Background

Antibiotics used to treat bacterial infections are becoming increasingly less efficacious due to the emergence of antibiotic-resistant pathogens (Högberg et al., 2010). In addition, such pathogens are often resistant to two or more antibiotics. As a consequence, first line therapies often involve the administration of either multiple or broad-spectrum antibiotics (Hagihara et al., 2012; Khameneh et al., 2016; Frieri et al., 2017). The overuse of antibiotics is thought to be the primary selection pressure driving the dissemination of resistance (World Health Organization, 2018). In addition, broad-spectrum treatments are known to cause long lasting alterations to the healthy gut microbiota, which is likely to have unforeseen health consequences (Willing et al., 2011). For these reasons, there is a need for alternative antimicrobials, such as bacteriocins, that could be used therapeutically.

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria to kill or inhibit other closely related bacteria for nutrients and/or niche competition (Eijsink et al., 2002). Bacteriocins comprise a very diverse group of peptides, from extensively post-translationally modified molecules (class I) to small unmodified peptides (class II) (Alvarez-Sieiro et al., 2016; Acedo et al., 2018). Most bacteriocins produced by Gram-positive bacteria are cationic (pI > 7) and hydrophobic/amphiphilic peptides (Diep and Nes, 2002). They are often of small size ranging from 40 to 70 amino acids, heat-stable, and do not lose activity after storage in organic solvents (2-propanol, acetonitrile, etc.). Most bacteriocins are protease-sensitive (especially unmodified peptides) and have narrow inhibitory spectra, targeting species or genera closely related to the producer (Nissen-Meyer and Nes, 1997), but some have wide inhibitory spectra (Field et al., 2015; Ovchinnikov et al., 2016). Unlike most antibiotics, bacteriocins normally exploit membrane proteins as receptors and disrupt the membrane integrity of sensitive cells upon binding, causing leakage of intracellular solutes and eventually cell death (Nes et al., 2007). Thus, due to different modes of action, bacteriocins are most often equally active against both antibiotic-sensitive pathogens and their antibiotic-resistant counterparts. Bacteriocins have many desirable properties for clinical use, such as high potency, low toxicity, specific inhibition spectrum, and the potential probiotic use of producer organisms (Cotter et al., 2013). However, bacteriocins have not been used in human clinical settings so far. There are a few challenges to the clinical use of bacteriocins, such as their sensitivity towards proteases and low solubility under physiological conditions. Another major factor is likely the insufficient investment spent on the discovery and characterization of new bacteriocins to find candidates more suitable for medical applications.

Here, we describe a set of methods for bacteriocin purification, identification, and characterization. The methods have been used to characterize several bacteriocins in our laboratory (Holo et al., 1991; Ovchinnikov et al., 2016; Desiderato et al., 2021; Goldbeck et al., 2021; Oftedal et al., 2021; Weixler et al., 2022). We believe that this scheme is a good starting point for most bacteriocins and could easily be optimized for special cases, such as multi-peptide bacteriocins, circular bacteriocins, or bacteriocins with relatively low isoelectric points.