Enumeration of thermotolerant Campylobacter spp. from poultry carcasses at the end of the slaughter-line

AIM: To enumerate Campylobacter on poultry carcasses at the end of the slaughter-line, and investigate the extent to which Campylobacter from a positive flock were transmitted to other flocks during slaughter. METHODS AND RESULTS: The presence (in caeca) and the level (from carcasses) of Campylobacter were determined. The isolates were fingerprinted by amplified fragment length polymorphism (AFLP). A total of three of 13 broiler flocks and three of four-layer flocks harboured caecal Campylobacter. Carcasses from the caeca-positive broiler flocks were Campylobacter positive with numbers ranging from 2.6 x 10(4) to 2.6 x 10(6) CFU per carcass. Two caeca-negative broiler flocks, slaughtered directly after the positive broiler flocks, had the first carcasses contaminated with Campylobacter, with numbers below 2 x 10(4) CFU per carcass of the same AFLP haplotypes as the preceding flock. Campylobacter was detected on carcasses from only one of the caeca-positive layer flocks in numbers below 2 x 10(4) CFU per carcass. No Campylobacter was detected on carcasses from a flock succeeding the positive-layer flocks. CONCLUSION: Carcasses from Campylobacter-positive broiler flocks were heavily contaminated with Campylobacter, and transmitted low levels of Campylobacter to carcasses from negative flocks, slaughtered directly after. Campylobacter-positive layer flocks had low numbers of Campylobacter on the carcasses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate limited cross-contamination of Campylobacter between flocks at the slaughterhouse, reducing the advantage of logistic slaughter.     

Influence of amino nitrogen in the culture medium enhances the production of δ-endotoxin and biomass of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var. <Emphasis Type="Italic">israelensis</Emphasis> for the large-scale production of the mosquito control agent

We reported here the role of amino nitrogen in the commercial production of Bacillus thuringiensis var. israelensis media design. The insect pathogen B. thuringiensis var. israelensis was cultured in different media containing varying initial levels of amino nitrogen sources obtained from three different commercial venders. The biomass, mosquito larval toxicity and spore count produced were measured during the fermentation process. The results showed that the higher level of initial amino nitrogen concentrations in the medium led to higher yield of biomass (dry weight 4.78 g l(-1)), larvicidal activity (LC(50) 18.52 ng ml(-1)) and spore count (3.24 x 10(11) CFU ml(-1)). Similarly decreasing the initial amino nitrogen concentration in the medium led to a decreased biomass (dry weight 1.64 g l(-1)), larvicidal activity (LC(50) 27.01 ng ml(-1)) and spore count (3.7 x 10(10) CFUml(-1)).     

Correlation of Campylobacter bacteriophage with reduced presence of hosts in broiler chicken ceca

Campylobacter jejuni and Campylobacter-specific bacteriophage were enumerated from broiler chicken ceca selected from 90 United Kingdom flocks (n = 205). C. jejuni counts in the presence of bacteriophage (mean log(10) 5.1 CFU/g) were associated with a significant (P < 0.001) reduction compared to samples with Campylobacter alone (mean log(10) 6.9 CFU/g).     

Introduction of the Serratia marcescens chiA gene into an endophytic Pseudomonas fluorescens for the biocontrol of phytopathogenic fungi

An endophytic strain of Pseudomonas fluorescens was isolated from micropropagated apple plantlets and introduced into beans (Phaseolus vulgaris) via their root tips. It was shown to be present as an endophyte in the roots at a level of 1.2 x 10(5) CFU/g fresh weight. The gene coding for the major chitinase of Serratia marcescens, chiA, was cloned under the control of the tac promoter into the broad-host-range plasmid pKT240 and the integration vector pJFF350. Pseudomonas fluorescens carrying tacchiA either on the plasmid or integrated into the chromosome is an effective biocontrol agent of the phytopathogenic fungus Rhizoctonia solani on bean seedlings under plant growth chamber conditions.     

