Antimicrobial peptide combinations against major infectious pathogens: in vitro and in silico approaches

Microbiology research has been investing significant efforts in the development of new and effective antimicrobial therapies against the growing number of multiresistant pathogens. Notably, the use of natural compounds, such as antimicrobial peptides (AMP), and the combination of different antimicrobials are being increasingly explored as means to improve individual agent actions while minimising microorganism resistance. This work contributes to these lines of research by investigating the potential of combining the actions of AMP with those of other agents against major infectious pathogens. To do so, this PhD project encompassed a twofold approach: the comprehensive in silico characterisation of existing experimental results on AMP combinations through bioinformatics approaches, and the in vitro (laboratorial) study of novel combinations. The in silico approach outputted a semi-automated curation workflow that supported the mining of scientific literature on AMP-related combinations and enabled the reconstruction of antimicrobial combination networks. Such reconstruction allowed the creation of a public database on AMP-related combinations (http://sing.ei.uvigo.es/antimicrobialCombination/) and sustains its update. Currently, this database contains data on antimicrobial combinations that have been experimentally tested against major pathogenic bacteria and fungi. Records describe species, strains, combination effects, testing methodologies, mode of growth, and expert observations. Researchers may explore the experimental findings for one or more organisms and look into indirect associations so to identify combinations with promising antimicrobial effects that justifies new testing. An adaptation of this workflow was further used to retrieve information on combinations involving three wellknown and FDA approved AMP, colistin (CST), polymyxin B (POL-B) and nisin (NIS). In-depth analysis of these combinations disclosed important observations on the potential of these peptides and their combination, namely in terms of their cellular targets and treated species. This information was also included in the database, which now includes a total of 3111 combinations, encompassing 350 AMP and 337 non- AMP agents. In the in vitro approach, and after a preliminary screening using a battery of AMP, CST was selected to be combined with three other AMP, namely temporin A (TEMP-A), citropin 1.1 (CIT-1.1) and a linear analogue of tachyplesin I (TP-I-L). These CST combinations were studied for their efficacy to prevent (prophylactic approach) or treat (therapeutic approach) planktonic and biofilm cultures of P. aeruginosa and S. aureus. These tests included single- and double-species biofilms, in order to mimic a more real-like infection, and also encompassed three strains for each species, comprising multi-resistant ones. The results from the in silico approach served as confirmation of these untested AMP combinations and the relevance of the two chosen pathogenic species, therefore bridging the gap between the two approaches. Results from the checkerboard assay showed overall synergy for planktonic P. aeruginosa and synergy/additiveness for planktonic S. aureus. The treatment of established 24 h-old biofilms was more difficult than the prevention of biofilm growth, especially for S. aureus and double-species biofilms. Nevertheless, synergic and additive outcomes were observed for higher concentrations, including for the doublespecies biofilms of multi resistant strains. Live/Dead inspection of the treated doublespecies biofilms revealed a substantial lack of cell vitality, which further strengthened the cells viability results. CST was less cytotoxic than the other peptides towards mammalian fibroblasts, but its combination was toxic for high concentrations. Ongoing work is tackling this toxicity issue, e.g. by combining AMP with matrix disrupting enzymes (MDE) such as DNAse I. Overall, CST-AMP combinations were able to reduce effective concentrations of each drug and highlighted the promise that lays in antimicrobial combination strategies to treat polymicrobial biofilm related infection. The outcomes derived from the two different approaches followed in this PhD project were complementary to each other, with the database aiding in the selection of the AMP combinations and species to be tested in vitro, which in turn outputted valuable information to be added to the database. Globally, the use of the two-fold approach (in silico + in vitro) in the AMP-combination study allowed not only the creation of an important resource for other researchers in the same field, but also pointed out AMP combinations that were deemed promising in the treatment of double-species biofilms of relevant pathogens.

Name thesis:degree_name

Tese de Doutoramento - Engenharia Química e Biológica

Grantor

Universidade do Minho

Year dc:date.issued

2017

Author dc:creator

• Jorge, Paula Alexandra Silva

Advisors dc:contributor.advisor

• Pereira, Maria Olívia
• Lourenço, Anália Maria Garcia

Statement dc:rights

• openAccess

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1822/48653

OAI record oai:identifier

http://repositorium.sdum.uminho.pt/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Arepositorium.uminho.pt%3A1822%2F48653