- Research article
- Open Access
Staphylococcus epidermidis recovered from indwelling catheters exhibit enhanced biofilm dispersal and “self-renewal” through downregulation of agr
© Dai et al.; licensee BioMed Central Ltd. 2012
- Received: 10 January 2012
- Accepted: 23 May 2012
- Published: 8 June 2012
In recent years, Staphylococcus epidermidis ( Se) has become a major nosocomial pathogen and the most common cause of infections of implanted prostheses and other indwelling devices. This is due in part to avid biofilm formation by Se on device surfaces. However, it still remains unknown that how the process of Se biofilm development is associated with relapsed infection in such patients.
We have identified clinical Se isolates displaying enhanced biofilm dispersal and self-renewal relative to reference strain. These isolates also exhibit enhanced initial cell attachment, extracellular DNA release, cell autolysis and thicker microcolonies during biofilm development relative to reference strain. Our genetic analyses suggest that these clinical isolates exhibit significant downregulation of RNAIII, the effector molecule of the agr quorum sensing system, and upregulation of the autolysin gene atlE. Isogenic deletion of the agr system in Se 1457 confirmed that agr negatively regulating atlE resulted in enhanced initial cell attachment, extracellular DNA release, cell autolysis and biofilm formation abilities. In contrast, double deletion of agr and atlE significantly abolished these features.
Collectively, these data reveal the role of agr system in long-term biofilm development and pathogenesis during Se caused indwelling devices-related relapsed infection.
- Staphylococcus epidermidis
- Extracellular DNA
In recent years, coagulase-negative Staphylococcus epidermidis ( Se) has become the leading cause of infections related to indwelling medical devices such as vascular catheters, prosthetic joints and artificial heart valves [1, 2]. Pathogenicity of Se is attributed to its formation of biofilm on the surface of medical devices, thereby enhancing Se resistance to antibiotics and host defenses in this setting [3, 4]. In general, Se biofilm formation is a two-step process, in which bacteria first adhere to the surface (initial attachment phase) and subsequently form cell–cell aggregates and a multilayered architecture (accumulative phase) [5, 6]. One autolysin protein, AtlE, facilitates bacterial attachment to the surface of medical devices and dictates pathogenesis for Se biofilm-associated infections in vivo [7, 8]. In the accumulative phase, the polysaccharide intercellular adhesin (PIA), a linear poly-Nacetyl-1,6-β-glucosamine (PNAG) encoded by the icaADBC locus, is the major pathogenic determinant for intercellular adhesion [9, 10]. In addition, we have previously demonstrated that extracellular DNA is generated during Se growth through AtlE-mediated lysis of a subpopulation of the bacteria; moreover, this process is required for initial bacterial attachment to surfaces and biofilm development . An important negative regulator of biofilm formation by Se and Staphylococcus aureus is the accessory gene regulator ( agr) quorum sensing system, and agr mutation promotes biofilm formation by increasing the capacity of Se for initial cell attachment [12–14]. The agr system of Se and S. aureus consists of 4 genes ( agrA agrC agrD, and agrB) that are cotranscribed (RNAII) and the gene for the effector molecule of the agr system, RNAIII, which also encodes the gene for δ-toxin ( hld) [12, 15].
Medical device-associated biofilms facilitate recalcitrant or recurrent infections despite use of appropriate antibiotics. However, there are only limited data about the long-term Se biofilm development, especially clinical isolates recovered from indwelling medical devices infection. It still remains unknown that how the process of Se biofilm development is associated with relapsed infection in such patients. Moreover, the molecular mechanisms causing such repeated infection also needs to be investigated. In the current study, we compared the long-term (~7 days) biofilm development and dispersal between Se clinical isolates causing indwelling medical devices infection and reference strain in the flow-chamber systems. We also compared the biofilm-related events (initial attachment, PIA synthesis, extracellular DNA release etc.) and biofilm-associated gene profiles in these clinical isolates and reference strain.
