triplicate. Figure 5. View largeDownload slide SEM of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with gentamicin (A and B), with clindamycin (C and D) and with vancomycin (E and F). Magnification: (A) ×3.700; (B) ×3.500; (C) ×1.900; (D) ×6.000; (E) ×4.300; (F) ×2.500. Specimens were analyzed by SEM (JSM-6010LV, JEOL GmbH, Freising, Germany). The experiment was carried out in technical triplicate. Figure 6. View largeDownload slide SEM of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with the combination of gentamicin and 25 μM of DFP (A and B), with clindamycin and 25 μM of DFP (C and D) and with vancomycin and 25 μM of DFP (E and F). Magnification: (A) ×2.700; (B) ×3.000; (C) ×2.300; (D) ×3.500; (E) ×800; and (F) ×2.200. Specimens were analyzed by SEM (JSM-6010LV, JEOL GmbH, Freising, Germany). The experiment was carried out in technical triplicate. Figure 6. View largeDownload slide SEM of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with the combination of gentamicin and 25 μM of DFP (A and B), with clindamycin and 25 μM of DFP (C and D) and with vancomycin and 25 μM of DFP (E and F). Magnification: (A) ×2.700; (B) ×3.000; (C) ×2.300; (D) ×3.500; (E) ×800; and (F) ×2.200. Specimens were analyzed by SEM (JSM-6010LV, JEOL GmbH, Freising, Germany). The experiment was carried out in technical triplicate. Our study thus indicates that the combination of the iron chelator DFP with conventional antibiotics can potentiate their antimicrobial activity towards biofilm producing CNS placed on titanium cover slips. We found that this interaction leads to a disruption of biofilms that likewise increases the penetration of antibiotics into bacteria where they can fulfill their antimicrobial activity. From our data it is suggestive that such a combination may be of benefit to eliminate bacteria residing on implanted devices that would be of enormous clinical benefit because bacterial biofilm formation on such surfaces is often associated with clinical failure of antibiotic therapies resulting in the need for surgical revision (Sendi and Zimmerli 2012). Iron chelators have been used to treat iron overload disorders for many years, and their pharmacological and safety profiles are well known (Hershko et al.2005; Kovacevic et al.2010). As iron is essential for bacterial growth and also involved in biofilm formation, the combination of iron chelation with antibiotic therapy may be safe and enhance the antimicrobial efficacy of antibiotics to eliminate biofilm producing bacteria on implanted devices; a hypothesis that has to be confirmed in vivo by using animal models of device related infections. Acknowledgements We would like to thank Andrea Windisch for the great technical support. FUNDING This study was supported by the Unit for Experimental Orthopedics, Department for Orthopedic Surgery, Medical University Innsbruck and by the Austrian Science Fund (FWF) funded doctoral program HOROS (W-1253, to SD, GW). Conflict of interest. None declared. REFERENCES Becker K, Heilmann C, Peters G. Coagulase-negative staphylococci. Clin Microbiol Rev  2014; 27: 870– 926. 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Google Scholar CrossRef Search ADS PubMed  Visca P, Bonchi C, Minandri F et al.   The dual personality of iron chelators: growth inhibitors or promoters? Antimicrob Agents Chemother  2013; 57: 2432– 3. Google Scholar CrossRef Search ADS PubMed  Weiss G, Carver PL. Role of divalent metals in infectious disease susceptibility and outcome. Clin Microbiol Infect   2018; 24: 16– 23. Google Scholar CrossRef Search ADS PubMed  © FEMS 2018. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Pathogens and Disease Oxford University Press

Iron chelation destabilizes bacterial biofilms and potentiates the antimicrobial activity of antibiotics against coagulase-negative Staphylococci

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2049-632X
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10.1093/femspd/fty052
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Abstract

