Current Pharmaceutical Design - Volume 21, Issue 1, 2015

Biofilms are microbial sessile communities characterized by cells that are attached to a substratum or interface or to each other, are embedded in a self-produced matrix of extracellular polymeric substances and exhibit an altered phenotype compared to planktonic cells. Biofilms are estimated to be associated with 80% of microbial infections and it is currently common knowledge that growth of micro-organisms in biofilms can enhance their resistance to antimicrobial agents. As a consequence antimicrobial therapy often fails to eradicate biofilms from the site of infection. For this reason, innovative anti-biofilm agents with novel targets and modes of action are needed. One alternative approach is targeting the bacterial communication system (quorum sensing, QS). QS is a process by which bacteria produce and detect signal molecules and thereby coordinate their behavior in a cell-density dependent manner. Three main QS systems can be distinguished: the acylhomoserine lactone (AHL) QS system in Gram-negative bacteria, the autoinducing peptide (AIP) QS system in Gram-positive bacteria and the autoinducer-2 (AI-2) QS system in both Gram-negative and -positive bacteria. Although much remains to be learned about the involvement of QS in biofilm formation, maintenance, and dispersal, QS inhibitors (QSI) have been proposed as promising antibiofilm agents. In this article we will give an overview of QS inhibitors which have been shown to play a role in biofilm formation and/or maturation.

Cyclic di-GMP is a second messenger found in almost all eubacteria that acts to regulate a wide range of functions including developmental transitions, adhesion and biofilm formation. Cyclic di-GMP is synthesised from two GTP molecules by diguanylate cyclases that have a GGDEF domain and is degraded by phosphodiesterases with either an EAL or an HD-GYP domain. Proteins with these domains often contain additional signal input domains, suggesting that their enzymatic activity may be modulated as a response to different environmental or cellular cues. Cyclic di-GMP exerts a regulatory action through binding to diverse receptors that include a small protein domain called PilZ, enzymatically inactive GGDEF, EAL or HD-GYP domains, transcription factors and riboswitches. In many bacteria, high cellular levels of cyclic di-GMP are associated with a sessile, biofilm lifestyle, whereas low levels of the nucleotide promote motility and virulence factor synthesis in pathogens. Elucidation of the roles of cyclic di-GMP signalling in biofilm formation has suggested strategies whereby modulation of the levels of the nucleotide or interference with signalling pathways may lead to inhibition of biofilm formation or promotion of biofilm dispersal. In this review we consider these approaches for the control of biofilm formation, beginning with an overview of cyclic di-GMP signalling and the different ways that it can act in regulation of biofilm dynamics.

Bacteria are able to colonize and thrive in a variety of different environments as a biofilm, but only within the last half century new insights have been gained in this complex biosystem. Bacterial biofilms play a major role in human health by forming a defensive barrier against antibacterial chemical therapeutics and other potential pathogens, and in infectious disease when the bacteria invade normally sterile compartments. Quorum sensing is the signaling network for cell-to-cell communication and utilized by bacteria to regulate biofilms and other cellular processes. This review will describe recent advances in quorum sensing and biofilms. Initially, it will focus on Streptococcus pneumoniae biofilm regulation and the involvement of the ComABCDE pathway. As part of this review an original analysis of the genotypic and phenotypic variation of the signaling molecule, ComC and its cognate receptor ComD, firstly within the pneumococcal species and then within the genus Streptococcus will be presented. Additionally, a pathway similar to ComABCDE, the BlpABCSRH that regulates bacteriocin and immunity protein production that inhibit the growth of competing bacteria will be described. This review will then examine a third quorum sensing mechanism in the pneumococcus, the LuxS/AI-2, and present a novel gene and protein sequence comparative analysis that indicates its occurrence is more universal across bacterial genera compared with the Com pathway, with more sequence similarities between bacterial genera that are known to colonize the mucosal epithelium.

