Isolation and Characterization of Lactobacillus brevis Phages (original) (raw)
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Journal of Food Protection, 2011
Beer quality can be compromised by the growth of certain lactobacilli, in particular Lactobacillus brevis and Lactobacillus plantarum, and various strategies have been used to control such bacterial spoilage. Biocontrol by means of bacteriophage is a reemerging approach for the suppression of spoilage bacteria in food and beverage matrices. A virulent phage capable of infecting L. brevis beer-spoilage strains was isolated and morphologically assessed by electron microscopy. The myophage SA-C12 was shown to be stable in beer and capable of controlling the growth of its host, L. brevis strain 56, in commercial beer. The results of this study indicate that bacteriophage-based treatments may be used as an alternative and natural strategy for the control of bacterial contamination of beer.
Biodiversity and Classification of Phages Infecting Lactobacillus brevis
Frontiers in Microbiology
Lactobacillus brevis is a lactic acid bacterium that is known as a food and beverage spoilage organism, and more specifically as a beer-spoiler. Phages of L. brevis have been described, but very limited data is available regarding temperate phages of L. brevis. Temperate phages may exert benefits to the host, while they may also be employed to combat beer spoilage. The current study reports on the incidence of prophage sequences present in nineteen distinct L. brevis genomes. Prophage induction was evaluated using mitomycin C exposure followed by genome targeted-PCR, electron microscopy and structural proteome analysis. The morphological and genome sequence analyses revealed significant diversity among L. brevis prophages, which appear to be dominated by members of the Myoviridae phage family. Based on this analysis, we propose a classification of L. brevis phages into five groups.
J. Am. Soc. Brew. Chem. 69(1): 2011 The aim of this study was to isolate and characterize bacteriophages against Pediococcus and Lactobacillus strains that cause spoilage in brewing processes. A number of beer-spoilage bacteria were isolated from breweries and characterized by 16S rRNA typing. Five distinct P. damnosus phages and four Lactobacillus phages, which lysed both L. brevis and L. paraplantarum, were isolated from municipal sewage and farmyard slurries. A rapid DNA isolation method was used to isolate DNA of sufficient purity for restriction endonuclease digestion from de Man, Rogosa, and Sharpe (MRS) broth. Phages were analyzed using restriction digest and transmission electron microscopy and shown to be in the family Siphoviridae, with genomes ranging in size from 40 to 50 kb. All phages were characterized and shown to be distinct. This study identifies five novel phages against P. damnosus. Phages for this species are very rare in the scientific literature. Four novel phages against L. brevis and L. paraplantarum are also identified. These phages may have an application in the biocontrol of beer-spoilage bacteria.
Bulgarian Journal of Agricultural Science, 2013
The modular evolution of phage genomes makes their classifi cation extremely diffi cult. Still in dairy industry, the identifi cation of the phage species that disturbs the fermentation process is of crucial importance in the selection of a strategy to avoid or eliminate phage infections. Phages attacking Lactobacillus delbrueckii are currently divided into four groups assigned as "a", "b", "c" and "d" which have substantial differences in their virion morphology and DNA homology. Lactobacillus delbrueckii ssp. bulgaricus phage Gb1 was isolated in 2005 from yoghurt production. Restriction fragments of phage Gb1 DNA were cloned into pIL253 in Lactococcus lctis IL1403 and PCR amplifi ed with vector-specifi c primers. The resulting amplicons were sequenced and identities with existing sequence databases were searched. All of the obtained sequences showed homology only to regions within the genome of phages LL-Ku and c5 with levels of nucleotide identity of 80-99%. No signifi cant sequence matches were found with other L. delbrueckii phages. The sequenced fragments from the genome of phage Gb1 were homologous to key genes in phages LL-Ku and c5 encoding a major head protein, the tape-measure protein, tail proteins, the single strand binding protein and a diesterase. As phages LL-Ku and c5 are typical representatives of group "b" L. delbrueckii bacteriophages that do not share DNA homology with L. delbrueckii phages from other groups we could classify phage Gb1 into group "b" L. delbrueckii phages. This is the fi rst report of a bacteriophage from this group isolated in Bulgaria. Further studies of phage Gb1 will facilitate the work on the selection of phage resistant starters and prevention of phage infections in yoghurt production.
Molecular characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages
Applied and environmental microbiology, 2014
In this study, three phages infecting Lactobacillus delbrueckii subsp. bulgaricus, named Ld3, Ld17, and Ld25A, were isolated from whey samples obtained from various industrial fermentations. These phages were further characterized in a multifaceted approach: (i) biological and physical characterization through host range analysis and electron microscopy; (ii) genetic assessment through genome analysis; (iii) mass spectrometry analysis of the structural components of the phages; and (iv), for one phage, transcriptional analysis by Northern hybridization, reverse transcription-PCR, and primer extension. The three obtained phage genomes display high levels of sequence identity to each other and to genomes of the so-called group b L. delbrueckii phages c5, LL-Ku, and phiLdb, where some of the observed differences are believed to be responsible for host range variations.