Mold-inhibitory activity of different yeast species during airtight storage of wheat grain

The yeast Pichia anomala J121 inhibits spoilage by Penicillium roqueforti in laboratory and pilot studies with high-moisture wheat in malfunctioning airtight storage. We tested the biocontrol ability of an additional 57 yeast species in a grain mini silo system. Most yeast species grew to CFU levels comparable to that of P. anomala J121 after 14 days of incubation (>10(6) CFU g(-1)). Of the 58 species, 38 (63 strains) had no mold-inhibitory effects (Pen. roqueforti levels >10(5) CFU g(-1)). Among these were 11 species (18 strains) that did not grow on the wheat grain. Several of the non-inhibiting yeast species have previously been reported as biocontrol agents in other postharvest environments. Weak inhibitory activity, reducing Pen. roqueforti levels to between 10(4) and 10(5) CFU g(-1), was observed with 11 species (12 strains). Candida silvicola and Pichia guillermondii reduced Pen. roqueforti to <10(4) CFU g(-1). Candida fennica, Candida pelliculosa, Candida silvicultrix, P. anomala (29 strains), Pichia burtonii, Pichia farinosa and Pichia membranifaciens strongly inhibited Pen. roqueforti (<10(3) CFU g(-1)) in the mini silos, but none had higher biocontrol activity than P. anomala strain J121. This report is the first of biocontrol activity of C. fennica and C. silvicultrix. The ability of 27 yeast species to grow to high CFU values without inhibiting mold growth suggests that nutrient competition may not be the main mode of action of P. anomala J121.     

Optimization of spray-drying conditions for the large-scale preparation of Bacillus thuringiensis var. israelensis after downstream processing

Reduction of water activity in the formulations of mosquito biocontrol agent, Bacillus thuringiensis var. israelensis is very important for long term and successful storage. A protocol for spray drying of B. thuringiensis var. israelensis was developed through optimizing parameters such as inlet temperature and atomization type. A indigenous isolate of B. thuringiensis var. israelensis (VCRC B-17) was dried by freeze and spray drying methods and the moisture content and mosquito larvicidal activity of materials produced by the two methods were compared. The larvicidal activity was checked against early fourth instars Aedes aegypti larvae. Results showed that the freeze-dried powders retained the larvicidal activity fairly well. The spray-dried powder moderately lost its larvicidal activity at different inlet temperatures. Between the two types of atomization, centrifugal atomization retained more activity than the nozzle type atomization. Optimum inlet temperature for both centrifugal and nozzle atomization was 160 degrees C. Keeping the outlet temperature constant at 70 degrees C the moisture contents for the spray-dried powders through centrifugal atomization and freeze-dried powders were 10.23% and 11.80%, respectively. The LC(50) values for the spray-dried and freeze-dried powders were 17.42 and 16.18 ng/mL, respectively. Spore count of materials before drying was 3 x 10(10) cfu/mL and after spray drying through nozzle and centrifugal atomization at inlet and outlet temperature of 160 degrees C/70 degrees C were 2.6 x 10(9) and 5.0 x 10(9) cfu/mL, respectively.     

Synergistic interaction between Beauveria bassiana- and Bacillus thuringiensis tenebrionis-based biopesticides applied against field populations of Colorado potato beetle larvae

Commercial biopesticides based on the fungal pathogen Beauveria bassiana strain GHA and the bacterial pathogen Bacillus thuringiensis tenebrionis were applied alone and in combination (tank mixed) against larval populations of the Colorado potato beetle, Leptinotarsa decemlineata, in small plots of potatoes over three field seasons. Interactions between the two products were evaluated in terms of pest-control efficacy. B. bassiana (formulated as Mycotrol) was applied at low and medium label rates of 1.25 and 2.5 x 10(13) conidia/ha, and B. thuringienis (formulated as Novodor) was applied at low and high label rates of 40.3 and 120.8 x 10(6) Leptinotarsa units/ha. Two weekly applications of the bacterial pesticide alone provided 50-85% control of beetle larvae within 14 days after the initial application, while applications of the mycopesticide alone produced no greater than 25% control. Maximum control, in nearly all tests, was produced by the combination of the two products. The combined treatments produced a statistically significant 6-35% greater reduction in larval populations than would have been predicted had the two biopesticides acted independently. This low-level synergistic interaction was observed during all field seasons and resulted from combinations at all rates, including, in one of two tests, the low rates of each product. These results indicate that B. thuringiensis and B. bassiana have strong potential for integrated biologically based management of Colorado potato beetle.     

