Biofilm dispersion : the key to biofilm eradication or opening Pandora’s box?

Biofilms are extremely difficult to eradicate due to their decreased antibiotic susceptibility. Inducing biofilm dispersion could be a potential strategy to help combat biofilm-related infections. Mechanisms of biofilm dispersion can basically be divided into two groups, i.e. active and passive dispersion. Active dispersion depends on a decrease in the intracellular c-di-GMP levels, leading to the production of enzymes that degrade the biofilm matrix and promote dispersion. In contrast, passive dispersion relies on triggers that directly release cells from the biofilm. In the present review, several active and passive dispersion strategies are discussed. In addition, the disadvantages and possible consequences of using dispersion as a treatment approach for biofilm-related infections are also reviewed

Biofilm Formation by Pathogenic Bacteria: Applying a Staphylococcus aureus Model to Appraise Potential Targets for Therapeutic Intervention

Carried in the nasal passages by up to 30% of humans, Staphylococcus aureus is recognized to be a successful opportunistic pathogen. It is a frequent cause of infections of the upper respiratory tract, including sinusitis, and of the skin, typically abscesses, as well as of food poisoning and medical device contamination. The antimicrobial resistance of such, often chronic, health conditions is underpinned by the unique structure of bacterial biofilm, which is the focus of increasing research to try to overcome this serious public health challenge. Due to the protective barrier of an exopolysaccharide matrix, bacteria that are embedded within biofilm are highly resistant both to an infected individual's immune response and to any treating antibiotics. An in-depth appraisal of the stepwise progression of biofilm formation by S. aureus, used as a model infection for all cases of bacterial antibiotic resistance, has enhanced understanding of this complicated microscopic structure and served to highlight possible intervention targets for both patient cure and community infection control. While antibiotic therapy offers a practical means of treatment and prevention, the most favorable results are achieved in combination with other methods. This review provides an overview of S. aureus biofilm development, outlines the current range of anti-biofilm agents that are used against each stage and summarizes their relative merits

Killing of organisms responsible for wound infections using a light-activated antimicrobial agent

Infected wounds are a major cause of hospital-acquired infections and these are difficult to treat due to the emergence of antibiotic-resistant bacteria. This project is concerned with evaluating a novel antimicrobial approach involving the photosensitizer indocyanine green (ICG) which generates reactive oxygen species when irradiated with near-infrared (NIR) light which enables good tissue penetration. The photo-susceptibility of common wound-infecting organisms to ICG coupled with NIR-light was investigated. All species were susceptible to killing. ICG at a concentration of 25 μg/mL enabled the killing of the Gram-positive species (Staphylococcus aureus and Streptococcus pyogenes), higher concentrations (100-200μg/mL) were necessary to achieve substantial kills of the Gram-negative species (Pseudomonas aeruginosa and Escherichia coli). Both high and low fluences were able to kill 99.999% of the Gram-positive bacteria. High fluence irradiation was necessary to kill 99.99% of the Gram-negative bacteria. The pulsed-mode of irradiation was as effective as the continuous-mode for killing the Gram-positive species. Yet only the continuous-mode of irradiation was able to kill P. aeruginosa. Biofilms of Staph. aureus and P. aeruginosa were susceptible to disruption and killing by ICG-photosensitization. A significant enhancement of lethal photosensitization of Staph. aureus was achievable using gold-nanoparticles and antioxidants. Significant kills (>99%) were achieved in the presence of serum and 100 μg/mL ICG. A low oxygen concentration reduced the kills to 96.77% and 71.62% for Staph. aureus and Strep. pyogenes respectively. Mechanistic studies revealed that killing was mediated mainly by reactive-oxygen species. In vivo studies in mice showed that ICG and continuous-NIR light could achieve kills of 96%, 93% and 78-91% for P. aeruginosa, Strep. pyogenes and Staph. aureus respectively. The results of these in vitro and in vivo studies imply that ICG-PDT could be an effective means of decreasing the microbial burden in wounds

