Exploiting bacterial lifestyles for the treatment of Pseudomonas aeruginosa biofilms

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‘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.
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