The DairySafe (Lactic Ferments and Biopreservation) research group has been closely connected to IPLA-CSIC since the group was created (1990), Dr Ana Rodríguez González having been its coordinator since then.

Biopreservation has been the group’s main line of research over the past 20 years, with a particular focus on developing food preservation strategies based on natural antimicrobials that improve food safety. The antimicrobials studied include bacteriocins produced by lactic acid bacteria, and bacteriophages that infect food pathogens, as well as phage-synthesised lytic proteins . The most significant objectives of this work include exploring the mode of action of these antimicrobials, the consequences of their use, and the possible resistance mechanisms of the target bacteria.

Over this period, we have isolated and characterised bacteriocin-producing lactic bacteria (nisin Z, lacticin 481, coagulin A and lactococcin 972). Producer strains of nisin Z act effectively as protective cultures in cheese production, while lactococcin 972 inhibits cell wall synthesis in lactococci through interaction with lipid II (an intermediate in peptidoglycan synthesis) and generates the two-component CesSR system which controls the cell wall’s immediate response to stress in Lactococcus lactis. Lactococcin 972 is being used in the adaptive evolution of lactococci to obtain more robust strains with improved technological aptitudes that are useful for the food industry. Coagulin A has a high level of activity against Listeria monocytogenes.

Bacteriophages, a natural enemy of bacteria, are a promising alternative to the use of chemical compounds such as biopreservatives and disinfectants in the food industry. The specificity of bacteriophages, which are active against one bacterial species and even against certain strains only, makes them completely harmless to humans, animals and the environment. In light of these insights, our group has been pioneering: the use of phage mixtures against Staphylococcus aureus in milk, curd and cheese; the combined use of phages and bacteriocins in milk to inhibit S. Aureus, and detecting the activity of recombinant phage endolysin in milk. It is worth noting that S. aureus is responsible for a wide range of infections in humans and animals, including toxic food infections caused by the consumption of food contaminated by enterotoxins. Phages and phage-synthesised lytic proteins are effective against S. aureus strains of human and animal origin – even those that are resistant to antibiotics – as well as against Staphylococcus epidermidis. They are equally effective whether the strains are biofilm producers or not. In fact, our group has characterised several phage-derived proteins that have lytic activity against biofilm producers, as well as a protein with polysaccharide depolymerase activity that breaks down the extracellular matrix of biofilms. The group has also characterised the phenotypic and transcriptional responses of biofilms exposed to low doses of bacteriophages and lytic proteins, in order to optimise their doses for different applications.

Our results clearly demonstrate the great potential of bacteriophages and their lytic proteins as alternatives to current antimicrobials and disinfectants for tackling S. aureus and S. Epidermidis infections and contamination, with applications in clinical and veterinary settings, as well as the food industry.

We have recently validated an impedance-based method for monitoring the ability of various pathogenic bacteria to form biofilm (Real Time Cell Analyser, RCTA). The method is quick, reliable and easy to use for several purposes, including analysing biofilm-producing strains, and searching for the antimicrobial compounds which inhibit the formation of this type of structure or which eliminate those that have already been formed.

Current and Future Priorities

Taking the knowledge acquired by the DairySafe group on new antimicrobials as a starting point, our main objective is to use these antimicrobials to eliminate pathogens that are of concern to hospitals. In this context, and due to the urgent need for alternatives to antibiotics, we intend to continue expanding our knowledge of the mechanisms that promote the action of new antimicrobials against antibiotic-resistant bacteria, and also against biofilm producers, given that these structures’ powerful protective effect makes them more difficult to eliminate. Our aim is to develop effective antimicrobials against this type of bacteria, as well as detection systems that enable early diagnosis of infections.

Food Safety

The group has many years of experience in developing food preservation strategies based on natural antimicrobials (biopreservation) that improve food health and hygiene standards, thereby reducing the frequency of foodborne outbreaks. Of these antimicrobials, we have paid particular attention to the bacteriocins produced by lactic acid bacteria (LAB), the bacteriophages that infect food pathogens, and phage-synthesised lytic proteins (endolysins).

Our work on bacteriocins has included designing protective cultures made up of producer strains, which have been successfully used in the production of dairy products.

Natural Antimicrobials: Genetic Regulation and Function

Understanding the mode of action of antimicrobial substances enables us to learn about the molecular mechanisms by which bacteria become resistant to them. Using the Lcn972 bacteriocin as a model, the group has studied both the mode of action and gene expression. This bacteriocin inhibits cell division: a completely different mode of action to the other bacteriocins produced by LAB. Lcn972 is the first bacteriocin described that interacts with Lipid II, without having been modified after transduction. It also generates the two-component CesSR system, which controls the cell wall’s immediate response to stress in Lactococcus lactis. Lnc972 is being used to study the adaptive evolution of L. lactis under conditions of cell-wall stress. This food-grade strategy is focused on obtaining more robust strains with improved technological aptitudes that are useful for the food industry, as well as strains that can be used as cell factories in the production of enzymes with clinical and food applications.

