Minimum Growth Temperature Research Articles

Temperature and nutrient availability control growth rate and fatty acid composition of facultatively psychrophilic Cobetia marina strain L-2.

A facultative psychrophilic bacterium, strain L-2, that grows at 0 and 5 degrees C as minimum growth temperatures in complex and defined media, respectively, was isolated. On the basis of taxonomic studies, strain L-2 was identified as Cobetia marina. The adaptability of strain L-2 to cold temperature was higher than that of the type strain and of other reported strains of the same species. When the bacterium was grown at 5-15 degrees C in a defined medium, it produced a high amount of trans-unsaturated fatty acids. By contrast, in a complex medium in the same temperature range it produced a low amount of trans-unsaturated fatty acids. In the complex medium at 5 degrees C, the bacterium exhibited a three-fold higher growth rate than that obtained in the defined medium. Following a temperature shift from 11 to 5 degrees C, strain L-2 grew better in complex than in defined medium. Furthermore, when the growth temperature was shifted from 0 to 5 degrees C both the growth rate and the yield of strain L-2 growing in complex medium was markedly enhanced. These phenomena suggest that an upshift of the growth temperature had a positive effect on metabolism. The effects of adding complex medium components to the defined medium on bacterial growth rate and fatty acid composition at 5 degrees C were also studied. The addition of yeast extract followed by peptone was effective in promoting rapid growth, while glutamate addition was less effective, resulting in a cis-unsaturated fatty acid ratio similar to that of cells grown in the complex medium. These results suggest that the rapid growth of strain L-2 at low temperatures requires a high content of various amino acids rather than the presence of a high ratio of cis-unsaturated fatty acids in the cell membrane.

Halomonas neptunia sp. nov., Halomonas sulfidaeris sp. nov., Halomonas axialensis sp. nov. and Halomonas hydrothermalis sp. nov.: halophilic bacteria isolated from deep-sea hydrothermal-vent environments

To assess the physiological and phylogenetic diversity of culturable halophilic bacteria in deep-sea hydrothermal-vent environments, six isolates obtained from low-temperature hydrothermal fluids, sulfide rock and hydrothermal plumes in North and South Pacific Ocean vent fields located at 1530-2580 m depth were fully characterized. Three strains were isolated on media that contained oligotrophic concentrations of organic carbon (0.002 % yeast extract). Sequencing of the 16S rRNA gene indicated that all strains belonged to the genus Halomonas in the gamma-subclass of the Proteobacteria. Consistent with previously described species, the novel strains were slightly to moderately halophilic and grew in media containing up to 22-27 % total salts. The isolates grew at temperatures as low as -1 to 2 degrees C and had temperature optima of 30 or 20-35 degrees C. Both the minimum and optimum temperatures for growth were similar to those of Antarctic and sea-ice Halomonas species and lower than typically observed for the genus as a whole. Phenotypic tests revealed that the isolates were physiologically versatile and tended to have more traits in common with each other than with closely related Halomonas species, presumably a reflection of their common deep-sea, hydrothermal-vent habitat of origin. The G+C content of the DNA for all strains was 56.0-57.6 mol%, and DNA-DNA hybridization experiments revealed that four strains (Eplume1(T), Esulfide1(T), Althf1(T) and Slthf2(T)) represented novel species and that two strains (Eplume2 and Slthf1) were related to Halomonas meridiana. The proposed new species names are Halomonas neptunia (type strain Eplume1(T)=ATCC BAA-805(T)=CECT 5815(T)=DSM 15720(T)), Halomonas sulfidaeris (type strain Esulfide1(T)=ATCC BAA-803(T)=CECT 5817(T)=DSM 15722(T)), Halomonas axialensis (type strain Althf1(T)=ATCC BAA-802(T)=CECT 5812(T)=DSM 15723(T)) and Halomonas hydrothermalis (type strain Slthf2(T)=ATCC BAA-800(T)=CECT 5814(T)=DSM 15725(T)).

