Mol Diagn, 2001 Dec, 6(4), 323 - 33 Biological agents: weapons of warfare and bioterrorism; Broussard LA; The use of microorganisms as agents of biological warfare is considered inevitable for several reasons, including ease of production and dispersion, delayed onset, ability to cause high rates of morbidity and mortality, and difficulty in diagnosis . Biological agents that have been identified as posing the greatest threat are variola major (smallpox), Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularaemia), filoviruses (Ebola hemorrrhagic fever and Marburg hemorrhagic fever), and arenaviruses Lassa (Lassa fever) and Junin (Argentine hemorrhagic fever) . The pathogenesis, clinical manifestations, diagnosis, and treatment of these agents are discussed . Rapid identification and diagnosis using molecular diagnostic techniques such as PCR is an essential element in the establishment of coordinated laboratory response systems and is the focus of current research and development . Molecular techniques for detection and identification of these organisms are reviewed. Appl Environ Microbiol, 2002 Jan, 68(1), 227 - 34 Optimization of the cell wall microenvironment allows increased production of recombinant Bacillus anthracis protective antigen from B . subtilis; Thwaite JE et al.; The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the trans side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases . In Bacillus subtilis, degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached . The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium . The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins . Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation . This paper describes the influence of the microenvironment on the trans side of the cytoplasmic membrane on the stability of rPA . Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is D-alanylated on the secretion and stability of rPA . The potential role of the dlt operon, responsible for D-alanylation, was investigated using a Bacillus subtilis strain encoding an inducible dlt operon . We show that, in the absence of D-alanylation, the yield of secreted rPA is increased 2.5-fold . The function of D-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of B . subtilis for the secretion of heterologous proteins. MMWR Morb Mortal Wkly Rep . 2001 Dec 7;50(48):1087. Use of onsite technologies for rapidly assessing environmental Bacillus anthracis contamination on surfaces in buildings. {Anthrax due to deliberate infection} van Dissel JT, Kullberg BJ, van den Berg PC, van Steenbergen JE. Afd . Infectieziekten, Leids Universitair Medisch Centrum, Postbus 9600, 2300 RC Leiden . j.t.van_dissel@lumc.nl Anthrax is a zoonosis which is particularly prevalent in cattle, goats and sheep and is caused by Bacillus anthracis, a Gram-positive spore forming aerobic microorganism . The endospores can survive outside of the body for many decades . The natural form of anthrax has a cutaneous, pulmonary and intestinal form . The pulmonary form can be rapidly fatal but is difficult to recognise due to an initially non-specific, flu-like clinical picture . As a result of spores being inhaled, a mediastinal lymphadenitis arises from which a systemic disease develops with a violent toxaemia, damage to the vascular endothelium, oedema, internal haemorrhages and circulatory collapse . Anthrax is diagnosed by demonstrating the presence of the bacteria in the cutaneous abnormality, in blood or another sterile body component such as cerebrospinal fluid, by means of a direct preparation, immunofluorescence or surface antigens, molecular diagnostics with PCR, or by means of culturing . B . anthracis is sensitive to quinolones, clindamycin and tetracyclines, and often to penicillin . Although naturally acquired cutaneous anthrax can be effectively treated with a short antibiotic cure, it is nevertheless advised in the USA to complete the full 60-day cure and to regard the cutaneous manifestation as a telltale sign of possible respiratory exposure . Anthrax is not transmitted from one person to another. Ned Tijdschr Geneeskd, 2001 Dec 8, 145(49), 2364 - 5 {Infectious diseases as a weapon: vigilance is needed}; Kingma JH et al.; The deliberate inclusion of Bacillus anthracis spores in mail has led to several cases of anthrax in the USA of which to date four have been fatal . Shortly before these incidents, the Health Council of the Netherlands had issued an advisory document which stressed the need for a well-established infrastructure and rigorous protocols so that an immediate and adequate response to cases of bioterrorism could be ensured . Physicians are expected to know the characteristics of the relevant diseases and to immediately notify the Health Inspectorate in the event of an infectious cluster which is unusual with respect to time, place or age, a cluster of a highly serious disease in healthy persons, or a cluster characterised by flaccid paralysis with bulbar signs. Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 14819 - 24 BETAWRAP: successful prediction of parallel beta -helices from primary sequence reveals an association with many microbial pathogens; Bradley P et al.; The amino acid sequence rules that specify beta-sheet structure in proteins remain obscure . A subclass of beta-sheet proteins, parallel beta-helices, represent a processive folding of the chain into an elongated topologically simpler fold than globular beta-sheets . In this paper, we present a computational approach that predicts the right-handed parallel beta-helix supersecondary structural motif in primary amino acid sequences by using beta-strand interactions learned from non-beta-helix structures . A program called BETAWRAP implements this method and recognizes each of the seven known parallel beta-helix families, when trained on the known parallel beta-helices from outside that family . BETAWRAP identifies 2,448 sequences among 595,890 screened from the National Center for Biotechnology Information (NCBI; nonredundant protein database as likely parallel beta-helices . It identifies surprisingly many bacterial and fungal protein sequences that play a role in human infectious disease; these include toxins, virulence factors, adhesins, and surface proteins of Chlamydia, Helicobacteria, Bordetella, Leishmania, Borrelia, Rickettsia, Neisseria, and Bacillus anthracis . Also unexpected was the rarity of the parallel beta-helix fold and its predicted sequences among higher eukaryotes . The computational method introduced here can be called a three-dimensional dynamic profile method because it generates interstrand pairwise correlations from a processive sequence wrap . Such methods may be applicable to recognizing other beta structures for which strand topology and profiles of residue accessibility are well conserved. J Bacteriol, 2002 Jan, 184(2), 370 - 80 Control of anthrax toxin gene expression by the transition state regulator abrB; Saile E et al.; Bacillus anthracis produces the anthrax toxin proteins protective antigen (PA), lethal factor (LF), and edema factor (EF) in a growth phase-dependent manner when cultured in liquid medium . Expression of the toxin genes pagA, lef, and cya peaks in late log phase, and steady-state levels of the toxin proteins are highest during the transition into stationary phase . Here we show that an apparent transition state regulator negatively regulates toxin gene expression . We identified two orthologues of the B . subtilis transition state regulator abrB in the B . anthracis genome: one on the chromosome and one on the 182-kb virulence plasmid pXO1 . The orthologue located on the chromosome is predicted to encode a 94-amino-acid protein that is 85% identical to B . subtilis AbrB . The hypothetical protein encoded on pXO1 is 41% identical to B . subtilis AbrB but missing 27 amino acid residues from the amino terminus compared to the B . subtilis protein . Deletion of the pXO1-encoded abrB orthologue did not affect toxin gene expression under the conditions tested . However, a B . anthracis mutant in which the chromosomal abrB gene was deleted expressed pagA earlier and at a higher level than the parent strain . Expression of a transcriptional pagA-lacZ fusion in the abrB mutant was increased up to 20-fold during early exponential growth compared to the parent strain and peaked in mid-exponential rather than late exponential phase . In contrast to the strong effect of abrB on pagA expression, lef-lacZ and cya-lacZ expression during early-log-phase growth was increased only two- to threefold in the abrB null mutant . Western hybridization analysis showed increased PA, LF, and EF synthesis by the mutant . As is true in B . subtilis, the B . anthracis abrB gene is negatively regulated by spo0A . Our findings tie anthrax toxin gene expression to the complex network of postexponential phase adaptive responses that have been well studied in B . subtilis. Emerg Infect Dis, 2001 Nov-Dec, 7(6), 933 - 44 Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States; Jernigan JA et al.; From October 4 to November 2, 2001, the first 10 confirmed cases of inhalational anthrax caused by intentional release of Bacillus anthracis were identified in the United States . Epidemiologic investigation indicated that the outbreak, in the District of Columbia, Florida, New Jersey, and New York, resulted from intentional delivery of B . anthracis spores through mailed letters or packages . We describe the clinical presentation and course of these cases of bioterrorism-related inhalational anthrax . The median age of patients was 56 years (range 43 to 73 years), 70% were male, and except for one, all were known or believed to have processed, handled, or received letters containing B . anthracis spores . The median incubation period from the time of exposure to onset of symptoms, when known (n=6), was 4 days (range 4 to 6 days) . Symptoms at initial presentation included fever or chills (n=10), sweats (n=7), fatigue or malaise (n=10), minimal or nonproductive cough (n=9), dyspnea (n=8), and nausea or vomiting (n=9) . The median white blood cell count was 9.8 X 10(3)/mm(3) (range 7.5 to 13.3), often with increased neutrophils and band forms . Nine patients had elevated serum transaminase levels, and six were hypoxic . All 10 patients had abnormal chest X-rays; abnormalities included infiltrates (n=7), pleural effusion (n=8), and mediastinal widening (seven patients) . Computed tomography of the chest was performed on eight patients, and mediastinal lymphadenopathy was present in seven . With multidrug antibiotic regimens and supportive care, survival of patients (60%) was markedly higher (<15%) than previously reported. Rapid Commun Mass Spectrom, 2001, 15(22), 2110 - 6 Detection of specific Bacillus anthracis spore biomarkers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Elhanany E et al.; Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied for the characterization of Bacillus anthracis spore biomarkers . B . anthracis spores were extracted under a simple procedure, followed by linear mode analysis, using sinapinic acid as the matrix . Several markers with a mass range of 4-7 kDa were detected in three B . anthracis strains: Vollum, Sterne and V770-NP1-R . Similar spectra were also obtained for spore extracts of two members of the B . cereus group: B . thuringiensis and B . cereus, but not for B . mycoides, B . subtilis or B . licheniformis, suggesting that these markers are specific to closely related members of the B . cereus group . When alpha-cyano-4-hydroxycinnamic acid was used as the matrix, at least four additional new markers within a mass range of 2-4 kDa could be detected in all B . anthracis spore extracts . These markers, corresponding to a molecular weight of 2528.3, 2792.4, 3077.4, and 3590.7 Da, have not been observed in extracts of the three closely related Bacillus species - B . cereus, B . thuringiensis and B . mycoides . These unique B . anthracis biomarkers, which were isotopically resolved and reproducibly detected in the highly accurate MALDI-TOFMS reflectron mode, may be useful as a basis for rapid and specific identification of B . anthracis strains . J Bacteriol, 2002 Jan, 184(1), 331 - 4 Plasmid-encoded autolysin in Bacillus anthracis: modular structure and catalytic properties; Mesnage S et al.; A Bacillus anthracis virulence plasmid-encoded peptidoglycan hydrolase (AmiA) with sequence similarity to N-acetylmuramoyl-L-alanine amidases hydrolyzes peptidoglycan independently of cell wall binding . Residues H341, E355, H415, and E486 are absolutely required for catalysis . Many AmiA paralogs are fused to different sorting signals, suggesting that these modular proteins result from domain shuffling. J Bacteriol, 2002 Jan, 184(1), 134 - 41 Bacillus anthracis pXO1 plasmid sequence conservation among closely related bacterial species; Pannucci J et al.; The complete sequencing and annotation of the 181.7-kb Bacillus anthracis virulence plasmid pXO1 predicted 143 genes but could only assign putative functions to 45 . Hybridization assays, PCR amplification, and