Bacillus cereus cell and spore properties as influenced by the micro-structure of the medium.

AIM: To investigate the effect of different growth conditions on Bacillus cereus cell and spore properties. METHODS AND RESULTS: Bacillus cereus was grown on agar plates with different surface water conditions (wet and dry) or viscosity. Cell populations displayed different types of behaviour, and heterogeneity was manifested in cell motility and dimension. Spore populations were heterogeneous regarding their properties, namely size and thermal resistance. The smallest spores were produced from flagellated cells, which also displayed jet-motility, growing on the wettest agar. Cytometric analysis also revealed within the smallest spores a sub-population labelled by propidium iodide (PI), indicating that spore populations were partly damaged. Nonmotile cells grown on diffusion-limiting media were elongated and produced the least thermal-resistant spores. CONCLUSIONS: The micro-structural properties of the media were found to influence cell and spore properties. Abundant surface water enabled flagellar motility and resulted in a heterogeneous cell and spore population, the latter including small and damaged spores. High viscosity gave rise to filamentous cells and more heat-sensitive spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information on conditions resulting in heterogeneous populations of damaged and heat-sensitive spores.

The food safety management system.

A comprehensive food safety strategy involves establishing risk management goals, food safety objectives and, for production systems, performance objectives and performance criteria. The working instructions for each step of the process should be validated for their effect before integration within a specific HACCP plan. The importance of realistic inactivation models to predict the hygienic equivalence of food processing operations is discussed.

Probabilistic representation of the exposure of consumers to Clostridium botulinum neurotoxin in a minimally processed potato product.

We have examined the potential of a well-specified, minimally processed potato product as a vehicle for the exposure of consumers to Clostridium botulinum neurotoxin. The product is a relatively simple combination of raw potato flakes, flour, starch and other minor ingredients and has an extended lifetime under refrigeration conditions. A combination of information and data, from a variety of sources that includes the manufacturer, has shown that the product is particularly safe with respect to non-proteolytic C. botulinum hazards. The model concentrates on a simple end point, the toxicity of an individual retail unit of the product at the point of consumer preparation, which is related to an individual risk. The probabilistic analysis was built using Bayesian Belief Network (BBN) techniques.

Prevalence of Clostridium species and behaviour of Clostridium botulinum in gnocchi, a REPFED of italian origin.

Sales and consumption of refrigerated processed foods of extended durability (REPFEDs) have increased many-fold in Europe over the last 10 years. The safety and quality of these convenient ready-to-eat foods relies on a combination of mild heat treatment and refrigerated storage, sometimes in combination with other hurdles such as mild preservative factors. The major hazard to the microbiological safety of these foods is Clostridium botulinum. This paper reports on the prevalence and behaviour of proteolytic C. botulinum and non-proteolytic C. botulinum in gnocchi, a potato-based REPFED of Italian origin. Attempts to isolate proteolytic C. botulinum and non-proteolytic C. botulinum from gnocchi and its ingredients were unsuccessful. Based on assessment of the adequacy of the methods used, it was estimated that for proteolytic C. botulinum there was < 25 spores/kg of gnocchi and < 70 spores/kg of ingredients. The total anaerobic microbial load of gnocchi and its ingredients was low, with an estimated 1 MPN/g in processed gnocchi. Most of the anaerobic flora was facultatively anaerobic. A few obligately anaerobic bacteria were isolated from gnocchi and its ingredients and belonged to different Clostridium species. The protection factor, number of decimal reductions in the probability of toxigenesis from a single spore, was determined for eight different gnocchi formulations by challenge test studies. For all gnocchi stored at 8 degrees C (as recommended by the manufacturer) or 12 degrees C (mild temperature abuse), growth and toxin production were not detected in 75 days. The protection factor was >4.2 for proteolytic C. botulinum, and >6.2 for non-proteolytic C. botulinum. When inoculated packs were stored at 20 degrees C (severe temperature abuse), toxin production in 75 days was prevented by the inclusion of 0.09% (w/w) sorbic acid (protection factors as above), however in the absence of sorbic acid the packs became toxic before the end of the intended shelf-life and the protection factors were lower. Providing sorbic acid (0.09% w/w) is included in the gnocchi, the safety margin would seem to be very large with respect to the foodborne botulism hazard.