Phage Therapy and Biocontrol

Bacteriophages and their endolysins are a promising alternative to the use of biocides, including preservatives and disinfectants (food industry) and antibiotics (health sector).

Bacteriophages specifically inhibit bacteria, making them completely harmless to other living beings and the environment. Our group has therefore been pioneering the use of phages and endolysins against Staphylococcus aureus, with the aim of tackling toxic food infections and health-related infections caused by this bacterium. Our phages and some phage-synthesised proteins (endolysins and virion-associated peptidoglycan hydrolases) are also effective against staphylococcus strains of human and animal origin, including Staphylococcus epidermidis and even antibiotic-resistant strains, whether they are biofilm producers or not.

The results obtained by our group clearly demonstrate the great potential of phage-based products for future applications in the clinical, veterinary and food sectors.

• Gutiérrez, D., Adriaenssens, E.M., Martínez, B., Rodríguez, A., Lavigne, R., Kropinski, A.M., García, P. 2014. Three proposed new bacteriophage genera of staphylococcal phages: “3alikevirus”, “77likevirus” and “Phietalikevirus”. Archives of Virology 159: 389-398 (doi 10.1007/s00705-013-1833-1). (A) (Q3). IF: 2.390
• Campelo, A.B., Roces, C., Mohedano, M.L., López, P., Rodríguez, A. and Martínez, B 2014. A bacteriocin gene cluster able to enhance plasmid maintenance in Lactococcus lactis. Microbial Cell Factories 13: 77. (Q1). IF: 4.221
• Diana Gutiérrez, Patricia Ruas-Madiedo, Beatriz Martínez, Ana Rodríguez and Pilar García. (2014). Effective removal of staphylococcal biofilms by the endolysin LysH5. PLoS ONE 9 (9):e107307 (Q1). IF: 3.234
• Rehaiem, A., Ben Belgacem, Z., Edalatian M.R., Martínez, B., Rodríguez, A., Manai, M. and Guerra, N.P. (2014). Assessment of potential probiotic properties and multiple bacteriocin encoding-genes of the technological performing strain Enterococcus faecium Food Control (doi:10.1016/j.foodcont.2013.09.044) (Q1) IF: 2.806
• Rodríguez-Rubio, L., García, P., Rodríguez, A., Billington, C., Hudson, J.A. and Martínez, B. (2015). Listeriaphages and coagulin C23 act synergistically to kill Listeria monocytogenes in milk under refrigeration conditions. International Journal of Food Microbiology 205: 68-72. doi: 10.1016/j.ijfoodmicro.2015.04.007 (Q1) IF: 3.445
• Milioni, B. Martínez, S. Degl’Innocenti, B. Turchi, F. Fratini, D. Cerri, R. Fischetti. (2015). A novel bacteriocin produced by Lactobacillus plantarum LpU4 as a valuable candidate for biopreservation in artisanal raw milk cheese. Dairy Science and Technology 95: 479–494 (Q3) IF: 1.435
• Diana Gutiérrez, DieterVandenheuvel, Beatriz Martínez, Ana Rodríguez, Rob Lavigne and Pilar García. Two phages, phiIPLA-RODI and phiIPLA-C1C, lyse mono- and dual-staphylococcal biofilms. Applied and Environmental Microbiology 81: 3336-3348. doi:10.1128/AEM.03560-14. (Q1) IF: 3.823
• Diana Gutiérrez, Yves Briers, Lorena Rodríguez-Rubio, Beatriz Martínez, Ana Rodríguez, Rob Lavigne and Pilar García. (2015). Role of the pre-neck appendage protein (Dpo7) from phage vB_SepiS-phiIPLA7 as an anti-biofilm agent in staphylococcal species. Frontiers in Microbiology 6: 1315 (doi: 10.3389/fmicb.2015.01315) (Q1) IF: 4.165
• Roces, C., Campelo, A.B., Escobedo, S., Wegmann, U., García, P., Rodríguez, A. and Martínez, B. (2016). Reduced binding of the endolysin LysTP712 to Lactococcus lactis ΔftsH contributes to phage resistance. Frontiers in Microbiology 7: 138 (doi: 10.3389/fmicb.2016.00138) (Q1) IF: 4.076
• Lorena Rodríguez-Rubio, Diana Gutiérrez, David M. Donovan, Beatriz Martínez, Ana Rodríguez and Pilar García. (2016). Phage lytic proteins: biotechnological applications beyond clinical antimicrobials. Critical Reviews in Biotechnology 36 (3): 542-552. (doi:10.3109/07388551.2014.993587). (Q1) IF: 6.542