Growth of Autobioluminescent Campylobacter jejuni in Response to Various Environmental Conditions

An autobioluminescent Campylobacter jejuni organism generated from the fusion of luxCDABE genes from Xenorhabdus luminescens to the flaA promoter of C. jejuni was used in conjunction with gel-stabilized gradient plates to map the responses of this organism to three environmental factors (pH, NaCl concentration, and temperature) and various concentrations of l-fucose, d-fucose, and sodium desoxycholate. The minimum, optimum, and maximum temperatures for the growth of auto-bioluminescent C. jejuni ATCC 35921 in solid media were found to be 30, 40, and 45°C, respectively. At its optimum growth temperature, C. jejuni ATCC 35921 was able to grow well at pHs of 5.5 to 8.0 and in the presence of up to 1.70 to 1.75% NaCl. At its minimum growth temperature, however, C. jejuni ATCC 35921 could grow only at pHs of 6.5 to 8.0 and in the presence of up to 0.5% NaCl. l-Fucose, d-fucose, and sodium desoxycholate were shown to inhibit the growth of C. jejuni. Autobioluminescent C. jejuni was also used to determine whether the flaA promoter responds to both environmental conditions and chemical stimulants. While sodium desoxycholate was found to down-regulate C. jejuniflaA promoter activity, l-fucose was found to up-regulate its activity. Sodium chloride, pH, and d-fucose were all shown to exert no significant effects on promoter activity. Bacterial bioluminescence in combination with two-dimensional gradient gels is a powerful tool for studying the behavior of bacteria exposed to various environments.

GROWTH KINETICS OF CLOSTRIDIUM PERFRINGENS IN COOKED BEEF1

ABSTRACTThe objective of this work was to investigate the growth kinetics of a three‐strain cocktail of Clostridium perfringens in cooked beef. The study was conducted by growing the heat‐activated spores in ground beef under isothermal conditions between 17–50C. A four‐parameter Gompertz equation was used as a primary model to fit the growth curves along with a modified Ratkowsky model to analyze the temperature dependence of the bacterial growth. Results indicated that the Gompertz model could accurately describe the growth of C. perfringens in cooked beef. The estimated theoretical minimum, optimum, and maximum growth temperatures of this organism in cooked beef were 9.8, 47.1, and 50.8C, respectively. A linear relationship between the durations of the lag and exponential phases of growth curves was observed in this study. Such a linear relationship can be used to generate a linear isothermal growth curve complete with the lag, exponential, and stationary phases without complex mathematical computation. The kinetic models and growth parameters obtained from this study potentially can be applied to the food industry to design appropriate cooling schedules and estimate the growth of C. perfringens in thermally processed beef products under temperature abuse conditions.

Glaciecola mesophila sp. nov., a novel marine agar-digesting bacterium.

Alteromonas-like strains KMM 241(T) and KMM 642, isolated from marine invertebrate specimens, were investigated to clarify their taxonomic position. The novel isolates were aerobic, Gram-negative, motile, slightly halophilic and heterotrophic and hydrolysed polysaccharides. They did not hydrolyse urea, gelatin or casein and produced acid weakly from carbohydrates. The DNA G + C content ranged between 44.6 and 44.8 mol%. DNA-DNA similarity between the two strains was 71%. Comparison of the 16S rRNA gene sequence of strain KMM 241(T) revealed 94.5-94.8% similarity to Glaciecola species. The novel strains shared several phenotypic and physiological properties with members of Glaciecola, but they differed in their lack of pigment production, their minimal and maximal growth temperatures and their ability to hydrolyse agar and carrageenan and in the utilization of organic compounds. On the basis of phenotypic and physiological characteristics as well as phylogenetic analysis, the isolates should be assigned to a novel species, Glaciecola mesophila sp. nov. The type strain is strain KMM 241(T) (=DSM 15026(T)).

