Polymeric Systems as Antimicrobial or Antifouling Agents Printed Edition of the Special Issue Published in International Journal of Molecular Sciences www.mdpi.com/journal/ijms Antonella Piozzi and Iolanda Francolini Edited by Polymeric Systems as Antimicrobial or Antifouling Agents Polymeric Systems as Antimicrobial or Antifouling Agents Special Issue Editors Antonella Piozzi Iolanda Francolini MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade Special Issue Editors Antonella Piozzi University of Rome Italy Iolanda Francolini University “La Sapienza” Italy Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal International Journal of Molecular Sciences (ISSN 1422-0067) from 2018 to 2019 (available at: https: //www.mdpi.com/journal/ijms/special issues/antimicrobial-polymers 2018). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year , Article Number , Page Range. ISBN 978-3-03928-456-6 (Pbk) ISBN 978-3-03928-457-3 (PDF) c © 2020 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND. Contents About the Special Issue Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Iolanda Francolini and Antonella Piozzi Polymeric Systems as Antimicrobial or Antifouling Agents Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 4866, doi:10.3390/ijms20194866 . . . . . . . . . . . . . . 1 Cristina Catt ` o and Francesca Cappitelli Testing Anti-Biofilm Polymeric Surfaces: Where to Start? Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 3794, doi:10.3390/ijms20153794 . . . . . . . . . . . . . . 6 Carmen Mabel Gonz ́ alez-Henr ́ ıquez, Mauricio A. Sarabia-Vallejos and Juan Rodr ́ ıguez Hernandez Antimicrobial Polymers for Additive Manufacturing Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 1210, doi:10.3390/ijms20051210 . . . . . . . . . . . . . . 66 Nor Fadhilah Kamaruzzaman, Li Peng Tan, Ruhil Hayati Hamdan, Siew Shean Choong, Weng Kin Wong, Amanda Jane Gibson, Alexandru Chivu and Maria de Fatima Pina Antimicrobial Polymers: The Potential Replacement of Existing Antibiotics? Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 2747, doi:10.3390/ijms20112747 . . . . . . . . . . . . . . 93 Minghan Chi, Manlin Qi, Lan A, Ping Wang, Michael D. Weir, Mary Anne Melo, Xiaolin Sun, Biao Dong, Chunyan Li, Junling Wu, Lin Wang and Hockin H. K. Xu Novel Bioactive and Therapeutic Dental Polymeric Materials to Inhibit Periodontal Pathogens and Biofilms Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 278, doi:10.3390/ijms20020278 . . . . . . . . . . . . . . . 124 Mark ́ eta Pazderkov ́ a, Petr Malo ˇ n, Vlastimil Z ́ ıma, Kateˇ rina Hofbauerov ́ a, Vladim ́ ır Kopeck ́ y Jr., Eva Koˇ ciˇ sov ́ a, Tom ́ aˇ s Pazderka, V ́ aclav ˇ Ceˇ rovsk ́ y and Lucie Bedn ́ arov ́ a Interaction of Halictine-Related Antimicrobial Peptides with Membrane Models Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 631, doi:10.3390/ijms20030631 . . . . . . . . . . . . . . . 153 Min Kyung Kim, Na Hee Kang, Su Jin Ko, Jonggwan Park, Eunji Park, Dong Won Shin, Seo Hyun Kim, Seung A. Lee, Ji In Lee, Seung Hyun Lee, Eun Gi Ha, Seung Hun Jeon and Yoonkyung Park Antibacterial and Antibiofilm Activity and Mode of Action of Magainin 2 against Drug- Resistant Acinetobacter baumannii Reprinted from: Int. J. Mol. Sci. 2018 , 19 , 3041, doi:10.3390/ijms19103041 . . . . . . . . . . . . . . 179 Malgorzata Anna Paduszynska, Magdalena Maciejewska, Katarzyna Ewa Greber, Wieslaw Sawicki and Wojciech Kamysz Antibacterial Activities of Lipopeptide (C 10 ) 2 -KKKK-NH 2 Applied Alone and in Combination with Lens Liquids to Fight Biofilms Formed on Polystyrene Surfaces and Contact Lenses Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 393, doi:10.3390/ijms20020393 . . . . . . . . . . . . . . . 193 Bruna Agrillo, Marco Balestrieri, Marta Gogliettino, Gianna Palmieri, Rosalba Moretta, Yolande T.R. Proroga, Ilaria Rea, Alessandra Cornacchia, Federico Capuano, Giorgio Smaldone and Luca De Stefano Functionalized Polymeric Materials with Bio-Derived Antimicrobial Peptides for “Active” Packaging Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 601, doi:10.3390/ijms20030601 . . . . . . . . . . . . . . . 212 v Olena Moshynets, Jean-Fran ̧ cois Bardeau, Oksana Tarasyuk, Stanislav Makhno, Tetiana Cherniavska, Oleg Dzhuzha, Geert Potters and Sergiy Rogalsky Antibiofilm Activity of Polyamide 11 Modified with Thermally Stable Polymeric Biocide Polyhexamethylene Guanidine 2-Naphtalenesulfonate Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 348, doi:10.3390/ijms20020348 . . . . . . . . . . . . . . . 