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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 reviewed

The original publication is available at www.springerlink.com ; Bacteriophages infecting Staphylococcus epidermidis were isolated by mitomycin C induction. Three distinct phages (vB-SepiS-phiIPLA5, vB-SepiS-phiIPLA6, and vB-SepiS-phiIPLA7)-defined by plaque morphology, structure, virion proteins pattern, DNA restriction bands, and host range-were obtained. One-step growth curves of bacteriophages under optimal growth conditions for S. epidermidis F12 revealed eclipse and latent periods of 5-10 and 10-15 min, respectively, with burst sizes of about 5 to 30 PFU per infected cell. Transmission electron microscopy revealed that the phages were of similar size and belonged to the Siphoviridae family. Phage phi-IPLA7 had the broadest host range infecting 21 out of 65 S. epidermidis isolates. Phage phi-IPLA5 seemed to be a virulent phage probably derived from phi-IPLA6. Phages phi-IPLA5 and phi-IPLA7 exhibited increasing plaques surrounded by a halo that could be indicative of a polysaccharide depolymerase activity. Viable counts, determined during the infection of S. epidermidis F12, confirmed that phi-IPLA5 had a potent lytic capability and reduced S. epidermidis population by 5.67 log units in 8 h of incubation; in the presence of the mixture of phi-IPLA6 and phi-IPLA7, however, a reduction of 2.27 log units was detected © 2010 Springer Science+Business Media, LLC. ; This research study was supported by grants AGL2009-13144-C02-01 from the Ministry of Education of Spain and IB08-052 from FICYT (Regional Government of Asturias). P.G. was a fellow of the Spanish Ministry of Education Ramón y Cajal Research Programme. ; Peer Reviewed

The interest in biopreservation of food has prompted the quest for new natural antimicrobial compounds from different origins. Bacteriocins have been widely recognized as natural food biopreservatives but lastest advances on bateriocin biology have opened new fields to explore. On the contrary, the use of bacteriophages and endolysins has only been considered in the last five years and recent developments have produced promising perspectives. This review provides an overview of the current and foreseen applications of bacteriocins, bacteriophages and phage-encoded endolysins along the food chain and highlights research topics to be addressed in the future. © 2010 Elsevier Ltd. ; The DairySafe group at IPLA-CSIC is financially supported by grants from the Spanish Government BIO2007-65061 and AGL2009-13144-C02-01 and the regional research program FICYTIB08-052. ; Peer Reviewed

The endolysin gene (lysH5) from the genome of the Staphylococcus aureus bacteriophage ΦH5 was cloned in Escherichia coli and characterized. The lysH5 gene encoded a protein (LysH5) whose calculated molecular mass and pI were 53.7 kDa and 8.7, respectively. Comparative analysis revealed that LysH5 significantly resembled other murein hydrolases encoded by staphylococcal phages. The modular organization of LysH5 comprised three putative domains, namely, CHAP (cysteine, histidine-dependent amidohydrolase/peptidase), amidase (l-muramoyl-l-alanine amidase), and SH3b (cell wall recognition). In turbidity reduction assays, the purified protein lysed bovine and human S. aureus, and human Staphylococcus epidermidis strains. Other bacteria belonging to different genera were not affected. The lytic activity was optimal at pH 7.0, 37˚C, and sensitive to high temperatures. The purified protein was able to kill rapidly S. aureus growing in pasteurized milk and the pathogen was not detected after 4 h of incubation at 37˚C. As far as we know, this is the first report to assess the antimicrobial activity of a phage endolysin which might be useful for novel biocontrol strategies in dairying. ; This research study was supported by grants AGL2006-03659/ALI from the Ministry of Education of Spain and PC06-009 from FICYT (Regional Government of Asturias). P.G. is a fellow of the Spanish Ministry of Education Ramón y Cajal Research Programme. J.O. is a fellow of the Spanish Ministry of Education (I3P Programme- CSIC). ; Peer reviewed

Bacteria found in diverse ecosystems grow in a community of aggregated cells that favors their survival and colonization. Different extracellular polymeric substances are used to entrap this multispecies community forming a biofilm, which can be associated to biotic and abiotic surfaces. This widespread and successful way of bacterial life, however, can lead to negative effects for human activity since many pathogen and spoiling bacteria form biofilms which are not easy to eradicate. Therefore, the search for novel anti-biofilm bio-active molecules is a very active research area for which simple, reliable, and fast screening methods are demanded. In this work we have successfully validated an impedance-based method, initially developed for the study of adherent eukaryotic cells, to monitor the formation of singlespecies biofilms of three model bacteria in real time. The xCelligence real time cell analyzer (RTCA) equipment uses specific microtiter E-plates coated with gold-microelectrodes that detect the attachment of adherent cells, thus modifying the impedance signal. In the current study, this technology allowed the distinction between biofilm-producers and non-producers of Staphylococcus aureus and Staphylococcus epidermidis, as well as the formation of Streptococcus mutans biofilms only when sucrose was present in the culture medium. Besides, different impedance values permitted discrimination among the biofilm-producing strains tested regardless of the nature of the polymeric biofilm matrix. Finally, we have continuously monitored the inhibition of staphylococcal biofilm formation by the bacteriophage phi-IPLA7 and the bacteriophage-encoded endolysin LysH5, as well as the removal of a preformed biofilm by this last antimicrobial treatment. Results observed with the impedance-based method showed high correlation with those obtained with standard approaches, such as crystal violet staining and bacteria enumeration, as well as with those obtained upon other abiotic surfaces (polystyrene and stainless steel). Therefore, this RTCA technology opens new opportunities in the biofilm research arena and its application could be further explored for other bacterial genera as well as for different bio-active molecules. ; This work was financed by the FEDER European Union funds through the projects AGL2015-64901-R and AGL201565673-R from the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad MINECO) and through the grants EQUIP11 and GRUPIN14-139 (Program of Science, Technology and Innovation, Principado de Asturias). DG and CH-C hold FPI-scholarships of MINECO. ; Peer Reviewed

