Effect of heat shock and recovery temperature on variability of single cell lag time of Cronobacter turicensis.

The effect of heat stress and subsequent recovery temperature on the individual cellular lag of Cronobacter turicensis was analysed using optical density measurements. Low numbers of cells were obtained through serial dilution and the time to reach an optical density of 0.035 was determined. Assuming the lag of a single cell follows a shifted Gamma distribution with a fixed shape parameter, the effect of recovery temperature on the individual lag of untreated and sublethally heat treated cells of Cr. turicensis were modelled. It was found that the shift parameter (Tshift) increased asymptotically as the temperature decreased while the logarithm of the scale parameter (θ) decreased linearly with recovery temperature. To test the validity of the model in food, growth of low numbers of untreated and heat treated Cr. turicensis in artificially contaminated infant first milk was measured experimentally and compared with predictions obtained by Monte Carlo simulations. Although the model for untreated cells slightly underestimated the actual growth in first milk at low temperatures, the model for heat treated cells was in agreement with the data derived from the challenge tests and provides a basis for reliable quantitative microbiological risk assessments for Cronobacter spp. in infant milk.

The microbiology of Bandji, palm wine of Borassus akeassii from Burkina Faso: identification and genotypic diversity of yeasts, lactic acid and acetic acid bacteria.

AIM: To investigate physicochemical characteristics and especially genotypic diversity of the main culturable micro-organisms involved in fermentation of sap from Borassus akeassii, a newly identified palm tree from West Africa. METHODS AND RESULTS: Physicochemical characterization was performed using conventional methods. Identification of micro-organisms included phenotyping and sequencing of: 26S rRNA gene for yeasts, 16S rRNA and gyrB genes for lactic acid bacteria (LAB) and acetic acid bacteria (AAB). Interspecies and intraspecies genotypic diversities of the micro-organisms were screened respectively by amplification of the ITS1-5.8S rDNA-ITS2/16S-23S rDNA ITS regions and repetitive sequence-based PCR (rep-PCR). The physicochemical characteristics of samples were: pH: 3.48-4.12, titratable acidity: 1.67-3.50 mg KOH g(-1), acetic acid: 0.16-0.37%, alcohol content: 0.30-2.73%, sugars (degrees Brix): 2.70-8.50. Yeast included mainly Saccharomyces cerevisiae and species of the genera Arthroascus, Issatchenkia, Candida, Trichosporon, Hanseniaspora, Kodamaea, Schizosaccharomyces, Trigonopsis and Galactomyces. Lactobacillus plantarum was the predominant LAB species. Three other species of Lactobacillus were also identified as well as isolates of Leuconostoc mesenteroides, Fructobacillus durionis and Streptococcus mitis. Acetic acid bacteria included nine species of the genus Acetobacter with Acetobacter indonesiensis as predominant species. In addition, isolates of Gluconobacter oxydans and Gluconacetobacter saccharivorans were also identified. Intraspecies diversity was observed for some species of micro-organisms including four genotypes for Acet. indonesiensis, three for Candida tropicalis and Lactobacillus fermentum and two each for S. cerevisiae, Trichosporon asahii, Candida pararugosa and Acetobacter tropicalis. CONCLUSION: fermentation of palm sap from B. akeassii involved multi-yeast-LAB-AAB cultures at genus, species and intraspecies level. SIGNIFICANCE AND IMPACT OF THE STUDY: First study describing microbiological and physicochemical characteristics of palm wine from B. akeassii. Genotypic diversity of palm wine LAB and AAB not reported before is demonstrated and this constitutes valuable information for better understanding of the fermentation which can be used to improve the product quality and develop added value by-products.

Genotypic diversity of lactic acid bacteria isolated from African traditional alkaline-fermented foods.

