Actividades antimicrobianas derivadas de fagos y su aplicación como bioconservantes alimentarios

Comunicación presentada en el XXII Congreso Nacional SEM 2009, celebrado en Almeria del 21 al 24 de septiembre de 2009.Staphylococcus aureus es un importante patógeno asociados a intoxicaciones de tipo alimentario, y a su vez, uno de los principales agentes causantes de la mastitis en el ganado vacuno.Peer reviewe

Synergy between the phage endolysin LysH5 and nisin to kill Staphylococcus aureus in pasteurized milk

Phage-encoded endolysins are recently considered as new biocontrol tools to inhibit pathogens in food. In this work, we have studied the ionic requirements for optimal lytic activity of LysH5, the endolysin encoded by the staphylococcal bacteriophage phi-SauS-IPLA88. LysH5 activity was inhibited by the presence of Mn++ and Zn++ and enhanced by Ca++, Mg++ and NaCl. When LysH5 was combined with nisin, a bacteriocin currently used as a biopreservative in food, a strong synergistic effect was observed. The Minimum Inhibitory Concentrations of nisin and LysH5 were reduced 64- and 16-fold, respectively, as determined in checkerboard microtitre tests. In addition, nisin enhanced 8-fold the lytic activity of LysH5 on cell suspensions. The synergy observed in vitro was confirmed in challenge assays in pasteurized milk contaminated with S. aureus Sa9. Clearance of the pathogen was only achieved by the combined activity of both antimicrobials. As far as we know, this is the first study that exploits the possibilities of hurdle technology combining a phage-encoded endolysin and the bacteriocin nisin for efficient S. aureus inhibition in milk.This research study was supported by grant AGL2009-13144-C02-01 from the Ministry of Science and Innovation of Spain and IB08-052 from FICYT (Regional Government of Asturias).Peer reviewe

Is single layer graphene a promising anode for sodium-ion batteries?

In an attempt to find an adequate carbon material to achieve a successful reversible adsorption of Na+ ions, single layer graphene, is experimentally investigated in this work, for the first time, as anode for sodium-ion batteries. To this end, single layer graphene that was grown on copper foil by chemical vapor deposition was subjected to extended galvanostatic cycling and to cyclic voltammetry in the potential range of 0-2.8 V versus Na/Na+. Regardless of the current density and electrolyte formulation used, the amount of Na+ ions adsorbed/desorbed reversibly per surface area (specific reversible cell capacity) was very modest and comparable to that obtained with bare copper electrodes of reference, thus suggesting that the reversible capacity of the single layer graphene electrode is mostly due to the electrochemical response of the copper substrate. These experimental results clearly agree with recent theoretical calculations showing that the adsorption of Na+ ions on the surface of single layer graphene is energetically unfavourable unless that surface includes significant defects density.Financial support from IBERDROLA FOUNDATION (www.fundacioniberdrola.org, Projects 2014-2015) and the Spanish Ministry of Economy and Competitiveness MINECO (under Projects ENE2011-28318-CO-02 and ENE2014-52189-C2-2-R) is gratefully acknowledged. A. Ramos and N. Cuesta, respectively, thank the Spanish Research Council for Scientific Research (CSIC) for a JAE-Doc contract, co-funded by the European Social Fund (ESF), and the Spanish Ministry of Economy and Competitiveness (MINECO) for a pre-doctoral grant (BES-2012-052711).Peer reviewe

Bacteriophages: The Enemies of Bad Bacteria Are Our Friends!

Some bacteria can enter the human body and make people ill. Usually, these diseases can be cured by antibiotics, but sometimes bacteria are resistant to them, meaning that the antibiotics do not kill the bacteria. In these cases, bacteria become very dangerous. Bacteriophages are viruses that infect bacteria but are harmless to humans. To reproduce, they get into a bacterium, where they multiply, and finally they break the bacterial cell open to release the new viruses. Therefore, bacteriophages kill bacteria. Here, we explain how bacteriophages can be used to treat infectious diseases or to remove bacteria from other places where they are unwanted.Peer reviewedPeer Reviewe

'Artilysation' of endolysin λSa2lys strongly improves its enzymatic and antibacterial activity against streptococci