225 Marian Szkudlarek, Elisabeth Heine, Helmut Keul, Uwe Beginn and Martin M ̈ oller Synthesis, Characterization, and Antimicrobial Properties of Peptides Mimicking Copolymers of Maleic Anhydride and 4-Methyl-1-pentene Reprinted from: Int. J. Mol. Sci. 2018 , 19 , 2617, doi:10.3390/ijms19092617 . . . . . . . . . . . . . . 244 Alexandra Mu ̃ noz-Bonilla, Daniel L ́ opez and Marta Fern ́ andez-Garc ́ ıa Providing Antibacterial Activity to Poly(2-Hydroxy Ethyl Methacrylate) by Copolymerization with a Methacrylic Thiazolium Derivative Reprinted from: Int. J. Mol. Sci. 2018 , 19 , 4120, doi:10.3390/ijms19124120 . . . . . . . . . . . . . . 266 Francesco Galiano, Raffaella Mancuso, Maria Grazia Guzzo, Fabrizio Lucente, Ephraim Gukelberger, Maria Adele Losso, Alberto Figoli, Jan Hoinkis and Bartolo Gabriele New Polymeric Films with Antibacterial Activity Obtained by UV-induced Copolymerization of Acryloyloxyalkyltriethylammonium Salts with 2-Hydroxyethyl Methacrylate Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 2696, doi:10.3390/ijms20112696 . . . . . . . . . . . . . . 279 Carolina Nascimento Galv ̃ ao, Luccas Missfeldt Sanches, Beatriz Ideriha Mathiazzi, Rodrigo Tadeu Ribeiro, Denise Freitas Siqueira Petri and Ana Maria Carmona-Ribeiro Antimicrobial Coatings from Hybrid Nanoparticles of Biocompatible and Antimicrobial Polymers Reprinted from: Int. J. Mol. Sci. 2018 , 19 , 2965, doi:10.3390/ijms19102965 . . . . . . . . . . . . . . 290 Yanna Gurianov, Faina Nakonechny, Yael Albo and Marina Nisnevitch Antibacterial Composites of Cuprous Oxide Nanoparticles and Polyethylene Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 439, doi:10.3390/ijms20020439 . . . . . . . . . . . . . . . 303 Katarzyna Czarnobaj, Magdalena Prokopowicz and Katarzyna Greber Use of Materials Based on Polymeric Silica as Bone-Targeted Drug Delivery Systems for Metronidazole Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 1311, doi:10.3390/ijms20061311 . . . . . . . . . . . . . . 319 Laura Sisti, Grazia Totaro, Nicole Bozzi Cionci, Diana Di Gioia, Annamaria Celli, Vincent Verney and Fabrice Leroux Olive Mill Wastewater Valorization in Multifunctional Biopolymer Composites for Antibacterial Packaging Application Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 2376, doi:10.3390/ijms20102376 . . . . . . . . . . . . . . 331 Iolanda Francolini, Ilaria Silvestro, Valerio Di Lisio, Andrea Martinelli and Antonella Piozzi Synthesis, Characterization, and Bacterial Fouling-Resistance Properties of Polyethylene Glycol-Grafted Polyurethane Elastomers Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 1001, doi:10.3390/ijms20041001 . . . . . . . . . . . . . . 345 Cristina Catt ` o, Francesco Secundo, Garth James, Federica Villa and Francesca Cappitelli α -Chymotrypsin Immobilized on a Low-Density Polyethylene Surface Successfully Weakens Escherichia coli Biofilm Formation Reprinted from: Int. J. Mol. Sci. 2018 , 19 , 4003, doi:10.3390/ijms19124003 . . . . . . . . . . . . . . 361 vi Fabienne Fa ̈ y, Ma ̈ elle Gouessan, Isabelle Linossier and Karine R ́ ehel Additives for Efficient Biodegradable Antifouling Paints Reprinted from: Int. J. Mol. Sci. 2019 , 20 , 361, doi:10.3390/ijms20020361 . . . . . . . . . . . . . . . 376 vii About the Special Issue Editors Antonella Piozzi In 1987, Antonella Piozzi graduated with a degree in Industrial Chemistry at the Sapienza University of Rome. In 1987, she was awarded an E.N.I. fellowship studying biomedical polymers, synthesis, and characterization. In 1992, she obtained a Ph.D. in Chemical Science and received a fellowship for the project Chimica Fine II from the National Research Council. From 1993 to 2001, she was appointed as a researcher in the Chemistry Department of the Sapienza University of Rome. From 1997 to 2001, she lectured for the courses Macromolecular Chemistry Laboratory and Industrial Chemistry Laboratory at the Sapienza University of Rome. In 2001, Antonella Piozzi was appointed Associate Professor of Industrial Chemistry at the Sapienza University of Rome. Her research experience includes the fields of macromolecular chemistry, biomaterials, and applied enzymology, including studies on the synthesis and physico-chemical characterization of polymers in industrial and medical applications. She has authored over 90 scientific publications