AIM: To identify and compare lactic acid bacteria (LAB) isolated from alkaline fermentations of cassava (Manihot esculenta Crantz) leaves, roselle (Hibiscus sabdariffa) and African locust bean (Parkia biglobosa) seeds for production of, respectively, Ntoba Mbodi, Bikalga and Soumbala. METHODS AND RESULTS: A total of 121 LAB were isolated, identified and compared by phenotyping and genotyping using PCR amplification of 16S-23S rDNA intergenic transcribed spacer (ITS-PCR), repetitive sequence-based PCR (rep-PCR) and DNA sequencing. The results revealed a diversity of genera, species and subspecies of LAB in African alkaline fermentations. The isolates were characterized as nonmotile (in most cases) Gram-positive rods, cocci or coccobacilli, catalase and oxidase negative. ITS-PCR allowed typing mainly at species level, with differentiation of a few bacteria at subspecies level. Rep-PCR permitted typing at subspecies level and revealed significant genotypic differences between the same species of bacteria from different raw materials. DNA sequencing combined with use of API 50CHL and API 20Strep systems allowed identification of bacteria as Weissella confusa, Weissella cibaria, Lactobacillus plantarum, Pediococcus pentosaceus, Enterococcus casseliflavus, Enterococcus faecium, Enterococcus faecalis, Enterococcus avium and Enterococcus hirae from Ntoba Mbodi; Ent. faecium, Ent. hirae and Pediococcus acidilactici from Bikalga and Soumbala. CONCLUSION: LAB found in African alkaline-fermented foods belong to a range of genera, species and subspecies of bacteria and vary considerably according to raw material. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study confirms that LAB survive in alkaline fermentations, a first crucial stage in determining their significance and possible value as probiotic bacteria.

Genotypic characterization of Brochothrix spp. isolated from meat, poultry and fish.

AIM: To study genotypic diversity of isolates of Brochothrix thermosphacta recovered from meat, poultry and fish. METHODS AND RESULTS: A total of 27 bacteria isolated from 19 samples of meat, poultry and fish were identified phenotypically and genotypically using PCR amplification of 16S-23S rDNA intergenic transcribed spacer (ITS-PCR), repetitive sequence-based PCR (rep-PCR) and 16S rDNA sequencing. Using ITS-PCR, all bacteria showed the same DNA profile as the reference strains of Br. thermosphacta, allowing typing of the isolates at species level. Using 16S rDNA sequencing, all isolates were identified, at genus and species level, as Br. thermosphacta. Identification as Br. campestris was observed with a lower, but very close, level of similarity. Rep-PCR was more discriminatory than ITS-PCR and allowed differentiation of four subgroups among the isolates. CONCLUSION: Minor genotypic differences among Br. thermosphacta strains from meat, poultry and fish were observed. SIGNIFICANCE AND IMPACT OF THE STUDY: A rudimentary exploration of genotypic differences of Br. thermosphacta from meat, poultry and fish resulted in preliminary confirmation of the suitability of ITS-PCR for typing Br. thermosphacta and confirmed the value of rep-PCR fingerprinting to discriminate between Br. thermosphacta strains.

Predictive modelling of growth and measurement of enzymatic synthesis and activity by a cocktail of selected Enterobacteriaceae and Aeromonas hydrophila.

The possibility was examined of developing a predictive model that would predict food spoilage by combining microbial growth (increase in cellular number) with extracellular enzymatic activity of a cocktail of five strains of Enterobacteriaceae: Escherichia coli, Enterobacter agglomerans, Klebsiella oxytoca, Klebsiella pneumoniae and Proteus vulgaris and one Aeromonas hydrophila strain. Estimations of growth and enzyme activity were made within a three-dimensional matrix of conditions: temperature 2-20 degrees C, pH value 4.0-7.5 and water activity (a(w)) 0.95-0.995. A mathematical model was constructed which predicted growth based on increases in cell number. However, although notable effects of extracellular lipases and proteases were detected, it was not possible to model enzymatic activity and prepare a combined model because the data did not follow the characteristic profile that would allow curve-fitting. Nevertheless, the model for microbial growth and information relating to enzyme activity will be made freely available in a database on the internet.

Predictive modelling of growth of Yersinia enterocolitica: the effects of temperature, pH and sodium chloride.

The growth responses of Yersinia enterocolitica as affected by NaCl concentration, pH value and storage temperature were studied in laboratory medium. Growth curves at concentrations of NaCl in the range 0.5-6.5% (w/v), pH values in the range 4.0-7.0 and storage temperatures in the range 5-30 degrees C were fitted using the Gompertz routine and the derived parameters modelled. Growth curves could then be regenerated for any set of conditions within the matrix studied and values for growth rate, generation time, lag time and time to 1000-fold increase predicted. The model was validated against data from the literature and was found to give realistic estimates for generation time in media and a range of foods including meat and meat products, milk, eggs, fish and tofu. All predictions were consistently 'fail-safe'.

Predictive modelling of growth and measurement of enzymatic synthesis and activity by a cocktail of Brochothrix thermosphacta.

