Nitroxoline: a broad-spectrum biofilm-eradicating agent against pathogenic bacteria

Nitroxoline: a broad-spectrum biofilm-eradicating agent against pathogenic bacteria

ARTICLE IN PRESS International Journal of Antimicrobial Agents ■■ (2016) ■■–■■ Contents lists available at ScienceDirect International Journal of An...

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ARTICLE IN PRESS International Journal of Antimicrobial Agents ■■ (2016) ■■–■■

Contents lists available at ScienceDirect

International Journal of Antimicrobial Agents j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j a n t i m i c a g

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Q2 Short Communication

Nitroxoline: a broad-spectrum biofilm-eradicating agent against pathogenic bacteria Q1 Yasmeen Abouelhassan a, Qingping Yang b, Hussain Yousaf a, Minh Thu Nguyen a,

Melanie Rolfe a, Gregory S. Schultz b, Robert W. Huigens III a,*



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Department of Medicinal Chemistry, College of Pharmacy, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610, USA Institute of Wound Research, Department of Obstetrics and Gynecology, College of Medicine, University of Florida, Gainesville, FL 32610, USA



Article history: Received 7 July 2016 Accepted 8 October 2016 Keywords: Nitroxoline Biofilm eradication Antibiotic tolerance Drug discovery Antibiofilm agents


Bacterial biofilms are surface-attached communities of slow-growing or non-replicating bacteria tolerant to conventional antibiotic therapies. Although biofilms are known to occur in ca. 80% of all bacterial infections, no therapeutic agent has been developed to eradicate bacteria housed within biofilms. We have discovered that nitroxoline, an antibacterial agent used to treat urinary tract infections, displays broad-spectrum biofilm-eradicating activities against major human pathogens, including drugresistant Staphylococcus aureus and Acinetobacter baumannii strains. In this study, the effectiveness of nitroxoline to eradicate biofilms was determined using an in vitro [minimum biofilm eradication concentration (MBEC) = 46.9 μM against A. baumannii] and ex vivo porcine skin model (2–3 log reduction in viable biofilm cells). Nitroxoline was also found to eradicate methicillin-resistant S. aureus (MRSA) persister cells in non-biofilm (stationary) cultures, whereas vancomycin and daptomycin were found to be inactive. These findings could lead to effective, nitroxoline-based therapies for biofilm-associated infections. © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

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1. Introduction

2. Materials and methods

Free-floating planktonic bacteria use a signalling process known as quorum sensing to co-ordinate virulent behaviours, including the formation of surface-attached biofilms [1]. Bacteria encased within a biofilm lead a slow- or non-growing (dormant) existence and have contrastingly different gene expression profiles compared with their planktonic counterparts [2–4]. As a result, biofilms are highly tolerant to conventional antibiotics and are the underlying cause of many recurring and chronic infections [5]. We have discovered halogenated quinolines and phenazines capable of eradicating biofilms of multiple Gram-positive pathogens [6–10], which led us to nitroxoline, a structurally related antibacterial agent used to treat urinary tract infections [11,12]. Nitroxoline operates through a metal chelation-dependent mechanism and has reported biofilm dispersal activity against Pseudomonas aeruginosa [13]. Here we report our findings of the broad-spectrum biofilm-eradicating activities of nitroxoline against multiple human pathogens. This phenotype is extremely rare as mitomycin C has been the only reported broad-spectrum biofilmeradicating agent that does not target bacterial membranes for destruction [14].

2.1. Bacterial strains Multidrug-resistant (MDR) Acinetobacter baumannii ATCC 1794, A. baumannii ATCC 19606, P. aeruginosa PAO1, methicillin-resistant Staphylococcus aureus (MRSA) ATCC BAA-1707, methicillin-resistant Staphylococcus epidermidis (MRSE) ATCC 35984 and vancomycinresistant Enterococcus faecium (VRE) ATCC 700221 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Methicillin-resistant S. aureus MRSA-1, MRSA-2 and MRSA-156 and methicillin-resistant S. epidermidis MRSE-1 were obtained as clinical isolates from patients treated at Shands Hospital (Gainesville, FL). UAEC-1 is an Escherichia coli isolate from UAMS Hospital (Fayetteville, AR). 2.2. Minimum inhibitory concentration (MIC) assays MIC assays were performed as previously described to determine antibacterial activities against pathogenic bacteria [6–10,15]. Results from MIC assays were obtained from three independent experiments (Table 1).

