Influence of temperature on biofilm formation by Listeria monocytogenes on various food-contact surfaces: relationship with motility and cell surface hydrophobicity

Aims:  To assess the ability of Listeria monocytogenes to form biofilm on different food-contact surfaces with regard to different temperatures, cellular hydrophobicity and motility.
Methods and Results:  Forty-four L. monocytogenes strains from food and food environment were tested for biofilm formation by crystal violet staining. Biofilm levels were significantly higher on glass at 4, 12 and 22°C, as compared with polystyrene and stainless steel. At 37°C, L. monocytogenes produced biofilm at significantly higher levels on glass and stainless steel, as compared with polystyrene. Hydrophobicity was significantly ( P  < 0·05) higher at 37°C than at 4, 12 and 22°C. Thirty (68·2%) of 44 strains tested showed swimming at 22°C and 4 (9·1%) of those were also motile at 12°C. No correlation was observed between swimming and biofilm production.
Conclusions:  L. monocytogenes can adhere to and form biofilms on food-processing surfaces. Biofilm formation is significantly influenced by temperature, probably modifying cell surface hydrophobicity.
Significance and Impacts of the Study:  Biofilm formation creates major problems in the food industry because it may represent an important source of food contamination. Our results are therefore important in finding ways to prevent contamination because they contribute to a better understanding on how L. monocytogenes can establish biofilms in food industry and therefore survive in the processing environment.     

Integration and decontamination of Bacillus cereus in Pseudomonas fluorescens biofilms

Aims:  The hypothesis that surrogate planktonic pathogens ( Bacillus cereus and polystyrene microspheres) could be integrated in biofilms and protected from decontamination was tested.
Methods and Results:  Pseudomonas fluorescens biofilms were grown on polyvinyl chloride coupons in annular reactors under low nutrient conditions. After biofilm growth, B. cereus spores and polystyrene microspheres (an abiotic control) were introduced separately. Shear stress at the biofilm surface was varied between 0·15 and 1·5 N m⁻². The amount of surrogate pathogens introduced ranged from approximately 10⁵ CFU ml⁻¹ to 10¹⁰ spheres ml⁻¹. The quantity of surrogate pathogens integrated in the biofilm was proportional to the amount introduced. In 14 of the 16 cases, 0·4–3·0% of the spores or spheres introduced were measured in the biofilms. The other two cases had 10% and 21% of the spores detected. Data suggested that the spores germinated in the system. The amount of surrogate pathogens detected in the biofilms was higher in the mid-shear range. Chlorine treatment reduced the quantity of both surrogate pathogens and biofilm organisms. In one experiment, the biofilms and B. cereus recovered when the chlorine treatment was terminated.
Conclusions:  Planktonic surrogate pathogens can be integrated in biofilms and protected from chlorination decontamination.
Significance and Impact of the Study:  This knowledge assists in understanding the impact of biofilms on harbouring potential pathogens in drinking-water systems and protecting the pathogens from decontamination.     

Heat resistance of Cronobacter species (Enterobacter sakazakii) in milk and special feeding formula

Aim:  To determine D - and z -values of Cronobacter species ( Enterobacter sakazakii ) in different reconstituted milk and special feeding formula and the effect of reconstitution of powdered milk and special feeding formula with hot water on the survival of the micro-organism.
Methods and Results:  Five Cronobacter species (four C. sakazakii isolates and C. muytjensii ) were heated in reconstituted milk or feeding formula pre-equilibrated at 52–58°C for various times or mixed with powdered milk or feeding formula prior to reconstitution with water at 60–100°C. The D -values of Cronobacter at 52–58°C were significantly higher in whole milk (22·10–0·68 min) than in low fat (15·87–0·62 min) or skim milk (15·30–0·51 min) and significantly higher in lactose-free formula (19·57–0·66 min) than in soy protein formula (17·22–0·63 min). The z -values of Cronobacter in reconstituted milk or feeding formula ranged from 4·01°C to 4·39°C. Water heated to ≥70°C and added to powdered milk and formula resulted in a > 4 log₁₀ reduction of Cronobacter .
Conclusions:  The heat resistance of Cronobacter should not allow the survival of the pathogen during normal pasteurization treatment. The use of hot water (≥70°C) during reconstitution appears to be an effective means to reduce the risk of Cronobacter in these products.
Significance and Impact of the Study:  This study supports existing data available to regulatory agencies and milk producers that recommended heat treatments are sufficient to substantially reduce risk from Cronobacter which may be present in these products.     

