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      Discrimination of Burkholderia mallei/pseudomallei from Burkholderia thailandensis by sequence comparison of a fragment of the ribosomal protein S21 ( rpsU) gene

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          Discrimination of Burkholderia ( B.) pseudomallei and B. mallei from environmental B. thailandensis is challenging. We describe a discrimination method based on sequence comparison of the ribosomal protein S21 ( rpsU) gene.The rpsU gene was sequenced in ten B. pseudomallei, six B. mallei, one B. thailandensis reference strains, six isolates of B. pseudomallei, and 37 of B. thailandensis. Further rpsU sequences of six B. pseudomallei, three B. mallei, and one B. thailandensis were identified via NCBI GenBank. Three to four variable base-positions were identified within a 120-base-pair fragment, allowing discrimination of the B. pseudomallei/mallei-cluster from B. thailandensis, whose sequences clustered identically. All B. mallei and three B. pseudomallei sequences were identical, while 17/22 B. pseudomallei strains differed in one nucleotide (78A>C). Sequences of the rpsU fragment of ‘out-stander’ reference strains of B. cepacia, B. gladioli, B. plantarii, and B. vietnamensis clustered differently.Sequence comparison of the described rpsU gene fragment can be used as a supplementary diagnostic procedure for the discrimination of B. mallei/pseudomallei from B. thailandensis as well as from other species of the genus Burkholderia, keeping in mind that it does not allow for a differentiation between B. mallei and B. pseudomallei.

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          Public Health Assessment of Potential Biological Terrorism Agents

