Evaluation of thermal inactivation of Escherichia coli using microelectrode ion flux measurements with osmotic stress.

AIMS: To elucidate the potential use of microelectrode ion flux measurements to evaluate bacterial responses to heat treatment. METHODS AND RESULTS: Escherichia coli K12 was used as a test bacterium to determine whether various heat treatments (55-70°C for 15 min) affected net ion flux across E. coli cell membranes using the MIFE™ system to measure net K(+) fluxes. No difference in K(+) fluxes was observed before and after heat treatments regardless of the magnitude of the treatment. Applying hyperosmotic stress (3% NaCl w/v) during flux measurement led to a net K(+) loss from the heat-treated E.coli cells below 65°C as well as from nonheated cells. In contrast, with E. coli cells treated at and above 65°C, hyperosmotic stress disrupted the pattern of K(+) flux observed at lower temperatures and resulted in large flux noise with random scatter. This phenomenon was particularly apparent above 70°C. Although E. coli cells lost the potential to recover and grow at and above 62°C, K(+) flux disruption was not clearly observed until 68°C was reached. CONCLUSIONS: No changes in net K(+) flux from heat-stressed E. coli cells were observed directly as a result of thermal treatments. However, regardless of the magnitude of heat treatment above 55°C, loss of viability indicated by enrichment culture correlated with disrupted K(+) fluxes when previously heated cells were further challenged by imposing hyperosmotic stress during flux measurement. This two-stage process enabled evaluation of the lethality of heat-treated bacterial cells within 2 h and may be an alternative and more rapid method to confirm the lethality of heat treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to confirm the lethality of thermal treatments and to specify minimal time/temperature combinations by a nonculture-dependent test offers an alternative system to culture-based methods.

Significance of the rdar and bdar morphotypes in the hydrophobicity and attachment to abiotic surfaces of Salmonella Sofia and other poultry-associated Salmonella serovars.

AIMS: To investigate the relative role of the red dry and rough (rdar) and brown dry and rough (bdar) morphotypes on hydrophobicity and ability to attach to abiotic surfaces of poultry-associated Salmonella strains with a focus on S. Sofia. METHODS AND RESULTS: Cellulose synthase gene null mutants were constructed in five Salmonella strains converting them from rdar to bdar morphotypes. One S. Sofia null mutant displayed reduced hydrophobicity and attachment to Teflon® relative to its parent strain. The S. Virchow and S. Infantis null mutants attached less well to glass relative to their parent strains. CONCLUSIONS: The rdar or bdar morphotype may influence S. Sofia persistence but did not explain why bdar strains predominate in this serotype. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides some insight into why some Salmonella strains survive in poultry environments and may ultimately contribute to their control.

Ecophysiology of food-borne pathogens: Essential knowledge to improve food safety.

The term ecophysiology suggests that a natural connection exists between microbial ecology and microbial physiology, the former being concerned with the responses of microbial populations to environmental influences, and the latter with activities within individual cells. In this contribution we choose to integrate these as far as possible and also indicate how understanding of both is benefiting from advances in molecular biology and informatics. We consider how microbial dispersal relates to microbial survival, recovery and proliferation, including the significance of random factors (stochasticity) in continuation of bacterial lineages, observing that minor environmental changes, can greatly influence the potential for food-borne disease. Homeostasis and membrane transport are identified as potential targets to control food-borne pathogens and the role of compatible solutes in stress protection is presented. Phenotypic variation in genetically homogeneous populations is highlighted as a major component of the overall microbial survival strategy. The marked influence and potential of predictive microbiology as an aid to food safety management is discussed, as is the need for greater knowledge of the ecophysiology of microbes in the growth/no growth region. The application of fundamental scientific principles, including thermodynamics, chemistry and microbial physiology is advocated as the basis for development of theory underpinning microbial ecophysiology. Advancing microbial food safety continues to require development of rapid, quantitative methods as an early warning system and mechanism to curtail microbial food-borne disease outbreaks. However, advances made by technologists and molecular biologists must be combined with knowledge of ecophysiology: e.g. biological rates will continue to constrain resolution of the recalcitrant problem of reducing the time required for enrichment processes. The discussion presented leads to the conclusion that microbial and molecular methods are appropriate for enumeration and prevalence studies but that predictive model development should continue for the purposes of comparative process control and to support the risk assessment paradigm. We conclude also that contributions of human error or complacency to microbial food-borne illness will continue to thwart the best efforts of microbiologists and technologists to reduce its incidence. Decision-support technologies reporting in real-time appear to have potential to make objective food safety decisions thereby reducing the impact of human indifference to the application of simple, but effective, food hygiene rules.