Endolysins constitute a promising class of antibacterials against Gram-positive bacteria. Recently, endolysins have been engineered with selected peptides to obtain a new generation of lytic proteins, Artilysins, with specific activity against Gram-negative bacteria. Here, we demonstrate that artilysation can also be used to enhance the antibacterial activity of endolysins against Gram-positive bacteria and to reduce the dependence on external conditions. Art-240, a chimeric protein of the anti-streptococcal endolysin λSa2lys and the polycationic peptide PCNP, shows a similar species specificity as the parental endolysin, but the bactericidal activity against streptococci increases and is less affected by elevated NaCl concentrations and pH variations. Time-kill experiments and time-lapse microscopy demonstrate that the killing rate of Art-240 is approximately two-fold higher compared to wildtype endolysin λSa2lys, with a reduction in viable bacteria of 3 log units after 10 min. In addition, lower doses of Art-240 are required to achieve the same bactericidal effect.This research study was supported by grants AGL2012-40194-C02-01 (Ministry of Science and Innovation, Spain), FEDER founds and GRUPIN14-139 (Program of Science, Technology and Innovation 2013–2017, Principado de Asturias, Spain), bacteriophage network FAGOMA and research grant 1.5.171.15N of the Research Foundation – Flanders (FWO). DG was a fellow of the Ministry of Science and Innovation, Spain. LR-R was a FWO Pegasus Marie Curie Fellow. PG, BM, RL and AR are members of the FWO Vlaanderen funded “Phagebiotics” research community (WO.016.14).Peer Reviewe

Reduced Binding of the Endolysin LysTP712 to Lactococcus lactis ΔftsH Contributes to Phage Resistance

Absence of the membrane protease FtsH in Lactococcus lactis hinders release of the bacteriophage TP712. In this work we have analyzed the mechanism responsible for the non-lytic phenotype of L. lactis ΔftsH after phage infection. The lytic cassette of TP712 contains a putative antiholin–pinholin system and a modular endolysin (LysTP712). Inducible expression of the holin gene demonstrated the presence of a dual start motif which is functional in both wildtype and L. lactis ΔftsH cells. Moreover, simulating holin activity with ionophores accelerated lysis of wildtype cells but not L. lactis ΔftsH cells, suggesting inhibition of the endolysin rather than a role of FtsH in holin activation. However, zymograms revealed the synthesis of an active endolysin in both wildtype and L. lactis ΔftsH TP712 lysogens. A reporter protein was generated by fusing the cell wall binding domain of LysTP712 to the fluorescent mCherry protein. Binding of this reporter protein took place at the septa of both wildtype and L. lactis ΔftsH cells as shown by fluorescence microscopy. Nonetheless, fluorescence spectroscopy demonstrated that mutant cells bound 40% less protein. In conclusion, the non-lytic phenotype of L. lactis ΔftsH is not due to direct action of the FtsH protease on the phage lytic proteins but rather to a putative function of FtsH in modulating the architecture of the L. lactis cell envelope that results in a lower affinity of the phage endolysin to its substrate.This work has been supported by grant BIO2013-46266R (MINECO, Spain). BM, PG, and AR also acknowledge funding by GRUPIN14-139 Plan de Ciencia, Tecnología e Innovación 2013-2017 (Principado de Asturias, Spain) and FEDER EU funds.Peer reviewe

Listeriaphages and coagulin C23 act synergistically to kill Listeria monocytogenes in milk under refrigeration conditions

© 2015 Elsevier B.V. Bacteriophages and bacteriocins are promising biocontrol tools in food. In this work, two Listeria bacteriophages, FWLLm1 and FWLLm3, were assessed in combination with the bacteriocin coagulin C23 to inhibit Listeria monocytogenes. Preliminary results under laboratory conditions demonstrated that both antimicrobials act synergistically when they were applied in suboptimal concentrations. The combined approach was further assessed in milk contaminated with 5×104CFU/ml L. monocytogenes 2000/47 and stored at 4°C for 10days. When used alone, phage FWLLm1 added at 5×106PFU/ml, FWLLm3 at 5×105PFU/ml and coagulin C23 at 584AU/ml kept L. monocytogenes 2000/47 counts lower than the untreated control throughout storage. However, when used in combination, inhibition was enhanced and in the presence of FWLLm1 and coagulin C23, L. monocytogenes 2000/47 counts were under the detection limits (less than 10CFU/ml) from day 4 until the end of the experiment. Resistant mutants towards phages and coagulin C23 could be obtained, but cross-resistance was not detected. Mutants resistant to FWLLm3 and coagulin C23 were also recovered from surviving colonies after cold storage in milk which may explain the failure of this combination to inhibit L. monocytogenes. Remarkably, the fraction of resistant mutants isolated from the combined treatment was lower than that from each antimicrobial alone, suggesting that synergy between bacteriocins and phages could be due to a lower rate of resistance development and the absence of cross-resistance.This research study was supported by the mobility grant PRI-AIBNZ-2011-1043 (Ministry of Science and Innovation, Spain) to BM and SPN12-01 (Royal Society of New Zealand) to CB. BM, PG and AR also acknowledge funding by grants BIO2010-17414 and AGL2012-40194-C02-01 (Ministry of Science and Innovation, Spain), and Plan de Ciencia, Tecnología e Innovación 2013–2017 (Principado de Asturias, Spain) and FEDER EU funds GRUPIN14-139. PG, BM and AR are members of the FWO Vlaanderen funded “Phagebiotics” research community (WO.016.14).Peer Reviewe

