Spatial distribution of bacterial colonies in a model cheese.

In most ripened cheeses, bacteria are responsible for the ripening process. Immobilized in the cheese matrix, they grow as colonies. Therefore, their distribution as well as the distance between them are of major importance for ripening steps since metabolites diffuse within the cheese matrix. No data are available to date about the spatial distribution of bacterial colonies in cheese. This is the first study to model the distribution of bacterial colonies in a food-type matrix using nondestructive techniques. We compared (i) the mean theoretical three-dimensional (3D) distances between colonies calculated on the basis of inoculation levels and considering colony distribution to be random and (ii) experimental measurements using confocal microscopy photographs of fluorescent colonies of a Lactococcus lactis strain producing green fluorescent protein (GFP) inoculated, at different levels, into a model cheese made by ultrafiltration (UF). Enumerations showed that the final numbers of cells were identical whatever the inoculation level (10(4) to 10(7) CFU/g). Bacterial colonies were shown to be randomly distributed, fitting Poisson's model. The initial inoculation level strongly influenced the mean distances between colonies (from 25 µm to 250 µm) and also their mean diameters. The lower the inoculation level, the larger the colonies were and the further away from each other. Multiplying the inoculation level by 50 multiplied the interfacial area of exchange with the cheese matrix by 7 for the same cell biomass. We finally suggested that final cell numbers should be discussed together with inoculation levels to take into account the distribution and, consequently, the interfacial area of colonies, which can have a significant influence on the cheese-ripening process on a microscopic scale.

Salmonella must be viable in order to attach to the surface of prepared vegetable tissues.

AIMS: The aims of the current study were to explore the site of bacterial attachment to vegetable tissues and to investigate the hypothesis that Salmonella must be living in order to attach to this site(s). METHODS AND RESULTS: Scanning electron micrographs of intact potato cells showed that Salm. serotype Typhimurium attached to cell-wall junctions; suggesting a high-level of site selectivity. Inactivation of Salm. Typhimurium using heat, ethanol, formalin or Kanamycin resulted in cells that could be no longer attached to these sites. Attachment of a Gfp(+) strain of Salm. Typhimurium to cell-wall material (CWM) was examined via flow cytometric analysis. Only live Salm. Typhimurium attached to the CWM. CONCLUSIONS: Salmonella serotype Typhimurium must be metabolically active to ensure attachment to vegetable tissues. Attachment preferentially occurs at the plant cell-wall junction and the cell-wall components found here, including pectate, may provide a receptor site for bacterial attachment. SIGNIFICANCE AND IMPACT OF THE STUDY: Further studies into individual plant cell-wall components may yield the specific bacterial receptor site in vegetable tissues. This information could in turn lead to the development of more targeted and effective decontamination protocols that block this site of attachment.

Image analysis as a mean to model growth of Escherichia coli O157:H7 in gel cassettes.

AIMS: The potential of image analysis for rapid and quantitative determination of the effect of environmental parameters such as temperature and pH on the growth of colonies of Escherichia coli O157:H7 derived from immobilized cells in gel cassettes was investigated. METHODS AND RESULTS: The organism was grown in brain heart infusion agar contained within a cassette formed between sheets of PVC film. The medium was adjusted to pH 5, 6 or 7 and incubated at 10, 20, 30 or 40 degrees C. The primary model of Baranyi was used to fit the growth data obtained by conventional plate counting and changes in colony area (2-dimensional spread of colonies) by light microscopy to derive estimates of maximum specific growth rates (micromax and Area micromax) in both cases. Growth rate values from both measurements were correlated and a secondary quadratic model was developed to predict micromax obtained via image analysis in response to environmental factors (temperature and pH). A progressive decrease of micromax and Area micromax was observed at lower temperatures and pH values. Immobilized cells failed to initiate growth at a pH of 5.0 and 10 degrees C. There was high correlation between micromax values estimated by conventional plate counting and Area micromax values from microscopic observations in gel cassettes, regardless of temperature and pH. The values of micromax derived indirectly from the correlation with Area micromax values fitted well to the secondary model and gave realistic predictions of maximum specific growth rate values estimated by standard plate counting. CONCLUSIONS: The micromax of E. coli O157:H7 determined by plate counting was linearly correlated with Area micromax estimated by light microscopy, enabling indirect determination of micromax via the Area micromax. The estimates of micromax via the image analysis technique may be further modelled in response to environmental factors such as temperature and pH to predict the response of the organism in intermediate conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Image analysis in combination with gel cassettes could be a potential tool for rapid and convenient data collection and construction of accurate mathematical models as an alternative to conventional plate counting methods.

