Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nmwavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.The October 2001 bioterrorist attack with Bacillus anthracis spores has sparked renewed interest in studying methods of bacterial spore inactivation and the mechanisms by which spores resist the lethal effects of various disinfection treatments. UV radiation at a 254-nm wavelength has been used as an efficient and cost-effective means of disinfecting surfaces (1,4,15,16,17), building air (3, 8, 13), and drinking water supplies (5). The most reliable method for testing the efficacy of UV disinfection equipment is biodosimetry, the use of a test organism to measure the biologically effective UV dose (2). Commonly used test organisms for UV biodosimetry studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of UV resistance, reproducible inactivation response, and ease of use (reviewed in references 9 and 11). In particular, exhaustive testing of spores of the B. subtilis strain ATCC 6633 has resulted in this strain serving as the current European biodosimetry standard for 254-nm UV disinfection of drinking water (5).Semilogarithmic plots of spore inactivation versus UV fluence (dose) produce a characteristic curve, consisting of a shoulder at low UV doses, followed by a curve reflecting exponential inactivation at higher UV doses (Fig. 1). Two parameters often used to describe spore resistance to UV are (i) the UV dose lethal for 90% of the population (LD 90 ) and (ii) the decimal reduction value (D value), defined as the UV dose which reduces spore viability by a factor of 10, measured from the exponential portion of the inactivation curve (6, 11). For example, from the published data of Hoyer (5) (Fig. 1), it can be calculated that spores of B. subtilis ATCC 6633 exhibit an LD 90 and a D value of 260 and 120 J/m 2 , respectively (Table 1). In stark contrast to the extensively characterized UV inactivation response of B. subtilis spores, much less work has been performed to characterize the UV inactivation kinetics of B. anthracis ...

Previously, spontaneous rifampin resistance mutations were isolated in cluster I of the rpoB gene, resulting in amino acid replacements (Q469R, H482R, H482Y, or S487L) in the Bacillus subtilis RNA polymerase ␤ subunit (W. L. Nicholson and H. Maughan, J. Bacteriol. 184:4936-4940, 2002). In this study, each amino acid change in the ␤ subunit was observed to result in its own unique spectrum of effects on growth and various developmental events, including sporulation, germination, and competence for transformation. The results thus establish the important role played by the RNA polymerase ␤ subunit, not only in the catalytic aspect of transcription, but also in the regulation of major developmental events in B. subtilis.The antibiotic rifampin (RIF) is a potent inhibitor of prokaryotic transcription initiation (33) and has long been used both to study bacterial transcription and as a highly clinically effective drug, particularly for the treatment of the infectious diseases tuberculosis and leprosy, caused by Mycobacterium tuberculosis and Mycobacterium leprae, respectively (22). Resistance to RIF (Rif r ) arises from mutations in the rpoB gene encoding the ␤ subunit of RNA polymerase (11), and the majority of Rif r mutations occur within a short (Ͻ100 bp) region within rpoB, designated cluster I in Escherichia coli (11,28). Cluster I homologues have also been studied in a number of bacteria outside the enteric paradigm, including M. tuberculosis (7,13,24,29,31,34,36), Streptomyces spp. (8, 9), and Bacillus subtilis (2,10,16,25).Several lines of evidence indicate profound fundamental connections between RIF resistance, RNA polymerase structure and function, and global gene expression. First, RIF has long been known to specifically block transcription initiation but not elongation (33). In the recently elucidated three-dimensional structure of RNA polymerase, the RIF binding site, including cluster I, was localized to the ␤ subunit in the DNA-RNA channel, ϳ12 Å downstream from the active site; RIF binding apparently physically blocks initiation when the nascent transcript is 2 to 3 nucleotides (nt) long (4, 15).Second, in addition to the Rif r phenotype, additional effects on gene expression have been noted in bacteria carrying mutations in rpoB cluster I. For example, binding of guanosine tetraphosphate (ppGpp) to RNA polymerase has long been recognized as an important modulator of global gene expression during growth, stationary phase, and the stringent response (reviewed in references 27 and 37). Although the exact location of the ppGpp binding site on RNA polymerase is at present unknown, it may reside in close proximity to the RIF binding site, because (i) in E. coli, Rif r mutations were isolated in rpoB which alleviated the toxic and growth-inhibitory effects of artificially induced ppGpp overproduction (32); (ii) certain mutations in cluster I of the B. subtilis rpoB gene confer both Rif r and hypersensitivity of B. subtilis RNA polymerase to the transcription termination factor NusG (10); and (iii) in Strep...

Stainless steel surfaces coated with paints containing a silver-and zinc-containing zeolite (AgION antimicrobial) were assayed in comparison to uncoated stainless steel for antimicrobial activity against vegetative cells and spores of three Bacillus species, namely, B. anthracis Sterne, B. cereus T, and B. subtilis 168. Under the test conditions (25°C and 80% relative humidity), the zeolite coating produced approximately 3 log 10 inactivation of vegetative cells within a 5-to 24-h period, but viability of spores of the three species was not significantly affected.Metals such as silver and zinc have long been recognized for their broad-spectrum antimicrobial properties, and a number of preparations containing silver and/or zinc have been formulated for reducing infections of indwelling catheters and stainless steel orthopedic devices (reviewed in references 1, 2, 7, 9, 11, and 13). An interesting advance in technology has been to trap silver and zinc ions within zeolites (inorganic ceramics) and to apply these compounds to various materials as longlasting antimicrobial treatments (8,14). Commercial concerns have claimed that application of silver-zinc zeolite coatings to stainless steel surfaces such as air ducts, countertops, or food preparation areas can reduce the bacterial load, hence lowering the risk of contamination by pathogenic or food spoilage microorganisms. However, controlled studies testing the efficacy of such preparations are scarce. Therefore, the present study was undertaken to test claims of the antibacterial properties of a particular silver-zinc zeolite preparation, the AgION antimicrobial, applied to stainless steel sheets. The challenge organisms used were species of the genus Bacillus, chosen for their environmental ubiquity, their ability to form resistant spores, and their known roles as agents of food poisoning (B. cereus, B. anthracis), food spoilage (B. subtilis), and bioterrorism (B. anthracis).The bacterial strains used, namely, B. cereus strain T (WN129), B. subtilis strain 168 (WN131), and B. anthracis Sterne (WN742), were from the communicating author's laboratory collection. Vegetative cells of all strains were produced as follows. Overnight cultures grown in liquid Luria-Bertani (LB) medium (5) were diluted to an optical density of 10 Klett units (Klett-Summerson colorimeter, no. 66 red filter) into 10 ml of fresh LB medium and incubated in a rotary shaker (New Brunswick G76; 300 rpm, 37°C) to 300 Klett units (ϳ10 8 CFU/ ml). (Note that to prevent sporulation during the course of experiments, B. anthracis Sterne vegetative cells were grown to only 160 Klett units [ϳ5 ϫ 10 7 CFU/ml] in LB.) Spores of all strains were produced by incubation in liquid Schaeffer's sporulation medium (12) at 37°C for 72 h in a rotary shaker with vigorous aeration. Spores were harvested by centrifugation (10,000 ϫ g for 10 min at 25°C), purified using the lysozyme and buffer-washing technique described by Nicholson and Setlow (6), heat shocked (80°C, 10 min), and stored at 4°C in phosphate-buffered s...