The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk

Aims:  The aim of this work was to investigate the germination and inactivation of spores of Bacillus species in buffer and milk subjected to high pressure (HP) and nisin.
Methods and Results:  Spores of Bacillus subtilis and Bacillus cereus suspended in milk or buffer were treated at 100 or 500 MPa at 40°C with or without 500 IU ml⁻¹ of nisin. Treatment at 500 MPa resulted in high levels of germination (4 log units) of B. subtilis spores in both milk and buffer; this increased to >6 logs by applying a second cycle of pressure. Viability of B. subtilis spores in milk and buffer was reduced by 2·5 logs by cycled HP, while the addition of nisin (500 IU ml⁻¹) prior to HP treatment resulted in log reductions of 5·7 and 5·9 in phosphate buffered saline and milk, respectively. Physical damage of spores of B. subtilis following HP was apparent using scanning electron microscopy. Treating four strains of B. cereus at 500 MPa for 5 min twice at 40°C in the presence of 500 IU ml⁻¹ nisin proved less effective at inactivating the spores of these isolates compared with B. subtilis and some strain-to-strain variability was observed.
Conclusions:  Although high levels of germination of Bacillus spores could be achieved by combining HP and nisin, complete inactivation was not achieved using the aforementioned treatments.
Significance and Impact of the Study:  Combinations of HP treatment and nisin may be an appealing alternative to heat pasteurization of milk.     

Lactic acid production by Lactobacillus delbrueckii in a dual reactor system using packed bed biofilm reactor

Aims:  An integrated dual reactor system for continuous production of lactic acid by Lactobacillus delbrueckii using biofilms developed on reticulated polyurethane foam (PUF) is demonstrated.
Methods and Results:  Lactobacillus delbrueckii was immobilized on PUF, packed in a bioreactor and used in lactic acid fermentation. The rate of lactic acid production was significantly high with a volumetric productivity of 5 g l⁻¹ h⁻¹ over extended period of time. When coupled to a bioreactor, the system could be operated as dual reactor for over 1000 h continuously without augmentation of inoculum and no compromise on productivity.
Conclusions:  Polyurethane foams offer an excellent support for biofilm formation.
Significance and Impact of the Study:  The system was very robust and could be operated for prolonged period at a volumetric productivity of 4–6 g l⁻¹ h⁻¹.     

Effects of moderately high pressure plus heat on the germination and inactivation of Bacillus cereus spores lacking proteins involved in germination

Aims:  To determine the germination and inactivation of Bacillus cereus spores lacking various germination proteins using moderately high pressure (MHP) and heat.
Methods:  The inactivation and germination of wild-type B. cereus spores in buffer by MHP (150 MPa) at various temperatures, as well as the MHP inactivation and germination of B. cereus spores lacking individual germinant receptors and monovalent cation antiporters, was determined.
Results:  Loss of individual germinant receptors had no large effects on spore inactivation or germination, although germination of receptor-deficient spores was generally slightly decreased. Loss of the GerN in particular the GerN and GerT antiporters also decreased spore germination by MHP, especially at 40 and 50°C.
Conclusions:  Both inactivation and germination of B. cereus spores by MHP increased with rise of temperature; however, mutant strains lacking individual germinant receptor had similar levels of germination as compared to wild-type spores. To evaluate the role of germinant receptors in MHP, a strain lacking a large number of germinant receptors is needed.
Significance and Impact of the Study:  The results of this work may lead to a better understanding of how MHP causes germination of spores of B. cereus .     

Production and characterization of pure Clostridium spore suspensions

Aims:  A general protocol was derived for optimizing the production of pure, high concentration Clostridium endospore suspensions.
Methods and Results:  Two sporulation methods were developed that yielded high concentrations of notably pure Clostridium sporogenes , C. hungatei and C. GSA-1 (Greenland ice core isolate) spore suspensions (10 ml of 10⁹ spores ml⁻¹ with >99% purity each). Each method was derived by evaluating combinations of three sporulation conditions, including freeze drying of inocula, heat shock treatment of cultures, and subsequent incubation at suboptimal temperatures that yielded the highest percentage of sporulation. Pure spore suspensions were characterized in terms of dipicolinic acid content, culturability, decimal reduction time ( D ) value for heat inactivation (100°C) and hydrophobicity.
Conclusions:  While some Clostridium species produce a high percentage of spores with heat shock treatment and suboptimal temperature incubation, other species require the additional step of freeze drying the inocula to achieve a high percentage of sporulation.
Significance and Impact of the Study:  Pure Clostridium spore suspensions are required for investigating species of medical and environmental importance. Defining the conditions for optimal spore production also provides insight into the underlying mechanisms of Clostridium sporulation.     

