Characterization of the murine macrophage response to infection with virulent and avirulent Burkholderia species.

BACKGROUND: Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Gram-negative facultative intracellular pathogens, which are the causative agents of melioidosis and glanders, respectively. Depending on the route of exposure, aerosol or transcutaneous, infection by Bp or Bm can result in an extensive range of disease - from acute to chronic, relapsing illness to fatal septicemia. Both diseases are associated with difficult diagnosis and high fatality rates. About ninety five percent of patients succumb to untreated septicemic infections and the fatality rate is 50 % even when standard antibiotic treatments are administered. RESULTS: The goal of this study is to profile murine macrophage-mediated phenotypic and molecular responses that are characteristic to a collection of Bp, Bm, Burkholderia thailandensis (Bt) and Burkholderia oklahomensis (Bo) strains obtained from humans, animals, environment and geographically diverse locations. Burkholderia spp. (N = 21) were able to invade and replicate in macrophages, albeit to varying degrees. All Bp (N = 9) and four Bm strains were able to induce actin polymerization on the bacterial surface following infection. Several Bp and Bm strains showed reduced ability to induce multinucleated giant cell (MNGC) formation, while Bo and Bp 776 were unable to induce this phenotype. Measurement of host cytokine responses revealed a statistically significant Bm mediated IL-6 and IL-10 production compared to Bp strains. Hierarchical clustering of transcriptional data from 84 mouse cytokines, chemokines and their corresponding receptors identified 29 host genes as indicators of differential responses between the Burkholderia spp. Further validation confirmed Bm mediated Il-1b, Il-10, Tnfrsf1b and Il-36a mRNA expressions were significantly higher when compared to Bp and Bt. CONCLUSIONS: These results characterize the phenotypic and immunological differences in the host innate response to pathogenic and avirulent Burkholderia strains and provide insight into the phenotypic alterations and molecular targets underlying host-Burkholderia interactions.

Characterization of the Burkholderia thailandensis SOS response by using whole-transcriptome shotgun sequencing.

The bacterial SOS response is a well-characterized regulatory network encoded by most prokaryotic bacterial species and is involved in DNA repair. In addition to nucleic acid repair, the SOS response is involved in pathogenicity, stress-induced mutagenesis, and the emergence and dissemination of antibiotic resistance. Using high-throughput sequencing technology (SOLiD RNA-Seq), we analyzed the Burkholderia thailandensis global SOS response to the fluoroquinolone antibiotic, ciprofloxacin (CIP), and the DNA-damaging chemical, mitomycin C (MMC). We demonstrate that a B. thailandensis recA mutant (RU0643) is ∼4-fold more sensitive to CIP in contrast to the parental strain B. thailandensis DW503. Our RNA-Seq results show that CIP and MMC treatment (P < 0.01) resulted in the differential expression of 344 genes in B. thailandensis and 210 genes in RU0643. Several genes associated with the SOS response were induced and include lexA, uvrA, dnaE, dinB, recX, and recA. At the genome-wide level, we found an overall decrease in gene expression, especially for genes involved in amino acid and carbohydrate transport and metabolism, following both CIP and MMC exposure. Interestingly, we observed the upregulation of several genes involved in bacterial motility and enhanced transcription of a B. thailandensis genomic island encoding a Siphoviridae bacteriophage designated E264. Using B. thailandensis plaque assays and PCR with B. mallei ATCC 23344 as the host, we demonstrate that CIP and MMC exposure in B. thailandensis DW503 induces the transcription and translation of viable bacteriophage in a RecA-dependent manner. This is the first report of the SOS response in Burkholderia spp. to DNA-damaging agents. We have identified both common and unique adaptive responses of B. thailandensis to chemical stress and DNA damage.

Live vaccine strain Francisella tularensis is detectable at the inoculation site but not in blood after vaccination against tularemia.