DNA sequencing were used to determine whether pXO1 open reading frame (ORF) sequences were present in other bacilli and more distantly related bacterial genera . Eighteen Bacillus species isolates and four other bacterial species were tested for the presence of 106 pXO1 ORFs . Three ORFs were conserved in most of the bacteria tested . Many of the pXO1 ORFs were detected in closely related Bacillus species, and some were detected only in B . anthracis isolates . Three isolates, Bacillus cereus D-17, B . cereus 43881, and Bacillus thuringiensis 33679, contained sequences that were similar to more than one-half of the pXO1 ORF sequences examined . The majority of the DNA fragments that were amplified by PCR from these organisms had DNA sequences between 80 and 98% similar to that of pXO1 . Pulsed-field gel electrophoresis revealed large potential plasmids present in both B . cereus 43881 (341 kb) and B . thuringiensis ATCC 33679 (327 kb) that hybridized with a DNA probe composed of six pXO1 ORFs. Prim Care, 2001 Dec, 28(4), 807 - 21, vii Vaccines, biological warfare, and bioterrorism; Polgreen PM et al.; This article presents a brief history of the use of biological agents in warfare and bioterrorism . Bacillus anthracis, smallpox virus, and Yersinia pestis, historically have been and currently are considered the most likely candidates for potential use under these circumstances . This article discusses the clinical syndromes these agents cause and the role of vaccines in protection against them. Vaccine, 2001 Dec 12, 20(5-6), 972 - 8 Anthrax vaccine: short-term safety experience in humans; Pittman PR et al.; Bacillus anthracis is the major terrorist and biological warfare agent of concern to civilian and military medical planners . The licensed anthrax vaccine, adsorbed (AVA) is believed to be an effective prophylactic medical countermeasure against this threat . Our objective in this report was to expand the safety database for this vaccine by assessing data on self-reported, short-term safety of AVA during more than 25 years of use, measured by local and systemic adverse events temporally associated with the administration of AVA . A minority of AVA recipients reported systemic and injection site reactions . Females reported a higher incidence of injection site and systemic adverse events than males . Data show a difference in incidence of local reactions between lots . A prospective, randomized, placebo-controlled study to actively examine reactogenicity is needed to more completely define the extent and nature of reactions associated with receipt of AVA in humans as well as to confirm the gender lot differences in local reaction rates. Int J Antimicrob Agents, 2001 Dec, 18(6), 559 - 62 In vitro resistance of Bacillus anthracis Sterne to doxycycline, macrolides and quinolones; Brook I et al.; Bacillus anthracis is a potential biological warfare agent . Its ability to develop resistance to antimicrobial agents currently recommended for the treatment of anthrax infection is a major concern . B . anthracis Sterne was grown from a live veterinary vaccine and used it to test for the development of resistance after 21 sequential subcultures in sub-inhibitory concentrations of doxycycline and three quinolones (ciprofloxacin, alatrofloxacin and gatifloxacin) and 15 sequential subcultures in sub-inhibitory concentrations of three macrolides (erythromycin, azithromycin and clarithromycin) . After 21 subcultures the minimal inhibitory concentrations (MICs) increased from 0.1 to 1.6 mg/l for ciprofloxacin, from 1.6 to 12.5 mg/l for alatrofloxacin, from 0.025 to 1.6 mg/l for gatifloxacin and from 0.025 to 0.1 mg/l for doxycycline . After 15 passages of sequential subculturing with macrolides, the MICs increased from 12.5 to 12.5 or 50.0 mg/l for azithromycin, from 0.2 to 1.6 or 0.4 mg/l for clarithromycin and from 6.25 to 6.25 or 50 mg/l for erythromycin . After sequential passages with a single quinolone or doxycycline, each isolate was cross-tested for resistance using the other drugs . All isolates selected for resistance to one quinolone were also resistant to the other two quinolones, but not to doxycycline . The doxycycline-resistant isolate was not resistant to any quinolone. Mol Microbiol, 2001 Nov, 42(4), 931 - 8 Fate of germinated Bacillus anthracis spores in primary murine macrophages; Guidi-Rontani C et al.; We investigated the fate of germinated Bacillus anthracis spores after their germination in Swiss murine peritoneal macrophages and in the cell line RAW264.7 . We found that the lethal toxin and the oedema toxin are germ-associated factors that are essential for the survival of the vegetative form in host cells . We also found that pX02 is not involved in this complex pathogenic process . By transmission electron microscopy, we showed the tight interaction between the exosporium of the spore and the phagosomal membrane of the macrophage . Our data strongly suggest that the B . anthracis toxinogenic, unencapsulated Sterne strain (7702) does not multiply within macrophages . These results contributed to reveal the strategies used by B . anthracis to survive within the host and to reach the external medium where they proliferate. J Clin Microbiol, 2001 Dec, 39(12), 4566 - 7 Molecular investigation of the Aum Shinrikyo anthrax release in Kameido, Japan; Keim P et al.; In 1993, the Aum Shinrikyo cult aerosolized Bacillus anthracis spores over Kameido, Japan . Spore samples were obtained from the release site, cultured, and characterized by molecular genetic typing . The isolates were consistent with strain Sterne 34F2, which is used in Japan for animal prophylaxis against anthrax. MMWR Morb Mortal Wkly Rep, 2001 Nov 16, 50(45), 1014 - 6 Update: Interim recommendations for antimicrobial prophylaxis for children and breastfeeding mothers and treatment of children with anthrax; Interim guidelines for investigation of and response to Bacillus anthracis exposures; Environmental Sampling . Environmental testing to detect B . anthracis on surfaces or in the air can be used to investigate known or suspected exposure events . The highest priority of an investigation is to evaluate the risk for exposure to aerosolized B . anthracis spores . Persons collecting and testing samples should 1) obtain adequate samples, 2) avoid cross-contamination during processing, and 3) ensure proficient laboratory testing and interpretation of test results . A positive laboratory test for B . anthracis from a sample of an environmental surface may be caused by cross-contamination from an exposure vehicle (e.g., contact with an envelope containing B . anthracis), background occurrence of B . anthracis spores in the environment, or previously aerosolized B . anthracis that has settled onto environmental surfaces . Laboratory test results of environmental surface samples should not be the only criterion for starting, continuing, or stopping antimicrobial prophylaxis for inhalational disease. MMWR Morb Mortal Wkly Rep, 2001 Nov 9, 50(44), 984 - 6 Considerations for distinguishing influenza-like illness from inhalational anthrax; Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach; Environmental Health Engineering Program, Department of Civil Engineering, Northwestern University, Evanston, IL 60208, USA . cveliuwt@nus.edu.sg The utility of a high-density oligonucleotide microarray (microchip) for identifying strains of five closely related bacilli (Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus medusa and Bacillus subtilis) was demonstrated using an approach that compares the non-equilibrium dissociation rates ('melting curves') of all probe-target duplexes simultaneously . For this study, a hierarchical set of 30 oligonucleotide probes targeting the 16S ribosomal RNA of these bacilli at multiple levels of specificity (approximate taxonomic ranks of domain, kingdom, order, genus and species) was designed and immobilized in a high-density matrix of gel pads on a glass slide . Reproducible melting curves for probes with different levels of specificity were obtained using an optimized salt concentration . Clear discrimination between perfect match (PM) and mismatch (MM) duplexes was achieved . By normalizing the signals to an internal standard (a universal probe), a more than twofold discrimination (> 2.4x) was achieved between PM and 1-MM duplexes at the dissociation temperature at which 50% of the probe-target duplexes remained intact . This provided excellent differentiation among representatives of different Bacillus species, both individually and in mixtures of two or three . The overall pattern of hybridization derived from this hierarchical probe set also provided a clear 'chip fingerprint' for each of these closely related Bacillus species. Acta Crystallogr D Biol Crystallogr, 2001 Dec, 57(Pt 12), 1881 - 4 Epub 2001 Nov 21. Crystallization and preliminary X-ray study of the edema factor exotoxin adenylyl cyclase domain from Bacillus anthracis in the presence of its activator, calmodulin; Drum CL et al.; Edema factor from Bacillus anthracis is a 92 kDa secreted adenylyl cyclase exotoxin and is activated by the host-resident protein calmodulin . Calmodulin is a ubiquitous intracellular calcium sensor in eukaryotes and activates edema factor nearly 1000-fold upon binding . While calmodulin has many known effectors, including kinases, phosphodiesterases, motor proteins, channels and type 1 adenylyl cyclases, no structures of calmodulin in complex with a functional enzyme have been solved . The crystallization and initial experimental phasing of crystals containing a complex of edema factor adenylyl cyclase domain and calmodulin are reported here . The edema factor-calmodulin complex crystallizes in three different space groups . A native data set in the I222 space group has been collected to 2.7 A and the self-rotation function solution suggests three edema factor-calmodulin complexes in each asymmetric unit . Initial 4 A phases were obtained by selenomethionyl MAD in combination with two heavy-atom derivatives . These phases were successfully extended to 2.7 A using NCS averaging. MMWR Morb Mortal Wkly Rep . 2001 Nov 2;50(43):961. Interim recommendations for protecting workers from exposure to Bacillus anthracis in work sites in which mail is handled or processed; Updated recommendations for antimicrobial prophylaxis among asymptomatic pregnant women after exposure to Bacillus anthracis; The antimicrobial of choice for initial prophylactic therapy among asymptomatic pregnant women exposed to Bacillus anthracis is ciprofloxacin, 500 mg twice a day for 60 days . In instances in which the specific B . anthracis strain has been shown to be penicillin-sensitive, prophylactic therapy with amoxicillin, 500 mg three times a day for 60 days, may be considered . Isolates of B . anthracis implicated in the current bioterrorist attacks are susceptible to penicillin in laboratory tests, but may contain penicillinase activity . Pencillins are not recommended for treatment of anthrax, where such penicillinase activity may decrease their effectiveness . However, penicillins are likely to be effective for preventing anthrax, a setting where relatively few organisms are present . Doxycycline should be used with caution in asymptomatic pregnant women and only when contraindications are indicated to the use of other appropriate antimicrobial drugs. MMWR Morb Mortal Wkly Rep, 2001 Nov 2, 50(43), 941 - 8 Update: Investigation of bioterrorism-related anthrax and interim guidelines for clinical evaluation of persons with possible anthrax; Centers for Disease Control and Prevention; Since October 3, 2001, CDC and state and local public health authorities have been investigating cases of bioterrorism-related anthrax . This report updates findings as of October 31, and includes interim guidelines for the clinical evaluation of persons with possible anthrax . A total of 21 cases (16 confirmed and five suspected) of bioterrorism-related anthrax have been reported among persons who worked in the District of Columbia, Florida, New Jersey, and New York City (Figure 1) . Until the source of these intentional exposures is eliminated, clinicians and laboratorians should be alert for clinical evidence of Bacillus anthracis infection . Epidemiologic investigation of these cases and surveillance to detect new cases of bioterrorism-associated anthrax continues. Nature, 2001 Nov 8, 414(6860), 225 - 9 Identification of the cellular receptor for anthrax toxin; Bradley KA et al.; The tripartite toxin secreted by Bacillus anthracis, the causative agent of anthrax, helps the bacterium evade the immune system and can kill the host during a systemic infection . Two components of the toxin enzymatically modify substrates within the cytosol of mammalian cells: oedema factor (OF) is an adenylate cyclase that impairs host defences through a variety of mechanisms including inhibiting phagocytosis; lethal factor (LF) is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinase and causes lysis of macrophages . Protective antigen (PA), the third component, binds to a cellular receptor and