Am J Pathol, 2003 Aug, 163(2), 701 - 9 Pathology and pathogenesis of bioterrorism-related inhalational anthrax; Guarner J et al.; During October and November 2001, public health authorities investigated 11 patients with inhalational anthrax related to a bioterrorism attack in the United States . Formalin-fixed samples from 8 patients were available for pathological and immunohistochemical (IHC) study using monoclonal antibodies against the Bacillus anthracis cell wall and capsule . Prominent serosanguinous pleural effusions and hemorrhagic mediastinitis were found in 5 patients who died . Pulmonary infiltrates seen on chest radiographs corresponded to intraalveolar edema and hyaline membranes . IHC assays demonstrated abundant intra- and extracellular bacilli, bacillary fragments, and granular antigen-staining in mediastinal lymph nodes, surrounding soft tissues, and pleura . IHC staining in lung, liver, spleen, and intestine was present primarily inside blood vessels and sinusoids . Gram's staining of tissues was not consistently positive . In 3 surviving patients, IHC of pleural samples demonstrated abundant granular antigen-staining and rare bacilli while transbronchial biopsies showed granular antigen-staining in interstitial cells . In surviving patients, bacilli were not observed with gram's stains . Pathological and IHC studies of patients who died of bioterrorism-related inhalational anthrax confirmed the route of infection . IHC was indispensable for diagnosis of surviving anthrax cases . The presence of B . anthracis antigens in the pleurae could explain the prominent and persistent hemorrhagic pleural effusions. Infect Immun, 2003 Aug, 71(8), 4563 - 79 Genome-based bioinformatic selection of chromosomal Bacillus anthracis putative vaccine candidates coupled with proteomic identification of surface-associated antigens; Ariel N et al.; Bacillus anthracis (Ames strain) chromosome-derived open reading frames (ORFs), predicted to code for surface exposed or virulence related proteins, were selected as B . anthracis-specific vaccine candidates by a multistep computational screen of the entire draft chromosome sequence (February 2001 version, 460 contigs, The Institute for Genomic Research, Rockville, Md.) . The selection procedure combined preliminary annotation (sequence similarity searches and domain assignments), prediction of cellular localization, taxonomical and functional screen and additional filtering criteria (size, number of paralogs) . The reductive strategy, combined with manual curation, resulted in selection of 240 candidate ORFs encoding proteins with putative known function, as well as 280 proteins of unknown function . Proteomic analysis of two-dimensional gels of a B . anthracis membrane fraction, verified the expression of some gene products . Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses allowed identification of 38 spots cross-reacting with sera from B . anthracis immunized animals . These spots were found to represent eight in vivo immunogens, comprising of EA1, Sap, and 6 proteins whose expression and immunogenicity was not reported before . Five of these 8 immunogens were preselected by the bioinformatic analysis (EA1, Sap, 2 novel SLH proteins and peroxiredoxin/AhpC), as vaccine candidates . This study demonstrates that a combination of the bioinformatic and proteomic strategies may be useful in promoting the development of next generation anthrax vaccine. Environ Microbiol, 2003 Aug, 5(8), 631 - 40 The hidden lifestyles of Bacillus cereus and relatives; Jensen GB et al.; Bacillus cereus sensu lato, the species group comprising Bacillus anthracis, Bacillus thuringiensis and B . cereus (sensu stricto), has previously been scrutinized regarding interspecies genetic correlation and pathogenic characteristics . So far, little attention has been paid to analysing the biological and ecological properties of the three species in their natural environments . In this review, we describe the B . cereus sensu lato living in a world on its own; all B . cereus sensu lato can grow saprophytically under nutrient-rich conditions, which are only occasionally found in the environment, except where nutrients are actively collected . As such, members of the B . cereus group have recently been discovered as common inhabitants of the invertebrate gut . We speculate that all members disclose symbiotic relationships with appropriate invertebrate hosts and only occasionally enter a pathogenic life cycle in which the individual species infects suitable hosts and multiplies almost unrestrained. Crit Rev Biochem Mol Biol, 2003, 38(3), 173 - 98 Bacillus species proteins involved in spore formation and degradation: from identification in the genome, to sequence analysis, and determination of function and structure; Jedrzejas MJ et al.; The members of Bacillus species are Gram-positive, ubiquitous spore-forming bacilli . Several genomic sequences have been made available during recent years, including Bacillus subtilis, a model organism among this genus, Bacillus anthracis, and their analyses provided a wealth of information about spore-forming bacteria . Some members of this species can cause serious diseases in livestock and humans . An important pathogen in this group of organisms is B . anthracis, which is the causative agent of anthrax . A summary of the B . subtilis genome information, based on the publicly released sequence, that allowed for the identification and characterization of new and novel proteins of this organism as well as similar proteins from other members of Bacillus species is provided . The primary goal for this work is to present a review of the genome sequence-identified genes that encode proteins involved in the sporulation, germination, and outgrowth processes . These three processes are essential for spore development and later its transformation into a vegetative cell . Additionally, for a few selected examples of the protein products of the identified genes, the application of bioinformatics and modeling tools is illustrated in order to determine their likely structure and function . Two three-dimensional models of the structures of such proteins, PrfA endonuclease and phosphatase PhoE, are presented together with the structure-based functional conclusions . The review of such studies provides an example of how the genomic sequence can be utilized in order to elucidate the structure and function of proteins, in particular proteins of the Bacillus species . Because only a limited number of proteins of Bacillus species organisms are involved in the synthesis and degradation of spores and have been characterized to date, this genome-based analysis may provide new insights into the developmental processes of bacterial organism. Nature, 2003 Jul 17, 424(6946), 329 - 34 Impairment of dendritic cells and adaptive immunity by anthrax lethal toxin; Agrawal A et al.; Anthrax poses a clear and present danger as an agent of biological terrorism . Infection with Bacillus anthracis, the causative agent of anthrax, if untreated can result in rampant bacteraemia, multisystem dysfunction and death . Anthrax lethal toxin (LT) is a critical virulence factor of B . anthracis, which occurs as a complex of protective antigen and lethal factor . Here we demonstrate that LT severely impairs the function of dendritic cells--which are pivotal to the establishment of immunity against pathogens--and host immune responses by disrupting the mitogen-activated protein (MAP) kinase intracellular signalling network . Dendritic cells exposed to LT and then stimulated with lipopolysaccharide do not upregulate co-stimulatory molecules, secrete greatly diminished amounts of proinflammatory cytokines, and do not effectively stimulate antigen-specific T cells in vivo . Furthermore, injections of LT induce a profound impairment of antigen-specific T- and B-cell immunity . These data suggest a role for LT in suppressing host immunity during B . anthracis infections, and represent an immune evasion strategy, where a microbe targets MAP kinases in dendritic cells to disarm the immune response. J Antimicrob Chemother, 2003 Aug, 52(2), 297 - 9 Epub 2003 Jul 15. Susceptibility of Bacillus anthracis to eleven antimicrobial agents including novel fluoroquinolones and a ketolide; Frean J et al.; OBJECTIVES: To determine the susceptibility of southern African strains of Bacillus anthracis to new, investigational agents as well as conventional antibiotics . MATERIALS AND METHODS: The MICs of 26 isolates of B . anthracis from South Africa and Zimbabwe, as well as the Sterne vaccine strain and a type culture strain, were determined by agar dilution . RESULTS: The most active antimicrobial agents were the novel ketolide ABT 773, new and conventional fluoroquinolones, and doxycycline; macrolides were intermediately active . The lack of activity of extended-spectrum cephalosporins against B . anthracis was confirmed . CONCLUSIONS: Susceptibility to conventional antibiotics was in keeping with previous studies . Two new fluoroquinolones and a ketolide showed promising in vitro activity that would support their further evaluation in animal models of anthrax. Cell Microbiol, 2003 Aug, 5(8), 523 - 32 Decreased glycogen synthase kinase 3-beta levels and related physiological changes in Bacillus anthracis lethal toxin-treated macrophages; Tucker AE et al.; The lethal factor (LF) component of Bacillus anthracis lethal toxin (LeTx) cleaves mitogen activated protein kinase kinases (MAPKKs) in a variety of different cell types, yet only macrophages are rapidly killed by this toxin . The reason for this selective killing is unclear, but suggests other factors may also be involved in LeTx intoxication . In the current study, DNA membrane arrays were used to identify broad changes in macrophage physiology after treatment with LeTx . Expression of genes regulated by MAPKK activity did not change significantly, yet a series of genes under glycogen synthase kinase-3-beta (GSK-3beta) regulation changed expression following LeTx treatment . Correlating with these transcriptional changes GSK-3beta was found to be below detectable levels in toxin-treated cells and an inhibitor of GSK-3beta, LiCl, sensitized resistant IC-21 macrophages to LeTx . In addition, zebrafish embryos treated with LeTx showed signs of delayed pigmentation and cardiac hypertrophy; both processes are subject to regulation by GSK-3beta . A putative compensatory response to loss of GSK-3beta was indicated by differential expression of three motor proteins following toxin treatment and Kif1C, a motor protein involved in sensitivity to LeTx, increased expression in toxin-sensitive cells yet decreased in resistant cells following toxin treatment . Differential expression of microtubule-associating proteins and a decrease in the level of cellular tubulin were detected in LeTx-treated cells, both of which can result from loss of GSK-3beta activity . These data provide new information on LeTx's overall influence on macrophage physiology and suggest loss of GSK-3beta contributes to cytotoxicity. Proc Natl Acad Sci U S A, 2003 Jul 22, 100(15), 8945 - 50 Epub 2003 Jul 11. Poly(gamma-D-glutamic acid) protein conjugates induce IgG antibodies in mice to the capsule of Bacillus anthracis: a potential addition to the anthrax vaccine; Schneerson R et al.; Both the protective antigen (PA) and the poly(gamma-d-glutamic acid) capsule (gamma dPGA) are essential for the virulence of Bacillus anthracis . A critical level of vaccine-induced IgG anti-PA confers immunity to anthrax, but there is no information about the protective action of IgG anti-gamma dPGA . Because the number of spores presented by bioterrorists might be greater than encountered in nature, we sought to induce capsular antibodies to expand the immunity conferred by available anthrax vaccines . The nonimmunogenic gamma dPGA or corresponding synthetic peptides were bound to BSA, recombinant B . anthracis PA (rPA), or recombinant Pseudomonas aeruginosa exotoxin A (rEPA) . To identify the optimal construct, conjugates of B . anthracis gamma dPGA, Bacillus pumilus gamma dLPGA, and peptides of varying lengths (5-, 10-, or 20-mers), of the d or l configuration with active groups at the N or C termini, were bound at 5-32 mol per protein . The conjugates were characterized by physico-chemical and immunological assays, including GLC-MS and matrix-assisted laser desorption ionization time-of-flight spectrometry, and immunogenicity in 5- to 6-week-old mice . IgG anti-gamma dPGA and antiprotein were measured by ELISA . The highest levels of IgG anti-gamma dPGA were elicited by decamers of gamma dPGA at 10 -20 mol per protein bound to the N- or C-terminal end . High IgG anti-gamma dPGA levels were elicited by two injections of 2.5 microg of gamma dPGA per mouse, whereas three injections were needed to achieve high levels of protein antibodies . rPA was the most effective carrier . Anti-gamma dPGA induced opsonophagocytic killing of B . anthracis tox-, cap+ . gamma dPGA conjugates may enhance the protection conferred by PA alone . gamma dPGA-rPA conjugates induced both anti-PA and anti-gamma dPGA. J Clin Microbiol, 2003 Jul, 41(7), 2908 - 14 Identification of Bacillus anthracis by rpoB sequence analysis and multiplex PCR; Ko KS et al.; Comparative sequence analysis was performed upon Bacillus anthracis and its closest relatives, B . cereus and B . thuringiensis . Portions of rpoB DNA from 10 strains of B . anthracis, 16 of B . cereus, 10 of B . thuringiensis, 1 of B . mycoides, and 1 of B . megaterium were amplified and sequenced . The determined rpoB sequences (318 bp) of the 10 B . anthracis strains, including five Korean isolates, were identical to those of Ames, Florida, Kruger B, and Western NA strains . Strains of the "B . cereus group" were separated into two subgroups, in which the B . anthracis strains formed a separate clade in the phylogenetic tree . However, B . cereus and B . thuringiensis could not be differentiated . Sequence analysis confirmed the five Korean isolates as B . anthracis . Based on the rpoB sequences determined in the present study, multiplex PCR generating either B . anthracis-specific amplicons (359 and 208 bp) or cap DNA (291 bp) in a virulence plasmid could be used for the rapid differential detection and identification of virulent B . anthracis. Int J Antimicrob Agents, 2003 Jul, 22(1), 70 - 2 Antimicrobial susceptibilities of 40 isolates of Bacillus anthracis isolated in Turkey; Esel D et al.; Forty clinical isolates of Bacillus anthracis were studied . The MIC(90) values of penicillin G, doxycycline, ciprofloxacin, gatifloxacin, and levofloxacin were 0.016, 0.03, 0.06, 0.06 and 0.12 mg/l, respectively . Susceptibilities suggest that the quinolones may also be considered as an alternative therapy for anthrax. Appl Environ Microbiol, 2003 Jul, 69(7), 4329 - 31 Inactivation of vegetative cells, but not spores, of Bacillus anthracis, B . cereus, and B . subtilis on stainless steel surfaces coated with an antimicrobial silver- and zinc-containing zeolite formulation; Galeano B et al.; 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 degrees 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. MedGenMed . 