Analysis of Burkholderia pseudomallei virulence and efficacy of potential therapeutics, The

2011 Summer.Includes bibliographical references.Burkholderia pseudomallei is the causative agent of the disease melioidosis and is classified as a category B Select Agent. There are currently many challenges associated with both the study of this pathogen and its treatment in the clinical setting. Prior to these studies, there was no attenuated B. pseudomallei strain available that was exempt of Select Agent regulations and approved for study outside of biosafety level 3 (BSL-3) containment, and consequently basic research on this pathogen was largely hindered. The first purpose of these studies was to extensively characterize the attenuation of two B. pseudomallei mutant strains using melioidosis animal models. The two mutants constructed were Bp82 and Bp190, Δ purM derivatives deficient in adenine and thiamine biosynthesis. These mutants were found to be fully attenuated in immune competent and immune deficient mouse and hamster melioidosis models. Bp82 is currently exempt of all Select Agent regulations and can be safely handled in the BSL-2 setting, greatly accelerating research on this priority pathogen. Since basic research on B. pseudomallei was not common in the Western world until its Select Agent classification, much is still unknown regarding the bacterial factors contributing to its virulence. A second purpose of this research was to determine whether resistance-nodulation-cell division (RND) efflux systems and iron acquisition siderophores impact the virulence of B. pseudomallei in a pneumonic murine melioidosis model. This was examined using a clinical isolate naturally devoid of a characterized efflux system and the gene cluster for malleobactin siderophore synthesis, and by the construction of isogenetic mutants. The two characterized B. pseudomallei efflux pumps, AmrAB-OprA and BpeAB-OprB, were both found to be completely dispensable during in vivo murine infection. The removal of one or both of these systems did not reduce lethality of the mutant strains. Unlike that observed with similar bacterial pathogens, the lethality of B. pseudomallei was also not reduced upon the removal of either the malleobactin or pyochelin siderophores. This finding indicates B. pseudomallei is likely capable of utilizing alternative systems for iron acquisition within the host. In addition to the challenges associated with the study of this pathogen, there are also many clinical challenges associated with melioidosis, providing a basis for the final two purposes of this research. One particular challenge is the high frequency of patient relapse, even after appropriate prolonged antibiotic therapy. A third purpose of this research was to determine whether traditional antibiotic therapy could be augmented by the co-administration of immunotherapy. Cationic liposome-DNA complexes (CLDC), which are potent activators of the innate immune system, were found to synergistically reduce intracellular B. pseudomallei concentrations in macrophages in vitro when combined with the antibiotic ceftazidime. In addition, this combination therapy also significantly increased mouse survival during both acute and chronic melioidosis. A similar enhancement to ceftazidime therapy was observed with recombinant IFN-γ, illustrating the potential of immunotherapy to improve clinical outcome and decrease patient relapse. The lack of an effective approved vaccine for human use is another substantial clinical challenge associated with melioidosis and its prevention. The final purpose of these studies was to develop an effective mucosal vaccine, offering both short-term protection from acute pneumonic disease and long-term protection from disseminated chronic melioidosis. CLDC was identified as a highly effective mucosal adjuvant within complexed to heat-killed B. pseudomallei, and this adjuvant offered moderate protection from acute disease when combined with Burkholderia protein subunits. The longest-term protection from lethal challenge in our murine model, lasting beyond 100 days, was elicited by the fully attenuated live Bp82 strain. Since this strain is both fully attenuated and exempt of Select Agent regulations, it has great potential clinically for high-risk persons as an effective live vaccine strain

Molecular Basis of Rare Aminoglycoside Susceptibility and Pathogenesis of Burkholderia pseudomallei Clinical Isolates from Thailand