Determination of peroxy radical-scavenging of lactic acid bacteria.

Responses of lactic acid bacteria (LAB) to peroxy radicals generated via thermal (40 degrees C) decomposition of the diazocompound 2,2,-azo-bis (2-amidinopropane) dihydrochloride (ABAP), were studied. In general, LAB displayed survival curves with shoulders and tails indicative of 'multihit' killing by exposure to peroxy radicals. One strain, Lactococcus lactis subsp. lactis DIP15, producing a slope of 0.0105 in the kinetic analysis when exposed to 4 mM ABAP, exhibited a measurable antioxidant capacity. The other LAB failed to show any significant antioxidant capacity. The antioxidant capacity of strain DIP15 remained constant after cells have been heat-treated, suggesting that compounds bearing free radical scavenging capacity are rather stable.

Influence of agar content on the growth parameters of Bacillus cereus.

Factors governing Bacillus cereus colony growth on agar media as modified by the agar content (1-7%, w/v) were studied. Agar had a significant effect on the radial growth rate which diminished as the agar content increased. Cell density in colonies (colony density) was found to decrease during the incubation time, with lower values occurring in the presence of 1% agar. Size and DNA content of the cells grown on 1 and 7% agar were similar. An increasing proportion of cell population growing on 7% agar produced spores during the 24-h incubation period. It was shown that 'water condition' on the agar surface could be associated with colony density, with 7% agar media presenting the thinnest nominal thickness of the liquid film. On the other hand, the partial drying phenomena of the agar media which occur during preparation and incubation, could not account for the observed differences in colony growth.

Prevalence and behaviour of Bacillus cereus in a REPFED of Italian origin.

This paper reports on the prevalence and behaviour of Bacillus cereus in gnocchi, a REPFED of Italian origin. A survey of gnocchi under varying storage conditions, revealed that, although B. cereus was found in 33% of the samples, the contamination level was lower than 10(2) CFU/g for the unstored and the refrigerated (8 degrees C) samples. Counts increased with increasing storage temperatures and prolonged storage times in samples prepared without sorbic acid. The effect of different formulations (sorbic, citric and lactic acid) and storage conditions (8, 12 and 20 degrees C) have been evaluated in a challenge testing with spores of B. cereus. Results indicate that the use of sorbic acid in association with citric or lactic acid to pH 5.0 is effective in inhibiting growth of B. cereus and the anticipated shelf life of the product is safe even if temperature abuse occurs. If sorbic acid is omitted, lactic acid can inhibit B. cereus growth during storage at 8 degrees C. On the contrary, when temperature abuse occurs (12 and 20 degrees C), lactic or citric acid are not able to prevent growth of B. cereus.

Growth of Bacillus cereus on solid media as affected by agar, sodium chloride, and potassium sorbate.

The effect of two independent variables: microstructure, as modified by the agar content (1.0, 4.0, 7.0%), and water activity (a(w)), as modified by the NaCl content (0.5, 2.5, 4.5%), in the absence or in the presence of potassium sorbate (0.0; 2,000 ppm) on Bacillus cereus growth on solid media was studied. The time to visible growth (TVG) and the radial growth rate (RGR) of colonies were evaluated. TVG was not affected by microstructure and K-sorbate, although when a(w) was reduced, TVG tended to increase. RGR depended on linear effects of microstructure and a(w) variables and their interaction. When K-sorbate was added to cultural media, RGR was reduced significantly. However, in the presence of K-sorbate, RGR was found to change only when a(w) vas varied.

Investigation of the ability of proteolytic Clostridium botulinum to multiply and produce toxin in fresh Italian pasta.

