Effect of aerosolization on subsequent bacterial survival.

To determine whether aerosolization could impair bacterial survival, Pseudomonas syringae and Erwinia herbicola were aerosolized in a greenhouse, the aerosol was sampled at various distances from the site of release by using all-glass impingers, and bacterial survival was followed in the impingers for 6 h. Bacterial survival subsequent to aerosolization of P. syringae and E. herbicola was not impaired 1 m from the site of release. P. syringae aerosolized at 3 to 15 m from the site of release at a temperature of 12 degrees C and a relative humidity of 80% survived 35- to 65-fold better than P. syringae released at 27 degrees C and a relative humidity of 40%. No difference was observed in the survival of P. syringae and E. herbicola following aerosolization at the same temperature and relative humidity. Bacteria sprayed directly onto bean and oat plants established stable populations at comparable numbers on both plants over an 8-day period following inoculation. Bacteria that inoculated adjacent plants by drifting downwind up to 5 m were detectable at an initial population of 10(2) CFU/g on oats and 10(5) CFU/g on beans 2 h after the spray. However, bacterial populations on both plants were undetectable within 48 h.     

Profiling bacterial survival through a water treatment process and subsequent distribution system

AIMS: To profile fractions of active bacteria and of bacteria culturable with routine heterotrophic plate count (HPC) methods through a typical water treatment process and subsequent distribution system. In doing so, investigate how water treatment affects both bacterial abundance and diversity, and reveal the identities of active bacteria not detected by traditional HPC culture. METHODS AND RESULTS: Profiling active fractions was performed by flow cytometric cell sorting of either membrane-intact (BacLight kit) or enzymatically active (carboxyfluorescein diacetate, CFDA) bacteria, followed by eubacterial 16S rDNA-directed PCR and denaturing gradient gel electrophoresis (DGGE). Water treatment significantly reduced active bacterial numbers detected by the BacLight kit and CFDA assay by 2.89 and 2.81 log respectively. Bacterial diversity was also reduced from > 20 DGGE bands in the active fractions of reservoir water to only two bands in the active fractions of finished water. These two bands represented Stenotrophomonas maltophila, initially culturable by HPC, and a Burkholderia-related species. Both species maintained measurable traits of physiological activity in distribution system bulk water but were undetected by HPC. CONCLUSIONS: Flow cytometric cell sorting with PCR-DGGE, to assess water treatment efficacy, identified active bacteria from a variety of major phylogenetic groups undetected by routine HPC. Following treatment S. maltophila and a Burkholderia-related species retained activity and entered distribution undetected by HPC. SIGNIFICANCE AND IMPACT OF THE STUDY: Methods used here demonstrate how water treatment operators can better monitor water treatment plant efficacy and assess distribution system instability by the detection and identification of active bacteria recalcitrant to routine HPC culture.     

Effect of thermal treatments in oils on bacterial spore survival

The heat resistance of Bacillus cereus F4165/75, Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores suspended in buffer (pH 7˙2), olive oil and a commercial oil (a mixture of rapeseed oil and soy oil) was investigated. Linear survivor curves were obtained with B. cereus spores in the three menstrua and with 62A and PA 3679 spores suspended in buffer. However, the inactivation kinetics of the clostridial spores suspended in oils were concave upward with a characteristic tailing-off for 62A spores suspended in olive oil. These deviations from the semi-log model could not be ascribed to a heterogeneity in heat resistance of the spore population or to the variation of a_w during heating. Spore resistance to heat increased in the order: buffer ⋖ commercial oil < olive oil. The greater heat resistance of oil-suspended spores was ascribed to the low a_w (0˙479 and 0˙492 for commercial oil and olive oil, respectively) and to the composition of the oils. The difference in z values ( ca 28°C in oils and 10°-12°C in buffer) suggested that the mechanism of inactivation differs for spores suspended in lipids and in aqueous systems. The thermodynamic data were consistent with this hypothesis.     

Effect of simulated lunar impact on the survival of bacterial spores

Bacterial spores encased in beads and fired into crushed basalt at statistically probable lunar capture velocities were not recoverable. At velocities near 2.4 km s^–1, the minimum lunar capture velocity, only 10⁴ of a total of 10⁹ spores were recovered.     

Effect of sunlight on bacterial survival in transparent air samplers.

Two types of commercially available microbiological air samplers (model STA-203, New Brunswick Scientific and All-Glass Impingers, Millipore) were used outdoors at an activated sludge treatment plant. The recovery of viable particles with both instruments was increased three- to eight-fold by shielding the trapping medium from the sun during the sampling process.     

