CORE Metadata, citation and similar papers at core.ac.uk Provided by Cranfield CERES CRANFIELD UNIVERSITY Cranfield Health PhD THESIS VASILIKI A. BLANA QUORUM SENSING: UNDERSTANDING THE ROLE OF BACTERIA IN MEAT SPOILAGE Supervisors: 1st Professor Naresh Magan and 2nd Professor George-John Nychas 2010 ABSTRACT Quorum sensing is a fundamental process to all of microbiology since it is ubiquitous in the bacterial world, where bacterial cells communicate with each other using low molecular weight signal molecules called autoinducers. Despite the fact that quorum sensing regulates numerous bacterial behaviours, very few studies have addressed the role of this phenomenon in foods. The microbial association of beef consists mainly of pseudomonads, Enterobacteriaceae, Brochothrix thermosphacta and lactic acid bacteria as revealed by minced beef samples purchased from retail shops, which fluctuates according to the storage conditions. Certain members of the microbial association, which are considered to produce signal molecules, have been found to be major contributors to meat spoilage. Pseudomonas fragi and Enterobacteriaceae strains, i.e., Hafnia alvei and Serratia liquefaciens are among the most common quorum sensing signal producers recovered from various food environments. N-acyl homoserine lactone (AHL) and autoinducer-2 (AI-2) signal molecules were found to be present in meat stored under different conditions (i.e., temperature and packaging), and correlated with the ephemeral spoilage organisms that comprise the microbial community generally associated with this product. The microbial association was strongly affected by the packaging condition, which consequently had an effect on quorum sensing signals detected throughout storage. The presence of signal molecules was detected in minced beef samples stored aerobically and under modified atmospheres, when pseudomonads and Enterobacteriaceae populations ranged from 107 to 109 CFU g-1, whereas in minced beef stored under modified atmospheres with the presence of volatile compounds of oregano essential oil where i Enterobacteriaceae population was inhibited, no signals were detected. Enterobacteriaceae appeared to be the main AHL producers since the Pseudomonas spp. did not produce detectable AHL signals with the biosensor strains used. Members of the microbial association i.e., Enterobacteriaceae and lactic acid bacteria, which were detected in high populations in minced meat stored aerobically and under modified atmospheres respectively, were capable of producing quorum sensing signal molecules, either AHLs or AI-2. H. alvei and Ser. liquefaciens were the most common identified AHL-producing bacteria, and Leuconostoc spp. was the most common AI-2-producing strain dominating in minced beef packaged under modified atmospheres. AHL-producing bacteria were isolated from all stages of storage, whereas AI-2 producing ones were recovered from relatively high storage temperatures and the initial stages of beef stored at chill temperatures. The presence of microbial quorum sensing signal molecules, obtained from AHL-producing H. alvei strain 718 and AI-2 producing Salmonella enterica subsp. enterica serovar Typhimurium strain 4/74, affected the growth of the spoilage bacteria Pseudomonas fluorescens and Ser. liquefaciens. AHL signal molecules encouraged the growth of both spoilage bacteria tested, while bacterial growth was reduced under low amounts of AI-2 molecules. These data indicate the involvement of quorum sensing signal molecules in modulating the ecology of these bacteria and suggest that they play a role in influencing the rate of spoilage of meat products. ii ACKNOWLEDGEMENTS I would like to express my greatest thanks to my supervisors Prof. Naresh Magan and Prof. George-John Nychas for their advices and support throughout this study. I would like to thank my two colleagues and best friends Anthoula Argyri and Agapi Doulgeraki for working together and supporting me during my study. Finally, I would like to thank my family, my parents Anastasios and Georgia and my brother Nick, for their support and encouragement all these years. iii Table of Contents ABSTRACT .............................................................................................................................i ACKNOWLEDGEMENTS ................................................................................................. iii LIST OF TABLES .............................................................................................................. vii LIST OF FIGURES ............................................................................................................... x ABBREVIATIONS .............................................................................................................xiv Chapter 1: Literature Review and Objectives ................................................ 1 1.1 Quorum Sensing ................................................................................................................ 2 1.1.1 The languages of bacteria ................................................................................................. 3 Quorum sensing in Gram-negative bacteria .................................................................. 3 Quorum sensing in Gram-positive bacteria ................................................................... 4 The universal LuxS language ........................................................................................ 5 1.1.2 Quorum sensing signal molecules ..................................................................................... 7 Autoinducer-1 ............................................................................................................... 8 Autoinducer-2 ............................................................................................................. 10 1.1.3 Methods for detecting quorum sensing signals in foods and bacteria ............................. 10 Autoinducer-1 ............................................................................................................. 11 Autoinducer-2 ............................................................................................................. 15 1.2 Role of Quorum Sensing in meat spoilage ..................................................................... 16 1.2.1 Spoilage of meat ............................................................................................................... 16 Microorganisms of the spoilage association .............................................................. 18 1.2.2 Quorum sensing in meat and meat products .................................................................... 19 1.3 Objectives ......................................................................................................................... 25 Chapter 2: Materials and Methods ................................................................ 27 2.1 Survey of minced beef ..................................................................................................... 28 2.1.1 Minced beef samples ........................................................................................................ 28 2.1.2 Microbiological analysis .................................................................................................. 28 2.1.3 pH measurement ............................................................................................................... 29 2.1.4 Sensory analysis ............................................................................................................... 29 2.1.5 Data analysis .................................................................................................................... 30 2.2 Microbiological analysis of beef meat ............................................................................ 30 2.2.1 Preparation of beef pieces ................................................................................................ 30 2.2.2 Microbiological analysis .................................................................................................. 30 2.2.3 pH measurement ............................................................................................................... 31 2.2.4 Sensory analysis ............................................................................................................... 31 2.2.5 Data analysis .................................................................................................................... 32 2.3 Detection of quorum sensing signals during minced beef spoilage .............................. 32 2.3.1 Bacterial strains and culture conditions .......................................................................... 32 2.3.2 Minced beef samples ........................................................................................................ 34 iv 2.3.3 Microbiological analysis .................................................................................................. 34 2.3.4 pH measurement ............................................................................................................... 35 2.3.5 Preparation of Cell-free Meat Extracts ............................................................................ 35 2.3.6 Preparation of Cell-free Meat Extract from “sterile” meat tissue ................................... 36 2.3.7 Well diffusion assay .........................................................................................................