Bacterial strains, growth media and reagents
4 Se clinical isolates, referred to as Se-1, Se-2, Se-3 and Se-4, were recovered from 4 different patients at the Zhongshan Hospital (Shanghai, China) with indwelling catheter-associated infections as defined by the presence of fever, bacterial growth from peripheral blood samples collected from catheter sites. Se biofilm-positive strain 1457 wild type and agr mutants were kindly provided by Dr. Min Li (Huashan Hospital, Shanghai, China), as described previously . The agr/ atlE double mutant was constructed as described previously . The mutation was confirmed by Southern blotting and direct sequencing (data not shown), and we also independently confirmed that the 1457 agr mutant or agr/ atlE double mutant does not affect bacterial growth (see Additional file 1: Figure S1). Se biofilm-positive ATCC 35984 (also referred as RP62A) and biofilm-negative ATCC 12228 reference strains were purchased from American Type Culture Collection (ATCC). Tryptic soy broth (TSB; Oxoid) medium containing 0.25% glucose was used to support biofilm formation in the microtitre plates. AB medium  supplemented with 0.3 mM glucose and 3% TSB was used for biofilm cultivation in the flow-chamber system. SYTO 9 and propidium iodide (PI) (Live_Dead reagents, Molecular Probes) were used at a concentration of 1 μM for staining live or dead bacteria in biofilms, respectively. DDAO [7-hydroxy-9 H-(1,3-dichloro-9,9-dimethylacridin-2-one)] (Molecular Probes) was used at a concentration of 1 μM to stain extracellular DNA in biofilms . TRITC (tetramethyl rhodamine isothiocyanate)-labeled wheat germ agglutinin (Molecular Probes, Eugene, OR) was used at a concentration of 0.1 mg/mL to stain the PIA in biofilms . Hemoglobin was purchased from Sigma and used as indicated concentrations. The Ethics Committee of the Zhongshan Hospital of Fudan University and the East Hospital of Tongji University both exempted this study from review because the current study only focused on bacteria.
Cultivation of bacterial biofilms
Biofilm cultivation in polystyrene microtitre plates was carried out as described previously . Briefly, overnight cultures of Se strains grown in TSB (0.25% glucose) medium were diluted 1:200. The diluted cultures were transferred to wells of polystyrene microtitre plates (200 μL per well) and incubated at 37 °C for 24 h. After washing, the wells were stained with 2% crystal violet for 5 min. Then, the plate was rinsed, air-dried, redissolved in ethanol and the absorbance was determined at 590 nm. For cultivation of Se biofilms in the flow-chamber system, the flow-chamber system was first assembled and prepared as described previously . Briefly, the flow chambers were inoculated by injecting 350 μL overnight culture diluted to OD600 = 0.001 into each flow channel with a small syringe. After inoculation, flow channels were left without flow for 1 h, after which medium flow (0.2 mm/s) was started using a Watson-Marlow 205 S peristaltic pump.
All microscopic observations and image acquisition were performed using a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss, Jena) equipped with detectors and filter sets for monitoring SYTO 9, PI, DDAO and TRITC fluorescence. Images were obtained using an x63/1.4i objective or an x40/1.3i objective. Simulated 3D images and sections were generated using the IMARIS software package (Bitplane).
Bacterial attachment assays
Initial cell attachment was tested as described previously . Briefly, cell suspensions from the mid-exponential phase of bacterial growth were diluted to OD600 = 0.1 in PBS, and then incubated in wells (1 mL per well) of cover-glass cell culture chambers (Nunc) for 30 min at 37°C, after which attached cells were calculated by microscopy.
Quantification of extracellular DNA
Extracellular DNA was quantified as described previously . Overnight cultures were diluted to OD600 = 0.001 in AB medium supplemented with 0.5% glucose, 0.05 mM PI and 10% TSB. The diluted cultures were transferred to wells of polystyrene microtitre plates (150 μL per well) and incubated for 24 h at 37°C, upon which PI absorbance was measured at 480 nm and cell density was measured by OD600 using a Wallac microtitre plate reader. Relative amounts of extracellular DNA per OD600 unit were calculated.
