Thermal limitations of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> efficacy: selection for application on warm-blooded vertebrates

Temperature is one of the main obstacles for on-host applications of entomopathogenic fungi for ectoparasite control. The effects of temperatures typical of the body surfaces of warm-blooded animals on the germination, growth and virulence of four strains of Metarhizium anisopliae toward engorged Rhipicephalus (Boophilus) annulatus females were evaluated. The M. anisopliae strains studied can be divided according to their thermal characteristics: (1) strains which germinate (90–100%), grow and infect ticks similarly at 25, 30 and 35°C; and (2) strains which recover their ability to germinate relatively quickly following a thermal shock (37 or 40°C for 6–48 h) before incubation at a favorable temperature. These latter strains could recover their infectivity after a short thermal shock (6 h at 37–40°C), but not after more prolonged exposure to these temperatures (48–72 h). These two thermal characteristics do not interact, but reflect the efficacy of strains used to control ectoparasites on warm-blooded vertebrates.     

Cj0596 is a periplasmic peptidyl prolyl cis-trans isomerase involved in Campylobacter jejuni motility, invasion, and colonization

Background  

Campylobacter jejuni is a gastrointestinal pathogen of humans, but part of the normal flora of poultry, and therefore grows well at the respective body temperatures of 37°C and 42°C. Proteomic studies on temperature regulation in C. jejuni strain 81–176 revealed the upregulation at 37°C of Cj0596, a predicted periplasmic chaperone that is similar to proteins involved in outer membrane protein folding and virulence in other bacteria.     

Direct ethanol production from cellulosic materials at high temperature using the thermotolerant yeast Kluyveromyces marxianus displaying cellulolytic enzymes

To exploit cellulosic materials for fuel ethanol production, a microorganism capable of high temperature and simultaneous saccharification–fermentation has been required. However, a major drawback is the optimum temperature for the saccharification and fermentation. Most ethanol-fermenting microbes have an optimum temperature for ethanol fermentation ranging between 28 °C and 37 °C, while the activity of cellulolytic enzymes is highest at around 50 °C and significantly decreases with a decrease in temperature. Therefore, in the present study, a thermotolerant yeast, Kluyveromyces marxianus, which has high growth and fermentation at elevated temperatures, was used as a producer of ethanol from cellulose. The strain was genetically engineered to display Trichoderma reesei endoglucanase and Aspergillus aculeatus β-glucosidase on the cell surface, which successfully converts a cellulosic β-glucan to ethanol directly at 48 °C with a yield of 4.24 g/l from 10 g/l within 12 h. The yield (in grams of ethanol produced per gram of β-glucan consumed) was 0.47 g/g, which corresponds to 92.2% of the theoretical yield. This indicates that high-temperature cellulose fermentation to ethanol can be efficiently accomplished using a recombinant K. marxianus strain displaying thermostable cellulolytic enzymes on the cell surface.     

The Influence of Water Activity and Temperature on Germination,Growth and Sporulation of <Emphasis Type="Italic">Stachybotrys chartarum</Emphasis> Strains

The objectives were to determine the influence of water activity (a_w, 0.997–0.92) and temperature (10–37°C) and their interactions on conidial germination, mycelial growth and sporulation of two strains of Stachybotrys chartarum in vitro on a potato dextrose medium. Studies were carried out by modifying the medium with glycerol and either spread plating with conidia to evaluate germination and germ tube extension or centrally inoculating treatment media for measuring mycelial growth rates and harvesting whole colonies for determining sporulation. Overall, germination of conidia was significantly influenced by a_w and temperature and was fastest at 0.997–0.98 a_w between 15 and 30°C with complete germination within 24 h. Germ tube extension was found to be most rapid at similar a_w levels and 25–30°C. Mycelial growth rates of both strains were optimal at 0.997 a_w between 25 and 30°C, with very little growth at 37°C. Sporulation was optimum at 30°C at 0.997 a_w. However, under drier conditions, this was optimum at 25°C. This shows that there are differences in the ranges of a_w x temperature for germination and growth and for sporulation. This may help in understanding the role of this fungal species in damp buildings and conditions under which immune-compromised patients may be at risk when exposed to such contaminants in the indoor air environment.     