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2.3. Minimum biofilm eradication concentration (MBEC) assays


* Corresponding author. Department of Medicinal Chemistry, College of Pharmacy, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610, USA. E-mail address: [email protected] (R.W. Huigens III).

Biofilm eradication assays were performed in 96-well plates (polystyrene for MRSA-2 and MRSE-1; polyvinyl chloride for A. 0924-8579/© 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Please cite this article in press as: Yasmeen Abouelhassan, et al., Nitroxoline: a broad-spectrum biofilm-eradicating agent against pathogenic bacteria, International Journal of Antimicrobial Agents (2016), doi: 10.1016/j.ijantimicag.2016.10.017

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Table 1 Summary of antibacterial and biofilm eradication assays performed in 96-well plates and in Calgary Biofilm Devices (CBDs). All values are reported in μM. Strain/isolate

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Acinetobacter baumannii 19606b A. baumannii 1794b UAEC-1 PAO1 MRSA-1 MRSA-2f MRSA-156 MRSA-BAA-1707 MRSE-1f MRSE 35984 VRE 700221




96-well plates (MIC/MBEC)


96-well plates (MIC/MBEC)


96-well plates (MIC/MBEC)


6.25/46.9c 4.69c/62.5 12.5/— 250/1500c 12.5/— 25/188c 12.5/— 9.38c/— 18.8c/125 18.8c/— 125/—

—d 125/46.9c 93.8c/62.5 — 250/188c 250/62.5 750c/125c 250/93.8c — 500/250 125/62.5

0.39/1000 0.39/— 0.39/— 0.78/1500c — — — — — — —

— 375c/375c 11.7c/46.9c — — — — — — — —

— — — — 0.39 0.78/>2000 0.78 0.39 0.59c/>2000 — >100/—

— — — — 15.6e/750c 11.7c/62.5 31.3/750c 23.5c/1500c — — >100/150c

MIC, minimum inhibitory concentration; MBEC, minimum biofilm eradication concentration; MBC, minimum bactericidal concentration. All data were obtained from three independent experiments. a Colistin served as the anti-Gram-negative comparator antibiotic and vancomycin served as the anti-Gram-positive comparator antibiotic in these experiments. b A. baumannii biofilms were grown on polyvinyl chloride plates. c Corresponds to the mid-point of a two-fold range in MIC, MBC or MBEC values. d Indicates not tested. e Corresponds to the mid-point value of a four-fold range in MBC experiments. f MRSA-2 and MRSE-1 biofilms were grown on polystyrene plates.

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baumannii and PAO1) [6] or in Calgary Biofilm Devices (CBDs) [7–10,16]. Biofilm eradication assays involve three phases separated by wash steps, including (i) initial biofilm establishment on well/peg surfaces without test compound; (ii) biofilm treatment with test compound; and (iii) recovery of viable biofilms in fresh medium alone. Both assays were used to demonstrate the biofilm eradication activities of nitroxoline. For polystyrene and polyvinyl chloride 96-well plate biofilm eradication assays, microtitre wells were inoculated with 100 μL of a 1:1000-fold exponential-phase culture [optical density at 600 nm (OD600) of ca. 1.0] and were incubated for 24 h at 37 °C to allow biofilms to establish on the surface of microtitre wells (phase 1). Following biofilm establishment, medium and planktonic cells were removed from microtitre plates and the plates were rinsed with water. Then, 100 μL of two-fold serial dilutions of test compound was added to the microtitre wells in fresh medium and was incubated for 24 h at 37 °C (phase 2). After this time, the contents of the microtitre wells were removed and 100 μL of fresh medium only was added to allow viable biofilms to recover and to disperse planktonic bacteria into the medium resulting in a turbid microtitre well (24-h incubation at 37 °C; phase 3). After this final phase, microtitre plates were examined for visible bacterial growth (turbidity) and the MBEC was recorded as the lowest concentration at which no turbidity could be observed (due to eradicated biofilms). For MRSA-2 and MRSE-1 biofilm eradication assays, 100 μL of a 1% gelatin (aqueous) solution was used to pre-treat polystyrene plates for a minimum of 1 h to enhance S. aureus and S. epidermidis biofilm formation, as previously reported [17,18]. Biofilm eradication assays involving CBDs (Innovotech Inc., Edmonton, AB, Canada) (Supplementary Fig. S1) were performed in an analogous manner to the previous microtitre plate assay; however, CBDs allow biofilms to be established and treated on pegs suspended from 96-well plate lids. We have previously reported these assay conditions [7–10,16]. CBD experiments with A. baumannii 1794, UAEC-1, all MRSA strains and MRSE 35984 were carried out on hydroxyapatite-coated P&G devices, whilst experiments with VRE 700221 were performed on non-coated P&G CBDs. 2.4. Ex vivo porcine skin biofilm model Biofilms were formed on porcine skin explants as previously described [17,18] (Supplementary Fig. S2) with slight modifications.

