高静压对枯草杆菌芽孢灭活作用的研究

采用高静压协同中温的方法,研究了连续式施压(continuous pressurization)和间歇式施压(intermittent pressurization)两种不同方式对枯草杆菌芽孢的灭活作用.实验设计了分阶段施压方式,即先低压200 MPa/5 min,再高压500 MPa/5 min,循环1～3次.结果表明,同200 MPa～500MPa连续施压30 min相比,间歇施压更有效地杀灭芽孢,并缩短了处理时间.经扫描电镜观察,芽孢外壳出现凹陷、皱褶等形态变化,这种间歇式的施压产生强烈的机械剪切力,造成芽孢损伤,及内容物的泄漏,甚至死亡.     

Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis

The rates of germination of Bacillus subtilis spores with L-alanine were increased markedly, in particular at low L-alanine concentrations, by overexpression of the tricistronic gerA operon that encodes the spore's germinant receptor for L-alanine but not by overexpression of gerA operon homologs encoding receptors for other germinants. However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K(+) (AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic gerB and gerK operons. Overexpression of the gerB or gerK operon or both the gerB and gerK operons also did not stimulate spore germination in AGFK. Overexpression of a mutant gerB operon, termed gerB*, that encodes a receptor allowing spore germination in response to either D-alanine or L-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations. However, the magnitudes of the increases in the germination rates with D-alanine or L-asparagine in spores overexpressing gerB* were well below the increases in the spore's levels of the GerBA protein. Germination of gerB* spores with D-alanine or L-asparagine did not require participation of the products of the gerK operon, but germination with these agents was decreased markedly in spores also overexpressing gerA. These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with D-alanine or L-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination. We also found that very high levels of overexpression of the gerA or gerK operon (but not the gerB or gerB* operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of gerA or gerK overexpression that were used to generate spores for analysis of rates of germination.     

Kinetics of inactivation of Bacillus subtilis spores by continuous or intermittent ohmic and conventional heating

Bacillus subtilis spores were suspended in 0.1% NaCl solution (ca. 10(7) CFU/mL) and treated by conventional or ohmic heating under identical temperature histories. Temperatures tested were in the range of 88 to 99 degrees C. Survival curves and calculated D values showed significantly higher lethality for spores by ohmic than conventional heating. The z or Ea values corresponding to the two heating methods, however, were not significantly different. Spores of B. subtilis were suspended in nutrient broth and treated with conventional and ohmic heating through a single- or a double-stage treatment. In case of double-stage treatment, heating was interrupted by a 20 min of incubation at 37 degrees C to induce a Tyndallization effect. Spore inactivation during double-stage treatment was greater for ohmic than conventional heating. The enhanced spore inactivation by ohmic, compared with conventional, heating resulted from a greater rate of spore death during the first stage of heating and greater decrease in count of viable spores immediately after the incubation period that intervened the heating process. Thus it is concluded that spore inactivation during ohmic heating was primarily due to the thermal effect but there was an additional killing effect caused by the electric current.     

Resporulation of outgrowing Bacillus subtilis spores.

Germinated spores of Bacillus subtilis were incubated in outgrowth medium and tested periodically for capacity to sporulate when suspended in sporulation medium. Concurrent measurements were made of deoxyribonucleic acid (DNA) content and numbers of cell division septa and nucleoids. Sporulation potential is shown to reach a peak at about 110 min at which time the chromosomes are probably well into the second round of replication. Experiments with nalidixic acid show that sporulation potential can be generated in the outgrowth medium even when DNA synthesis is largely prevented. Further experiments show that nalidixic acid apparently does not prevent the formation of DNA initiation complexes, which can subsequently function after resuspension in the sporulation medium. The results support those previously obtained with a temperature-sensitive DNA mutant which indicated that sporulation could only be induced at a specific stage of chromosome replication, and then only if the cells are in a state of nutritional "step-down".     

Germination and inactivation of Bacillus subtilis spores induced by moderate hydrostatic pressure