Bovicin HC5 reduces thermal resistance of Alicyclobacillus acidoterrestris in acidic mango pulp

Aims:  To test the effect of bovicin HC5 against vegetative cells and endospores of Alicyclobacillus acidoterrestris DSMZ 2498 in synthetic media and in acidic mango pulp.
Methods and Results:  Alicyclobacillus acidoterrestris was grown in synthetic medium at 40°C and pH 4·0. The effect on vegetative cells was assayed by adding bovicin HC5 to synthetic medium (40–160 AU ml⁻¹) or to mango pulp (100 AU ml⁻¹) at various pH values and determining the effect on growth (OD_600nm) and viable cell number, respectively. The effect of bovicin HC5 on spore germination and thermal sensitivity of A. acidoterrestris was tested in mango pulp (pH 4·0) containing 80 AU ml⁻¹ of bovicin HC5. Bovicin HC5 was bactericidal against vegetative cells of A. acidoterrestris at different pH values and showed sporicidal activity against endospores of this bacterium. When spores of A. acidoterrestris were heat treated in the presence of bovicin HC5, D -values decreased 77% to 95% compared to untreated controls at temperatures ranging from 80 to 95°C.
Conclusion:  Bovicin HC5 was bactericidal and sporicidal against A. acidoterrestrsi DSMZ 2498.
Significance and Impact of the Study:  These results indicated that bovicin HC5 has potential to prevent spoilage of acidic fruit juices by thermocidophilic spore-forming bacteria.     

Survival and growth of Cronobacter species (Enterobacter sakazakii) in wheat-based infant follow-on formulas

Aims:  To determine the survival and growth characteristics of Cronobacter species ( Enterobacter sakazakii ) in infant wheat-based formulas reconstituted with water, milk, grape juice or apple juice during storage.
Methods and Results:  Infant wheat-based formulas were reconstituted with water, ultra high temperature milk, pasteurized grape or apple juices. The reconstituted formulas were inoculated with Cronobacter sakazakii and Cronobacter muytjensii and stored at 4, 25 or 37°C for up to 24 h. At 25 and 37°C, Cronobacter grew more (>5 log₁₀) in formulas reconstituted with water or milk than those prepared with grape or apple juices ( c. 2–3 log₁₀). The organism persisted, but did not grow in any formulas stored at 4°C. Formulas reconstituted with water and milk decreased from pH 6·0 to 4·8–5·0 after 24 h, whereas the pH of the formulas reconstituted with fruit juices remained at their initial pH values, c. pH 4·8–5·0.
Conclusions:  Cronobacter sakazakii and C. muytjensii can grow in reconstituted wheat-based formulas. If not immediately consumed, these formulas should be stored at refrigeration temperatures to reduce the risk of infant infection.
Significance and Impact of the Study:  The results of this study will be of use to regulatory agencies and infant formula producers to recommend storage conditions that reduce the growth of Cronobacter in infant wheat-based formulas.     

Influences of milk components on biofilm formation of Cronobacter spp. (Enterobacter sakazakii)

Aim:  To determine the critical component(s) of skim milk for biofilm formation of Cronobacter species.
Methods and Results:  Biofilm forming ability of 72 Cronobacter strains in skim milk preparation was assayed by crystal violet staining. The results revealed that whey protein and casein are more important determinants of skim milk for biofilm formation than lactose, although there was a wide variation in biofilm forming ability. Biofilm structure and capsular material of six strains exhibiting different biofilm forming ability was investigated via electron microscopes. Scanning electron microscopy showed visually that while the strong biofilm formers (E27B, FSM 30 and 2·82) resulted in almost complete coagulation of skim milk, the weak biofilm formers (55, FSM 290 and 2·84) caused less coagulation. No capsule was clearly delineated in transmission electron micrographs of either strong or weak biofilm formers.
Conclusion:  These results indicate that, for biofilm formation of Cronobacter species in skim milk, nitrogen source is probably a more important determinant than carbohydrate, and that strong biofilm formers are responsible for substantial coagulation of skim milk.
Significance and Impact of the Study:  This study provides information for better understanding of the underlying mechanisms by which Cronobacter species form biofilm in infant formula milk.     