Briefly, porcine skin explants were inoculated with 20 μL of bacterial culture (OD600 = 0.2–0.4) and were incubated on agar for 48 h at 37 °C to establish biofilms on porcine skin explants. Following biofilm establishment, porcine skin explants were treated with test compound in phosphate-buffered saline (PBS) at 400 μM for 72 h (at 37 °C). After this time, porcine skin explants were rinsed with PBS to remove planktonic cells and the remaining biofilm cells were removed via sonication from the porcine explants. Subsequent colony counts were performed to determine relative biofilm viabilities between treated and untreated (vehicle-only) samples.

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2.5. MRSA-2 stationary cell kill kinetics MRSA-2 stationary killing was performed as previously described [8]. Briefly, an MRSA-2 overnight culture was diluted in fresh medium and was allowed to grow for 4 h to reach stationary phase. Test compound was then added to stationary cultures and aliquots were removed to perform colony counts to determine CFU/mL (viable bacteria) at pre-determined time points.

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2.6. Minimum biofilm inhibitory concentration (MBIC) determination In PVC 96-well plates, two-fold serial dilutions of test compounds were added in LB medium. Then, 1:1000-fold exponentialphase culture (OD600 of ca. 0.8) in LB was added to each well and was allowed to incubate at 37 °C for 24 h. After this time, the contents from the 96-well plates were removed and the wells were rinsed with water, followed by the addition of 120 μL of crystal violet to stain the biofilms (0.1% for A. baumannii and 1% for PAO1; 10min incubation at room temperature). The plates were then rinsed and 120 μL of ethanol was added to dissolve the crystal violetstained biofilms. Minimum concentrations required to inhibit 80% of biofilm formation (MBIC80) were determined (OD540) by comparing compound-treated versus untreated wells and the resulting data were used to generate dose–response curves using GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA). Note: these experiments were performed to determine whether the antibiofilm activities of nitroxoline were dependent on or independent of their antibacterial activities (Supplementary Table S1; Supplementary Fig. S3).

Please cite this article in press as: Yasmeen Abouelhassan, et al., Nitroxoline: a broad-spectrum biofilm-eradicating agent against pathogenic bacteria, International Journal of Antimicrobial Agents (2016), doi: 10.1016/j.ijantimicag.2016.10.017

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Fig. 1. (A) Acinetobacter baumannii 19606 biofilm eradication using 96-well plates and (B) multidrug-resistant (MDR) A. baumannii 1794 biofilm eradication using the Calgary Biofilm Device (CBD). Concentration ranges from 2 to 2000 μM for nitroxoline and colistin. MBEC, minimum biofilm eradication concentration; DMSO, dimethyl sulphoxide.