INTRODUCTION: Live vaccine strain (LVS) Francisella tularensis is a live, attenuated investigational tularemia vaccine that has been used by the US Army for decades to protect laboratory workers. Postvaccination bacterial kinetic characteristics of LVS at the inoculation site and in the blood are unknown and, therefore, were assessed in a prospective study. LVS vaccination of laboratory workers provided the opportunity to compare culture with polymerase chain reaction (PCR) for the detection of F. tularensis in human clinical samples. METHODS: Blood and skin swab samples were prospectively collected from volunteers who received the LVS tularemia vaccine at baseline (negative controls) and at 5 specified time points (days 1, 2, 7 or 8, 14 or 15, and 35 after vaccination). Bacterial culture and PCR of whole blood samples (17 volunteers) and inoculation site swabs (41 volunteers) were performed. RESULTS: The culture and PCR results of all blood samples were negative. Results of real-time PCR from the inoculation site samples were positive for 41 (100%) of 41 volunteers on day 1, for 40 (97.6%) of 41 volunteers on day 2, for 24 (58.5%) of 41 on day 7 or 8, for 6 (16.7%) of 36 on day 14 or 15, and for 0 (0%) of 9 on day 35. Positive results of bacterial cultures of the inoculation site samples occurred significantly less frequently, compared with PCR testing, with 4 (9.8%) of 41 volunteers having positive results on day 1 (P<.001) and 4 (9.8%) of 41 on day 2 (P<.001); all results from subsequent days were negative. CONCLUSIONS: F. tularensis LVS genomic DNA was detected in the majority of samples from the inoculation site up to 1 week after LVS vaccination, with real-time PCR being more sensitive than culture. Our data suggest that bacteremia does not occur after LVS vaccination in normal, healthy human volunteers.

Cynomolgus macaque as an animal model for severe acute respiratory syndrome.

BACKGROUND: The emergence of severe acute respiratory syndrome (SARS) in 2002 and 2003 affected global health and caused major economic disruption. Adequate animal models are required to study the underlying pathogenesis of SARS-associated coronavirus (SARS-CoV) infection and to develop effective vaccines and therapeutics. We report the first findings of measurable clinical disease in nonhuman primates (NHPs) infected with SARS-CoV. METHODS AND FINDINGS: In order to characterize clinically relevant parameters of SARS-CoV infection in NHPs, we infected cynomolgus macaques with SARS-CoV in three groups: Group I was infected in the nares and bronchus, group II in the nares and conjunctiva, and group III intravenously. Nonhuman primates in groups I and II developed mild to moderate symptomatic illness. All NHPs demonstrated evidence of viral replication and developed neutralizing antibodies. Chest radiographs from several animals in groups I and II revealed unifocal or multifocal pneumonia that peaked between days 8 and 10 postinfection. Clinical laboratory tests were not significantly changed. Overall, inoculation by a mucosal route produced more prominent disease than did intravenous inoculation. Half of the group I animals were infected with a recombinant infectious clone SARS-CoV derived from the SARS-CoV Urbani strain. This infectious clone produced disease indistinguishable from wild-type Urbani strain. CONCLUSIONS: SARS-CoV infection of cynomolgus macaques did not reproduce the severe illness seen in the majority of adult human cases of SARS; however, our results suggest similarities to the milder syndrome of SARS-CoV infection characteristically seen in young children.

Using real-time PCR to specifically detect Burkholderia mallei.

Burkholderia mallei is the causative agent of human and animal glanders and is a category B biothreat agent. Rapid diagnosis of B. mallei and immediate prophylactic treatment are essential for patient survival. The majority of current bacteriological and immunological techniques for identifying B. mallei from clinical samples are time-consuming, and cross-reactivity with closely related organisms (i.e. Burkholderia pseudomallei) is a problem. In this investigation, two B. mallei-specific real-time PCR assays targeting the B. mallei bimA(ma) gene (Burkholderia intracellular motility A; BMAA0749), which encodes a protein involved in actin polymerization, were developed. The PCR primer and probe sets were tested for specificity against a collection of B. mallei and B. pseudomallei isolates obtained from numerous clinical and environmental (B. pseudomallei only) sources. The assays were also tested for cross-reactivity using template DNA from 14 closely related Burkholderia species. The relative limit of detection for the assays was found to be 1 pg or 424 genome equivalents. The authors also analysed the applicability of assays to detect B. mallei within infected BALB/c mouse tissues. Beginning 1 h post aerosol exposure, B. mallei was successfully identified within the lungs, and starting at 24 h post exposure, in the spleen and liver. Surprisingly, B. mallei was not detected in the blood of acutely infected animals. This investigation provides two real-time PCR assays for the rapid and specific identification of B. mallei.