The ability of proteolytic Clostridium botulinum (types A, B, and F) to produce toxin in filled fresh Italian pasta (tortelli) packed under a modified atmosphere was investigated. Four types of tortelli (filled with artichoke, meat, ricotta-spinach, or salmon) were inoculated with a suspension of heat-shocked spores to give an initial concentration of approximately 10(3) spores per piece. Samples were incubated at both 12 and 20 degrees C for up to 50 days and examined at selected time intervals for the presence of toxin by an ELISA and the mouse test. Toxin was not detected in any tortelli stored at 12 degrees C. When storage was at 20 degrees C, toxin was detected in the salmon-filled tortelli at day 30, in the meat and ricotta-spinach tortelli at day 50, but not in the artichoke-filled tortelli at day 50.

Influence of structural properties and kinetic constraints on Bacillus cereus growth.

The influence of structural properties and kinetic constraints on the behavior of Bacillus cereus was investigated on agar media. Dimensional criteria were used to study the growth in bacterial colonies. The architecture of the agar gel as modified by the agar content was found to influence the colony size, and smaller colonies were observed on media containing 50 to 70 g of agar liter-1. Except at low nutrient levels, colonies responded to nutrient gradients by decreasing in size the farther away they were from the nutrient source, and the decrease in colony size was influenced by the agar content. The diffusivities of glucose and a protein (insulin-like growth factor) were not affected by the gel architecture, suggesting that other factors, such as mechanical factors, could influence microbial growth in the agar systems used. Increasing the viscosity of the liquid phase of the agar media by adding polyvinylpyrrolidone resulted in a reduction in colony size. When the agar concentration was increased, the colony areas were not influenced by the viscosity of the system.

Comparison between office and ambulatory blood pressure in young and elderly subjects with isolated systolic hypertension.

BACKGROUND: It has been claimed that isolated systolic hypertension (ISH) in the elderly is not a sustained condition but a short-lasting increase in office systolic blood pressure magnified by arterial stiffness. DESIGN: Office and ambulatory blood pressures werecompared at baseline and after 3 months of observation of young and elderly subjects with ISH. METHODS: The study was carried out in 39 young (mean age 27.1+/-9.8 years) and 37 elderly patients (mean age 72.5+/-5.7 years). Office blood pressure was defined as the mean of six readings. All subjects underwent two non-invasive 24 h blood pressure monitorings performed 3 months apart and echocardiography (n = 50). RESULTS: The difference between office and mean 24 h systolic/diastolic blood pressure was 27.9/8.2 mmHg in the young and 18.9/6.9 mmHg in the elderly patients (P < 0.01 for systolic blood pressure). Twenty-four-hour (P < 0.001), daytime (P = 0.001) and night-time (P < 0.001) systolic blood pressures were higher in the elderly and the difference between daytime and night-time systolic blood pressure was greater in the young (P < 0.05). Office and ambulatory heart rates were significantly higher in the young subjects. The elderly patients showed a greater left ventricular wall thickness ( P = 0.005 for posterior wall; P < 0.005 for septum), relative wall thickness (P = 0.01) and left ventricular mass index (P = 0.001) and impaired left ventricular filling rate ( P = 0.05), whereas systolic performance and stroke volume were no different in the two groups. Due to the higher heart rate, cardiac output was greater in the young (P = 0.03). CONCLUSION: These data show that larger differences between office and ambulatory systolic blood pressure are not unique to elderly patients with ISH. Increased ambulatory blood pressure levels and a decreased nocturnal blood pressure fall were associated with left ventricular structural and functional abnormalities in the elderly subjects.

Effect of evening versus morning benazepril on 24-hour blood pressure: a comparative study with continuous intraarterial monitoring.

