Our results indicate that we have successfully constructed a STAMP with dual antimicrobial specificities controlled by the targeting peptides present in the molecule, namely KH for Pseudomonas spp. and M8 for S. mutans. In a closed multispecies system (), the dual specificity of M8(KH)-20 was readily discernable: the population of the culture ‘shifted’ away from targeted organisms following MH-STAMP treatment. The targeted bacteria were eliminated and the population of untargeted organisms increased, to varying degrees, above input CFU/mL. Additionally, interruption of KH or M8 in the MH-STAMP construct with the non-binding peptide BL-1 resulted in the expected elimination of only one targeted species. These results support the hypothesis that functional MH-STAMPs can be constructed from a wide-spectrum AMP base.
The emergence of metagenomics and the development of more sensitive molecular diagnostics has driven an increase in the understanding of human-associated microbial ecologies and host–microbe interactions [19
]. At mucosal surfaces, it has become clear that human bodies harbour an abundance of residential flora that may impact innate and humoral immunity, nutrient availability, protection against pathogens and even host physiology [22
]. Furthermore, findings have indicated that shifts in the diversity of normal flora are associated with negative clinical consequences, for example the overgrowth of S. mutans
in the oral cavity during cariogenesis (linked to the uptake of sucrose) or the antibiotic-assisted colonisation of the intestine by Clostridium difficile
]. Other population shifts may be linked to axilla odour (Corynebacterium
] or even host obesity. Given the quantity and diversity of microbes present, pathogenesis at mucosal surfaces is not likely to be associated with the overgrowth of a single strain or species. More often, it is a population shift resulting in the predominance of two or more species, for example the persistence of Burkholderia cepacia
and P. aeruginosa
in cystic fibrosis airways or Treponema denticola
and Porphymonas gingivalis
and other ‘red cluster’ organisms in gingivitis [30
]. In many cases (such as the latter), these species may have only distant phylogenetic relationships and display differential susceptibilities to antibiotic therapies resulting in persistent disease progression despite treatment [32
]. Currently available treatments for infections of mucosal surfaces are largely non-specific (traditional small molecule antibiotics, mechanical removal) and thus are not effective in retaining flora or shifting the constituent balance back to a health-associated composition [34
]. There is a need for a therapeutic treatment that can selectively target multiple pathogens, regardless of their phylogenetic relationship, and MH-STAMPs may help achieve this goal.
In monoculture experiments (), our results suggest that inclusion of M8 or KH in the MH-STAMP drove activity towards S. mutans
or P. aeruginosa
, but also that the presence of a targeting domain reduced the activity of the parent AMP BD2.20 against untargeted organisms. In contrast, the results of our MIC assays () indicate little difference in activity between BD2.20 and any MH-STAMP. Against untargeted organisms, the M8 and KH regions are likely to have a negative, but not completely inhibitory, impact on BD2.20 activity. Given the long duration of activity and the lower inoculum size in the MIC assay (compared with experiments in ), it is likely that all BD2.20-containing peptides could reach equal levels of growth inhibition, despite large and target-specific differences in antimicrobial speed. This pattern of results was also observed when comparing the MICs of targeted and untargeted organisms utilising STAMPs against S. mutans
and Pseudomonas mendocina
Although more rigorous studies and a more medically relevant combination of pathogen targets are necessary, these findings suggest that it is possible to design an AMP-based therapeutic with multiple and defined fidelities in vitro. MH-STAMPs may help improve human health through the promotion of healthy microbial constituencies.