mediates delivery of the enzymatic components to the cytosol . Here we describe the cloning of the human PA receptor using a genetic complementation approach . The receptor, termed ATR (anthrax toxin receptor), is a type I membrane protein with an extracellular von Willebrand factor A domain that binds directly to PA . In addition, a soluble version of this domain can protect cells from the action of the toxin. MMWR Morb Mortal Wkly Rep, 2001 Oct 26, 50(42), 909 - 19 Update: Investigation of bioterrorism-related anthrax and interim guidelines for exposure management and antimicrobial therapy, October 2001; Centers for Disease Control and Prevention; Since October 3, 2001, CDC and state and local public health authorities have been investigating cases of bioterrorism-related anthrax . This report updates previous findings, provides new information on case investigations in two additional areas, presents the susceptibility patterns of Bacillus anthracis isolates, and provides interim recommendations for managing potential threats and exposures and for treating anthrax. MMWR Morb Mortal Wkly Rep, 2001 Oct 19, 50(41), 889 - 93 Update: Investigation of anthrax associated with intentional exposure and interim public health guidelines, October 2001; Human anthrax in India: urgent need for effective prevention; Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry-605 006, IndiaAnthrax is a zoonotic illness caused by Bacillus anthracis . Sporadic cases continue to be reported from many parts of the world . From India, both sporadic cases and outbreaks are being reported regularly . The Union Territory of Pondicherry (a former French colony) lies on the coast of Bay of Bengal, where the incidence of anthrax is on the rise with 28 cases being detected in the year 1999 and 2000 alone . So far, about 34 human cases have been encountered in this region . Recently, an increase in the number of anthrax cases has been noted in veterinary and human practice in this area . Most cases have occurred in agricultural labourers who gave history of handling animal meat or skin of infected animals . The meningitic form of the disease has a very bad prognosis . Patients with this form of disease died despite treatment with high dose penicillin . The typical bacilli were seen in the CSF in all cases of anthrax meningitis and was diagnostic of the condition . The cutaneous form of illness had a benign course and responded favourably to penicillin treatment . Awareness among clinicians and mandatory reporting of cases to public health departments along with public education will help control morbidity and mortality due to anthrax . Effective immunization of animals is the other important control measure for anthrax. Hist Med Vet, 1997, 22(3), 49 - 63 Not Available Theves G. Anthrax is an infectious disease of herbivores, especially sheep and cattle, but also of horses, of pigs, of dogs, of wild animals and of humans . Bacillus anthracis causes the disease . This bacterium needs plenty of oxygen to procreate and to produce resistant spores, which remains viable in the soil during 3.5 years, at times during 15-20 years . The author tries to follow step by step the evolution of the ideas concerning the origin and the pathology as well as of the veterinarians measures against this disease during the 19th and 20th centuries . Many studies of the endemic anthrax were made from the beginning of the second half of the 19th century . Thanks to Louis Pasteur (1822-1895) in France and Robert Koch (1843-1910) in Germany, the anthrax could be identified with a soil-disease between 1870 and 1880 . The statistics concerning the anthrax in the Grand-Duchy of Luxembourg compared to the geological and pedological structures of the soil, have fully confirmed the scientific findings at the end of the 19th century . Nowadays the anthrax has disappeared from our landscape thanks to the preventive inoculation and to the industrial use of the animal cadavers. Crit Rev Microbiol, 2001, 27(3), 167 - 200 Anthrax toxin; Bhatnagar R et al.; Anthrax is primarily a disease of herbivores caused by gram-positive, aerobic, spore-forming Bacillus anthracis . Humans are accidental hosts through the food of animal origin and animal products . Anthrax is prevelant in most parts of the globe, and cases of anthrax have been reported from almost every country . Three forms of the disease have been recognized: cutaneous (through skin), gastrointestinal (through alimentary tract), and pulmonary (by inhalation of spores) . The major virulence factors of Bacillus anthracis are a poly-D glutamic acid capsule and a three-component protein exotoxin . The genes coding for the toxin and the enzymes responsible for capsule production are carried on plasmid pXO1 and pXO2, respectively . The three proteins of the exotoxin are protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa) . The toxins follow the A-B model with PA being the B moeity and LF/EF, the alternative A moeities . LF and EF are individually nontoxic, but in combination with PA form two toxins causing different pathogenic responses in animals and cultured cells . PA + LF forms the lethal toxin and PA + EF forms the edema toxin . During the process of intoxication, PA binds to the cell surface receptor and is cleaved at the sequence RKKR (167) by cell surface proteases such as furin generating a cell-bound, C-terminal 63 kDa protein (PA63) . PA63 possesses a binding site to which LF or EF bind with high affinity . The complex is then internalized by receptor-mediated endocytosis . Acidification of the vesicle leads to instertion of PA63 into the endosomal membrane and translocation of LF/EF across the bilayer into the cytosol where they exert their toxic effects . EF has a calcium- and calmodulin-dependent adenylate cyclase activity . Recent reports indicate that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and 2), and this cleavage inactivates MAPKK1 and thus inhibits the mitogen-activated protein kinase signal transduction pathway . We describe in detail the studies so far done on unraveling the molecular mechanisms of pathogenesis of Bacillus anthracis. Toxicon, 2001 Nov, 39(11), 1747 - 55 Toxins of Bacillus anthracis; Brossier F et al.; Bacillus anthracis, a gram positive bacterium, is the causative agent of anthrax . This organism is capsulogen and toxinogenic . It secretes two toxins which are composed of three proteins: the protective antigen (PA), the lethal factor (LF) and the edema factor (EF) . The lethal toxin (PA+LF) provokes a subit death in animals, the edema toxin (PA+EF) induces edema . The edema and the lethal factors are internalised into the eukaryotic target cells via the protective antigen . EF and LF exert a calmoduline dependent adenylate cyclase and a metalloprotease activity respectively . Progress in the structure-function relationship of these three proteins, their regulation mechanisms and their roles in pathogenesis and immunoprotection will be exposed. Environ Microbiol, 2001 Aug, 3(8), 493 - 501 Distribution of S-layers on the surface of Bacillus cereus strains: phylogenetic origin and ecological pressure; Mignot T et al.; Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis have been described as members of the Bacillus cereus group but are, in fact, one species . B . anthracis is a mammal pathogen, B . thuringiensis an entomopathogen and B . cereus a ubiquitous soil bacterium and an occasional human pathogen . In two clinical isolates of B . cereus, in some B . thuringiensis strains and in B . anthracis, an S-layer has been described . We investigated how the S-layer is distributed in B . cereus, and whether phylogeny or ecology could explain its presence on the surface of some but not all strains . We first developed a simple biochemical assay to test for the presence of the S-layer . We then used the assay with 51 strains of known genetic relationship: 26 genetically diverse B . cereus and 25 non-B . anthracis of the B . anthracis cluster . When present, the genetic organization of the S-layer locus was analysed further . It was identical in B . cereus and B . anthracis . Nineteen strains harboured an S-layer, 16 of which belonged to the B . anthracis cluster . All 19 were B . cereus clinical isolates or B . thuringiensis, except for one soil and one dairy strain . These findings suggest a common phylogenetic origin for the S-layer at the surface of B . cereus strains and, presumably, ecological pressure on its maintenance. Appl Environ Microbiol, 2001 Oct, 67(10), 4863 - 73 Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereus and Bacillus thuringiensis Soil Isolates; Ticknor LO et al.; We examined 154 Norwegian B . cereus and B . thuringiensis soil isolates (collected from five different locations), 8 B . cereus and 2 B . thuringiensis reference strains, and 2 Bacillus anthracis strains by using fluorescent amplified fragment length polymorphism (AFLP) . We employed a novel fragment identification approach based on a hierarchical agglomerative clustering routine that identifies fragments in an automated fashion . No method is free of error, and we identified the major sources so that experiments can be designed to minimize its effect . Phylogenetic analysis of the fluorescent AFLP results reveals five genetic groups in these group 1 bacilli . The ATCC reference strains were restricted to two of the genetic groups, clearly not representative of the diversity in these bacteria . Both B . anthracis strains analyzed were closely related and affiliated with a B . cereus milk isolate (ATCC 4342) and a B . cereus human pathogenic strain (periodontitis) . Across the entire study, pathogenic strains, including B . anthracis, were more closely related to one another than to the environmental isolates . Eight strains representing the five distinct phylogenetic clusters were further analyzed by comparison of their 16S rRNA gene sequences to confirm the phylogenetic status of these groups . This analysis was consistent with the AFLP analysis, although of much lower resolution . The innovation of automated genotype analysis by using a replicated and statistical approach to fragment identification will allow very large sample analyses in the future. Biochim Biophys Acta, 2001 Sep 28, 1537(2), 132 - 46 The cell envelope-bound metalloprotease (camelysin) from Bacillus cereus is a possible pathogenic factor; Fricke B et al.; A novel membrane proteinase of the nosocomial important bacteria species Bacillus cereus (synonyms: camelysin, CCMP) was purified up to homogeneity as was shown by mass spectrometry in its amphiphilic form . Camelysin is a neutral metalloprotease with a molecular mass of 19 kDa . Its unique N-terminus Phe-Phe-Ser-Asp-Lys-Glu-Val-Ser-Asn-Asn-Thr-Phe-Ala-Ala-Gly-Thr-Leu-Asp-Leu-Thr-Leu-Asn-Pro-Lys-Thr-Leu-Val-Asp-(Ile-Lys-Asp)- was not detected in the protein data bases during BLAST searches, but in the partially sequenced genome of Bacillus anthracis, coding for an unknown protein . Cleavage sites of the membrane proteinase for the insulin A- and B-chains were determined by mass spectrometry and N-terminal sequencing . Camelysin prefers cleavage sites in front of aliphatic and hydrophilic amino acid residues (-OH, -SO3H, amido group), avoiding bulky aromatic residues . The internally quenched fluorogenic substrates of the matrix metalloproteases 2 and 7 were cleaved with the highest efficiency at the Leu-decrease-Gly or Leu-decrease-Ala bond with the smaller residue in the P1' position . The protein specificity is broad--all various kinds of casein were cleaved as well as acid-soluble collagen, globin and ovalbumin; intact insulin was destroyed only to a low extent . Actin, collagen type I, fibrinogen, fibrin, alpha2-antiplasmin and alpha1-antitrypsin were cleaved . The protease formed SDS-stable complexes with Glu-plasminogen and antithrombin III, visible after SDS electrophoresis by gold staining and Western blot . The CCMP-plasminogen complex caused a partial activation of plasminogen to plasmin . Camelysin interacts with proteins of the blood coagulation cascade and could facilitate the penetration of fibrin clots and of the extracellular matrix during bacterial invasion. J Appl Microbiol, 2001 Sep, 91(3), 421 - 6 A simple and sensitive detection system for Bacillus anthracis in meat and tissue; Cheun HI et al.; AIMS: To detect and isolate Bacillus anthracis from meat and tissue by rapid and simple procedures . METHODS AND RESULTS: Bacillus anthracis Pasteur II cells were added to 1 g lymph node and pig meat, which were then cut into small pieces and suspended in PBS . Aliquots were spread on Bacillus cereus selective agar (BCA) plates to isolate B . anthracis cells, and incubated in trypticase soy broth . The enrichment culture was used for nested PCR with B . anthracis specific primers, which were to confirm the presence of B . anthracis chromosomal DNA and the pXO1/pXO2 plasmids . CONCLUSION: One cell of B . anthracis was detected by nested PCR from 1 g of the samples, and was also isolated on BCA plates according to colony morphology within two days . SIGNIFICANCE AND