Date of Award

6-2018

Document Type

Master Thesis

Degree Name

Master of Science

Department

Biological Sciences

First Advisor

Dr. Máire Begley

Second Advisor

Dr. Paul Cotter

Third Advisor

Prof. Colin Hill

CIT Disciplines

4.2 ANIMAL AND DAIRY SCIENCE; Animal and dairy science; 1.6 BIOLOGICAL SCIENCES; Microbiology

Abstract

Listeria monocytogenes is a foodborne pathogen, which continues to be problematic to the food industry due to its ability survive within food products and persist within food processing environments. The growing trend towards natural food preservatives and antimicrobial agents increases the need for the development of novel, natural anti-Listeria agents. Evidence presented in the literature suggests that the human gut microbiota is a reservoir of novel antimicrobial agents. It was therefore hypothesized that novel anti-Listeria agents are produced by human gut-derived bacteria. The objective of this study was to isolate and characterize gut bacteria with anti-Listeria monocytogenes activity. A total of 23 fecal banks of human origin were screened for antagonistic activity against L. monocytogenes 10403S. The results of the initial screening have shown that 1,569 gut-derived isolates have demonstrated various levels of anti-Listeria activity. Following an extensive screen 59 gut-derived isolates were initially shortlisted and were identified mostly as Enterococcus spp. By a combination of MALDI-TOF MS analysis and 16S rRNA gene sequencing. The data collected allowed a further shortlisting of strains from 59 to 16 based on differences observed in the colony morphology, the size and appearance of the zones of inhibition observed in the deferred antagonism assays and well diffusion assays as well as spectrum of activity of each isolate against a number of indicator strains. The antimicrobials produced by 11 out of 16 shortlisted gut derived isolates were identified as peptides using a proteinase K assay. The anti-Listeria activity of the shortlisted isolates was examined in a model broth co-culture experiment. Based on the results obtained the antimicrobials produced by two Enterococcus faecium isolates were selected for further investigation. The activity of the antimicrobials was investigated in dairy food homogenate models and stainless steel biofilm assays. The findings of the food trials have shown a reduction of Listeria numbers by ~0.6 Log CFU/mL in natural yogurt treated with cell free supernatant (CFS) and a reduction of ~1.5 log CFU/mL observed in cheddar cheese treated with whole cell extract (WCE) following a 3 hr incubation. The anti-biofilm assays demonstrated a reduction in biofilm formation of stainless steel by 70% when treated with CFS and 92.42% when treated with WCE. v The experiments presented in this thesis confirm that bacteria from the human gut produce anti-Listeria compounds which can control or reduce the numbers of Listeria in model co-cultures and in food homogenates. The antimicrobials isolated in this study have also shown the ability to reduce Listeria biofilm formation. Future efforts should focus on identifying the antimicrobials produced by all of the short-listed strains (through a combination of genome sequencing and peptide purifications). It is hoped that any novel antimicrobial that is discovered have an application in the food industry to reduce the significant threat posed by L. monocytogenes. These antimicrobials could be used as a natural preservative in food products or packaging or incorporated into a spray to control biofilm in the food processing environment.

Comments

The work presented in this thesis was funded by a grant from the Innovation Center for U.S. Dairy and the National Dairy Council.

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

Access Level

info:eu-repo/semantics/openAccess

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