Effect of the osmotic conditions during sporulation on the subsequent resistance of bacterial spores

The causes of Bacillus spore resistance remain unclear. Many structures including a highly compact envelope, low hydration of the protoplast, high concentrations of Ca-chelated dipicolinic acid, and the presence of small acid-soluble spore proteins seem to contribute to resistance. To evaluate the role of internal protoplast composition and hydration, spores of Bacillus subtilis were produced at different osmotic pressures corresponding to water activities of 0.993 (standard), 0.970, and 0.950, using the two depressors (glycerol or NaCl). Sporulation of Bacillus subtilis was slower and reduced in quantity when the water activity was low, taking 4, 10, and 17 days for 0.993, 0.970, and 0.950 water activity, respectively. The spores produced at lower water activity were smaller and could germinate on agar medium at lower water activity than on standard spores. They were also more sensitive to heat (97 degrees C for 5-60 min) than the standard spores but their resistance to high hydrostatic pressure (350 MPa at 40 degrees C for 20 min to 4 h) was not altered. Our results showed that the water activity of the sporulation medium significantly affects spore properties including size, germination capacity, and resistance to heat but has no role in bacterial spore resistance to high hydrostatic pressure.     

ErratumTo: Effect of the osmotic conditions during sporulation on the subsequent resistance of bacterial spores

A set of mutants was generated by targeted deletion of the hsdR loci of two type I restriction modification systems (RMS) identified in Bacillus licheniformis DSM13. Single as well as double knock-outs resulted in strains being readily transformable with plasmids isolated from Bacilli. Introduction of shuttle plasmids isolated from Escherichia coli was routinely possible when the double mutant B. licheniformis MW3 (ΔhsdR1, ΔhsdR2) was used in transformation experiments. Growth and secretion of extracellular enzymes were not affected in any of the mutants. Thus, along with an optimized transformation protocol, this study makes available an urgently needed transformation system for this industrially exploited species. This work is dedicated to Prof. Dr. Hans Jürgen Rehm on the occasion of his 80th birthday.     

Effect of simultaneous application of heat and pressure on the survival of bacterial spores

C.G. MALLIDIS AND D. DRIZOU. 1991. The effect of simultaneous application of moderate hydrostatic pressure (10–300 atm) and heat on the survival of the Bacillus stearothermophilus spores in a flow-through system was investigated. A high heterogeneity of the sensitization of spores to heat by pressure was found. A higher degree of reduction of heat resistance was observed at the low than at the high temperatures tested. The simultaneous application of moderate pressure and heat can not be applied for the preservation of liquid foods due to the extreme heterogeneity of spore sensitization to heat by pressure.     

Effect of simultaneous application of heat and pressure on the survival of bacterial spores

The effect of simultaneous application of moderate hydrostatic pressure (10-300 atm) and heat on the survival of the Bacillus stearothermophilus spores in a flow-through system was investigated. A high heterogeneity of the sensitization of spores to heat by pressure was found. A higher degree of reduction of heat resistance was observed at the low than at the high temperatures tested. The simultaneous application of moderate pressure and heat can not be applied for the preservation of liquid foods due to the extreme heterogeneity of spore sensitization to heat by pressure.     

R-plasmid effects on bacterial multiplication and survival

The multiplication of Escherichia coli C containing either the plasmid R46 or its non-selftransmissible derivative was studied in the presence or absence of the isogenic R^– parent strain. Neither plasmid conferred any detectable effect on the host's ability to multiply. Similarly under conditions of prolonged incubation neither plasmid conferred a disadvantage on its host when the bacteria were grown in pure culture. However, when the incubation of mixed R⁺/R^– cultures was prolonged, the possession of either R-plasmid resulted in small but reproducible differences which favoured the R^– strain.     

Effect of medium on growth and subsequent survival,after freeze-drying,ofLactobacillus delbrueckii subsp.bulgaricus

Summary Growth ofLactobacillus delbrueckii subsp.bulgaricus, grown at a constant pH (5.7) in papain-or pepsin-treated whey was superior to that in whey or whey permeate, but inferior to growth in milk; it was not significantly different from that found in a 1:1 whey: milk medium. When 2% sodium citrate was added to the fermented medium, the cell recovery levels (59–75%) upon centrifugation were not significantly different in papain-treated media compared to untreated substrates. Cultures obtained from papain-treated milk or whey showed similar mortality levels following freeze-drying (99%) to those grown on untreated milk or whey. Addition of Tween 80 to the growth medium improved survival rate by a factor of ten.     