Bacillus cereus emetic toxin production in cooked rice

Three mesophilic strains of Bacillus cereus known to produce emetic toxin were used to model germination, growth and emetic toxin production in boiled rice cultures at incubation temperatures ranging from 8°C to 30°C. Minimum temperatures for germination and growth in boiled rice were found to be 15°C for all strains. Toxin production at 15°C was found to be significantly greater ( P<0·01; reciprocal toxin titre of 373±124) than at 20°C and 30°C (reciprocal toxin titres 112±37 and 123±41, respectively). Toxin production became detectable after 48 h incubation at 15°C, with a maximum titre reached by 96 h. At 20°C and 30°C, toxin production was detected at 24 h incubation, with a maximum titre reached by 72 h. Toxin production at 15°C was detectable at lower bacterial counts (6·2 log 10 cfu g −1), than with incubation at 20°C and 30°C (>7·0 log 10 cfu g −1). In this study, the lower temperature limit for germination and growth on solid laboratory medium was found to be 12°C for all strains, i.e. 3°C lower than that observed in boiled rice.

Phytophthora palmivora a New Pathogen of Lavender in Italy.

Root rot caused by Phytophthora nicotianae is considered the most serious disease of lavender in commercial cultivations in Italy. In summer 2001, in the Gela area (Sicily), ≈60% of 34,000 2-year-old landscape shrubs of English lavender (L. angustifolia) grown in a clay loam soil showed symptoms of dieback associated with root rot. Plants had been transplanted from pots in May and watered using a trickle irrigation system. A species of Phytophthora was isolated consistently from roots of symptomatic plants using potato dextrose agar (PDA) containing benomyl, nystatin, pentachloronitrobenzene, rifampicin, ampicillin, and hymexazol. The species was identified as P. palmivora on the basis of morphological and cultural characters. Ten representative single-zoospore isolates were characterized. On agar media, the isolates produced elliptical to ovoid, papillate sporangia, with a mean length/breadth ratio of 1.8. Sporangia, produced on sporangiophores forming simple sympodia (as many as 20 sporangia per sympodium), were caducous with a short pedicel (mean pedicel length = 5 μm) and a conspicuous basal plug. In addition to typical sporangia, all isolates produced sporocysts, i.e., subglobose, nonpapillate sporangia (2). The minimum temperature for mycelium growth on PDA was 10°C, the optimum was 27°C, and the maximum was 35°C. All isolates were A1 mating type. Antheridia were amphyginous. The identification was confirmed by electrophoresis of mycelial proteins on a polyacrylamide slab gel. Electrophoretic banding patterns of total soluble proteins and eight isozymes of the isolates from lavender were identical to those of a reference isolate of P. palmivora from olive (1). Conversely, the electrophoretic phenotype of the isolates from lavender was distinct from those of reference isolates of other species, including P. cactorum, P. capsici, P. citrophthora, P. nicotianae, and P. tropicalis. The pathogenicity of a representative isolate of P. palmivora from lavender was tested in the greenhouse using 6-month-old plants of English lavender, Rosea, a commercial cultivar very susceptible to root rot caused by P. nicotianae (3). Inoculum was produced on a mixture of vermiculite and autoclaved oat seeds (4) and mixed with soil (sand/lime/peat 1:1:1) at a concentration of 4% (vol/vol). Plants were transplanted to pots filled with infested soil. Control plants were grown in pots containing noninfested soil. After transplanting, all pots were flooded for 24 h by plugging the drain hole. Three months after transplanting all plants grown in pots containing infested soil showed extensive root necrosis and dieback symptoms. Control plants remained healthy. P. palmivora was recovered from roots of symptomatic plants. To our knowledge, this is the first report from Italy of P. palmivora on lavender. Root rot caused by P. palmivora may be a potential problem for commercial cultivation of lavender.