Abstract OBJECTIVES The ability of certain bacteria to form biofilms underlies their capacity to cause medical device-associated infections. Most bacteria need the metal iron for their proliferation but also to form biofilms. The aim of this in vitro study was to investigate whether iron restriction upon application of the iron chelator deferiprone (DFP) impacts on bacterial biofilm formation and whether such an intervention can exert synergistic effects towards the antibacterial activity of three antibiotic compounds against coagulase-negative staphylococci (CNS) residing on titanium plates. METHODS Bacteria were seeded on titanium discs and cultured to obtain biofilms. Biofilms were then exposed to DFP and/or antibiotic treatment with clindamycin, gentamycin or vancomycin. Fluorescence microscopy and scanning electron microscopy (SEM) were used for morphological analysis of the biofilms before and after treatment. RESULTS Whereas DFP alone had only a moderate inhibitory effect on biofilm growth, the combination of DFP with the respective antibiotics resulted in a significant decline of bacterial numbers by two to three logs as compared to the effect of antibiotics alone. Fluorescence staining and SEM demonstrated severe damage to even complete destruction of biofilms after combined treatment with DFP and antibiotics that was not the case upon sole treatment with antibiotics. CONCLUSION Iron chelation is able to potentiate the antibacterial activity of conventional antibiotics by destroying bacterial biofilms that recommends this combination as a promising strategy for the treatment of chronic device infections with biofilm producing CNS. device-related infections, coagulase-negative staphylococci, biofilms, antibiotic resistance, iron chelation INTRODUCTION Coagulase-negative staphylococci (CNS) account for the majority of foreign body-related infections (Becker, Heilmann and Peters 2014) but often such bacteria have a reduced susceptibility or even resistance to conventional antibiotics (Hoyle and Costerton 1991; Giormezis et al.2014;Benito et al.2016). One additional mechanism contributing to this phenomenon and negatively affecting the antimicrobial susceptibility of CNS is the potential of these bacteria to produce biofilms when attached to foreign body surfaces (Giormezis et al.2014; Benito et al.2016). Biofilm formation enables bacteria in general to resists and survive under conditions with limited access to nutrients or in hostile environments (Hoyle and Costerton 1991). The ability to form biofilms equips certain bacteria with the capacity to cause chronic medical device-associated infections. Biofilm formation also explains why some normal flora organisms traditionally considered ‘a-pathogenic’ become virulent when they grow in the presence of foreign bodies (Tande and Patel 2014). The presence of biofilm results in tolerance of bacteria to antibiotics because many of them cannot sufficiently penetrate through biofilms (Singh et al.2010; Brauner et al.2016). Iron is an essential element for both eukaryotes and prokaryotes because it is involved in many central metabolic processes, and a sufficient supply of iron is central for cellular proliferation and growth. Thus, the control over iron homeostasis is considered to be a central battlefield in host-pathogen interaction and infection (Soares and Weiss 2015). Bacteria such as staphylococci have developed iron acquisition systems to guarantee a sufficient supply of iron needed for their growth and proliferation. These iron acquisition systems include cell-surface-associated heme-iron extraction and transfer mechanisms to obtain heme from hemoproteins, the production of high-affinity iron scavenging siderophores to capture iron from transferrin and lactoferrin, and ferric iron reductases and transporters for the acquisition of free inorganic iron (Sheldon and Heinrichs 2015). A sufficient availability of iron is not only linked to bacterial growth and pathogenicity, but iron also plays an important role in the biofilm formation process (Lin et al.2012; Farrand et al.2015). Iron restriction strategies can limit the growth of bacteria in vitro and in vivo (Luo et al.2014; Lebeaux et al.2015; Liu et al.2017). Iron availability for bacteria can be restricted upon application of iron chelators that impair microbial proliferation (Neupane and Kim 2009; Thompson et al.2012; Nairz et al.2013). There are three classes of iron chelators currently available for clinical application. Deferiprone (DFP), as a bi-dentate chelator, and desferasirox that is a tri-dentate molecule, do not bind all six sites on the iron molecule capable of catalyzing free radical reactions. In contrast, deferoxamine is a hexa-dentate molecule capable of binding to all six iron coordination sites (Ma et al.2012; Horwitz and Horwitz 2014). DFP has a good tissue penetration (Thompson et al.2012), a certain antimicrobial activity (Kontoghiorghes et al.2010; Visca et al.2013; Ma, Gao and Maresso 2015; Gokarn and Pal 2018) and has also been shown to transport iron among cellular compartments (Sohn et al.2011). In addition to affecting