The recent boom in phage therapy and phage biocontrol requires the design of suitable cocktails of genetically different bacteriophages. The current methods for typing phages need significant quantities of purified DNA, may require a priori genetic information and are cost and time consuming. We have evaluated the randomly amplified polymorphic DNA (RAPD)-PCR technique to produce unique and reproducible band patterns from 26 different bacteriophages infecting Staphylococcus epidermidis, Staphylococcus aureus, Lactococcus lactis, Escherichia coli, Streptococcus thermophilus, Bacillus subtilis and Lactobacillus casei bacterial strains. Initially, purified DNA and phage suspensions of seven selected phages were used as a template. The conditions that were found to be optimal 8 μM of 10-mer primers, 3 μM magnesium oxalacetate and 5% dimethyl sulfoxide. The RAPD genomic fingerprints using a phage titer suspension higher than 109 PFU mL−1 were highly reproducible. Clustering by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm correlated largely with genetically different phages infecting the same bacterial species, although closely related phages with a similar DNA restriction pattern were indistinguishable. The results support the use of RAPD-PCR for quick typing of phage isolates and preliminary assessment of their genetic diversity bypassing tedious DNA purification protocols and previous knowledge of their sequence. ; This work was supported by grants AGL2009-13144-C02-01 from the Ministry of Education of Spain, IB08-052 from FICYT (Regional Government of Asturias) and PIE200970I090 (CSIC, Spain). ; Peer reviewed

Throughout history, humans have consistently developed strategies to prevent food-associated illnesses. However, despite our multiple technological advances, food safety is still an issue of concern. Moreover, there is a demand for gaining access to less processed and naturally preserved food. Food biopreservation, understood as the use of natural antimicrobials already present in food with a long history of safe consumption, is seen as a plausible strategy to reduce the intensity of current preservation technologies (e.g., presence of chemically synthesized food preservatives). In that sense, the combined use of several antimicrobial strategies, known as hurdle technology, has been often chosen as a means to improve the efficacy of food biopreservation. This review intends to summarize the most recent examples of the combined use of bacteriocins and bacteriophages to extend food shelf-life and reduce the risks associated with the presence of foodborne bacteria along the food chain. However, while the efficacy of bacteriocins has been extensively documented, bacteriophages have only started to be assessed as potential food biopreservatives more recently. Within this context, we would like to consider whether these two types of natural antimicrobials would help each other to overcome bottlenecks in food biopreservation. ; Research at the DairySafe group is funded by grant PID2019-105311RB-I00 (MCIN/ AEI/10.13039/501100011033) to P.G. and A.R., grant PID2020-119697RB-I00 (MCIN/ AEI/10.13039/501100011033) to B.M. and A.R and grant AYUD/2021/52120 (Program of Science, Technology and Innovation 2021–2023 (FICYT and FEDER EU), Principado de Asturias, Spain). C.R is a fellow of the program "Ayudas Severo Ochoa" of the Principado de Asturias, Spain (BP20 006). A.C.D. is a fellow from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No. 813439. ; Peer reviewed

Staphylococcus aureus is considered a priority pathogen due to its increasing acquisition of antibiotic resistance determinants. Additionally, this microbe has the ability to form recalcitrant biofilms on different biotic and inert surfaces. In this context, bacteriophages and their derived lytic proteins may be a forward-looking strategy to help combat staphylococcal biofilms. However, these antimicrobials exhibit individual limitations that may be overcome by combining them with other compounds. This work investigates the combination of a phage-derived lytic protein, CHAPSH3b, and the virulent bacteriophage phiIPLA-RODI. The obtained results show the synergy between both antimicrobials for the treatment of 24-h-old S. aureus biofilms, with greater reductions in viable cell counts observed when phage and lysin are applied together compared to the individual treatments. Time-kill curves and confocal microscopy revealed that the fast antibacterial action of CHAPSH3b reduces the population up to 7 hours after initial exposure, which is subsequently followed by phage predation, limiting regrowth of the bacterial population. Moreover, at least 90% of bacteriophage insensitive mutants are susceptible to the lytic protein. Therefore, CHAPSH3b might help curtail the development of phage resistance during treatment. The combination of the lysin and phiIPLA-RODI also showed promising results in an ex vivo pig skin model of wound infection. Overall, the results of this study demonstrate that the combination of phage-derived lytic proteins and bacteriophages can be a viable strategy to develop improved antibiofilm products. ; This research study was supported by funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No. 813439. In addition, grants IDI/2018/000119 (Program of Science, Technology and Innovation 2018-2020 and FEDER EU, Principado de Asturias, Spain) and EU ANIWHA ERA-NET (BLAAT ID: 67)/PCIN-2017-001 (AEI/FEDER UE) also funded this work. A.C.D. is a fellow from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No. 813439. V.M. and D.G are funded by the Research Foundation–Flanders (FWO) under grant G066919N, awarded to Y.B. and R.L.