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3. Results Nitroxoline demonstrated broad-spectrum antibacterial activities (Table 1) against A. baumannii (MIC = 4.69–6.25 μM; 0.9–1.2 mg/L), E. coli UAEC-1 (MIC = 12.5 μM; 2.4 mg/L), MRSA strains (MIC = 9.38–25 μM; 1.8–4.8 mg/L) and MRSE-1 (MIC = 18.8 μM; 3.6 mg/L); however, nitroxoline displayed weak antibacterial activities against VRE 700221 (MIC = 125 μM; 24 mg/L) and PAO1 (MIC = 250 μM; 48 mg/L). Encouraged by the broad-spectrum antibacterial activities of nitroxoline against planktonic bacteria, we then moved to bacterial biofilms to determine eradication activities. The biofilm eradication activities of nitroxoline were initially investigated in vitro by treating bacterial biofilms established on the bottom of 96well plates and with the CBD. The CBD apparatus allows for the simultaneous determination of planktonic and biofilm cell eradication in a single assay, resulting in a more accurate comparison between the planktonic [i.e. minimum bactericidal concentration (MBC)] and biofilm (i.e. MBEC) activities. Nitroxoline was found to have broad-spectrum biofilm eradication activities in both in vitro assays (Table 1). Interestingly, A. baumannii biofilms proved to be most sensitive to the eradication activities of nitroxoline (MBEC = 46.9–62.5 μM; 8.9–11.9 mg/L) (Table 1; Fig. 1). In 96-well plates and CBDs, nitroxoline was found to be >20-fold more potent than colistin against A. baumannii biofilms. In addition, nitroxoline demonstrated biofilm eradication activities in 96-well plate and CBD assays against MRSA strains (MBEC = 62.5–188 μM) (Table 1), MRSE-1 and MRSE 35984 with MBECs of 125 μM and 250 μM, respectively (Table 1). Nitroxoline outperformed vancomycin in staphylococcal biofilm eradication whilst demonstrating similar planktonic and biofilm cell eradication activities (MBC/MBEC values; Table 1). In these assays, vancomycin was more selective in killing planktonic cells compared with biofilm cells, demonstrating much more potent MBC values than MBEC values, typical of conventional antibiotics (growth inhibitors). Nitroxoline also proved to effectively eradicate E. coli UAEC-1 (MBEC = 62.5 μM) and VRE 700221 (MBEC = 62.5 μM) biofilms in CBD assays whilst demonstrating significantly less potent biofilm eradication activities against PAO1 (Table 1). After investigating the biofilm eradication activities of nitroxoline in vitro, we moved to a more sophisticated ex vivo (wound) biofilm infection model using porcine skin explants. Since biofilms formed in vivo are different from those formed on plastic in vitro surfaces, we chose the ex vivo porcine biofilm model as a better representative for biofilms grown on tissues. For these experiments, we modified published protocols using our ex vivo model [19,20].

Porcine skin explants were inoculated and were allowed to incubate on agar for 48 h, producing a relatively immature but established bacterial biofilm (ca. 107 CFU/mL biofilm cells) on the porcine skin explants. The infected porcine skin explants were then washed to remove planktonic bacteria and were treated with nitroxoline at 400 μM for 72 h. Similar to in vitro assays, nitroxoline effectively reduced the viability of established A. baumannii, E. coli UAEC-1, MRSA-2 and MRSE 35984 biofilms in the order of 2–3 log reduction (99–99.9% killing) of viable biofilm cells using this ex vivo model (Fig. 2A). In addition, nitroxoline was more effective at killing biofilm cells than colistin against A. baumannii and E. coli biofilms and than vancomycin against MRSA-2 and MRSE 35984 biofilms in these porcine skin experiments. Interestingly, in MIC assays, these bacterial strains proved to be highly sensitive (MIC < 0.78 μM) to these conventional antibiotics (Table 1). Following biofilm eradication studies, kill kinetic experiments were performed against MRSA-2 stationary-phase cultures rich in persister cells with nitroxoline (200 μM; 8× MIC), vancomycin (200 μM; 256 × MIC) and daptomycin (200 μM; 43× MIC). In addition, the membrane-lysing antimicrobial peptide mimic QAC-10 (quaternary ammonium cation-10 [21–24]) was used as a positive control owing to its ability to rapidly eradicate MRSA-2 persister cells in stationary-phase cultures [8]. Nitroxoline demonstrated a slow yet effective ability to eradicate stationary (persister) cells by causing a >4-log reduction (>99.99%) of viable MRSA-2 stationary phase cells after 24 h (Fig. 2B). Front-running MRSA therapies, i.e. daptomycin and vancomycin, were found to be ineffective at eradicating MRSA stationary cells at 200 μM. Despite these high concentrations of daptomycin and vancomycin (i.e. 43× and 256× their corresponding MICs), these antibiotics were unable to eradicate MRSA persisters, illustrating the innate tolerance that persister cells display towards antibiotics. QAC-10 rapidly eradicated MRSA stationary cultures, typical of membrane-lysing agents [21–24].

