# A study of key meat spoilage bacteria

Stanborough, T ORCID: 0000-0001-5945-2536 2018 , 'A study of key meat spoilage bacteria', PhD thesis, University of Tasmania.

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## Abstract

Fresh meat is a highly nutritious commodity, conducive to the rapid growth of microorganisms and spoilage. Despite the economic and social importance associated with preventing meat spoilage and minimising food waste, the physiology of many microorganisms involved in meat spoilage remains poorly studied. Such fundamental knowledge is of great importance to enable further improvements in the control of meat contamination and spoilage, and to facilitate shelf-life extension. In this context, the broad objective of this thesis was to improve current knowledge of the biology of important meat spoilage bacteria, Brochothrix thermosphacta and psychrotrophic pseudomonads Pseudomonas fragi and Pseudomonas lundensis.
In contrast to the well-studied and phylogenetically related pathogen Listeria monocytogenes, genome analyses of B. thermosphacta were lacking. Thus, draft genomes of 12 B. thermosphacta strains were analysed and compared to genomes of Brochothrix campestris and L. monocytogenes to identify genes that play a role in spoilage and persistence of B. thermosphacta throughout the meat production chain. The 12 strains shared a high degree of genomic similarity. Genes/pathways likely involved in the production of organoleptically unpleasant compounds such as acetoin, butanediol and isobutyric, isovaleric and 2-methylbutyric acid were identified in both Brochothrix species, while amino acid decarboxylase genes were not found, and phenotypic testing confirmed their absence. Orthologues of key Listeria virulence genes were absent from the Brochothrix genomes, however auxiliary virulence genes such as factors involved in surface protein anchoring (e.g. sortase A), and key stress response regulatory genes were identified, establishing parallels to and differences from this related foodborne pathogen.
Gram-positive bacteria utilise class A sortases to attach a diversity of proteins to the cell wall, including factors involved in the adhesion of pathogens to host cells and tissues. As a starting point for understanding how B. thermosphacta interacts with its environment, a truncated and tagged variant of the B. thermosphacta sortase A (His$$_6$$-BtSrtA) was generated and catalytic activity of His$$_6$$-BtSrtA was investigated. His$$_6$$-BtSrtA recognised and cleaved LPXTG sorting motifs and attached SrtA pseudo-substrates to rhodamine-labelled tri-glycine, demonstrating in vitro SrtA bioconjugation activity. Genome examination identified 11 potential SrtA substrate proteins, two of which contained protein domains associated with adherence of pathogens to host extracellular matrix proteins and cells, suggesting the B. thermosphacta SrtA may be indirectly involved in its attachment to meat surfaces.
Despite their importance as aerobic meat spoilers, genomic studies of the species P. fragi and P. lundensis were missing, and limited knowledge existed of their metabolic potential at the strain level. Thus, the genomes and metabolic activity of 13 P. fragi and seven P. lundensis were analysed. Genome investigations showed that the 20 isolates may belong to more than two species with possible spoilage potential; they revealed a high degree of diversity among the P. fragi and indicated that genetic flexibility and diversity may be traits of both species. Growth of the P. lundensis isolates on a beef paste was characterised by the production of large amounts of 1-undecene, 5-methyl-2-hexanone and methyl-2-butenoic acid, while P. fragi isolates produced extensive amounts of methyl and ethyl acetate. Some of the P. fragi produced extremely low levels of volatile organic compounds, suggesting not all strains have the same spoilage potential.
It is not understood how P. fragi competes so successfully with other bacteria in foods. As little is known about iron uptake systems of P. fragi and iron is a limiting factor in many environments, the possibility of siderophore-mediated iron uptake as an iron acquisition system for P. fragi was explored. A vibrioferrin siderophore gene cluster was identified in the 13 P. fragi and experiments were conducted with a representative strain of this group (F1801). Chromeazurol S assays showed P. fragi F1801 produced siderophores under iron limitation. Disruption of the vibrioferrin receptor (pvuA) caused polar effects on downstream vibrioferrin biosynthetic genes. This led to impaired siderophore production of the ΔpvuA mutant and growth defects under severe iron-restriction, demonstrating that the identified vibrioferrin-mediated iron acquisition system is required for growth of this bacterium under iron-starvation.
Meat tenderness is an important quality attribute and considerable interest exists for the development of novel technologies to improve meat tenderness. Low field strength pulsed electric field (PEF) has come under investigation for its potential to tenderise meat due to its ability to cause limited muscle cell disruption and enhanced proteolysis, which may also promote meat spoilage owing to an increased availability of precursor metabolites of microbial spoilage. A brief study was conducted to determine the effects of a low field strength PEF (0.25 kV/cm, 3000 pulses) on the aerobic microbiota of beef. The microbial load of naturally contaminated PEF-treated and -untreated samples was compared and potential damage to muscle cells and enhanced proteolysis was assessed with reversed phase-high performance liquid chromatography (RP-HPLC). Until three days post-treatment, little difference was detected in the mean log cfu/g of treated and untreated samples. Differences in the sample means were observed at later time points, and on day-eight and -nine these differences were statistically significant (P<0.05), indicating that PEF may promote microbial growth. RP-HPLC showed no differences in the profiles of phosphate buffered saline-soluble compounds derived from the meat surface of treated and untreated samples, demonstrating evidence for muscle cell leakage and enhanced proteolysis was not obtained with this method.
In summary, the genomic and physiological studies of key meat spoilage bacteria that form this thesis enhance knowledge of these poorly studied bacteria by: 1) providing insight into genomic and metabolic diversity of strains of these bacteria 2) demonstrating how strains of these bacteria may or may not contribute to spoilage by the production of various malodourous compounds 3) providing a starting point for further studies on surface protein attachment of B. thermosphacta and understanding mechanisms with which this bacterium interacts with its environment and 4) by revealing a siderophore-mediated iron acquisition system of P. fragi.