The possibility was examined of developing a predictive model that combined microbial growth (increase in cellular number) and extracellular enzyme activity of a cocktail of three strains of Brochothrix thermosphacta. Estimations of growth and enzyme activity were made within a three-dimensional matrix of conditions: temperature 2-20 degrees C, pH value 4.0-7.5 and water activity (a(w)) 0.95-0.995. A model which predicted growth based on increases in cell number was constructed. No extracellular lipases were detected, but slight proteolytic reactions were observed. Although it was not possible to model protease activity, the growth model and information relating to enzyme activity will be made freely available in a database on the Internet.

Predictive modelling of growth and enzyme production and activity by a cocktail of Pseudomonas spp., Shewanella putrefaciens and Acinetobacter sp.

The possibility was examined of developing a predictive model that combined microbial growth (increase in cellular number) with extracellular lipolytic and proteolytic enzyme activity of a cocktail of four strains of Pseudomonas spp. and one strain each of Acinetobacter sp. and Shewanella putrefaciens. Environmental conditions within the following matrix of conditions were examined: temperature 2-20 degrees C, pH value 4.0-7.5 and water activity (a(w)) 0.95-0.995 and a model was constructed, which predicted growth based on increase in cell number. Data on lipase production and protease activity were generated and will be available as a database, but no function could be identified, which was a good fit to these data, since most enzymatic production and activity occurred, as expected, during transition from exponential to stationary phase. Even at lower cell numbers, in more unfavourable conditions, hydrolysing effects were detectable, which made it difficult to construct a model combining both microbiological and enzymatic data.

A survey of the microbiological quality of bottled water sold in the UK and changes occurring during storage.

Eight brands of domestic and imported bottled water were microbiologically analysed within three hours of purchase at a local supermarket. Viable numbers of microorganisms were estimated on Plate Count Agar (PCA) and PCA diluted to quarter and tenth strengths (1/4 PCA and 1/10 PCA) and incubated at temperatures of 10, 15, 25 and 37 degrees C. Plate count agar diluted to 1/4 and 1/10 incubated at 25 degrees C yielded the highest initial counts, up to 10(4) cfu ml(-1). Pseudomonas spp. was the predominant species. After 6 months of storage at room temperature (18-25 degrees C), few quantitative and qualitative differences were found in the microflora.

Preparation and validation of a growth model for Bacillus cereus: the effects of temperature, pH, sodium chloride and carbon dioxide.

The growth responses of a vegetative inoculum of Bacillus cereus as influenced by varying conditions of temperature, pH value and sodium chloride concentration (% w/v) and carbondioxide concentration (% v/v) were determined in laboratory medium. Growth curves in concentrations of NaCl in the range 0.5-10.5% (w/v), pH values in the range 4.5-7.0, CO2 concentrations in the range 10-80% (v/v) and storage temperatures from 10 degrees C to 30 degrees C were fitted using the regime of Baranyi et al. (1993). A response surface model was prepared and predictions of doubling time, growth rate, lag time and time to 1000-fold increase could be obtained for any set of conditions within the matrix studied. This model is included in Food MicroModel Version 1. Predicted doubling times from the model were compared to observed doubling times in the literature and the model was found to give realistic estimates of doubling time for a range of foods including milk, meat and poultry and carbohydrate-based products.

Predictive modelling of growth of Staphylococcus aureus: the effects of temperature, pH and sodium chloride.

The growth responses of Staphylococcus aureus as affected by NaCl concentration, pH value and storage temperature were studied in laboratory medium. Growth curves at concentrations of NaCl in the range 0.5-13.5% (w/v), pH values in the range 4.0-7.0 and storage temperatures in the range 10-30 degrees C were fitted using the Gompertz routine and the derived parameters modelled. Growth curves could then be regenerated for any set of conditions within the matrix studied and values for growth rate, generation time, lag time and time to 1000-fold increase predicted. The model was validated against data from the literature and was found to give realistic estimates of generation time for media and a range of foods including milk, cheese, starch-based foods and cooked meats but not for mayonnaise or Wiltshire bacon. All predictions were consistently 'fail-safe'.

Predictive modelling of growth of Escherichia coli O157:H7: the effects of temperature, pH and sodium chloride.

The growth responses of Escherichia coli O157:H7 as affected by NaCl concentration, pH value and storage temperature were studied in laboratory medium. Growth curves at concentrations of NaCl in the range 0.5-6.5% (w/v), pH values in the range 4.0-7.0 and storage temperatures in the range 10-30 degrees C were fitted using the Gompertz routine and the derived parameters modelled. Growth curves could then be regenerated for any set of conditions within the matrix studied and values for growth rate, generation time, lag time and time to 1000-fold increase predicted. The model was validated against data from the literature and was found to give realistic estimates for generation time in media and a range of foods including meat and poultry, milk, cheese and tempeh. All predictions were consistently 'fail-safe'.