Studies of the biofilm life cycle can identify novel targets and strategies for improving biofilm control measures. Of particular interest are dispersal events, where a subpopulation of cells is released from the biofilm community to search out and colonize new surfaces. Recently, the simple gas and ubiquitous biological signaling molecule nitric oxide (NO) was identified as a key mediator of biofilm dispersal conserved across microbial species. Here, we review the role and mechanisms of NO mediating dispersal in bacterial biofilms, and its potential for novel therapeutics. In contrast to previous attempts using high dose NO aimed at killing pathogens, the use of low, non-toxic NO signals (picomolar to nanomolar range) to disperse biofilms represents an innovative and highly favourable approach to improve infectious disease treatments. Further, several NO-based technologies have been developed that offer a versatile range of solutions to control biofilms, including: (i) NO-generating compounds with short or long half-lives and safe or inert residues, (ii) novel compounds for the targeted delivery of NO to infectious biofilms during systemic treatments, and (iii) novel NO-releasing materials and surface coatings for the prevention and dispersal of biofilms. Overall the use of low levels of NO exploiting its signaling properties to induce dispersal represents an unprecedented and promising strategy for the control of biofilms in clinical and industrial contexts.

Biofilms are formed by the attachment of single or mixed microbial communities to a variety of biological and/or synthetic surfaces. Biofilm micro-organisms benefit from many advantages of the polymicrobial environment including increased resistance against antimicrobials and protection against the host organism’s defence mechanisms. These benefits stem from a number of structural and physiological differences between planktonic and biofilm-resident microbes, but two main factors are the presence of extracellular polymeric substances (EPS) and quorum sensing communication. Once formed, biofilms begin to synthesise EPS, a complex viscous matrix composed of a variety of macromolecules including proteins, lipids and polysaccharides. In terms of drug delivery strategies, it is the EPS that presents the greatest barrier to diffusion for drug delivery systems and free antimicrobial agents alike. In addition to EPS synthesis, biofilm-based micro-organisms can also produce small, diffusible signalling molecules involved in cell density-dependent intercellular communication, or quorum sensing. Not only does quorum sensing allow microbes to detect critical cell density numbers, but it also permits co-ordinated behaviour within the biofilm, such as iron chelation and defensive antibiotic activities. Against this backdrop of microbial defence and cell density-specific communication, a variety of drug delivery systems have been developed to deliver antimicrobial agents and antibiotics to extracellular and/or intracellular targets, or more recently, to interfere with the specific mechanisms of quorum sensing. Successful delivery strategies have employed lipidic and polymeric-based formulations such as liposomes and cyclodextrins respectively, in addition to inorganic carriers e.g. metal nanoparticles. This review will examine a range of drug delivery systems and their application to biofilm delivery, as well as pharmaceutical formulations with innate antimicrobial properties such as silver nanoparticles and microemulsions.

The increasing prevalence of persistent biofilm infections, such as wound infections, chronic lung infections or medical device- related infections, which usually tolerate conventional antibiotic treatment, calls for the development of new therapeutic strategies. To date, antimicrobial peptides (AMPs) are considered as promising agents in the fight against multidrug-resistant bacterial biofilm infections, since many of them have been shown to prevent biofilm formation or even kill preexisting, mature biofilms of several Grampositive and Gram-negative bacteria in addition to their bactericidal actions to planktonic cells. In this mini-review, we summarize in vitro and in vivo antibiofilm properties of natural and synthetic cationic AMPs against clinically relevant bacterial pathogens. Furthermore, the benefits and challenges in the use of AMPs for the medical treatment of bacterial biofilm infections are discussed.

