Radiation sterilization of surgical instruments with a consideration of metal shielding on sterilization efficiency.

The feasibility of the use of radiation for sterilization of surgical instruments was evaluated. Two aspects were considered: radiation biology of relevant microorganisms, that is, bacterial spores and viruses, and shielding and radiation protection by the metal of the instruments. After proper cleaning and hot water machine washing, surgical instruments carry few, if any contaminants; however, subsequent handling increases the contamination load. Although large instruments may attenuate as much as 30% of the incident radiation, spores dried on the metal are sensitized to irradiation by some 40%. A dose of 25 kGy (2.5 Mrad) is adequate to inactivate a potential contamination load of approximately 10(7) bacterial spores or approximately 10(4) viruses. Therefore, 25 kGy will provide a high sterility assurance level, and can be recommended with a considerable degree of confidence for hospital-based sterilization of surgical instruments.

Gamma processing of Arabic bread for immune system-compromised cancer patients.

Arabic bread prepared from local Saudi flour contained a total of up to 10(4) organisms per g. Most of these were bacterial spores that survived the baking process (1.3 X 10(2) to 3.5 X 10(3] and a small number of yeasts and molds (10 to 40 cells per g). The organisms in Arabic bread appear to be harmless to healthy individuals. However, for immune system-compromised cancer patients and bone marrow transplant recipients, it is prudent to irradiate the bread to reduce microbial contamination. The decimal reduction doses (10% survival) for the most radiation-resistant organisms (spore formers) in bread were 0.11 to 0.15 Mrad. Accordingly, 0.6 Mrad was sufficient to reduce the number of spores in Arabic bread by a factor of 10,000, i.e., to less than 1/g. This treatment constitutes radiation pasteurization (radicidation), and to this extent, provides a margin of microbiological safety. Sensory evaluation by the nine-point hedonic scale showed no detectable loss of organoleptic quality of bread up to 0.6 Mrad, while irradiation to 2.5 Mrad induced unacceptable organoleptic changes.

Genetic control of heat resistance and thermotolerance by recA and uvrA in E. coli K12.

Several recA and uvrA derivatives of E. coli K12 AB1157 develop a transient increase in heat resistance, i.e. induced thermotolerance after a brief exposure to 43.5 degrees C (less than 1 h). Thermotolerance was identified from the appearance of an inflection in the survival curve or from the loss of heat resistance in the presence of chloramphenicol (CAM) or rifampicin. Heat resistance and induced thermotolerance were enhanced by recA and uvrA gene functions and their contribution was roughly as follows: AB1157 (recA+ uvrA+) greater than AB2463 (recA- uvrA+) greater than AB1886 (recA+ uvrA-) greater than AB2480 (recA- uvrA-). In heat resistance, uvrA and recA contributed approximately equally and their effects were additive. Induced thermotolerance developed sooner and was maintained at a higher level in the presence of uvrA as compared with recA. Since uvrA-dependent excision repair is scheduled prior to recA-dependent (postreplication) repair, induction of thermotolerance may be linked to DNA repair. Although recA and uvrA play a distinct role, they are not essential, and thermotolerance can develop in the absence of either one or both of these gene functions. Furthermore, since thermotolerance can be induced in recA mutants (AB2463 and AB2480), its biochemical pathway must be different from that of the recA-dependent SOS system.

Effect of radiation and freezing on [3H]DNA of Yersinia enterocolitica.

Freezing of the enteropathogenic bacterium Yersinia enterocolitica to -18 and -75 degrees C caused 7 and 42% cell death, respectively, and 0.329 and 0.588 single-strand breaks per 10(8) daltons of DNA, respectively, while radiation to one D10 dose (10% cell survival) combined with freezing to 2 to 0, -18, and -75 degrees C induced 0.05, 0.75, and 5.04 single-strand breaks, respectively. The increase in the effectiveness of radiation with respect to the yield of single-strand breaks at -18 and -75 degrees C is contrary to expectation and seems to be due to arrest of repair of single-strand breaks by these low temperatures.

Role of AP endonuclease in DNA breakage and cell inactivation of Escherichia coli subjected to mild heat (52 degrees C).