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4. Discussion Bacterial biofilms are the underlying cause of persistent and recurring infections [2–4]. Unfortunately, our antibiotic arsenal is not poised to effectively treat biofilm-associated infections as these agents were initially discovered as growth inhibitors against rapidly-dividing bacteria [5,8–10]. In addition, pharmaceutical companies have either reduced or eliminated antibacterial discovery programmes [25] despite the need for biofilm-eradicating therapies.

Please cite this article in press as: Yasmeen Abouelhassan, et al., Nitroxoline: a broad-spectrum biofilm-eradicating agent against pathogenic bacteria, International Journal of Antimicrobial Agents (2016), doi: 10.1016/j.ijantimicag.2016.10.017

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Fig. 2. (A) Biofilm eradication with nitroxoline (Nitrox), colistin and vancomycin (Vanc) using an ex vivo porcine skin model against Acinetobacter baumannii 1794, methicillinresistant Staphylococcus aureus MRSA-2, methicillin-resistant Staphylococcus epidermidis (MRSE) 35984 and Escherichia coli UAEC-1. All compounds were tested at 400 μM. (B) Kinetics of MRSA-2 stationary cell eradication using nitroxoline, vancomycin, daptomycin and QAC-10. *Corresponds to the lowest detection level of viable stationary cells tested in this experiment. DMSO, dimethyl sulphoxide.

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Nitroxoline was previously reported to demonstrate biofilm dispersion activities against P. aeruginosa [13]; however, our MBEC, MRSA stationary killing, ex vivo biofilm killing and MBIC80:MIC ratios (Supplementary Table S1) against multiple pathogenic bacteria suggest that any antibiofilm activities demonstrated by nitroxoline are likely due to biofilm eradication and not a mechanism independent of its antibacterial activities, such as biofilm attachment and dispersion. These findings are timely as mitomycin C is the only non-membrane-active broad-spectrum biofilm-eradicating agent reported in the literature [14]. During these investigations, nitroxoline outperformed conventional antibiotics (i.e. colistin for A. baumannii and vancomycin for MRSA/MRSE) in biofilm/MRSA stationary cell eradication experiments and in an ex vivo porcine skin infection model, suggesting that nitroxoline may be an effective topical treatment for biofilm-associated skin wounds. The biofilm-related therapeutic applications for nitroxoline-based therapies are numerous (i.e. device-related biofilm infections, wound infections) and could be streamlined as this agent is an approved drug. Importantly, nitroxoline is a structurally simple molecule and could be used as a synthetic template for extensive drug development efforts.

5. Conclusion We have demonstrated that nitroxoline possesses broad-spectrum biofilm eradication activities against several human pathogens, including MDR A. baumannii, E. coli, MRSA, MRSE and VRE. As persister cells lead to high levels of antibiotic tolerance in biofilm infections, finding new agents that operate through novel modes of action that lead to the eradication of persister cells in biofilms is critical, thus our findings are timely. Nitroxoline-based therapies could lead to significant advances in the treatment of persistent biofilmassociated bacterial infections. Funding: This work was funded by the College of Pharmacy and Q4 the Division of Sponsored Research at the University of Florida Q5 (Gainesville, FL) [start-up funds]. Competing interests: None declared. Ethical approval: Not required. Appendix. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ijantimicag.2016.10.017.

Please cite this article in press as: Yasmeen Abouelhassan, et al., Nitroxoline: a broad-spectrum biofilm-eradicating agent against pathogenic bacteria, International Journal of Antimicrobial Agents (2016), doi: 10.1016/j.ijantimicag.2016.10.017

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