Effect of carbon and nitrogen sources on growth and biological efficacy of Pseudomonas fluorescens and Bacillus subtilis against Rhizoctonia solani, the causal agent of bean damping-off

One of the most important environmental factors that regulate the growth and antagonistic efficacy of biocontrol agents is the medium. The aim of this paper was to find the nitrogen and carbon sources that provide maximum biomass production of strains P-5 and P-6 (Pseudomonas fluorescens), B-3 and B-16 (Bacillus subtilis) and minimum cost of media, whilst maintaining biocontrol efficacy. All of the strains were grown in seven liquid media (pH=6.9) including: sucrose + yeast extract, molasses of sugar beet + yeast extract in 2:1 and 1:1 w/w ratios, molasses of sugar beet + urea, nutrient broth, molasses and malt extract, at an initial inoculation of 1 x 10(5) CFU ml(-1). Cells from over night cultures used to inoculate soil at 1 x 10(9) CFU cm(-3) soil. At the same time, fungal inoculum (infected millet seed with Rhizoctonia solani) was added to soil at the rate of 2 g kg(-1) soil. Results indicated that growth of P-6, B-3 and B-16 in molasses + yeast extract (1:1 w/w) medium was significantly higher than in the other media. Molasses + yeast extract (1:1 and 2:1 w/w) media supported rapid growth and high cell yields in P-5. In greenhouse condition, results indicated that the influence of the media on the biocontrol efficacy of P-5, P-6, B-3 and B-16 was the same and Pseudomonas fluorescens P-5 in molasses and malt extract media reduced the severity of disease up to 72.8 percent. On the other hand, there were observed significant differences on bean growth after one month in greenhouse. P-5 in molasses + yeast extract (1:1 w/w) medium had the most effects on bean growth promotion. In this study molasses media showed good yield efficacy in all of the strains. The high sucrose concentration in molasses justifies the high biomass in all of the strains. Also, the low cost of molasses allows its concentration to be increased in media. On the other hand, yeast extract was the best organic nitrogen source for antagonist bacteria but it is expensive for an industrial process. So it should be replaced by another industrial product instead of yeast extract, which confirm by an economic and technological study. The results obtained in this study could be used to provide a reliable basis to increase the population of biocontrol agents in fermentation process.     

Biocontrol of Rhizoctonia solani damping-off disease in cucumber with Bacillus pumilus SQR-N43

Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Micrographs were used to investigate the ability of Bacillus pumilus (B. pumilus) SQR-N43 to control Rhizoctonia solani (R. solani) Q1 in cucumbers. The root colonization ability of B. pumilus SQR-N43 was analyzed in vivo with a green fluorescent protein (GFP) tag. A pot experiment was performed to assess the in vivo disease-control efficiency of B. pumilus SQR-N43 and its bio-organic fertilizer. Results indicate that B. pumilus SQR-N43 induced hyphal deformation, enlargement of cytoplasmic vacuoles and cytoplasmic leakage in R. solani Q1 mycelia. A biofilm on the root surface was formed when the roots were inoculated with 10(7)-10(8)cells g(-1) of soil of GFP-tagged B. pumilus SQR-N43. In the pot experiment, the biocontrol reduced the concentration of R. solani. In contrast to applications of only B. pumilus SQR-N43 (N treatment), which produced control efficiencies of 23%, control efficiencies of 68% were obtained with applications of a fermented organic fertilizer inoculated with B. pumilus SQR-N43 (BIO treatment). After twenty days of incubation, significant differences in the number of CFUs and the percentage of spores of B. pumilus SQR-N43 were recorded between the N treatment (2.20×10(7)CFU g(-1) of soil and 79%, respectively) and the BIO treatment (1.67×10(8)CFU g(-1) of soil and 52%, respectively). The results indicate that B. pumilus SQR-N43 is a potent antagonist against R. solani Q1. The BIO treatment was more effective than the N treatment because it stabilized the population and increased the active form of the antagonist.     