In a single-blind, in-patient, crossover study, the influence on the circadian blood pressure (BP) profile of the 9:00 a.m. versus the 9:00 p.m. acute administration of a single dose of benazepril 10 mg, a new angiotensin-converting-enzyme inhibitor, was assessed in 10 hypertensive patients by means of 24-hour intraarterial ambulatory BP monitoring. Mean 24-hour BP for the three treatments (placebo, benazepril a.m., benazepril p.m.) were 155/93, 131/83 and 138/86 mmHg, respectively. No significant differences between the two benazepril schedules were found in terms of either 24-hour or day-time and night-time mean BP values. However, hourly averages showed that benazepril a.m. had a more sustained antihypertensive effect than benazepril p.m., where a loss of efficacy was observed 19 hours after the administration. BP responses to static and dynamic exercise and to cold pressor test were unchanged after both benazepril schedules, as were BP peaks. These results demonstrate that acute benazepril administration markedly reduces systolic and diastolic BP. The morning administration is preferable because it more effectively covers the whole 24 hours than an evening dose.

Intra-arterial blood pressure monitoring in the evaluation of the hypertensive athlete.

To compare the blood pressure (BP) changes during a long-distance run with those during bicycle ergometry, nine normotensive and 18 hypertensive joggers were studied by means of ambulatory intra-arterial monitoring. In all subjects the ergometric test caused a progressive increase in systolic and little change in diastolic BP. Exertional BP levels were closely related to pre-exercise baseline values (P less than 0.001). A different BP pattern was observed during track running, as a sharp rise in systolic BP reaching maximum values 2-4 min after the start was recorded. Subsequently, systolic BP progressively declined throughout the run, only to increase again during the final sprint. Diastolic BP fell markedly at the onset of the run and then remained substantially stable throughout. A poor relationship was observed between the BP values at peak exercise and baseline levels (P less than 0.05) as the normotensives showed a significantly higher BP response than the hypertensives. On the contrary, during the ergometric test a parallel increase in BP was recorded in the normotensive and the hypertensive joggers. No correlation was found between the BP response to track running and to bicycle ergometry. These results indicate that the BP response to a standard stress test is not predictive of the BP changes determined by a long-distance run. The BP increase with strenuous effort seems to be reduced in hypertensive individuals, probably because of latent impairment of cardiac performance.

Blood pressure changes during heavy-resistance exercise.

To study the mechanisms of the blood pressure changes during weight-lifting, three hypertensive and five normotensive body-builders underwent continuous intra-arterial monitoring. In two subjects (one normotensive and one hypertensive), intrathoracic and intra-abdominal pressures were also measured. Extremely high blood pressure elevations of up to 345/245 mmHg were observed during the lifts. Squatting caused the highest pressure rises and single-arm curls the lowest. Both the intrathoracic and the intra-abdominal pressures increased greatly during each lift and closely paralleled the changes in intra-arterial pressure. A close correlation was found between the blood pressure increase during the exercise and during a hand-grip test (r = 0.95, P less than 0.001). These results suggest that a pronounced increase in intra-thoracic and intra-abdominal pressures is a major determinant of the blood pressure elevations occurring during weight-lifting. The pressor reflex which accompanies static contractions and the individual baseline blood pressure levels also seem to affect the height of the pressure peaks.

Beats modulate blood pressure during running.

Blood pressure (BP) changes during running were studied in 25 subjects with intraarterial monitoring. Periodic pulse pressure variations ranging from 20 to 200 mm Hg were recorded throughout the exercise. To prove that these pressure oscillations were due to a "beat" phenomenon 10 athletes ran with a Teruflex container filled with saline: pressure changes up to +/- 62 mm Hg were recorded in the container. These pressure waves were added by computer to the sphygmic waves recorded intraarterially in the same subject during bicycle ergometry: the resultant tracing showed a beat-shaped pattern similar to that recorded during running.

[Cold pressor test in the evaluation of autonomic neuropathy]. / Il cold pressor test nella valutazione della neuropatia autonomica.

Neurogenic orthostatic hypotension is due to interruption of the baroreceptor reflex. To optimize the pharmacological therapy in this clinical setting the lesion should be carefully pinpointed and the degree of denervation established. To this purpose 7 patients with autonomic failure underwent ambulatory intraarterial monitoring, which showed the reversal of the normal 24-hour blood pressure (BP) pattern. During daytime BP gradually rose from its lowest point early in the morning to a peak during late evening. No BP fall was observed during sleep. Tilting test, Valsalva maneuver, hyperventilation, cold pressor test, handgrip and pharmacological tests showed interruption of the efferent pathway of the reflex. The BP fall 60 seconds after assuming the orthostatic position significantly correlated with the BP response to the cold pressor test (r = 0.89; p less than 0.01), while it did not with the BP changes during hand grip and hyperventilation and with the orthostatic increase of heart rate. Therefore, the BP response to the cold pressor test seems the most reliable indicator of the degree of autonomic dysfunction in the lesions of the efferent pathway of the reflex.

