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Mayo B.,Institute Productos Lacteos Of Asturias Ipla Csic | Rachid C.T.C.C.,Federal University of Rio de Janeiro | Alegria A.,Institute Productos Lacteos Of Asturias Ipla Csic | Leite A.M.O.,Federal University of Rio de Janeiro | And 2 more authors.
Current Genomics | Year: 2014

Understanding the Maxam-Gilbert and Sanger sequencing as the first generation, in recent years there has been an explosion of newly-developed sequencing strategies, which are usually referred to as next generation sequencing (NGS) techniques. NGS techniques have high-throughputs and produce thousands or even millions of sequences at the same time. These sequences allow for the accurate identification of microbial taxa, including uncultivable organisms and those present in small numbers. In specific applications, NGS provides a complete inventory of all microbial operons and genes present or being expressed under different study conditions. NGS techniques are revolutionizing the field of microbial ecology and have recently been used to examine several food ecosystems. After a short introduction to the most common NGS systems and platforms, this review addresses how NGS techniques have been employed in the study of food microbiota and food fermentations, and discusses their limits and perspectives. The most important findings are reviewed, including those made in the study of the microbiota of milk, fermented dairy products, and plant-, meat- and fishderived fermented foods. The knowledge that can be gained on microbial diversity, population structure and population dynamics via the use of these technologies could be vital in improving the monitoring and manipulation of foods and fermented food products. They should also improve their safety. © 2014 Bentham Science Publishers.


Rodriguez-Rubio L.,Institute Productos Lacteos Of Asturias Ipla Csic | Martinez B.,Institute Productos Lacteos Of Asturias Ipla Csic | Rodriguez A.,Institute Productos Lacteos Of Asturias Ipla Csic | Donovan D.M.,U.S. Department of Agriculture | Garcia P.,Institute Productos Lacteos Of Asturias Ipla Csic
Applied and Environmental Microbiology | Year: 2012

Virion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against live S. aureus cells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysed S. aureus cells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and human S. aureus strains, the methicillin-resistant S. aureus (MRSA) strain N315, and human Staphylococcus epidermidis strains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections. © 2012, American Society for Microbiology.


Gutierrez D.,Institute Productos Lacteos Of Asturias Ipla Csic | Martinez B.,Institute Productos Lacteos Of Asturias Ipla Csic | Rodriguez A.,Institute Productos Lacteos Of Asturias Ipla Csic | Garcia P.,Institute Productos Lacteos Of Asturias Ipla Csic
BMC Genomics | Year: 2012

Background: Staphylococcus epidermidis is a commensal bacterium but can colonize the hospital environment due to its ability to form biofilms favouring adhesion to host tissues, medical devices and increasing resistance to antibiotics. In this context, the use of phages to destroy biofilms is an interesting alternative.Results: The complete genomes of two Staphylococcus epidermidis bacteriophages, vB_SepiS-phiIPLA5 and vB_SepiS-phiIPLA7, have been analyzed. Their genomes are 43,581 bp and 42,123 bp, and contain 67 and 59 orfs. Bioinformatic analyses enabled the assignment of putative functions to 36 and 29 gene products, respectively, including DNA packaging and morphogenetic proteins, lysis components, and proteins necessary for DNA recombination, regulation, modification and replication. A point mutation in vB_SepiS-phiIPLA5 lysogeny control-associated genes explained its strictly lytic behaviour. Comparative analysis of phi-IPLA5 and phi-IPLA7 genome structure resembled those of S. epidermidis φ{symbol}PH15 and φ{symbol}CNPH82 phages. A mosaic structure of S. epidermidis prophage genomes was revealed by PCR analysis of three marker genes (integrase, major head protein and holin). Using these genes, high prevalence (73%) of phage DNA in a representative S. epidermidis strain collection consisting of 60 isolates from women with mastitis and healthy women was determined. Putative pectin lyase-like domains detected in virion-associated proteins of both phages could be involved in exopolysaccharide (EPS) depolymerization, as evidenced by both the presence of a clear halo surrounding the phage lysis zone and the phage-mediated biofilm degradation.Conclusions: Staphylococcus epidermidis bacteriophages, vB_SepiS-phiIPLA5 and vB_SepiS-phiIPLA7, have a mosaic structure similar to other widespread S. epidermidis prophages. Virions of these phages are provided of pectin lyase-like domains, which may be regarded as promising anti-biofilm tools. © 2012 Gutiérrez et al.; licensee BioMed Central Ltd.