2003 Mar 21;5(1):1. Anthrax-protective effects of yeast beta 1,3 glucans; Kournikakis B et al.; CONTEXT: The recent events increasing the threat of bioterrorism have prompted a widespread search for defenses against this peril . OBJECTIVE: To evaluate the anthrax-protective effect of beta1,3-glucan immune modulators (PGG-glucan and WGP beta glucan) in an experimental animal model . DESIGN: Beta1,3-glucan immune modulators were administered by subcutaneous injection to Balb/c mice 2 days prior to anthrax challenge . WGP beta glucan was administered by daily oral gavage for 7 days prior to challenge, or in drinking water for 10 days postchallenge with a lethal dose of Bacillus anthracis spores . Survival, survival time, and microbial bioburden relative to an infected, untreated control group were assessed . RESULTS: A single injected dose of PGG-glucan or WGP beta glucan immune modulators given 2 days before challenge significantly: (a) increased the survival rate of infected mice (2.5-fold), (b) diminished the bacterial load in the lungs of infected mice (4-8-fold), and (c) increased the proportion of bacteria-free animals 10 days after challenge (2-fold) . In mice prophylactically administered oral WGP beta glucan for 1 week prior to infection, survival increased from 50% to 100%; therapeutic administration of oral WGP beta glucan for 10 days postinfection increased survival from 30% up to 90% in treatment groups . CONCLUSIONS: These results demonstrate the potential for beta1,3-glucan immune modulators to provide a significant degree of protection against anthrax, a potential biological warfare (BW) agent in a mouse model of anthrax infection . Further studies are needed to optimize protection, evaluate activity in combination with other treatment options, demonstrate activity in a validated primate model of infection, and determine if protection is effective against other potential BW agents. Antimicrob Agents Chemother, 2003 Jul, 47(7), 2362 - 5 In vitro selection and characterization of Bacillus anthracis mutants with high-level resistance to ciprofloxacin; Price LB et al.; Mutants of attenuated Bacillus anthracis with high-level ciprofloxacin resistance were isolated using a three-step in vitro selection . Ciprofloxacin MICs were 0.5 micro g/ml for first-step mutants, which had one of two gyrA quinolone resistance-determining region (QRDR) mutations . Ciprofloxacin MICs were 8 and 16 microg/ml for second-step mutants, which had one of three parC QRDR mutations . Ciprofloxacin MICs for third-step mutants were 32 and 64 microg/ml . Mutants for which MICs were 64 microg/ml had one of two additional mutations within the gyrA QRDR or one of two mutations within the gyrB QRDR . Mutants for which MICs were 32 microg/ml had no additional target modifications but showed evidence of enhanced ciprofloxacin efflux. Infect Immun, 2003 Jul, 71(7), 3954 - 9 Macrophage-mediated germination of Bacillus anthracis endospores requires the gerH operon; Weiner MA et al.; The gerHABC operon of Bacillus anthracis, encoding a gerA-like family member of germinant sensors, was shown to be required for endospore germination in the presence of macrophages and in macrophage-conditioned media . The loss of the germination phenotype in macrophage cultures of B . anthracis gerH-null endospores was restored by complementation in trans with a wild-type copy of gerH expressed under the control of its own promoter . Using endospores from both the parental strain B . anthracis Sterne and an isogenic gerH-null strain, we partially characterized germinants secreted by macrophages into the extracellular medium. Infect Immun, 2003 Jul, 71(7), 3914 - 9 Detection of a luxS-signaling molecule in Bacillus anthracis; Jones MB et al.; Quorum-sensing regulation of density-dependent genes has been described for numerous bacterial species . The partially annotated genome sequence of Bacillus anthracis contains an open reading frame (BA5047) predicted to encode an ortholog of luxS, required for synthesis of the quorum-sensing signaling molecule autoinducer-2 (AI-2) . To determine whether B . anthracis produces AI-2, the Vibrio harveyi luminescence bioassay was used . Cell-free conditioned media from vaccine (Sterne) strain 34F(2) induced luminescence in V . harveyi reporter strain BB170, indicating its production of AI-2 . Cloned BA5047, expressed in Escherichia coli DH5 alpha cells, restored AI-2 activity to these cells . To evaluate whether BA5047 is essential for AI-2 synthesis, it was deleted through allelic exchange with marker rescue; the resulting mutant had no functional luxS activity and had reduced growth in vitro . In the wild-type strain, AI-2 activity was greatest during the exponential phase of growth . In total, these data indicate that BA5047 is a functional luxS ortholog in B . anthracis necessary for growth-phase-specific AI-2 expression . Thus, B . anthracis may utilize extracellular signaling molecules to regulate density-dependent gene expression. Infect Immun, 2003 Jul, 71(7), 3831 - 6 Salmonella enterica serovar typhimurium expressing a chromosomally integrated copy of the Bacillus anthracis protective antigen gene protects mice against an anthrax spore challenge; Garmory HS et al.; Protective immunity against infection with Bacillus anthracis is almost entirely based on a response to the protective antigen (PA), the binding moiety for the two other toxin components . We cloned the PA gene into an auxotrophic mutant of Salmonella enterica serovar Typhimurium as a fusion with the signal sequence of the hemolysin (Hly) A gene of Escherichia coli to allow the export of PA via the Hly export system . To stabilize the export cassette, it was also integrated into the chromosome of the live Salmonella carrier . When S . enterica serovar Typhimurium with the chromosomally integrated PA gene was given intravenously to A/J mice, they developed high levels of antibody to PA . These mice were protected against intraperitoneal challenge with 100 or 1,000 50% lethal doses of B . anthracis strain STI . This work contributes to the development of a Salmonella-based orally delivered anthrax vaccine. Int J Syst Evol Microbiol, 2003 May, 53(Pt 3), 695 - 704 Phylogenetic relationships between Bacillus species and related genera inferred from comparison of 3' end 16S rDNA and 5' end 16S-23S ITS nucleotide sequences; Xu D et al.; The nucleotide sequences of the 3' end of the 16S rDNA and the 16S-23S internal transcribed spacer (ITS) of 40 Bacillaceae species were determined . These included 21 Bacillus, 9 Paenibacillus, 6 Brevibacillus, 2 Geobacillus, 1 Marinibacillus and 1 Virgibacillus species . Comparative sequence analysis of a 220 bp region covering a highly conserved 150 bp sequence located at the 3' end of the 16S rRNA coding region and a conserved 70 bp sequence located at the 5' end of the 16S-23S ITS of the 40 species and six sequences available in GenBank were used to infer the phylogenetic relationships between all 46 taxa . When a maximal distance (D(max), where D refers to the number of nucleotide substitutions per site) of 0.31 was introduced as a threshold to determine groupings, 10 phylogenetically distinct clusters were revealed . Twenty-six Bacillus species were separated in seven groups (I, II, III, IV, V, VI and X), but Bacillus circulans remained ungrouped . All six Brevibacillus species under study were in Group VII . The nine Paenibacillus species fell into two distinct groups (VIII and IX) . Species with D(max) values within 0.05 were considered to be very closely related . These were Bacillus psychrophilus and Bacillus psychrosaccharolyticus in Group II; 'Bacillus maroccanus' and Bacillus simplex in Group II; Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus mojavensis and Bacillus subtilis in Group VI; Bacillus fusiformis and Bacillus sphaericus in Group VI; Brevibacillus brevis and Brevibacillus formosus in Group VII; Paenibacillus gordonae and Paenibacillus validus in Group VIII; and Bacillus anthracis, Bacillus cereus, Bacillus mycoides and Bacillus thuringiensis in Group X . The phylogenetic classification presented here is, in general, in agreement with current classifications based on phenotypic and molecular data . Our findings suggest, however, that in some cases, further divisions or, conversely, further groupings might be warranted . Should current classifications be re-examined in the light of our results, D(max) values of 0.31 and 0.05, as exemplified here, may prove useful threshold values for the grouping of Bacillaceae into taxa akin to genera and species, respectively . These D(max) thresholds may also reveal, in a different way, bacterial species for which further characterization might be warranted for proper classification and/or reassignment. Lett Appl Microbiol, 2003, 37(1), 17 - 20 Destruction of Bacillus anthracis strain Sterne 34F2 spores in postal envelopes by exposure to electron beam irradiation; Niebuhr SE et al.; AIMS: To determine the irradiation dose necessary to reduce the populations of Bacillus anthracis spores in a dry medium in postal envelopes . METHODS AND RESULTS: Bacillus anthracis Sterne 34F2 spores were dispersed in non-fat dry milk and then placed into standard business postal envelopes . The spores were treated with a sequence of irradiation doses to determine the decimal reduction value (D10) in kiloGrays (kGy) . The average D10 value was 3.35 +/- 0.02 kGy . CONCLUSIONS: An irradiation dose of 40.2 kGy would be required to result in a process equivalent to the thermal canning process (12 D10 reduction) to eliminate Clostridium botulinum spores . SIGNIFICANCE AND IMPACT OF THE STUDY: Irradiation is an effective means of reducing or eliminating B . anthracis spores in a dry medium in postal envelopes. FEMS Microbiol Lett, 2003 Jun 6, 223(1), 21 - 4 The Bacillus cereus bceT enterotoxin sequence reappraised; Hansen BM et al.; Bacillus cereus is a known opportunistic human pathogen belonging to the B . cereus group . Establishment of the pathogenesis most likely involves several gene products . One of these gene products, a single gene component named bceT, has been cloned and described from B . cereus B-4ac {Agata et al., Microbiology 141 (1995) 983-988} . However, our sequences of the bceT region from 16 B . cereus group strains showed inconsistency with the published bceT sequence . Only part of the bceT sequence had homology to our sequences . This initiated a more thorough investigation of the bceT sequence . Restriction site search and database searches intimated that the cloned bceT was created by an incidental joining of four DNA fragments during ligation . One of these fragments had 93% homology to an open reading frame (ORF 101) located within the pathogenic island of the Bacillus anthracis pXO1 virulence plasmid . We suggest that the reported enterotoxic activity of the original cloned bceT construct could be due to either the fusion gene or the fragment with homology to ORF 101 in pXO1. Vaccine, 2003 Jun 20, 21(21-22), 3011 - 8 Evaluation of the compatibility of a second generation recombinant anthrax vaccine with aluminum-containing adjuvants; Jendrek S et al.; Recombinant protective antigen (rPA) is the active pharmaceutical ingredient in a second generation anthrax vaccine undergoing pre-clinical evaluation . This rPA vaccine differs from the currently licensed vaccine, anthrax vaccine adsorbed (AVA), in that the sole component is a recombinant form of protective antigen (PA) . Unlike AVA the rPA vaccine contains no lethal factor (LF) or edema factor (EF), components of the two bipartite toxins, nor many other Bacillus anthracis-related contaminating proteins that are present in AVA . The proposed clinical protocol involves