The growth of Bacillus stearothermophilus on stainless steel

AIMS: To determine the potential for Bacillus stearothermophilus cells to form biofilms of significance in dairy manufacture. METHODS AND RESULTS: The ability of isolates of B. stearothermophilus from dairy manufacturing plants to attach to stainless steel surfaces was demonstrated by exposing stainless steel samples to suspensions of spores or vegetative cells and determining the numbers attaching using impedance microbiology. Spores attached more readily than vegetative cells. The attachment of cells to stainless steel was increased 10-100-fold by the presence of milk fouling the stainless steel. The growth of B. stearothermophilus as a biofilm on stainless steel surfaces was determined using a continuously flowing experimental reactor. Vegetative cells were released in greater numbers than spores from biofilms of most strains studied. Biofilms of one strain (B11) were studied in detail. Biofilms of > 106 cells cm-2 formed in the reactor and released approximately 106 cells ml-1 into milk passing over the biofilm. A doubling time of 25 min was calculated for this organism grown as a biofilm. CONCLUSION: The formation of biofilms of thermophilic Bacillus species within the plant appears to be a likely cause of contamination of manufactured dairy products. Methods to control the formation of biofilms in dairy manufacturing plants are required to reduce the contamination of dairy products with thermophilic bacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilms of B. stearothermophilus growing in dairy manufacturing plants can explain the contamination of dairy products with these bacteria.     

Influence of phosphorus on biofilm formation in model drinking water distribution systems

Aims:  This study investigated the effects of phosphorus on biofilm formation via annular reactor systems in terms of biofilm cell growth, exopolysaccharide (EPS) production, biofilm structure and cell metabolic potential.
Methods and Results:  Drinking water biofilms were developed in annular reactors with supplement of carbon and different levels of phosphorus. The biofilm formation was monitored over a period of 30 days. Biofilm related parameters were examined by various methods, which included heterotrophic plate count, total carbohydrate content, confocal laser scanning microscopy and GN2 microplate assay. Our results showed that phosphorus addition can promote the biofilm cell growth (cell count increased about 1 log with addition of 30 and 300 μg l⁻¹ of phosphorus). However, the addition of 30 and 300 μg l⁻¹ of phosphorus caused 81% and 77% decrease in EPS production, respectively. The results of biofilm structure analysis showed that the addition of 30 and 300 μg l⁻¹ of phosphorus can induce thicker and less homogeneous biofilms with more biomass. Furthermore, the addition of 30 and 300 μg l⁻¹ of phosphorus dramatically increased the biofilm cell metabolic potential. The addition of 3 μg l⁻¹ of phosphorus was found to have minor effects on the parameters examined.
Conclusions:  The results indicate phosphorus addition to drinking water distribution system (DWDS) has a complicated effect on the biofilm formation.
Significance and Impact of the Study:  As the addition of phosphorus at certain levels can affect the biofilm growth in DWDS, care should be taken when phosphate-based corrosion inhibitors are used in the DWDS.     

Spore Formation and Toxin Production in Clostridium difficile Biofilms

The ability to grow as a biofilm can facilitate survival of bacteria in the environment and promote infection. To better characterize biofilm formation in the pathogen Clostridium difficile, we established a colony biofilm culture method for this organism on a polycarbonate filter, and analyzed the matrix and the cells in biofilms from a variety of clinical isolates over several days of biofilm culture. We found that biofilms readily formed in all strains analyzed, and that spores were abundant within about 6 days. We also found that extracellular DNA (eDNA), polysaccharide and protein was readily detected in the matrix of all strains, including the major toxins A and/or B, in toxigenic strains. All the strains we analyzed formed spores. Apart from strains 630 and VPI10463, which sporulated in the biofilm at relatively low frequencies, the frequencies of biofilm sporulation varied between 46 and 65%, suggesting that variations in sporulation levels among strains is unlikely to be a major factor in variation in the severity of disease. Spores in biofilms also had reduced germination efficiency compared to spores obtained by a conventional sporulation protocol. Transmission electron microscopy revealed that in 3 day-old biofilms, the outermost structure of the spore is a lightly staining coat. However, after 6 days, material that resembles cell debris in the matrix surrounds the spore, and darkly staining granules are closely associated with the spores surface. In 14 day-old biofilms, relatively few spores are surrounded by the apparent cell debris, and the surface-associated granules are present at higher density at the coat surface. Finally, we showed that biofilm cells possess 100-fold greater resistance to the antibiotic metronidazole then do cells cultured in liquid media. Taken together, our data suggest that C. difficile cells and spores in biofilms have specialized properties that may facilitate infection.     