Estimation of fungal growth rates in soil using 14C-acetate incorporation into ergosterol

A technique to estimate fungal growth rates in field samples was tested in soil. The technique is based on the addition of 14C-acetate to a soil slurry and the subsequent uptake and incorporation of the labelled acetate into the fungus specific substance ergosterol by the fungi. The addition of fungal inhibitors decreased acetate incorporation rates, while bacterial inhibitors did not. Fungus-free soil exhibited no incorporation of acetate into ergosterol, indicating that the method was specific for measuring fungal activity. Incorporation rates were linear up to 18 h after the addition of acetate indicating that changing the conditions (adding acetate as a solution to a soil slurry) did not affect the incorporation rate. Problems associated with saturation of the incorporation of the added acetate were encountered, which together with uncertain conversion factors made calculations of absolute growth rates difficult. However, for relative comparisons the technique worked well. This was exemplified by measuring the relationship between temperature and growth rate of the soil fungal community, where an optimum temperature between 25 and 30°C and an apparent minimum temperature for fungal growth of −11°C were found. The technique was also used to indicate which nutrients limited instantaneous fungal growth in soil by adding carbon, nitrogen and phosphorus in different combinations and measuring the rate of acetate incorporation into ergosterol 2 days later. Carbon appeared to be the limiting nutrient for fungal growth in both an agricultural soil and a forest humus soil.

Host and geographical factors influence the thermal niche of enteric bacteria isolated from native Australian mammals.

The thermal profiles of 118 bacterial strains, representing six species of the family Enterobacteriaceae, isolated from a variety of native Australian mammals were determined under in vitro conditions. Each of the bacterial species had a unique thermal profile and differed in their minimum or maximum temperature for growth and in their response to changing temperatures. The taxonomic classification of the host from which the bacterial strains were isolated explained a significant amount of the variation in thermal profile among strains of a species. Host effects were detected at all taxonomic levels: order, family, genus, and species. The locality (State or Territory) or climate zone from which the strain was collected explained a significant amount of the variation in the thermal profile of Citrobacter freundii, Enterobacter cloacae and Klebsiella pneumoniae strains. Genetically similar strains, as determined by allozyme profiles, had similar thermal profiles for the bacterial species Hafnia alvei and Escherichia coli. The results of this study indicate that there are potentially many aspects of host biology that may determine the thermal profile of these bacteria.

Thermotoga petrophila sp. nov. and Thermotoga naphthophila sp. nov., two hyperthermophilic bacteria from the Kubiki oil reservoir in Niigata, Japan.

Two hyperthermophilic bacteria, strains RKU-1T and RKU-10T, which grew optimally at 80 degrees C, were isolated from the production fluid of the Kubiki oil reservoir in Niigata, Japan. They were strictly anaerobic, rod-shaped fermentative heterotrophs. Based on the presence of an outer sheath-like structure (toga) and 16S rDNA sequences, they were shown to belong to the genus Thermotoga. Cells of strain RKU-1T were 2-7 microm by 0.7-1.0 microm, with flagella. They grew at 47-88 degrees C on yeast extract, peptone, glucose, fructose, ribose, arabinose, sucrose, lactose, maltose, starch and cellulose as sole carbon sources. Cells of strain RKU-10T were 2-7 microm by 0.8-1.2 microm, with flagella. They grew at 48-86 degrees C on yeast extract, peptone, glucose, galactose, fructose, mannitol, ribose, arabinose, sucrose, lactose, maltose and starch as sole carbon sources. While strains RKU-1T and RKU-10T reduced elemental sulfur to hydrogen sulfide, their final cell yields and specific growth rates decreased in the presence of elemental sulfur. Thiosulfate also inhibited growth of strain RKU-1T but not strain RKU-10T. The G+C contents of the DNA from strains RKU-1T and RKU-10T were 46.8 and 46.1 mol%. Phenotypic characteristics and 165 rDNA sequences of the isolates were similar to those of Thermotoga maritima and Thermotoga neapolitana, both being hyperthermophilic bacteria isolated from hydrothermal fields. However, the isolates differed from these species in their minimum growth temperatures, utilization of some sugars, sensitivity to rifampicin and the effects of elemental sulfur and thiosulfate on growth. The low levels (less than 31%) of DNA reassociation between any two of these hyperthermophilic Thermotoga strains indicated that the isolates were novel species. Analysis of the gyrB gene sequences supported the view that the isolates were genotypically different from these reference species. The isolates were named Thermotoga petrophila sp. nov., with type strain RKU-1T (= DSM 13995T = JCM 10881T), and Thermotoga naphthophila sp. nov., with type strain RKU-10T (= DSM 13996T = JCM 10882T).