adsorption of the rPA to an aluminum-based adjuvant . The adsorptive characteristics of rPA and two aluminum-containing adjuvants were examined in a physiological buffer with and without EDTA . Based on the pI of rPA (pI=5.6) and the zero charge point of aluminum hydroxide adjuvant (11.5) and aluminum phosphate adjuvant (4.5), it was predicted and demonstrated that rPA bound in a more efficient manner to aluminum hydroxide adjuvant than to aluminum phosphate adjuvant in the physiological buffer . Binding of the rPA to the aluminum hydroxide adjuvant was decreased by increasing amounts of phosphate in the buffer . The adsorptive capacity for rPA onto aluminum hydroxide adjuvant in the physiological buffer and in water were calculated to be 0.46 mg rPA/mg aluminum in DPBS and 0.73 mg rPA/mg aluminum in water . This study also demonstrated that upon desorption from the aluminum hydroxide adjuvant the rPA was physically intact and free of detectable aggregates . Further, the eluted material was biologically active in an in vitro cytotoxicity assay . Desorption was only possible after an overnight incubation of 2-8 degrees C and not after a room temperature incubation reflecting increased contact with the aluminum hydroxide adjuvant over time . These data suggest that the interaction between rPA and aluminum hydroxide adjuvant is predominantly electrostatic in character. EMBO Rep, 2003 Jul, 4(7), 692 - 8 Horizontal transfer of drug-resistant aminoacyl-transfer-RNA synthetases of anthrax and Gram-positive pathogens; Brown JR et al.; The screening of new antibiotics against several bacterial strains often reveals unexpected occurrences of natural drug resistance . Two examples of this involve specific inhibitors of Staphylococcus aureus isoleucyl-transfer-RNA synthetase 1 (IleRS1) and, more recently, Streptococcus pneumoniae methionyl-tRNA synthetase 1 (MetRS1) . In both cases, resistance is due to the presence of a second gene that encodes another synthetase (IleRS2 or MetRS2) . Here, we show that both S . pneumoniae MetRS2 and S . aureus IleRS2 have closely related homologues in the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax . Furthermore, similar to drug-resistant pathogens, strains of B . anthracis and its closest relative, B . cereus, also have wild-type ileS1 and metS1 genes . Clostridium perfringens, the causative agent of gangrene, also has two metS genes, whereas Oceanobacillus iheyensis isolated from deep-sea sediments has a single ileS2-type gene . This study shows the importance of understanding complex evolutionary networks of ancient horizontal gene transfer for the development of novel antibiotics. Appl Environ Microbiol, 2003 Jun, 69(6), 3317 - 26 Fluorescent heteroduplex assay for monitoring Bacillus anthracis and close relatives in environmental samples; Merrill L et al.; A fluorescent heteroduplex method was developed to assess the presence of 16S rRNA gene (rDNA) sequences from Bacillus anthracis and close relatives in PCR-amplified 16S rDNA sequence mixtures from environmental samples . The method uses a single-stranded, fluorescent DNA probe, 464 nucleotides in length, derived from a B . anthracis 16S rRNA gene . The probe contains a unique, engineered deletion such that all probe-target duplexes are heteroduplexes with an unpaired G at position 343 (deltaG343) . Heteroduplex profiles of sequences >/=85% similar to the probe were produced using an ABI 377 sequencer in less than 3 h . The method divides strains of the Bacillus cereus-Bacillus thuringiensis-B . anthracis group into two subgroups . Each subgroup is defined by a specific 16S rRNA gene sequence type . Sequence type A, containing one mismatch with the probe, occurs in B . anthracis and a small number of closely related clonal lineages represented mostly by food-borne pathogenic isolates of B . cereus and B . thuringiensis . Sequence type B, containing two mismatches with the probe, is found in the majority of B . cereus and B . thuringiensis strains examined to date . Sequence types A and B, when hybridized to the probe, generate two easily differentiated heteroduplexes . Thus, from heteroduplex profiles, the presence of B . cereus-B . thuringiensis-B . anthracis subgroups in environmental samples can be inferred unambiguously . The results show that fluorescent heteroduplex analysis is an effective profiling technique for detection and differentiation of sequences representing small phylogenetic or functional groups in environmental samples. Biosens Bioelectron, 2003 Aug 15, 18(9), 1115 - 23 A handheld real time thermal cycler for bacterial pathogen detection; Higgins JA et al.; The handheld advanced nucleic acid analyzer (HANAA) is a portable real time thermal cycler unit that weighs under 1 kg and uses silicon and platinum-based thermalcycler units to conduct rapid heating and cooling of plastic reaction tubes . Two light emitting diodes (LED) provide greater than 1 mW of electrical power at wavelengths of 490 nm (blue) and 525 nm (green), allowing detection of the dyes FAM and JOE/TAMRA . Results are displayed in real time as bar graphs, and up to three, 4-sample assays can be run on the charge of the 12 V portable battery pack . The HANAA was evaluated for detection of defined Escherichia coli strains, and wild-type colonies isolated from stream water, using PCR for the lac Z and Tir genes . PCR reactions using SYBR Green dye allowed detection of E . coli ATCC 11775 and E . coli O157:H7 cells in under 30 min of assay time; however, background fluorescence associated with dye binding to nonspecific PCR products was present . DNA extracted from three isolates of Bacillus anthracis Ames, linked to a bioterrorism incident in Washington DC in October 2001, were also successfully tested on the HANAA using primers for the vrrA and capA genes . Positive results were observed at 32 and 22 min of assay time, respectively . A TaqMan probe specific to the aroQ gene of Erwinia herbicola was tested on the HANAA and when 500 cells were used as template, positive results were observed after only 7 min of assay time . Background fluorescence associated with the use of the probe was negligible . The HANAA is unique in offering real time PCR in a handheld format suitable for field use; a commercial version of the instrument, offering six reaction chambers, is available as of Fall 2002. Ned Tijdschr Tandheelkd, 2003 May, 110(5), 198 - 9 {Anthrax}; Bol P; Anthrax is a severe infectious disease by Bacillus anthracis . It can cause massacres among large herbivores, but means also a threat to humans . The latter develop mainly cutaneous anthrax, which they mostly survive . Inhalation can lead to more severe infections which, without medical intervention, are virtually always lethal . At the moment the disease draws much attention since it is thought to be a potential weapon in the hands of bioterrorists. Curr Microbiol, 2003 Jul, 47(1), 55 - 8 Three Bacillus anthracis bacteriophages from topsoil; Walter MH et al.; Three Bacillus anthracis bacteriophages from Iowa topsoil are characterized as to latent period, morphology, structural proteins, DNA size, and restriction endonuclease digestion . Electron micrographs indicate that the three isolates include two members of the Myoviridae and one smaller phage belonging to the Podoviridae . Phages Nk and DB resemble Myoviridae phage SP50 in morphology, but host range studies, protein, and DNA analysis indicate that both differ from SP50 . Phage MH is very similar to phage phi 29, but differs in terms of host range, structural protein, and DNA characteristics. J Microbiol Methods, 2003 Aug, 54(2), 143 - 52 Carbohydrates and glycoproteins of Bacillus anthracis and related bacilli: targets for biodetection; Fox A et al.; The spore is the form released in a bioterrorism attack . There is a real need for definition of new targets for Bacillus anthracis that might be incorporated into emerging biodetection technologies . Particularly of interest are macromolecules found in B . anthracis that are (1) spore-specific, (2) readily accessible on the spore surface and (3) distinct from those present in related organisms . One of the few biochemical methods to identify the spores of B . anthracis is based on the presence of rhamnose and 3-O-methyl rhamnose as determined by gas chromatography-mass spectrometry . Related organisms additionally contain 2-O-methyl rhamnose and fucose . Carbohydrates and glycoproteins of the B . cereus group of organisms and the related B . subilis group are reviewed here . It is hypothesized that the spore-specific carbohydrate is a component of the newly described glycoprotein of the exosporium of B . anthracis . Further work to define the protein and carbohydrate components of the glycoprotein of B . anthracis could be highly useful in developing new technologies for rapid biodetection. Emerg Infect Dis, 2003 Jun, 9(6), 689 - 96 Isolated case of bioterrorism-related inhalational anthrax, New York City, 2001; Holtz TH et al.; On October 31, 2001, in New York City, a 61-year-old female hospital employee who had acquired inhalational anthrax died after a 6-day illness . To determine sources of exposure and identify additional persons at risk, the New York City Department of Health, Centers for Disease Control and Prevention, and law enforcement authorities conducted an extensive investigation, which included interviewing contacts, examining personal effects, summarizing patient's use of mass transit, conducting active case finding and surveillance near her residence and at her workplace, and collecting samples from co-workers and the environment . We cultured all specimens for Bacillus anthracis . We found no additional cases of cutaneous or inhalational anthrax . The route of exposure remains unknown . All environmental samples were negative for B . anthracis . This first case of inhalational anthrax during the 2001 outbreak with no apparent direct link to contaminated mail emphasizes the need for close coordination between public health and law enforcement agencies during bioterrorism-related investigations. Emerg Infect Dis, 2003 Jun, 9(6), 681 - 8 Bioterrorism-related inhalational anthrax in an elderly woman, Connecticut, 2001; Griffith KS et al.; On November 20, 2001, inhalational anthrax was confirmed in an elderly woman from rural Connecticut . To determine her exposure source, we conducted an extensive epidemiologic, environmental, and laboratory investigation . Molecular subtyping showed that her isolate was indistinguishable from isolates associated with intentionally contaminated letters . No samples from her home or community yielded Bacillus anthracis, and she received no first-class letters from facilities known to have processed intentionally contaminated letters . Environmental sampling in the regional Connecticut postal facility yielded B . anthracis spores from 4 (31%) of 13 sorting machines . One extensively contaminated machine primarily processes bulk mail . A second machine that does final sorting of bulk mail for her zip code yielded B . anthracis on the column of bins for her carrier route . The evidence suggests she was exposed through a cross-contaminated bulk mail letter . Such cross-contamination of letters and postal facilities has implications for managing the response to future B . anthracis-contaminated mailings. Emerg Infect Dis, 2003 Jun, 9(6), 623 - 7 Inactivation of Bacillus anthracis spores; Spotts Whitney EA et al.; After the intentional release of Bacillus anthracis through the U.S . Postal Service in the fall of 2001, many environments were contaminated with B . anthracis spores, and frequent inquiries were made regarding the science of destroying these spores . We conducted a survey of the literature that had potential application to the inactivation of B . anthracis spores . This article provides a tabular summary of the results. Infect Immun, 2003 Jun, 71(6), 3183 - 9 Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin; Shannon JG et al.; We characterized the expression of a putative toxin of Bacillus anthracis, a member of the cholesterol-dependent cytolysin (CDC) family, which includes listeriolysin O, perfringolysin O, and streptolysin O . We named this cytotoxin anthrolysin O (ALO) . Although B . anthracis expresses minimal hemolytic activity in clinical settings, we show that Sterne strain 7702 expresses hemolytic activity when grown in brain heart infusion broth or in other rich bacteriologic media, but it secretes barely detectable amounts of hemolysin when grown in Luria-Bertani (LB) broth . Glucose supplementation of LB broth increases the amount of secreted hemolytic activity . Expression of hemolytic activity is maximal during mid- to late-log phase and decreases in the stationary phase . These observations are supported, in part, by semiquantitative reverse transcriptase PCR of alo mRNA . Hemolytic activity in growth supernatants was increased in the presence of reducing agent and almost totally inhibited in a dose-dependent manner by cholesterol; both of these activities are characteristic of a CDC toxin . A mutant of Sterne strain 7702, strain UT231, in which the alo gene was deleted and replaced by a kanamycin cassette, secreted barely detectable hemolytic activity into the growth medium . When strain UT231 was complemented in trans with native alo on a low-copy-number plasmid {strain UT231(pUTE554)}, it regained the ability to secrete hemolytic activity, indicating that ALO is the major hemolysin secreted by this strain of B . anthracis in rich media in vitro . To further support the alo gene product being a hemolysin, recombinant B . anthracis ALO (rALO) purified from Escherichia coli was extremely active against washed human erythrocytes, with complete hemolysis detected at approximately 30 molecules of rALO per erythrocyte . Considering the virulence roles of CDCs for other gram-positive bacteria, we speculate that ALO may have a role in anthrax virulence. Antimicrob Agents Chemother, 2003 Jun, 47(6), 2040 - 2 Biochemical characterization of beta-lactamases Bla1 and Bla2 from Bacillus anthracis; Materon IC et al.; The Sterne and Ames strains of Bacillus anthracis carry chromosomal genes bla1 and bla2, which confer beta-lactam resistance when expressed in Escherichia coli . MIC measurements and steady-state kinetic analyses indicate that Bla1 possesses penicillinase activity while Bla2 possesses penicillinase, cephalosporinase, and carbapenem-hydrolyzing activities. Antimicrob Agents Chemother, 2003 Jun, 47(6), 1784 - 9 Variable sensitivity to bacterial methionyl-tRNA synthetase inhibitors reveals subpopulations of Streptococcus pneumoniae with two distinct methionyl-tRNA synthetase genes; Gentry DR et al.; As reported previously (J . R . Jarvest et al., J . Med . Chem . 