Immobilization in alginate as a technique for the preservation of Bacillus thuringiensis var. israelensis for long-term preservation

Technique for immobilization using sodium alginate as the matrix to preserve Bacillus thuringiensis var. israelensis isolates for long time storage was developed. Two strains of B. thuringiensis var. israelensis viz., VCRC B-17 and WHO standard strain IPS-82 were immobilized in alginate matrix and preserved at 4 degrees C and when tested both were found to have maintained excellent viability and mosquito larvicidal activity for 10 years. Mosquito larvicidal activity of B-17 and IPS-82 alginate beads, in term of LC(50) values before storage was 72.07 ng/ml and 47.07 ng/ml, respectively and after storage at 4 degrees C for a period of 1 to 10 years the values ranged from 69.88 to 73.86 ng/ml with a mean of 72.38 ng/ml and 45.32 to 48.60 ng/ml with a mean of 47.49 ng/ml, respectively. Similarly spore count of the beads of the respective strains was 4.37 x 10(8) and 3.33 x 10(10) CFU/mg before storage. After storage at 4 degrees C for a period of 1 to 10 years the counts of the beads of the respective strains ranged from 4.23 x 10(8) to 4.83 x 10(8) CFU/mg (mean of 4.49 x 10(8) CFU/mg) and 3.2 x 10(10) to 3.87 x 10(10) CFU/mg (mean of 3.54 x 10(10) CFU/mg). The alginate matrix immobilization technique has many advantages over free cells are that they enhance the stability of both spores and toxin against several physicochemical conditions and confer reduced susceptibility to contamination.     

Fate of Escherichia coli O157:H7 as affected by pH or sodium chloride and in fermented, dry sausage.

The influence of pH adjusted with lactic acid or HCl or sodium chloride concentration on survival or growth of Escherichia coli O157:H7 in Trypticase soy broth (TSB) was determined. Studies also determined the fate of E. coli O157:H7 during the production and storage of fermented, dry sausage. The organism grew in TSB containing less than or equal to 6.5% NaCl or at a pH of 4.5 to 9.0, adjusted with HCl. When TSB was acidified with lactic acid, the organism grew at pH 4.6 but not at pH 4.5. A commercial sausage batter inoculated with 4.8 x 10(4) E. coli O157:H7 per g was fermented to pH 4.8 and dried until the moisture/protein ratio was less than or equal to 1.9:1. The sausage chubs were then vacuum packaged and stored at 4 degrees C for 2 months. The organism survived but did not grow during fermentation, drying, or subsequent storage at 4 degrees C and decreased by about 2 log10 CFU/g by the end of storage. These studies reveal the importance of using beef containing low populations or no E. coli O157:H7 in sausage batter, because when initially present at 10(4) CFU/g, this organism can survive fermentation, drying, and storage of fermented sausage regardless of whether an added starter culture was used.     

Iron Stress and Pyoverdin Production by a Fluorescent Pseudomonad in the Rhizosphere of White Lupine (Lupinus albus L.) and Barley (Hordeum vulgare L.)