Applied and Environmental Microbiology, 2014
Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 ؎ 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.
Kelly et al ASBC Beer phages.pdf
J. Am. Soc. Brew. Chem. 69(1): 2011 The aim of this study was to isolate and characterize bacteriophages against Pediococcus and Lactobacillus strains that cause spoilage in brewing processes. A number of beer-spoilage bacteria were isolated from breweries and characterized by 16S rRNA typing. Five distinct P. damnosus phages and four Lactobacillus phages, which lysed both L. brevis and L. paraplantarum, were isolated from municipal sewage and farmyard slurries. A rapid DNA isolation method was used to isolate DNA of sufficient purity for restriction endonuclease digestion from de Man, Rogosa, and Sharpe (MRS) broth. Phages were analyzed using restriction digest and transmission electron microscopy and shown to be in the family Siphoviridae, with genomes ranging in size from 40 to 50 kb. All phages were characterized and shown to be distinct. This study identifies five novel phages against P. damnosus. Phages for this species are very rare in the scientific literature. Four novel phages against L. brevis and L. paraplantarum are also identified. These phages may have an application in the biocontrol of beer-spoilage bacteria.
Journal of Dairy Science, 1997
Lactobacillus delbrueckii ssp. bulgaricus strain CRL 539 was shown to be lysogenic and inducible with mitomycin C. The conditions were determined for an optimal induction of temperate bacteriophage lb539 with mitomycin C as well as the sensitivity of lb539 to physical and chemical agents. Electron microscopy of lysates revealed bacteriophage particles with an isometric head of 47 nm and a noncontractile tail of 159 nm. Phage lb539 was classified within Bradley's B1 phage group and the Siphoviridae family. The host range of lb539 encompassed mainly Lactobacillus delbrueckii ssp. lactis strains; strain LKT (CNRZ 700) was the most sensitive for detection of lb539 lysates induced by mitomycin C. The lb539 genome is a linear, double-stranded DNA molecule of approximately 35 kbp. The presence of submolar fragments in restriction enzyme digests suggests that lb539 DNA may contain a pac site. Dotblot experiments showed that the lb539 genome hybridized with the genomes of phages mv4 and LL-H, which are type phages of group a of L. delbrueckii ssp. phages. Restriction enzyme patterns and morphological features showed lb539 to be distinct from mv4 and LL-H.
Journal of Applied Microbiology, 2004
Aims: Three indigenous Lactobacillus delbrueckii subsp. bulgaricus bacteriophages and their adsorption process were characterized. Methods and Results: Phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed low burst size and short latent periods. A remarkably high sensitivity to pH was also demonstrated. Indigenous phage genomes were linear and double-stranded DNA molecules of approx. 31-34 kbp, with distinctive restriction patterns. Only one phage genome appeared to contain cohesive ends. Calcium ions did not influence phage adsorption, but it was necessary to accelerate cell lysis and improve plaque formation. The adsorption kinetics were similar on viable and nonviable cells, and the adsorption rates were high between 0 and 50°C. SDS and proteinase K treatments did not influence the phage adsorption but mutanolysin and TCA reduced it appreciably. No significant inhibitory effect on phage adsorption was observed for the saccharides tested. This study also revealed the irreversibility of phage adsorption to their hosts. Conclusions, Significance and Impact of the Study: The study increases the knowledge on phages of thermophilic lactic acid bacteria.
Characterization of a new virulent phage (MLC-A) of Lactobacillus paracasei
Journal of dairy science, 2006
A new virulent bacteriophage (MLC-A) was recently isolated in Argentina from a probiotic dairy product containing a strain of Lactobacillus paracasei. Observation of the lysate with an electron microscope revealed bacteriophage particles with an icosahedral capsid of 57 +/- 2 nm; with a collar and a noncontractile tail of 156 +/- 3 nm terminating with a baseplate to which a tail fiber was attached. Therefore, phage MLC-A belongs to the Siphoviridae family. This phage was able to survive the pasteurization process and was resistant to alcohols and sodium hypochlorite (400 mg/kg). Only peracetic acid could inactivate high-titer suspensions of phages in a short time. The maximum rates of phage adsorption to its host cells were obtained at 30 degrees C with a pH between 5 and 7, and in the presence of calcium or magnesium ions. The host range of phage MLC-A encompassed L. paracasei and Lactobacillus casei strains, but it was not able to infect Lactobacillus rhamnosus or Lactobacillus ga...