Although free-swimming planktonic bacteria historically have been the typical focus of microbiological studies, the natural state of many or most bacteria is one where they instead are associated with surfaces and/or each other. For many pathogenic as well as nuisance bacteria, including biofouling bacteria, it consequently is within the context of this biofilm state that antibacterial strategies must be implemented. For reasons that are not fully understood, however, biofilm-associated bacteria tend to be less susceptible to treatments with standard chemical antibacterial agents than are planktonic bacteria, and this appears to be especially an issue with the use of lessharsh agents such as antibiotics. Within a variety of contexts the development of less- or selectively toxic antibacterial agents capable of clearing biofilms therefore would be welcome. In this review we consider the use of three categories of such agents as anti-biofilm antibacterials. These are lytic viruses of bacteria, that is, bacteriophages, effecting phage-mediated biocontrol of bacteria (a.k.a., phage therapy); purified phage-encoded enzymes that digest bacterial cell-wall material (endolysins or simply lysins); and a second category of phage-encoded enzymes that digest the extracellular polymeric substance (EPS) that are particularly notable components of bacterial biofilms (EPS depolymerases). These agents have been shown to reduce the bacterial density of a diversity of biofilms and, in many cases, tend to be lacking in inherent toxicity against the tissues of animals. Here we consider these phage-based anti-biofilm strategies with emphasis on ecological aspects of their action and with particular consideration of EPS depolymerases.

Staphylococci, in particular Staphylococcus aureus and Staphylococcus epidermidis, are a leading cause of healthcareassociated infections. Patients who have a medical device inserted are at particular risk of an infection with these organisms as staphylococci possess a wide range of immune evasion mechanisms, one of which being their ability to form biofilm. Once embedded in a biofilm, bacteria are inherently more resistant to treatment with antibiotics. Despite advances in our understanding of the pathogenesis of staphylococcal biofilm formation, medical devices colonised with biofilms frequently require removal. New and novel approaches to prevent and treat biofilm infections are urgently required. In recent years, progress has been made on approaches that include antiadhesive strategies to prevent surface adhesion or production of bacterial adhesins, dissolution of already established biofilm, targeting of biofilm matrix for degradation and interference with biofilm regulation. Several obstacles need to be overcome in the further development of these and other novel anti-biofilm agents. Most notably, although in vitro investigation has progressed over recent years, the need for biofilm models to closely mimic the in vivo situation is of paramount importance followed by controlled clinical trials. In this review we highlight the issues associated with staphylococcal colonisation of medical devices and potential new treatment options for the prevention and control of these significant infections.

The translation of promising preclinical treatments into effective drugs for Alzheimer's disease (AD) has been challenging. One of the most potent risk factors for sporadic AD is carrier status of the epsilon 4 allele of the apolipoprotein E gene (E4). E4 carriers show a differential response to several therapies which are being investigated as AD treatments, including acetylcholinesterase inhibitors and therapeutics with vascular and metabolic targets. The differential treatment responses of E4 carriers may partially explain why some treatments show a null effect in clinical trials. Understanding the reasons behind these responses is not only important for clinical practice, but may also help us elucidate mechanisms for this neurodegenerative disease.

Malignant gliomas and metastatic tumors are the most common forms of brain tumors. From a clinical perspective, neuroimaging plays a significant role, in diagnosis, treatment planning, and follow-up. To date MRI is considered the current clinical gold standard for imaging, however, despite providing superior structural detail it features poor specificity in identifying viable tumors in brain treated with surgery, radiation, or chemotherapy. In the last years functional neuroimaging has become largely widespread thanks to the use of molecular tracers employed in cellular metabolism which has significantly improved the management of patients with brain tumors, especially in the post-treatment phase. Despite the considerable progress of molecular imaging in oncology its use in the diagnosis of brain tumors is still limited by a few wellknown technical problems. Because 18F-FDG, the most common radiotracer used in oncology, is avidly accumulated by normal cortex, the low tumor/background signal ratio makes it difficult to distinguish the tumor from normal surrounding tissues. By contrast, radiotracers with higher specificity for the tumor are labeled with a short half-life isotopes which restricts their use to those centers equipped with a cyclotron and radiopharmacy facility. 11C-Choline has been reported as a suitable tracer for neuroimaging application. The recent availability of choline labeled with a long half-life radioisotope as 18F increases the possibility of studying this tracer's potential role in the staging of brain tumors. The present review focuses on the possible clinical applications of PET/CT with choline tracers in malignant brain tumors and brain metastases, with a special focus on malignant gliomas.