A response surface study on the role of some environmental factors affecting the growth of Saccharomyces cerevisiae.

The combined effect of pH, ethanol and fructose on the growth of Saccharomyces cerevisiae at 25 degrees C was studied by the standard Response Surface Methodology. Canonical analysis of the obtained response surface led to the conclusion that the effects of ethanol and fructose can be described by a single factor, the value of water activity, which can be calculated from the other two. Therefore the number of explanatory variables can be reduced. The computational usefulness of the transformation bw = square root of (1-aw) is demonstrated.

Predictive modelling of growth of Listeria monocytogenes. The effects on growth of NaCl, pH, storage temperature and NaNO2.

The effect of NaCl concentration (5.0 115.0 g/l). pH value (4.0-7.2), temperature (1-35 degrees C) and NaNO2 concentration (0 200 mg/l) on the growth responses of Listeria monocytogenes, in laboratory medium was investigated. The growth curves generated within this matrix of conditions were fitted using the function of Baranyi and Roberts (1994) and the growth responses modelled using a quadratic polynomial to produce response surfaces. Growth curves could then be regenerated for any set of conditions within the experimental matrix and values predicted for the growth rate, doubling time, lag time and time to 1000-fold increase. The model was validated using data from published literature and was found to give realistic predictions for doubling times in foods, including meat and meat products, milk, dairy products and vegetables. Predictions from this model (Baranyi and Roberts. 1994) compared favourably with those from the models of Buchanan and Phillips (1990), Murphy et al. (1996) and the Food MicroModel.

Mathematical modelling of the growth, survival and death of Yersinia enterocolitica.

This paper discusses the development of a novel mathematical equation capable of handling the growth, survival and death of microorganisms. The equation was used in combination with second-order polynomials to fit a response surface to data representing the growth, survival and death of Yersinia enterocolitica as affected by temperature (0-30 degrees C), sodium chloride (0-10.5% w/v), pH (3.5-7.0) and undissociated lactic acid (0-9.63 g/l). The resulting predictive model showed a good correlation over the entire range of the experimental data. Predictions from the model have been compared with independent data from both published literature and inoculated food studies. Analysis of the results showed a good correlation, particularly for data from milk and meat products.

Predictive modelling of Escherichia coli O157:H7: inclusion of carbon dioxide as a fourth factor in a pre-existing model.

Two models for Escherichia coli O157:H7 are compared, one with growth-controlling factors pH (4.5-7.0), temperature (10-30 degrees C) and NaCl concentration (0.5-6.5% w/v) and the other with the same factors and ranges, but with the addition of carbon dioxide (CO2: 10-80% v/v). Validation of the four-factor model, to include food packed in modified atmospheres containing CO2, was not possible due to lack of published data. However, where CO2 concentration was entered as 0%, only minor differences occurred between the predictions from the two models for the same conditions of pH, NaCl and temperature; consequently reliable, safe predictions using the four-factor model, with CO2 concentration recorded as 0%, can be made for foods packed in air. At temperatures from 10 to 30 degrees C, it was found that lower (10 and 20%) concentrations of CO2 had little effect on lag times and growth rates, and higher concentrations still permitted growth of E. coli O157:H7 under a wide range of conditions of NaCl concentration, pH value and temperature, suggesting that the organism is relatively CO2-tolerant.

Modelling the growth, survival and death of microorganisms in foods: the UK food micromodel approach.

Techniques for the development of mathematical models in the area of predictive microbiology have greatly improved recently, allowing better and more accurate descriptions of microbial responses to particular environmental conditions, thus enabling predictions of those responses to be made with greater confidence. Recognising the potential value of applying these techniques in the food industry, the Ministry of Agriculture, Fisheries and Food (MAFF) initiated a nationally coordinated five-year programme of research into the growth and survival of microorganisms in foods, with the aim of developing a computerised Predictive Microbiology Database in the UK. This initiative has resulted in the systematic generation of data, through protocols which ensure consistency of methodology, so that data in the database are truly comparable and compatible, and lead to reliable predictive models. The approaches taken by scientists involved in this programme are described and the various stages in the development of mathematical models summarized. It is hoped that this initiative and others being developed in the USA, Australia, Canada and other countries, will encourage a more integrated approach to food safety which will influence all stages of food production and, eventually, result in the development of an International Predictive Microbiology Database.