After cultured for 1 d or 6 d, biofilm cells were resuspended in 500 mL buffer containing 25% sucrose, 10 mM Tris/HCl (pH 7.5) and 0.2 mg lysostaphin (Sigma-Aldrich). After incubation at 37°C for 10 min, total RNA was isolated using the RNeasy Mini kit according to the manufacturer’s instructions (QIAGEN). cDNA was synthesized from equivalent concentrations of total RNA using the SuperScript III First-Strand Synthesis SuperMix Kit (Invitrogen) according to the manufacturer’s instructions. Coding sequences for bacterial genes (and gyrB for internal controls) were amplified using iQ SYBR Green Supermix (Bio-rad). Custom primer sequences used for amplification experiments are included in Additional file 2: Table S1. Amplification was carried out using an iCycler IQ Real-Time PCR Detection System, and cycle threshold (Ct) values determined in duplicate for target gene transcripts and gyrB for each experiment. “No template” (water) and “no-RT” controls were used to ensure minimal background DNA contamination. Fold changes for experimental groups relative to assigned controls were calculated using automated iQ5 2.0 software (Bio-rad).
PCR and sequencing
Genomic DNA was extracted by using Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer’s instructions. The primers included in Additional file 2: Table S1 were designed from conserved sequences of agr, which are common to agr groups I, II and III, to amplify a 1022 bp fragment . The PCR production was purified by using QIAquick PCR Purification Kit (Qiagen) then sequenced (Operon), and alignment analysis was performed by using Vector NTI Advance 9 software (Invitrogen).
Cell autolysis assays
Autolysis assays for Se strains were performed as described previously . Briefly, cell samples (50 mL) were collected from exponential-phase cultures growing in TSB medium (OD600 = 0.6 ~ 0.8) containing 1 M NaCl, and cells were pelleted by centrifugation. The cells were washed twice with 50 mL ice-cold water and resuspended in 50 mL 0.05 M Tris/HCl (pH 7.2) containing 0.05% (v/v) Triton X-100. The cells were then incubated at 30°C with shaking, and OD600 was measured at 30 min intervals. The lysis rate induced by Triton X-100 was calculated as: OD0-ODt/OD0.
Se isolates associated with catheter infection exhibit more avid self-renewal in long-term cultured biofilm assays
Se isolates associated with catheter infection exhibit greater extracellular DNA content and capacity for cell attachment
The agr system regulates initial cell attachment and cell autolysis during biofilm formation for se
Agr mutant increases initial cell attachment and cell death during biofilm formation through upregulation of atlE
Agr regulates se release of extracellular DNA and autolysis through suppression of atlE
Chemical inhibition of agr increases biofilm formation, initial attachment and cell autolysis through upregulation of atlE
Se biofilm formation on implanted medical devices may result in recurrent or refractory infection unless the devices are removed, and removal and replacement of these devices incurs significant cost and risk for the patient. Flow-chamber systems simulate blood or other body-fluid flow in the vasculature of patients . Using this and other complimentary approaches, we found that clinical Se isolates from patients with implanted catheter infections display greater microcolony densities, spontaneous cell death, and self-renewal capacity during biofilm development relative to reference strains. Bacteria in biofilms are 100 ~ 1000 times more resistant to antibiotics than planktonic cells [21–23], although our study does not directly address antibiotic sensitivity for our clinical isolates. Staphylococcal biofilm dispersal is associated with severe infection, including endocarditis, pneumonia and sepsis [24–26]. In addition, dispersal cells help bacteria establish new biofilms in more suitable niches, resulting in infection within multiple tissues . Of interest, we collected the detached and “flow-out” cells in the flow-chamber systems for our clinical isolates and found living cells capable of forming new biofilms as quickly as their parent cells (Qin et al., unpublished data).