Effect of relative inoculum concentration on Listeria monocytogenes growth in co-culture.

Growth suppression of multi-species bacterial populations in batch cultures by a single 'dominant' strain has been referred to in the literature as the 'Jameson Effect'. The effect is often attributed to production of specific inhibitors of growth by one species against another. To explore its basis, we hypothesised that the Jameson Effect is often a non-specific interaction and that growth inhibition of species in co-culture can be controlled by manipulation of inoculum concentration and growth rate so as to enable a selected species to achieve stationary phase first. The hypothesis was assessed by co-culturing pairs of bacterial species under conditions selected to ensure that both strains grew at a similar rate and manipulating the initial concentration of each species. Specifically, the effect of inoculum concentration on the growth kinetics of Listeria monocytogenes when co-cultured in complex laboratory media with Escherichia coli, Pseudomonas fluorescens or a commercial strain of the lactic acid bacterium, Lactobacillus plantarum, was studied. Starting numbers of the second strain were either higher than ( approximately 10(6) cfu mL(-1)), equal to ( approximately 10(4) cfu mL(-1)) or lower than ( approximately 10(3) cfu mL(-1)) L. monocytogenes. In most trials, the initial inoculum concentration governed which species became dominant and suppressed the growth of the other strain. L. monocytogenes was suppressed by all other strains when its inoculum level was lower. Conversely, when L. monocytogenes was initially present at higher concentration than either P. fluorescens or L. plantarum, their growth was suppressed. E. coli, however, was not suppressed by L. monocytogenes even when the E. coli density was initially lower. While simple competition for nutrients could explain most of the observations, in some co-culture experiments pH reduction also seemed to play a role in inhibition of growth of some species. In other cases, available growth substrates were apparently not utilised by both co-cultured species, and the Jameson Effect did not occur. Thus, while it appears that under many conditions the Jameson Effect may be largely due to non-specific inhibition, more complex interactions between co-cultured strains involving species-specific pH limits for growth and differential utilisation of growth substrates may also occur and confound the simple Jameson Effect.

Salmonella Sofia differs from other poultry-associated Salmonella serovars with respect to cell surface hydrophobicity.

Salmonella enterica is one of the most important foodborne pathogens. Salmonella enterica subsp. II 4,12:b:- (Salmonella Sofia) is commonly found in Australian poultry. It has been suggested that physicochemical properties such as surface charge and hydrophobicity may affect bacterial attachment to surfaces and their ability to persist in food systems. A possible link between hydrophobicity cell surface charge and persistence of Salmonella from the poultry system was examined. Hydrophobicity of Salmonella Sofia (n = 14), Salmonella Typhimurium (n = 6), Salmonella Infantis (n = 3), and Salmonella Virchow (n = 2) was assayed using hydrophobic interaction chromatography, bacterial adherence to hydrocarbons (BATH), using xylene or hexadecane, and the contact angle method (CAM). Cellular surface charge (CSC) of the isolates was determined using zeta potential measurements. The majority (12 of 14) of Salmonella Sofia isolates were found to be hydrophobic when assayed using BATH with xylene, except isolates S1635 and S1636, and the other serovars were found to be hydrophilic. Salmonella Sofia isolates were not significantly different (P > 0.05) from isolates of other serovars as measured by hydrophobic interaction, BATH with hexadecane, or the CAM. No significant differences (P > 0.05) in zeta potential measurements were observed between isolates. Principal component analysis using results from all four measures of hydrophobicity allowed clear differentiation between isolates of the serovar Salmonella Sofia (except S1635 and S1636) and those of other Salmonella serovars. Differences in physicochemical properties may be a contributing factor to the Salmonella Sofia serovar's ability to attach to surfaces and persist in a food system.

Predictive microbiology: Quantitative science delivering quantifiable benefits to the meat industry and other food industries.