Radiographic appearance of the odontoid lateral mass interspace in the occipitoatlantoaxial complex.

STUDY DESIGN: This is an anatomic study in which the odontoid lateral mass interspace is evaluated radiographically in various positions of upper cervical spine rotation. OBJECTIVES: The objectives are to determine whether odontoid lateral mass interspace asymmetry exists in the ligamentously intact cervical spine and to define odontoid lateral mass interspace behavior during atlantoaxial rotation. SUMMARY OF BACKGROUND DATA: Controversy exists regarding the behavior of the odontoid lateral mass interspace during atlantoaxial rotation. The meaning of interspace asymmetry varies depending on the author and the method of study. Atlas fractures account for 7% of cervical spine fractures and are frequently associated with other cervical fractures, so an understanding of the radiographic anatomy is crucial to the evaluation of these patients. METHODS: Ten human cadaveric cervical spines were dissected of all but ligamentous soft tissue and were mounted and prepared for radiographic study in neutral position and in varying degrees of right and left rotation. Radiographs were evaluated for odontoid lateral mass interspace asymmetry in neutral and in rotated positions, and data were analyzed. RESULTS: Measurable asymmetry could be shown when comparing neutral positions and when comparing rotated with neutral positions. A statistically significant difference could not be proven in this asymmetry, although clinical significance is suggested by the analysis of the data. CONCLUSION: Measurable asymmetry can be present in the neutrally positioned, ligamentously intact atlanto-axial complex and is not necessarily indicative of instability. This agrees with the concept of the "neutral zone" as described by White and Panjabi in Clinical Biomechanics of the Spine. There is a trend for increasing odontoid lateral mass interspace on the side to which the head is rotated, and this odontoid lateral mass interspace is measurably different from the contralateral odontoid lateral mass interspace, indicating that odontoid lateral mass interspace asymmetry is not a good indicator for cervical instability in the otherwise asymptomatic individual.

Observational study of temperature, moisture, pH and bacteria in straw bedding, and faecal consistency, cleanliness and mastitis in cows in four dairy herds.

A study of four dairy farms showed that much of the straw stored for bedding was too wet (over 15 per cent moisture content). Most of the beds, including their top surfaces, were damp (above 75 per cent relative humidity). The temperature of the surface of most of the straw beds was related to the air temperature, many being below 15 degrees C, but below the surface the temperatures of most beds reached between 15 degrees C and 45 degrees C within about a week of their being renewed. Bacterial counts also reached a plateau within one to two weeks. The pH of the top layers of straw was usually between 8.5 and 9.5. Adding lime daily to the top layer of the straw failed to raise the pH to levels at which Escherichia coli and Streptococcus uberis do not survive. Most of the counts of E coli and faecal streptococci in the top layers of straw were above 10(6) colony-forming units/g. Counts of E coli and S uberis were much higher in the beds of early lactation cows than in those of dry cows. Many of the early lactation cows were heavily and persistently contaminated with faeces. Dry cows were much cleaner. Groups of cows with firmer faeces were also cleaner. The farm with the lowest incidence of mastitis had the cleanest cows and the most satisfactory beds.

Assessing the effects of exposure to environmental stress on some functional properties of Bifidobacterium animalis ssp. lactis.

This study assessed the effects of exposing a strain of Bifidobacterium animalis ssp. lactis to acid, bile and osmotic stresses on antagonistic properties, biofilm formation and antibiotic susceptibility/resistance profile. Exposure to each stress factor appeared to have no significant effect on the antagonism against Escherichia coli NCTC 12900 and Salmonella enterica serovar Enteritidis PT4. No suppression in biofilm formation due to exposure to stress was observed. Bile and osmotic stresses resulted in significantly higher biofilm formation. Expression of an exopolysaccharide synthesis gene, gtf 01207, was significantly higher when the B. animalis ssp. lactis strain was exposed to osmotic stress. Susceptibility of the B. animalis ssp. lactis strain to chloramphenicol, erythromycin, ampicillin and vancomycin, and resistance to tetracycline remained unchanged when exposed to each stress. The expression of a tetracycline resistance gene, tet(W), was significantly higher when exposed to each stress. These results may suggest that the potential for the B. animalis ssp. lactis strain to provide probiotic benefit, after exposure to the stressful conditions of the gastrointestinal tract, remains intact.