Gutierrez D.,Institute Productos Lacteos Of Asturias Ipla Csic | Ruas-Madiedo P.,Institute Productos Lacteos Of Asturias Ipla Csic | Martinez B.,Institute Productos Lacteos Of Asturias Ipla Csic | Rodriguez A.,Institute Productos Lacteos Of Asturias Ipla Csic | Garcia P.,Institute Productos Lacteos Of Asturias Ipla Csic
PloS one | Year: 2014

Staphylococcal biofilms are a major concern in both clinical and food settings because they are an important source of contamination. The efficacy of established cleaning procedures is often hindered due to the ability of some antimicrobial compounds to induce biofilm formation, and to the presence of persister cells, a small bacterial subpopulation that exhibits multidrug tolerance. Phage lytic enzymes have demonstrated antimicrobial activity against planktonic and sessile bacteria. However, their ability to lyse and/or select persister cells remains largely unexplored so far. In this work, the lytic activity of the endolysin LysH5 against Staphylococcus aureus and Staphylococcus epidermidis biofilms was confirmed. LysH5 reduced staphylococcal sessile cell counts by 1-3 log units, compared with the untreated control, and sub-inhibitory concentrations of this protein did not induce biofilm formation. LysH5-surviving cells were not resistant to the lytic activity of this protein, suggesting that no persister cells were selected. Moreover, to prove the lytic ability of LysH5 against this subpopulation, both S. aureus exponential cultures and persister cells obtained after treatment with rifampicin and ciprofloxacin were subsequently treated with LysH5. The results demonstrated that besides the notable activity of endolysin LysH5 against staphylococcal biofilms, persister cells were also inhibited, which raises new opportunities as an adjuvant for some antibiotics.


Alegria A.,Polish Academy of Sciences | Alegria A.,Institute Productos Lacteos Of Asturias Ipla Csic | Szczesny P.,Polish Academy of Sciences | Szczesny P.,University of Warsaw | And 3 more authors.
Applied and Environmental Microbiology | Year: 2012

Oscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g., Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, and Enterococcus, identified by all three methods, other, subdominant bacteria belonging to the families Bifidobacteriaceae and Moraxellaceae (mostly Enhydrobacter), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures. © 2012, American Society for Microbiology.


Delgado S.,Institute Productos Lacteos Of Asturias Ipla Csic | Cabrera-Rubio R.,Centro Superior Of Investigacion En Salud Publica Csisp | Mira A.,Centro Superior Of Investigacion En Salud Publica Csisp | Suarez A.,Hospital Of Cabuenes | Mayo B.,Institute Productos Lacteos Of Asturias Ipla Csic
Microbial Ecology | Year: 2013

Stomach mucosa biopsies and gastric juices samples of 12 healthy persons were analysed by culturing in selective- and non-selective-rich media. Microbial DNA from four mucosal samples was also amplified by nested PCR using universal bacterial primers, and the 16S rDNA amplicons pyrosequenced. The total number of cultivable microorganisms recovered from the samples ranged from 102 to 104 cfu/g or ml. The isolates were identified at the species level by PCR amplification and sequencing of the 16S rDNA. Isolates belonged mainly to four genera; Propionibacterium, Lactobacillus, Streptococcus and Staphylococcus. A total of 15,622 high-quality 16S rDNA sequence reads were obtained by pyrosequencing from the four mucosal samples. Sequence analysis grouped the reads into 59 families and 69 genera, revealing wide bacterial diversity. Considerable differences in the composition of the gastric microbiota were observed among the subjects, although in all samples the most abundant operational taxonomic units belonged to Streptococcus, Propionibacterium and Lactobacillus. Comparison of the stomach microbiota with that present in other parts of the human gastrointestinal tract revealed distinctive microbial communities. This is the first study in which a combination of culture and culture-independent techniques has been used to explore the bacterial diversity of the human stomach. © 2013 Springer Science+Business Media New York.