• Gutiérrez, D., Rodríguez-Rubio, L., García, P., Billington, C., Premarante, A., Rodríguez, A. and Martínez, B. (2016). Phage sensitivity and prophage carriage in Staphylococcus aureus isolated from foods in Spain and New Zealand. International Journal of Food Microbiology 230: 16-20. (Q1) IF: 3.339
• Gutiérrez D, Rodríguez-Rubio L, Martínez B, Rodríguez A and García P (2016) Bacteriophages as Weapons against Bacterial Biofilms in the Food Industry. Frontiers in Microbiology. 7: 825. (doi: 10.3389/fmicb.2016.00825) (Q1) IF: 4.076
• Lorena Rodríguez-Rubio, Wai-Ling Chang, Diana Gutiérrez, Rob Lavigne, Beatriz Martínez, Ana Rodríguez, Sander K. Govers Abram Aertsen, Christine Hirl, Manfred Biebl, Yves Briers, Pilar García. (2016). ‘Artilysation’ of endolysinλSa2lys strongly improves its enzymatic and antibacterial activity against streptococci. Scientific Reports 6: 35382. doi: 10.1038/srep35382. (Q1) IF: 4.259
• Diana Gutiérrez, Claudio Hidalgo-Cantabrana, Ana Rodríguez, Pilar García, Patricia Ruas-Madiedo (2016). Monitoring in Real Time the Formation and Removal of Biofilms from Clinical Related Pathogens Using an Impedance-Based Technology. PLoS ONE 11 (8): e0163966 doi:10.1371/journal.pone.0163966. (Q1) IF: 2.806
• Diana Gutiérrez, Lorena Rodríguez-Rubio, Lucía Fernández, Beatriz Martínez, Ana Rodríguez, Pilar García. (2017). Applicability of commercial phage-based products against Listeria monocytogenes for improvement of food safety in Spanish dry-cured ham and food contact surfaces. Food Control 73: 1474-1482. http:// dx.doi.org /10.1016/j.foodcont.2016.11.007. (Q1) IF: 3.496
• Silvia González, Lucía Fernández, Ana Belén Campelo, Diana Gutiérrez, Beatriz Martínez, Ana Rodríguez, Pilar García. (2017). The behavior of Staphylococcus aureus dual-species biofilms treated with bacteriophage phiIPLA-RODI depends on the accompanying microorganism. Applied and Environmental Microbiology 83(3): e02821. (Q1) IF: 3.807
• Abedon, S.; García, P.; Mullany, P. and Aminov, R. (2017). Editorial: Phage therapy: past, present and future. Frontiers in Microbiology. 8: 981. doi: 10.3389/fmicb.2017.00981 (Q2) IF: 4.076
• Lucía Fernández, Silvia González, Ana Belén Campelo, Beatriz Martínez, Ana Rodríguez and Pilar García. (2017). Low-level predation by lytic phage phiIPLA-RODI promotes biofilm formation and triggers the stringent response in Staphylococcus aureus. Scientific Reports 7, 40965; doi: 10.1038/srep40965 (Q1) IF: 4.259
• Gutiérrez D.; Fernández L.; Martínez B.; Ruas-Madiedo P., García P.; Rodríguez A. (2017). Real-Time Assessment of Staphylococcus aureus Biofilm Disruption by Phage-derived Proteins. Frontiers in Microbiology 8: 1632 (Q2) IF: 4.076
• Fernández, L.; González, S; Campelo, A.B.; Martínez, B.; Rodríguez, A. and García, P. (2017). Downregulation of autolysin-encoding genes by phage-derived lytic proteins inhibits biofilm formation in Staphylococcus aureus. Antimicrobials Agents and Chemotherapy 61 (5): e02724-16. https://doi.org/10.1128/AAC.02724-16 (Q1) IF: 4.302

• Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion associated peptidoglycan hydrolase: fusions, deletions and synergy with LysH5

Inventors: David M. Donovan, Lorena Rodríguez-Rubio, Beatriz Martínez, Ana Rodríguez, Pilar García.
Application No: US13298966
Patent Assignment: 501810442 (6 February 2012)
Priority Country: US
Owner: CSIC – USDA (United States Department of Agriculture)
Priority Date: 17/11/2011
US Patent No 8,986,695, issued on 24/03/2015

• Chimeric protein with high antimicrobial activity

Inventors: Diana Gutiérrez, Beatriz Martínez, Pilar García, Ana Rodríguez, Lorena Rodríguez-Rubio, Rob Lavigne
Application No: PCT/EP2018/082715 (27 November 2018)
Reference Number: EP1641.1323
Country: Spain
Organisation: CSIC