In this study, we investigated the mechanisms of spore inactivation by high pressure at moderate temperatures to optimize the sterilization efficiency of high‐pressure treatments. Bacillus subtilis spores were first subjected to different pressure treatments ranging from 90 to 550 MPa at 40°C, with holding times from 10 min to 4 h. These treatments alone caused slight inactivation, which was related to the pressure‐induced germination of the spores. After these pressures treatments, the sensitivity of these processed spores to heat (80°C/10 min) or to high pressure (350 MPa/40°C/10 min) was tested to determine the pressure‐induced germination rate and the advancement of the spores in the germination process. The subsequent heat or pressure treatments were applied immediately after decompression from the first pressure treatment or after a holding time at atmospheric pressure. As already known, the spore germination is more efficient at low pressure level than at high pressure level. Our results show that this low germination efficiency at high pressure seemed not to be related either to a lower induction or a difference in the induction mechanisms but rather to an inhibition of enzyme activities which are involved in germination process. In fact, high pressure was necessary and very efficient in inducing spore germination. However, it seemed to slow the enzymatic digestion of the cortex, which is required for germinated spores to be inactivated by pressure. Although these results indicate that high‐pressure treatments are more efficient when the two treatments are combined, a small spore population still remained dormant and was not inactivated with any holding time or pressure level. Biotechnol. Bioeng. 2010;107: 876–883. © 2010 Wiley Periodicals, Inc.     

Effects of chlorhexidine gluconate on the development of spores of Bacillus subtilis

The effects of sublethal concentrations of the membrane-active agent chlorhexidine gluconate (CHG) on the growth rate and sporulation of Bacillus subtilis vegetative MB₂ cells have been investigated. CHG increased the mean generation time (Mgt) of vegetative cells in casein medium. It also affected spore development: as CHG concentrations increased, spore index (SI) values decreased and sensitivity to both toluene and heat increased.     

Effects of aqueous and alcoholic povidone-iodine on spores of Bacillus subtilis

G orman , S.P., S cott , E.M. & H utchinson , E.P. 1985. Effects of aqueous and alcoholic povidone-iodine on spores of Bacillus subtilis. Journal of Applied Bacteriology , 59 , 99–105.
Spores of Bacillus subtilis NCTC 10073 were examined for susceptibility to two proprietary brands of povidone-iodine: an aqueous solution, Betadine and an alcoholic solution, Videne. Spores were converted to ion-exchange (Ca, H) and coat-defective (SLS-, UME-, UMS-, UDT- and UDS-treated) forms. The resistance of these to povidone-iodine was compared and related to uptake. Effects on spore protoplasts and cortex in relation to hexosamine release were also examined. The degree of spore penetration and site of action of povidone-iodine is discussed.     

Effects of aqueous and alcoholic povidone-iodine on spores of Bacillus subtilis

Spores of Bacillus subtilis NCTC 10073 were examined for susceptibility to two proprietary brands of povidone-iodine: an aqueous solution, Betadine and an alcoholic solution, Videne. Spores were converted to ion-exchange (Ca, H) and coat-defective (SLS-, UME-, UMS-, UDT- and UDS-treated) forms. The resistance of these to povidone-iodine was compared and related to uptake. Effects on spore protoplasts and cortex in relation to hexosamine release were also examined. The degree of spore penetration and site of action of povidone-iodine is discussed.     

Thermal inactivation kinetics of Bacillus subtilis spores suspended in buffer and in oils

The heat resistance of Bacillus subtilis 5230 and A spores freeze dried and suspended in buffer or oils was investigated. As expected, spores were more resistant to heat when suspended in oils than in buffer. This was ascribed to the low a _w of oils and to their content of free fatty acids. Linear survivor curves were obtained for spores suspended in buffer at 105°C or above and for B. subtilis A spores suspended in a vegetable oil. However, the survivor curves of the spores suspended in mineral oil (strain 5230) or olive oil (both strains) were concave upward with a characteristic tailing. The tailing could not be ascribed to spore clumping or to a specific heat injury that can be circumvented by Ca-dipicolinate. It is possibly due to another mechanism of injury or to the activation at high temperature of a normally dormant germination system.     

Triple fixation of Bacillus subtilis dormant spores.

A triple-fixation method with a sequential application of 5% glutaraldehyde, 1% osmium tetroxide, and 2% potassium permanganate gave superior preservation of the ultrastructure of Bacillus subtilis dormant spores with a thick spore coat.     

Effects of DNA-polymerase-defective and recombination-deficient mutations on the ultraviolet sensitivity of Bacillus subtilis spores.

The DNA of UV-irradiated Bacillus subtilis spores, which contains 5-thyminyl-5,6-dihydrothymine (TDHT) as the major thymine photoproduct, is known to be repaired during germination by two complementary mechanisms: (I) the well-known excision repair, and (2) a special process, "spore repair", which destroys TDHT in situ without rendering it acid-soluble. In the absence of both mechanisms TDHT is not removed, and spores are highly UV-sensitive. When either of two mutations (pol-59 and pol-151) giving defective DNA polymerase, or one (rec-A1) giving a recombination deficiency are introduced into strains defective in one of these known TDHT removal processes, the chemically measured elimination of TDHT from spore DNA is unaltered, but spore UV-sensitivity is increased. The pol mutations produce their greatest sensitivity increase in spores of strains already deficient for the in situ destruction of TDHT, while the rec mutation gives its maximum sensitivity increase to spores of strains lacking excision. These facts argue that the pol mutations interfere mostly with excision repair (presumably its later resynthesis step), shile the rec mutation impairs "spore repair" in some step occurring subsequent to the TDHT destruction in situ. With either of these impairments of the later repair steps, DNA of UV-irradiated and germinated spores is considerably degraded, unless germination is carried out in the presence of chloramphenicol.     