45:1952-1962, 2002), potent inhibitors (at nanomolar concentrations) of Staphylococcus aureus methionyl-tRNA synthetase (MetS; encoded by metS1) have been derived from a high-throughput screening assay hit . Optimized compounds showed excellent activities against staphylococcal and enterococcal pathogens . We report on the bimodal susceptibilities of S . pneumoniae strains, a significant fraction of which was found to be resistant (MIC, > or =8 mg/liter) to these inhibitors . Using molecular genetic techniques, we have found that the mechanism of resistance is the presence of a second, distantly related MetS enzyme, MetS2, encoded by metS2 . We present evidence that the metS2 gene is necessary and sufficient for resistance to MetS inhibitors . PCR analysis for the presence of metS2 among a large sample (n = 315) of S . pneumoniae isolates revealed that it is widespread geographically and chronologically, occurring at a frequency of about 46% . All isolates tested also contained the metS1 gene . Searches of public sequence databases revealed that S . pneumoniae MetS2 was most similar to MetS in Bacillus anthracis, followed by MetS in various non-gram-positive bacterial, archaeal, and eukaryotic species, with streptococcal MetS being considerably less similar . We propose that the presence of metS2 in specific strains of S . pneumoniae is the result of horizontal gene transfer which has been driven by selection for resistance to some unknown class of naturally occurring antibiotics with similarities to recently reported synthetic MetS inhibitors. Biochemistry, 2003 May 27, 42(20), 6078 - 89 Structural studies on the hairpins at the 3' untranslated region of an anthrax toxin gene; Shiflett PR et al.; Three proteins, namely, protective antigen (PA), edema factor (EF), and lethal factor (LF), encoded by the pX01 plasmid of Bacillus anthracis play a major role in the pathogenesis of target host cells . PA combines with EF and LF to form bipartite PA-EF and PA-LF toxins and facilitates intracellular delivery of EF and LF both of which cause cytotoxicity to the host . Since the level of PA is crucial to pathogenesis by anthrax toxins, it is important to understand how the host environment regulates the expression of the PA (or pagA) gene by utilizing the 5' and 3' untranslated regions (UTR) . The 5' UTR sequence determines the initiation of transcription, whereas the 3' UTR sequence determines the efficient termination and stability of the transcript . Although, the role of the 5'UTR sequence of pagA has been investigated, little is known about the role of the 3' UTR . Since hairpin formation at the 3'UTR of a gene is an established mechanism for efficient termination and stability of the transcript, we carried out structural studies, including gel electrophoresis, circular dichroism, and two-dimensional nuclear magnetic resonance spectroscopy, to determine whether the 3' UTR sequences of pagA also form hairpin structures . Our results unequivocally demonstrate that both the coding and the noncoding 3' UTR sequences form stable hairpin structures . It is quite likely that the hairpins at the 3'UTR may contribute to efficient termination and stability of the pagA transcript. J Bacteriol, 2003 Jun, 185(11), 3373 - 8 Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium; Todd SJ et al.; The exosporium is the outermost layer of spores of Bacillus cereus and its close relatives Bacillus anthracis and Bacillus thuringiensis . For these pathogens, it represents the surface layer that makes initial contact with the host . To date, only the BclA glycoprotein has been described as a component of the exosporium; this paper defines 10 more tightly associated proteins from the exosporium of B . cereus ATCC 10876, identified by N-terminal sequencing of proteins from purified, washed exosporium . Likely coding sequences were identified from the incomplete genome sequence of B . anthracis or B . cereus ATCC 14579, and the precise corresponding sequence from B . cereus ATCC 10876 was defined by PCR and sequencing . Eight genes encode likely structural components (exsB, exsC, exsD, exsE, exsF, exsG, exsJ, and cotE) . Several proteins of the exosporium are related to morphogenetic and outer spore coat proteins of B . subtilis, but most do not have homologues in B . subtilis . ExsE is processed from a larger precursor, and the CotE homologue appears to have been C-terminally truncated . ExsJ contains a domain of GXX collagen-like repeats, like the BclA exosporium protein of B . anthracis . Although most of the exosporium genes are scattered on the genome, bclA and exsF are clustered in a region flanking the rhamnose biosynthesis operon; rhamnose is part of the sugar moiety of spore glycoproteins . Two enzymes, alanine racemase and nucleoside hydrolase, are tightly adsorbed to the exosporium layer; they could metabolize small molecule germinants and may reduce the sensitivity of spores to these, limiting premature germination. Lett Appl Microbiol, 2003, 36(6), 418 - 22 Molecular detection of anthrax spores on animal fibres; Levi K et al.; AIMS: To develop a rapid, specific and sensitive diagnostic test for the detection of the spores of Bacillus anthracis on commercial samples of animal fibres (e.g . wool and cashmere) . METHODS AND RESULTS: Extraction of DNA from spores using a mechanical disruption method based on bead beating was evaluated but subsequently abandoned as it compromised the sensitivity of the overall protocol . A multiplex PCR and two nested amplification reactions designed for B . anthracis were developed during this study . CONCLUSIONS: A simple selective incubation step in combination with multiplex PCR was found to be more effective than generic DNA extraction coupled to a sensitive nested amplification reaction . SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid diagnostic test could be applied to the analysis of commercial fibre samples for the detection of anthrax as required by health and safety legislation resulting in considerable savings in time and expense. J Appl Microbiol, 2003, 94(6), 1108 - 19 Genetic relationship in the 'Bacillus cereus group' by rep-PCR fingerprinting and sequencing of a Bacillus anthracis-specific rep-PCR fragment; Cherif A et al.; AIMS: To evaluate the genetic relationship in the Bacillus cereus group by rep-PCR fingerprinting . METHODS AND RESULTS: A collection of 112 strains of the six species of the B . cereus group was analysed by rep-PCR fingerprinting using the BOX-A1R primer . A relative genetic distinctness was found among the species . Cluster analysis of the rep-PCR patterns showed clusters of B . thuringiensis strains quite separate from those of B . cereus strains . The B . anthracis strains represented an independent lineage in a B . cereus cluster . The B . mycoides, B . pseudomycoides and B . weihenstephanensis strains were clustered into three groups at some distance from the other species . Comparison of sequences of AC-390, a typical B . anthracis rep-PCR fragment, from 27 strains of B . anthracis, B . cereus, B . thuringiensis and B . weihenstephanensis, representative of different clusters identified by rep-PCR fingerprinting, confirmed that B . anthracis diverges from its related species . CONCLUSIONS: The genetic relationship deduced from the rep-PCR patterns indicates a relatively clear separation of the six species, suggesting that they can indeed be considered as separate units . SIGNIFICANCE AND IMPACT OF THE STUDY: rep-PCR fingerprinting can make a contribution in the clarification of the genetic relationships between the species of the B . cereus group. Natl Toxicol Program Tech Rep Ser, 1987 Apr, 318, 1 - 190 NTP Toxicology and Carcinogenesis Studies of Ampicillin Trihydrate (CAS No . 7177-48-2) in F344/N Rats and B6C3F1 Mice (Gavage Studies); National Toxicology Program ; Ampicillin trihydrate is a broad-spectrum semi-synthetic penicillin that is effective in the treatment of gram-positive and gram-negative bacterial infections produced by Streptococcus, Bacillus anthracis, Haemophilus influenzae, Neisseria gonorrhoeae, and Escherichia coli . This antibiotic is used in the treatment of upper respiratory tract infections, genital and urinary tract infections, and otitis media in children . Toxicology and carcinogenesis studies of ampicillin trihydrate (97%-99% pure) were conducted by administering the chemical in corn oil by gavage to groups of 50 F344/N rats and 50 B6C3F1 mice of each sex, 5 days per week for 103 weeks . Male and female rats received doses of 0, 750, or 1,500 mg/kg, and male and female mice received doses of 0, 1,500, or 3,000 mg/kg . Doses selected for the 2-year studies were based on the lack of body weight effects and histopathologic effects at 2,400 mg/kg in the 14-day studies and 3,000 mg/kg in the 13-week studies . Clinical signs in the 13-week studies included diarrhea at 3,000 mg/kg in male and female rats and male mice . Corn oil suspensions containing more than 300 mg ampicillin trihydrate/ml were too viscous to be administered by gavage; therefore, a high dose of 1,500 mg/kg was selected for rats and a high dose of 3,000 mg/kg was selected for mice . During the 2-year studies, mean body weights of male and female rats were similar to or slightly increased over those of the corresponding vehicle control groups . Mean body weights of low dose and high dose male mice were similar to those of the corresponding vehicle group during year 1 of the study but were slightly below those of the vehicle control group during the last half of the study . Mean body weights of low dose and high dose female mice were greater than those of the vehicle controls throughout most of the study . No significant differences in survival were observed in groups of rats or mice of either sex . Clinical signs observed in dosed rats included diarrhea, excessive urination, and chromodacryorrhea and in dosed mice included increased salivation and decreased activity . In male rats, administration of ampicillin trihydrate was associated with an increased incidence of mononuclear cell leukemia (vehicle control, 5/50; low dose, 14/50; high dose, 13/50) . Malignant lymphomas were observed in one additional vehicle control male rat and two low dose male rats . Lymphocytic leukemia was seen in one high dose rat . High dose male rats showed increased incidences of pheochromocytomas of the adrenal gland medulla (13/50; 12/50; 23/49) . Malignant pheochromocytomas were observed in 1/50 vehicle control, 5/50 low dose, and 1/49 high dose male rats . The incidence of adrenal gland medullary hyperplasia was not increased in male rats (14/50; 10/50; 8/49) . There were increased incidences of C-cellhyperplasia of the thyroid gland in low dose male and high dose female rats . High dose male rats showed increased incidences of hyperkeratosis and acanthosis of the forestomach . In male and female mice, ampicillin trihydrate administration was associated with increased incidences of forestomach lesions, including ulcers, inflammation, hyperkeratosis, acanthosis, and evidence of fungal infection . Ampicillin trihydrate was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 in the presence or absence of Aroclor 1254-induced male Syrian hamster or male Sprague-Dawley rat liver S9 when tested according to preincubation protocol . Ampicillin trihydrate was not mutagenic in L5178Y mouse lymphoma cells with or without metabolic activation . Ampicillin trihydrate did not cause chromosomal aberrations or sister-chromatid exchanges in Chinese hamster ovary cells with or without metabolic activation . An audit was conducted for these 2-year studies . Animal/carcass identification discrepancies were observed in rats and mice . The most common findings were the failure to clip some toes in rats and opened ear holes in mice . A review of the inlife data (including body weights, clinical observations, and dosing records) indicrecords) indicated that animals had not been interchanged among groups . The data are considered adequate to support the conclusions . Under the conditions of these 2-year gavage studies, there was equivocal evidence of carcinogenicity of ampicillin trihydrate for male