Listeria monocytogenes - inactivation by essential oils and high pressure, and contribution of genetic variation to stress resistance

<p> </p><p>A variety of non-thermal mild preservation techniques have lately been introduced in food production, or are currently being investigated thoroughly for potential use in food processing. Two of these novel preservation techniques are the use of essential oil compounds isolated from plants and of High Hydrostatic Pressure (HHP) treatment. Applications of these preservation methods, alone or combined, have shown to reduce the viable numbers of microorganisms occurring in foods, and in addition, are able to preserve the nutritional and sensory characteristics of the product. The objectives of this thesis were to asses the antimicrobial potential of these combined processing techniques against the foodborne bacterium <em>Listeria monocytogenes</em> , which can cause serious disease with a relatively high mortality rate. In addition, to get more insight in the primary cellular targets that are affected upon HHP treatment of <em>L. monocytogenes</em> , we examined the mechanism underlying increased HHP tolerance of an isolate of this organism.</p><strong><p>Chapter 1</strong> gives a short introduction on <em>L. monocytogenes.</em> Furthermore, mild and novel preservation techniques in food processing are addressed, with special emphasis on the use of essential oil compounds from plants, and the use of High Hydrostatic Pressure (HHP). General characteristics and the mode of action of these preservation techniques are described, followed by resistance mechanisms that bacteria use to counteract their potentially lethal action. This chapter is concluded with the combined use of these preservation methods, and an outline of this thesis.</p><p>In <strong>Chapter 2</strong> , it is demonstrated that various plant-derived volatiles, namely, S-carvone, carvacrol, cinnamaldehyde, thymol, and decanal, have the ability to reduce the viable numbers of <em>L. monocytogenes</em> if used in combination with mild heat treatment, while these treatments alone were not affecting the viability of this pathogen. These findings show that essential oil compounds can play an important role in minimally processed foods, and can be used in the concept of Hurdle Technology to reduce the intensity of heat treatment or possibly other individual hurdles.</p><p>In <strong>Chapter 3</strong> , the combined antimicrobial action of carvacrol or thymol in combination with HHP was investigated at different temperatures. These treatments showed a synergistic action, with greater antimicrobial effects upon pressurisation at 1 <sup>o</SUP>C than at 8°C or 20°C. Furthermore, addition of carvacrol to cells after exposure to sub-lethal HHP treatment showed reductions in viable numbers that were similar to those using simultaneous treatment with carvacrol and HHP. Synergism was also observed between carvacrol and HHP in semi-skimmed milk that was artificially contaminated with <em>L. monocytogenes</em> . <strong></strong> The study demonstrates the synergistic antimicrobial effect of essential oils in combination with HHP. Together with the results described in Chapter 2, these data indicate the potential of essential oils to be employed in food processing if combined with other techniques.</p><p>A major problem that successful application of HHP treatment faces is the variable piezotolerance of bacterial species, strains within a species, and even cells within pure cultures that are not homogenous. In <strong>Chapter 4</strong> , we investigated the characteristics of a <em>L. monocytogenes</em> ScottA isolate, named AK01, that was derived from a wild type (wt) culture after a single pressurisation treatment. The survival of exponential and stationary phase cells of AK01 was at least 2 logs higher than that of the wt over a broad range of pressures (150-500 MPa), while both strains showed higher piezotolerance in the stationary than in the exponential phase of growth. In semi-skimmed milk, exponential phase cells of both strains showed lower reductions upon pressurisation than in buffer, but again, AK01 was more piezotolerant than the wt. The piezotolerance of AK01 was retained for at least 40 generations in rich medium, suggesting a stable phenotype. Interestingly, cells of AK01 lacked flagella and were elongated, and this strain showed slightly lower maximum specific growth rates at 8°C, 22°C and 30°C than the wt. Moreover, the piezotolerant strain AK01 showed increased resistance to heat, acid, and H <sub>2</sub> O <sub>2</sub> compared with the wt. The difference in HHP tolerance between the piezotolerant strain and the wt strain could not be attributed to differences in membrane fluidity, since strain AK01 and the wt had identical <em>in situ</em> lipid melting curves, as determined by Fourier Transfer Infrared spectroscopy.</p><p>In <strong>Chapter 5</strong> , the mechanisms underlying the altered phenotypic characteristics of AK01 are further examined. In this chapter, high piezotolerance, immobility, and reduced virulence were conclusively linked to a single amino acid deletion in the highly conserved glycine-rich region of the regulatory protein CtsR ( <u>C</u> lass <u>t</u> hree <u>s</u> tress gene <u>r</u> epressor). CtsR negatively regulates the expression of Class III heat shock genes ( <em>clpP</em> , <em>clpE</em> and the <em>clpC</em> operon). The Clp proteins prevent the accumulation of misfolded proteins that might be toxic to the cell<FONT FACE="Times New Roman">. In the mutant strain AK01, expressing the CtsRΔGly protein, we observed upregulation of</font><em>clpP</em> , which demonstrates the involvement of Class III heat shock genes in increased survival upon HHP treatment and other stresses. Replacement of the wt <em>ctsR</em> gene with the <em>ctsR</em><FONT FACE="Times New Roman">Δ</font><em>Gly</em> gene in a wt background resulted in a mutant strain that also lost its motile character, and importantly, also showed attenuated virulence. <strong></strong> The deletion of three base pairs in the region of <em>ctsR</em> that normally encodes four glycines is seemingly not an isolated incident, since we detected the same mutation in the majority of other spontaneous piezotolerant isolates of <em>L. monocytogenes</em> . This suggests a functional role for this mutation in <em>L. monocytogenes</em> to survive unforeseen events that do not allow for adaptation of normal cells, but require genetic diversity.</p><p>In conclusion, this work demonstrated some of the potentials that novel techniques, like the use of essential oils and HHP, can offer alone or combined with other novel or traditional techniques, like heat treatment. We furthermore investigated risks that could emerge from the use of those novel techniques. The occurrence of the piezotolerant strain AK01 of <em>L. monocytogenes</em> demonstrated the existence of bacterial heterogeneity within a supposedly pure wt population with regard to HHP resistance. The variance of piezotolerance within a microbial population, but also between different strains, is an important parameter that determines successful application of HHP treatment. This will be an important aspect for risk assessment of HHP treated foods that are, or will become, available on the market. Tailored HHP treatments, possibly in combination with additional mild preservatives, will be required for different products, and increased knowledge about the effects of these treatments will enable the food industry to produce safe foods at minimal costs