Induction of high-affinity iron transport during root colonization by Pseudomonas fluorescens Pf-5 (pvd-inaZ) was examined in lupine and barley growing in microcosms. P. fluorescens Pf-5 (pvd-inaZ) contains a plasmid carrying pvd-inaZ; thus, in this strain, ice nucleation activity is regulated by pyoverdin production. Lupine or barley plants were grown for 18 or 8 days, respectively, in soil amended with 2% calcium carbonate and inoculated with P. fluorescens Pf-5 (pvd-inaZ) at a density of 4 x 10(sup8) CFU g (dry weight) of soil(sup-1). A filter paper blotting technique was used to sample cells from the rhizosphere in different root zones, and then the cells were resuspended for enumeration and measurement of ice nucleation activity. The population density of P. fluorescens Pf-5 (pvd-inaZ) in the rhizosphere decreased by one order of magnitude in both lupine and barley over time. The ice nucleation activity ranged from -3.4 to -3.0 log ice nuclei CFU(sup-1) for lupine and -3.0 to -2.8 log ice nuclei CFU(sup-1) for barley, was similar in all root zones, and did not change over time. An in vitro experiment was conducted to determine the relationship between ice nucleation activity and pyoverdin production in P. fluorescens Pf-5 (pvd-inaZ). An ice nucleation activity of approximately -3.0 log ice nuclei CFU(sup-1) was measured in the in vitro experiment at 25 to 50 (mu)M FeCl(inf3). By using the regression between ice nucleation activity and pyoverdin production determined in vitro and assuming a P. fluorescens Pf-5 (pvd-inaZ) population density of 10(sup8) CFU g of root(sup-1), the maximum possible pyoverdin accumulation by P. fluorescens Pf-5 (pvd-inaZ) in the rhizosphere was estimated to be 0.5 and 0.8 nmol g of root(sup-1) for lupine and barley, respectively. The low ice nucleation activity measured in the rhizosphere suggests that nutritional competition for iron in the rhizosphere may not be a major factor influencing root colonization by P. fluorescens Pf-5 (pvd-inaZ).     

Nutritive value of deep stacked and ensiled broiler litter for sheep

Broiler litter was deep stacked and ensiled with water to achieved 40% moisture before being added, with or without 5% sugarcane molasses or with rumen contents, to a basal diet. The influence of stacking and ensiling of broiler litter on the numbers of Salmonella, Shigella, Proteus and total number of colony forming units (CFU) was investigated. Nutritive value of broiler litter processed by deep stacking and ensiling was evaluated in a digestibility trial. The experiment was conducted with 30 wethers allotted to five diets. A basal diet (20% corn grain, 23% wheat bran, 37% cotton seed cake, 18% wheat straw and 2% dicalcium phosphate) was given alone (1) or with broiler litter processed by deep stacking (2), ensiling (3), ensiling with 5% added molasses (4), or ensiling with rumen contents (1:1, wet basis) (5). For Diets 2–5, the ratio of basal diet to silage was 1:1, dry basis. For the digestion trial, diets were given at 20 g dry matter (DM) kg⁻¹ body weight per day. Initial samples of broiler litter showed 2.5 × 10⁸ CFU and Salmonella, Shigella and Proteus were present. The processes of deep stacking and ensiling were equally effective in achieving a complete elimination of all the pathogens. Apparent digestibilities of organic matter (OM) and crude protein (CP) (559.1 g kg⁻¹ and 608.7 g kg⁻¹ DM) were lower (P < 0.05) for diet 2 (deep stacked litter) than for the other waste-containing diets (OM: 578.7 g kg⁻¹, 582.9 g kg⁻¹, 594.1 g kg⁻¹; CP: 688.6 g kg⁻¹, 675.8 g kg⁻¹, 709.0 g kg⁻¹ DM, for Diets 3, 4 and 5, respectively). Among the waste-containing diets, cellulose digestibility (398.7 g kg⁻¹ DM) was higher (P < 0.05) for Diet 5 (ensiled litter-rumen contents). The results indicate that deep stacking and ensiling are equally feasible and effective for eliminating the pathogens and processed broiler litter can be incorporated in the diet of ruminants at levels of up to 50% without any adverse effect on the health of the animals.     