F344/N rats as shown by increased incidences of pheochromocytomas of the adrenal medulla and by marginally increased incidences of mononuclear cell leukemia . There was no evidence of carcinogenicity for female F344/N rats receiving 750 or 1,500 mg/kg or for male and female B6C3F1 mice receiving 1,500 or 3,000 mg/kg per day . Nonneoplastic lesions of the forestomach were seen in male rats and male and female mice . Synonyms and trade names: Acillin; Amcap; Amcill; Aminobenzylpencillin trihydrate; a-Aminobenzylpencillin trihydrate; Amperil; Ampichel; Ampikel; Ampinova; Amplin; Cymbi; Divercillin; Liffampil; Morepen; Pen A; Pensyn; Polycillin; Princillin; Principen; Ro-ampen; Trafarbiot J Dermatolog Treat, 2003 Jan, 14(1), 51 - 3 Cutaneous anthrax associated with facial palsy: case report and literature review; Faghihi G et al.; BACKGROUND: Anthrax is primarily an animal disease . Bacillus anthracis, the causal agent in anthrax, is a Gram-positive rod . Humans can acquire anthrax by industrial exposure to infected animals or animal products . METHODS: Reported here is the case of a 48-year-old male farm worker from Iran with a history of direct contact with herds . He presented after 6 days of fever with toxicity and a crusted ulcer on the face that was later confirmed bacteriologically to be cutaneous anthrax . He was treated with large doses of intravenous penicillin and corticosteroids along with multiple subcutaneous epinephrine injections that were used to control the infection and massive facial edema . RESULTS: After 14 days, he partially recovered; however, ipsilateral facial nerve palsy developed and persisted despite therapeutic efforts . CONCLUSION: It is not possible to conclude whether early diagnosis and treatment of anthrax results in a lower risk of complications . Facial palsy can be added to the list of variable complications of the cutaneous effects of anthrax. Emerg Infect Dis, 2003 May, 9(5), 520 - 5 Endemic gastrointestinal anthrax in 1960s Lebanon: clinical manifestations and surgical findings; Kanafani ZA et al.; Anthrax is an ancient disease caused by the gram-positive Bacillus anthracis; recently, it has gained much attention because of its potential use in biologic warfare . Anthrax infection occurs in three forms: cutaneous, inhalational, and gastrointestinal . The last type results from ingestion of poorly cooked contaminated meat . Intestinal anthrax was widely known in Lebanon in the 1960s, when a series of >100 cases were observed in the Bekaa Valley . We describe some of these cases, introduce the concept of the surgical management of advanced intestinal anthrax, and describe some of the approaches for treatment. Pharmacoepidemiol Drug Saf, 2003 Apr-May, 12(3), 177 - 82 Increased US prescription trends associated with the CDC Bacillus anthracis antimicrobial postexposure prophylaxis campaign; Shaffer D et al.; PURPOSE: We evaluated national outpatient antimicrobial prescription trends in relation to the first United States case of inhalational anthrax due to the intentional delivery of Bacillus anthracis (B . anthracis) spores . METHODS: We queried IMS HEALTH's National Prescription Audit Plus7 database for two 6-month periods (July-December) in 2001 and 2000 to describe outpatient prescription trends of antimicrobials recommended during the Centers for Disease Control and Prevention's (CDC) postexposure prophylaxis campaign . RESULTS: Overall, antimicrobial utilization for the referent 6-month time frame was greater in 2000 compared to 2001 . In contrast, ciprofloxacin utilization was greater in 2001 during October, the month following the index case, increasing by more than 40% over utilization in October 2000 . Similarly, doxycycline utilization increased by 30% during October/November . This corresponded to relative increases in US utilization for ciprofloxacin of approximately 160,000 prescriptions for the month of October and for doxycycline of approximately 96,000 prescriptions during October and 120,000 prescriptions for November . CONCLUSIONS: We conclude more widespread prescribing of ciprofloxacin and doxycycline occurred in response to the first US bioterrorist-associated anthrax attacks than was warranted based upon confirmed or suspected B . anthracis exposure alone. Appl Environ Microbiol, 2003 May, 69(5), 2755 - 64 Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis; Radnedge L et al.; The three species of the group 1 bacilli, Bacillus anthracis, B . cereus, and B . thuringiensis, are genetically very closely related . All inhabit soil habitats but exhibit different phenotypes . B . anthracis is the causative agent of anthrax and is phylogenetically monomorphic, while B . cereus and B . thuringiensis are genetically more diverse . An amplified fragment length polymorphism analysis described here demonstrates genetic diversity among a collection of non-anthrax-causing Bacillus species, some of which show significant similarity to B . anthracis . Suppression subtractive hybridization was then used to characterize the genomic differences that distinguish three of the non-anthrax-causing bacilli from B . anthracis Ames . Ninety-three DNA sequences that were present in B . anthracis but absent from the non-anthrax-causing Bacillus genomes were isolated . Furthermore, 28 of these sequences were not found in a collection of 10 non-anthrax-causing Bacillus species but were present in all members of a representative collection of B . anthracis strains . These sequences map to distinct loci on the B . anthracis genome and can be assayed simultaneously in multiplex PCR assays for rapid and highly specific DNA-based detection of B . anthracis. Pol Merkuriusz Lek, 2003 Feb, 14(80), 156 - 8 {Anthrax as a pathogen for a biological weapon}; Dybowska D et al.; The authors presented an outline of modern knowledge about anthrax . Some aspects of the use of Bacillus anthracis as a biological terrorist agent are also described. FEMS Immunol Med Microbiol, 2003 May 15, 36(1-2), 83 - 6 Characterisation of the immune response to the UK human anthrax vaccine; Baillie L et al.; The UK human anthrax vaccine consists of the alum-precipitated culture supernatant of Bacillus anthracis Sterne . In addition to protective antigen (PA), the key immunogen, the vaccine also contains a number of other bacteria- and media-derived proteins . These proteins may contribute to the transient side effects experienced by some individuals and could influence the development of the PA-specific immune response . Bacterial cell-wall components have been shown to be potent immunomodulators . B . anthracis expresses two S-layer proteins, EA1 and Sap, which have been demonstrated to be immunogenic in animal studies . These are also immunogenic in man so that convalescent and post-immunisation sera contain specific antibodies to Ea1, and to a lesser extent, to Sap . To determine if these proteins are capable of modifying the protective immune response to PA, A/J mice were immunised with equivalent amounts of recombinant PA and S-layer proteins in the presence of alhydrogel . IgG isotype profiles were determined and the animals were subsequently challenged with spores of B . anthracis STI . The results suggest that there was no significant shift in IgG isotype profile and that the presence of the S-layer proteins did not adversely affect the protective immune response induced by PA. J Biol Chem, 2003 Aug 1, 278(31), 29261 - 6 Epub 2003 Apr 29. Calcium dependence of the interaction between calmodulin and anthrax edema factor; Ulmer TS et al.; Edema factor (EF), a toxin from Bacillus anthracis (anthrax), possesses adenylyl cyclase activity and requires the ubiquitous Ca2+-sensor calmodulin (CaM) for activity . CaM can exist in three major structural states: an apo state with no Ca2+ bound, a two Ca2+ state with its C-terminal domain Ca2+-loaded, and a four Ca2+ state in which the lower Ca2+ affinity N-terminal domain is also ligated . Here, the interaction of EF with the three Ca2+ states of CaM has been examined by NMR spectroscopy and changes in the Ca2+ affinity of CaM in the presence of EF have been determined by flow dialysis . Backbone chemical shift perturbations of CaM show that EF interacts weakly with the N-terminal domain of apoCaM . The C-terminal CaM domain only engages in the interaction upon Ca2+ ligation, rendering the overall interaction much tighter . In the presence of EF, the C-terminal domain binds Ca2+ with higher affinity, but loses binding cooperativity, whereas the N-terminal domain exhibits strongly reduced Ca2+ affinity . As judged by chemical shift differences, the N-terminal CaM domain remains bound to EF upon subsequent Ca2+ ligation . This Ca2+ dependence of the EF-CaM interaction differs from that observed for most other CaM targets, which normally interact only with the Ca2+-bound CaM domains and become active following the transition to the four Ca2+ state. Nature, 2003 May 1, 423(6935), 87 - 91 Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis; Ivanova N et al.; Bacillus cereus is an opportunistic pathogen causing food poisoning manifested by diarrhoeal or emetic syndromes . It is closely related to the animal and human pathogen Bacillus anthracis and the insect pathogen Bacillus thuringiensis, the former being used as a biological weapon and the latter as a pesticide . B . anthracis and B . thuringiensis are readily distinguished from B . cereus by the presence of plasmid-borne specific toxins (B . anthracis and B . thuringiensis) and capsule (B . anthracis) . But phylogenetic studies based on the analysis of chromosomal genes bring controversial results, and it is unclear whether B . cereus, B . anthracis and B . thuringiensis are varieties of the same species or different species . Here we report the sequencing and analysis of the type strain B . cereus ATCC 14579 . The complete genome sequence of B . cereus ATCC 14579 together with the gapped genome of B . anthracis A2012 enables us to perform comparative analysis, and hence to identify the genes that are conserved between B . cereus and B . anthracis, and the genes that are unique for each species . We use the former to clarify the phylogeny of the cereus group, and the latter to determine plasmid-independent species-specific markers. Nature, 2003 May 1, 423(6935), 81 - 6 The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria; Read TD et al.; Bacillus anthracis is an endospore-forming bacterium that causes inhalational anthrax . Key virulence genes are found on plasmids (extra-chromosomal, circular, double-stranded DNA molecules) pXO1 (ref . 2) and pXO2 (ref . 3) . To identify additional genes that might contribute to virulence, we analysed the complete sequence of the chromosome of B . anthracis Ames (about 5.23 megabases) . We found several chromosomally encoded proteins that may contribute to pathogenicity--including haemolysins, phospholipases and iron acquisition functions--and identified numerous surface proteins that might be important targets for vaccines and drugs . Almost all these putative chromosomal virulence and surface proteins have homologues in Bacillus cereus, highlighting the similarity of B . anthracis to near-neighbours that are not associated with anthrax . By performing a comparative genome hybridization of 19 B . cereus and Bacillus thuringiensis strains against a B . anthracis DNA microarray, we confirmed the general similarity of chromosomal genes among this group of close relatives . However, we found that the gene sequences of pXO1 and pXO2 were more variable between strains, suggesting plasmid mobility in the group . The complete sequence of B . anthracis is a step towards a better understanding of anthrax pathogenesis. Anal Chem, 2003 Apr 15, 75(8), 1880 - 6 Microchip-based purification of DNA from biological samples; Breadmore MC et al.; A microchip solid-phase extraction method for purification of DNA from biological samples, such as blood, is demonstrated . Silica beads were packed into glass microchips and the beads immobilized with sol-gel to provide a stable and reproducible solid phase onto which DNA could be adsorbed . Optimization of the DNA loading conditions established a higher DNA recovery at pH 6.1 than 7.6 . This lower pH also allowed for the flow rate to be increased, resulting in a decrease in extraction time from 25 min to less than 15 min . Using this procedure, template genomic DNA from human whole blood was purified on the microchip platform with the only sample preparation being mixing of the blood with load buffer prior to loading on the microchip device . Comparison between the microchip SPE (microchipSPE) procedure and a commercial microcentrifuge method showed comparable amounts of PCR-amplifiable DNA could be isolated from cultures of Salmonella typhimurium . The greatest potential of the microchipSPE device was illustrated by purifying DNA from spores from the vaccine strain of Bacillus anthracis, where eventual integration of SPE, PCR, and separation on a single microdevice could potentially enable complete detection of the infectious agent in less than 30 min. Arch Neurol, 2003 Apr, 60(4), 483 - 8 Neurologic complications of anthrax: a review of the literature; Meyer MA; A review of the literature suggests that the major neurologic symptom complex of infection by Bacillus anthracis is a fulminant and rapidly fatal hemorrhagic meningoencephalitis and that the reported initial mode of entry can be via the cutaneous or inhalation route . For febrile patients with acute neurologic deterioration with associated findings of dark necrotic pustules on the extremities, gram-positive rods in the cerebrospinal fluid, and multifocal areas of unexplained intracerebral hemorrhage on computed tomographic scans, anthrax should be considered within the differential diagnosis . A low cerebrospinal fluid glucose level has been reported, with gram-positive rods often noted on the gram stain of the cerebrospinal fluid in severely affected patients . Reports indicate that death usually occurs within a week. Infect Immun, 2003 May, 71(5), 2736 - 43 Global effects of virulence gene regulators in a Bacillus anthracis strain with both virulence plasmids; Bourgogne A et al.; Control of anthrax toxin and capsule synthesis, the two major virulence factors of Bacillus anthracis, has been associated with two regulatory genes, atxA and acpA, located on virulence plasmids pXO1 and pXO2, respectively . We used transcriptional profiling to determine whether atxA and/or acpA control genes other than those already described and to investigate functional similarities of the regulators . Transcription was assessed in a pXO1(+) pXO2(+) parent strain and in isogenic mutants in which one or both regulatory genes were deleted . We determined that in addition to the toxin and capsule genes, atxA controls expression of numerous other genes on both plasmids and the chromosome . Generally, plasmid-encoded genes were more highly regulated than chromosomal genes, and both positive and negative effects were observed . Certain atxA-regulated genes were affected synergistically in an atxA acpA mutant . Yet overall, acpA appears to be a minor regulator with fewer targets than atxA . In contrast to previous reports of acpA function in attenuated strains, acpA had a minimal influence on capsule gene transcription and capsule synthesis in a genetically complete strain . Surprisingly, acpA expression was positively affected by atxA, although atxA-activated capsule gene transcription is not acpA dependent . The newly discovered atxA-regulated targets include genes predicted to encode secreted proteins and proteins with roles in transcriptional regulation and signaling . Regulation of chromosomal genes by atxA is particularly intriguing, given that many of the target genes have homologues in other Bacillus species that lack atxA homologues . Given the global effect of atxA on gene expression in B . anthracis, previous assumptions regarding reduced virulence of strains harboring single plasmids must be reassessed and the potential roles of newly identified atxA-regulated genes should be investigated. Emerg Infect Dis, 2003 Apr, 9(4), 503 - 5 Fear of bioterrorism and implications for public health preparedness; Dworkin MS et al.; After the human anthrax cases and exposures in 2001, the Illinois Department of Public Health received an increasing number of environmental and human samples (1,496 environmental submissions, all negative for Bacillus anthracis) . These data demonstrate increased volume of submissions to a public health laboratory resulting from fear of bioterrorism. Proc Natl Acad Sci U S A, 2003 Apr 29, 100(9), 5170 - 4 Epub 2003 Apr 16. Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor; Scobie HM et al.; Bacillus anthracis secretes two bipartite toxins thought to be involved in anthrax pathogenesis and resulting death of the host . The current model for intoxication is that protective antigen (PA) toxin subunits bind a single group of cell-surface anthrax toxin receptors (ATRs), encoded by the tumor endothelial marker 8 (TEM8) gene . The ATR/TEM8-PA interaction is mediated by the receptor's extracellular domain related to von Willebrand factor type A or integrin inserted domains (VWA/I domains) . A metal ion-dependent adhesion site (MIDAS) located within this domain of the ATR/TEM8 protein chelates a divalent cation critical for PA binding . In this report, we identify a second PA receptor encoded by capillary morphogenesis gene 2 (CMG2), which has 60% amino acid identity to ATR/TEM8 within the VWA/I domain, as well as a conserved MIDAS motif . A recombinant CMG2 protein bound PA and mediated toxin internalization when expressed on receptor-deficient cells . Binding between the CMG2 VWA/I domain and PA was shown to be direct and metal-dependent, although the cation specificity of this interaction is different than that observed with ATR/TEM8 . Northern blot analysis revealed that CMG2 is widely expressed in human tissues, indicating that this receptor is likely to be relevant for disease pathogenesis . Finally, a soluble version of the CMG2 VWA/I domain inhibited intoxication of cells expressing endogenous toxin receptors when it was added to PA at a 3:1 ratio . These studies distinguish CMG2 as a second anthrax toxin receptor and identify a potent antitoxin that may prove useful for the treatment of anthrax. J Bacteriol, 2003 May, 185(9), 2910 - 9 The YSIRK-G/S motif of staphylococcal protein A and its role in efficiency of signal peptide processing; Bae T et al.; Many surface proteins of pathogenic gram-positive bacteria are linked to the cell wall envelope by a mechanism requiring a C-terminal sorting signal with an LPXTG motif . Surface proteins of Streptococcus pneumoniae harbor another motif, YSIRK-G/S, which is positioned within signal peptides . The signal peptides of some, but not all, of the 20 surface proteins of Staphylococcus aureus carry a YSIRK-G/S motif, whereas those of surface proteins of Listeria monocytogenes and Bacillus anthracis do not . To determine whether the YSIRK-G/S motif is required for the secretion or cell wall anchoring of surface proteins, we analyzed variants of staphylococcal protein A, an immunoglobulin binding protein with an LPXTG sorting signal . Deletion of the YSIR sequence or replacement of G or S significantly reduced the rate of signal peptide processing of protein A precursors . In contrast, cell wall anchoring or the functional display of protein A was not affected . The fusion of cell wall sorting signals to reporter proteins bearing N-terminal signal peptides with or without the YSIRK-G/S motif resulted in hybrid proteins that were anchored in a manner similar to that of wild-type protein A . The requirement of the YSIRK-G/S motif for efficient secretion implies the existence of a specialized mode of substrate recognition by the secretion pathway of gram-positive cocci . It seems, however, that this mechanism is not essential for surface protein anchoring to the cell wall envelope. J Food Prot, 2003 Apr, 66(4), 691 - 9 Bacillus anthracis: current knowledge in relation to contamination of food; Erickson MC et al.; In this article, information related to anthrax and its etiologic agent, Bacillus anthracis, in food is reviewed . The major topics discussed include the taxonomic relationship of B . anthracis to other Bacillus species, methods used for the recovery of the organism from surfaces and foods, routes of infection, the pathogenesis of the organism, the microbial ecology of the vegetative cell and spore in foods and the environment, chemical and physical treatments for spore inactivation, and the control of the disease in animals. AIHA J (Fairfax, Va), 2003 Mar-Apr, 64(2), 189 - 95 Volatile organic compounds produced during irradiation of mail; Smith PA et al.; In 2001, Bacillus anthracis spores were delivered through the United States postal system in a series of bioterrorist acts . Controls proposed for this threat included sanitization with high-energy electrons . Solid phase microextraction was used with gas chromatography/mass spectrometry for field sampling and analysis of volatile compounds apparently produced from polymeric materials such as cellulose and plastics, immediately following processing of mail at a commercial irradiation facility . Solid phase microextraction and direct sampling of air into a cryogenically cooled temperature programmable inlet were used in the laboratory for gas chromatography/mass spectrometry analysis of air in contact with irradiated mail, envelopes only (packaged identically to mail), and air inside irradiated plastic mail packaging bags (with neither mail nor envelopes) . Irradiated mail or envelope systems produced hydrocarbons such as propane, butane, pentane, hexane, heptane, methylpentanes, and benzene; and oxygen-containing compounds such as acetaldehyde, acrolein, propionaldehyde, furan, 2-methylfuran, methanol, acetone, 2-butanone, and ethanol . In addition to hydrocarbons, methyl and ethyl nitrate were detected in irradiated bags that contained only air, suggesting reactive nitrogen species formed from air irradiation reacted with hydroxy-containing compounds to give nitro esters . The similarities of volatile compounds in irradiated systems containing paper to those observed by researchers studying cellulose pyrolysis suggests common depolymerization and degradation mechanisms in each case . These similarities should guide additional work to examine irradiated mail for chemical compounds not detectable by methods used here. Curr Opin Pulm Med, 2003 May, 9(3), 221 - 6 Inhalational anthrax and bioterrorism; Quintiliani R Jr et al.; Until recently, inhalational anthrax was considered an infectious disease curiosity for medical specialists and veterinarians . This attitude abruptly changed following the intentional release of Bacillus anthracis spores via the US Postal Service in October 2001 . Because of its rarity, few physicians were familiar with its clinical manifestations, treatment and prophylaxis . In this report, we try to fill this informational gap by reviewing these issues based on additional data culled from this recent bioterrorism-related epidemic . Moreover, we have purposely emphasized its clinical manifestations, searching for common findings that may alert the physician to suspect and rapidly diagnose this infection . To improve survival rates, prompt diagnosis of inhalational anthrax is crucial, since even a brief delay in therapy of this fulminating infection almost uniformly results in death. Appl Opt, 2003 Apr 1, 42(10), 1757 - 62 Detection of anthrax simulants with microcalorimetric spectroscopy: Bacillus subtilis and Bacillus cereus spores; Arakawa ET et al.; Recent advances in the development of ultrasensitive micromechnical thermal detectors have led to the advent of novel subfemtojoule microcalorimetric spectoscopy (CalSpec) . On the basis of principles of photothermal IR spectroscopy combined with efficient thermomechanical transduction, CalSpec provides acquisition of vibrational spectra of microscopic samples and absorbates . We use CalSpec as a method of identifying nanogram quantities of biological micro-organisms . Our studies focus on Bacillus subtilis and Bacillus cereus spores as simulants for Bacillus anthracis spores . Using CalSpec, we measured IR spectra of B . subtilis and B . cereus spores present on surfaces in nanogram quantities (approximately 100-1,000 spores) . The spectra acquired in the wavelength range of 690-4000 cm(-1) (2.5-14.5 microm) contain information-rich vibrational signatures that reflect the different ratios of biochemical makeup of the micro-organisms . The distinctive features in the spectra obtained for the two types of microorganism can be used to distinguish between the spores of the Bacillus family . As compared with conventional IR and Fourier-transform IR microscopic spectroscopy techniques, the advantages of the present technique include significantly improved sensitivity (at least a full order of magnitude), absence of expensive IR detectors, and excellent potential for miniaturization. Appl Environ Microbiol, 2003 Apr, 69(4), 2372 - 6 Production of diarrheal enterotoxins and other potential virulence factors by veterinary isolates of bacillus species associated with nongastrointestinal infections; Rowan NJ et al.; With the exceptions of Bacillus cereus and Bacillus anthracis, Bacillus species are generally perceived to be inconsequential . However, the relevance of other Bacillus species as food poisoning organisms and etiological agents in nongastrointestinal infections is being increasingly recognized . Eleven Bacillus species isolated from veterinary samples associated with severe nongastrointestinal infections were assessed for the presence and expression of diarrheagenic enterotoxins and other potential virulence factors . PCR studies revealed the presence of DNA sequences encoding hemolysin BL (HBL) enterotoxin complex and B . cereus enterotoxin T (BceT) in five B . cereus strains and in Bacillus coagulans NB11 . Enterotoxin HBL was also harbored by Bacillus polymyxa NB6 . After 18 h of growth in brain heart infusion broth, all seven Bacillus isolates carrying genes encoding enterotoxin HBL produced this toxin . Cell-free supernatant fluids from all 11 Bacillus isolates demonstrated cytotoxicity toward human HEp-2 cells; only one Bacillus licheniformis strain adhered to this test cell line, and none of the Bacillus isolates were invasive . This study constitutes the first demonstration that Bacillus spp . associated with serious nongastrointestinal infections in animals may harbor and express diarrheagenic enterotoxins traditionally linked to toxigenic B . cereus. Appl Environ Microbiol, 2003 Apr, 69(4), 2330 - 9 Detection of frequency resonance energy transfer pair on double-labeled microsphere and Bacillus anthracis spores by flow cytometry; Zahavy E et al.; Development of an