published in international scientific journals, 4 book chapters, and more than 60 conference proceedings. She is a member of the Editorial Board of the International Journal of Molecular Science , section Biomaterial Science. Iolanda Francolini Since 2004, Iolanda Francolin i has be en a researcher at Sapienza University of Rome where she studies the synthesis and characterization of polymers for biomedical applications and serves as a lecturer of the science and technologies of polymers. She obtained a degree in Industrial Chemistry cum laudem in 2000 from the Sapienza University of Rome. In 2001, she completed ultrastructural analysis of polymer surfaces and microbial biofilms at the Istituto Superiore di Sanit` a (National Institute of Health) of Rome. In 2003, she was a visiting scientist for one year at the Center for Biofilm Engineering, Montana, USA, where she worked on the development of polyacrylates releasing drugs on demand. In 2005, she obtained a Ph.D. degree at the Sapienza University of Rome in Chemical and Industrial Processes, researching antimicrobial functionalized polyurethanes. Her research interests include synthesis of antimicrobial and antifouling polymers, drug-releasing polymers, and magnetic core/shell nanocomposites for drug targeting. She has been an invited speaker at several international conferences and Guest Editor of several Special Issues of International Journal of Molecular Science Currently, she is member of the Editorial Board of the International Journal of Molecular Science , section Biomaterial Science, and has authored 64 scientific publications in international Journals, 3 book chapters, and more than 60 conference proceedings, with an H-index of 21. ix International Journal of Molecular Sciences Editorial Polymeric Systems as Antimicrobial or Antifouling Agents Iolanda Francolini * and Antonella Piozzi * Department of Chemistry, Sapienza University of Rome, P.le Aldo Moro, 5-00185 Rome, Italy * Correspondence: iolanda.francolini@uniroma1.it (I.F.); antonella.piozzi@uniroma1.it (A.P.) Received: 25 September 2019; Accepted: 29 September 2019; Published: 30 September 2019 The rapid increase in the emergence of antibiotic-resistant bacterial strains combined with a dwindling rate of discovery of novel antibiotic molecules has lately created an alarming issue worldwide [ 1 ]. Resistant genes in microorganisms may be inherited from forerunners or acquired through genetic mutations or gene exchange [ 2 ]. Although the occurrence of resistance in microbes is a natural process, the overuse of antibiotics is known to improve the rate of resistance evolution [ 3 ]. Indeed, under antibiotic treatment, susceptible bacteria inevitably die, while resistant microorganisms proliferate under reduced competition. Therefore, the out-of-control use of antibiotics causes the elimination of drug-susceptible species that would naturally limit the expansion of resistant ones. On top of that, the ability of many microbial species to grow as biofilm has further complicated the treatment of infections with conventional antibiotics. Indeed, microbial biofilms, that is microbial communities growing attached to abiotic surfaces (medical devices, surgical instruments, industrial pipelines, etc.) and tissues [ 4 ], are known to be an optimal environment to amplify both naturally occurring and induced antibiotic-resistance phenomena [ 5 ]. That together with other defense mechanisms significantly increases biofilm antibiotic tolerance. A number of corrective measures are currently under exploration to reverse or slow down antibiotic resistance evolution, among which the development of polymer-based antimicrobial compounds has emerged as one of the most promising solutions [ 6 , 7 ]. Indeed, antimicrobial polymers benefit from a non-specific mode of action, primarily targeting the microbial membrane, and generally display less propensity to promote antimicrobial resistance. Most of the so far investigated polymeric biocides are able to interact with the bacterial cell membrane causing membrane disassembly and leakage of intracellular material [ 8 , 9 ]. Interestingly, some antimicrobial polymers have also been reported to potentiate the activity of conventional antibiotics [10]. A plethora of di ff erent polymer systems has been designed to prevent or treat biofilm formation, including: (i) cationic polymers [ 11 , 12 ]; (ii) antibacterial peptide-mimetic