Characterization of beer-spoiling Lactobacillus brevis and their associated bacteriophages

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Feyereisen, Marine
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University College Cork
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Certain bacteria have acquired the ability to survive and even grow in beer, despite the antimicrobial nature of this harsh liquid environment. Bacterial growth in beer is often attributed to lactic acid bacteria (LAB) that are found in many environments, yet are commonly associated with plant material. While most LAB species bestow positive attributes to foods and food fermentations, others have the potential to survive and even thrive in inhospitable environments, including beer. Lactobacillus brevis strains are among the most frequently encountered LAB isolates in spoiled beer. In finished beer products, the presence of Lb. brevis is considered undesirable as they typically impart undesirable organoleptic properties on the product including off-flavors, odors and changes in the physical appearance and viscosity. The goal of the current study was to identify and characterize the molecular players that allow certain Lb. brevis to grow in and spoil in beer. It was also aimed to isolate phages that infect strains of Lb. brevis in order to assess their potential application in the brewing industry for the purpose of preventing or diminishing bacterial spoilage. The findings described in this thesis have improved our understanding of Lb. brevis as a diverse species and as a beer-spoiling microorganism. The genomic features of 19 Lb. brevis strains (encompassing the genomes of six Lb. brevis strains sequenced in this study and thirteen Lb. brevis strains available in public databases) were evaluated in a comparative genome analysis of the species, paying particular attention to evolutionary aspects and adaptation to beer. Moreover, novel molecular players were identified revealing the importance of chromosomal genes for general stress response (pH tolerance), and the relevance of plasmid-encoded genes for beer-specific stress response (such as tolerance to hop compounds). The response of beer-spoiling Lb. brevis strains to stress factors associated with beer was assessed using a transcriptomic approach comparing growth in nutritive media with or without the imposition of various beer-associated stressors including ethanol, low pH and hops. This analysis allowed the identification of a chromosomal gene encoding a presumed manganese transporter involved in low pH tolerance in Lb. brevis. Furthermore, the importance of plasmids for beer-spoiling Lb. brevis strains was investigated resulting in the identification of a plasmid-encoded putative glycosyltransferase involved in hop tolerance and thus growth in beer. Furthermore, to alleviate the problem of beer spoilage associated with Lb. brevis, the possibility of employing lytic phages capable of infecting such strains can be envisaged as a bio-sanitation approach. This thesis reports on the isolation of five Lb. brevis-infecting virulent phages, which were shown to exhibit a high level of genetic and morphological diversity. Interestingly, certain phage isolates displayed activity against Lb. brevis beer-spoiling strains preventing them from growing optimally, thus providing a phage-mediated approach to control bacterial spoilage of beer. Moreover, the incidence of prophages among Lb. brevis strains was studied in order to understand their potential benefit for their bacterial carrier. Prophage-encoded phage resistance systems such as abortive infection system (Abi), confer to the host resistance to phage infection, thus increasing its overall fitness. Two adjacent genes encoding an Abi system were identified on the prophage sequence of a beer-spoiling Lb. brevis strain. This Abi system exhibits activity against phages infecting Lb. brevis and Lactococcus lactis strains. The presence of such prophage-encoded systems highlights the importance of temperate phages for Lb. brevis strains and, in some cases, may explain their resistance to phage infection. With the study and characterization of virulent and temperate Lb. brevis phages, we have significantly extended our knowledge on Lb. brevis phages. Furthermore, it has provided novel insights into the diversity of Lb. brevis phages and their potential application as a bio-sanitation tool in the beer brewing industry.
Lactobacillus brevis , Lactic acid bacteria , Beer , Beer spoilage , Phage , Fermentation
Feyereisen, M. 2019. Characterization of beer-spoiling Lactobacillus brevis and their associated bacteriophages. PhD Thesis, University College Cork.
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