ultrasensitive biosensor for biological hazards in the environment is a major need for pollutant control and for the detection of biological warfare . Fluorescence methods combined with immunodiagnostic methods are the most common . To minimize background noise, arising from the unspecific adsorption effect, we have adapted the FRET (frequency resonance energy transfer) effect to the immunofluorescence method . FRET will increase the selectivity of the diagnosis process by introducing a requirement for two different reporter molecules that have to label the antigen surface at a distance that will enable FRET . Utilizing the multiparameter capability of flow cytometry analysis to analyze the double-labeling/FRET immunostaining will lead to a highly selective and sensitive diagnostic method . This work examined the FRET interaction of fluorescence-labeled avidin molecules on biotin-coated microspheres as a model system . As target system, we have used labeled polyclonal antibodies on Bacillus anthracis spores . The antibodies used were purified immunoglobulin G (IgG) molecules raised in rabbits against B . anthracis exosoporium components . The antibodies were fluorescence labeled by a donor-acceptor chromophore pair, alexa488 as a donor and alexa594 as an acceptor . On labeling the spores with alexa488-IgG as a donor and alexa594-IgG as an acceptor, excitation at 488 nm results in quenching of the alexa-488 fluorescence (E(q) = 35%) and appearance of the alexa594 fluorescence (E(s) = 22%), as detected by flow cytometry analysis . The FRET effect leads to a further isolated gate (FL1/FL3) for the target spores compared to competitive spores such as B . thuringiensis subsp . israelensis and B . subtilis . This new approach, combining FRET labeling and flow cytometry analysis, improved the selectivity of the B . anthracis spores by a factor of 10 with respect to B . thuringiensis subsp . israelensis and a factor of 100 with respect to B . subtilis as control spores. J Bacteriol, 2003 Apr, 185(8), 2418 - 31 Methionine regeneration and aminotransferases in Bacillus subtilis, Bacillus cereus, and Bacillus anthracis; Berger BJ et al.; The conversion of ketomethiobutyrate to methionine has been previously examined in a number of organisms, wherein the aminotransferases responsible for the reaction have been found to be members of the Ia subfamily (L . C . Berger, J . Wilson, P . Wood, and B . J . Berger, J . Bacteriol . 183:4421-4434, 2001) . The genome of Bacillus subtilis has been found to contain no subfamily Ia aminotransferase sequences . Instead, the analogous enzymes in B . subtilis were found to be members of the If subfamily . These putative aspartate aminotransferases, the yugH, ywfG, ykrV, aspB, and patA gene products, have been cloned, expressed, and characterized for methionine regeneration activity . Only YkrV was able to convert ketomethiobutyrate to methionine, and it catalyzed the reaction only when glutamine was used as amino donor . In contrast, subcellular homogenates of B . subtilis and Bacillus cereus utilized leucine, isoleucine, valine, alanine, phenylalanine, and tyrosine as effective amino donors . The two putative branched-chain aminotransferase genes in B . subtilis, ybgE and ywaA, were also cloned, expressed, and characterized . Both gene products effectively transaminated branched-chain amino acids and ketoglutarate, but only YbgE converted ketomethiobutyrate to methionine . The amino donor preference for methionine regeneration by YbgE was found to be leucine, isoleucine, valine, phenylalanine, and tyrosine . The B . subtilis ybgE gene is a member of the family III of aminotransferases and falls in a subfamily designated here IIIa . Examination of B . cereus and Bacillus anthracis genome data found that there were no subfamily IIIa homologues in these organisms . In both B . cereus and B . anthracis, two putative branched-chain aminotransferases and two putative D-amino acid aminotransferases were discovered as members of subfamily IIIb . These four sequences were cloned from B . cereus, expressed, and characterized . Only the gene product from the sequence designated Bc-BCAT2 was found to convert ketomethiobutyrate to methionine, with an amino donor preference of leucine, isoleucine, valine, phenylalanine, and tyrosine . The B . anthracis homologue of Bc-BCAT2 was also cloned, expressed, and characterized and was found to be identical in activity . The aminooxy compound canaline was found to be an uncompetitive inhibitor of B . subtilis YbgE and also inhibited growth of B . subtilis and B . cereus in culture. Biochem Biophys Res Commun, 2003 Apr 11, 303(3), 855 - 62 Anaerobic induction of Bacillus anthracis hemolytic activity; Klichko VI et al.; A number of genes in Bacillus anthracis encode for proteins homologous to the membrane-damaging factors known as pathogenic determinants in different bacteria . B . anthracis, however, has been traditionally considered non-hemolytic, and the recently identified hemolytic genes have been suggested to be transcriptionally silent . We found that the hemolytic genes of B . anthracis, collectively designated as anthralysins (Anls), could be induced in strict anaerobic conditions . We also demonstrate that Anl genes are expressed at the early stages of infection within macrophages by vegetating bacilli after spore germination . Cooperative and synergistic enhancement of the pore-forming and phospholipase C (PLC) activities of the Anls was found in hemolytic tests on human, but not sheep, red blood cells (RBC) . These findings imply Anls as B . anthracis pathogenic determinants and highlight oxygen limitation as environmental factor controlling their expression at both early and late stages of infection. Mol Gen Mikrobiol Virusol, 2003, (1), 6 - 14 {Genodiagnosis and molecular typing of the pathogens for plague, cholera, and anthrax}; Kutyrev VV et al.; The paper contains a survey of published data about the use of DNA-diagnostics in indicating and identifying the causative agents of highly dangerous infections like plague, cholera and anthrax . A discussion of data about the genetic relationship between strains of the mentioned causative agents isolated from different sources by using the molecular-typing methods as well as about the evolution ties between strains of different origins is in the focus of attention . Results of comparative studies of nucleotide sequences of genomes or of individual genomes in different Yersinia pestis, Vibrio cholerae and Bacillus anthracis strains, which are indicative of the evolution of their pathogenicity, are also under discussion. J Microbiol Methods, 2003 May, 53(2), 263 - 71 Discovery of phage display peptide ligands for species-specific detection of Bacillus spores; Turnbough CL Jr; Short peptides are capable of tight and specific binding to physiological or fortuitous receptors on the surface of cells . These peptides can be used to tag or capture target cells in an assortment of detector platforms . As part of an effort to identify small-molecule ligands for advanced detectors for spores of Bacillus anthracis, the causative agent of anthrax, we are screening (or biopanning) commercial phage display peptide libraries for peptides that bind tightly and selectively to spores of several Bacillus species . In addition to B . anthracis, these species include B . cereus, B . subtilis, and B . globigii . This review summarizes the methods used in our studies, the results from the biopanning experiments, and the characterization of the spore-binding peptides identified to date . Briefly, several unique families of peptides, with consensus sequences< or = seven-amino-acids long, were identified that exhibit preferential binding to spores (but not vegetative cells) of either one or only a few Bacillus species . At least one peptide family binds well to spores of multiple strains of B . anthracis, while binding poorly or not at all to spores of phylogenetically similar species . This review also discusses other points of interest regarding the use of peptide ligands for spore detection and for the detection of other types of cells. J Microbiol Methods, 2003 May, 53(2), 141 - 7 Application of the real-time PCR for the detection of airborne microbial pathogens in reference to the anthrax spores; Makino S et al.; To establish the rapid detection method of airborne bacterial spores, we examined Bacillus anthracis spores by real-time PCR . One hundred liters of air were trapped on a filter of an air monitor device . After it was suspended in PBS, spores of B . anthracis were artificially added . The suspension was also heated at 95 degrees C for 15 min and used for real-time PCR using anthrax-specific primers . A single cell of B . anthracis was detected by real-time PCR within 1 h . Our results provide evidence that anthrax spores from the atmosphere can be detected rapidly, suggesting that real-time PCR provides a flexible and powerful tool to prevent epidemics. Rinsho Byori, 2002 Nov, Suppl 123, 79 - 87 {Bacillus anthracis, prions, Coxiella burnetii}; Machida K; The detection kits for Bacillus anthracis, an isoform of host prion and Coxiella burnetii, using genetic technology are still not generalized . For B . anthracis target genes for detection would be genes of toxins . Although it is difficult to detect prions related to prion diseases before death, some mutants of prion gene in leukocytes and 14-3-3 proteins in cerebrospinal fluid are detectable in living patients suffering from Creutzfeldt-Jakob disease . The gene of superoxide dismutase in Coxiella burnetii is a useful target for detecting this Rickettsiae. Ophthalmologe, 2003 Mar, 100(3), 216 - 21 {Anthrax--an old disease becomes topical: cutaneous anthrax and the eye}; Kalpadakis P et al.; BACKGROUND: Anthrax disease and its eye manifestations were rare in the last 100 years, but the threat of terrorist actions has revived its topicality . MATERIALS AND METHODS: After an introductory historical review, the pathogenesis of this disease with regard to the virulence of Bacillus anthracis is reported . On the basis of photos displaying the course of the disease, the symptoms particularly of the cutaneous form of this disease as well as the diagnostic possibilities are described . RESULTS AND CONCLUSIONS: The current status of therapy and research for more effective treatment is discussed, with particular emphasis on the development of new substances with antitoxin properties and better vaccines . Bacillus anthracis is once again an actual threat, and therefore it is necessary for doctors to familiarize themselves with the current knowledge of this infection. JAMA, 2003 Mar 12, 289(10), 1274 - 7 Efficacy of selected hand hygiene agents used to remove Bacillus atrophaeus (a surrogate of Bacillus anthracis) from contaminated hands; Weber DJ et al.; CONTEXT: The intentional use of Bacillus anthracis transmitted via the US mail in October-November 2001 resulted in 22 people developing inhalation or cutaneous anthrax . Glove use with handwashing prior to and after contact with potential contaminated environmental surfaces and cutaneous lesions has been recommended . However, only limited data are available on the susceptibility of B anthracis to antiseptics . OBJECTIVE: To evaluate the efficacy of several hand antiseptics (interventions) and soap and water (control) against Bacillus atrophaeus, a surrogate of B anthracis . DESIGN, SETTING, AND PARTICIPANTS: Challenge study conducted among healthy adult volunteers, using the Standard Test Method for Evaluation of the Effectiveness of Health Care Professional Handwash Formulations (American Society for Testing and Materials E 1174-94) to determine the efficacy of various hand hygiene products at wash times of 10, 30, and 60 seconds . Volunteers were excluded if they had eczema, psoriasis, or other chronic skin conditions; nonintact skin; or allergies to any study agent . Study agents were a waterless rub containing 61% ethyl alcohol, a 2% chlorhexidine gluconate preparation, and an antibacterial microfiber towel that releases hypochlorite . A nonantimicrobial soap was used as a control . MAIN OUTCOME MEASURE: Reduction of B atrophaeus spores (log10 CFU/mL) on contaminated hands . RESULTS: Washes of 10, 30, and 60 seconds with either soap and water or 2% chlorhexidine gluconate eliminated 1.5 to 2.0 log10 CFUs/mL of B atrophaeus spores at wash 3 . Mean reductions (95% confidence intervals) with 10-, 30-, and 60-second washes with soap and water were 2.4 (2.2-2.5), 2.3 (2.2-2.4), and 2.1 (1.9-2.4) log(10) CFUs/mL, respectively; and with 2% chlorhexidine gluconate, 2.1 (2.0-2.3), 1.8 (1.5-2.0), and 1.7 (1.5-1.9) log10 CFUs/mL, respectively . Handwashing with chlorine-containing towels was increasingly effective as the wipe time increased; reductions at 10, 30, and 60 seconds were 1.3 (1.1-1.5), 1.6 (1.2-2.0), and 2.2 (2.1-2.2) log10 CFUs/mL, respectively . A waterless rub containing 61% ethyl alcohol was ineffective in eliminating B atrophaeus spores at all times tested (0 {-0.1 to 0.1}, -0.2 {-0.3 to -0.1}, and 0 {-0.2 to 0.2} log10 CFUs/mL) . CONCLUSIONS: In this evaluation of hand hygiene agents, handwashing with soap and water, 2% chlorhexidine gluconate, or chlorine-containing towels reduced the amount of B atrophaeus spore contamination, whereas use of a waterless rub containing ethyl alcohol was not effective in removing spores. J Clin Microbiol, 2003 Mar, 41(3), 1252 - 5 Novel sample preparation method