T.F. Brocklehurst

Salmonella enterica Serovar Typhimurium and Listeria monocytogenes Acid Tolerance Response Induced by Organic Acids at 20°C: Optimization and Modeling

ABSTRACT An acid tolerance response (ATR) has been demonstrated in Listeria monocytogenes and Salmonella enterica serovar Typhimurium in response to low pH poised (i.e., adapted) with acetic or lactic acids at 20°C and modeled by using dynamic differential equations. The ATR was not immediate or prolonged, and optimization occurred after exposure of L. monocytogenes for 3 h at pH 5.5 poised with acetic acid and for 2 h at pH 5.5 poised with lactic acid and after exposure of S. enterica serovar Typhimurium for 2 h at pH 5.5 poised with acetic acid and for 3 h at pH 5.5 poised with lactic acid. An objective mechanistic analysis of the acid inactivation data yielded estimates of the duration of the shoulder (ts), the log-linear decline (kmax), and the magnitude of a critical component (C). The magnitude of kmax gave the best agreement with estimates of conditions for optimum ATR induction made from the raw data.

NMR Water Relaxation, Water Activity and Bacterial Survival in Porous Media

Working from the simple assumption that bulk, surface and bound water in a heterogeneous system are characterised by different intrinsic nuclear magnetic resonance (NMR) relaxation rates and water activities several non-trivial relationships are derived between NMR water relaxation times and water activity. These relationships are tested for randomly packed beds of silica and Sephadex particles both in the fast and slow diffusive exchange regimes. It is shown that Archies law for electrical conductivity is obeyed by these porous beds and this observation is used to establish further relationships between NMR relaxation rates and electrical conductivity. By comparing the rates of recovery of viable cells of Salmonella typhimurium with the NMR, sorption isotherm and conductivity data it is shown that the bacterial cells are monitoring the local water activity in the fluid immediately surrounding them and not the experimental, global water activity coefficient. The implications of this observation for food preservation are considered.

The effects of cell immobilization, pH and sucrose on the growth of Listeria monocytogenes Scott A at 10°C

A study into the growth of Listeria monocytogenes Scott A investigated the effects of cell immobilization, varying sucrose concentrations (as a humectant) and alteration of initial pH at 10°C. Three conclusions were drawn from this study. Firstly, it was found that the growth rate of the organism was affected by immobilization, increasing sucrose concentrations (up to 59% w/v) and the lowering of the pH (pH 7.0 to 4.6). Secondly it was found that the lower limits of growth, in terms of pH and water activity, were different for immobilized and planktonic cultures. Lastly, it was observed that the viable population increased when planktonic cells were incubated at 10°C in combination with either low pH (pH 4.6 and 4.8) or high sucrose concentrations (59% w/v). However, this was not observed with immobilized cells, which failed to initiate growth when incubated under these conditions. Such influences of immobilization on the effects of food preservation methods are an important consideration in the predictive modelling of the survival and growth of bacteria in foods.

Water availability and the survival of Salmonella typhimurium in porous systems

The survival of Salmonella Typhimurium LT2 in randomly packed beds of glass beads, microporous silica particles and Sephadex microspheres is examined. It is shown that the decrease in the percentage cell recovery in these porous materials at reduced water content is not correlated with the global water activity as determined by conventional vapour pressure measurements but rather with the osmotic shock induced by the sudden redistribution of water and air among the microscopic pores in the matrix surrounding the cells. For this reason the bacterial survival and growth data correlates best with physical measurements, such as NMR and electrical conductivity, which are sensitive to the microscopic air-water distribution. The implications of this observation in food safety and preservation are discussed.

Microgradients in bacterial colonies : use of fluorescence ratio imaging : a non-invasive technique

Fluorescence ratio imaging is a non-invasive technique for studying the formation of microgradients in immobilised bacterial colonies. These gradients can be quantified easily when combined with the gel cassette system designed at the Institute of Food Research, Norwich, UK. Colonies of Lactobacillus curvatus were observed using this technique and relevant pH gradients were present when the colonies reached a diameter of about 100 microm. These pH gradients were due to production of lactic acid by L. curvatus cells in the colonies. The spatial resolution of the images was about 1.5 microm (scale of bacterial cells) and therefore very suitable for observing local effects in colonies which ranged in sizes from 1 to 500 microm.