Burkholderia pseudomallei is the etiologic agent of melioidosis, an emerging tropical disease. Because of low infectious dose, broad-host-range infectivity, intrinsic antibiotic resistance and historic precedent as a bioweapon, B. pseudomallei was listed in the United States as a Select Agent and Priority Pathogen of biodefense concern by the US Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases. The mechanisms governing antibiotic resistance and/or susceptibility and virulence in this bacterium are not well understood. Most clinical and environmental B. pseudomallei isolates are highly resistant to aminoglycosides, but susceptible variants do exist. The results of our studies with three such variants from Thailand reveal that lack of expression or deletion of an efflux pump is responsible for this susceptibility. The large deletion present in one strain not only removes an efflux pump but also several putative virulence genes, including an entire siderophore gene cluster. Despite this deletion, the strain is fully virulent in an acute mouse melioidosis model. In summary, our findings shed light on mechanisms of antibiotic resistance and pathogenesis. They also validate the previously advocated use of laboratory-constructed, aminoglycoside susceptible efflux pump mutants in genetic manipulation experiments

Immunotherapy Markedly Increases the Effectiveness of Antimicrobial Therapy for Treatment of Burkholderia pseudomallei Infection▿ †

Burkholderia pseudomallei is a soil bacterium that is endemic in southeast Asia and northern Australia and that can cause both acutely lethal pneumonia and chronic systemic infections in humans. The effective treatment of infection with B. pseudomallei requires rapid diagnosis and prolonged treatment with high doses of antimicrobials, and even with appropriate antibiotic therapy, patient relapses are common. Thus, new approaches to the treatment of B. pseudomallei infections are needed. In the present study, we asked whether active immunotherapy with gamma interferon (IFN-γ), a key cytokine regulating the intracellular replication of B. pseudomallei, could increase the effectiveness of conventional antimicrobial therapy for B. pseudomallei infection. Macrophage infection assays and in vivo pulmonary challenge models were used to assess the inhibitory effects of combined treatment with IFN-γ and ceftazidime on B. pseudomallei infection. We found that treatment with even very low doses of IFN-γ and ceftazidime elicited strong synergistic inhibition of B. pseudomallei growth within infected macrophages. In vivo, active immunotherapy markedly potentiated the effectiveness of low-dose ceftazidime therapy for the treatment of infected mice in a pulmonary challenge model of B. pseudomallei. Combined treatment was associated with a significant reduction in the bacterial burden and a significant lessening of bacterial dissemination. We concluded, therefore, that immunotherapy with either endogenous or exogenous IFN-γ could significantly increase the effectiveness of conventional antimicrobial therapy for the treatment of acute B. pseudomallei infection

<i>B. pseudomallei</i> 1710b malleobactin mutant strains mimic the siderophore phenotypes of the <i>B. pseudomallei</i> 708a clinical isolate.

<p><b>A</b>. <i>Burkholderia</i> GBrowse map of the <i>B. pseudomallei</i> 1710b genomic region corresponding to the extent of the deletion found in the <i>B. pseudomallei</i> 708a clinical isolate <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001715#pntd.0001715-Trunck1" target="_blank">[32]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001715#pntd.0001715-Winsor1" target="_blank">[62]</a>. The extents of the <i>amrRAB-oprA</i> genes ecoding the AmrAB-OprA efflux pump and AmrR repressor and the malleobactin synthesis gene cluster (<i>mbaS-mbaF</i>) are noted with horizontal green lines. Vertical black lines and gene locus numbers indicate the borders of 1710b genomic region deletions constructed in this study. The shorthand nomenclatures for strains indicating the genomic region deletions contained in them are bolded. <b>B.</b> Quantitative CAS siderophore assays indicate similar amounts of secondary siderophore production by 1710b malleobactin minus strains and <i>B. pseudomallei</i> 708a. Supernatants from overnight cultures grown in low-iron TSBFC medium were tested by quantitative CAS assays for siderophore production adjusted for cell density by OD₆₀₀ of a 1∶10 dilution. Means and standard deviations of two measurements each from three independent experiments are shown. <b>C.</b> Bacterial colony and CAS halo diameters were measured daily for 4 days on CAS agar plates spotted and incubated as described above. Red bars indicate colony diameter and blue bars halo diameter. Means and standard deviations of two measurements each from three independent experiments are shown. <b>D.</b> CAS plate assays indicate similar secondary siderophore production by 1710b malleobactin deficient strains and <i>B. pseudomallei</i> 708a. Five µl samples of overnight cultures grown in low-iron TSBFC medium were spotted onto CAS agar plates and incubated at 37°C for 4 days prior to photographing.</p