Predictive microbiology is considered in the context of the conference theme "chance, innovation and challenge", together with the impact of quantitative approaches on food microbiology, generally. The contents of four prominent texts on predictive microbiology are analysed and the major contributions of two meat microbiologists, Drs. T.A. Roberts and C.O. Gill, to the early development of predictive microbiology are highlighted. These provide a segue into R&D trends in predictive microbiology, including the Refrigeration Index, an example of science-based, outcome-focussed food safety regulation. Rapid advances in technologies and systems for application of predictive models are indicated and measures to judge the impact of predictive microbiology are suggested in terms of research outputs and outcomes. The penultimate section considers the future of predictive microbiology and advances that will become possible when data on population responses are combined with data derived from physiological and molecular studies in a systems biology approach. Whilst the emphasis is on science and technology for food safety management, it is suggested that decreases in foodborne illness will also arise from minimising human error by changing the food safety culture.

Effect of suspension media on nonthermal inactivation of Escherichia coli.

AIMS: To investigate the influence of suspension media on the survival of Escherichia coli M23 exposed to nonthermal, lethal stresses. METHODS AND RESULTS: Populations of E. coli M23 suspended in minimal medium (MM) or in different nutrient-rich broths were exposed to water activity 0.90 and/or pH 3.5 and inactivation was determined by culture-based enumeration. In response to the osmotic or acid challenges, E. coli M23 displayed enhanced survival in MM rather than in complex broth. That trend was reversed when populations were exposed to low water activity in combination with low pH. Comparison of microbial survival in three complex media indicated that even relatively small differences in composition influenced inactivation. In most media the combination of lethal stresses resulted in a synergism, which enhanced bacterial inactivation; however, an exception (tryptone soya broth) was observed. CONCLUSIONS: The suspension medium strongly influences the inactivation of E. coli M23 by osmotic and/or acid stresses. This should be considered when comparing studies of microbial survival that use different media and when broth-derived data are intended to represent specific environments (e.g. food matrices). SIGNIFICANCE AND IMPACT OF THE STUDY: The specific effects of synthetic media need to be appreciated when studying bacterial inactivation in conditions relevant to food-manufacturing regimes.

Information systems in food safety management.

Information systems are concerned with data capture, storage, analysis and retrieval. In the context of food safety management they are vital to assist decision making in a short time frame, potentially allowing decisions to be made and practices to be actioned in real time. Databases with information on microorganisms pertinent to the identification of foodborne pathogens, response of microbial populations to the environment and characteristics of foods and processing conditions are the cornerstone of food safety management systems. Such databases find application in: Identifying pathogens in food at the genus or species level using applied systematics in automated ways. Identifying pathogens below the species level by molecular subtyping, an approach successfully applied in epidemiological investigations of foodborne disease and the basis for national surveillance programs. Predictive modelling software, such as the Pathogen Modeling Program and Growth Predictor (that took over the main functions of Food Micromodel) the raw data of which were combined as the genesis of an international web based searchable database (ComBase). Expert systems combining databases on microbial characteristics, food composition and processing information with the resulting "pattern match" indicating problems that may arise from changes in product formulation or processing conditions. Computer software packages to aid the practical application of HACCP and risk assessment and decision trees to bring logical sequences to establishing and modifying food safety management practices. In addition there are many other uses of information systems that benefit food safety more globally, including: Rapid dissemination of information on foodborne disease outbreaks via websites or list servers carrying commentary from many sources, including the press and interest groups, on the reasons for and consequences of foodborne disease incidents. Active surveillance networks allowing rapid dissemination of molecular subtyping information between public health agencies to detect foodborne outbreaks and limit the spread of human disease. Traceability of individual animals or crops from (or before) conception or germination to the consumer as an integral part of food supply chain management. Provision of high quality, online educational packages to food industry personnel otherwise precluded from access to such courses.

Viable count estimates of lag time responses for Salmonella typhimurium M48 subjected to abrupt osmotic shifts.

