Biomedical implants such as catheters, valves, and joints have revolutionized medicine, but they also increase the infection risk [1], [2], [3], [4]. Indeed, implant infection is one of the most frequent and severe complications associated with the use of biomaterials [5]. Infections are caused by pathogenic bacteria developing into biofilms [6], [7] and antibiotics are often used to prevent or treat microbial infections. However, current antibiotics are effective for bacteria present in an unshielded, planktonic, and single cell-state, but the majority of conventional antibiotics are insufficient to treat established biofilm infections. Surgical replacement of these infected devices therefore becomes the only treatment, leading to increased patient morbidity and mortality, and posting a huge financial burden on healthcare services in the USA [8], [9]. Another concern with the use of antibiotics is the rapid emergence and dissemination of resistance among bacterial pathogens [10], [11], [12]. Development of a new strategy to improve anti-infection properties of biomaterials and increase the efficacy of antibiotics is critically important for the application of biomaterials as implants [13], [14].

Pathogenic bacteria adhere, grow, and produce extracellular polymeric substances (EPS) to form a biofilm matrix on devices, resulting in microbial infection. Bacterial adhesion and biofilm formation are influenced by many factors from the microorganism itself, the substrate, and the local environment. More specifically, these factors may include the type of bacteria (Gram-positive or Gram-negative, surface energy and charge, outer membrane molecular details, etc.), the biomaterial surface factors (chemical composition, surface roughness and topography, surface energy and charge, etc.) and environmental factors (osmolarity, pH, carbon availability, iron availability, oxygen, flow conditions, temperature, etc.). These properties can also be interconnected and in turn affected by other parameters [15]. During the transition from planktonic cells to the sessile biofilm community, bacterial cells undergo a series of physiological, metabolic, and phenotypic changes driven by the molecular signaling messengers in cells [16]. Compelling evidence has demonstrated that the molecular mechanism of biofilm formation involves bacterial intracellular nucleotide second messenger signaling which monitors and responds to changing environments [17], [18], [19], [20], [21], [22], [23], [24]. Interference with the nucleotide signaling pathway provides a novel approach to control pathogenic bacterial adhesion and biofilm formation on biomaterial surfaces thereby combating microbial infection [25], [26], [27], [28].

Microorganisms can use cyclic nucleotide second messengers including cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and cyclic dimeric adenosine monophosphate (c-di-AMP) in the transduction of signals and for regulating motility and adhesion [29]. Among these messengers, c-di-GMP is a general and key signaling molecule in many bacterial cells that regulates whether the cells produce an extracellular matrix and form biofilm or assume a planktonic lifestyle [30], [31], [32], [33], [34], [35], [36]. The c-di-GMP effectors utilize a range of mechanisms to relay signals to cellular processes and impact exopolysaccharide synthesis, motility, transcription, and subcellular or cell-surface protein localization, thereby regulating biofilm formation [17]. For example, Escherichia coli has shown that c-di-GMP binding to the PilZ domain of the effector protein YcgR stimulates its interaction with the flagellar motor and/or switch complex to positively affect the motile-to-sessile transition[37]. In Pseudomonas aeruginosa, a number of c-di-GMP metabolizing enzymes have been identified that participate in reversible and irreversible attachment and biofilm maturation. The high levels of c-di-GMP in P. aeruginosa were reported to increase the production of EPS and promote biofilm formation while low levels of c-di-GMP lead to increased motility and biofilm dispersal [38]. A recent in vivo study of biofilm formation by various P. aeruginosa strains containing different levels of c-di-GMP in a rabbit model of pleural empyema demonstrated a positive correlation between biofilm formation and c-di-GMP levels in bacteria [39]. The same group also studied P. aeruginosa biofilms in a knee septic arthritis model and reached the same conclusion [40]. These results provide evidence for the critical role of c-di-GMP signaling in the establishment of biofilm and the pathogenicity for P. aeruginosa.

Since c-di-GMP is synthesized from two molecules of GTP (guanosine 5’-triphosphate) by di-guanylate cyclases (DGCs) and is degraded into pGpG and/or GMP by phosphodiesterases (PDEs) [22], [41], small molecules that are either DGC-inhibitors or PDE activators have been developed to interrupt signaling and reduce the c-di-GMP content in bacteria for combating biofilms [25], [42], [43]. These small molecules apply less pressure on bacterial survival but interfere with bacterial virulence such as biofilm formation, thereby increasing the efficacy of antibiotics in standard therapy but reducing the risk for resistance development[44]. In this study, we tethered two small molecules that can interfere with bacterial intracellular nucleotide signaling pathways and interrupt biofilm formation on polymeric biomaterial surfaces in a way that increases the efficacy of antibiotics on biofilms.

Small molecules derivates of 4-arylazo-3,5-diamino-1 H-pyrazole such as SP02 and SP03 (named in this study, Scheme 1) are new anti-biofilm agents which were recently reported [45], [46]. They were screened from 50,000 chemical compounds and identified as novel activators of the PDE BifA present in Pseudomonas spp. The identified compounds were found to significantly reduce the c-di-GMP levels in P. aeruginosa, leading to inhibition of biofilms and dispersal of established biofilms. In this study, we expand these studies to staphylococcal biofilms as staphylococci are the most commonly diagnosed microorganisms in microbial infection on indwelling devices[47], [48]. Specifically, we used Staphylococcus epidermidis RP62A as a model bacterium and investigated the effects of these small molecules on nucleotide levels in S. epidermidis as well as the ability of the bacterium to form and exist as biofilms in the presence of these small molecules. Further, we tethered these small molecules on polyurethane (PU) biomaterial surfaces and studied the feasibility of using small molecule-modified polymer surfaces to interrupt biofilm formation and increase the efficacy of antibiotics. Results demonstrate that these small molecules affect nucleotide second messenger signaling, resulting in decreased biofilm formation, followed by an increased susceptibility to antibiotic therapy for this important bacterial strain, and demonstrating a template for assessing other bacteria involved in biofilm formation.