<i>B. pseudomallei</i> 1710b siderophore synthesis and hemin uptake mutant can utililize ferritin as an iron source.

<p><b>A.</b> Microtiter plates containing 200 µl of M9-glucose minimal medium with 200 µM 2,2′-dipyridyl (open white symbols), or 200 µM 2,2′-dipyridyl and 10 µg/ml ferritin (green symbols) were inoculated with strain 1710b. The optical density at 600 nm (OD₆₀₀) was measured hourly. OD₆₀₀ means and standard deviation of three cultures from a single experiment are shown for all strains <b>B</b>. Δ141-kb ΔPCH ΔHMU ΔHEM was grown in the same media and using the same conditions as described above in <b>A</b> for 1710b, except that two different 2,2′-dipyridyl concentrations were used: 100 µM (open green symbols and open white symbols) and 200 µM (closed green symbols).</p

A <i>B. pseudomallei</i> 1710b malleobactin and pyochelin deficient double mutant exhibits siderophore activity.

<p><b>A. </b><i>Burkholderia</i> GBrowse map of the <i>B. pseudomallei</i> 1710b pyochelin synthesis and uptake gene cluster <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001715#pntd.0001715-Winsor1" target="_blank">[62]</a>. Gene names are labeled. Dashed lines bordered by vertical solid black lines indicate the extents of deletions. <b>B.</b> Detection of apo-pyochelin in the culture supernatant of pyochelin synthesis gene cluster mutants by mass spectrometry. TSBFC overnight cultures were filtered through 0.2 µM membranes, acidified and extracted with ethyl acetate. Extracts were dried under N₂ and suspended in methanol. 5 µl samples were injected to detect the 325.068 M+H apo-pyochelin ion. The apo-pyochelin ion is indicated with arrows and isotope distribution ions are labeled. Note the different intensity scales in the top and bottom panels. <b>C.</b> To illustrate the presence of residual siderophore activity in Δ141-kb ΔPCH mutants, 5 µl samples from iron-limited TSBFC overnight cultures were spotted onto CAS agar plates and incubated at 37°C for 4 days prior to photographing.</p

<i>B. pseudomallei</i> strains used in this study.

a<p>Abbreviations: <i>FRT</i>, Flp recombinase target; Km, kanamycin; R, resistance; S, sensitive/susceptible; of Zeo, zeocin.</p>b<p><i>mbaS</i> is annotated as <i>psbS</i> in the 1710b genome annotation (GenBank accession number NC_007434.1).</p>c<p>All strains containing Δ(BURPS1710b_2054–BURPS1710b_2155)::<i>FRT</i> are aminoglycoside susceptible because the deletion of the genes encoding the AmrRAB-OprA efflux pump.</p

Total number of pneumococci recovered from individual rats' lungs five hours after an intranasal challenge with 1 × 10cfu of pneumolysin sufficient (Ply+) or pneumolysin deficient (Ply-)

<p><b>Copyright information:</b></p><p>Taken from "Cirrhosis-induced defects in innate pulmonary defenses against "</p><p>http://www.biomedcentral.com/1471-2180/7/94</p><p>BMC Microbiology 2007;7():94-94.</p><p>Published online 23 Oct 2007</p><p>PMCID:PMC2140065.</p><p></p> Horizontal bars represent the mean values for each group