Detección y caracterización de péptidos con actividad antimicrobiana en aislados de Staphylococcus. Aplicación al ámbito agroalimentario y en salud pública stars

Resumo

Bacteriocins have been considered an effective tool to address the problem of antibiotic resistance. Staphylococcus is normally found as commensal microorganisms of the nasal and skin microbiota of animals and humans, although some species can be opportunistic pathogens, especially S. aureus. Currently, Staphylococcus is gaining attention as a bacteriocin-producing genus, especially coagulase-negative species. In this work, the antimicrobial activity (AA) of 1205 staphylococcal isolates, both coagulase-positive (CoPS, n=284) and -negative (CoNS, n=921), from 5 different origins and 29 different species against indicator bacteria of public health and food safety relevance, including multi-resistant (MDR) and zoonotic bacteria, were by the spot-on-lawn method. Thus, 92 antimicrobial-producing staphylocci (Bac+ isolates) were detected from various sources (nº of producing isolates/nº of tested isolates): humans (24/83), wild-life animals (34/662), pets (15/50), food (17/278) and environment (2/132). Notably, AA was only detected against Gram-positive (G+) indicator bacteria. Then, 37 Bac+ staphylococci were selected for characterisation based on their species, origin, phenotypic/genotypic characteristics, and inhibitory activity profile. AA was assessed in cell-free supernatants (CFS) and butanol extracts (BT). Nine out of 37 (24%) staphylococci showed AA in CFS while more than 90% of BT extracts revealed AA against at least one of the indicators tested. Moreover, the bacteriocin micrococcin P1 (MP1) was identified by whole genome sequencing (WGS) and mass spectrometry, in 3 CoNS recovered from meat and water, from species infrequently implicated in infections (2 S. sciuri, 1 S. hominis) and 1 SCoP (S. aureus) from surface water. This bacteriocin showed high activity (both in CFS and BT) against indicators of many different genera and species of G+ bacteria, some of them of great relevance in biomedicine, veterinary medicine, and food safety. In addition, relevant bacteriocins mainly lanthipeptides were detected including epilancin 15X, BSA and bacCH91 (previously described in the literature) and other unknown lanthipeptides identified in three CoNS isolates from food. On the other hand, WGS studies allowed the detection of Class II bacteriocin gene clusters such as bacSp2222 (2 S. pseudintermedius, 1 human/1 dog), lactococcin 972 present in high frequency in this collection; circular bacteriocins in 3 producing isolates (2 CoNS and 1 CoPS) and lugdunin, a non-ribosomal antimicrobial peptide detected in 2 S. lugdunensis isolates (recovered from human). As for MP1-producing staphylococci, it is to highlight the antimicrobial activity detected against methicillin-resistant S. aureus (MRSA) both in competition studies as well as their MP1-prepurified extracts (BT), which showed potent inhibitory and bacteriostatic activity in liquid medium and in combination with antibiotics (synergy with clindamycin, oxacillin, and chloramphenicol) has been demonstrated. On the other hand, the potential of antimicrobial-producing staphylococci in the modulation of the nasotracheal microbiota has been evaluated using the stork model. For this purpose, the AA of the Staphylococcus was analysed against the G+ isolates present in the same sample and against a representative collection of G+ bacteria from the nasotracheal microbiota of storks. It is worth mentioning the detection of 9 isolates, especially CoNS (n=7), which are of great interest due to their AA and their potential for microbial modulation. Although further characterisation and bacteriocin purification is necessary, this work opens the door to the interest of CoNS isolates producing bacteriocins or extracts in the modulation of the nasal microbiota as a novel application in the prevention of diseases caused by relevant pathogens such as MRSA. Finally, in this thesis, the antimicrobial activity of 15 bacteriocin-producing staphylococcal isolates was studied against 27 pathogenic microorganisms used as indicators and distributed in categories based on their potential areas of application: a) mastitis and dairy industry; b) poultry pathogens and zoonotic agents; c) swine zoonoses; d) food safety; e) aquaculture; f) wine industry; and g) mushroom cultivation. These studies showed potent AA against G+ indicators (≥60% of the indicators were inhibited) while no inhibitory capacity was detected against G- indicators. It is worth noting the increase observed in the AA of the BT extracts, compared with Spot-on-lawn and CFS, both considering the percentage of inhibited indicators and the intensity of the halos. On the other hand, possible bacterial combinations have been identified for synergic applications against a specific pathogen. Based on all the above, the results of hemolysis, gelatinase activity, antibiotic resistance and virulence of the bacteriocin-producing staphylococcal isolates included in this work, the three MP1-producing CoNS isolates can be proposed as potential candidates for future applications.