Molecular Epidemiology of Campylobacter jejuni in Broiler Flocks Using Randomly Amplified Polymorphic DNA-PCR and 23S rRNA-PCR and Role of Litter in Its Transmission

Poultry has long been cited as a reservoir for Campylobacter spp., and litter has been implicated as a vehicle in their transmission. Chicks were raised on litter removed from a broiler house positive for Campylobacter jejuni. Litter was removed from the house on days 0, 3, and 9 after birds were removed for slaughter. Chicks were raised on these three litters under controlled conditions in flocks of 25. None of these birds yielded C. jejuni in their cecal droppings through 7 weeks. Two successive flocks from the same Campylobacter-positive broiler house were monitored for Campylobacter colonization. Campylobacter jejuni prevalence rates were determined for each flock. Randomly amplified polymorphic DNA (RAPD)-PCR and 23S rRNA-PCR typing methods were used to group isolates. A high prevalence (60%) of C. jejuni in flock 1 coincided with the presence of an RAPD profile not appearing in flock 2, which had a lower rate of prevalence (28%). A 23S rRNA-PCR typing method was used to determine if strains with different RAPD profiles and different prevalence rates contained different 23S sequences. RAPD profiles detected with higher prevalence rates contained a spacer in the 23S rRNA region 100% of the time, while RAPD profiles found with lower prevalence rates contained an intervening sequence less than 2% of the time. Data suggest varying colonizing potentials of different RAPD profiles and a source other than previously used litter as a means of transmission of C. jejuni. These molecular typing methods demonstrate their usefulness, when used together, in this epidemiologic investigation.     

Identification of beta-exotoxin production, plasmids encoding beta-exotoxin, and a new exotoxin in Bacillus thuringiensis by using high-performance liquid chromatography.

An improved high-performance liquid chromatography separation was developed to detect and quantify beta-exotoxin production in Bacillus thuringiensis culture supernatants. Exotoxin production was assigned to a plasmid in five strains, from three subspecies (B. thuringiensis subsp. thuringiensis serotype 1, B. thuringiensis subsp. tolworthi serotype 9, and B. thuringiensis subsp. darmstadiensis serotype 10). A new exotoxin, called type II beta-exotoxin in this report, was discovered in B. thuringiensis subsp. morrisoni serotype 8ab, purified, and partially characterized. This material is more specific than type I beta-exotoxin and is very active against the Colorado potato beetle, Leptinotarsa decemlineata.     

Spatial variation in Bacillus thuringiensis/cereus populations within the phyllosphere of broad-leaved dock (Rumex obtusifolius) and surrounding habitats

The aim of this study was to determine the prevalence and toxin gene diversity of Bacillus thuringiensis/B. cereus in the phyllosphere of broad-leaved dock (Rumex obtusifolius) at a small spatial scale. B. thuringiensis/cereus populations were isolated from the phyllosphere of dock and neighbouring grass and in neighbouring soil using commercially available selective media which avoided the disadvantageous heat-shock selection procedure. The maximum density of B. thuringiensis/cereus in the dock phyllosphere was 1.9 x 10(4) CFU g(-1) but the between-leaf variation in numbers was found to follow a lognormal distribution. B. thuringiensis/cereus was also found at significant densities in soil and the phyllosphere of grass adjacent to the dock plants. PCR screening indicated that genes encoding cry1 toxin were present in the plasmids of 36.9% of B. thuringiensis/cereus isolates tested, 11.9% contained cry2, and none of the dock leaf isolates tested contained cry3, cry4, cry7 or cry8 genes. The diversity of cry genes is similar to that found from other studies focused on other parts of the world. This work is the first concerning the prevalence of B. thuringiensis/cereus on leaves in the UK, finding population sizes of previously unrecorded levels and a greater relative proportion of B. thuringiensis. We have also illustrated that before any ecological function can be investigated, suitable sampling scales need to be considered -- here we have shown that the minimum sampling unit should be individual leaves, to account for the log-normal distribution.     