Growth and sporulation of entomopathogenic Beauveria bassiana,Metarhizium anisopliae,Isaria farinosa and Isaria fumosorosea strains in relation to water activity and temperature interactions

This study examined six strains of Beauveria bassiana s.l. and Isaria farinosa, one strain of Isaria fumosorosea and five strains of Metarhizium anisopliae s.l. to identify the ability for (1) growth and (2) sporulation under interacting environmental factors of water activity (a_w) and temperature stress. Growth on Sabouraud Dextrose Agar (SDA; water activity, a_w = 0.995) or SDA modified with glycerol to 0.98, 0.96 and 0.94 a_w was measured at four different temperatures (25, 30, 35 and 37°C). All M. anisopliae strains grew at 25–35°C and 0.995 a_w while only two strains tolerated extreme water stress at 0.94 a_w.Three strains of B. bassiana were able to grow at 25–37°C and 0.995 a_w. Only one strain of I. farinosa was able to grow at 25–37°C and 0.995 a_w. A_w and temperature interactions resulted in different strain-dependent responses, in terms of growth and sporulation. Only one strain of I. farinosa and three of M. anisopliae grew at 0.94 a_w and none of the B. bassiana strains tolerated such water stress. At 0.96 and 0.94 a_w and 35–37°C, sporulation by all the strains of the three species were significantly affected. Under elevated temperatures and drought stress, very few of these strains of entomopathogenic fungi are able to grow and sporulate. Indeed, the B. bassiana strains were unable to tolerate the extreme conditions examined. Resilience to such abiotic interactions is critical for selecting strains for formulations. Tolerance to water and temperature stress could be good criteria for selection of strains with secondary spread potential for use as part of an integrated pest management system where secondary cycling may be important, especially in sub-tropical and tropical environments.     

Heart valve allograft decontamination with antibiotics: impact of the temperature of incubation on efficacy

Heart valve allografts are typically processed at 4°C in North America, including the step of antibiotic decontamination. In our own experience with heart valve banking, we often observe persistent positive cultures following decontamination at wet ice temperature. We hypothesized that warmer temperatures of incubation might increase the efficacy of the decontamination procedure. In a first series of experiments, 12 different bacterial species were grown overnight, frozen in standardized aliquots and used directly to inoculate antibiotic cocktail aliquots at 10⁵ colony-forming units (CFU)/ml. The antibiotic cocktail contains vancomycin (50 μg/ml), gentamicin (80 μg/ml) and cefoxitin (240 μg/ml) in Dulbecco’s Modified Eagle’s Medium. Inoculated aliquots were incubated at 4, 22 and 37°C and CFUs were determined at regular intervals up to 24 h post-inoculation. In a second set of experiments, 10 heart valves were spiked with 5000 CFU/ml and incubated with antibiotics at 4 and 37°C for 24 h. The final rinse solutions of these heart valves were filtered and tested for bacterial growth. After 24 h of incubation, CFUs of all 12 bacterial species were reduced by a factor of only one to two logs at 4°C whereas log reductions of 3.7 and 5.0 or higher were obtained at 22 and 37°C, respectively. Most microorganisms, including Staphylococcus epidermidis, Lactococcus lactis lactis and Propionibacterium acnes survived well the 24-h antibiotic treatment at 4°C (<1 Log reduction). All 10 heart valves that were spiked with microorganisms had positive final rinse solutions after antibiotic soaking at 4°C, whereas 8 out of 10 cultures were negative when antibiotic decontamination was done at 37°C. These experiments show that a wet ice temperature greatly reduces the efficacy of the allograft decontamination process as microorganisms survived well to a 24-h 4°C antibiotic treatment. This could explain the high rate of positive post-processing cultures obtained with our routine tissue decontamination procedure. Increasing the decontamination temperature from 4 to 37°C may significantly reduce the incidence of post-disinfection bacterial contamination of heart valves.     

The effect of growth media and physical treatments on the adhesion properties of canine probiotics

Aims

The manufacturing processes have been reported to influence the properties of probiotics with potential impact on health properties. The aim was to investigate the effect of different growth media and inactivation methods on the properties of canine‐originated probiotic bacteria alone and in combination mixture.

Methods and Results

Three established dog probiotics, Lactobacillus fermentum VET9A, Lactobacillus plantarum VET14A and Lactobacillus rhamnosus VET16A, and their combination mixture were evaluated for their adhesion to dog mucus. The effect of different growth media, one reflecting laboratory and the other manufacturing conditions, and inactivation methods (95°C, 80°C and UV irradiation) on the mucus adhesion of the probiotic strains was characterized. Evaluation of dog probiotics was supported by cell visualization using transmission electron microscopy (TEM). Higher adhesion percentage was reported for probiotic strains growing in laboratory rather than in manufacturing conditions (P < 0·05). Inactivation by heat (95°C, 80°C) decreased the adhesion properties when strains were cultivated in soy‐based growth media compared with those grown in MRS broth (P < 0·05). TEM observations uncovered differences in cell‐surface components in nonviable forms of probiotic strains as compared with their viable forms.