          As part of a Congressional initiative begun in 1999 to upgrade national public health capabilities for response to acts of biological terrorism, the Centers for Disease Control and Prevention (CDC) was designated the lead agency for overall public health planning. A Bioterrorism Preparedness and Response Office has been formed to help target several areas for initial preparedness activities, including planning, improved surveillance and epidemiologic capabilities, rapid laboratory diagnostics, enhanced communications, and medical therapeutics stockpiling (1). To focus these preparedness efforts, however, the biological agents towards which the efforts should be targeted had to first be formally identified and placed in priority order. Many biological agents can cause illness in humans, but not all are capable of affecting public health and medical infrastructures on a large scale. The military has formally assessed multiple agents for their strategic usefulness on the battlefield (2). In addition, the Working Group on Civilian Biodefense, using an expert panel consensus-based process, has identified several biological agents as potential high-impact agents against civilian populations ( 3 – 7 ). To guide national public health bioterrorism preparedness and response efforts, a method was sought for assessing potential biological threat agents that would provide a reviewable, reproducible means for standardized evaluations of these threats. In June 1999, a meeting of national experts was convened to 1) review potential general criteria for selecting the biological agents that pose the greatest threats to civilians and 2) review lists of previously identified biological threat agents and apply these criteria to identify which should be evaluated further and prioritized for public health preparedness efforts. This report outlines the overall selection and prioritization process used to determine the biological agents for public health preparedness activities. Identifying these priority agents will help facilitate coordinated planning efforts among federal agencies, state and local emergency response and public health agencies, and the medical community. Overview of Agent Selection and Prioritization Process On June 3-4, 1999, academic infectious disease experts, national public health experts, Department of Health and Human Services agency representatives, civilian and military intelligence experts, and law enforcement officials (see footnote) met to review and comment on the threat potential of various agents to civilian populations. The following general areas were used as criteria: 1) public health impact based on illness and death; 2) delivery potential to large populations based on stability of the agent, ability to mass produce and distribute a virulent agent, and potential for person-to-person transmission of the agent; 3) public perception as related to public fear and potential civil disruption; and 4) special public health preparedness needs based on stockpile requirements, enhanced surveillance, or diagnostic needs. Participants reviewed lists of biological warfare or potential biological threat agents and selected those they felt posed the greatest threat to civilian populations. The following unclassified documents containing potential biological threat agents were reviewed: 1) the Select Agent Rule list, 2) the Australian Group List for Biological Agents for Export Control, 3) the unclassified military list of biological warfare agents, 4) the Biological Weapons Convention list, and 5) the World Health Organization Biological Weapons list ( 8 – 12 ). Participants with appropriate clearance levels reviewed intelligence information regarding classified suspected biological agent threats to civilian populations. Genetically engineered or recombinant biological agents were considered but not included for final prioritization because of the inability to predict the nature of these agents and thus identify specific preparedness activities for public health and medical response to them. In addition, no information was available about the likelihood for use of one biological agent over another. This aspect, therefore, could not be considered in the final evaluation of the potential biological threat agents. Participants discussed and identified agents they felt had the potential for high impact based on subjective assessments in the four general categories. After the meeting, CDC personnel then attempted to identify objective indicators in each category that could be used to further define and prioritize the identified high-impact agents and provide a framework for an objective risk-matrix analysis process for any potential agent. The agents were evaluated in each of the general areas according to the objective parameters and were characterized by the rating schemes outlined in the Appendix. Final category assignments (A, B, or C) of agents for public health preparedness efforts were then based on an overall evaluation of the ratings the agents received in each of the four areas. Results Based on the overall criteria and weighting, agents were placed in one of three priority categories for initial public health preparedness efforts: A, B, or C (Table 1). Agents in Category A have the greatest potential for adverse public health impact with mass casualties, and most require broad-based public health preparedness efforts (e.g., improved surveillance and laboratory diagnosis and stockpiling of specific medications). Category A agents also have a moderate to high potential for large-scale dissemination or a heightened general public awareness that could cause mass public fear and civil disruption. Table 1 Critical biological agent categories for public health preparedness Biological agent(s) Disease Category A Variola major Smallpox Bacillus anthracis Anthrax Yersinia pestis Plague Clostridium botulinum (botulinum toxins) Botulism Francisella tularensis Tularemia Filoviruses and Arenaviruses (e.g., Ebola virus, Lassa virus) Viral hemorrhagic fevers Category B Coxiella burnetii Q fever Brucella spp. Brucellosis Burkholderia mallei Glanders Burkholderia pseudomallei Melioidosis Alphaviruses (VEE, EEE, WEEa) Encephalitis Rickettsia prowazekii Typhus fever Toxins (e.g., Ricin, Staphylococcal enterotoxin B) Toxic syndromes Chlamydia psittaci Psittacosis Food safety threats (e.g., Salmonella spp., Escherichia coli O157:H7) Water safety threats (e.g., Vibrio cholerae, Cryptosporidium parvum) Category C Emerging threat agents (e.g., Nipah virus, hantavirus) aVenezuelan equine (VEE), eastern equine (EEE), and western equine encephalomyelitis (WEE) viruses Most Category B agents also have some potential for large-scale dissemination with resultant illness, but generally cause less illness and death and therefore would be expected to have lower medical and public health impact. These agents also have lower general public awareness than Category A agents and require fewer special public health preparedness efforts. Agents in this category require some improvement in public health and medical awareness, surveillance, or laboratory diagnostic capabilities, but presented limited additional requirements for stockpiled therapeutics beyond those identified for Category A agents. Biological agents that have undergone some development for widespread dissemination but do not otherwise meet the criteria for Category A, as well as several biological agents of concern for food and water safety, are included