The pharmaceutical development of the bacteriocin lacticin 3147 into a next generation antibiotic

Antimicrobial resistance (AMR) is more prevalent in society than ever before. The number of microorganisms resistant and becoming resistant to antimicrobial drugs is constantly on the rise. This resistance is endangering the effectiveness of a range of drugs that have saved millions of lives. With 10 million deaths per year projected by 2050 as a result of AMR, an alternative to traditional antibiotics is essential. Bacteriocins are a class of antimicrobial peptides produced by bacteria. They are active against a range of clinically relevant Gram-positive and Gram-negative bacteria. Despite bacteriocins having been discussed in the literature for over a century, comprehensive physicochemical characterisation to enable the selection of formulation strategies for their use as antimicrobial therapeutics has rarely been carried out. The bacteriocin lacticin 3147 is composed of two peptides, Ltnα and Ltnβ, which work together to form pores in the membrane of Gram-positive bacteria. Lacticin 3147 is active against various clinically-relevant bacteria such as Listeria monocytogenes and antimicrobial-resistant bacteria such as Closteroides difficile in the colon and methicillin-resistant Staphylococcus aureus (MRSA). L. monocytogenes infections can cause life-long effects in the elderly and vulnerable and can cause severe complications in pregnant women. C. difficile causes one of the most common healthcare-associated infections and can be fatal in vulnerable groups such as the elderly. Superficial wound infections, like S. aureus and MRSA infections, inhibit the healing of chronic wounds which have a health care cost of around 20 billion dollars per year in the US. Thus lacticin 3147 has significant potential as an alternative to traditional antibiotics. 

In order to select a formulation strategy for the oral delivery of lacticin 3147 to the colon to treat L. monocytogenes and C. difficile infections, its physicochemical properties were characterised. As lacticin 3147 is not produced commercially, it was first produced and purified in-house. Lacticin 3147 was found to be degraded by intestinal proteases and to have poor aqueous solubility, thus encapsulation strategies were employed to enable its use as an antimicrobial for treating these bacterial infections locally in the gastrointestinal tract. Lacticin 3147 displayed activity in aqueous solutions at a range of pH values and in gastric and intestinal fluids. Exposure to trypsin and α-chymotrypsin resulted in complete inactivation, implying that lacticin 3147 would need to be protected from these enzymes to achieve successful local delivery to the gastrointestinal tract. The amount of lacticin 3147 dissolved, i.e. its solution concentration, in water or buffered solutions at pH 1.6 and 7.4 was low and varied with time but increased and was stabilized in gastrointestinal fluids by the phospholipid and bile salt components present in that environment. 

As mesoporous silicates (MPS) were used previously to protect the bacteriocins, bactofencin  A and nisin A, from enzyme degradation they were trialled as a delivery system for lacticin 3147 during this work. MCM-41 and MSE were produced and characterised but no lacticin  3147 adsorption occurred onto the MPSs. It was proposed that this was due to lacticin 3147’s  affinity for its hydrophobic adsorption buffer. Subsequently, a lipid-based system was  proposed due to the increased solution concentrations and the stability of lacticin 3147 noted  in the presence of the phospholipid components of gastrointestinal fluids. Thus, the feasibility  of a solid lipid nanoparticle (SLN) delivery system for local administration of lacticin 3147 to  the colon was investigated. Bacteriocin activity was observed after encapsulation and release  from the lipid matrix. Moreover, activity was seen after exposure to degrading enzymes.  

Lacticin 3147 was next encapsulated into SLNs both individually (single occupancy, SLNα + SLNβ) and together (double occupancy SLNαβ) via a nanoprecipitation technique. This achieved SLNs of uniform size with an encapsulation efficiency above 87% for both peptides at loadings of 9 or 18 mg/g of lipid under single occupancy or double occupancy respectively. SLNαβ dispersions displayed more potent activity than SLNα + SLNβ dispersions. Thus, the SLNαβ dispersion was chosen for further analysis. SLNαβ dispersions showed no cytotoxicity to endothelial cells. The SLN release media (fasted state simulated intestinal fluid; FaSSIF) retained activity at 1 h and 3 h indicating that lacticin 3147 may be sufficiently protected from proteases present in the duodenum. Finally, a reconstituted freeze-dried SLNαβ dispersion was stable and achieved 99.99% bacterial killing at 3.125 µg/ml lacticin 3147. Thus, an SLN?based lacticin 3147 delivery system was developed, potentially enabling oral administration of the bacteriocin to the colon to treat local infections such as C. difficile.

Finally, a lacticin 3147 formulation for topical treatment of S. aureus chronic wound infections was investigated. An SLN gel with the desired physicochemical properties was produced and characterised. A lacticin 3147 SLN gel (SLNαβ gel) and a free lacticin 3147 hydrogel (freeαβ hydrogel) were formulated. Both gels demonstrated sustained activity when compared to the free lacticin 3147 aqueous solution (freeαβ aqueous). The SLNαβ gel, however, displayed more activity at each time point and longer sustained activity when compared to the freeαβ hydrogel. 

In this thesis, lacticin 3147 was produced, purified and characterised, then formulated in various ways to display its potential as an alternative to traditional antibiotics, both as an orally administered antimicrobial for the treatment of C. difficile in the colon and as a topical antimicrobial treatment for S. aureus infected wounds.