Interestingly, expression of RNAIII, a gene for the effector molecule of the agr system, was significantly reduced in all 4 Se clinical isolates, suggesting that the functions of agr quorum-sensing system were impaired in these isolates. Besides its regulatory function, RNAIII also encodes a δ-toxin, which effectively reduces cell attachment and subsequent biofilm formation of a Se agr mutant . Our work does not address how RNAIII transcripts might be downregulated in our clinical isolates. Ongoing studies have found some potential spontaneous mutations present in the agr conserved region from Se-1, Se-2, Se-3 when compared with ATCC35984 strain (see Additional file 5: Figure S4), while Se-4 displayed some larger fragment variations for unknown reasons (Qin et al., unpublished data). However, it still requires further investigations to identify these potential spontaneous mutations responsible for RNAIII transcripts downregulation in these clinical isolates. Interestingly, about ~25% of S. aureus and ~17% of Se clinical isolates are naturally occurring agr mutants [19, 28]. One recent study indicated that Se agr mutant showed increased biofilm development and colonization in a rabbit model . In addition, nonfunctional agr occurred more frequently among strains isolated from infections of joint prostheses, which includes some mutations caused by insertion of an IS256 element . Moreover, polymorphisms within the agr locus for staphylococci are associated with its pathogenicity [19, 29, 30]. We have also observed that agr-positive (with normal RNAIII transcription) Se clinical isolates retain capacity for self-renewal in long-term culture (Qin et al., unpublished data), suggesting that other mechanisms are responsible for self-renewal for these isolates. Another recent study reported that addition of a cyclic autoinducing peptide (AIP) to activate agr in S. aureus agr–positive strains mediated dramatic detachment of S. aureus biofilms through an increase in expression of Aur metalloprotease and the SplABCDEF serine proteases . However, it is unclear whether these proteases may have similar functions in biofilms formed by agr–positive Se strains.
Expression of the gene encoding autolysin, atlE, was significantly increased in all 4 our clinical isolates. Previous data indicate that atlE expression is essential for initial cell attachment and biofilm formation by Se[7, 11, 13]. We previously reported that isogenic deletion of atlE in Se 1457 significantly reduced cell attachment, extracellular DNA release, cell autolysis and final biofilm formation . We and others found that atlE transcripts were significantly increased in Se 1457 agr mutants, which exhibited enhanced cell attachment, extracellular DNA release, cell death ( atlE-mediated autolysis) and subsequent biofilm formation . In contrast, we found that Se 1457 agr/atlE double mutant seriously impaired these features mentioned above in the current study. In fact, we think that increased densities of microcolonies in Se mutant mature biofilms will cause more cell death and detachment due to nutrition deficiency, oxygen stress or other reasons required further investigation. In addition, other mechanisms have also been recently reported to be related with staphylococcal extracellular DNA release and biofilm dissemination, including the cidA murein hydrolase regulator  and the β subclass of phenol-soluble modulins (PSMs) .
Taken together, our studies illuminate a novel mechanism for enhancing Se biofilm self-renewal: repression of agr and induction of atlE expression, which results in increased cell autolysis/death, biofilm dispersal and final promoting new biofilm formation. Better understanding the process and mechanisms of Se biofilm self-renewal in patients will help us develop more effective strategies against Se biofilm-related infection.
This work was supported by grants from the National Natural Science Foundation for Young Scientist of China (81101791 to Z.Q.). Z.Q. was also supported by the DANIDA fellowship during his visit at DTU. L.Y. was supported by a grant from the Danish Research Council for Independent Research (09-073917).