Growth and toxin profiles of Bacillus cereus isolated from different food sources

Eleven strains of Bacillus cereus isolated from milk and meat products have been used to study growth and sporulation profiles in detail. Polymerase chain reaction (PCR) using primers detecting cold shock protein A gene signatures ( cspA), showed that none of the strains were the newly suggested species in the B. cereus group, B. weihenstephanensis, comprising psychrotolerant cereus strains, although one of the strains grew at 4°C, two at 6°C and seven grew at 7°C. One of the two strains that grew at 6°C had a maximum growth temperature of 42°C, while the remaining 10 strains all grew at temperature of 43°C or higher. Only three strains grew at 48°C. At 42°C, the generation time varied between 11 and 34 min. Spore germination was much faster for the two strains that grew at 6°C than for the other nine strains in milk at 7°C and 10°C. All strains were cytotoxic and contained the non-haemolytic enterotoxin gene ( nhe), 10 strains contained the enterotoxin T gene ( bceT), and only six had the gene ( hbl) encoding haemolytic enterotoxin. Two strains showed some microheterogeneity in the nhe operon, but contained all three genes. We can conclude that true B. cereus strains can have growth profiles as expected for B. weihenstephanensis, and that nhe and bceT were not correlated with growth profiles. However, the two psychrotolerant strains with minimal growth temperature of 4°C and 6°C did not contain hbl, as judged from our PCR results.

Vegetative growth and conidium production by Monilinia oxycocci in vitro

The effects of various natural agar media, light, and temperature on mycelium growth of and conidium production by Monilinia oxycocci were examined in the laboratory under controlled conditions. Vegetative growth and sporulation were affected differentially by these factors. Optimum vegetative growth and formation of stromata occurred on potato dextrose agar at 20–24 C in the dark. The minimum and maximum temperatures for mycelium growth were 4 and 32 C, respectively. Production of conidia was best on V8 juice agar. The greatest numbers of conidia were produced at 16 C, but abundant conidium production was most consistent at 20 C. The influence of light on sporulation was inconsistent among trials and differed among media and isolates. However, on V8 juice agar at 20 C, production of conidia was most abundant in the dark. In general, considerable variability occurred with M. oxycocci throughout the study—among replicates of individual isolates, among isolates within a trial, and among trials with the same isolates. The highly variable nature of M. oxycocci in vitro appears to be an inherent characteristic of this plant pathogenic fungus.

Influence of the modelling approach on the estimation of the minimum temperature for growth in Belehrádek-type models

This paper compares three different approaches for describing the growth rate dependence on sub-optimal temperatures {e.g., the square-root model described by Ratkowsky et al. (1982. J. Bacteriol. 154, 1222–1226): √μ=b.(T−Tmin1) or μ=b2. (T−Tmin1)2, the model originally found by Belehrádek (1926a. Nature11, 117–118): μ=a. (T−T0)αand a dimensionless analysis described previously, (Dantigny 1998. J. Ind. Microbiol. Biotechnol.21, 215–218.):μdim=μμopt=T−TminTopt−Tminα=[Tdim]α}.Data sets, growth rate vs temperature, have been taken from the literature for various organisms (e.g. Lactobacillus plantarum, Yersinia enterolitica and Acinetobacter).Firstly, this paper analyses the effect of using dimensionless (e.g. Tdimand μdim) or natural variables (e.g. T and μ) on the estimation of the minimum temperature for growth (e.g. T0and Tmin) and the α value. Secondly, the Belehrádek model is compared to the square-root model by using the natural variables. It has been demonstrated that the use of the square-root model leads to an under-estimation of the minimum temperature for growth when the α -value is significantly less than 2. In such a case, it has been highlighted that the dimensionless approach provides a closer estimation of the experimental minimum temperature for growth than the square-root model.