polymers [ 13 , 14 ]; (iii) polymers or composites able to load and release bioactive molecules [ 15 – 17 ]; and (iv) antifouling polymers, able to repel microbes by physical or chemical mechanisms [ 18 ]. The potential fields of application of antimicrobial polymers are numerous. They may play a predominant role in the design and fabrication of medical devices as well as in food packaging and as drug carriers. This special issue collected nineteen papers, of which four were reviews and fifteen were original articles. All of the four reviews were essentially focused on the application of antimicrobial polymers in the biomedical field [ 19 – 22 ]. The review by Catt ò and Cappitelli [ 19 ] provided an overview of the most common methods for testing the antibiofilm activity of polymeric surfaces. The authors underlined how there is a general lack of standardized in vitro methods as well as controlled in vivo studies, which may question the relevance of obtained results. In this regard, simplified guidelines were proposed in the review to help readers choose the most appropriate tests for their objectives. The review by Gonz á lez-Henr í quez and colleagues was instead focused on the manufacturing of 3D-printed objects based on antimicrobial polymers for the production of personalized devices, including implants and drug dosage forms [ 20 ]. In the first part of the review, a particular manufacturing Int. J. Mol. Sci. 2019 , 20 , 4866; doi:10.3390 / ijms20194866 www.mdpi.com / journal / ijms 1 Int. J. Mol. Sci. 2019 , 20 , 4866 technology to produce 3D-objects, that is “additive manufacturing”, was described, and illustrative examples of fabrication of 3D-objects using natural and synthetic antimicrobial polymers were discussed. The potentiality of antimicrobial polymers to replace existing antibiotics was reviewed by Kamaruzzaman et al. [ 21 ], who provided the latest updates in the context of ESKAPE ( Enterecoccus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , and Enterobacter spp.) pathogens. Finally, the state of the art of antibacterial polymers against periodontal pathogens was reviewed by Chi et al. [ 22 ], who paid particular attention to polymeric systems for functional guided tissue regeneration (GTR) membrane, polymer composites for decay restoration, and photosensitizer (PS) modification for photodynamic therapy enhancement. As for the 15 original articles of this special issue, they can be sketchily divided in two broad categories, namely studies focused on polymers able to kill microorganisms ( antimicrobial system s) and studies focused on materials with fouling resistance properties ( antifouling systems ). Among the antimicrobial systems, readers can find antimicrobial peptides [ 23 – 26 ], cationic polymers [27–31], and inorganic / polymer composites [32–34]. Basically, four antimicrobial peptides were investigated: (i) Halictine-1, for which the correlation between changes in primary / secondary structure and antimicrobial activity was studied through various membrane-mimicking models [ 23 ]; (ii) Magainin 2, whose antibiofilm activity was tested against Acitenobacter baumannii strains [ 24 ]; (iii) the lipopeptide (C 10 ) 2 -KKKK-NH 2 , whose potentiality, alone and in combination with lens liquids, in the prophylaxis of contact lens-related eye infections was studied [ 25 ]; and iv) a bactenecin-derivative peptide named 1018K6, which was conjugated to polyethylene terephthalate (PET) to obtain an active packaging for the food industry [26]. As far as cationic polymers are concerned, a thermally stable cationic polymer biocide was obtained by Moshynets and colleagues [ 27 ], through polymerization of guanidine hydrochloride and hexamethylenediamine. Such polymer biocide was then incorporated into Polyamide 11 film to obtain contact-active composites. Interesting antibiofilm activities were found against two biofilm-forming model bacterial strains, E. coli K12 and S. aureus ATCC 25923 [27]. The synthesis of peptides-mimicking amphiphilic cationic copolymers based on maleic anhydride and 4-methyl-1-pentene was reported by Szkudlarek et al. [ 28 ]. The copolymers were then quaternized with either methyl iodide or dodecyl iodide to stabilize polymer cationic charges. Of particular relevance was the minimum inhibitory concentration (MIC) of quaternized copolymers, which was found to be lower than Nisin on a molar basis. Cationic acrylic