the growth of bacteria, iron chelators may also impact on iron-dependent biofilm formation and thereby improve the therapeutic efficacy of antibiotics by promoting their penetration. To study this hypothesis we used a model of titanium discs colonized with biofilm forming CNS. We examined the effects of DFP, three antibiotic classes alone and combination thereof towards biofilm stability and antimicrobial efficacy. MATERIAL AND METHODS Bacterial strains Staphylococcus epidermidis (ATCC 12228) and a CNS (S. epidermidis) isolated from an implant-related infections patient were used in this study. The strains were suspended in Mueller–Hinton bouillon to a Mc Farland (McF, Mc Farland Densitometer, Den 1B, Grant Instruments (Cambridge) Ltd, Shepreth, GB) turbidity of 0.5 (1.5 × 108 CFU/mL). Antibiotics Gentamicin (gentamicin sulphate, dry powder, Heraeus Medical GmbH, Wehrheim, Germany), clindamycin (clindamycin phosphate, Dalacin-C® Phosphat (600 mg/4 mL), Pfizer Corporation Austria GmbH, Vienna, Austria) and vancomycin (vancomycin hydrochloride, Vancocin® (1g dry powder), AstroPharma GmbH, Vienna, Austria) were used. The concentration used for each antibiotic is described in Table 1. Table 1. Comparison of the MIC for S. epidermidis ATCC 12228 and a clinical CNS strain. Concentration (mg/L)  Antibiotic  S. epidermidis ATCC 12228  Clinical CNS  Gentamicin  0.064  0.064  Clindamycin  0.032  0.125  Vancomycin  2  2  Concentration (mg/L)  Antibiotic  S. epidermidis ATCC 12228  Clinical CNS  Gentamicin  0.064  0.064  Clindamycin  0.032  0.125  Vancomycin  2  2  View Large Table 1. Comparison of the MIC for S. epidermidis ATCC 12228 and a clinical CNS strain. Concentration (mg/L)  Antibiotic  S. epidermidis ATCC 12228  Clinical CNS  Gentamicin  0.064  0.064  Clindamycin  0.032  0.125  Vancomycin  2  2  Concentration (mg/L)  Antibiotic  S. epidermidis ATCC 12228  Clinical CNS  Gentamicin  0.064  0.064  Clindamycin  0.032  0.125  Vancomycin  2  2  View Large Iron chelator As iron chelator we used the commercially available and clinically used compound DFP (Sigma Aldrich, St. Louis, MI, USA), a 10 mM stock solution was stored at −20°C and a 25 μM working solution was prepared prior to the tests upon dilution with phosphate buffered saline (PBS, 9.55 g/l Dulbecco, Lonza). We chose to treat the biofilms first with DFP for 6 h and subsequently treat the biofilms with the antibiotics for 24 h. This treatment simulates an adequate clinical treatment where DFP would be applied locally by using, for example, a vacuum system direct in the wound and afterwards the patient would be systemically treated with the adequate antibiotic compound. Establishment of iron chelator doses To find an appropriate dose of DFP for subsequent experiments we exposed S. epidermidis ATCC 12228 and the CNS clinical isolate to different concentrations of DFP. For generation of biofilms 2 mL of bacterial suspensions (1.5 × 108 CFU/mL) of each strain were grown on multi-wells plates (VWR®, Radnor. Pennsylvania, USA). The plates were incubated on a shaker (Edmund Bühler GmbH, Hechingen, Germany) at 37°C (30 cycles/min) for 72 h for the obtainment of biofilms. The growth medium was not changed during this period. Thereafter, the media containing the bacteria were removed and the wells were washed with PBS for the removal of planktonic cells. All the wells containing biofilm were incubated with different concentrations of DFP (2.5, 25 and 250 μM) dissolved in 1 mL of medium at 37°C for 6 h. After incubation the wells were washed in PBS once, fresh PBS was added and the plates were sonicated for 1 min at high intensity (Bactosonic, Bandelin electronic GmbH & Co. KG, Berlin, Germany). After sonication the fluid was transferred to a Mueller–Hinton agar plate (10 μL) and incubated for 24 h at 36°C. After incubation the number of colony forming units (CFU) was defined (Fig. 1). The control wells were sonicated after the incubation of the biofilms with DFP and the CFU was counted. Figure 1. View largeDownload slide Effects of increasing DFP dosages on two isolates of S. epidermidis in vitro (average/SD). The CFU for the control group is significantly higher than all DFP treated groups. Staphylococcus epidermidis ATCC 12228 (P < 0.0001); CNS patient isolate (P < 0.005). The experiment was carried out in technical triplicate. Figure 1. View largeDownload slide Effects of increasing DFP dosages on two isolates of S. epidermidis in vitro (average/SD). The CFU for the control group is significantly higher than all DFP treated groups. Staphylococcus epidermidis ATCC 12228 (P < 