The molecular mechanism of DNA injury by mild heat was investigated using matched isogenic mutants of E. coli. On heating at 52 degrees C for 1 h, the number of DNA single-strand breaks (SSBs) detected by the alkaline sucrose gradient sedimentation technique was consistently smaller in mutants NH5016 and BW2001, both deficient in the AP (apurinic/apyrimidinic) endonuclease of exonuclease III, as compared with their wild-type parent AB1157. The greater number of SSBs in the wild type was accompanied by more extensive cell death as compared with the AP-deficient mutants. Heating of endonuclease-free DNA systems, viz., T4 phage and T4 DNA, at 52 degrees C for up to 4 h did not result in any detectable SSB. Apparently, cellular injury by mild heat is self-inflicted through an AP-endonuclease-mediated process and hence depends on the cell's genetic complement of AP endonuclease. Mild heat is believed to activate the nucleolytic attack, and the resultant DNA-strand breaks, if not repaired, will eventually lead to cell death.

Effect of temperature on spore germination and vegetative cell growth of Clostridium botulinum.

Spore germination and vegetative growth of Clostridium botulinum type E strain VH at 2 to 50 degrees C were studied. At all of these temperatures, germination began immediately after the addition of the spores to the germination medium. Microscopic observations during germination revealed three types of spores: phase bright (ungerminated), phase variable (partially germinated), and phase dark (fully germinated). At all temperatures except 50 degrees C, there was a pronounced lag between the initial appearance of phase-variable spores and their eventual conversion to phase-dark spores. The number of partially germinated spores increased steadily, reaching 40 to 60% by 18 to 21 h of incubation. During this time, phase-dark, fully germinated spores developed slowly and did not exceed 28% in any of the samples. At 18 to 26 h of incubation, the rate of full germination increased abruptly four-fold. There was extensive and relatively rapid germination at 2 degrees C, the lowest temperature tested, yielding about 60% phase-variable spores by 18 h, which became phase-dark by 26 h of incubation. The optimum temperature for partial and full germination was consistently 9 degrees C. Germination at 50 degrees C was exceptionally rapid and was completed within 1 to 2 h, although 40% remained phase bright. Vegetative cells showed detectable growth at 6 to 41 degrees C, with a distinct optimum at 32.5 degrees C. No growth occurred at 50 degrees C, and only marginal growth was observed at 6 to 14 degrees C. The psychrophilic nature of the germination process coupled with the cold tolerance of vegetative growth appears to give C. botulinum type E an advantage in cold climates as well as in cold-stored foods.

Inactivation and injury of Yersinia enterocolitica by radiation and freezing.

Cell inactivation and cell injury by irradiation and freezing of the potentially enteropathogenic, food-borne gram-negative rod Yersinia enterocolitica strain WA was investigated. The radiation dose necessary to kill 90% of the initial population, i.e., one D-value, was 10.0, 14,3, and 24.0 krad when irradiation was carried out at 2 to 0, -18, and -75 degrees C, respectively. On the other hand, cell injury, i.e., inability to form colonies in agar containing 2.5% NaCl, was 32, 42 and 54% when cells were irradiated to one D-value at 2 to 0, -18, and -75 degrees C, respectively. Freezing alone (without irradiation) at -18 and -75 degrees C for 1 h resulted in 7 and 42% cell inactivation and 55 and 83% cell injury, respectively. These data show that given the same extent of cell inactivation, freezing caused substantially greater cell injury than radiation. For purposes of radiation sterilization, doses of 100 and 150 krad would be sufficient to inactivate 10 log cycles of Y. enterocolitica strain WA if irradiated at 2 to 0 and -18 degrees C, respectively. Presence of 2.5% NaCl may result in a further 50% reduction of the dose required to achieve sterility.

Role of chelation and water binding of calcium in dormancy and heat resistance of bacterial endospores.

The possible relationship between the water binding by bacterial endospores and their dormancy and heat resistances has been examined in terms of the coordination characteristics of the spore-bound calcium. Stabilities of the calcium complexes of typical cytoplasmic and structural spore components were determined by potentiometric equilibrium pH measurements in model systems consisting of DPA, glycine, alanine, glutamic acid, alanyl-glutamic acid, triglycine, and tetraglycine. The Ca++-form and H+-form spores of Clostridium botulinum 33A were investigated in vivo with respect to their water sorption and heat-resistance characteristics. The results suggest that the complexing of calcium and Ca(II)-DPA may be biologically significant for spore resistance and dormancy at the following three levels: (1) complexing with spore cytoplasmic pool constituents consistent with the idea of a metal-chelate cross-linked cytoplasm or spore cement stabilizing the essential biological macromolecules, (2) complexing with structural components of the spore as indicated by the interaction with model peptides, and (3) coordination with water to produce an apparently dehydrated environment in the spore as evident from the much greater water-sorption capacity of the Ca++-form spores vs the much smaller water sorption of the H+-form spores. Interestingly enough, DPA itself, in the absence of metal ion, showed some interaction with di-, tri-, and tetrapeptides and a weak but detectable interaction with amino acids. Although the exact mode of the DPA-peptide interaction is not clear, it is attractive to speculate about its possible involvement in the control of spore dormancy and resistance.