Formaldehyde gas inactivation of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials

AIMS: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using formaldehyde gas. METHODS AND RESULTS: B. anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to approx. 1100 ppm formaldehyde gas for 10 h. Formaldehyde exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with B. subtilis (galvanized metal and painted wallboard paper) and G. stearothermophilus (industrial carpet and painted wallboard paper). Formaldehyde gas inactivated>or=50% of the biological indicators and spore strips (approx. 1x10(6) CFU) when analyzed after 1 and 7 days. CONCLUSIONS: Formaldehyde gas significantly reduced the number of viable spores on both porous and nonporous materials in which the two surrogates exhibited similar log reductions to that of B. anthracis on most test materials. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using formaldehyde gas.     

Purification and properties of glucose-6-phosphate dehydrogenase from Bacillus subtilis spores.

Glucose-6-phosphate dehydrogenase [D-glucose-6-phosphate: NADP oxidoreductase, EC. 1. 1. 1. 49] obtained from spores of Bacillus subtilis PCI 219 strain was partially purified by filtration on Sephadex G-200, ammonium sulfate fractionation and chromatography on DEAE-Sephadex A-25 (about 54-fold). The optimum pH for stability of this enzyme was about 6.3 and the optimum pH for the reaction about 8.3. The apparent Km values of the enzyme were 5.7 X 10(-4) M for glucose-6-phosphate and 2.4 X 10(-4) M for nicotinamide adenine dinucleotide phosphate (NADP). The isoelectric point was about pH 3.9. The enzyme activity was unaffected by the addition of Mg++ or Ca++. The inactive glucose-6-phosphate dehydrogenase obtained from the spores heated at 85 C for 30 min was not reactivated by the addition of ethylenediaminetetraacetic acid, dipicolinic acid or some salts unlike inactive glucose dehydrogenase.     

Photodynamic inactivation of Bacillus subtitis spores

The effect of strong visible light on spores of Bacillus subtilis has been studied. With the illumination conditions used, spores were killed exponentially and viability decreased to less than 13% survivors after 24 h. This decrease did not take place when the cells were kept under strictly anaerobic conditions. The killing is, therefore, attributable to a photodynamic effect. The presence in the medium of exogenous photosensitizers did not increase the effect of light and this is attributable to the impermeability of spores to these agents. Once germinated, the spores were killed at a much faster rate, even when outgrowth was stopped. The highest sensitivity was reached 90 min after onset of outgrowth.     

Thermal inactivation and injury of Bacillus stearothermophilus spores.

Aqueous spore suspensions of Bacillus stearothermophilus ATCC 12980 were heated at different temperatures for various time intervals in a resistometer, spread plated on antibiotic assay medium supplemented with 0.1% soluble starch without (AAMS) or with (AAMS-S) 0.9% NaCl, and incubated at 55 degrees C unless otherwise indicated. Uninjured spores formed colonies on AAMS and AAMS-S; injured spores formed colonies only on AAMS. Values of D, the decimal reduction time (time required at a given temperature for destruction of 90% of the cells), when survivors were recovered on AAMS were 62.04, 18.00, 8.00, 3.33, and 1.05 min at 112.8, 115.6, 118.3, 121.1, and 123.9 degrees C, respectively. Recovery on AAMS-S resulted in reduced decimal reduction time. The computed z value (the temperature change which will alter the D value by a factor of 10) for spores recovered on AAMS was 8.3 degrees C; for spores recovered on AAMS-S, it was 7.6 degrees C. The rates of inactivation and injury were similar. Injury (judged by salt sensitivity) was a linear function of the heating temperature. At a heating temperature of less than or equal to 118.3 degrees C, spore injury was indicated by the curvilinear portion of the survival curve (judged by salt sensitivity), showing that injury occurred early in the thermal treatment as well as during logarithmic inactivation (reduced decimal reduction time). Heat-injured spores showed an increased sensitivity not only to 0.9% NaCl but also to other postprocessing environmental factors such as incubation temperatures, a pH of 6.6 for the medium, and anaerobiosis during incubation.