Survival of bacteria during aerosolization

One form of commercial application of microorganisms, including genetically engineered microorganisms is as an aerosol. To study the effect of aerosol-induced stress on bacterial survival, nonrecombinant spontaneous antibiotic-resistant mutants of four organisms, Enterobacter cloacae, Erwinia herbicola, Klebsiella planticola, and Pseudomonas syringae, were sprayed in separate experiments in a greenhouse. Samples were collected over a distance of 15 m from the spray site for enumeration. Spores of Bacillus subtilis were used as tracers to estimate the effects of dilution on changes in population over distance. Viable counts of P. syringae, Enterobacter cloacae, and K. planticola decreased significantly over a distance of 15 m. Erwinia herbicola showed no significant decline in counts over the same distance. The degree of survival of P. syringae during aerosolization was dependent on ambient environmental conditions (i.e., temperature, relative humidity), droplet size of the aerosol, and prior preparative conditions. Survival was greatest at high relative humidities (70 to 80%) and low temperatures (12 degrees C). Survival was reduced when small droplet sizes were used. The process of washing the cells prior to aerosolization also caused a reduction in their survival. Results from these experiments will be useful in developing sound methodologies to optimize enumeration and for predicting the downwind dispersal of airborne microorganisms, including genetically engineered microorganisms.     

Effect of water activity and protective solutes on growth and subsequent survival to air-drying of Lactobacillus and Bifidobacterium cultures

Probiotic cultures of Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei and Lactobacillus acidophilus were grown in media having water activities (a _w) adjusted between 0.99 and 0.94 with NaCl or with a mixture of glycerol and sucrose in order to find conditions of osmotic stress which would still allow for good growth. Cultures grown at a _w?=?0.96 or 0.99 were then recovered by centrifugation, added to a sucrose–phosphate medium and air-dried. In some assays, a 2-h osmotic stress was applied to the cell concentrate prior to air-drying. Assays were also carried out where betaine, glutamate and proline (BGP) supplements were added as protective compounds to the growth or drying media. For most strains, evidence of osmotic stress and benefits of BGP supplementation on growth occurred at a _w?=?0.96. Growing the cells in complex media adjusted at a _w?=?0.96 did not enhance their subsequent survival to air-drying, but applying the 2-h osmotic stress did. Addition of the BGP supplements to the growth medium or in the 2-h stress medium did not enhance survival to air-drying. Furthermore, addition of BGP to a sucrose–phosphate drying medium reduced survival of the cultures to air-drying. This study provides preliminary data for producers of probiotics who wish to use air-drying in replacement of freeze-drying for the stabilization of cultures.     

Effect of superovulation induction on embryonic development on day 5 and subsequent development and survival after nonsurgical embryo transfer in pigs

To evaluate the effects of eCG dosage on recovery and quality of Day 5 embryos and on subsequent development and survival after embryo transfer, batches of 5 to 10 donor sows were treated with 1000 or 1500 IU eCG. Recipients from the same batch were synchronously treated with 800 IU eCG. Ovulation was induced with 750 IU hCG (72 h after eCG) in donors and recipients. Donors were inseminated and embryos were collected at 162 h after hCG (120 h after ovulation). Ovulation rate was lower using 1000 IU eCG (28.5+/-11.7; n=48) than 1500 IU eCG (45.7+/-20.3; n=32; P<0.0001). Embryo recovery rate (82.9+/-16.9%) and percentage expanded blastocysts (56.2+/-31.4%) were similar (P>0.05). Expanded blastocysts from each group of sows were pooled into 2 groups within eCG treatment, containing embryos from normally ovulating sows (< or = 25 corpora lutea [CL]) or from superovulated sows (> 25 CL). Average diameter and number of cells of a random sample of the expanded blastocysts per pool were recorded. The average diameter of blastocysts (160.5+/-11.5 microm) was not affected by eCG dosage or ovulation rate (P>0.10). The average number of cells per embryo was higher in the 1000 IU eCG group (84.3+/-15.3) than in the 1500 IU eCG group (70.2+/-1.9; P<0.05) but was similar for normal and superovulated donors within each eCG group (P>0.10). Of the 4 groups, litters of 28 to 30 blastocysts were nonsurgically transferred to 27 synchronous recipients. Pregnant recipients were slaughtered on Day 37 after hCG treatment to evaluate embryonic development and survival. Pregnancy rate for the 1000 and 1500 IU eCG donor groups was 71% (10/14) and 46% (6/13; P>0.10), respectively. The number of implantations and fetuses for the 1000 IU eCG groups was 12.9+/-3.0 and 11.1+/-2.7, and 14.2+/-7.0 and 10.5+/-4.6, respectively, for the 1500 IU eCG groups (P>0.10). After post-priory categorizing the litters of blastocysts to below or above the average diameter (158 microm) of the transferred embryos, irrespective of eCG dosage or ovulation rate, the pregnancy rate was 43% (6/14) and 77% (10/13; P<0.10), respectively. Post-priory categorizing the transferred litters to below or above the average number of cells per embryo litter, irrespective of eCG dosage or ovulation rate, showed no differences in pregnancy rates or number of implantations and fetuses (P>0.10). It was concluded that eCG dosage affects embryonic development at Day 7 after hCG, and this effect was not due to ovulation rate. Embryonic survival after nonsurgical transfer was not related to eCG dosage but tended to be related to the diameter of the blastocysts.