Quantification of hurdles: predicting the combination of effects -- Interaction vs. non-interaction.

Combination of disparate as well as related antimicrobial effects constitutes the concept of hurdle technology. Quantification of combined effects, including claims of synergy, can be accomplished using surface response modelling, as is frequently done and reported. The Gamma hypothesis, however, states that the relative effects of different antimicrobial factors combine independently. Studies performed using time to detection have shown that the Gamma hypothesis is an adequate foundation for the analysis of multi-factor environmental stresses placed on microorganisms, including pH, weak acids and temperature. Data from the combined action of Na acetate and pH on Aeromonas hydrophila, Na acetate/pH , K sorbate/pH and combined Na acetate/K sorbate at pH 6.5, 6.0 and 5.5 on Escherichia coli and the combined action of Na acetate/pH and temperature on Enterobacter sakazakii were examined using nominal logistic modelling, response surface modelling (RS) and by using a Gamma model. The Gamma model can be used in a predictive manner unlike the RS models and the parameters of the RS models can be approximated from the fit of the Gamma model to the observed data. The expansion of the Gamma model explains the occurrence of the statistically significant cross terms of the RS polynomials. The emphasis within the literature of seeking interactions or synergies between environmental factors should be replaced with one emphasising the falsification of the Gamma approach. This can be done by examining the relative ratios of the gamma factors when in combination, but this also requires the use of appropriate functions to do this.

An explanation for the effect of inoculum size on MIC and the growth/no growth interface.

The inoculum effect (IE) is the phenomenon observed where changes in the inoculum size used in an experiment alters the outcome with respect to, for example, the minimum inhibitory concentration of an antimicrobial or the growth/no growth boundary for a given set of environmental conditions. Various hypotheses exist as to the cause of the IE such as population heterogeneity and quorum sensing, as well as the null hypothesis - that it is artefactual. Time to detection experiments (TTD) were carried out on different initial inoculum sizes of several bacterial species (Aeromonas hydrophila, Enterobacter sakazakii, Salmonella Poona, Escherichia coli and Listeria innocua) when challenged with different pH and with combined pH and sodium acetate. Data were modelled using a modification to a Gamma model (Lambert and Bidlas 2007, Int. J. Food Microbiology 115, 204-213), taking into account the inoculum size dependency on the TTD obtained under ideal conditions. The model suggests that changes in minimum inhibitory concentration (MIC) or in the growth-no growth boundary with respect to inoculum size are due to using a smaller or larger inoculum (i.e. is directly related to microbial number) and is not due to other, suggested, phenomena. The model used further suggests that the effect of a changing inoculum size can be modelled independently of any other factor, which implies that a simple 1 to 2-day experiment measuring the TTD of various initial inocula can be used as an adjunct to currently available models.

Comparing the antimicrobial effectiveness of NaCl and KCl with a view to salt/sodium replacement.

A study using a small range of pathogenic bacterial species (Aeromonas hydrophila, Enterobacter sakazakii, Shigella flexneri, Yersinia enterocolitica and 3 strains of Staphylococcus aureus) has shown that potassium chloride has an equivalent antimicrobial effect on these organisms when calculated on a molar basis. Combined NaCl and KCl experiments were carried out and data was analysed using a modification to the Lambert and Lambert [Lambert, R.J.W., and Lambert, R., 2003. A model for the efficacy of combined inhibitors. Journal of Applied Microbiology 95, 734-743.] model for combined inhibitors and showed that in combination KCl is a direct 1:1 molar replacement for the antimicrobial effect of common salt. If this is a general finding then, where salt is used to help preserve a product, partial or complete replacement by KCl is possible.

The taxonomy of Enterobacter sakazakii: proposal of a new genus Cronobacter gen. nov. and descriptions of Cronobacter sakazakii comb. nov. Cronobacter sakazakii subsp. sakazakii, comb. nov., Cronobacter sakazakii subsp. malonaticus subsp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov. and Cronobacter genomospecies 1.

BACKGROUND: Enterobacter sakazakii is an opportunistic pathogen that can cause infections such as necrotizing enterocolitis, bacteraemia, meningitis and brain abscess/lesions. When the species was defined in 1980, 15 biogroups were described and it was suggested that these could represent multiple species. In this study the taxonomic relationship of strains described as E. sakazakii was further investigated. RESULTS: Strains identified as E. sakazakii were divided into separate groups on the basis of f-AFLP fingerprints, ribopatterns and full-length 16S rRNA gene sequences. DNA-DNA hybridizations revealed five genomospecies. The phenotypic profiles of the genomospecies were determined and biochemical markers identified. CONCLUSION: This study clarifies the taxonomy of E. sakazakii and proposes a reclassification of these organisms.