Correlation of Campylobacter Bacteriophage with Reduced Presence of Hosts in Broiler Chicken Ceca

Campylobacter jejuni and Campylobacter-specific bacteriophage were enumerated from broiler chicken ceca selected from 90 United Kingdom flocks (n = 205). C. jejuni counts in the presence of bacteriophage (mean log₁₀ 5.1 CFU/g) were associated with a significant (P < 0.001) reduction compared to samples with Campylobacter alone (mean log₁₀ 6.9 CFU/g).     

Immigration of Bacillus thuringiensis to bean leaves from soil inoculum or distal plant parts

AIMS: We addressed the process of immigration of Bacillus thuringiensis from soil to leaves and its capacity to grow on bean diffusate medium (BDM), a medium designed to simulate the nutrient composition of the phylloplane. METHODS AND RESULTS: Two different B. thuringiensis strains were inoculated into soils, onto seeds or onto lower leaves of bean plants to determine if they were able to disperse to upper leaves under controlled conditions. While B. thuringiensis isolates were commonly recovered from leaves exposed to such inocula, populations were very low (<10 CFU cm(-2) of leaf). In addition, the number of cells of B. thuringiensis recovered decreased with increasing distance from the soil or from the inoculated leaves. Moreover, B. thuringiensis colonies did not grow well on BDM. CONCLUSIONS: This indicates that B. thuringiensis disperses poorly from the soil or the seed to the leaves or between leaves of the same plant under controlled conditions. Bacillus thuringiensis apparently has greater nutrient requirements than other bacterial species that are prominent inhabitants of the phylloplane. SIGNIFICANCE AND IMPACT OF THE STUDY: Finding the mechanisms that favour bacteria colonization on leaves will in turn help to improve the efficacy of biocontrol agents against the target pests.     

Production of Bacillus thuringiensis spores in total cell retention culture and two-stage continuous culture using an internal ceramic filter system

The production of Bacillus thuringiensis spores was investigated in a bioreactor incorporating a ceramic membrane filter to improve spore concentration and volumetric productivity. Two cultivation methods were used in this study: a total cell retention culture (TCRC), and a two-stage continuous culture with partial cell bleeding. In the TCRC, fed by 50 g/L of glucose, a spore concentration of 1.6 x 10(10) CFU/mL was obtained with a spore percentage of greater than 95% and a maximum cell mass of 82.2 g/L. The volumetric productivity was four times higher than that obtained from batch cultivation. In the two-stage continuous culture with partial cell bleeding spore concentration was strongly dependent on the bleed ratio. The spore concentration of 1.8 x 10(9) CFU/mL and the spore percentage of 70% were obtained at the second stage when a bleed ratio of 0.33 and a dilution rate of 0.23 h(-1) were used. (c) 1993 John Wiley & Sons, Inc.     

Bacillus thuringiensis pulmonary infection: critical role for bacterial membrane-damaging toxins and host neutrophils

The occurrence of Bacillus thuringiensis bacteremia in a neutropenic patient suffering from severe pulmonary disease addressed the question of whether the aggressive behavior of B. thuringiensis depended on the host status and/or on the membrane-damaging toxins the isolate produced. After intratracheal injection, BALB/c mice developed pneumonia followed by fatal dissemination into deep organs, with mice rendered neutropenic by cyclophosphamide injection being extremely more susceptible to infection than normal animals. In animals infected with isogenic strains of B. thuringiensis progressively more defective in membrane-damaging toxins (407 Cry->IP2>MP02), an increase in the 50% lethal dose was registered (3.9x10(5), 1.1x10(6), 1.2x10(7)CFU). Consistently, after non-lethal dose application, only 407 Cry- replicated intrapulmonary, reaching a bacterial burden 4.7-fold and 40.9-fold higher than IP2 (P=0.018) and MP02 (P=0.008) at 48h post-inoculation. Notably, the time-course of infection was similar in animals infected with viable bacilli or spores, with neutropenic mice always being more susceptible to infection. The overall results indicate that B. thuringiensis may be responsible for opportunistic infections and strongly suggest that membrane-damaging toxins contribute to intrapulmonary bacterial persistence favoring dissemination.