Bacteriophage infection of Escherichia coli leads to the formation of membrane vesicles via both explosive cell lysis and membrane blebbing

Membrane vesicles (MVs) are membrane-bound spherical nanostructures that prevail in all three domains of life. In Gram-negative bacteria, MVs are thought to be produced through blebbing of the outer membrane and are often referred to as outer membrane vesicles (OMVs). We have recently described another mechanism of MV formation in Pseudomonas aeruginosa that involves explosive cell-lysis events, which shatters cellular membranes into fragments that rapidly anneal into MVs. Interestingly, MVs are often observed within preparations of lytic bacteriophage, however the source of these MVs and their association with bacteriophage infection has not been explored. In this study we aimed to determine if MV formation is associated with lytic bacteriophage infection. Live super-resolution microscopy demonstrated that explosive cell lysis of Escherichia coli cells infected with either bacteriophage T4 or T7, resulted in the formation of MVs derived from shattered membrane fragments. Infection by either bacteriophage was also associated with the formation of membrane blebs on intact bacteria. TEM revealed multiple classes of MVs within phage lysates, consistent with multiple mechanisms of MV formation. These findings suggest that bacteriophage infection may be a major contributor to the abundance of bacterial MVs in nature

Exploiting bacterial lifestyles for the treatment of Pseudomonas aeruginosa biofilms

University of Technology Sydney. Faculty of Science.‘Pseudomonas aeruginosa’ is a Gram-negative opportunistic pathogen commonly associated with respiratory infections. ‘P. aeruginosa’ has mechanisms of both intrinsic and adaptive resistance that render this bacterium very difficult to eradicate. The formation of biofilms, which is considered a mechanism of adaptive resistance, is the cause of refractory chronic infections of both the upper and lower respiratory tract. ‘P. aeruginosa’ infections have now become a serious global health concern due to the variety of mechanisms of resistance to the most commonly used antibiotics. This highlights the importance of developing new antibiotics and alternative strategies to combat these infections. It has been previously shown in the Whitchurch laboratory that ‘P. aeruginosa’ modifies its lifestyle to survive exposure to β-lactam antibiotics. Upon exposure to this class of antibiotics, planktonic rod-shaped cells transition to a viable cell wall deficient (CWD) spherical morphotype that lack a functional cell wall and has a disrupted outer membrane. This transition can be rapidly reversed after antibiotic removal, indicating that this response may be an alternative mechanism of antibiotic tolerance that enables ‘P. aeruginosa’ to survive exposure to β-lactam antibiotics. The CWD morphological structure lacks the presence of the outer membrane and this can be exploited to induce cell lysis by compounds that induce pores in the cytoplasmic membrane. In fact, it has been shown that the addition of antimicrobial peptides, such as LL-37 and nisin, rapidly and efficiently kills planktonic β-lactam-induced CWD cells of ‘P. aeruginosa’. However, as ‘P. aeruginosa’ infections are primarily associated with biofilms, it is essential to investigate whether the CWD morphotype can be exploited as an alternative strategy for the treatment of ‘P. aeruginosa’ biofilms. This Thesis therefore aims to determine whether the CWD spherical morphotype could be induced in ‘P. aeruginosa’ biofilms by the β-lactam antibiotic meropenem and whether the addition of the antimicrobial peptide nisin could efficiently lyse these CWD cells in the biofilm. Moreover, given the need to develop novel strategies to specifically target upper respiratory tract infections caused by ‘P. aeruginosa’, a combination of meropenem with nisin was formulated as a nasal spray treatment. This Thesis demonstrates that the CWD spherical morphotype could also occur in both static and flow cell biofilms after exposure to 5x MIC meropenem. The combination treatment of meropenem with nisin showed efficacy in eradicating static biofilms and significantly decreased biofilm viability. This Thesis also demonstrates that the combination of meropenem and nisin was suitable for nasal delivery and effective in reducing the viability of biofilms when used in a nasal spray form. Overall the results presented in this Thesis demonstrate that it is possible to exploit the transition to the CWD spherical morphotype to treat ‘P. aeruginosa’ biofilms that form in the nasal cavity and opens a new perspective for the development of alternative strategies to defeat antibiotic-resistant bacteria