Garcia P.,Institute Productos Lacteos Of Asturias Ipla Csic | Martinez B.,Institute Productos Lacteos Of Asturias Ipla Csic | Rodriguez L.,Institute Productos Lacteos Of Asturias Ipla Csic | Rodriguez A.,Institute Productos Lacteos Of Asturias Ipla Csic
International Journal of Food Microbiology | Year: 2010

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. © 2010 Elsevier B.V.


Ladero V.,Institute Productos Lacteos Of Asturias Ipla Csic | Fernandez M.,Institute Productos Lacteos Of Asturias Ipla Csic | Cuesta I.,Institute Productos Lacteos Of Asturias Ipla Csic | Alvarez M.A.,Institute Productos Lacteos Of Asturias Ipla Csic
Food Microbiology | Year: 2010

Tyramine is the most abundant biogenic amine in fermented dairy products, in which it is produced through the microbial enzymatic decarboxylation of tyrosine. This activity has been detected in a variety of lactic acid bacteria mainly belonging to the genera Enterococcus and Lactobacillus. This paper describes a culture-independent qPCR method, based on the specific amplification of the tdc gene, for the detection, quantification and identification of bacteria with the ability to produce tyramine. This method was found to be specific and to show a wide dynamic range, thus allowing the quantification of these tdc+ bacterial groups among the complex microbiota of cheese. tdc qPCR was used to follow the development of tdc+ microbiota during the manufacture of a blue-veined cheese (Cabrales) made from raw milk. In this type of cheese, tdc+ enterococci seem to be responsible for the high concentrations of tyramine detected. The method was also used to identify and quantify tdc+ enterococci and lactobacilli in 18 commercially available cheeses. Different types and numbers of these microorganisms were found. Their relationships with the concentration of tyramine and technological factors are discussed. © 2010 Elsevier Ltd.


Garcia P.,Institute Productos Lacteos Of Asturias Ipla Csic | Rodriguez L.,Institute Productos Lacteos Of Asturias Ipla Csic | Rodriguez A.,Institute Productos Lacteos Of Asturias Ipla Csic | Martinez B.,Institute Productos Lacteos Of Asturias Ipla Csic
Trends in Food Science and Technology | Year: 2010

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.


Bueno E.,Institute Productos Lacteos Of Asturias Ipla Csic | Garcia P.,Institute Productos Lacteos Of Asturias Ipla Csic | Martinez B.,Institute Productos Lacteos Of Asturias Ipla Csic | Rodriguez A.,Institute Productos Lacteos Of Asturias Ipla Csic
International Journal of Food Microbiology | Year: 2012

Bacteriophages are regarded as natural antibacterial agents in food since they are able to specifically infect and lyse food-borne pathogenic bacteria without disturbing the indigenous microbiota. Two Staphylococcus aureus obligately lytic bacteriophages (vB_SauS-phi-IPLA35 and vB_SauS-phi-SauS-IPLA88), previously isolated from the dairy environment, were evaluated for their potential as biocontrol agents against this pathogenic microorganism in both fresh and hard-type cheeses. Pasteurized milk was contaminated with S. aureus Sa9 (about 106CFU/mL) and a cocktail of the two lytic phages (about 106PFU/mL) was also added. For control purposes, cheeses were manufactured without addition of phages. In both types of cheeses, the presence of phages resulted in a notorious decrease of S. aureus viable counts during curdling. In test fresh cheeses, a reduction of 3.83log CFU/g of S. aureus occurred in 3h compared with control cheese, and viable counts were under the detection limits after 6h. The staphylococcal strain was undetected in both test and control cheeses at the end of the curdling process (24h) and, of note, no re-growth occurred during cold storage. In hard cheeses, the presence of phages resulted in a continuous reduction of staphylococcal counts. In curd, viable counts of S. aureus were reduced by 4.64log CFU/g compared with the control cheeses. At the end of ripening, 1.24log CFU/g of the staphylococcal strain was still detected in test cheeses whereas 6.73log CFU/g was present in control cheeses. Starter strains were not affected by the presence of phages in the cheese making processes and cheeses maintained their expected physico-chemical properties. © 2012 Elsevier B.V.

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