Conclusions

Manufacturing process conditions such as growth media and pretreatment methods may significantly affect the adhesive ability of the tested strains.

Significance and Impact of the Study

Growth conditions, growth media, pretreatment methods and different probiotic combinations should be carefully considered for quality control of existing probiotics and for identification of new probiotics for dogs. These may also have an impact on health benefits for the host.     

Improvement of l-valine production at high temperature in Brevibacterium flavum by overexpressing ilvEBNrC genes

Brevibacterium flavum ATCC14067 was engineered for l-valine production by overexpression of different ilv genes; the ilvEBN^rC genes from B. flavum NV128 provided the best candidate for l-valine production. In traditional fermentation, l-valine production reached 30.08 ± 0.92 g/L at 31°C in 72 h with a low conversion efficiency of 0.129 g/g. To further improve the l-valine production and conversion efficiency based on the optimum temperatures of l-valine biosynthesis enzymes (above 35°C) and the thermotolerance of B. flavum, the fermentation temperature was increased to 34, 37, and 40°C. As a result, higher metabolic rate and l-valine biosynthesis enzymes activity were obtained at high temperature, and the maximum l-valine production, conversion efficiency, and specific l-valine production rate reached 38.08 ± 1.32 g/L, 0.241 g/g, and 0.133 g g⁻¹ h⁻¹, respectively, at 37°C in 48 h fermentation. The strategy for enhancing l-valine production by overexpression of key enzymes in thermotolerant strains may provide an alternative approach to enhance branched-chain amino acids production with other strains.     

Test method development to evaluate hot,humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and B. thuringiensis Al Hakam spores

Aims

To develop test methods and evaluate the survival of Bacillus anthracis ?Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air.

Methods and Results

Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH‐adjusted bleach decontamination.

Conclusions

Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ?Sterne and B. thuringiensis Al Hakam spores with similar kinetics.

Significance and Impact of the Study

Hot, humid air is a potential alternative to conventional chemical decontamination.     

Physiological diversity within the <Emphasis Type="Italic">kluyveromyces marxianus</Emphasis> species

The Kluyveromyces marxianus strains CBS 6556, CBS 397 and CBS 712^T were cultivated on a defined medium with either glucose, lactose or sucrose as the sole carbon source, at 30 and 37°C. The aim of this work was to evaluate the diversity within this species, in terms of the macroscopic physiology. The main properties evaluated were: intensity of the Crabtree effect, specific growth rate, biomass yield on substrate, metabolite excretion and protein secretion capacity, inferred by measuring extracellular inulinase activity. The strain Kluyveromyces lactis CBS 2359 was evaluated in parallel, since it is the best described Kluyveromyces yeast and thus can be used as a control for the experimental setup. K. marxianus CBS 6556 presented the highest specific growth rate (0.70 h⁻¹) and the highest specific inulinase activity (1.65 U mg⁻¹ dry cell weight) among all strains investigated, when grown at 37°C with sucrose as the sole carbon source. The lowest metabolite formation and highest biomass yield on substrate (0.59 g dry cell weight g sucrose⁻¹) was achieved by K. marxianus CBS 712^T at 37°C. Taken together, the results show a systematic comparison of carbon and energy metabolism among three of the best known K. marxianus strains, in parallel to K. lactis CBS 2359.     

Effect of temperature on <Emphasis Type="SmallCaps">d</Emphasis>-arabitol production from lactose by <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

d-Arabitol production from lactose by Kluyveromyces lactis NBRC 1903 has been studied by following the time courses of concentrations of cell mass, lactose, d-arabitol, ethanol, and glycerol at different temperatures. It was found that temperature is a key factor in d-arabitol production. Within temperatures ranging from 25 to 39°C, the highest d-arabitol concentration of 99.2 mmol l⁻¹ was obtained from 555 mmol l⁻¹ of lactose after 120 h of batch cultivation at 37°C. The yield of d-arabitol production on cell mass growth increased drastically at temperatures higher than 35°C, and the yield reached 1.07 at 39°C. Increasing the cell mass concentration two-fold after 24 h of culture growth at 37°C, the d-arabitol concentration further increased to 168 mmol l⁻¹. According to the distribution of the metabolic products, metabolic changes related to growth phase were also discussed. The stationary-phase K. lactis cells in the batch culture that is started with exposing the precultured inoculum to high osmotic stress, high oxidative stress, and high heat stress are found to be preferable for d-arabitol production.     