copolymers based on poly(2-hydroxy ethyl methacrylate) (HEMA), a largely employed biocompatible polymer, were investigated in 2 of the 15 studies of this special issue [ 29 , 30 ]. Specifically, Muñoz-Bonilla et al. [ 29 ] copolymerized HEMA with a methacrylic monomer bearing a thiazole side group susceptible to quaternization, while Galiano et al. [ 30 ] used UV-induced polymerization to copolymerize HEMA with two cationic acryloyloxyalkyltriethylammonium bromides (C-11 or C-12 alkyl chain linker). In both studies, copolymers exhibited significant activity versus Gram-positive ( S. aureus ) and Gram-negative ( P. aeruginosa and E. coli ) bacteria and, as expected, copolymer antimicrobial activity increased with increasing of the cationic unit content. Cationic poly(methylmethacrylate)-based nanoparticles were instead prepared by Galv ã o et al. [ 31 ]. The layering of such nanoparticles onto model surfaces (silicon wafers, glass, and polystyrene sheets) resulted in a significant reduction (ca. 7 logs) of the number of E. coli and S. aureus adhered onto the coated-surfaces compared to pristine surfaces. Always in the framework of antimicrobial systems, three types of antibacterial inorganic / polymer composites were reported in this special issue [ 32 – 34 ]. Antibacterial cuprous oxide nanoparticles (Cu 2 ONPs) were loaded into linear low-density polyethylene (LLDPE) by Gurianov et al. [ 32 ] to develop materials for tap water and wastewater disinfection. Inorganic silica materials functionalized with various types of organic groups (3-aminopropyl, 3-mercaptopropyl, or 3-glycidyloxypropyl groups) were used as bone-targeted delivery systems for metronidazole [ 33 ]. Antibacterial and antioxidant phenol molecules, extracted from olive mill wastewater, were intercalated into the host structure 2 Int. J. Mol. Sci. 2019 , 20 , 4866 of ZnAl layered double hydroxide and employed for the preparation of poly(butylene succinate) composites by Sisti et al. [ 34 ]. These composites showed interesting properties for application in food packaging. Finally, three studies of this special issue were focused on development of antifouling systems following di ff erent approaches [ 35 – 37 ]. Francolini and colleagues [ 35 ] functionalized segmented polyurethanes, one of the most important class of biomedical polymers, with polyethylene glycol (PEG), known to possess strong antifouling properties. Findings showed how PEG-functionalization not only positively a ff ected polyurethane ability to resist to Staphylococcus epidermidis adhesion but also improved mechanical properties of the polymer with clear advantages for practical applications. Catt ò et al. [ 36 ] immobilized the protease α -Chymotrypsin, supposed to degrade the biofilm matrix, on a low-density polyethylene surface. Interestingly, enzyme immobilization significantly weakened E. coli biofilm formation a ff ecting thickness, roughness, and surface area coverage but not bacterial viability, thus reducing the risk of drug resistance development. Finally, Faÿ and coworkers [ 37 ] developed antifouling paints by the use of three additives (Tween 80, Span 85, and PEG-silane) as surface modifiers. In conclusion, antimicrobial polymers may have a pivotal role in the global e ff ort to find solutions against drug resistant infections. In the last 20 years, great scientific and technological advances have been made in this area, mainly thanks to the increased knowledge on mechanisms involved in materials / bacteria interaction as well as on the complexities of biofilm biology. Such knowledge was and still is the inspiration for biomaterials scientists to develop materials able to control biofilm formation. Despite that, a massive amount of work still remains to be done to address unsolved challenges, such as long-term stability, functionality, and biocompatibility of antimicrobial polymers. Translational research is also strongly needed in the near future, in order to make possible the transition of antimicrobial polymers from the bench to the patient bedside. Conflicts of Interest: The authors declare no conflict of interest. References 1. Chokshi, A.; Sifri, Z.; Cennimo, D.; Horng, H. Global Contributors to Antibiotic Resistance. J. Glob. Infect. Dis. 2019 , 11 , 36–42. [PubMed] 2. Blair, J.M.; Webber, M.A.; Baylay, A.J.; Ogbolu, D.O.; Piddock, L.J. Molecular mechanisms of antibiotic resistance. Nat. Rev. Microbiol. 