Impact of carbohydrates and carnocyclin A on growth and gene expression of Listeria monocytogenes

Abstract

L. monocytogenes is the foodborne pathogen that causes listeriosis, which has a high fatality rate of 20 to 30%. L. monocytogenes is often associated with ready-to-eat food products. Therefore, techniques to control growth of this organism are needed for the safety of these foods. Biopreservation techniques such as bacteriocins are being researched as an alternative to chemical preservatives to control L. monocytogenes. Carnocyclin A is one of the bacteriocins (class 1b) that can inhibit L. monocytogenes. The aim of this research was to investigate the impact of carbohydrates on the development of resistance in strains of L. monocytogenes exposed to carnocyclin A in cooked ground beef. To determine the impact of carbohydrates and carnocyclin A, the strains of L. monocytogenes were grown in cooked ground beef supplemented with 3 different carbohydrates (fructose, dextrose and sucrose) with or without carnocyclin A and the growth was observed. The L. monocytogenes isolates from the cooked ground beef were screened for resistance to carnocyclin A. For the bacteriocin resistant isolates of L. monocytogenes, reverse transcription q-PCR was carried out to determine the impact of carbohydrates and carnocyclin A on the expression of genes involved in resistance. To determine the impact of carnocyclin A on the genomes of L. monocytogenes, whole genome sequencing was performed to investigate the SNPs (single nucleotide polymorphisms) of the parent strains (carnocyclin A sensitive) and resistant isolates of L. monocytogenes. The resistance of L. monocytogenes to carnocyclin A is both strain and carbohydrate dependent, as evidenced by the different growth patterns. The downregulation of the Mannose PTS system in the presence of dextrose for the resistant L. monocytogenes J1-177 strain suggests that the carbohydrate transport systems are used as receptor molecules for carnocyclin A. However, the upregulation of the Mannose PTS system, sucrose phosphorylase, β-glucoside PTS system in other resistant isolates (L. monocytogenes J1-177 and L. monocytogenes C1-056 isolated from meat supplemented with sucrose) suggests that mechanism of resistance to carnocyclin A is also dependant on carbohydrate and strain. The high number of SNPs present in the resistant isolates from cooked ground beef indicate that there is hypermutation of L. monocytogenes in response to carnocyclin A. In addition, as the SNPs occurred not only in genes related to the carbohydrate transport systems, but also in genes associated with cell wall and virulence, this suggests a more general stress response to the presence of bacteriocins. Overall, the L. monocytogenes resistance to carnocyclin A is mediated by factors such as strain individuality and carbohydrate source, and mechanisms of resistance are broad rather than specific. The results presented in this thesis will contribute to a more comprehensive understanding on how carnocyclin A and the carbohydrates available in food products can impact the resistance to bacteriocins and the mechanisms of resistance in L. monocytogenes. This can then inform the use of bacteriocins in the food industry, particularly towards more effective control strategies for L. monocytogenes in ready-to-eat meat products.