title: Investigating the molecular mechanisms of piezotolerance in Escherichia coli and the antimicrobial effect of combined high hydrostatic pressure and supercritical carbon dioxide in Escherichia coli and Listeria monocytogenes
Creators: name: Olowomofe, Tolulope Tomiye
Publisher: name: University of Reading
Year: 2023

description: High hydrostatic pressure (HHP) food processing is a novel minimal technology that involves 
hydrostatic pressure in the inactivation of foodborne pathogens towards achieving stable food shelf 
life, microbial safety and nutritious food products. Escherichia coli is an emerging foodborne
pathogen sensitive to lethal levels of HHP, but some pathogenic strains have developed 
piezotolerance compromising HHP efficiency and presenting a potential risk to food safety. Gene 
composition and regulation are essential in bacterial adaptation to food processing conditions.
Genetic information that enabled piezotolerance in foodborne bacteria is inadequate. Identifying
gene and molecular mechanisms that play significant roles in piezotolerance could enable the 
design of efficient HHP treatment and tackle microbial food safety issues. The conventional 
bagging method majorly applied in HHP is challenging. Designing techniques for rapid HHP
assessment of bacterial cultures will encourage the unravelling of many genes relevant to 
piezotolerance. The research aimed to apply functional genomics in conventional microbiology 
studies to: identify genes and molecular mechanisms relevant to piezotolerance, design hurdle 
technology of synergistic combination of HHP and supercritical carbon dioxide and adopt the 
technology in designing conditions for bacterial inactivation in ready-to-eat salmon. Bagging of
E. coli using bubble wrap was investigated. Three needle sizes were determined for making holes 
in bubble wraps. Moreso, nail polish, chemical sealants, and a physical sealant, Grey septum were 
evaluated for sealing holes made by the selected needle. The counting of E. coli colonies in agar 
containing tetrazolium salt in bubble wrap in HHP studies was investigated. Conventional dilution
of E. coli in agar and broth before HHP and plating afterwards was evaluated. E. coli K-12 (WT) 
and its mutants were screened in HHP at 300 MPa for 5 min using the bubble wrap-septum 
technique, and viability was determined with the conventional diluting and plating method.
Twenty-six presumptive piezosensitive and twenty-four presumptive piezotolerant mutants were 
selected. Further assessment of these mutants in HHP revealed that twenty-eight and eleven genes 
confer piezotolerance and piezosensitivity, respectively, with associated molecular mechanisms.
E. coli K-12 and selected mutants were treated in the designed hurdle technology of synergistic 
combination of Supercritical carbon dioxide (Sc-CO2) and HHP of 2.3X volume of CO2 at 150 
MPa for 10 min at 35 oC. Ten genes relevant to Sc-CO2 tolerance were revealed, including zntA, 
yaiS, ybiA, yafX, cpxA, ylaC, rseA, ydcE, ynfC and rpoS. The WT inactivation was higher in
combined treatment than in HHP only. Similarly, its inactivation increased at 150 to 300 MPa, and 
in increased holding times, 10 to 20 min, p&lt;0.05. The concentration of WT, 8 log CFU mL-1
contaminated on 1 to 3g salmon weights reduced significantly with the designed hurdle of Sc-CO2 and HHP than HHP only. The logarithm concentration reduction of E. coli was highest on 3g
salmon, 5.6 log CFU mL-1
in the combined treatment after 20 min at 300 MPa, p&lt;0.05. Similarly, 
the inactivation of L. monocytogenes contaminated on 1 to 3g of salmon was higher using the 
combined treatment than HHP only. The logarithm concentration reduction of L. monocytogenes
was highest on 3g salmon, &gt; 5.0 log CFU mL-1
in the combined treatment after 20 min at 300 MPa.
The designed hurdle method is promising for fish processing. However, the process needs 
modification to preserve the fish’s natural colour, enhancing its organoleptic acceptability. This 
study can provide a standard guideline to gain information on all genes in E. coli and other 
foodborne pathogens relevant to HHP and to design efficient hurdle technology of Sc-CO2+HHP
for their control, ensuring the safety of ready-to-eat food products.