Gamma study of pH, nitrite, and salt inhibition of Aeromonas hydrophila.

The gamma hypothesis states that there are no interactions between antimicrobial environmental factors. The time to growth of Aeromonas hydrophila challenged with pH, NaNO(2), and salt combinations at 30 degrees C was investigated. Data were examined using a model based on the gamma hypothesis (the gamma model), which takes into account variance-stabilizing transformations and which gives biologically relevant parameters. At high concentrations of NaNO(2) and at pHs of >6.0, the antimicrobial action of the nitrite ion has a strong influence (MIC = 2,033 mg liter(-1)), whereas at pHs of <6, nitrous acid is dominant (MIC = 1.5 mg liter(-1)). This change is not due to a "synergy" between pH and the nitrite ion but is due to the shift in the equilibrium concentrations of nitrous acid and nitrite in solution caused by pH. In combination with salt, the parameters found for the action of Na nitrite were identical to those found when it was examined in isolation. Therefore, pH, NaNO(2), and salt act independently on the growth of A. hydrophila. By expanding the gamma model with a cardinal temperature model, the results of fitting the model of Palumbo et al. (J. Food Prot. 54:429-435, 1994) to randomly produced environmental conditions could be reproduced, suggesting that temperature also has an independent effect.

A study of the Gamma hypothesis: predictive modelling of the growth and inhibition of Enterobacter sakazakii.

Although the temperature growth profile of the opportunistic pathogen Enterobacter sakazakii is known, few other environmental factors affecting growth have been analysed. Using a model based on the Gamma hypothesis--that antimicrobial factors in mixtures exert independent effects--a range of weak acids (lactic, acetic, propionic, citric, sorbic and benzoic), pH, salt and temperature and some of their combinations were examined. The weak acids examined inhibited principally with the acid-form of the weak acid, however, benzoic, sorbic and propionic acids also displayed an inhibitory contribution from their respective anionic forms. In all cases pH could be considered an independent inhibiting factor. The minimum pH and maximum salt concentration for growth were calculated to be 3.89 and 9.1% respectively. In combination, there was no suggestion of any interactive effect between them. Studies performed on combinations of Na acetate/pH between 25 and 41 degrees C showed that temperature did not affect the relative inhibitory effects of the weak acid/pH mixtures. The results of this study support the Gamma hypothesis suggesting that there are no synergistic interactions between inhibitory factors and that growth can be predicted from a library of known effects. More importantly to the food industry, the results can be used to design good quality shelf-life challenge tests by reducing the number of studies required.

An investigation of the Gamma hypothesis: a predictive modelling study of the effect of combined inhibitors (salt, pH and weak acids) on the growth of Aeromonas hydrophila.

The Gamma hypothesis, that multiple inhibitory factors combine independently, is the underlying hypothesis for the quantification of the Hurdle concept used in food manufacture. The literature, however, is confused as to whether interactive effects exist and under which circumstances they occur, if at all. Using the method of time to detection (TTD), the inhibitory effect of pH, salt and specific weak acids (acetic, propionic, sorbic and benzoic) and combinations of these with respect to the growth of Aeromonas hydrophila (ATCC 7966) were analysed. A model based on the relative rate to detection described all combinations analysed as having independent effects on the TTD. No synergistic interactions were found between pH and salt, between pH and individual weak acids or between combinations of weak acids and pH for any of the systems under study. This study supports the validity of the Gamma concept -- that individual environmental effects act independently and should, in turn, facilitate attempts to model the growth of other microorganisms under a variety of conditions.

Salmonella detection in probiotic products.

The presence of large amounts of probiotic bacteria in a sample may interfere with the detection of undesirable microorganisms. To illustrate this, infant formula with various strains of probiotic bacteria and the corresponding probiotic culture powders and premixes were artificially contaminated with low levels of Salmonella. Recovery of Salmonella was generally very poor when the conventional pre-enrichment procedure using buffered peptone water (BPW) was applied. However, this problem was overcome by adding antimicrobial compounds to selectively suppress the growth and/or metabolic activity of the probiotic bacteria and increasing the buffering capacity of the pre-enrichment broth. It is recommended that these analytical constraints are already addressed during the development phase of new probiotic products.