Thermophilic fungi: their physiology and enzymes.

Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20 degrees C and a maximum temperature of growth extending up to 60 to 62 degrees C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45 degrees C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62 degrees C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.

Effects of salts on Debaryomyces hansenii and Saccharomyces cerevisiae under stress conditions

The effect of Na + and K + on growth and thermal death of Debaryomyces hansenii and Saccharomyces cerevisiae were compared under stress conditions as those commonly found in food environments. At the supraoptimal temperature of 34°C both cations at concentrations of 0.5 M stimulated growth of D. hansenii, while K + had no effect and Na + inhibited growth of S. cerevisiae. At 8°C, close to the minimum temperature for growth in both species, both cations inhibited both yeasts, this effect being more pronounced with Na + in S. cerevisiae. At extreme pH values (7.8 and 3.5) both cations at concentrations of 0.25 M stimulated D. hansenii while Na + inhibited S. cerevisiae. K + inhibited this yeast at pH 3.5. Thermal inactivation rates, measured at 38°C in D. hansenii and at 48°C in S. cerevisiae, decreased in the presence of both cations. This protective effect could be observed in a wider range of concentrations in D. hansenii. These results call the attention to the fact that not all yeasts have the same behaviour on what concerns synergy or antagonism of salt together with other stress factors and should be taken into consideration in the establishment of food preservation procedures.

Studies of Archaebacterial Bipolar Tetraether Liposomes by Perylene Fluorescence

Membrane packing and dynamics of bipolar tetraether liposomes composed of the polar lipid fraction E (PLFE) from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius have been studied by perylene fluorescence. At a probe-to-PLFE lipid ratio of 1:400, we have detected an unusual fluorescence intensity increase with increasing temperature, while the fluorescence lifetime changed little. As the ratio was decreased, the intensity anomaly was diminished. At 1:3200 and 1:6400, the anomaly disappeared. A remarkable perylene intensity anomaly was also observed in bilayers composed of saturated monopolar diester phosphatidylcholines at their main phase transition temperatures. These results suggest that the intensity anomaly may be due to probe aggregation caused by tight membrane packing. At the same probe-to-lipid ratio (1:400), however, 1,2-diphytanoyl- sn-glycero-3-phosphocholine (DPhPC) and 1,2-diphytanoyl- sn-glycero-3-phosphoglycerol (DPhPG) liposomes did not exhibit any intensity anomaly with increasing temperature. This suggests that DPhPC and DPhPG liposomes are more loosely packed than PLFE liposomes; thus the branched methyl groups are not the contributing factor of the tight membrane packing found in PLFE liposomes. Using a multiexcitation method, we have also determined the average ( R), in-plane ( R ip), and out-of-plane ( R op) rotational rates of perylene in PLFE liposomes at various temperatures (20–65°C). R and R ip, determined at two different probe-to-lipid ratios (1:400 and 1:3200), both undergo an abrupt increase when the temperature is elevated to ∼48°C. These data suggest that PLFE liposomes are rigid and tightly packed at low temperatures, but they begin to possess appreciable “membrane fluidity” at temperatures close to the minimum growth temperature (∼50°C) of thermoacidophilic archaebacteria.