2015 , 13 , 42–51. [CrossRef] [PubMed] 3. Aslam, B.; Wang, W.; Arshad, M.I.; Khurshid, M.; Muzammil, S.; Rasool, M.H.; Nisar, M.A.; Alvi, R.F.; Aslam, M.; Qamar, M.U.; et al. Antibiotic resistance: A rundown of a global crisis. Infect. Drug Resist. 2018 , 11 , 1645–1658. [CrossRef] [PubMed] 4. Costerton, J.W.; Lewandowski, Z.; Caldwell, D.E.; Korber, D.R.; Lappin-Scott, H.M. Microbial biofilms. Annu. Rev. Microbiol. 1995 , 49 , 711–745. 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Cristina Catt ò and Francesca Cappitelli * Department of Food Environmental and Nutritional Sciences, Universit à degli Studi di Milano, via Celoria 2, 20133 Milano, Italy * Correspondence: francesca.cappitelli@unimi.it Received: 24 July 2019; Accepted: 2 August 2019; Published: 3 August 2019 Abstract: Present day awareness of biofilm colonization on polymeric surfaces has prompted the scientific community to develop an ever-increasing number of new materials with anti-biofilm features. However, compared to the large amount of work put into discovering potent biofilm inhibitors, only a small number of papers deal with their validation, a critical step in the translation of research into practical applications. This is due to the lack of standardized testing methods and / or of well-controlled in vivo studies that show biofilm prevention on polymeric surfaces; furthermore, there has been little correlation with the reduced incidence of material deterioration. Here an overview of the most common methods for studying biofilms and for testing the anti-biofilm properties of new surfaces is provided. Keywords: anti-biofilm surfaces; polymeric surfaces; biofilm methods; biofilm analysis; biofilm devices 1. Introduction Polymeric materials, given their low cost, high specificity and adaptability [ 1 ], are currently used for a very broad range of applications ranging from structural materials to coatings, health care [ 2 ], packaging [ 3 , 4 ], communication [ 5 ], heritage [ 6 , 7 ], energy [ 8 ], transportation [ 9 ] and the agri-food industry [ 10 ]. Indeed, the very easy manipulation of molecular structure and chemical composition allows the production of innovative, advanced materials with specific chemical, biological, and physical features [ 1 ]. Polymer materials can be lightweight, hard, strong, and flexible, and can have peculiar thermal, electrical, and optical properties [ 1 ]. Consequently, in the last decade, material science has been experiencing an ever-growing active demand for innovative polymers of notable importance in present-day life. Although polymeric materials play an invaluable role in providing solutions for a wide range of applications they are also easily colonized by biofilm, microorganisms that live in a self-organized, cooperative community attached to a substratum and covered by a self-produced matrix of extracellular polymeric substances (EPS) [ 11 ]. On the global scale, the impact of biofilm on present-day life is incalculable, with the spending of billions of dollars throughout the di ff erent sectors of the economy [ 12 ]. Biofilms are potentially able to contaminate all polymeric structural and infrastructural elements, systems, and devices, such as plumbing, medical implants, food processing facilities, and heating and air conditioning systems [ 13 ]. The result is a reduced industrial yield as well as the physical degradation of industrial systems such as pipe obstruction and corrosion [ 14 ]. In food-processing plants and drinking water networks, biofilm is a persistent source of microbial contamination that can a ff ect the quality and safety of food products and water [ 15 – 17 ]. The worst biofilm reputation is most probably that of biofilm associated with medical implants, causing more than 60% of all microbial infection in humans [ 18 , 19 ]. Indeed, infection can give rise to complications, such as life-threatening systemic infections, contributing to post-operative morbidity, mortality, protracted Int. J. Mol. Sci. 2019 , 20 , 3794; doi:10.3390 / ijms20153794 www.mdpi.com / journal / ijms 6 Int. J. Mol. Sci. 2019 , 20 , 3794 hospitalization and re-operation rate, diagnostic tests and treatments increase, resulting in medical and financial burden [ 20 , 21 ]. It has been estimated that catheter-associated urinary tract infections cause approximately 40% of worldwide hospital-acquired infections, there being approximately 900,000 cases each year in the