Resolving Bio-Nano Interactions of E.coli Bacteria-Dragonfly Wing Interface with Helium Ion and 3D-Structured Illumination Microscopy to Understand Bacterial Death on Nanotopography

Obtaining a comprehensive understanding of the bactericidal mechanisms of natural nanotextured surfaces is crucial for the development of fabricated nanotextured surfaces with efficient bactericidal activity. However, the scale, nature, and speed of bacteria-nanotextured surface interactions make the characterization of the interaction a challenging task. There are currently several different opinions regarding the possible mechanisms by which bacterial membrane damage occurs upon interacting with nanotextured surfaces. Advanced imaging methods could clarify this by enabling visualization of the interaction. Charged particle microscopes can achieve the required nanoscale resolution but are limited to dry samples. In contrast, light-based methods enable the characterization of living (hydrated) samples but are limited by the resolution achievable. Here we utilized both helium ion microscopy (HIM) and 3D structured illumination microscopy (3D-SIM) techniques to understand the interaction of Gram-negative bacterial membranes with nanopillars such as those found on dragonfly wings. Helium ion microscopy enables cutting and imaging at nanoscale resolution while 3D-SIM is a super-resolution optical microscopy technique that allows visualization of live, unfixed bacteria at ~100 nm resolution. Upon bacteria-nanopillar interaction, the energy stored due to the bending of natural nanopillars was estimated and compared with fabricated vertically aligned carbon nanotubes. With the same deflection, shorter dragonfly wing nanopillars store slightly higher energy compared to carbon nanotubes. This indicates that fabricated surfaces may achieve similar bactericidal efficiency as dragonfly wings. This study reports in situ characterization of bacteria-nanopillar interactions in real-time close to its natural state. These microscopic approaches will help further understanding of bacterial membrane interactions with nanotextured surfaces and the bactericidal mechanisms of nanotopographies so that more efficient bactericidal nanotextured surfaces can be designed, fabricated, and their bacteria-nanotopography interactions can be assessed in situ.peerReviewe

Characterizing the mechanism of action of an ancient antimicrobial, manuka honey, against pseudomonas aeruginosa using modern transcriptomics

Manuka honey has broad-spectrum antimicrobial activity, and unlike traditional antibiotics, resistance to its killing effects has not been reported. However, its mechanism of action remains unclear. Here, we investigated the mechanism of action of manuka honey and its key antibacterial components using a transcriptomic approach in a model organism, Pseudomonas aeruginosa. We show that no single component of honey can account for its total antimicrobial action, and that honey affects the expression of genes in the SOS response, oxidative damage, and quorum sensing. Manuka honey uniquely affects genes involved in the explosive cell lysis process and in maintaining the electron transport chain, causing protons to leak across membranes and collapsing the proton motive force, and it induces membrane depolarization and permeabilization in P. aeruginosa. These data indicate that the activity of manuka honey comes from multiple mechanisms of action that do not engender bacterial resistance. IMPORTANCE The threat of antimicrobial resistance to human health has prompted interest in complex, natural products with antimicrobial activity. Honey has been an effective topical wound treatment throughout history, predominantly due to its broad-spectrum antimicrobial activity. Unlike traditional antibiotics, honey-resistant bacteria have not been reported; however, honey remains underutilized in the clinic in part due to a lack of understanding of its mechanism of action. Here, we demonstrate that honey affects multiple processes in bacteria, and this is not explained by its major antibacterial components. Honey also uniquely affects bacterial membranes, and this can be exploited for combination therapy with antibiotics that are otherwise ineffective on their own. We argue that honey should be included as part of the current array of wound treatments due to its effective antibacterial activity that does not promote resistance in bacteria