The effects of growth dynamics upon pH gradient formation within and around subsurface colonies of Salmonella typhimurium

pH measurements made in and around submerged colonies of Salmonella typhimurium grown within a model gelatin gel system using pH‐sensitive micro‐ and macroelectrodes indicated some pH heterogeneity occurring in and around the bacterial colony. Inoculation density, initial pH and glucose concentration were all found to influence colony diameter and metabolism of Salmonella colonies. Colony growth in the presence of glucose, at pH 7.0 with an inoculation density of 1 cell ml‐1 led to a pH fall of 1–2 pH units after 2 d. At pH 5.0, with glucose, colony growth rates were much slower than at pH 7.0, and the pH change varied by less than one pH unit often becoming alkaline. In the absence of glucose, only small pH changes were observed within the medium, although growth rates were similar to those in glucose‐containing media. At the higher inoculation density (ca 1000 cells ml‐1), isolated pH changes were not observed. Morphological changes, such as the production of annular rings, were noted in stationary phase colonies as was alkali production in colonies. These results are discussed in relation to observations with surface colonies.

The effect of transient temperatures on the growth of Salmonella typhimurium LT2 in gelatin gel.

The growth of colonies of Salmonella typhimurium derived from single immobilised cells was studied while subjected to constant and sinusoidally-varying temperatures. The bacteria grew in microbiological culture media adjusted to different pH and sodium chloride (NaCl) concentration and solidified with gelatin that was contained within a cassette formed between sheets of PVC film that allowed gaseous exchange. At pH 7.0 and 0.5% (w/v) NaCl and either 12 degrees C or 20 degrees C, S. typhimurium grew at a rate similar to that in liquid medium. The decrease in growth rate at 20 degrees C at a lower pH or higher NaCl concentration was greater in the case of immobilised cells than for cells in liquid medium. The change in the numbers of viable bacteria was measured with time under sinusoidally-varying temperatures between 4 and 22 degrees C and between 12 and 22 degrees C of period in the range 12 to 480 min. The experimental growth curves were compared with predictions based on isothermal growth in liquid medium. The discrepancies between experiment and prediction were greater for gels stressed by NaCl or pH than for gels at pH 7.0 and containing 0.5% (w/v) NaCl, consistent with the isothermal observations.

The Lactic Acid-Induced Acid Tolerance Response in Salmonella enterica Serovar Typhimurium Induces Sensitivity to Hydrogen Peroxide†

ABSTRACT Transcriptome analyses of Salmonella enterica serovar Typhimurium revealed that 15 genes were significantly up-regulated after 2 h of adaptation with lactic acid. cadB was the most highly up-regulated gene and was shown to be an essential component. Lactic acid-adapted cells exhibited sensitivity to hydrogen peroxide, likely due to down-regulation of the OxyR regulon.

The microstructure and distribution of micro-organisms within mature Serra cheese

The distribution of micro‐organisms in mature Serra, a traditional Portuguese cheese made from unpasteurised ewes’ milk without added starter culture, was examined by light microscopy and electron microscopy. Four populations of micro‐organisms were recognized according to their position within the cheese: (i) those present as apparently axenic colonies within the curd matrix; (ii) bacteria growing along curd junctions; (iii) yeasts and bacteria present in the smear on the surface of the cheese and (iv) bacteria found in cracks which penetrated the outer part of the cheese from the rind. Two types of crystals were observed, together with contaminants of vegetable origin and somatic cells originating from the milk.

A third mode of surface-associated growth: immobilization of Salmonella enterica serovar Typhimurium modulates the RpoS-directed transcriptional programme

Although the growth of bacteria has been studied for more than a century, it is only in recent decades that surface-associated growth has received attention. In addition to the well-characterized biofilm and swarming lifestyles, bacteria can also develop as micro-colonies supported by structured environments in both food products and the GI tract. This immobilized mode of growth has not been widely studied. To develop our understanding of the effects of immobilization upon a food-borne bacterial pathogen, we used the IFR Gel Cassette model. The transcriptional programme and metabolomic profile of Salmonella enterica serovar Typhimurium ST4/74 were compared during planktonic and immobilized growth, and a number of immobilization-specific characteristics were identified. Immobilized S.Typhimurium did not express motility and chemotaxis genes, and electron microscopy revealed the absence of flagella. The expression of RpoS-dependent genes and the level of RpoS protein were increased in immobilized bacteria, compared with planktonic growth. Immobilized growth prevented the induction of SPI1, SPI4 and SPI5 gene expression, likely mediated by the FliZ transcriptional regulator. Using an epithelial cell-based assay, we showed that immobilized S.Typhimurium was significantly less invasive than planktonic bacteria, and we suggest that S.Typhimurium grown in immobilized environments are less virulent than planktonic bacteria. Our findings identify immobilization as a third type of surface-associated growth that is distinct from the biofilm and swarming lifestyles of Salmonella.