Blood pressure changes during physical exercise (the beat phenomenon).

We have previously reported that blood pressure during running shows a peculiar pattern attributable to the beat phenomenon. To elucidate the mechanism behind this phenomenon in 10 amateur athletes, intra-arterial blood pressure was continuously recorded using the Oxford technique. During the run, each athlete carried on his chest a container filled with saline kept under pressure, connected to a second transducer. In the container, pressure waves ranging in amplitude from +/- 10 to +/- 62 mmHg were recorded. Their frequency was equal to that of the athletes' strides. When these waves were added by computer to the blood pressure tracing recorded during a bicycle ergometric test, the resultant harmonic proved to be similar to the tracing observed during running. The present results demonstrate that the running-induced blood pressure pattern is the sum of the accessory wave generated by the rhythmic aortic shocks produced by running locomotion and the normal sphygmic wave.

[Validity of the dynamic exertion test for the diagnosis of arterial hypertension. The exertion test in hypertension]. / Validità del test da sforzo dinamico per la diagnosi di ipertensione arteriosa. Il test da sforzo nell'ipertensione.

To assess the role of dynamic stress test in hypertension three normotensive and ten borderline or mild hypertensive subjects were studied by means of the intraarterial Oxford technique. In all of the subjects the intraarterial monitoring lasted for 12 hours; during the recording a graded maximal bicycle exercise test was performed. Five patients considered hypertensive on the basis of their office blood pressure readings showed normal average 12-hour intraarterial recordings. Systolic blood pressure increase during ergometry ranged from 22 to 57 mmHg; exertional pressure readings were strictly related to average 12 hours intraarterial blood pressure (r = 0.92; p less than 0.001). No correlation was found between resting blood pressure and the exertional pressure taken with the auscultatory method. These data indicate that the level that blood pressure attains during ergometry primarily depends on the resting blood pressure values, and therefore do not justified the separation between normo and hyperreactive subjects as some authors have done. In conclusion, dynamic exercise may be a useful diagnostic test for hypertension but has no predictive value for either the development of high blood pressure or the progression from borderline to more severe forms of hypertension.

[Behavior of arterial pressure during sports activity: track running]. / Comportamento della pressione arteriosa durante l'attività sportiva: corsa piana.

In 6 normotensive and 14 borderline or mild hypertensive runners the intraarterial blood pressure (BP) changes determined by a maximal bicycle ergometric test and by an exhaustive run performed in outdoor conditions were recorded by means of the portable Oxford system. During running systolic BP attained higher values than during bicycle ergometry, while diastolic BP decreased during running and remained unchanged during ergometry. The highest BP values were recorded soon after starting running, then they progressively declined to increase once again during the final sprinting. Beat to beat analysis of the pressure tracings showed a peculiar pattern of the phasic waves throughout the run: continuous cyclic oscillations of pulse pressure were detected, as the variations of systolic blood pressure were not paralleled by those of the diastolic. The frequency of these rhythmic oscillations varied from 6-7 to 24-27/minute and was not related to respiration rate. The shape of these oscillations prompted us to investigate whether they were due to the "beat" phenomenon, that is to the combined effect of two waves with a nearly equal frequency. To verify this hypothesis 10 athletes during the run carried around the chest a Teruflex container filled with saline. The amplitude of the BP changes recorded in the container ranged from +/- 10 to +/- 50 mmHg. They were added by means of a computer to the BP tracing recorded during bicycle ergometry. The resultant wave was similar to that recorded in the radial artery during running.(ABSTRACT TRUNCATED AT 250 WORDS)