- Raad II, Bodey GP: Infectious complications of indwelling vascular catheters. Clin Infect Dis. 1992, 15 (2): 197-208. 10.1093/clinids/15.2.197.PubMedView ArticleGoogle Scholar
- Rupp ME, Archer GL: Coagulase-negative staphylococci: pathogens associated with medical progress. Clin Infect Dis. 1994, 19 (2): 231-243. 10.1093/clinids/19.2.231. quiz 244-235PubMedView ArticleGoogle Scholar
- von Eiff C, Peters G, Heilmann C: Pathogenesis of infections due to coagulase-negative staphylococci. Lancet Infect Dis. 2002, 2 (11): 677-685. 10.1016/S1473-3099(02)00438-3.PubMedView ArticleGoogle Scholar
- Vadyvaloo V, Otto M: Molecular genetics of Staphylococcus epidermidis biofilms on indwelling medical devices. Int J Artif Organs. 2005, 28 (11): 1069-1078.PubMedGoogle Scholar
- Gotz F: Staphylococcus and biofilms. Mol Microbiol. 2002, 43 (6): 1367-1378. 10.1046/j.1365-2958.2002.02827.x.PubMedView ArticleGoogle Scholar
- Mack D, Becker P, Chatterjee I, Dobinsky S, Knobloch JK, Peters G, Rohde H, Herrmann M: Mechanisms of biofilm formation in Staphylococcus epidermidis and Staphylococcus aureus: functional molecules, regulatory circuits, and adaptive responses. Int J Med Microbiol. 2004, 294 (2–3): 203-212.PubMedView ArticleGoogle Scholar
- Heilmann C, Hussain M, Peters G, Gotz F: Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface. Mol Microbiol. 1997, 24 (5): 1013-1024. 10.1046/j.1365-2958.1997.4101774.x.PubMedView ArticleGoogle Scholar
- Rupp ME, Fey PD, Heilmann C, Gotz F: Characterization of the importance of Staphylococcus epidermidis autolysin and polysaccharide intercellular adhesin in the pathogenesis of intravascular catheter-associated infection in a rat model. J Infect Dis. 2001, 183 (7): 1038-1042. 10.1086/319279.PubMedView ArticleGoogle Scholar
- Mack D, Fischer W, Krokotsch A, Leopold K, Hartmann R, Egge H, Laufs R: The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear beta-1,6-linked glucosaminoglycan: purification and structural analysis. J Bacteriol. 1996, 178 (1): 175-183.PubMedPubMed CentralGoogle Scholar
- Mack D, Riedewald J, Rohde H, Magnus T, Feucht HH, Elsner HA, Laufs R, Rupp ME: Essential functional role of the polysaccharide intercellular adhesin of Staphylococcus epidermidis in hemagglutination. Infect Immun. 1999, 67 (2): 1004-1008.PubMedPubMed CentralGoogle Scholar
- Qin Z, Ou Y, Yang L, Zhu Y, Tolker-Nielsen T, Molin S, Qu D: Role of autolysin-mediated DNA release in biofilm formation of Staphylococcus epidermidis. Microbiology. 2007, 153 (Pt 7): 2083-2092.PubMedView ArticleGoogle Scholar
- Vuong C, Saenz HL, Gotz F, Otto M: Impact of the agr quorum-sensing system on adherence to polystyrene in Staphylococcus aureus. J Infect Dis. 2000, 182 (6): 1688-1693. 10.1086/317606.PubMedView ArticleGoogle Scholar
- Vuong C, Gerke C, Somerville GA, Fischer ER, Otto M: Quorum-sensing control of biofilm factors in Staphylococcus epidermidis. J Infect Dis. 2003, 188 (5): 706-718. 10.1086/377239.PubMedView ArticleGoogle Scholar
- Yarwood JM, Bartels DJ, Volper EM, Greenberg EP: Quorum sensing in Staphylococcus aureus biofilms. J Bacteriol. 2004, 186 (6): 1838-1850. 10.1128/JB.186.6.1838-1850.2004.PubMedPubMed CentralView ArticleGoogle Scholar
- Peng HL, Novick RP, Kreiswirth B, Kornblum J, Schlievert P: Cloning, characterization, and sequencing of an accessory gene regulator (agr) in Staphylococcus aureus. J Bacteriol. 1988, 170 (9): 4365-4372.PubMedPubMed CentralGoogle Scholar
- Clark JD, Maaloe O: DNA replication and the cell cycle in Escherichia coli cells. J Mol Biology. 1967, 23 (2): 99-112.View ArticleGoogle Scholar