for safe and rapid detection of Bacillus anthracis spores in environmental powders and nasal swabs; Luna VA et al.; Bacillus anthracis spores have been used as a biological weapon in the United States . We wanted to develop a safe, rapid method of sample preparation that provided safe DNA for the detection of spores in environmental and clinical specimens . Our method reproducibly detects B . anthracis in samples containing <10 spores. J Clin Microbiol, 2003 Mar, 41(3), 1212 - 8 Bacillus anthracis virulence in Guinea pigs vaccinated with anthrax vaccine adsorbed is linked to plasmid quantities and clonality; Coker PR et al.; Bacillus anthracis is a bacterial pathogen of great importance, both historically and in the present . This study presents data collected from several investigations and indicates that B . anthracis virulence is associated with the clonality and virulence of plasmids pXO1 and pXO2 . Guinea pigs vaccinated with Anthrax Vaccine Adsorbed were challenged with 20 B . anthracis isolates representative of worldwide genetic diversity . These same isolates were characterized with respect to plasmid copy number by using a novel method of quantitative PCR developed for rapid and efficient detection of B . anthracis from environmental samples . We found that the copy numbers for both pXO1 and pXO2 differed from those in previously published reports . By combining the data on survival, plasmid copy numbers, and clonality, we developed a model predicting virulence . This model was validated by using a randomly chosen set of 12 additional B . anthracis isolates . Results from this study will be helpful in future efforts to elucidate the basis for variation in the virulence of this important pathogen. J Bacteriol, 2003 Mar, 185(6), 1903 - 10 Identification of the immunodominant protein and other proteins of the Bacillus anthracis exosporium; Steichen C et al.; Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose-fitting, balloon-like layer called the exosporium . Although the exosporium serves as the source of surface antigens and a primary permeability barrier of the spore, its molecular structure and function are not well characterized . In this study, we identified five major proteins in purified B . anthracis (Sterne strain) exosporia . One protein was the recently identified collagen-like glycoprotein BclA, which appears to be a structural component of the exosporium hair-like nap . Using a large panel of unique antispore monoclonal antibodies, we demonstrated that BclA is the immunodominant antigen on the B . anthracis spore surface . We also showed that the BclA protein and not a carbohydrate constituent directs the dominant immune response . In addition, the length of the central (GXX)(n) repeat region of BclA appears to be strain specific . Two other unique proteins, BxpA and BxpB, were identified . BxpA is unusually rich in Gln and Pro residues and contains several different tandem repeats, which also exhibit strain-specific variation . In addition, BxpA was found to be cleaved approximately in half . BxpB appears to be glycosylated or associated with glycosylated material and is encoded by a gene that (along with bclA) may be part of an exosporium genomic island . The other two proteins identified were alanine racemase and superoxide dismutase, both of which were reported to be associated with the surface of other Bacillus spores . Possible functions of the newly identified proteins are discussed. Int J Antimicrob Agents, 2003 Feb, 21(2), 200 - 6 Bioterrorism--a new challenge for public health; Venkatesh S et al.; The opening years of the new millennium have presented a new and worrisome possibility to the public, including travellers: the threat of deadly infectious diseases from biological agents being deliberately released . The possibility of bioterrorism had always seemed remote but the recent anthrax attacks by mail have made this threat of immediate relevance . The deliberate use of Bacillus anthracis with the intent to harm civilian populations has raised public health concerns about potential exposure to intentionally released Variola virus and other biological agents . There is an urgent need for countries to examine their preparedness to respond to biological weapons attacks . Given the emotional shock of even an alleged threat of a biological release, it will be wise for governments to consider how to address such dangers as an integral part of the national response to other threats to public health and well being . Physicians and other health professionals, including those providing guidance to international and domestic travellers, need to have a clear understanding of the possible agents and the appropriate therapy or prophylaxis . This paper attempts to give a perspective on the threat of bioterrorism, the consequences of its use, the likely biological agents that may be used, and the clinical presentation and management of diseases caused by some agents most likely to be used. Presse Med, 2003 Feb 1, 32(4), 167 - 73 {Anthrax in the era of biowarfare}; Bossi P et al.; THE CONDITIONS OF INFECTION: Anthrax is a zoonosis due to Bacillus anthracis . Human contamination usually results from contact with an infected animal or product, or direct exposure to the bacteria . The latter represents one of the principle agents that can be used in biowarfare by spraying the spores . VARIOUS POSSIBILITIES: The inhaled form of the disease, characterised by hemorrhagic necrosis of the mediastinum adenopathies and septic shock, is the form that would probably be observed during a terrorist attack . The cutaneous and digestive forms are also possible . EVOLUTION: The clinical diagnosis, easy in the cutaneous form, is difficult in the other, rapidly progressive forms . Many guidelines have been published with recommendations for the treatment and prophylaxis of anthrax . Prognosis remains poor in the systemic form of the disease. J Clin Pathol, 2003 Mar, 56(3), 182 - 7 Bacillus anthracis; Spencer RC; The events of 11 September 2001 and the subsequent anthrax outbreaks have shown that the West needs to be prepared for an increasing number of terrorist attacks, which may include the use of biological warfare . Bacillus anthracis has long been considered a potential biological warfare agent, and this review will discuss the history of its use as such . It will also cover the biology of this organism and the clinical features of the three disease forms that it can produce: cutaneous, gastrointestinal, and inhalation anthrax . In addition, treatment and vaccination strategies will be reviewed. J Biol Chem, 2003 May 16, 278(20), 18056 - 62 Epub 2003 Feb 26. Production of Bacillus anthracis protective antigen is dependent on the extracellular chaperone, PrsA; Williams RC et al.; Protective antigen (PA) is a component of the Bacillus anthracis lethal and edema toxins and the basis of the current anthrax vaccine . In its heptameric form, PA targets host cells and internalizes the enzymatically active components of the toxins, namely lethal and edema factors . PA and other toxin components are secreted from B . anthracis using the Sec-dependent secretion pathway . This requires them to be translocated across the cytoplasmic membrane in an unfolded state and then to be folded into their native configurations on the trans side of the membrane, prior to their release from the environment of the cell wall . In this study we show that recombinant PA (rPA) requires the extracellular chaperone PrsA for efficient folding when produced in the heterologous host, B . subtilis; increasing the concentration of PrsA leads to an increase in rPA production . To determine the likelihood of PrsA being required for PA production in its native host, we have analyzed the B . anthracis genome sequence for the presence of genes encoding homologues of B . subtilis PrsA . We identified three putative B . anthracis PrsA proteins (PrsAA, PrsAB, and PrsAC) that are able to complement the activity of B . subtilis PrsA with respect to cell viability and rPA secretion, as well as that of AmyQ, a protein previously shown to be PrsA-dependent. Protein Expr Purif, 2003 Feb, 27(2), 325 - 30 Expression and purification of the Bacillus anthracis protective antigen domain 4; Krishnanchettiar S et al.; The protective antigen (PA) of Bacillus anthracis plays a crucial role in the pathogenesis of the anthrax disease . The fourth domain of PA (PA-D4) is responsible for initial binding of the anthrax toxin to the cellular receptor, and thus, is an attractive target for structure-based drug therapies . A synthetic gene for PA-D4 has been prepared by recursive PCR . PA-D4 has been expressed as a fusion protein in Escherichia coli . PA-D4 has been purified to near homogeneity and its identity has been verified by mass spectrometry . The recombinant PA-D4 exhibits CD and NMR spectra that suggest that it is folded and amenable for biophysical studies . Moreover, recombinant PA-D4 binds to HeLa cells, which suggests that recombinant PA-D4 is functional to bind to its cellular receptor . Infect Immun, 2003 Mar, 71(3), 1491 - 6 Venezuelan equine encephalitis virus-vectored vaccines protect mice against anthrax spore challenge; Lee JS et al.; Anthrax, a disease usually associated with herbivores, is caused by the bacterium Bacillus anthracis . The current vaccine licensed for human use requires a six-dose primary series and yearly boosters and causes reactogenicity in up to 30% of vaccine recipients . A minimally reactogenic vaccine requiring fewer inoculations is warranted . Venezuelan equine encephalitis (VEE) virus has been configured for use as a vaccine vector for a wide variety of immunogens . The VEE vaccine vector is composed of a self-replicating RNA (replicon) containing all of the VEE virus nonstructural genes and a multiple-cloning site in place of the VEE structural genes . Four different anthrax vaccines were constructed by cloning the protective antigen (PA) gene from B . anthracis into the VEE vaccine vector . The anthrax vaccines were produced by assembling the vectors into propagation-deficient VEE replicon particles in vitro . A/J mice inoculated subcutaneously with three doses of the mature 83-kDa PA vaccine were completely protected from challenge with the Sterne strain of B . anthracis . Similar results were obtained with vaccines composed of the PA gene fused to either the B . anthracis secretory sequence or to a tissue plasminogen activator secretory sequence in three additional mouse strains . Mice were unprotected from challenge after inoculation with the carboxy-terminal 63-kDa PA vaccine . These results suggest that these VEE-vectored vaccines may be suitable as candidate vaccines against anthrax. J Bacteriol, 2003 Mar, 185(5), 1555 - 63 Polymorphism in the collagen-like region of the Bacillus anthracis BclA protein leads to variation in exosporium filament length; Sylvestre P et al.; We recently identified a Bacillus anthracis glycoprotein which is a structural constituent of the exosporium filaments (P . Sylvestre, E . Couture-Tosi, and M . Mock, Mol . Microbiol . 45:169-178, 2002) . This Bacillus collagen-like protein (BclA) contains an internal collagen-like region (CLR) of GXX repeats which includes a large proportion of GPT triplets . Here, we report that the polymorphic marker Ceb-Bams13, for which there are nine alleles (P . Le Fleche et al., BMC Microbiol . 1:2, 2001), maps within the open reading frame encoding BclA . The bclA gene in 11 B . anthracis strains representative of seven Ceb-Bams13 alleles was sequenced and compared to the Ames bclA gene sequence . The amino- and carboxy-terminal sequences surrounding the CLR are conserved . The CLR itself is highly polymorphic: it contains between 17 and 91 GXX repeats and one to eight copies of the 21-amino-acid sequence (GPT)(5)GDTGTT, named the BclA repeat . The length of the filament on the spore surface differed between the strains . We exchanged the bclA gene between strains with different CLRs and examined the spore surfaces by electron microscopy analysis . The length of the BclA CLR is responsible for the variation in filament length. Mol Microbiol, 2003 Feb, 47(4), 917 - 27 A plasmid-encoded regulator couples the synthesis of toxins and surface structures in Bacillus anthracis; Mignot T et al.; Transcription of the major Bacillus anthracis virulence genes is triggered by CO2, a signal believed to reflect the host environment . A 180 kb plasmid, pXO1, carries the anthrax toxin genes and the genes responsible for their regulation, pagR and atxA; the latter encodes a major trans-activator . It has long been known that pXO1 genes have major effects on the physiology of B . anthracis, probably through regulatory cross-talk between plasmid and chromosomal genes . Accordingly, we found that the chromosomal S-layer genes, sap and eag, are regulated by pXO1 genes so that only eag is significantly expressed in the presence of CO2 . This effect results from the product of pagR acting as the most downstream element of a signalling cascade initiated by AtxA . In vitro evidence showed that PagR is a transcription factor that controls the S-layer genes by direct binding on their promoter regions . This work provides evidence that AtxA is a master regulator that co-ordinates the response to host signals by orchestrating positive and negative controls over genes located on all genetic elements. J Clin Microbiol, 2003 Feb, 41(2), 896 - 9 PCR assay to detect Ba