in this category. Biological agents that are currently not believed to present a high bioterrorism risk to public health but which could emerge as future threats (as scientific understanding of these agents improves) were placed in Category C. These agents will be addressed nonspecifically through overall bioterrorism preparedness efforts to improve the detection of unexplained illnesses and ongoing public health infrastructure development for detecting and addressing emerging infectious diseases (13). Agents were categorized based on the overall evaluation of the different areas considered. Table 2 shows the evaluation schemes as applied to agents in Categories A and B. For example, smallpox would rank higher than brucellosis in the public health impact criterion because of its higher untreated mortality (approximately 30% for smallpox and ≤2% for brucellosis); smallpox has a higher dissemination potential because of its capability for person-to-person transmission. Smallpox also ranks higher for special public health preparedness needs, as additional vaccine must be manufactured and enhanced surveillance, educational, and diagnostic efforts must be undertaken. Inhalational anthrax and plague also have higher public health impact ratings than brucellosis because of their higher morbidity and mortality. Although mass production of Vibrio cholera (the biological cause of cholera) and Shigella spp. (the cause of shigellosis) would be easier than the mass production of anthrax spores, the public health impact of widespread dissemination would be less because of the lower morbidity and mortality associated with these agents. Although the infectious doses of these bacteria are generally low, the total amount of bacteria that would be required and current water purification and food-processing methods would limit the effectiveness of intentional large-scale water or food contamination with these agents. Table 2 Criteria and weightinga used to evaluate potential biological threat agents Disease Public health impact Dissemination
potential Public
perception Special preparation Category Disease Death P-Db P - Pc Smallpox + ++ + +++ +++ +++ A Anthrax ++ +++ +++ 0 +++ +++ A Plagued ++ +++ ++ ++ ++ +++ A Botulism ++ +++ ++ 0 ++ +++ A Tularemia ++ ++ ++ 0 + +++ A VHFe ++ +++ + + +++ ++ A VEf ++ + + 0 ++ ++ B Q Fever + + ++ 0 + ++ B Brucellosis + + ++ 0 + ++ B Glanders ++ +++ ++ 0 0 ++ B Melioidosis + + ++ 0 0 ++ B Psittacosis + + ++ 0 0 + B Ricin toxin ++ ++ ++ 0 0 ++ B Typhus + + ++ 0 0 + B Cholerag + + ++ +/- +++ + B Shigellosisg + + ++ + + + B aAgents were ranked from highest threat (+++) to lowest (0).
bPotential for production and dissemination in quantities that would affect a large population, based on availability, BSL requirements, most effective route of infection, and environmental stability.
cPerson-to-person transmissibility.
dPneumonic plague.
eViral hemorrhagic fevers due to Filoviruses (Ebola, Marburg) or Arenaviruses (e.g., Lassa, Machupo).
fViral encephalitis.
gExamples of food- and waterborne diseases. Discussion Although use of conventional weapons such as explosives or firearms is still considered the most likely means by which terrorists could harm civilians (14), multiple recent reports cite an increasing risk and probability for the use of biological or chemical weapons ( 15 – 18 ). Indeed, the use of biological and chemical agents as small- and large-scale weapons has been actively explored by many nations and terrorist groups ( 19 , 20 ). Although small-scale bioterrorism events may actually be more likely in light of the lesser degrees of complexity to be overcome, public health agencies must prepare for the still-possible large-scale incident that would undoubtedly lead to catastrophic public health consequences. The selection and prioritization of the potential biological terrorism agents described in this report were not based on the likelihood of their use, but on the probability that their use would result in an overwhelming adverse impact on public health. Most evaluations of potential risk agents for biological warfare or terrorism have historically been based on military concerns and criteria for troop protection. However, several characteristics of civilian populations differ from those of military populations, including a wider range of age groups and health conditions, so that lists of military biological threats cannot simply be adopted for civilian use. These differences and others may greatly increase the consequences of a biological attack on a civilian population. Civilians may also be more vulnerable to food- or waterborne terrorism, as was seen in the intentional Salmonella contamination of salad bars in The Dalles, Oregon, in 1984 (21). Although food and water systems in the United States are among the safest in the world, the occurrence of nationwide outbreaks due to unintentional food or water contamination demonstrates the ongoing need for vigilance in protecting food and water supplies ( 22 , 23 ). Overall, many other factors must be considered in defining and focusing multiagency efforts to protect civilian populations against bioterrorism. Category A agents are being given the highest priority for preparedness. For Category B, public health preparedness efforts will focus on identified deficiencies, such as improving awareness and enhancing surveillance or laboratory diagnostic capabilities. Category C agents will be further assessed for their potential to threaten large populations as additional information becomes available on the epidemiology and pathogenicity of these agents. In addition, special epidemiologic and laboratory surge capacity will be maintained to assist in the investigation of naturally occurring outbreaks due to Category C “emerging” agents. Linkages established with established programs for food safety, emerging infections diseases, and unexplained illnesses will augment the overall bioterrorism preparedness efforts for many Category B and C agents. The above categories of agents should not be considered definitive. The prioritization of biological agents for preparedness efforts should continue. Agents in each category may change as new information is obtained or new assessment methods are established. Disease elimination and eradication efforts may result in new agents being added to the list as populations lose their natural or vaccine-induced immunity to these agents. Conversely, the priority status of certain agents may be reduced as the identified public health and medical deficiencies related to these agents are addressed (e.g., once adequate stores of smallpox vaccine and improved diagnostic capabilities are established, its rating within the special preparedness needs category would be reduced, as would its overall rating within the risk-matrix evaluation process). To meet the ever-changing response and preparedness challenges presented by bioterrorism, a standardized and reproducible evaluation process similar to the one outlined above will continue to be used to evaluate and prioritize currently identified biological critical agents, as well as new agents that may emerge as threats to civilian populations or national security. Supplementary Material Appendix Risk-Matrix Analysis Process Used to Evaluate Potential Biological Threat Agents
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            The molecular and cellular basis of pathogenesis in melioidosis: how does Burkholderia pseudomallei cause disease?