- Jager S, Mack D, Rohde H, Horstkotte MA, Knobloch JK: Disintegration of Staphylococcus epidermidis biofilms under glucose-limiting conditions depends on the activity of the alternative sigma factor sigmaB. Appl Environ Microbiol. 2005, 71 (9): 5577-5581. 10.1128/AEM.71.9.5577-5581.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Moller S, Sternberg C, Andersen JB, Christensen BB, Ramos JL, Givskov M, Molin S: In situ gene expression in mixed-culture biofilms: evidence of metabolic interactions between community members. Appl Environ Microbiol. 1998, 64 (2): 721-732.PubMedPubMed CentralGoogle Scholar
- Li M, Guan M, Jiang XF, Yuan FY, Xu M, Zhang WZ, Lu Y: Genetic polymorphism of the accessory gene regulator (agr) locus in Staphylococcus epidermidis and its association with pathogenicity. J Med Microbiol. 2004, 53 (Pt 6): 545-549.PubMedView ArticleGoogle Scholar
- Pynnonen M, Stephenson RE, Schwartz K, Hernandez M, Boles BR: Hemoglobin promotes Staphylococcus aureus nasal colonization. PLoS Pathog. 2011, 7 (7): e1002104-10.1371/journal.ppat.1002104.PubMedPubMed CentralView ArticleGoogle Scholar
- Evans RC, Holmes CJ: Effect of vancomycin hydrochloride on Staphylococcus epidermidis biofilm associated with silicone elastomer. Antimicrob Agents Chemother (Bethesda). 1987, 31 (6): 889-894.View ArticleGoogle Scholar
- Prosser BL, Taylor D, Dix BA, Cleeland R: Method of evaluating effects of antibiotics on bacterial biofilm. Antimicrob Agents Chemother (Bethesda). 1987, 31 (10): 1502-1506.View ArticleGoogle Scholar
- Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A: The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol. 1999, 37 (6): 1771-1776.PubMedPubMed CentralGoogle Scholar
- Pitz AM, Yu F, Hermsen ED, Rupp ME, Fey PD, Olsen KM: Vancomycin susceptibility trends and prevalence of heterogeneous vancomycin-intermediate Staphylococcus aureus in clinical methicillin-resistant S. aureus isolates. J Clin Microbiol. 2011, 49 (1): 269-274. 10.1128/JCM.00914-10.PubMedPubMed CentralView ArticleGoogle Scholar
- Adair CG, Gorman SP, Feron BM, Byers LM, Jones DS, Goldsmith CE, Moore JE, Kerr JR, Curran MD, Hogg G, et al: Implications of endotracheal tube biofilm for ventilator-associated pneumonia. Intensive Care Med. 1999, 25 (10): 1072-1076. 10.1007/s001340051014.PubMedView ArticleGoogle Scholar
- Wang R, Khan BA, Cheung GY, Bach TH, Jameson-Lee M, Kong KF, Queck SY, Otto M: Staphylococcus epidermidis surfactant peptides promote biofilm maturation and dissemination of biofilm-associated infection in mice. J Clin Invest. 2011, 121 (1): 238-248. 10.1172/JCI42520.PubMedPubMed CentralView ArticleGoogle Scholar
- Boles BR, Horswill AR: Staphylococcal biofilm disassembly. Trends Microbiol. 2011, 19 (9): 449-455. 10.1016/j.tim.2011.06.004.PubMedPubMed CentralView ArticleGoogle Scholar
- Otto M: Staphylococcus aureus and Staphylococcus epidermidis peptide pheromones produced by the accessory gene regulator agr system. Peptides. 2001, 22 (10): 1603-1608. 10.1016/S0196-9781(01)00495-8.PubMedView ArticleGoogle Scholar
- Vuong C, Kocianova S, Yao Y, Carmody AB, Otto M: Increased colonization of indwelling medical devices by quorum-sensing mutants of Staphylococcus epidermidis in vivo. J Infect Dis. 2004, 190 (8): 1498-1505. 10.1086/424487.PubMedView ArticleGoogle Scholar
- Moore PC, Lindsay JA: Genetic variation among hospital isolates of methicillin-sensitive Staphylococcus aureus: evidence for horizontal transfer of virulence genes. J Clin Microbiol. 2001, 39 (8): 2760-2767. 10.1128/JCM.39.8.2760-2767.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Boles BR, Horswill AR: Agr-mediated dispersal of Staphylococcus aureus biofilms. PLoS Pathog. 2008, 4 (4): e1000052-10.1371/journal.ppat.1000052.PubMedPubMed CentralView ArticleGoogle Scholar
- Rice KC, Mann EE, Endres JL, Weiss EC, Cassat JE, Smeltzer MS, Bayles KW: The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus. Proc Natl Acad Sci USA. 2007, 104 (19): 8113-8118. 10.1073/pnas.0610226104.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.