0.0001); CNS patient isolate (P < 0.005). The experiment was carried out in technical triplicate. Antibiotic susceptibility tests Standard susceptibility tests for the estimation of minimal inhibitory concentration (MIC) of the antibiotics were carried out using E-test stripes (bioMérieux Austria GmbH, Vienna, Austria). The bacterial strains were plated on Mueller–Hinton agar plates (10 μL of 1.5 × 108 CFU/mL solution) with a sterile loop and afterwards the E-test stripes for each antibiotic (Lannacher Heilmittel GmbH, Lannach, Austria) were placed on the plates. The plates were incubated at 37°C for 24 h. After incubation the zones of inhibition were read from each stripe for the obtainment of MIC. The estimation of MIC for each antibiotic helped us establish the concentration used for subsequent experiments. Biofilm growth The bacterial suspensions were incubated on sterile titanium alloy discs (TiMo12Zr6Fe2, diameter 7 mm; Stryker GmbH & Co KG, Duisburg, Germany) to allow for biofilm formation. Specifically, 2 mL of bacterial suspension (1.5 × 108 CFU/mL) of each strain was added to different multi-well plates (VWR®, Radnor. Pennsylvania, USA) containing sterile titanium discs. The plates containing the inoculated discs were incubated under humidity control on a shaker (Edmund Bühler GmbH, Hechingen, Germany) at 37°C (30 cycles/min) for 72 h for the obtainment of biofilms. The growth medium was not changed during this period. CFU quantification After the growth of the biofilms, the discs containing bacteria with biofilms were removed from the media and washed in PBS for the removal of planktonic cells. The discs were then placed in a new multi-well plate (VWR®, Radnor. Pennsylvania, USA) containing fresh Mueller–Hinton medium. The discs containing biofilm were incubated in 1 mL of 25 μM of DFP for 6 h at 37°C or medium serving as control. As control the discs containing biofilms were also treated with 1 mL of 25 μM of ferric chloride or concomitantly with 25 μM of ferric chloride and 25 μM of DFP. After the incubation with DFP or control the discs were exposed to antibiotic treatment. For the antibiotic treatment the discs were incubated with gentamicin, clindamycin and vancomycin at the indicated dosages (section 2.2) at 37°C for 24 h. After that the discs were washed in PBS once and sonicated in fresh PBS for 1 min at high intensity (Bactosonic, Bandelin electronic GmbH & Co. KG, Berlin, Germany). The sonication fluid was transferred to a Mueller–Hinton agar plate (10 μL) and incubated for 24 h at 37°C. After incubation the number ofCFU was determined. Fluorescence microscopy A Live/Dead Double Staining Kit (EMD Millipore Corporation, San Diego, USA) for determination of bacterial viability was used. The staining solution was prepared in a 2 mL Eppendorf tube. The 1 mL PBS was mixed with 1 μL solution A (1 mM Cyto-Dye-calcein AM) and 1 μL solution B (2.5 mg/mL propidium iodide). The solution was then covered with aluminium foil to protect from light. The biofilms were grown in a microtiter plate containing glass discs (cover slips: 12 mm) with 2 mL of an inoculum of S. epidermidis ATCC 12228 and the CNS in Mueller–Hinton medium (McF turbidity of 0.5 each). Here cover slips were used instead of metal discs to allow the microscopy of the biofilms by fluorescence light microscopy. The discs were incubated for 72 h at 37°C in a moist chamber in an incubator shaker. After incubation, the discs containing biofilm were incubated in 1 mL of 25 μM of DFP for 6 h at 37°C or medium serving as control. After the incubation with DFP or control the discs were exposed to antibiotic treatment. For the antibiotic treatment the discs were incubated with gentamicin, clindamycin and vancomycin at the indicated dosages at 37°C for 24 h. After the treatment with antibiotics, the discs were washed with PBS for the removal of planktonic cells and were covered with the staining solution and placed upside down on glass slides. The slides were incubated in darkness for 15 min at 37°C. The fluorescence microscopy was done with an Axio Scope A1 (Carl Zeiss Microscopy GmbH, Jena, Germany) and images were captured by an AxioCamERc 5s (Carl Zeiss Microscopy GmbH, Jena, Germany). Fluorescence from propidium iodide was detected using a filter with excitation wavelength of 540–580 nm and an emission filter of 600–660 nm. Fluorescence from Cyto was detected using a filter with excitation wavelength of 465–495 nm and an emission filter of 515–555 nm. Scanning electron microscopy After biofilm growth and subsequent treatments, the discs were removed from