Destruction of Bacillus licheniformis spores by microwave irradiation

Aims:  To investigate the sporicidal mechanisms of microwave irradiation on Bacillus licheniformis spores.
Methods and Results:  We measured spore viability and the release of DNA and proteins, and performed transmission electron microscopy (TEM). A microwave oven (0·5 kW) was modified to output power at 2·0 kW, which allowed a shorter sterilization cycle. A 2·0 kW microwave treatment at the boiling temperature for 1 min did not kill all spores, but killed most spores. The spore inactivation rate was faster than that of boiling and 0·5 kW microwave oven. In contrast to boiling and 0·5 kW microwave treatments, the 2·0 kW microwave resulted in significant leakage of proteins and DNA from spores due to injury to the spore structure. TEM revealed that 2·0 kW microwave irradiation affected spore cortex hydrolysis and swelling, and ruptured the spore coat and inner membrane.
Conclusions:  These results suggest that 2·0 kW microwave irradiation ruptures the spore coat and inner membrane, and is significantly different from boiling.
Significance and Impact of the Study:  This study provides information on the sporicidal mechanisms of microwave irradiation on B. licheniformis spores.     

Effects of temperature and heat activation on germination of individual spores of Bacillus subtilis

Phase intensity changes of individual germinating spores of Bacillus subtilis were determined by phase-contrast light microscopy and image analysis. Two germination phases were investigated. The length of the time period before a change in phase brightness was evident and the duration of the phase intensity change until a constant greylevel was maintained. The incubation temperature (37 and 20 °C) and heat activation (10 min at 65 °C) had a distinct effect on both phases. At 37 °C, spores of B. subtilis 604 started to show a decrease in brightness in l -alanine buffer after 3–39 min and needed 10–39 min to complete the phase change. At 20 °C, lag times of 10–100 min were observed and the spores needed 30–100 min to reach a constant greylevel. Heat activation and subsequently exposure to l -alanine buffer at 20 °C reduced the lag phase to 6–90 min and the phase change was finished after 30–60 min. Our results indicate enzymatic involvement before and during the phase intensity change of germinating spores.     

Interactions of Salmonella enterica with lettuce leaves

Aims:  To investigate the interactions of Salmonella enterica with abiotic and plant surfaces and their effect on the tolerance of the pathogen to various stressors.
Methods and Results:  Salmonella strains were tested for their ability to form biofilm in various growth media using a polystyrene plate model. Strong biofilm producers were found to attach better to intact Romaine lettuce leaf tissue compared to weak producers. Confocal microscopy and viable count studies revealed preferential attachment of Salmonella to cut-regions of the leaf after 2 h at 25°C, but not for 18 h at 4°C. Storage of intact lettuce pieces contaminated with Salmonella for 9 days at 4°C resulted only in small changes in population size. Exposure of lettuce-associated Salmonella cells to acidic conditions (pH 3·0) revealed increased tolerance of the attached vs planktonic bacteria.
Conclusions:  Biofilm formation on polystyrene may provide a suitable model to predict the initial interaction of Salmonella with cut Romaine lettuce leaves. Association of the pathogen with lettuce leaves facilitates its persistence during storage and enhances its acid tolerance.
Significance and Impact of the Study:  Understanding the interactions between foodborne pathogens and lettuce might be useful in developing new approaches to prevent fresh produce-associated outbreaks.     

Resting spore formation of aphid-pathogenic fungus Pandora nouryi depends on the concentration of infective inoculum