Relation between radiation resistance and salt sensitivity of spores of five strains of Clostridium botulinum types A, B, and E.

The NaCl tolerance of different strains of Clostridium botulinum varies over a wide range, and the patterns of NaCl inhibition differ distinctly and characteristically from strain to strain. The more radiation-resistant strains, such as 33A, 62A, and 7272A, are more resistant to NaCl, whereas the more radiation-sensitive strains, such as 51B and 1304E, are more sensitive to NaCl. This rule appears to hold irrespective of whether the spores were unirradiated controls or whether they were radiation damaged prior to exposure to NaCl in the recovery media. The data seem to indicate that radiation doses in the shoulder portion of the radiation survival curves did not noticeably sensitive the spores to NaCl, whereas radiation doses in the exponential-decline portion of the survival curve invariably produced a distinct sensitization. Thus, strains 33A and 62A were not sensitized to NaCl by 0.3 to 0.4 Mrad, i.e., in the shoulder portion of the survival curve. Radiation-sensitive strain 51B, which shows no distinct shoulder in its survival curve, was sensitized to NaCl by 0.1 Mrad, the lowest radiation dose employed in this study. These observations seem to suggest a possible relationship between deoxyribonucleic acid repair capacity and salt tolerance.

Direct enzymatic repair of deoxyribonucleic acid single-strand breaks in dormant spores.

With the alkaline sucrose gradient centrifugation method, it was found that dormant spores of Clostridium botulinum subjected to 300 krads of gamma radiation showed a distinct decrease in deoxyribonucleic acid (DNA) fragment size, indicating induction of single-strand breaks (SSB). A two- to threefold difference in radiation resistance of spores of two strains of C. botulinum, 33A (37% survival dose [D(37)] = 110 krads) and 51B (D(37) = 47 krads), was accompanied by relatively larger DNA fragments (molecular weight 7.9 x 10(7)) obtained during extraction from the radiation-resistant strain 33A and smaller DNA fragments (molecular weight 1.8 x 10(7)) obtained under identical conditions from radiation-sensitive strain 51B. The apparent number of DNA SSB produced by 300 krads in strains 33A and 51B was 0.37 and 3.50, respectively, per 10(8) daltons of DNA. Addition of 0.02 M ethylenediaminetetraacetic acid (EDTA) to spore suspensions during irradiation doubled the apparent number of SSB in strain 33A but had no effect on strain 51B. In vivo, 0.02 M EDTA present during irradiation to 100 to 300 krads decreased survival of spores of 33A by about 30% but had little or no effect on 51B. Survival of 33A was also reduced by about 45% when the spores were irradiated while frozen in dry ice (-75 C) and, after irradiation, immediately exposed to 0.03 M EDTA for 1 h to inhibit repair in the dormant spores. These results suggest that the highly radiation-resistant strain 33A may be able to accomplish repair of SSB during irradiation or after irradiation under nonphysiological conditions, i.e., in the dormant state. This repair can be inhibited by EDTA. Sedimentation patterns show that DNA from spores of both strains 33A and 51B did not show any postirradiation repair during the first 6 h of germination, as opposed to Bacillus subtilis spores, which exhibit repair immediately after germination. These observations suggest the existence of direct repair in physiological dormant spores of strain 33A in the cryptobiotic resting state in the absence of germination. The repair seems to be similar to that of polynucleotide ligase activity shown to be operative in some vegetative cells. Apparently radiation-sensitive strains such as 51B and B. subtilis are generally poor in DNA repair enzyme activity under conditions of spore dormancy, which may account for the approximately threefold difference in radiation sensitivity or DNA fragility of different strains, or both.

Photoprotection by dipicolinate against inactivation of bacterial spores with ultraviolet light.

The resistance of three types of Bacillus cereus T spores to ultraviolet radiation corresponded to their dipicolinic acid (DPA) content. Photoprotection against ultraviolet light was observed in DPA-containing spores and in DPA-less spores irradiated through calcium dipicolinate.