Generally, relative lag times (RLT; lag time divided by generation time) become extended as conditions become less favourable for growth. Mellefont et al. (2003, 2004) [Mellefont, L.A., McMeekin, T.A., Ross, T., 2003. The effect of abrupt osmotic shifts on the lag phase duration of foodborne bacteria. Int. J. Food Microbiol. 83(3), 281-293; Mellefont, L.A., McMeekin, T.A., Ross, T., 2004. The effect of abrupt osmotic shifts on the lag phase duration of physiologically distinct populations of Salmonella typhimurium. Int. J. Food Microbiol. 92, 111-120] reported that abrupt osmotic shifts of Salmonella typhimurium M48 from optimal to low aw led to unexpectedly small RLTs at low aw. In this study, RLTs resulting from similar osmotic shifts were estimated by viable count (VC) and compared to turbidimetric estimates to test the hypothesis that the 'downturn' in RLT is an artefact of the turbidimetric technique used. No 'downturn' in RLT was observed with VC data and RLTs increased with increasing magnitude of osmotic shift. Anomalous turbidimetric estimates of lag time at low aw were confirmed as the likely source of the 'downturn' in RLT. The abrupt osmotic shifts resulted in a complex pattern of microbial population behaviour. Immediately after transfer from optimal aw to low aw, inactivation of a portion of the population occurred for all the conditions tested. The degree of inactivation became progressively larger with larger shifts in aw. The initial decline in population was followed by a period during which no change in numbers occurred, followed by growth that appeared, in most cases, to be exponential. At the lowest aws tested (< or =0.954), the growth response after the initial decline was at a rate slower than that of exponential phase growth. Due to the use of non-selective media containing pyruvate (to eliminate oxygen radicals), the observed patterns of inactivation, lag and regrowth at most aw conditions are unlikely to result from a temporary loss of culturability, but may represent inactivation of a portion of the population.

Temperature step changes: a novel approach to control biofilms of Streptococcus thermophilus in a pilot plant-scale cheese-milk pasteurisation plant.

A pilot plant-scale cheese-milk pasteurisation plant was designed and constructed to study the development of biofilms of Streptococcus thermophilus during pasteurisation of milk, and to investigate methods for preventing this growth from occurring. Under base run conditions, S. thermophilus grew on surfaces in the cool-down sections of the pilot plant, between 50 and 35 degrees C (bulk milk temperature), and could be detected in the product stream after 8-10 h production, reaching levels of 10(6) CFU ml-1 after 16 h. Thermoduric bacteria also grew in the heating sections of the pilot plant, although to a lesser extent, as did non-thermoduric bacteria that originated in the raw milk. The novel application of temperature step changes, implemented periodically to the growth region of S. thermophilus, successfully controlled the development of biofilms of these organisms. The growth of bacteria on the heating side of the pilot plant was also prevented by the implementation of these same step changes. The optimum step change conditions required to achieve a 20-h production run without detectable growth of S. thermophilus comprised a step change to 55 degrees C, applied for 10 min, with a 60-min interval between step changes.

The effect of abrupt osmotic shifts on the lag phase duration of physiologically distinct populations of Salmonella typhimurium.

Relative lag time (RLT), i.e. lag time divided by generation time, was used to characterise the lag phase response of exponential and stationary phase Salmonella typhimurium subjected to NaCl-mediated hyperosmotic shifts. Abrupt hyperosmotic shifts induced lag phases. The RLT, however, varied with the physiological history of the inoculum and the magnitude of the shift. Turbidimetric data showed that exponential phase cells had larger RLTs (up to approximately 8 units) than stationary phase cells (up to 2-4 units). Inocula containing exponential and stationary phase cells mixed in known proportions gave intermediate results. For viable count data, there was little difference in RLT between exponential and stationary phase cells. The RLT response determined turbidimetrically was reproducible for exponential phase cells, but less so for stationary phase cells. It is suggested that there may be a lower limit for resolution of RLT, in the range 0-2 units, and that this may account for the lack of reproducibility in RLTs of stationary phase cells. It is hypothesised that stationary phase cells have enhanced resistance to osmotic stress and are able to exploit new growth environments at low a(w) more rapidly than exponential phase cells, resulting in shorter lag phases. However, the data indicate that turbidimetry may not accurately describe the lag phase response of exponential phase cells subjected to large osmotic shifts. Viable count data is required to investigate this hypothesis further.

The effect of abrupt osmotic shifts on the lag phase duration of foodborne bacteria.