Effects of Surface Topography on Bacterial Biofilm Formation

Biofilms are multicellular structures with bacterial cells attached to a surface and embedded in an extracellular matrix. With high-level resistance to antimicrobial agents, biofilms are the cause of chronic infections associated with implanted medical devices such as breast implants, orthopedic devices, pace markers, and many others. Besides the prevalence, biofilm infections are associated with high mortality, presenting an urgent need for more effective controls. Several strategies such as coating with antimicrobial agents and changing chemical, physical, and biological properties of biomaterials have been attempted, but bacteria have remarkable capabilities to overcome unfavorable conditions over time and long-term biofilm control remains challenging. In addition, most approaches are based on empirical experiments rather than rational designs, limiting their effects, especially in vivo. In this study, we engineered surface topography in two ways (static and dynamic) to better understand and control bacterial biofilm formation. For the static surface topography, a high-throughput approach to study bacterial attachment on PDMS surfaces with different textures was developed. By testing bacterial adhesion to samples with square-shaped recessive patterns with varying size and inter-pattern distance, surface features that promote biofilm formation were identified. E. coli attachment did not exhibit a monotonic, linear relationship with surface area, but depended on the 3D topography. For dynamic surface topography, we used shape memory polymers (SMPs) to obtain on-demand dynamic changes in substratum topography. Our results show that shape recovery of tert-butyl acrylate (tBA) based one-way SMP caused 99.9% detachment of 48 h Pseudomonas aeruginosa PAO1 biofilms. Interestingly, P. aeruginosa PAO1 biofilm cells detached by shape recovery showed 2,479 times higher antibiotic susceptibility compared to the original biofilm cells. The released biofilm cells also presented 4.1 times higher expression of the gene rrnB, encoding ribosomal RNA, and 11.8 times more production of adenosine triphosphate (ATP) than the control biofilm cells. To further develop this technology for long-term biofilm control, we synthesized reversible SMP with different molecular weights of poly(ɛ-caprolactone) diisocyanatoethyl dimethacrylate (PCLDIMA), with 25 wt.% of butyl acrylate (BA) as a linker, and 1 wt.% of benzoyl peroxide (BPO) as a thermal initiator. Among various combinations of molecular weight, 2:1 wt. ratio mixture of 15,000 g/mol PCLDIMA and 2,000 g/mol PCLDIMA showed a transition temperature of 36.7°C. The created rSMP has repeatable and reversible shape recovery for more than 3 cycles. With 18% stretch, 61.0±6.6% of 48 h P. aeruginosa PAO1 biofilm cells were removed in each shape recovery cycle on average, with a total of 94.3±1.0% biofilm removal after three consecutive shape recovery cycles. In summary, the results of this study demonstrated that surface topography has potent effects on bacterial adhesion and biofilm formation. We believe that these results not only provide important information for understanding the risk of medical devices but also helps the design of control methods for preventing chronic infections associated with implanted medical devices

A Comparative Evaluation of Endodontic Irrigation Methods for Removal of the Smear Layer

The purpose of this in vitro study is to compare the effect of various irrigation systems on smear layer removal in curved root canals. Root canal irrigation plays an important role in the debridement and disinfection of the root canal system. It has been well documented that the flushing component of the irrigants is as important as the tissue dissolving capability. Therefore, the efficacy of the irrigant might also be influenced by the method by which it is introduced. Fifty-one recently extracted molar teeth with root curvatures of more than 30° were selected according to Schneider\u27s method. The teeth were decoronated to obtain a standardized root length of 12 mm. The root tips were sealed with hot glue and embedded into a silicone mold. The canal preparations were performed by using ProTaper™ and ProFile™ systems up to #35,04. Sodium hypochlorite (NaOCl 6%) and ethylenediaminetetraacetic acid (EDTA 17%) were used as root canal irrigants according to Yamada protocol. To maintain irrigation consistency, a programmable syringe pump was connected to each system. After finishing the cleaning and shaping of the curved canals, the final cleansing of the root canal space, with proper irrigation solutions, were accompanied by activation systems. Five different treatment modalities were tested; Group 1: Traditional irrigation, Group 2: EndoActivator™, Group 3: Passive ultrasonic irrigation (PUI), Group 4: EndoVac™, Group 5: Saline. The root halves (n=102) were imaged with the FEI Quanta 200 scanning electron microscope™ (SEM). Over 7000 magnified images were reviewed and scored by three board certified Endodontists in a double-blind manner. The data was analyzed by using the Cochran-Mantel-Haenszel method, Pairwise Comparisons and Intra-class correlation coefficients. The EndoVac™ system (an apical negative pressure irrigation system) was found to be significantly more effective (p\u3c0.05) than the other groups in all sections observed, this would include the apical, middle and coronal sections for the elimination of the smear layer as well as the debris removal and improved tubule visibility. The negative pressure delivery systems may provide cleaner surfaces in the canals of curved roots of at least 30 degree or more

The Host-Microbiota Axis in Chronic Wound Healing

Chronic, non-healing skin wounds represent a substantial area of unmet clinical need, leading to debilitating morbidity and mortality in affected individuals. Due to their high prevalence and recurrence, chronic wounds pose a significant economic burden. Wound infection is a major component of healing pathology, with up to 70% of wound-associated lower limb amputations preceded by infection. Despite this, the wound microbiome remains poorly understood. Studies outlined in this thesis aimed to characterise the wound microbiome and explore the complex interactions that occur in the wound environment. Wound samples were analysed using a novel long-read nanopore sequencing-based approach that delivers quantitative species-level taxonomic identification. Clinical wound specimens were collected at both the point of lower-extremity amputation and via a pilot clinical trial evaluating extracorporeal shockwave therapy (ESWT) for wound healing. Combining microbial community composition, host tissue transcriptional (RNAseq) profiling, with clinical parameters has provided new insight into healing pathology. Specific commensal and pathogenic organisms appear mechanistically linked to healing, eliciting unique host response signatures. Patient- and site-specific shifts in microbial abundance and communitycomposition were observed in individuals with chronic wounds versus healthy skin. Transcriptional profiling (RNAseq) of the wound tissue revealed important insight into functional elements of the host-microbe interaction. Finally, ESWT was shown to confer beneficial effects on both cellular and microbial aspects of healing. High-resolution long-read sequencing offers clinically important genomic insights, including rapid wide-spectrum pathogen identification and antimicrobial resistance profiling, which are not possible using current culture-based diagnostic approaches. Thus, data presented in this thesis provides important new insight into complex host-microbe interactions within the wound microbiome, providing new and exciting future avenues for diagnostic and therapeutic approaches to wound management