media, washed for removal of planktonic cells and treated in 1 mL glutaraldehyde 2.5% for fixation. After fixating for 24 h at 4°C, the discs were dehydrated with an ascending alcohol series (50%–70%–80%–99.9% ethanol). Each step lasted for 5 min. After the last step the discs were placed in an incubator at 37°C for 1 h. The dried discs were placed on aluminum pins and fixed with conductive carbon cement (Leit-C; Göcke, Plano GmbH, Wetzlar, Germany). The pins were sputtered with gold (Agar Sputter Coater, Agar Scientific Ltd, Stansted, GB) for 1 min, thickness 10 nm approximately and analyzed by scanning electron microscopy (SEM, JSM-6010LV, JEOL GmbH, Freising, Germany). Data analysis The results were evaluated by using GraphPad Prism 7.00 (GraphPad Software, Inc., La Jolla, CA, USA). The results obtained for the establishment of the iron chelator dosis were statistically analysed with two-way Analysis of Variance (ANOVA) and Bonferroni's multiple comparisons test. For the difference between CFU counting within each group of strains one-way ANOVA with Turkey's multiple comparison test was carried out. Multiple t tests were carried out to compare the different treatments for the different bacterial strains. RESULTS AND DISCUSSION Susceptibility tests The MICs for gentamicin, clindamycin and vancomycin for S. epidermidis ATCC 12228 strains and a clinical isolate of CNS are shown in Table 1. These MICs were then used for the subsequent combination experiments with DFP. Establishment of iron chelator doses We then investigated the antibacterial effects of increasing dosages of DFP towards biofilm producing bacterial strains. Whereas exposure of S. epidermidis ATCC 12228 to DFP resulted in a moderate, dose-dependent reduction of biofilm formation already at a concentration of 2.5 μM, this effect was only weak with the clinical isolate of CNS and not increased by escalating dosages of DFP (Fig. 1). We thus chose the dosage of 25 μM DFP for subsequent in vitro experiments (range 10–400 μM). The results were statistically analysed with two-way ANOVA and Bonferroni's multiple comparisons test. Combined effects of iron chelation and antibiotics As control the discs containing biofilms were also treated with ferric chloride and concomitantly with ferric chloride and DFP. This treatment showed that the effect of DFP can be over-come by concomitant addition of iron to the medium (Fig. 2A and B). We then studied the effect of antibiotics with/without DFP on bacterial proliferation in vitro. The addition of antibiotics at MIC as well as DFP application when used alone resulted in a significant reduction of both bacterial isolates by several logs, however, no complete killing was observed. After exposure of biofilm producing bacteria to 25 μM of DFP for 6 h prior to the treatment with the antibiotics, a complete elimination of bacteria was observed for the S. epidermidis ATCC 12228 (Fig. 2A). We also found a significant reduction in bacterial numbers for the clinical CNS isolate (from 107 to 103 CFU/μL) upon combined treatment with DFP and antibiotics as compared to sole treatment with the respective compounds (Fig. 2B). This indicated that the addition of the iron chelator DFP amplified the antibacterial activity of conventional antibiotics against S. epidermidis. Our results are in good agreement with observations made with biofilm producing Pseudomonas aeruginosas using the metal chelator ethylenediamine tetraacetic acid (EDTA) [14]. Similarly, EDTA enhanced the antimicrobial activity of selected antibiotics against strains isolated from infected intravascular catheters (Lebeaux et al.2015). While our studies in addition highlight the importance of the specific chelation of iron we provide further details on the underlying mechanisms of this synergistic effect by disrupting biofilm integrity. Gokarn and Pal (2018) hypothesized that iron deprivation caused by iron chelators in bacteria may result in inhibition or inactivation of proteins and enzymes involved in vital functions. Siderophores may increase the biofilm permeability by scavenging iron thereby increasing the bacteria's antibiotic susceptibility (Gokarn and Pal 2018). Hypothesizing that altering bacterial iron homeostasis could help developing new antimicrobial strategies, Ma, Gao and Maresso (2015) found that E. coli is eliminated by sub-lethal doses of antibiotics when combined with the iron chelator DFP. This process is associated with increased permeability of the outer bacterial membrane, with an iron starvation response of bacteria, as measured by upregulation of the