The lactic acid-induced acid tolerance response in Salmonella enterica serovar Typhimurium induces sensitivity to hydrogen peroxide.

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 Acetic Acid Tolerance Response induces cross-protection to salt stress in Salmonella typhimurium.

Salmonella typhimurium induces an Acid Tolerance Response (ATR) upon exposure to mildly acidic conditions in order to protect itself against severe acid shock. This response can also induce cross-protection to other stresses such as heat and salt. We investigated whether both the acetic acid induced and lactic acid induced ATR in S. typhimurium provided cross-protection to a salt stress at 20 degrees C. Acid-adapted cells were challenged with both a sodium chloride (NaCl) and potassium chloride (KCl) shock and their ability to survive ascertained. Acetic acid adaptation provided cells with protection against both NaCl and KCl stress. However, lactic acid adaptation did not protect against either osmotic stressor and rendered cells hypersensitive to NaCl. These results have implications for the food industry where hurdle technology means multiple sub-lethal stresses such as mild pH and low salt are commonly used in the preservation of products.

Modeling the rate of attachment of Listeria monocytogenes, Pantoea agglomerans, and Pseudomonas fluorescens to, and the probability of their detachment from, potato tissue at 10 degrees C.

The rate of attachment of bacteria to, and their subsequent detachment from, the cut surface of raw potato tissue was measured and modeled by using mathematical approaches that allowed detailed objective comparisons of adhesion processes under different conditions. Attachment was rapid and reached equilibrium after contact for 60 min. A new method to measure the probability of detachment was developed and modeled, revealing that the probability of detachment for Pseudomonas fluorescens remained unchanged for contact times between less than 5 s and 60 min. Listeria monocytogenes, however, was more easily removed initially, with the probability of detachment decreasing over the first 2 min of contact but remaining constant and equivalent to that for Pseudomonas fluorescens thereafter. For all of the bacteria tested, the number of bacteria attached after 2 min of contact was proportional to the inoculum concentration raised to the power of 0.79.

Batch growth of Salmonella typhimurium LT2: stoichiometry and factors leading to cessation of growth.

Salmonella typhimurium LT2 was grown in batch culture (trypticase soy broth, with 0.3%(w/v) yeast extract, 1% (w/v) glucose and 0.5% (w/v/) NaCl, 20 degrees C) at a range of initial pH (4.4, 4.8, 5.0 and 7.0). The consumption of oxygen and glucose was found to be independent of initial pH, and stoichiometric with growth. Mean yield coefficients of 6.9 x 10(-15) and 15.5 x 10(-15) mol oxygen/cell were estimated. Calculation of the instantaneous state of carbon during the cultivation showed stoichiometric conversion of glucose into biomass, carbon dioxide and organic acids. The concentration of the undissociated form of the primary acidic product (acetic acid) was shown to be the factor limiting growth.

Salmonella enterica serovar Typhimurium and Listeria monocytogenes acid tolerance response induced by organic acids at 20 degrees C: optimization and modeling.

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 degrees 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 (t(s)), the log-linear decline (k(max)), and the magnitude of a critical component (C). The magnitude of k(max) gave the best agreement with estimates of conditions for optimum ATR induction made from the raw data.

Modelling microbial growth in structured foods: towards a unified approach.