IMPACT OF THE STUDY: These results could be useful for detecting animals with latent anthrax, and meat contaminated with B . anthracis, rapidly and simply. Lett Appl Microbiol, 2001 Sep, 33(3), 237 - 40 Detection of anthrax spores from the air by real-time PCR; Makino SI et al.; AIMS: To detect and isolate Bacillus anthracis from the air by a simple and rapid procedure . METHODS AND RESULTS: One hundred litres of air were filtered through an air monitor device . After the membrane was suspended in PBS, spores of B . anthracis were added . The suspension was plated on Bacillus cereus selective agar (BCA) plates to detect B . anthracis colonies . The suspension was also heated at 95 degrees C for 15 min and used for real-time PCR using a Light Cycler system and anthrax-specific primers . CONCLUSION: A single cell of B . anthracis was detected by real-time PCR within 1 h and was also isolated on a BCA plate within two d . SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provide evidence that anthrax spores from the atmosphere can be detected rapidly, suggesting that real-time PCR and a Light Cycler provides a flexible and powerful tool to prevent epidemics. Infect Immun, 2001 Oct, 69(10), 6532 - 6 Purification of anthrax edema factor from Escherichia coli and identification of residues required for binding to anthrax protective antigen; Kumar P et al.; The structural gene for anthrax edema factor (EF) was expressed in Escherichia coli under the control of a powerful T5 promoter to yield the 89-kDa recombinant protein that reacted with anti-EF antibodies . Recombinant EF was purified to homogeneity by a two-step procedure involving metal chelate affinity chromatography and cation-exchange chromatography . From 1 liter of culture, 2.5 mg of biologically active EF was easily purified . This is the first report of purification of anthrax EF from E . coli . EF purified from E . coli was biologically and functionally as active as its Bacillus anthracis counterpart . The recombinant protein could compete with lethal factor for binding to protective antigen . Sequence analysis revealed a stretch of seven amino acids, Val Tyr Tyr Glu Ile Gly Lys, present both in EF (residues 136 to 142) and lethal factor (residues 147 to 153) . To investigate the role of these seven residues in binding to protective antigen, the residues were individually mutated to alanine in EF . Mutations in residues Tyr137, Tyr138, Ile140, and Lys142 of EF specifically blocked its interaction with anthrax protective antigen . The adenylate cyclase activity of the mutants remained unaffected . The results suggested that residues Tyr137, Tyr138, Ile140, and Lys142 are required for binding of EF to anthrax protective antigen, which facilitates its entry into susceptible cells. Annu Rev Microbiol, 2001, 55, 647 - 71 Anthrax; Mock M et al.; Bacillus anthracis was shown to be the etiological agent of anthrax by R . Koch and L . Pasteur at the end of the nineteenth century . The concepts on which medical microbiology are based arose from their work on this bacterium . The link between plasmids and major virulence factors of B . anthracis was not discovered until the 1980s . The three toxin components are organized in two A-B type toxins, and the bacilli are covered by an antiphagocytic polyglutamic capsule . Structure-function analysis of the toxins indicated that the common B-domain binds to a ubiquitous cell receptor and forms a heptamer after proteolytic activation . One enzyme moiety is an adenylate cyclase and the other is a Zn(2+) metalloprotease, which is able to cleave MAPKKs . The capsule covers an S-layer sequentially composed of two distinct proteins . Knowledge of the toxins facilitates the design of safer veterinary vaccines . Spore-structure analysis could contribute to the improvement of human nonliving vaccines . The phylogeny of B . anthracis within the Bacillus cereus group is also reviewed. Vaccine, 2001 Sep 14, 19(32), 4768 - 73 In vitro correlate of immunity in a rabbit model of inhalational anthrax; Pitt ML et al.; A serological correlate of vaccine-induced immunity was identified in the rabbit model of inhalational anthrax . Animals were inoculated intramuscularly at 0 and 4 weeks with varying doses of Anthrax Vaccine Adsorbed (AVA) ranging from a human dose to a 1:256 dilution in phosphate-buffered saline (PBS) . At 6 and 10 weeks, both the quantitative anti-protective antigen (PA) IgG ELISA and the toxin-neutralizing antibody (TNA) assays were used to measure antibody levels to PA . Rabbits were aerosol-challenged at 10 weeks with a lethal dose (84-133 LD(50)) of Bacillus anthracis spores . All the rabbits that received the undiluted and 1:4 dilution of vaccine survived, whereas those receiving the higher dilutions of vaccine (1:16, 1:64 and 1:256) had deaths in their groups . Results showed that antibody levels to PA at both 6 and 10 weeks were significant (P<0.0001) predictors of survival. AJNR Am J Neuroradiol, 2001 Aug, 22(7), 1303 - 5 CT and MR findings of anthrax meningoencephalitis: report of two cases and review of the literature; Kim HJ et al.; Anthrax meningoencephalitis is a rare complication of infection with Bacillus anthracis and generally produces a hemorrhagic meningoencephalitis . We present the CT and MR imaging findings in two patients demonstrating subarachnoid, intracerebral, and intraventricular hemorrhage with leptomeningeal enhancement. Vaccine, 2001 Aug 14, 19(31), 4409 - 16 Passive transfer of protection against Bacillus anthracis infection in a murine model; Beedham RJ et al.; Passive transfer of lymphocytes and sera from mice immunised using two different formulations containing recombinant protective antigen (rPA) have been used to further elucidate the mechanism of protection against Bacillus anthracis infection . The results demonstrated that an antibody response maybe important in protection against B . anthracis infection, under the conditions tested . The results provide further data for the development of an improved anthrax vaccine. J Med Microbiol, 2001 Aug, 50(8), 702 - 11 Susceptibility of irradiated mice to Bacillus anthracis sterne by the intratracheal route of infection; Brook I et al.; The susceptibility of sublethally irradiated mice to pulmonary infection with Bacillus anthracis was investigated in a mouse model . Female B6D2F1/J mice were challenged intratracheally with 4.3 x 10(6), 3.7 x 10(7) and 4.4 x 10(8) cfu of B . anthracis Sterne spores 4 days after 60Co gamma-radiation at a dose of 0, 1, 2, 3, 4, 5, 6 or 7 Gy . Bacterial cultures were obtained from lung, spleen homogenates and heart blood . A biphasic mode of mortality was observed, with a constant response of up to 3 or 4 Gy (up to 18% mortality), after which a sharp increase in mortality occurred (up to 100%) . When irradiation was delayed beyond 15 days after inoculation, the susceptibility to B . anthracis infection and subsequent mortality disappeared . B . anthracis was recovered from the organs and blood of up to 89% of the animals . However, organisms of enteric origin were also isolated mixed with B . anthracis from up to 36% of the animals exposed to 3, 5 or 7 Gy . Inoculation of B . anthracis delta-Sterne-1 that lacks lethal toxin and oedema toxin also induced infection with B . anthracis, but not translocation of enteric micro-organisms . The synergic adverse effect of exposure to gamma-radiation followed by intratracheal challenge with B . anthracis was observed above 4 Gy . The lethal toxin of B . anthracis may enhance the emergence of polymicrobial infection with B . anthracis and enteric micro-organisms. Appl Environ Microbiol, 2001 Aug, 67(8), 3720 - 7 Utilization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis; Qi Y et al.; The potential use of Bacillus anthracis as a weapon of mass destruction poses a threat to humans, domesticated animals, and wildlife and necessitates the need for a rapid and highly specific detection assay . We have developed a real-time PCR-based assay for the specific detection of B . anthracis by taking advantage of the unique nucleotide sequence of the B . anthracis rpoB gene . Variable region 1 of the rpoB gene was sequenced from 36 Bacillus strains, including 16 B . anthracis strains and 20 other related bacilli, and four nucleotides specific for B . anthracis were identified . PCR primers were selected so that two B . anthracis-specific nucleotides were at their 3' ends, whereas the remaining bases were specific to the probe region . This format permitted the PCR reactions to be performed on a LightCycler via fluorescence resonance energy transfer (FRET) . The assay was found to be specific for 144 B . anthracis strains from different geographical locations and did not cross-react with other related bacilli (175 strains), with the exception of one strain . The PCR assay can be performed on isolated DNA as well as crude vegetative cell lysates in less than 1 h . Therefore, the rpoB-FRET assay could be used as a new chromosomal marker for rapid detection of B . anthracis. Appl Environ Microbiol, 2001 Aug, 67(8), 3665 - 70 Bacillus spore inactivation methods affect detection assays; Dang JL et al.; Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism . One great concern is the detection of Bacillus anthracis, the causative agent of anthrax . Assays for detection in the laboratory often employ inactivated preparations of spores or nonpathogenic simulants . This study uses several common biodetection platforms to detect B . anthracis spores that have been inactivated by two methods and compares those data to detection of spores that have not been inactivated . The data demonstrate that inactivation methods can affect the sensitivity of nucleic acid- and antibody-based assays for the detection of B . anthracis spores . These effects should be taken into consideration when comparing laboratory results to data collected and assayed during field deployment. Lett Appl Microbiol, 2001 Aug, 33(2), 100 - 5 Evaluation of spore extraction and purification methods for selective recovery of viable Bacillus anthracis spores; Dragon DC et al.; AIMS: To investigate methods of improving anthrax spore detection with PLET . METHODS AND RESULTS: Comparisons were made of PLET and blood-supplemented PLET to recover and distinguish spores of a variety of Bacillus species . Heat and ethanol purification of spores, and spore extraction from soil with water and high specific gravity sucrose plus non-ionic detergent, were also carried out . CONCLUSION: PLET was more selective and suitable than blood-supplemented PLET for detection of anthrax spores in the environmental specimens . However, PLET is not an optimal spore recovery medium . Purification of spores with ethanol was as effective as heat purification . High specific gravity sucrose plus detergent extraction solutions may be more sensitive than extraction with water . SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights shortcomings with the standard PLET isolation of anthrax spores and describes ways in which the procedure may be improved. Biochem Biophys Res Commun, 2001 Jul 27, 285(4), 1025 - 33 Enhanced expression of the recombinant lethal factor of Bacillus anthracis by Fed-Batch culture; Gupta P et al.; High cell density cultivation has been one of the most effective ways to increase cell as well as the product yields . The structural gene for the 90-kDa lethal factor (LF) isolated from Bacillus anthracis was expressed as fusion protein with 6x histidine residues under the transcriptional regulation of the T5 promoter in Escherichia coli . Various strategies were tried to scale up the expression of the recombinant lethal factor by bioprocess optimization using fed batch culture technique in a 14 litre fermentor . The media, a defined mixture of salts, trace elements, vitamins, etc . along with a specified carbon source was used for the growth . The pH of the media was maintained at 6.8 while the temperature was changed from 37 to 28 degrees C during the cultivation . During the growth and induction phases, the DO was maintained above 20% by automatic control of agitation . The specific growth rate was controlled by utilizing an exponential feeding profile determined from mass balance equations . As a result of control of specific growth rate at two different levels, there was about twenty five fold increase in biomass compared to the biomass in the shake flask . E . coli cells yielded a soluble cytosolic protein with an apparent molecular mass of 90 kDa . The protein was purified to homogeneity using metal chelate affinity chromatography, followed by anion exchange on FPLC using Mono-Q column . In solution, trypsin cleaved protective antigen bound to native and recombinant LF with comparable affinity . The recombinant LF resembled the LF purified from B . anthracis in the macrophage lysis assay, using a murine macrophage cell line J774A.1 sensitive to anthrax toxin . It was possible to achieve a yield of 50 mg of the purified protein from 1 litre culture broth . Vaccine, 2001 Jul 20, 19(30), 4214 - 8 Detection of anthrax vaccine virulence factors by polymerase chain reaction; Fasanella A et al.; In Italy, an attenuated Bacillus anthracis strain, named 'Carbosap', is used for immunization against ovine and bovine anthrax . Analysis on 'Carbosap', Sterne vaccine strain F34 and Pasteur vaccine strain SS104, were performed using primers specific for the sequences, encoding the toxic factors, located on plasmids pXO1 and pXO2 and primers specific for the chromosome . The results obtained from polymerase chain reaction (PCR) assay revealed the presence of both plasmids pXO1 and pXO2 in 'Carbosap' strain . This study showed that the 'Carbosap' vaccine strain has a different plasmid pattern in comparison to Pasteur vaccine strain SS104 and Sterne vaccine strain F34. Appl Environ Microbiol, 2001 Jul, 67(7), 3021 - 8 Use of long-range repetitive element polymorphism-PCR to differentiate Bacillus anthracis strains; Brumlik MJ et al.; The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level . Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B . anthracis strains . A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb . We were able to characterize five genetically distinct groups by examining 105 B . anthracis strains of diverse geographical origins . All B . anthracis strains produced fingerprints comprising seven to eight bands, referred to as "skeleton" bands, while one to three "diagnostic" bands differentiated between B . anthracis strains . LR REP-PCR fingerprints of B . anthracis strains showed very little in common with those of other closely related species such as B . cereus, B . thuringiensis, and B . mycoides, suggesting relative heterogeneity among the non-B . anthracis strains . Fingerprints from transitional non-B . anthracis strains, which possessed the B . anthracis chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct B . anthracis groups that we have identified . The LR REP-PCR method described in this report provides a simple means of differentiating B . anthracis strains. Cutis, 2001 Jun, 67(6), 488 - 92 Cutaneous anthrax in eastern Turkey; Caksen H et al.; Anthrax, caused by the spore-forming bacterium Bacillus anthracis, is rarely seen in industrial nations but is common in developing countries . Cutaneous anthrax (CA), the most common form of the disease, accounts for 95% of cases and usually develops on exposed sites . This study reviews the clinical and laboratory findings of 21 patients diagnosed with CA during 2 separate epidemics in the Van region of Turkey . All patients had a history of direct contact with infected cattle . The patients, aged 1.5 to 64 years, included 13 females and 8 males . Of the patients, 9 were 15 years or younger . Skin lesions were localized on the hands and fingers in 15 patients, on the face in 3 patients, on the face and finger in 1 patient, on the chest and finger in 1 patient, and on the eyelid in 1 patient . Gram-positive bacillus were noted on Gram stains of material obtained from skin lesions in 2 patients . All but one patient was successfully treated with penicillin; the unresponsive patient was treated with cefuroxime and required plastic reconstructive surgery because of a skin defect on the eyelid. Infect Immun, 2001 Jul, 69(7), 4509 - 15 Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein; Price BM et al.; The ability of genetic vaccination to protect against a lethal challenge of anthrax toxin was evaluated . BALB/c mice were immunized via gene gun inoculation with eucaryotic expression vector plasmids encoding either a fragment of the protective antigen (PA) or a fragment of lethal factor (LF) . Plasmid pCLF4 contains the N-terminal region (amino acids {aa} 10 to 254) of Bacillus anthracis LF cloned into the pCI expression plasmid . Plasmid pCPA contains a biologically active portion (aa 175 to 764) of B . anthracis PA cloned into the pCI expression vector . One-micrometer-diameter gold particles were coated with plasmid pCLF4 or pCPA or a 1:1 mixture of both and injected into mice via gene gun (1 microg of plasmid DNA/injection) three times at 2-week intervals . Sera were collected and analyzed for antibody titer as well as antibody isotype . Significantly, titers of antibody to both PA and LF from mice immunized with the combination of pCPA and pCLF4 were four to five times greater than titers from mice immunized with either gene alone . Two weeks following the third and final plasmid DNA boost, all mice were challenged with 5 50% lethal doses of lethal toxin (PA plus LF) injected intravenously into the tail vein . All mice immunized with pCLF4, pCPA, or the combination of both survived the challenge, whereas all unimmunized mice did not survive . These results demonstrate that DNA-based immunization alone can provide protection against a lethal toxin challenge and that DNA immunization against the LF antigen alone provides complete protection. Biochem Biophys Res Commun, 2001 Jun 15, 284(3), 568 - 73 Effect of pH on stability of anthrax lethal factor: correlation between denaturation and activity; Gupta P et al.; Anthrax is caused by Gram positive bacterium Bacillus anthracis . Pathogenesis is result of production of three protein components, protective antigen (PA), lethal factor (LF), and edema factor (EF) . PA in combination with LF (lethal toxin) is lethal to animals, while PA in combination with EF (edema toxin), causes edema . PA, LF, and EF are very thermolabile . Differential scanning calorimetry (DSC) was used to unravel the energetics of LF denaturation as a function of pH ranging from 7.8 to 5.5 . Transition temperature (T(m)) of LF was found to be approximately equal to 42 degrees C and onset of denaturation occurs at approximately equal to 30 degrees C . The ratio of calorimetric to van't Hoff's enthalpy was nearly equal to unity at pH 7.0, indicative of presence of single structural domain in LF at pH 7.0, unlike PA which has been structurally observed to consist of 4 domains . It was found by cytotoxicity studies using J774A.1 macrophage like cells that LF was most stable at pH approximately 6.5 . This paper reports for the first time the denaturation of LF at different pH values at 37 degrees C and tries to establish a correlation between denaturation and loss of LF activity at different pH values . Microbiology, 2001 Jun, 147(Pt 6), 1677 - 85 The role of antibodies to Bacillus anthracis and anthrax toxin components in inhibiting the early stages of infection by anthrax spores; Welkos S et al.; Vaccines which are efficacious against anthrax, such as the human vaccine, Anthrax Vaccine Absorbed (AVA), contain the protective antigen (PA) component of the anthrax toxins as the major protective immunogen . Although AVA protects against inhalational anthrax, the immune responses to and role in protection of PA and possibly other antigens have yet to be fully elucidated . Sera from animals immunized with a toxin-producing, unencapsulated live vaccine strain of Bacillus anthracis have been reported to have anti-spore activities associated with the antitoxin humoral response . The authors performed studies to determine whether anti-PA antibody (Ab)-containing preparations stimulated spore uptake by phagocytes and suppressed the germination of spores in vitro . AVA- and PA-immune sera from several species enhanced the phagocytosis by murine peritoneal macrophages of spores of the virulent Ames and the Sterne vaccine strains . Antitoxin Abs appeared to contribute significantly, although not solely, to the enhanced uptake . Rabbit antisera to PA purified from either Sterne or a PA-producing pX01-cured recombinant, affinity-purified anti-PA IgG, and monkey antisera to AVA were used to assess the role of anti-PA ABS: Rabbit anti-PA Abs promoted the uptake of spores of the PA-producing strains Sterne, Ames and RP42, a mutant of Sterne producing only PA, but not of the pX01-Sterne-1 strain, Ames strain, or RP4, a mutant of Sterne with deletions in the loci encoding PA and the oedema factor (EF) toxin component and producing only the lethal factor toxin component . Rabbit anti-PA and monkey anti-AVA Abs also significantly inhibited spore germination in vitro compared to preimmune serum or medium . Spore-associated proteins recognized by anti-PA Abs were detected by electron microscopy and confirmed by immunoblotting of spore coat extracts . Thus, the anti-PA Ab-specific immunity induced by AVA has anti-spore activity and might have a role in impeding the early stages of infection with B . anthracis spores. Mt Sinai J Med, 2001 May, 68(3), 213 - 5 Toxemic shock, hematuria, hypokalemia, and hypoproteinemia in a case of cutaneous anthrax; Khajehdehi P; A 20-year-old woman who had daily contact with domestic herbivores presented with a painless and pruritic lesion in her neck; the lesion ulcerated to a black necrotic eschar from which Bacillus anthracis grew . Rapidly expanding edema at the site of the ulcer was followed by shock, hematuria, hypokalemia, and hypoproteinemia . The latter symptoms - unusual for cutaneous anthrax - responded to intravenous penicillin therapy. Mod Pathol, 2001 May, 14(5), 482 - 95 Quantitative pathology of inhalational anthrax I: quantitative microscopic findings; Grinberg LM et al.; Forty-one cases of documented inhalational anthrax from the Sverdlovsk epidemic of 1979 traced to release of aerosols of Bacillus anthracis at a secret biologic-agent production facility were evaluated by semiquantitative histopathologic analysis of tissue concentrations of organisms, inflammation, hemorrhage, and other lesions in the mediastinum, mediastinal lymph nodes, bronchi, lungs, heart, spleen, liver, intestines, kidneys, adrenal glands, and central nervous system . These data were correlated with clinical, epidemiologic, and demographic data . The patients' courses, with a variable incubation period and short nonspecific course (4 days before hospitalization) with rapid demise (1 day of hospitalization before death), correlated with systemic bacterial infection and lesions . Bacillus anthracis were identified in all cases in which there was no antibiotic treatment or there was treatment for fewer than 21 hours . The lesions that were the most severe and apparently of longest duration were in the mediastinal lymph nodes and mediastinum . There and elsewhere, peripheral transudate surrounded fibrin-rich edema; necrosis of arteries and veins was the most likely source of large hemorrhages displacing tissue or infiltrating tissue, respectively; and apoptosis of lymphocytes was observed . Respiratory function was compromised by mediastinal expansion, large pleural effusions, and hematogenous and retrograde lymphatic vessel spread of B . anthracis to the lung with consequent pneumonia . The central nervous system and intestines manifested similar hematogenous spread, vasculitis, hemorrhages, and edema . These pathologic findings are consistent with previous experimental studies showing transport of inhaled spores to mediastinal lymph nodes, where germination and growth lead to local lesions and systemic spread, with resulting edema and cell death, owing to the effects of edema toxin and lethal toxin . The identification of the vascular lesions as a basis for the prominent hemorrhages is a novel observation for human inhalational anthrax. Biochem Biophys Res Commun, 2001 May 4, 283(2), 308 - 15 Constitutive expression of protective antigen gene of Bacillus anthracis in Escherichia coli; Chauhan V et al.; The fatal bacterial infection caused by inhalation of the Bacillus anthracis spores results from the synthesis of protein toxins-protective antigen (PA), lethal factor (LF), and edema factor (EF)--by the bacterium . PA is the target-cell binding protein and is common to the two effector molecules, LF and EF, which exert their toxic effects once they are translocated to the cytosol by PA . PA is the major component of vaccines against anthrax since it confers protective immunity . The large-scale production of recombinant protein-based anthrax vaccines requires overexpression of the PA protein . We have constitutively expressed the protective antigen protein in E . coli DH5alpha strain . We have found no increase in degradation of PA when the protein is constitutively expressed and no plasmid instability was observed inside the expressing cells . We have also scaled up the expression by bioprocess optimization using batch culture technique in a fermentor . The protein was purified using metal-chelate affinity chromatography . Approximately 125 mg of recombinant protective antigen (rPA) protein was obtained per liter of batch culture . It was found to be biologically and functionally fully active in comparison to PA protein from Bacillus anthracis . This is the first report of constitutive overexpression of protective antigen gene in E . coli. J Toxicol Clin Toxicol, 2001, 39(1), 85 - 100 Use of anthrax vaccine in the United States: recommendations of the Advisory Committee on Immunization Practices; Delayed life-threatening reaction to anthrax vaccine; Pittsburgh Poison Center, Children's Hospital of Pittsburgh, Pennsylvania 15213, USABACKGROUND: Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis . Due to the current world threat of unpredictable biological terrorism, the Department of Defense has mandated the systematic vaccination of all US military personnel against this warfare agent . Many may experience al mild flu-like illness and soreness at the injection site, but systemic reactions are rare . CASE REPORT: We report a delayed and potentially serious life-threatening adverse reaction to anthrax vaccine . A previously healthy 34-year-old male was transported to the emergency department with dyspnea, diaphoresis, pallor, and urticarial wheals on his face, arms, and torso after the administration of the third dose of anthrax vaccine . All symptoms resolved after pharmacological intervention and the patient was discharged . Pharmaco-epidemiological data indicate that 30% of anthrax vaccine recipients experience mild local reactions . With large numbers of military personnel being vaccinated, emergency physicians may encounter more vaccine-related adverse reactions. Microbiology, 2001 May, 147(Pt 5), 1343 - 51 A general strategy for identification of S-layer genes in the Bacillus cereus group: molecular characterization of such a gene in Bacillus thuringiensis subsp . galleriae NRRL 4045; Mesnage S et al.; Despite its possible role in virulence, there has been little molecular characterization of members of the S-layer protein family of the Bacillus cereus group . It is hypothesized that the components of the S-layers are likely to display similar anchoring structures in Bacillus thuringiensis and Bacillus anthracis . Based on inferred sequence similarities, a DNA fragment encoding the cell-wall-anchoring domain of an S-layer component of BAC: thuringiensis subsp . galleriae NRRL 4045 was isolated . The complete gene was identified and sequenced . An ORF of 2463 nt was identified, which was predicted to encode a protein of 821 aa, SlpA . The mature SlpA protein (792 residues) carries three S-layer homology (SLH) motifs towards its amino terminus, each about 50 aa long . These motifs were sufficient to bind Bac . thuringiensis and Bac . anthracis cell walls in vitro by interacting with peptidoglycan-associated polymers, confirming a common wall-anchoring mechanism . The slpA null-allele mutant was constructed and shown to possess no other abundant surface protein . It is proposed that the method described in this paper could be applied to the identification and deletion of any Bac . cereus S-layer gene and is of great value for the molecular and functional characterization of these genes. Genomics, 2001 Apr 15, 73(2), 223 - 31 Genetic, physical, and transcript map of the Ltxs1 region of mouse chromosome 11; Watters JW et al.; Lethal factor (LF) is a toxin secreted by Bacillus anthracis that plays an important role in the pathogenesis of anthrax . Intoxication with LF results in a macrophage-specific cytolysis that is not well understood . Interestingly, inbred mouse strains exhibit dramatic differences in the susceptibility of their cultured macrophages to killing by LF, and a gene that influences this phenotype, called Ltxs1, has been mapped to mouse chromosome 11 . Here we report a high-resolution genetic map that confines the Ltxs1 region to a 0.51-cM interval between D11Mit90 and D11Die37/D11Die38 . We have also constructed a complete physical map of YAC and BAC clones covering the Ltxs1 region . In conjunction with synteny homology searching, BLAST searches of sequences obtained from the clones in the physical map have revealed 14 known genes and five ESTs that reside in the critical interval . Additionally, a region of 100 kb or more is deleted in the Ltxs1 interval of some strains . Our genetic, physical, and transcript map provides an important resource for the molecular cloning of Ltxs1 . Vaccine, 2001 Apr 30, 19(23-24), 3241 - 7 Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin; Fellows PF et al.; The efficacy of a licensed human anthrax vaccine (Anthrax Vaccine Adsorbed (AVA)) was tested in guinea pigs, rabbits, and rhesus macaques against spore challenge by Bacillus anthracis isolates of diverse geographical origin . Initially, groups of Hartley guinea pigs were vaccinated at 0 and 4 weeks with AVA, then challenged intramuscularly at 10 weeks with spores from 33 isolates of B . anthracis . Survival among the vaccinated groups varied from 6 to 100%, although there were no differences in mean time to death among the groups . There was no correlation between isolate virulence and variable number tandem repeat category or protective antigen genotype identified . New Zealand white rabbits were then vaccinated with AVA at 0 and 4 weeks, and challenged at 10 weeks by aerosol with spores from six of the isolates that were highly virulent in vaccinated guinea pigs . AVA completely protected the rabbits from four of the isolates, and protected 90% of the animals from the other two isolates . Subsequently, two of these six isolates were then used to challenge rhesus macaques, previously vaccinated with AVA at 0 and 4 weeks, and challenged at 10 weeks by aerosol . AVA protected 80 and 100% of the animals from these two isolates . These studies demonstrated that, although AVA confers variable protection against different B . anthracis isolates in guinea pigs, it is highly protective against these same isolates in both rabbits and rhesus macaques. BMC Microbiol . 2001;1(1):2 . Epub 2001 Mar 30. A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis; Le Fleche P et al.; BACKGROUND: Some pathogenic bacteria are genetically very homogeneous, making strain discrimination difficult . In the last few years, tandem repeats have been increasingly recognized as markers of choice for genotyping a number of pathogens . The rapid evolution of these structures appears to contribute to the phenotypic flexibility of pathogens . The availability of whole-genome sequences has opened the way to the systematic evaluation of tandem repeats diversity and application to epidemiological studies . RESULTS: This report presents a database of tandem repeats from publicly available bacterial genomes which facilitates the identification and selection of tandem repeats . We illustrate the use of this database by the characterization of minisatellites from two important human pathogens, Yersinia pestis and Bacillus anthracis . In order to avoid simple sequence contingency loci which may be of limited value as epidemiological markers, and to provide genotyping tools amenable to ordinary agarose gel electrophoresis, only tandem repeats with repeat units at least 9 bp long were evaluated . Yersinia pestis contains 64 such minisatellites in which the unit is repeated at least 7 times . An additional collection of 12 loci with at least 6 units, and a high internal conservation were also evaluated . Forty-nine are polymorphic among five Yersinia strains (twenty-five among three Y . pestis strains) . Bacillus anthracis contains 30 comparable structures in which the unit is repeated at least 10 times . Half of these tandem repeats show polymorphism among the strains tested . CONCLUSIONS: Analysis of the currently available bacterial genome sequences classifies Bacillus anthracis and Yersinia pestis as having an average (approximately 30 per Mb) density of tandem repeat arrays longer than 100 bp when compared to the other bacterial genomes analysed to date . In both cases, testing a fraction of these sequences for polymorphism was sufficient to quickly develop a set of more than fifteen informative markers, some of which show a very high degree of polymorphism . In one instance, the polymorphism information content index reaches 0.82 with allele length covering a wide size range (600-1950 bp), and nine alleles resolved in the small number of independent Bacillus anthracis strains typed here. Med Dosw Mikrobiol, 2000, 52(4), 383 - 8 {Survival of Bacillus anthracis spores in various tannery baths}; Mendrycka M et al.; The influence of tannery baths: liming, deliming, bating, pickling, tanning, retannage on the survival and on the germination dynamism of B . anthracis spores (Sterne strain) was investigated . The periods and the conditions of this influence were established according to technological process of cow hide tannage . Practically after every bath some part of the spores remained vital . The most effective killing of spores occurred after pickling, liming and deliming . Inversely, the most viable spores remained after bating and retannage process . The lack of correlation that was observed between survival and germination of spores after retannage bath can be explained by different mechanism of spores germination inhibition and their killing. J Biol Chem, 2001 Jun 22, 276(25), 22090 - 4 Epub 2001 Mar 16. A dominant negative mutant of Bacillus anthracis protective antigen inhibits anthrax toxin action in vivo; Singh Y et al.; PA63, a proteolytically activated 63-kDa form of anthrax protective antigen (PA), forms heptameric oligomers and has the ability to bind and translocate the catalytic moieties, lethal factor (LF), and edema factor (EF) into the cytosol of mammalian cells . Acidic pH triggers oligomerization and membrane insertion by PA63 . A disordered amphipathic loop in domain II of PA (2beta2-2beta3 loop) is involved in membrane insertion by PA63 . Because conditions required for membrane insertion coincide with those for oligomerization of PA63 in mammalian cells, residues constituting the 2beta2-2beta3 loop were replaced with the residues of the amphipathic membrane-inserting loop of its homologue iota-b toxin secreted by Clostridium perfringens . It was hypothesized that such a molecule might assemble into hetero-heptameric structures with wild-type PA ultimately leading to the inhibition of cellular intoxication . The mutation blocked the ability of PA to mediate membrane insertion and translocation of LF into the cytosol but had no effect on proteolytic activation, oligomerization, or binding LF . Moreover, an equimolar mixture of purified mutant PA (PA-I) and wild-type PA showed complete inhibition of toxin activity both in vitro on J774A.1 cells and in vivo in Fischer 344 rats thereby exhibiting a dominant negative effect . In addition, PA-I inhibited the channel-forming ability of wild-type PA on the plasma membrane of CHO-K1 cells thereby indicating protein-protein interactions between the two proteins resulting in the formation of mixed oligomers with defective functional activity . Our findings provide a basis for understanding the mechanism of translocation and exploring the possibility of the use of this PA molecule as a therapeutic agent against anthrax toxin action in vivo. Lett Appl Microbiol, 2001 Mar, 32(3), 139 - 45 Molecular characterization of Bacillus anthracis using multiplex PCR, ERIC-PCR and RAPD; Shangkuan YH et al.; AIMS: To investigate the molecular characterization of Bacillus anthracis strains by multiplex PCR, enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and random amplification of polymorphic DNA (RAPD) . METHODS AND RESULTS: Three primers were used to amplify the cya, cap and cereolysinAB genes in the multiplex PCR . Two distinct ERIC-PCR and RAPD fragments, which separated B . anthracis into two groups, were used as probes in Southern hybridization experiments . The probes hybridized only to the cya+ B . anthracis strains identified by the multiplex PCR . Nucleotide sequence analysis of the two cloned fragments showed they were from the pXO1 plasmid of B . anthracis . CONCLUSION: Multiplex PCR simultaneously identified isolates of the Bacillus cereus group and the B . anthracis virulence factors . ERIC-PCR and RAPD, combined with the Southern hybridization analyses, differentiated B . anthracis strains and separated them from the closely related B . cereus group bacteria . SIGNIFICANCE AND IMPACT OF THE STUDY: ERIC-PCR and RAPD assay could be effective in differentiating virulent from avirulent B . anthracis . Our results also show that the amplification of the large plasmids was allowed in the ERIC-PCR and RAPD assay. Proc Natl Acad Sci U S A, 2001 Mar 27, 98(7), 4089 - 94 Epub 2001 Mar 20. Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways; Duesbery NS et al.; Lethal factor is a protease, one component of Bacillus anthracis exotoxin, which cleaves many of the mitogen-activated protein kinase kinases (MEKs) . Given the importance of MEK signaling in tumorigenesis, we assessed the effects of anthrax lethal toxin (LeTx) on tumor cells . LeTx was very effective in inhibiting mitogen-activated protein kinase activation in V12 H-ras-transformed NIH 3T3 cells . In vitro, treatment of transformed cells with LeTx caused them to revert to a nontransformed morphology, and inhibited their abilities to form colonies in soft agar and to invade Matrigel without markedly affecting cell proliferation . In vivo, LeTx inhibited growth of ras-transformed cells implanted in athymic nude mice (in some cases causing tumor regression) at concentrations that caused no apparent animal toxicity . Unexpectedly, LeTx also greatly decreased tumor neovascularization . These results demonstrate that LeTx potently inhibits ras-mediated tumor growth and is a potential antitumor therapeutic. J Food Prot, 2001 Mar, 64(3), 348 - 54 Rapid identification of Bacillus cereus based on the detection of a 28.5-kilodalton cell surface antigen; Chen CH et al.; Conventional procedures for the identification of suspect Bacillus cereus isolated on mannitol-egg yolk-polymyxin (MYP) agar may need several days . To facilitate the identification of the bacterium, an enzyme-linked immunosorbent assay (ELISA) was developed . The assay was based on the detection of a 28.5-kDa cell surface antigen of B . cereus . Bacterial colonies grown on MYP agar or nutrient agar were suspended in phosphate-buffered saline (pH 7.2) containing 0.1% Teepol . The cell suspensions were heated at 100 degrees C for 5 min and added to the microtiter plates coated with antibodies against the 28.5-kDa antigen . After washing, the same antibodies labeled with horseradish peroxidase were used as secondary antibodies to reveal the signal of antigen-antibody reaction . For 38 strains of B . cereus and 127 strains of non-B . cereus bacteria (including 79 isolates of Bacillus spp.) tested, the sensitivity and specificity of the ELISA were 100 and 88.2%, respectively . Strains producing false-positive results were members of the B . cereus group (i.e., Bacillus anthracis, Bacillus mycoides, and Bacillus thuringiensis), which are genetically and biochemically similar to B . cereus . Similar ELISA results were obtained by using antibodies against another cell surface antigen with a molecular mass of 20 kDa . If members of the B . cereus group were recognized as a single species, the sensitivity and specificity of the ELISA were 100 and 99.1%, respectively . The ELISA could be used as a rapid method for presumptive identification of B . cereus grown on MYP agar. Diagn Microbiol Infect Dis, 2001 Feb, 39(2), 77 - 83 Rapid identification of pathogenic bacteria by single-enzyme amplified fragment length polymorphism analysis; Velappan N et al.; Despite major progress in their treatment and prevention, bacterial infections remain a significant cause of morbidity and mortality worldwide . In responding to a disease outbreak, rapid and accurate identification of the bacterial species involved is of paramount importance . Strain level discrimination is desirable to allow selection of treatment modalities, and in the case of a deliberate release, for identification of the source . Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis was used to perform species and strain identification of subgroup I Bacilli, Yersinia, Staphylococci and Escherichia coli . By careful selection of AFLP primers, it was possible to obtain reproducible and sensitive identification to strain level, even within the highly monomorphic species Bacillus anthracis . SE-AFLP fragments can be analyzed using standard gel electrophoresis, and can be easily scored by visual inspection, due to the low complexity of the fingerprint obtained by this method . These features make SE-AFLP suitable for use in either field or laboratory applications. Ann N Y Acad Sci, 2000, 916, 240 - 52 Rapid recovery and identification of anthrax bacteria from the environment; Kiel JL et al.; Bacillus anthracis has been recognized as a highly likely biological warfare or terrorist agent . We have designed culture techniques to rapidly isolate and identify "live" anthrax from suspected environmental release . A special medium (3AT medium) allows for discrimination between closely related bacilli and non-pathogenic strains . Nitrate was found to be a primary factor influencing spore formation in Bacillus anthracis . Nitrate reduction in anthrax is not an adaptation to saprophytic environmental existence, but it is a signal to enhance environmental survival upon the death of the anthrax host, which can be mimicked in culture. Mol Gen Mikrobiol Virusol, 2000, (4), 12 - 5 {study of the virulence of a recombinant strain of Bacillus anthracis, obtained as a result of transductive transfer of chromosomal genes from a strain of Bacillus cereus}; Mikshis NI et al.; Auxotrophic markers of B . anthracis strains differing them from other Bacillus representatives have been determined . Chromosome genes from prototrophic B . cereus strain were transduced into auxotrophic B . anthracis strain . The properties of transductants were studied in order to establish common transfer of chromosomal determinants responsible for realization of various signs . Transduction mating between species resulted in construction of prototroph B . anthracis strains (pX01- pX02+), whose derivatives are characterized by decreased virulence for laboratory animals. Cell Microbiol, 2000 Dec, 2(6), 453 - 63 Early Bacillus anthracis-macrophage interactions: intracellular survival survival and escape; Dixon TC et al.; This study describes early intracellular events occurring during the establishment phase of Bacillus anthracis infections . Anthrax infections are initiated by dormant endospores gaining access to the mammalian host and becoming engulfed by regional macrophages (Mphi) . During systemic anthrax, late stage events include vegetative growth in the blood to very high titres and the synthesis of the anthrax exotoxin complex, which causes disease symptoms and death . Experiments focus on the early events occurring during the first few hours of the B . anthracis infectious cycle, from endospore germination up to and including release of the vegetative cell from phagocytes . We found that newly vegetative bacilli escape from the phagocytic vesicles of cultured Mphi and replicate within the cytoplasm of these cells . Release from the Mphi occurs 4-6 h after endospore phagocytosis, timing that correlates with anthrax infection of test animals . Genetic analysis from this study indicates that the toxin plasmid pXO1 is required for release from the Mphi, whereas the capsule plasmid pXO2 is not . The transactivator atxA, located on pXO1, is also found to be essential for release, but the toxin genes themselves are not required . This suggests that Mphi release of anthrax bacilli is atxA regulated . The putative 'escape' genes may be located on the chromosome and/or on pXO1. Cell Microbiol, 2000 Jun, 2(3), 259 - 64 Translocation of Bacillus anthracis lethal and oedema factors across endosome membranes; Guidi-Rontani C et al.; The two exotoxins of Bacillus anthracis, the causative agent of anthrax, are the oedema toxin (PA-EF) and the lethal toxin (PA-LF) . They exert their catalytic activities within the cytosol . The internalization process requires receptor-mediated endocytosis and passage through acidic vesicles . We investigated the translocation of EF and LF enzymatic moieties across the target cell membrane . By selective permeabilization of the plasma membrane with Clostridium perfringens delta-toxin, we observed free full-size lethal factor (LF) within the cytosol, resulting from specific translocation from early endosomes . In contrast, oedema factor (EF) remained associated with the membranes of vesicles. Curr Opin Microbiol, 2001 Feb, 4(1), 78 - 81 Bacillus anthracis, a bug with attitude! Baillie L, Read TD. The sequencing of the Bacillus anthracis genome and virulence plasmids represents the greatest advance in anthrax research in the past 100 years . The data will provide the foundation of all future work on this organism and will be invaluable to researchers in their battle to understand the basis of the host-microbe interaction. Biochem Biophys Res Commun, 2001 Jan 12, 280(1), 158 - 63 Involvement of residues 147VYYEIGK153 in binding of lethal factor to protective antigen of Bacillus anthracis; Gupta P et al.; Anthrax toxin is a complex of protective antigen (PA, 735 aa), lethal factor (LF, 776 aa), and edema factor (EF, 767 aa) . PA binds to cell surface receptors and is cleaved by cell surface proteases into PA63, while LF and EF compete for binding to PA63 . The PA63-LF/EF complex is internalized into the cytosol and causes different pathogenic responses in animals and cultured cells . 1-300 amino acid residues of LF have been viewed as the region responsible for the high affinity binding of LF to PA . Amino acid analysis of LF and EF revealed a common stretch of 7 amino acids (147VYYEIGK153) . In the present study, each amino acid of this stretch was replaced by alanine at a time . Y148A, Y149A, I151A, and K153A mutants were found to be deficient in their ability to lyse J774A.1 cells and their binding ability to PA63 was drastically reduced . We propose that these four amino acids play a crucial role in the process of binding of LF to PA63 . J Invertebr Pathol, 2001 Jan, 77(1), 13 - 21 The mammalian safety of Bacillus thuringiensis-based insecticides; Siegel JP; The United States Environmental Protection Agency between the years 1961 and 1995 registered 177 products containing viable Bacillus thuringiensis (Bt) . Numerous laboratory studies have demonstrated that Bt and Bt products are noninfectious and are toxic to mammals only at a dose > or =10(8) colony forming units (cfu) per mouse (a human equivalent based on the weight of >10(11) cfu) . In contrast, as few as three vegetative cells of Bacillus anthracis can kill mice (a human equivalent of >10(3) cfu) . There are only two literature reports of Bt infection in man between the year 1997 and the present, and all infected individuals had experienced either extensive burns or a blast injury, which predisposed them to infection . Two epidemiology studies conducted during large-scale aerial Bt serovar kurstaki spray campaigns reported no increased incidence of illness . Some recent papers have expressed concern about the production of Bacillus cereus enterotoxins by Bt isolates . Laboratory studies found no evidence of illness in rats and sheep fed Bt products, nor have epidemiology studies found increased incidence of diarrhea during Bt aerial spray campaigns . Increases in human antibody levels following exposure to Bt products have been reported but there was no increased incidence in asthma or other illness . Based on laboratory studies and field experience, Bt insecticides have an excellent safety record. Infect Immun, 2001 Feb, 69(2), 1175 - 7 Macrophage-derived cell lines do not express proinflammatory cytokines after exposure to Bacillus anthracis lethal toxin; Erwin JL et al.; We present evidence that Bacillus anthracis lethal toxin (LT) suppresses rather than induces proinflammatory cytokine production in macrophages . Suppression is observed with extremely low levels of LT and involves inhibition of transcription of cytokine messenger RNA . Thus, LT may contribute to anthrax pathogenesis by suppressing the inflammatory response. MMWR Recomm Rep, 2000 Dec 15, 49(RR-15), 1 - 20 Use of anthrax vaccine in the United States; Advisory Committee on Immunization Practices; These recommendations concern the use of aluminum hydroxide adsorbed cell-free anthrax vaccine (Anthrax Vaccine Adsorbed {AVA}, BioPort Corporation, Lansing, MI) in the United States for protection against disease caused by Bacillus anthracis . In addition, information is included regarding the use of chemoprophylaxis against B . anthracis. Int J Med Microbiol, 2000 Oct, 290(4-5), 421 - 7 Lethal factor of Bacillus anthracis cleaves the N-terminus of MAPKKs: analysis of the intracellular consequences in macrophages; Pellizzari R et al.; The lethal toxin of Bacillus anthracis consists of two proteins, PA and LF, which together induce lethal effects in some animal species and cause macrophage lysis . LF is a zinc-binding protein with metalloprotease activity . With a two-hybrid system approach we identified MAP kinase kinases (MAPKKs) Mekl and Mek2 as proteins interacting with LF . LF was shown to cleave Mek1 and Mek2 and an additional MAPKK family member MKK3, within their N-terminal region . We examined macrophage cell lines and primary peritoneal cells with different sensitivities to LF but did not find a direct correlation between MAPKKs cleavage and cell death . On the other hand, sublytic doses of LF cleave MAPKKs and cause a reduction in the LPS/IFNgamma-induced production of proinflammatory mediators . These findings are discussed with respect to the possible role of LF in the initial phase of infection. Int J Med Microbiol, 2000 Oct, 290(4-5), 313 - 6 Characterization of a plasmid region involved in Bacillus anthracis toxin production and pathogenesis; Sirard JC et al.; The germination of spores within the host is the initial step of anthrax infection . We have shown, using immunofluorescence staining, confocal scanning laser microscopy and image cytometry analysis, that the alveolar macrophage is the primary site of B . anthracis germination in a murine inhalation infection model . B . anthracis germinated inside macrophages, in vesicles derived from the phagosomal compartment . We have demonstrated that the toxin genes and their trans-activator, AtxA, are expressed within the macrophages after germination . It was also shown that the pXO1 plasmid strongly enhanced capsule formation and that this influence