ferric uptake regulator Fur, and a rise in the level of intracellular oxygen radicals (Ma, Gao and Maresso 2015). Figure 2. View largeDownload slide Effect of combined antibiotic and DFP treatment on bacterial growth. Staphylococcus epidermidis ATCC 12228 (A) and clinical isolated CNS (B) were treated with 25 μM of DFP pure, 25 μM ferric chloride, 25 μM ferric chloride + 25 μM DFP, 0.064 mg/L gentamicin, 0032 mg/L clindamycin and 2 mg/L vancomycin pure, or combination of DFP and antibiotics for 6 h and CFU were then quantified. Within the group of S. epidermidis ATCC 12228, the CFU counting for 25 μM of DFP pure, 0.064 mg/L gentamicin, 0032 mg/L clindamycin and 2 mg/L vancomycin pure, or combination of both were significantly lower in comparison with the control group (P < 0.0001). Significant higher bacteria killing could be observed on strains treated with 25 μM DFP + antibiotics in comparison with antibiotics alone (P < 0.0001). The same could be observed within the group of CNS patient isolate (P < 0.0001). Comparing the treatments between the strains, 25 μM of DFP alone*, 25 μM DFP + 0.064 mg/L gentamicin**, and 25 μM DFP + 0032 mg/L clindamycin* showed significantly less CFUs for S. epidermidis ATCC 12228 in comparison with CNS patient isolate (** = P < 0.05; * = P < 0.0001). The combined results (average/SD) of three independent experiments performed in technical triplicates are shown. Figure 2. View largeDownload slide Effect of combined antibiotic and DFP treatment on bacterial growth. Staphylococcus epidermidis ATCC 12228 (A) and clinical isolated CNS (B) were treated with 25 μM of DFP pure, 25 μM ferric chloride, 25 μM ferric chloride + 25 μM DFP, 0.064 mg/L gentamicin, 0032 mg/L clindamycin and 2 mg/L vancomycin pure, or combination of DFP and antibiotics for 6 h and CFU were then quantified. Within the group of S. epidermidis ATCC 12228, the CFU counting for 25 μM of DFP pure, 0.064 mg/L gentamicin, 0032 mg/L clindamycin and 2 mg/L vancomycin pure, or combination of both were significantly lower in comparison with the control group (P < 0.0001). Significant higher bacteria killing could be observed on strains treated with 25 μM DFP + antibiotics in comparison with antibiotics alone (P < 0.0001). The same could be observed within the group of CNS patient isolate (P < 0.0001). Comparing the treatments between the strains, 25 μM of DFP alone*, 25 μM DFP + 0.064 mg/L gentamicin**, and 25 μM DFP + 0032 mg/L clindamycin* showed significantly less CFUs for S. epidermidis ATCC 12228 in comparison with CNS patient isolate (** = P < 0.05; * = P < 0.0001). The combined results (average/SD) of three independent experiments performed in technical triplicates are shown. Effect of combination therapy on biofilm integrity We then performed fluorescence staining for investigating the effects of single and combined treatments on biofilm integrity and the presence of live/dead bacteria. Biofilm producing bacteria residing on titanium discs treated with DFP prior to antibiotic exposure showed more dead bacteria (red areas) as compared to biofilms treated with antibiotics alone. Staphylococcus epidermidis ATCC 12228 cultivated under control conditions showed formation of biofilms, which cover the complete surface of cover slips (Fig. 3A). All live bacteria were stained in green to show viability. The same could be observed for biofilms formed by the patient isolate; here the biofilms were formed as clumps of live bacteria (Fig. 3B). The biofilms treated with 25 μM of DFP resulted in impaired growth and more dead bacteria for both strains (Fig. 3C and D). Staphylococcus epidermidis ATCC 12228 biofilms treated with gentamicin showed less live bacteria (Fig. 3E) in contrast to biofilms cultivated from the patient CNS strain. Here the biofilms were massive and no dead bacteria were observed (Fig. 3F). Clindamycin was effective against S. epidermidis ATCC 12228 (Fig. 3G) resulting in reduced biofilm growth and partial bacterial killing as reflected by the red areas. The same could be observed on the image of the CNS patient isolate (Fig. 3H). Vancomycin exposure caused killing of S. epidermidis ATCC 12228 biofilms (Fig. 3I), but only to a limited extend with the patient isolate of CNS (Fig. 3J). The combination of DFP and antibiotics showed more profound effects on the killing of bacteria residing within biofilms. Gentamicin treatment combined with DFP showed more dead S. epidermidis (Fig. 3K) and CNS (Fig. 3L) in comparison to gentamicin alone (Fig. 3E and F). The same could be observed for the combination of clindamycin and DFP (Fig. 3M and N) as well as for vancomycin plus DFP (Fig. 3O and P). Figure 3. View largeDownload slide Live and dead fluorescence staining of S. epidermidis ATCC 12228; 10x (A) and patient isolated CNS; 40x (B) biofilms. Live and dead fluorescence staining of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with 25 μM of DFP; 10x and 10x (C and D), with gentamicin; 10x and 40x (E and F), with clindamycin; 40x and 40x (G and H), with vancomycin; 40x and 40x (I and J), with the combination of gentamicin and 25 μM of DFP; 40x and 40x (K and L), with clindamycin and 25 μM of DFP; 40x and 40x (M and N) and with vancomycin and 25 μM of DFP; 40x and 40x (O and P). Live/dead staining using green-fluorescent nucleic acid stain and propidium iodide solution; green staining = living cells; yellow staining = damaged cells; red staining = dead cells. One out of three independent experiments performed in technical triplicate is shown. Figure 3. View largeDownload slide Live and dead fluorescence staining of S. epidermidis ATCC 12228; 10x (A) and patient isolated CNS; 40x (B) biofilms. Live and dead fluorescence staining of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with 25 μM of DFP; 10x and 10x (C and D), with gentamicin; 10x and 40x (E and F), with clindamycin; 40x and 40x (G and H), with vancomycin; 40x and 40x (I and J), with the combination of gentamicin and 25 μM of DFP; 40x and 40x (K and L), with clindamycin and 25 μM of DFP; 40x and 40x (M and N) and with vancomycin and 25 μM of DFP; 40x and 40x (O and P). Live/dead staining using green-fluorescent nucleic acid stain and propidium iodide solution; green staining = living cells; yellow staining = damaged cells; red staining = dead cells. One out of three independent experiments performed in technical triplicate is shown. To further verify the effects of single and combined therapies observed upon CFU quantification and fluorescence microscopy, we next performed SEM of bacterial biofilms placed on titanium cover slips. The control groups showed biofilm growth for both strains. Massive biofilms showing channels and slime formation were observed for both strains. Staphylococcus epidermidis ATCC 12228 showed larger and more structured biofilms (Fig. 4A) in comparison to the patient isolate that presented with small slime-rich islands containing the biofilms (Fig. 4B). The treatment with DFP did not allow the growth of massive biofilms. In this case only small biofilms were observed in S. epidermidis ATCC 12228 cultures (Fig. 4C) and the CNS patient isolate (Fig. 4D). In contrast to the samples treated with DFP alone, the samples treated with iron or iron plus DFP showed more biofilm formation. In these cases cluster of cells was observed distributed on the surface of the discs (Fig. 4E and H). Little biofilm formation was observed on the strains treated with gentamicin (Fig. 5A and B) in comparison to the control groups. In the groups treated with clindamycin alone, small biofilms could be observed for both strains (Fig. 5C and D), whereas only minimum biofilm formation could be observed in the groups treated with vancomycin. In this latter case, still some small biofilms or free cells could be observed on the surface of the discs (Fig. 5E and F). In comparison to the strains treated with gentamicin alone, the strains treated with the combination of gentamicin and DFP hardly any biofilm formation was evident (Fig. 6A and B). Complete killing of bacteria and biofilm disruption was achieved by combined treatment of DFP with either clindamycin (Fig. 6C and D) or vancomycin (Fig. 6E and F). These results, as well as the results of fluorescence staining (above) on biofilm integrity, strongly suggest that iron chelator treatment enhances the susceptibly of biofilm forming staphylococci to conventional antibiotics that can be largely explained by disruption of biofilm formation by the iron chelator which then enables a better penetration of antibiotics into bacteria with subsequent promotion of bacterial killing. In addition, a direct, albeit smaller antibacterial effect of iron chelators due to limitation of bacterial iron availability may also play a role in this setting and may further contribute to the synergistic action of DFP and antibiotics (Cassat and Skaar 2013; Weiss and Carver 2018). Figure 4. View largeDownload slide SEM of S. epidermidis ATCC 12228 (A) and patient isolated CNS (B) biofilms. SEM of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with 25 μM of DFP (C and D—magnification X 1.600), with 25 μM of ferric chloride (E and F); and with 25 μM of ferric