Historically, the ability of foods to support the growth of spoilage organisms and food-borne pathogens has been assessed by inoculating a food with an organism of interest, and following its growth over a period of time. Information gained from such challenge tests, together with knowledge of the organoleptic stability of the product, can then be used to determine an appropriate shelf-life for the food. Whilst this approach may be seen as the "gold-standard" of microbiological assessment of food, it is both time-consuming and costly. A major advance to complement challenge testing was the development of predictive modelling, when it was demonstrated that the growth of a wide range of organisms of interest could be quite accurately modelled as a function of only a few environmental parameters-primarily temperature, pH and water activity (a(w)), with perhaps other factors such as nitrite, organic acids and oxygen. This approach to predictive microbiology is embodied in software tools such as the UK Food MicroModel and the Pathogen Modeling Program from the USA. Whilst modelling of this form yields accurate predictions of the growth of organisms in the majority of foods, there are occasions when there are discrepancies between the model and the observed growth. These discrepancies are most often described as "fail-safe", i.e. the observed growth is slower than predicted by the model. This paper examines the role of food structure in the development of microbial populations and communities, and describes the methodologies we propose to begin to tackle some of these complex and interlinked issues.

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.

Adenylates and adenylate-energy charge in submerged and planktonic cultures of Salmonella enteritidis and Salmonella typhimurium.

Adenine nucleotide values and adenylate energy charge (AEC) were measured during the growth of Salmonella enteritidis and Salmonella typhimurium as submerged colonies in agarose gel and gelatin gel, and as planktonic cells in broth. Growth in all three systems showed similar trends with a ten-fold decrease in total adenylate pool during exponential growth, before attaining a fairly stable value throughout stationary phase. AEC values were generally low, (approximately 0.66), but did rise slightly during stationary phase. The large proportion of dead cells during early exponential phase may have contributed to the adenosine diphosphate and adenosine monophosphate pools, through cell lysis or excretion, and it is suggested that this was likely to account for the low values of AEC. In agarose and gelatin gelled cultures the percentage of adenosine triphosphate (ATP) in relation to the total adenylates showed random fluctuations. This was contrary to the broth culture where percentage ATP was highest after 12 h and the data formed a smooth curve. These data demonstrated that considerable physiological heterogeneity exists within a colony of bacteria growing in a gel matrix and by analogy a food material also, and that AEC is a poor indicator of cell viability in such systems.

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.

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 effect of transient temperatures on the growth of Salmonella typhimurium LT2. II: Excursions outside the growth region.

The effect of fluctuating temperatures on microbial growth is important in the passage of foods from production to consumption. Suspensions of Salmonella typhimurium have been subjected to sinusoidally time-varying temperatures of periods from 60 to 240 min between 4 degrees and 22 degrees C, that is within and below the growth temperature range. The suspensions were prepared with two concentrations of sodium chloride and adjusted to two different values of pH. The change in the numbers of viable bacteria was measured with time and the experimental growth curves and average generation times compared with predictions based on isothermal growth data. Generally, the experimental average generation times exceeded the predictions by not more than 10%. In enumerating viable bacteria in the suspensions containing 3.5% (w/v) sodium chloride it was necessary to use sodium chloride in the diluent and recovery medium in order to recover the bacteria quantitatively.

Growth of food-borne pathogenic bacteria in oil-in-water emulsions: I--Methods for investigating the form of growth.

Methods are presented for investigating the site and form of growth of bacteria in model oil-in-water emulsions and in dairy cream. Following growth of the bacteria, the continuous aqueous phase is gelled using agarose and the oil phase removed using a mixture of chloroform and methanol. Using this method, the authors have found that Listeria monocytogenes, Salmonella typhimurium and Yersinia enterocolitica grow in the form of colonies in concentrated oil-in-water emulsions. Colonies of L. monocytogenes and Y. enterocolitica also form in artificially-inoculated fresh and tinned dairy cream. If information about the precise site of growth is not required, the authors have discovered that intact colonies can be liberated from the model emulsions by dissolving away the oil phase with chloroform:methanol.

Growth of food-borne pathogenic bacteria in oil-in-water emulsions: II--Effect of emulsion structure on growth parameters and form of growth.