is mediated by AtxA . This indicates the existence of a regulon where AtxA is the regulatory protein acting on genes located on different plasmids . We identified a tricistronic germination operon gerX located between the pag and atxA genes on the 40-kb toxin-encoding fragment of pXO1 . Analysis of a gerX null mutant indicated that gerX-encoded proteins are involved in the virulence of B . anthracis. Biochem J, 2000 Dec 15, 352 Pt 3, 739 - 45 Susceptibility of mitogen-activated protein kinase kinase family members to proteolysis by anthrax lethal factor; Vitale G et al.; The lethal factor (LF) produced by toxigenic strains of Bacillus anthracis is a Zn(2+)-endopeptidase that cleaves the mitogen-activated protein kinase kinases (MAPKKs) MEK1, MEK2 and MKK3 . Using genetic and biochemical approaches, we have extended the study of LF proteolytic specificity to all known MAPKK family members and found that LF also cleaves MKK4, MKK6 and MKK7, but not MEK5 . The peptide bonds hydrolysed by LF within all MAPKKs were identified . Cleavage invariably occurs within the N-terminal proline-rich region preceding the kinase domain, thus disrupting a sequence involved in directing specific protein-protein interactions necessary for the assembly of signalling complexes . Alignment of the sequences flanking the site of cleavage reveals the occurrence of some consensus motifs: position P2 and P1' are occupied by hydrophobic residues and at least one basic residue is present between P4 and P7 . The implications of these findings for the biochemical activity and functional specificity of LF are discussed. Appl Environ Microbiol, 2000 Dec, 66(12), 5460 - 8 Homoduplex and heteroduplex polymorphisms of the amplified ribosomal 16S-23S internal transcribed spacers describe genetic relationships in the "Bacillus cereus group"; Daffonchio D et al.; Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis are closely related in phenotype and genotype, and their genetic relationship is still open to debate . The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to describe the genetic relationships within the "B . cereus group," advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacrylamide gel electrophoresis and silver staining . One hundred forty-one strains belonging to the six species were investigated, and 73 ITS-HHP pattern types were distinguished by MDE, a polyacrylamide matrix specifically designed to resolve heteroduplex and single-strand conformation polymorphisms . The discriminating bands were confirmed as ITS by Southern hybridization, and the homoduplex or heteroduplex nature was identified by single-stranded DNA mung bean nuclease digestion . Several of the ITS-HHP types corresponded to specific phenotypes such as B . anthracis or serotypes of B . thuringiensis . Unweighted pair group method arithmetic average cluster analysis revealed two main groups . One included B . mycoides, B . weihenstephanensis, and B . pseudomycoides . The second included B . cereus and B . thuringiensis, B . anthracis appeared as a lineage of B . cereus. Cytometry, 2000 Dec 1, 41(4), 237 - 44 The flow cytometry of Bacillus anthracis spores revisited; Stopa PJ; BACKGROUND: The potential use of Bacillus anthracis spores as a weapon of terror has rekindled interest in the rapid detection and identification of the spores of these bacteria . Prior efforts to utilize flow cytometry (FCM) for this purpose resulted in tedious and time-consuming protocols . Advances in rapid immunoassays suggest a reinvestigation of the use of FCM because this may allow for the development of a rapid and sensitive system for detection and/or identification of spores in suspect samples . METHODS: In this study, antiserum was raised in goats using three different strains of B . anthracis spores as the immunogen . The resultant antibodies were purified, labeled with fluorescein, and evaluated for use in an immunoassay on a Coulter Epics XL flow cytometer . In the protocol that was developed, fluorescein-labeled antibodies are simply mixed with the sample, allowed to incubate, and then analyzed on the flow cytometer . Washes and centrifugation were eliminated . RESULTS: The results showed that a rapid (5 min) and sensitive immunological analysis was feasible . The detection limit (approximately 10(3) colony-forming units {CFU}/ ml) varied with strain, but there was no difference in the detection limit between live and irradiated spores . In addition, the power of FCM was utilized to minimize false-positive reactions among similar species of Bacillus by placing constraints on scatter and fluorescence intensity . The data also suggest that scatter might be useful to determine spore viability . CONCLUSION: This study shows that FCM may be an effective platform on which to perform immunological analysis for the detection and/or presumptive identification of B . anthracis spores . Published 2000 Wiley-Liss, Inc. Lancet, 2000 Nov 4, 356(9241), 1574 - 5 Injectional anthrax in a heroin skin-popper; Ringertz SH et al.; Anthrax is rare in western Europe but may arise sporadically in people exposed to animal products from endemic areas . A heroin-injecting drug user presented with a severe soft-tissue infection at the injection site, septic shock, and meningitis . A gram-positive endospore-forming aerobic rod was isolated from the soft tissue and cerebrospinal fluid; confirmation of Bacillus anthracis was made by PCR . Since contaminated heroin was the probable source of infection, this case is of concern and warrants surveillance. Proc Natl Acad Sci U S A, 2000 Nov 7, 97(23), 12800 - 3 Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death; Raoult D et al.; Medieval Black Death is believed to have killed up to one-third of the Western European population during the 14th century . It was identified as plague at this time, but recently the causative organism was debated because no definitive evidence has been obtained to confirm the role of Yersinia pestis as the agent of plague . We obtained the teeth of a child and two adults from a 14th century grave in France, disrupted them to obtain the pulp, and applied the new "suicide PCR" protocol in which the primers are used only once . There were no positive controls: Neither Yersinia nor Yersinia DNA were introduced in the laboratory . A negative result is followed by a new test using other primers; a positive result is followed by sequencing . The second and third primer pair used, coding for a part of the pla gene, generated amplicons whose sequence confirmed that it was Y . pestis in 1 tooth from the child and 19/19 teeth from the adults . Negative controls were negative . Attempts to detect the putative alternative etiologic agents Bacillus anthracis and Rickettsia prowazekii failed . Suicide PCR avoids any risk of contamination as it uses a single-shot primer-its specificity is absolute . We believe that we can end the controversy: Medieval Black Death was plague. Acta Crystallogr D Biol Crystallogr, 2000 Nov, 56 ( Pt 11), 1449 - 51 Expression, crystallization and preliminary X-ray diffraction studies of recombinant Bacillus anthracis lethal factor; Bernardi L et al.; The lethal factor (LF) produced by Bacillus anthracis is a Zn(2+)-dependent endopeptidase which specifically cleaves the N-terminal tail of several MAP kinase kinases (MAPKKs) . The recombinant expression, purification and crystallization of LF and of an inactive mutant consisting of a single amino-acid substitution in the conserved catalytic site are reported here . Both proteins crystallize in the cubic space group I432. Cell Biol Toxicol, 2000, 16(3), 165 - 74 Dehydroepiandrosterone and melatonin prevent Bacillus anthracis lethal toxin-induced TNF production in macrophages; Shin S et al.; The lethal toxin of Bacillus anthracis, which is composed of two separate proteinaceous exotoxins, namely protective antigen and lethal factor, is central to the pathogenesis of anthrax . Low levels of this toxin are known to induce release of cytokines such as tumor necrosis factor alpha (TNF-alpha) . In the present study we investigated the effect of dehydroepiandrosterone (DHEA), melatonin (MLT), or DHEA + MLT on production of lethal toxin-induced TNF-alpha in mouse peritoneal macrophages . We found that treatment with DHEA significantly inhibited the TNF-alpha production caused by anthrax lethal toxin . Exposure of MLT to anthrax lethal toxin-treated macrophages also decreased the release of TNF-alpha to the extracellular medium as compared to the control . However, combined use of DHEA and MLT also inhibited TNF-alpha release, but not more than single therapies . These results suggest that DHEA and MLT may have a therapeutic role in reducing the increased cytokine production induced by anthrax lethal toxin. J Appl Microbiol, 2000 Sep, 89(3), 452 - 62 Comparison of PCR-RFLP, ribotyping and ERIC-PCR for typing Bacillus anthracis and Bacillus cereus strains; Shangkuan YH et al.; PCR-RFLP analysis of the vrrA gene and cerAB gene was used to investigate the genomic diversity in 21 strains of Bacillus anthracis and 28 strains of Bacillus cereus, and was compared with results obtained by ribotyping and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) analysis . VrrA-typing divided the B . anthracis into four groups . Except for one Pasteur vaccine strain, the vrrA PCR-RFLP profiles of the B . anthracis were separated into three groups, which were different from those of the B . cereus strains . Ribotyping separated the B . anthracis isolates into seven ribotypes, and a common fragment of an approximately 850 bp band from the ERIC-PCR fingerprints separated most B . anthracis strains into two groups . VrrA/cerAB PCR-RFLP, ribotyping and ERIC-PCR generated 18, 22 and 23 types, respectively, from B . cereus strains . The results suggest that a combination of all three methods provides a high resolution typing method for B . anthracis and B . cereus . Compared with ribotyping and ERIC-PCR, PCR-RFLP is simple to perform and has potential as a rapid method for typing and discriminating B . anthracis strains from other B . cereus group bacteria. J Clin Microbiol, 2000 Oct, 38(10), 3780 - 4 Bacillus anthracis diversity in Kruger National Park; Smith KL et al.; The Kruger National Park (KNP), South Africa, has a recorded history of periodic anthrax epidemics causing widespread disease among wild animals . Bacillus anthracis is the causative agent of anthrax, a disease primarily affecting ungulate herbivores . Worldwide there is little diversity among B . anthracis isolates, but examination of variable-number tandem repeat (VNTR) loci has identified six major clones, with the most dissimilar types split into the A and B branches . Both the A and B types are found in southern Africa, giving this region the greatest genetic diversity of B . anthracis worldwide . Consequently, southern Africa has been hypothesized to be the geographic origin of B . anthracis . In this study, we identify the genotypic types of 98 KNP B . anthracis isolates using multiple-locus VNTR analysis . Two major types are evident, the A branch and the B branch . The spatial and temporal distribution of the different genotypes indicates that anthrax epidemic foci are independent, though correlated through environmental cues . Kruger B isolates were found on significantly higher-calcium and higher-pH soils than were Kruger type A . This relationship between genotype and soil chemistry may be due to adaptive differences among divergent anthrax strains . While this association may be simply fortuitous, adaptation of A types to diverse environmental conditions is consistent with their greater geographic dispersal and genetic dissimilarity. Ugeskr Laeger, 2000 Sep 4, 162(36), 4786 - 9 {Inhalation anthrax}; Hansen TH; The use of Bacillus anthracis as a biological weapon has the potential of causing considerable loss of human life compared to other pathogens . Inhalational anthrax has a very high mortality and can be induced by spraying an aerosol of anthrax spores . Research in recent years has increased our knowledge, especially of pathogenesis and treatment . A short review is presented here. Infect Immun, 2000 Oct, 68(10), 5731 - 4 Protective antigen-mediated antibody response against a heterologous protein produced in vivo by Bacillus anthracis; Brossier F et al.; Bacillus anthracis secretes a lethal toxin composed of two proteins, the lethal factor (LF) and the protective antigen (PA), which interact within the host or in vitro at the surfaces of eukaryotic cells . Immunization with attenuated B . anthracis strains induces an antibody response against PA and LF . The LF-specific response is potentiated by the binding of LF to PA . In this study, we investigated the capacity of PA to increase the antibody response against a foreign antigen . We constructed a chimeric gene encoding the PA-binding part of LF (LF254) fused to the C fragment of tetanus toxin (ToxC) . The construct was introduced by allelic exchange into the locus encoding LF . Two recombinant B . anthracis strains secreting the hybrid protein LF254-ToxC were