            Melioidosis, a febrile illness with disease states ranging from acute pneumonia or septicaemia to chronic abscesses, was first documented by Whitmore & Krishnaswami (1912). The causative agent, Burkholderia pseudomallei, was subsequently identified as a motile, gram-negative bacillus, which is principally an environmental saprophyte. Melioidosis has become an increasingly important disease in endemic areas such as northern Thailand and Australia (Currie et al., 2000). This health burden, plus the classification of B. pseudomallei as a category B biological agent (Rotz et al., 2002), has resulted in an escalation of research interest. This review focuses on the molecular and cellular basis of pathogenesis in melioidosis, with a comprehensive overview of the current knowledge on how B. pseudomallei can cause disease. The process of B. pseudomallei movement from the environmental reservoir to attachment and invasion of epithelial and macrophage cells and the subsequent intracellular survival and spread is outlined. Furthermore, the diverse assortment of virulence factors that allow B. pseudomallei to become an effective opportunistic pathogen, as well as to avoid or subvert the host immune response, is discussed. With the recent increase in genomic and molecular studies, the current understanding of the infection process of melioidosis has increased substantially, yet, much still remains to be elucidated.
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              Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: isolation of motility mutants and molecular characterization of the flagellin structural gene.

              Burkholderia pseudomallei is a human and animal pathogen in tropical regions, especially Southeast Asia and northern Australia. Currently little is known about the genetics and molecular biology of this organism. In this report, we describe the mutagenesis of B. pseudomallei with the transposon Tn5-OT182. B. pseudomallei 1026b transposon mutants were obtained at a frequency of 4.6 x 10(-4) per initial donor cell, and the transposon inserted randomly into the chromosome. We used Tn5-OT182 to identify the flagellin structural gene, fliC. We screened 3,500 transposon mutants and identified 28 motility mutants. Tn5-OT182 integrated into 19 unique genetic loci encoding proteins with homology to Escherichia coli and Salmonella typhimurium flagellar and chemotaxis proteins. Two mutants, MM35 and MM36, contained Tn5-OT182 integrations in fliC. We cloned and sequenced fliC and used it to complement MM35 and MM36 in trans. The fliC transcriptional start site and a sigmaF-like promoter were identified by primer extension analysis. We observed a significant difference in the expression of two distinct fliC-lacZ transcriptional fusions during bacterial growth, suggesting the presence of a latent intragenic transcriptional terminator in fliC. There was no significant difference in the virulence of 1026b compared to that of MM36 in diabetic rats or Syrian hamsters, suggesting that flagella and/or motility are probably not virulence determinants in these animal models of B. pseudomallei infection. A phylogenetic analysis based on the flagellins from a variety of bacterial species supported the recent transfer of B. pseudomallei from the genus Pseudomonas to Burkholderia.

                Author and article information

                European Journal of Microbiology and Immunology
                Akadémiai Kiadó
                1 June 2012
                13 June 2012
                : 2
                : 2 ( otherID: K022535X4502 )
                : 148-156
                [ 1 ] German Armed Forces Hospital Hamburg Department of Tropical Medicine at the Bernhard Nocht Institute Hamburg Germany
                [ 2 ] University of Rostock Hospital Institute for Medical Microbiology, Virology and Hygiene Rostock Germany
                [ 3 ] Mahidol University Department of Microbiology and Immunology, and Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine Bangkok Thailand
                [ 4 ] Institut de Recherches Biomédicales des Armées Département de Microbiologie La Tronche France
                [ 5 ] Federal Research Institute for Animal Health Friedrich Loeffler Institute Jena Germany
                [ 6 ] German Armed Forces Hospital Hamburg Department of Tropical Medicine at the Bernhard Nocht Institute Bernhard Nocht Strasse 74 D-20359 Hamburg Germany
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