The interactive effects of temperature and osmotic potential on the growth of marine isolates of <Emphasis Type="Italic">Fusarium solani</Emphasis>

The mycelial growth of 18 Fusarium solani strains isolated from sea beds of the south-eastern coast of Spain was tested on potato-dextrose-agar adjusted to different osmotic potentials with either KCl or NaCl (−1.50 to −144.54 bars) in 10 °C intervals ranging from 15 to 35 °C. Fungal growth was determined by measuring colony diameter after 4 days of incubation. Mycelial growth was maximal at 25 °C. The quantity and frequency pattern of mycelial growth of F. solani differ significantly at 15 and 25 °C, with maximal growth occurring at the highest water potential tested (−1.50 bars); and at 35 °C, with a maximal mycelial growth at −13.79 bars. The effect of water potential was independent of salt composition. The general growth pattern of F. solani showed declining growth at potentials below −41.79 bars. Fungal growth at 35 °C was always higher than that grow at 15 °C, of all the water potentials tested. Significant differences observed in the response of mycelia to water potential and temperature as main and interactive effects. The viability of cultures was increasingly inhibited as the water potential dropped, but some growth was still observed at −99.56 bars. These findings could indicate that marine strains of F. solani have a physiological mechanism that permits survival in environments with low water potential. The observed differences in viability and the magnitude of growth could indicate that the biological factors governing potential and actual growth are affected by osmotic potential in different ways.     

Entomopathogenic fungi as a potential control agent against the lesser mealworm, <Emphasis Type="Italic">Alphitobius diaperinus</Emphasis> in broiler houses

We tested the pathogenicity of 18 Metarhizium anisopliae (Metschn.) Sorokin isolates and 22 Beauveria bassiana (Balsamo) Vuillemin isolates against Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) larvae and adults. The efficacy of the most virulent isolate—M. anisopliae K—was evaluated in containers with a concrete bottom covered with wood shavings, under simulated poultry house conditions. Application of conidia of this isolate to the shavings or directly to the concrete bottom reduced the yield of larvae in 8–15 time compared with the control. In another test, the mortality of mature larvae placed on previously inoculated shavings or bottom reached 80–90% within 14 days, compared with 14% in the control. The residual activity of conidia kept at 28°C retained its initial level during 14 days post-inoculation, but declined after three weeks. Based on our data M. anisopliae has considerable potential for the control of A. diaperinus.

Impact of some environmental factors on growth and production of ochratoxin A of/by <Emphasis Type="Italic">Aspergillus tubingensis,A. niger</Emphasis>, and <Emphasis Type="Italic">A. carbonarius</Emphasis> isolated from moroccan grapes

The effects of temperature, water activity (a_w), incubation time, and their combinations on radial growth and ochratoxin A (OTA) production of/by eight Aspergillus niger aggregate strains (six A. tubingensis and two A. niger) and four A. carbonarius isolated from Moroccan grapes were studied. Optimal conditions for the growth of most studied strains were shown to be at 25°C and 0.95 a_w. No growth was observed at 10°C regardless of the water activity and isolates. The optimal temperature for OTA production was in the range of 25°C∼30°C for A. carbonarius and 30°C∼37°C for A. niger aggregate. The optimal a_w for toxin production was 0.95∼0.99 for A. carbonarius and 0.90∼0.95 for A. niger aggregate. Mean OTA concentration produced by all the isolates of A. niger aggregate tested at all sampling times shows that maximum amount of OTA (0.24 μg/g) was produced at 37°C and 0.90 a_w. However, for A. carbonarius, mean maximum amounts of OTA (0.22 μg/g) were observed at 25°C and 0.99 a_w. Analysis of variance showed that the effects of all single factors (a_w, isolate, temperature and incubation time) and their interactions on growth and OTA production were highly significant.     

Autoinduction of a genetic locus encoding putative acyltransferase in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

A genetic locus, encoding putative acyltransferase, was induced by autoinducers in Corynebacterium glutamicum. The autoinducers were maximally produced by the bacterium after 24 h culture. Those molecules are resistant to proteinase K treatment (300 μg ml⁻¹) for 30 min at 37°C or at 121°C for 15 min, and remained stable after extensive storage at 4°C. Autoinducers in the cell-free culture fluids from Corynebacterium ammoniagenes and Pseudomonas aeruginosa also induced the expression of acyltransferase in C. glutamicum, suggesting possible cross-recognition of the autoinducers by C. glutamicum. C. glutamicum thus possesses an autoinduction system which secretes autoinducers during growth, triggering the expression of downstream genes, exemplified by the putative acyltransferase gene.     