The effects of osmotic environment and inoculum history on lag times were examined. Abrupt osmotic shifts of cultures were found to induce lag phases in a variety of foodborne bacteria. Relative lag times (RLT; the ratio of lag time to generation time) were used to differentiate the effects of the shift from those of the outgrowth environment. In general, osmotic downshifts induced larger RLTs than equivalent upshifts. An observed reduction in RLT at very low a(w), however, was unexpected. For an osmotic downshift, differences were observed in the RLT response of the Gram-negative and -positive strains tested. RLTs were usually extended for Gram-negative organisms as conditions became less favourable for growth. In comparison, RLT remained relatively unaffected for Gram-positive organisms. The observations reported in this study demonstrate that lag time can be understood in terms of the amount of work to be done to adjust to new environmental conditions and the rate at which that work is done, and are consistent with known strategies for osmoregulation employed by the various organisms studied.

Modelling the effects of temperature, water activity, pH and lactic acid concentration on the growth rate of Escherichia coli.

An extended square root-type model describing Escherichia coli growth rate was developed as a function of temperature (7.63-47.43 degrees C), water activity (0.951-0.999, adjusted with NaCl), pH (4.02-8.28) and lactic acid concentration (0-500 mM). The new model, based on 236 growth rate data, combines and extends previously published square root-type models and incorporates terms for upper and lower limiting temperatures, upper and lower limiting pH, minimum inhibitory concentrations of dissociated and undissociated lactic acid and lower limiting water activity. A term to describe upper limiting water activity was developed but could not be fitted to the E. coli data set because of the difficulty of generating data in the super-optimal water activity range (i.e. >0.998). All data used to generate the model are presented. The model provides an excellent description of the experimental data.

Performance evaluation of a model describing the effects of temperature, water activity, pH and lactic acid concentration on the growth of Escherichia coli.

A square root-type model for Escherichia coli growth in response to temperature, water activity, pH and lactic acid was developed by Ross et al. [Int. J. Food Microbiol. (2002).]. Predicted generation times from the model were compared to the literature data using bias and accuracy factors, graphical comparisons and plots of residuals for data obtained from both liquid growth media and foods. The model predicted well for 1025 growth rate estimates reported in the literature after poor quality or unrepresentative data (n=215) was excluded, with a bias factor of 0.92, and an accuracy factor of 1.29. In a detailed comparison to two other predictive modes for E. coli growth, Pathogen Modeling Program (PMP) and Food MicroModel (FMM), the new model generally performed better. The new model consistently gave better predictions than the other models at generation times

Predictive microbiology: providing a knowledge-based framework for change management.

This contribution considers predictive microbiology in the context of the Food Micro 2002 theme, "Microbial adaptation to changing environments". To provide a reference point, the state of food microbiology knowledge in the mid-1970s is selected and from that time, the impact of social and demographic changes on microbial food safety is traced. A short chronology of the history of predictive microbiology provides context to discuss its relation to and interactions with hazard analysis critical control point (HACCP) and risk assessment. The need to take account of the implications of microbial adaptability and variable population responses is couched in terms of the dichotomy between classical versus quantal microbiology introduced by Bridson and Gould [Lett. Appl. Microbiol. 30 (2000) 95]. The role of population response patterns and models as guides to underlying physiological processes draws attention to the value of predictive models in development of novel methods of food preservation. It also draws attention to the paradox facing today's food industry that is required to balance the "clean, green" aspirations of consumers with the risk, to safety or shelf life, of removing traditional barriers to microbial development. This part of the discussion is dominated by consideration of models and responses that lead to stasis and inactivation of microbial populations. This highlights the consequence of change on predictive modelling where the need is now to develop interface and non-thermal death models to deal with pathogens that have low infective doses for general and/or susceptible populations in the context of minimal preservation treatments. The challenge is to demonstrate the validity of such models and to develop applications of benefit to the food industry and consumers as was achieved with growth models to predict shelf life and the hygienic equivalence of food processing operations.

Predictive microbiology: towards the interface and beyond.