The growth rates and yields of Listeria monocytogenes and Yersinia enterocolitica were determined in liquid culture media, and in model oil-in-water emulsions that contained 30, 70 or 83% (v/v) hexadecane. In emulsions with a mean droplet size of 2 microns containing 83% (v/v) hexadecane, the growth of both organisms resulted in decreased yields. Additionally, in these emulsions adjusted to pH 5.0 or 4.4 the growth rate of L. monocytogenes was significantly less than in other model systems which had an aqueous phase of equivalent chemical composition. Microscopic examination of the 83% (v/v) emulsion showed that its microstructure immobilized the bacteria, which were constrained to grow as colonies. Bacteria behaved similarly in model emulsions of either hexadecane or sunflower oil. Manipulation of the droplet size distribution of the emulsions changed the form and rate of growth of bacteria within them.

The effect of step changes in sucrose concentration on the growth of Salmonella typhimurium LT2.

Water activity is a method of preservation that can affect microbial growth in foods and that may fluctuate during their processing, distribution and storage. Sucrose has been used to change the water activity of microbiological culture media. Suspensions of Salmonella typhimurium LT2 in the exponential phase of growth have been subjected to step changes in sucrose concentration at 20 degrees C. The changes in the numbers of viable bacteria were measured with time and the experimental growth curves compared with predictions based on growth data obtained at constant sucrose concentrations. Steps down in sucrose concentration showed some apparent loss of viability after the step followed by growth at a rate close to the expected value. Steps up in sucrose concentration resulted in a greater apparent loss of viability after the step and either growth or the inducement of lag, depending on the final concentration of sucrose. A series of small steps up in sucrose concentration to 45% (w/v) was able to sustain growth where it was not possible by inoculation directly into this concentration. Improved recovery of bacteria subject to osmotic stress was possible with a medium containing sodium chloride.

The effect of transient temperatures on the growth of Salmonella typhimurium LT2. I: Cycling within the growth region.

The effect of fluctuating temperatures on microbial growth is important in the passage of foods through the food chain. Suspensions of Salmonella typhimurium were subjected to sinusoidally time-varying temperatures of periods from 40 to 480 min within their growth temperature range. The change in the numbers of viable bacteria was measured with time and the experimental growth curves and average generation times compared with predictions based on isothermal growth data. The experimental average generation times exceeded the predictions by less than 30%, although the discrepancy increased with cycle frequency. Instantaneous growth rates obtained for the 480 min cycles were in agreement with those predicted from isothermal behaviour.

The influence of pH, temperature and organic acids on the initiation of growth of Yersinia enterocolitica.

The influence of incubation temperature, and of acetic, lactic and citric acids on the minimum pH for the initiation of growth of six strains of Yersinia enterocolitica was determined. The strains included two of serotype O : 9, two of serotype O : 3, and one each of serotypes O : 8 and O : 5, 27. In a culture medium acidified with HCl to pH values between 4.0 and 6.0 at intervals of approximately 0.1 unit the minimum pH at which growth was detected after incubation at 20 degrees, 10 degrees, 7 degrees and 4 degrees C for 21 d was in the ranges 4.18-4.36, 4.26-4.50, 4.36-4.83 and 4.42-4.80, respectively. The minimum pH for growth was also determined in media that contained 17, 33 and 50 mmol/l acetic acid adjusted to pH values between 5.1 and 5.9 at intervals of approximately 0.2 unit, 24, 48 and 95 mmol/l citric acid adjusted to pH values between 4.1 and 4.9 at intervals of approximately 0.2 unit, and 22, 44, and 111 mmol/l lactic acid adjusted to pH values between 4.3 and 5.7 at intervals of approximately 0.4 or 0.5 unit. The effect of these concentrations of organic acids was, in most cases, to increase the minimum pH that allowed growth. The order of effectiveness of the organic acids in raising the minimum pH for growth was acetic greater than lactic greater than citric and the minimum inhibitory concentrations were greater at higher temperatures.