chlodire and 25 μM of DFP (G and H). Magnification: (A) ×3.000; (B) ×5.500; (C) ×4.000; (D) ×1.600; (E) ×8.000; (F) ×4.500; (G) ×8.500; (H) ×3.500. Specimens were analyzed by SEM (JSM-6010LV, JEOL GmbH, Freising, Germany). One out of three independent experiments perforemd in technical triplicates is shown. Figure 4. View largeDownload slide SEM of S. epidermidis ATCC 12228 (A) and patient isolated CNS (B) biofilms. SEM of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with 25 μM of DFP (C and D—magnification X 1.600), with 25 μM of ferric chloride (E and F); and with 25 μM of ferric chlodire and 25 μM of DFP (G and H). Magnification: (A) ×3.000; (B) ×5.500; (C) ×4.000; (D) ×1.600; (E) ×8.000; (F) ×4.500; (G) ×8.500; (H) ×3.500. Specimens were analyzed by SEM (JSM-6010LV, JEOL GmbH, Freising, Germany). One out of three independent experiments perforemd in technical triplicates is shown. Figure 5. View largeDownload slide SEM of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with gentamicin (A and B), with clindamycin (C and D) and with vancomycin (E and F). Magnification: (A) ×3.700; (B) ×3.500; (C) ×1.900; (D) ×6.000; (E) ×4.300; (F) ×2.500. Specimens were analyzed by SEM (JSM-6010LV, JEOL GmbH, Freising, Germany). The experiment was carried out in technical triplicate. Figure 5. View largeDownload slide SEM of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with gentamicin (A and B), with clindamycin (C and D) and with vancomycin (E and F). Magnification: (A) ×3.700; (B) ×3.500; (C) ×1.900; (D) ×6.000; (E) ×4.300; (F) ×2.500. Specimens were analyzed by SEM (JSM-6010LV, JEOL GmbH, Freising, Germany). The experiment was carried out in technical triplicate. Figure 6. View largeDownload slide SEM of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with the combination of gentamicin and 25 μM of DFP (A and B), with clindamycin and 25 μM of DFP (C and D) and with vancomycin and 25 μM of DFP (E and F). Magnification: (A) ×2.700; (B) ×3.000; (C) ×2.300; (D) ×3.500; (E) ×800; and (F) ×2.200. Specimens were analyzed by SEM (JSM-6010LV, JEOL GmbH, Freising, Germany). The experiment was carried out in technical triplicate. Figure 6. View largeDownload slide SEM of S. epidermidis ATCC 12228 and patient isolated CNS biofilms treated with the combination of gentamicin and 25 μM of DFP (A and B), with clindamycin and 25 μM of DFP (C and D) and with vancomycin and 25 μM of DFP (E and F). Magnification: (A) ×2.700; (B) ×3.000; (C) ×2.300; (D) ×3.500; (E) ×800; and (F) ×2.200. Specimens were analyzed by SEM (JSM-6010LV, JEOL GmbH, Freising, Germany). The experiment was carried out in technical triplicate. Our study thus indicates that the combination of the iron chelator DFP with conventional antibiotics can potentiate their antimicrobial activity towards biofilm producing CNS placed on titanium cover slips. We found that this interaction leads to a disruption of biofilms that likewise increases the penetration of antibiotics into bacteria where they can fulfill their antimicrobial activity. From our data it is suggestive that such a combination may be of benefit to eliminate bacteria residing on implanted devices that would be of enormous clinical benefit because bacterial biofilm formation on such surfaces is often associated with clinical failure of antibiotic therapies resulting in the need for surgical revision (Sendi and Zimmerli 2012). Iron chelators have been used to treat iron overload disorders for many years, and their pharmacological and safety profiles are well known (Hershko et al.2005; Kovacevic et al.2010). As iron is essential for bacterial growth and also involved in biofilm formation, the combination of iron chelation with antibiotic therapy may be safe and enhance the antimicrobial efficacy of antibiotics to eliminate biofilm producing bacteria on implanted devices; a hypothesis that has to be confirmed in vivo by using animal models of device related infections. Acknowledgements We would like to thank Andrea Windisch for the great technical support. FUNDING This study was supported by the Unit for Experimental Orthopedics, Department for Orthopedic Surgery, Medical University Innsbruck and by the Austrian Science Fund (FWF) funded doctoral program HOROS (W-1253, to SD, GW). Conflict of interest. None declared. REFERENCES Becker K, Heilmann C, Peters G. Coagulase-negative staphylococci. Clin Microbiol Rev  2014; 27: 870– 926. 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Pathogens and DiseaseOxford University Press

Published: May 31, 2018

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