Item Type: Thesis - PhD Stanborough, T Meat spoilage; sortase; siderophore; Brochothrix thermosphacta; Pseudomonas fragi ; Pseudomonas lundensis Copyright 2018 the author Chapter 2 appears to be the equivalent of a post-print version of an article published as: Stanborough, T., Fegan, N., Powell, S. M., Tamplin, M., Chandry, P. S., 2017. Insight into the genome of Brochothrix thermosphacta, a problematic meat spoilage bacterium, Applied and environmental microbiology, 83(5), e02786-16Chapter 3 appears to be the equivalent of a pre-copyedited, author-produced version of an article accepted for publication in FEMS microbiology letters following peer review. The version of record: Stanborough, T., Suryadinata, R., Fegan, N., Powell, S. M., Tamplin, M., Nuttall, S. D., Chandry, P. S., 2018. Characterisation of a putative Brochothrix thermosphacta sortase A enzyme., FEMS microbiology letters, 365(17), 1-9, is available online at: https://doi.org/10.1093/femsle/fny184Chapter 4 appears to be the equivalent of a post-print version of an article published as: Stanborough, T., Fegan, N., Powell, S. M., Singh, T., Tamplin, M., Chandry, P. S., 2018. Genomic and metabolic characterization of spoilage-associated Pseudomonas species, International journal of food microbiology, 268, 61-72Chapter 5 appears to be the equivalent of a pre-copyedited, author-produced version of an article accepted for publication in FEMS microbiology letters following peer review. The version of record: Stanborough, T., Fegan, N., Powell, S. M., Tamplin, M., Chandry, P. S., 2018. Vibrioferrin production by the food spoilage bacterium Pseudomonas fragi, FEMS microbiology letters, 365(6), 1-9, is available online at: https://doi.org/10.1093/femsle/fnx279 View statistics for this item