Phage lytic proteins: Biotechnological applications beyond clinical antimicrobials

Most bacteriophages encode two types of cell wall lytic proteins: endolysins (lysins) and virion-associated peptidoglycan hydrolases. Both enzymes have the ability to degrade the peptidoglycan of Gram-positive bacteria resulting in cell lysis when they are applied externally. Bacteriophage lytic proteins have a demonstrated potential in treating animal models of infectious diseases. There has also been an increase in the study of these lytic proteins for their application in areas such as food safety, pathogen detection/diagnosis, surfaces disinfection, vaccine development and nanotechnology. This review summarizes the more recent developments, outlines the full potential of these proteins to develop new biotechnological tools and discusses the feasibility of these proposals.Peer Reviewe

Phage sensitivity and prophage carriage in Staphylococcus aureus isolated from foods in Spain and New Zealand

Bacteriophages (phages) are a promising tool for the biocontrol of pathogenic bacteria, including those contaminating food products and causing infectious diseases. However, the success of phage preparations is limited by the host ranges of their constituent phages. The phage resistance/sensitivity profile of eighty seven Staphylococcus aureus strains isolated in Spain and New Zealand from dairy, meat and seafood sources was determined for six phages (Φ11, K, ΦH5, ΦA72, CAPSa1 and CAPSa3). Most of the S. aureus strains were sensitive to phage K (Myoviridae) and CAPSa1 (Siphoviridae) regardless of their origin. There was a higher sensitivity of New Zealand S. aureus strains to phages isolated from both Spain (ΦH5 and ΦA72) and New Zealand (CAPSa1 and CAPSa3). Spanish phages had a higher infectivity on S. aureus strains of Spanish dairy origin, while Spanish strains isolated from other environments were more sensitive to New Zealand phages. Lysogeny was more prevalent in Spanish S. aureus compared to New Zealand strains. A multiplex PCR reaction, which detected ΦH5 and ΦA72 sequences, indicated a high prevalence of these prophages in Spanish S. aureus strains, but were infrequently detected in New Zealand strains. Overall, the correlation between phage resistance and lysogeny in S. aureus strains was found to be weak.This research was supported by grants AGL2012-40194-C02-01, PRI-AIBNZ-2011-1043 (Ministry of Science and Innovation, Spain), SPN12-01 (Royal Society of New Zealand), GRUPIN14-139 (FEDER funds and program of Science, Technology and Innovation 2013-2017, Principado de Asturias, Spain). DG is a fellow of the Ministry of Science and Innovation, Spain.Peer reviewe

Role of the pre-neck appendage protein (Dpo7) from phage vB_SepiS-phiIPLA7 as an anti-biofilm agent in staphylococcal species

Staphylococcus epidermidis and Staphylococcus aureus are important causative agents of hospital-acquired infections and bacteremia, likely due to their ability to form biofilms. The production of a dense exopolysaccharide (EPS) matrix enclosing the cells slows the penetration of antibiotic down, resulting in therapy failure. The EPS depolymerase (Dpo7) derived from bacteriophage vB_SepiS-phiIPLA7, was overexpressed in Escherichia coli and characterized. A dose dependent but time independent response was observed after treatment of staphylococcal 24 h-biofilms with Dpo7. Maximum removal (>90%) of biofilm-attached cells was obtained with 0.15 μM of Dpo7 in all polysaccharide producer strains but Dpo7 failed to eliminate polysaccharide-independent biofilm formed by S. aureus V329. Moreover, the pre-treatment of polystyrene surfaces with Dpo7 reduced the biofilm biomass by 53-85% in the 67% of the tested strains. This study supports the use of phage-encoded EPS depolymerases to prevent and disperse staphylococcal biofilms, thereby making bacteria more susceptible to the action of antimicrobials.This research study was supported by grants AGL2012-40194-C02-01 (Ministry of Science and Innovation, Spain), GRUPIN14-139 (Program of Science, Technology and Innovation 2013–2017 and FEDER EU funds, Principado de Asturias, Spain) and bacteriophage network FAGOMA. DG is a fellow of the Ministry of Science and Innovation, Spain. PG, BM, RL, and AR are members of the FWO Vlaanderen funded “Phagebiotics” research community (WO.016.14).Peer Reviewe