United States alone, at an annual cost ranging from 296 million to 2.3 billion dollars [22,23]. Strategies to alleviate the e ff ects of biofilm formation on polymeric material have focused on cleaning and disinfection treatments aimed at killing microbial sessile cells already present on the surface. However, such treatments are not totally e ff ective as biofilm microorganisms have features that provide successful conditions for microbial life, including enhanced resistance to antibiotic and biocide treatments [ 24 , 25 ]. Indeed, biofilm-associated resistance is due to several factors like the physiological state of the sessile cells themselves and their physical structure, as well as the presence of EPS that act as a barrier for such cells [ 26 ]. Furthermore, resistance towards many antibiotics has increased in several pathogenic microbial taxa, reducing the chances to treat e ff ectively infections and increasing the risk of complications and fatal outcomes [27]. Consequently, in the past 20 years, studies in the field have addressed the development of preventive strategies, rather than approaches that kill microorganisms after their surface colonization. Indeed, the development of polymeric materials that can prevent microbial adhesion or weaken biofilm structure has emerged as a promising approach to overcome material-associated biofilm problems [ 28 ]. However, despite promising results, many experimental polymeric anti-biofilm surfaces reported in the literature have never been translated into real applications, nor have all newly created anti-biofilm surfaces undergone the critical step of validation of their anti-biofilm performance [ 29 ]. The in vivo testing of new anti-biofilm materials is an arduous task due to limited experimental control. It has been shown that in vivo assays can partially predict biofilm outcomes in humans, though there can be poor correlation with the clinical outcome [ 30 , 31 ]. Furthermore, and this is no less important, it is becoming more and more di ffi cult to get approval for animal studies. Indeed, in most countries, the approval for animal experiments depends on convincing in vitro evidence of e ffi cacy [29,32]. Tissue cultures have been used as a surrogate for in vivo biofilm studies, but the construction of a three-dimensional tissue culture is labour intensive and expensive. Moreover, experiments can only be conducted for short periods of time (i.e., in less than 24 hours) due to the cytotoxic e ff ects on the cells, this cytotoxicity being due to both the biofilm itself and to the anti-biofilm surface, thus reducing the utility of these studies as biofilms generally take multiple days to reach maturity [20]. Therefore, attention here is paid to in vitro evaluation methods, which are a compromise between the reality of the in vivo ecosystem and the simplification of the system. However, a well-devised model and studies allow researchers to get relevant results [ 33 ]. Whereas there are several in vitro industrial standard tests to evaluate the antimicrobial (i.e., killing of microorganisms) e ffi cacy of medical and non-medical products, there are no accepted standardized assays and validated methods to properly assess the activity of anti-biofilm material [ 34 ]. Indeed, current in vitro evaluation standard tests, especially tailored for specific action mechanisms that lead to cell death, are inadequate for today’s di ff erent advanced anti-biofilm surface designs. Note that the surface evaluation standard tests available today are mostly intended to test the ability of the material to abate microbial viability, without taking into consideration the di ff erences in the mechanism of action [ 29 ]. Indeed, the tests attempt to evaluate biofilm inhibition and eradication without proper investigation of the variability in biofilm architecture and the complexity of its development [ 34 ]. This deficiency in methodology has adversely a ff ected the translation of research into practical industrial and medical applications, and to regulatory agencies that assess the real-life usefulness of anti-biofilm surfaces. Over the last couple of decades, a variety of simplified in vitro systems have been proposed to study biofilm formation [ 35 ]. Therefore, given the lack of standardized procedures to test anti-biofilm properties of materials, the only solution to test novel anti-biofilm surfaces for clinical purposes is to