Application of nonlinear regression analysis to the estimation of kinetic parameters for two enterotoxigenic strains ofBacillus cereus spores

Two enterotoxigenic Bacillus cereus strains, isolated from cooked chilled foods containing vegetables, were characterized in relation to safe food production by studying their heat resistance characteristics. The kinetic parameters of thermal resistance were determined by one-step nonlinear regression and were compared with those calculated by using two linear regressions. Results showed that the new analysis methodology applied in this study can improve the estimation ofD - and z - values. Great differences were found in the thermal resistance of the two strains. TheD90°C were 4·04 and 39 min for the AV TZ415 and AV Z421 strains respectively. Growth studies indicated that the two strains were able to grow at refrigeration temperatures, but the minimum growth temperature for the AV Z421 strain was 10°C, while the less heat-resistant strain, AV TZ415, grew at 5°C.

Cold shock proteins and low-temperature response of Streptococcus thermophilus CNRZ302.

Low-temperature adaptation and cryoprotection were studied in the thermophilic lactic acid bacterium Streptococcus thermophilus CNRZ302. S. thermophilus actively adapts to freezing during a pretreatment at 20 degrees C, resulting in an approximately 1, 000-fold increased survival after four freeze-thaw cycles compared to mid-exponential-phase cells grown at an optimal temperature of 42 degrees C. No adaptation is observed when cells are exposed to a temperature (10 degrees C) below the minimal growth temperature of the strain (just below 15 degrees C). By two-dimensional gel electrophoresis several 7-kDa cold-induced proteins were identified, which are the major induced proteins after a shift to 20 degrees C. These cold shock proteins were maximally expressed at 20 degrees C, while the induction level was low after cold shock to 10 degrees C. To confirm the presence of csp genes in S. thermophilus, a PCR strategy was used which yielded products of different sizes. Sequence analysis revealed csp-like sequences that were up to 95% identical to those of csp genes of S. thermophilus ST1-1, Streptococcus dysgalactiae, Streptococcus pyogenes, and Lactococcus lactis. Northern blot analysis revealed a seven- to ninefold induction of csp mRNA after a temperature shift to 20 degrees C, showing that this thermophilic bacterium indeed contains at least one cold-inducible csp gene and that its regulation takes place at the transcriptional level.

A Polyphasic Study on the Taxonomic Position of Industrial Sour Dough Yeasts

The sour dough bread making process is extensively used to produce wholesome palatable rye bread. The process is traditionally done using a back-slopping procedure. Traditional sour doughs in Finland comprise of lactic acid bacteria and yeasts. The yeasts present in these doughs have been enriched in the doughs due to their metabolic activities, e.g. acid tolerance. We characterized the yeasts in five major sour bread bakeries in Finland. We found that most of the commercial sour doughs contained yeasts which were similar to Candida milleri on the basis of 18S rDNA and EF-3 PCR-RFLP patterns and metabolic activities. Some of the bakery yeasts exhibited extensive karyotype polymorphism. The minimum growth temperature was 8 degrees C for C. milleri and also for most of sour dough yeasts.

Variability in Growth Characteristics of Different E. coli O157:H7 Isolates, and its Implications for Predictive Microbiology

In growth experiments 75 clinical isolates of Escherichia coli O157:H7 were studied for the variability in seven growth characteristics: minimum, optimum and maximum growth temperature, minimum and optimum pH, minimum water activity and optimum specific growth rate. With these characteristics, growth can be predicted for any given set of conditions (temperature, acidity and water activity), when the “gamma model” is used as predictive microbiology model. The optimum specific growth rate of the 75 strains, as conceptually defined by the model, had a mean value of 4.71 (ln[emsp4 ](count)/h), with a standard deviation of 0.39. It could not be shown that the mean optimum specific growth rate differs significantly per strain, so the variability found will predominantly be the result of other sources of variation. In contrast, the experimental results suggest that the differences in minimum temperature of growth may be partially strain specific. As variability in growth is crucial for quantitative risk assessment, the results were implemented in the gamma model. Predictions at three sets of growth conditions were compared with predictions of the Pathogen Modeling Program (PMP) (USDA) and some published experimental results. This comparison showed that growth rates higher than those published and outside the 95% confidence interval predicted by the PMP are feasible. Although it needs further development and additional tests, our approach offers a promising strategy to predict the variability in growth.