antibiotics Review Phages for Biofilm Removal Celia Ferriol-González 1 and Pilar Domingo-Calap 1,2,* 1 Department of Genetics, Universitat de València, 46100 Valencia, Spain; [email protected] 2 Institute for Integrative Systems Biology, I2SysBio, Universitat de València-CSIC, 46910 Valencia, Spain * Correspondence: [email protected]; Tel.: +34-963-543-261 Received: 29 March 2020; Accepted: 19 May 2020; Published: 21 May 2020 Abstract: Biofilms are clusters of bacteria that live in association with surfaces. Their main characteristic is that the bacteria inside the biofilms are attached to other bacterial cells and to the surface by an extracellular polymeric matrix. Biofilms are capable of adhering to a wide variety of surfaces, both biotic and abiotic, including human tissues, medical devices, and other materials. On these surfaces, biofilms represent a major threat causing infectious diseases and economic losses. In addition, current antibiotics and common disinfectants have shown limited ability to remove biofilms adequately, and phage-based treatments are proposed as promising alternatives for biofilm eradication. This review analyzes the main advantages and challenges that phages can offer for the elimination of biofilms, as well as the most important factors to be taken into account in order to design effective phage-based treatments. Keywords: biofilm; bacteriophage; phage therapy; antibiotic resistance 1. Introduction Although bacteria are commonly found in nature as individual cells, they can also form multicellular structures called biofilms [1]. Biofilms are complex clusters of bacteria, containing one or more species. They are bound by extracellular polymeric substances (EPS) and attached to surfaces such as living tissue, medical devices, food, industrial equipment, or pipes, among others [2–5]. This extracellular matrix is the immediate bacterial environment within the biofilm, produced predominantly by the bacteria themselves. The EPS matrix consists mainly of exopolysaccharides, but may also contain proteins, nucleic acids, and lipids. This polymeric network connects and immobilizes the cells within the biofilm, providing mechanical stability and adhesion to surfaces. Each biofilm has its own architecture, determined mainly by the matrix. In addition, an aqueous channel system connects the embedded cells, allowing them to access nutrients. The biofilm matrix also contains extracellular enzymes, which act as an external digestive system to help extract nutrients (Figure1)[2,6,7]. Biofilm formation is a cooperative group behavior that begins with the adhesion of the first cells to a given surface. In this first step, cell motility can help bacteria to reach the surface, but is not essential to the process. The mechanisms of motility include flagella, fimbriae, and other surface proteins. Once adhered to the surface, cells begin to divide and the formation of the EPS matrix fixes the initial adhesion [7,8]. Coordination of the different bacteria within the biofilm is necessary for this and involves chemical communication between the cells. Quorum sensing is a mechanism of cell–cell communication, which consists of synchronizing gene expression in response to population cell density. The quorum sensing system allows bacteria to detect population density based on the accumulation of specific signaling molecules [9]. When population density is high, the accumulation of signals triggers different processes, modulating survival strategies through the differential expression of genes, including those involved in virulence [10–12]. Therefore, population density is an important determinant for coordinating the change to a biofilm lifestyle, or for activating the maturation of Antibiotics 2020, 9, 268; doi:10.3390/antibiotics9050268 www.mdpi.com/journal/antibiotics Antibiotics 2020, 9, x FOR PEER REVIEW 2 of 16 Antibiotics 2020, 9, 268 2 of 16 an important determinant for coordinating the change to a biofilm lifestyle, or for activating the maturationbiofilm disruption. of biofilm The disruption. signaling The molecules signaling involved molecules can involved be from can the be same from or the diff sameerent or species different [13]. speciesBacterial [13]. biofilms Bacterial exhibit biofilms a division exhibit of labor, a division also related of labor, to quorum also sensingrelated andto quorum determination sensing ofand the determinationfate of the biofilm of the formation fate of the process biofilm [9, 14formation]. Finally, process in mature [9,14]. biofilms, Finally, some in mature cells disperse, biofilms, allowing some cellsthe colonizationdisperse, allowing of new the surfaces colonization and the of formation new surfaces of new and biofilms the formation [10,15]. of new biofilms [10,15]. Figure 1. Schematic representation of biofilm formation. 1. Planktonic bacteria establish their initial Figureadhesion 1. Schematic to a surface. representation 2. The cells start of biofilm to produce formation. an extracellular 1. Planktonic polymeric bacteria substances establish (EPS)their initial matrix adhesionand divide. to a 3. surface. The bacterial 2. The cells population start to grows,produc increasinge an extracellular the bacterial polymeric density, substances and activating (EPS) matrixquorum andsensing divide.signaling-depending 3. The bacterial population processes. grows, 4. Quorum increasing sensing theregulates bacterial thedensity, development and activating of specialized quorum sensingcells and signaling-depending division of labor. The processes. biofilm matrix 4. Quorum contains sensing extracellular regulates enzymes the development and water channelsof specialized (WC), cellsthat and facilitate division access of labor. to nutrients. The biofilm 5. Activation matrix contains of biofilm extracellular disruption. en Somezymes cells and can water disperse channels and (WC),initiate that new facilitate biofilms. access Note to that nutrients. biofilms 5. can Activa be formedtion of bybiofilm multiple disruption. species of Some bacteria. cells can disperse and initiate new biofilms. Note that biofilms can be formed by multiple species of bacteria. The formation of bacterial biofilms is often considered a virulence factor [11]. Antibiotics are not suitableThe formation for removing of bacterial biofilms, biofilms mainly is because often considered of the antibiotic a virulence tolerance factor of [11]. the Antibiotics bacteria within are not the suitablebiofilms. for While removing drug biofilms, resistance mainly is often because referred of to the as antibiotic a genetic processtolerance resulting of the bacteria from spontaneous within the biofilms.mutations While or horizontal drug resistance gene transfer, is often tolerance referred isto a as phenotypically a genetic process defined resulting process from by which spontaneous bacteria mutationssurvive the or e horizontalffect of a particular gene transfer, antibiotic tolerance in a given is a phenotypically environment [16 defined]. 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