This review considers the concept and history of predictive microbiology and explores aspects of the modelling process including kinetic and probability modelling approaches. The "journey" traces the route from reproducible responses observed under close to optimal conditions for growth, through recognition and description of the increased variability in responses as conditions become progressively less favourable for growth, to defining combinations of factors at which growth ceases (the growth/no growth interface). Death kinetics patterns are presented which form a basis on which to begin the development of nonthermal death models. This will require incorporation of phenotypic, adaptive responses and may be influenced by factors such as the sequence in which environmental constraints are applied. A recurrent theme is that probability (stochastic) approaches are required to complement or replace kinetic models as the growth/no growth interface is approached and microorganisms adopt a survival rather than growth mode. Attention is also drawn to the interfaces of predictive microbiology with microbial physiology, information technology and food safety initiatives such as HACCP and risk assessment.

Differentiation of the effects of lethal pH and water activity: food safety implications.

The influence of a second lethal stress (SLS) was investigated when populations of Escherichia coli M23 OR.H- were exposed to either a low water activity (aw) of 0.90 or a pH of 3.50 after 24 h at 25 degrees C. Regardless of the initial stress, E. coli M23 OR.H- populations initially demonstrated biphasic inactivation kinetics consisting of a rapid first phase of death followed by a slower second phase. When cultures initially exposed to aw 0.90 experienced an SLS of pH 3.50, a second rapid inactivation period was observed before a subpopulation of more resistant cells emerged. This subpopulation was able to persist for approximately 50 h after imposition of the SLS. In contrast, E. coli M23 OR.H- cells first exposed to a pH of 3.50 were inactivated rapidly to levels below the limits of detection upon imposition of an SLS of aw 0.90. It is hypothesized that pH stress constitutes a large energy drain on the cell and subsequently sensitizes it to other environmental constraints requiring expenditure of metabolic energy.

Sterol and squalene content of a docosahexaenoic-acid-producing thraustochytrid: influence of culture age, temperature, and dissolved oxygen.

Thraustochytrid strain ACEM 6063, rich in omega-3 polyunsaturated fatty acids, was cultured at 15 degrees C and 20 degrees C in high (>40%) and low (<5%) dissolved oxygen (DO), and at 25 degrees C in low-DO media. Samples were taken 4, 2, and 0 days before each culture reached peak biomass (T(-4), T(-2), and T(p), respectively). Twenty sterols, 13 of which were identified, were detected. Predominant were cholest-5-en-3 beta-ol, 24-ethylcholesta-5,22E-dien-3 beta-ol, 24-methylcholesta-5,22E-dien-3 beta-ol, and 2 coeluting sterols, one of which was 24-ethylcholesta-5,7,22-trien-3 beta-ol. These 4 sterols comprised 50% to 90% of total sterols. Cultures grown at high DO had simpler sterol profiles than those grown at low DO. Only the 4 sterols mentioned above were present at more than 3% of total sterols in high-DO cultures. In low-DO cultures, up to 6 additional sterols were present at more than 3% of total sterols. Culture age, temperature, and DO influenced squalene and sterol content. Total sterols (as a proportion of total lipids) decreased with increasing culture age. If organisms such as ACEM 6063 are to be used for commercial production of lipid products for human consumption, both their sterol content and factors influencing sterol production need to be characterized thoroughly.

Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs.

The effect of different extraction techniques on the recovery of fatty acids from freeze-dried biomass of two lipid-producing microheterotrophs was examined. Two procedures were used: the extraction of lipids from biomass followed by transesterification of the fatty acids (extraction-transesterification); and the direct transesterification of biomass to produce fatty acid methyl esters (i.e. without the initial extraction step). Variable factors in the extraction-transesterification experiment were the sequence in which solvents were added to the samples, the relative amount of methanol in the solvent mix, and sonication of biomass while in the solvent mix. Variable factors in the direct transesterification experiment were sample size, and reaction duration. Statistical analysis of data (level of significance P<0.05) showed that: (1) extraction of total fatty acids prior to transesterification was significantly more efficient when solvents were added in the order of increasing polarity; (2) neither sonication nor increasing the proportion of methanol in the extraction solvent significantly affected extraction of fatty acids prior to transesterification; (3) efficiency of direct transesterification of fatty acids increased significantly with reaction time; (4) efficiency of direct transesterification of fatty acids was not significantly affected by sample size; (5) the most efficient method for extraction of fatty acids prior to transesterification yielded significantly less fatty acids than the most effective direct transesterification method. While the study examined only two strains, our results suggest that fatty acid analysis methodology for microheterotrophs under consideration for biotechnological exploitation requires optimisation and validation.