Evaluation of Metarhizium anisopliae strains as potential biocontrol agents of the tick Rhipicephalus (Boophilus) microplus and the cotton stainer Dysdercus peruvianus

The negative aspects of traditional pest control have led to the investigation of alternative methods such as biological control. Metarhizium anisopliae, well known as an entomopathogenic fungus capable of actively invading and killing its hosts and thus a candidate biopesticide, is here tested against two agricultural pests of economic/social importance and also evaluated for its chitinolytic secretion and capacity to grow and sporulate at different temperatures. None of the isolates was able to grow below 4 °C or above 37 °C. Chitinolytic activity under artificial growth conditions revealed that Rhipicephalus (Boophilus) microplus cuticle induces N-acetyl-β-d-glucosaminidase and endochitinase activities more efficiently than Dysdercus peruvianus cuticle and that glucose did not repress those activities. Bioassays were carried out with R. microplus females and fourth instar D. peruvianus. Six isolates of M. anisopliae were pathogenic to the engorged female cattle ticks. E6, GC47 and CG97 were the most virulent isolates for both arthropod models although differences were seen among them. M. anisopliae strains caused 90–100 % mortality on the fourth post-infection day in R. microplus. D. peruvianus females were more sensitive to fungal infection than males, and the most virulent strains caused 50 % mortality on the third to fourth day post-infection. Our studies suggest that M. anisopliae strain CG47 is a candidate for commercial pesticide formulations due to its capacity to kill both hosts and its ability to sporulate at higher temperatures.     

Exposure of Fischerella [Mastigocladus] to high and low temperature extremes: strain evaluation for a thermal mitigation process

In conjunction with a proposed algal cultivation scheme utilizing thermal effluent, twelve Fischerella strains were tested for tolerance to temperatures above and below their growth range. Exposure to 65 °C or 70 °C for 30 min caused bleaching and death of most or all cells. Effects of 60 °C exposure for periods of up to 2 h ranged from undetectable to severe for the various strains. Chlorophyll a content typically decreased 21–22% immediately following 60 °C or 65 °C (1 h) exposure. However, the 60 °C-shocked cultures regained normal Chl a content after 24 h at 45 °C, whereas Chl a in 65 °C-shocked cultures immediately lost visible autofluorescence and was later degraded. Exposure to 15 °C virtually stopped growth of all strains during a 48 h exposure period. Most strains grew as rapidly as 45 °C controls when restored to 45 °C, while a few strains recovered more slowly. Comparison with dark-incubated controls indicated that photooxidative damage did not occur during cold shock. Certain strains exhibited relatively rapid recovery from both heat and cold exposure, thus meeting the temperature tolerance criteria for the proposed algal cultivation process.     

Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor

Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion culture at 37°C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in the range of 26–37°C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below 32°C resulted in growth suppression. However, specific productivity of FSH, q _FSH, increased as culture temperature decreased, and the maximum q _FSH of 43.4 ng/10⁶ cells/h was obtained at 28°C, which is 13-fold higher than that at 37°C. Based on the results obtained from batch cultures, we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 × 10⁷ cells/ml at 37°C and culture temperature shifted down to 28°C to obtain a high FSH titer. Soon after the maximum FSH titer of 21 μg/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28°C is critical for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising.     

Effect of temperature and various nitrogen sources on L(+)-lactic acid production by Lactobacillus casei

 Two homofermentative strains, Lactobacillus casei NRRL B-441 and Lactobacillus casei subsp. rhamnosus NRRL B-445 were selected for further study from 17 lactic acid bacterial strains screened for lactic acid production. The effect of temperature on lactic acid production with the selected strains was investigated by adapting both strains to four different temperatures. The production of L(+)-lactic acid by both strains was most efficient at 37°C, although with L. casei the highest lactic acid concentration was obtained at 41°C. The maximal volumetric productivity with L. casei was 4.1 g l^-1 h^-1 and with L. casei subsp. rhamnosus 3.5 g l^-1 h^-1. The composition of the medium was studied in order to replace the costly yeast extract with less expensive sources of nitrogen and amino acids. From 11 different nitrogen sources investigated at 37°C, barley malt sprouts (88 g l^-1 lactic acid in 66 h) and grass extract (74 g l^-1 lactic acid in 73 h) were the best economic alternatives. The effect of different combinations of yeast extract, peptone and malt sprouts was further studied by using statistical experimental design, and an empirical second-order polynomial model was constructed on the basis of the results. With the right combination most of the yeast extract could be substituted by barley malt sprouts for efficient lactic acid production. A method for extraction of nutrients and growth factors from malt sprouts is also described. Received: 25 September 1995/Accepted: 24 October 1995