Resting spore formation of some aphid-pathogenic Entomophthorales is important for the seasonal pattern of their prevalence and survival but this process is poorly understood. To explore the possible mechanism involved in the process, Pandora nouryi (obligate aphid pathogen) interacted with green peach aphid Myzus persicae on cabbage leaves under favourable conditions. Host nymphs showered with primary conidia of an isolate (LC₅₀: 0.9–6.7 conidia mm⁻² 4–7 days post shower) from air captures in the low-latitude plateau of China produced resting spores (azygospores), primary conidia or both spore types. Surprisingly, the proportion of mycosed cadavers forming resting spores ( P _cfrs ) increased sharply within the concentrations ( C ) of 28–240 conidia mm⁻², retained high levels at 240–1760, but was zero or extremely low at 0.3–16. The P _cfrs – C relationship fit well the logistic equation P _cfrs  = 0.6774/[1 + exp(3.1229–0.0270 C )] ( r ² = 0.975). This clarified for the first time the dependence of in vivo resting spore formation of P. nouryi upon the concentration of infective inoculum. A hypothesis is thus proposed that some sort of biochemical signals may exist in the host–pathogen interaction so that the fungal pathogen perceives the signals for prompt response to forthcoming host-density changes by either producing conidia for infecting available hosts or forming resting spores for surviving host absence in situ .     

Antimicrobial effect and shelf-life extension by combined thermal and pulsed electric field treatment of milk

Aims:  The impact of a combined hurdle treatment of heat and pulsed electric fields (PEF) was studied on native microbiota used for the inoculation of low-fat ultra-high temperature (UHT) milk and whole raw milk. Microbiological shelf-life of the latter following hurdle treatment or thermal pasteurization was also investigated.
Methods and Results:  UHT milk was preheated to 30°C, 40°C or 50°C over a 60-s period, pulsed for 50  μ s or 60  μ s at a field strength of 40 kV cm⁻¹ or for 33  μ s at 50 kV cm⁻¹. Heat and PEF reduced the microbial count by a maximum of 6·4 log in UHT milk (50°C; 50 kV cm⁻¹, 33  μ s) compared to 6·0 log ( P  ≥ 0·05) obtained by thermal pasteurization (26 s, 72°C). When raw milk was treated with a combination of hurdles (50°C; 40 kV cm⁻¹, 60  μ s) a 6·0 log inactivation of microbiota was achieved and microbiological milk shelf-life was extended to 21 days under refrigeration (4°C) vs 14 days in thermally pasteurized milk. Native microbiota was decreased by 6·7 log following conventional pasteurization.
Conclusions:  The findings suggest that heat and PEF achieved similar inactivation of native microbiota in milk and longer stabilization of microbiological shelf-life than thermal pasteurization.
Significance and Impact of the Study:  A hurdle approach of heat and PEF could represent a valid milk processing alternative to conventional pasteurization. Hurdle treatment might also preserve native milk quality better due to less thermal exposure.     

Thermal acclimation of swimming performance in newt larvae: the influence of diel temperature fluctuations during embryogenesis

1.  Thermal acclimation is one of the basic strategies by which organisms cope with thermal heterogeneity of the environment. Under predictable variation in environmental temperatures, theory predicts that selection favours acclimation of thermal performance curves over fixed phenotypes.
2.  We examined the influence of diel fluctuations in developmental temperatures on the thermal sensitivity of the maximal swimming capacity in larvae of the alpine newt, Triturus alpestris .
3.  We incubated newt eggs under three thermal regimes with varying daily amplitudes (1, 5 and 9 °C) and similar means (17·6–17·9 °C), and accordingly we measured the swimming speed of hatched larvae at three experimental temperatures (12, 17 and 22 °C), which they would normally experience in their natural habitat.
4.  Embryonic development under low and middle temperature fluctuations produced larvae with similar swimming speeds across experimental temperatures. In contrast, the most fluctuating regime induced development of phenotypes, which at 12 °C swam faster than larvae developed under moderate diel fluctuations.
5.  Our results provide evidence that diel temperature fluctuations induce acclimation of thermal dependence of locomotor performance. In ectotherms experiencing diel cycles in environmental temperatures, this plastic response may act as an important pacemaker in the evolution of thermal sensitivity.     

Isolation and properties of psychrotrophic and psychrophilic, pectolytic strains of Clostridium

Fourteen strains of pectolytic clostridia have been isolated that were capable of growth at 5–10°C in 7 d; two strains were psychrophiles and failed to grow at 20°C in 14 d and the remainder were psychrotrophs. The bacteria formed pectate lyase enzymes and were capable of degrading potato tissue; they are therefore a potential cause of soft rot of potatoes stored at low temperatures. Doubling times for representative strains were 15–19 h at 10°C and 21–53 h at 5°C. Twelve strains were classified with Group I Clostridium species and two strains with Group II. In the case of one strain the mature spores were not released from the sporangium. Electron microscopy of ultrathin sections of this strain showed the presence of disorganized lamellar structures associated with the spore coat.