Development of a polymerase chain reaction assay for the specific identification of Burkholderia mallei and differentiation from Burkholderia pseudomallei and other closely related Burkholderiaceae.

Burkholderia mallei and Burkholderia pseudomallei, the etiologic agents responsible for glanders and melioidosis, respectively, are genetically and phenotypically similar and are category B biothreat agents. We used an in silico approach to compare the B. mallei ATCC 23344 and B. pseudomallei K96243 genomes to identify nucleotide sequences unique to B. mallei. Five distinct B. mallei DNA sequences and/or genes were identified and evaluated for polymerase chain reaction (PCR) assay development. Genomic DNAs from a collection of 31 B. mallei and 34 B. pseudomallei isolates, obtained from various geographic, clinical, and environmental sources over a 70-year period, were tested with PCR primers targeted for each of the B. mallei ATCC 23344-specific nucleotide sequences. Of the 5 chromosomal targets analyzed, only PCR primers designed to bimA(Bm) were specific for B. mallei. These primers were used to develop a rapid PCR assay for the definitive identification of B. mallei and differentiation from all other bacteria.

Real-time PCR assays targeting a unique chromosomal sequence of Yersinia pestis.

BACKGROUND: Yersinia pestis, the causative agent of the zoonotic infection plague, is a major concern as a potential bioweapon. Current real-time PCR assays used for Y. pestis detection are based on plasmid targets, some of which may generate false-positive results. METHODS: Using the yp48 gene of Y. pestis, we designed and tested 2 real-time TaqMan minor groove binder (MGB) assays that allowed us to use chromosomal genes as both confirmatory and differential targets for Y. pestis. We also designed several additional assays using both Simple-Probe and MGB Eclipse probe technologies for the selective differentiation of Yersinia pseudotuberculosis from Y. pestis. These assays were designed around a 25-bp insertion site recently identified within the yp48 gene of Y. pseudotuberculosis. RESULTS: The Y. pestis-specific assay distinguished this bacterium from other Yersinia species but had unacceptable low-level detection of Y. pseudotuberculosis, a closely related species. Simple-Probe and MGB Eclipse probes specific for the 25-bp insertion detected only Y. pseudotuberculosis DNA. Probes that spanned the deletion site detected both Y. pestis and Y. pseudotuberculosis DNA, and the 2 species were clearly differentiated by a post-PCR melting temperature (Tm) analysis. The Simple-Probe assay produced an almost 7 degrees C Tm difference and the MGB Eclipse probe a slightly more than 4 degrees C difference. CONCLUSIONS: Our method clearly discriminates Y. pestis DNA from all other Yersinia species tested and from the closely related Y. pseudotuberculosis. These chromosomal assays are important both to verify the presence of Y. pestis based on a chromosomal target and to easily distinguish it from Y. pseudotuberculosis.

Absence of oropharyngeal vaccinia virus after vaccinia (smallpox) vaccination.

BACKGROUND: With the resumption of the vaccinia (smallpox) vaccination, questions regarding transmission risk prompted this study to determine whether vaccinia virus could be detected in the oropharynx of adults recently vaccinated with vaccinia (smallpox) vaccine. German, Russian, and American studies on the oropharyngeal presence of vaccinia virus revealed conflicting results in different age groups. OBJECTIVE: To measure vaccinia viral particle or antigen presence in the oropharynx of adult health care workers after vaccination with vaccinia (smallpox) vaccine using viral culture and high-sensitivity assays (polymerase chain reaction [PCR] and electrochemiluminescence) and to determine whether there is an association between the presence of vaccinia virus and adverse reactions. METHODS: A total of 155 adults (primary vaccinees and revaccinees) were enrolled for 1 baseline and 5 subsequent throat swabs. The swabs were evaluated using viral culture, PCR, and electrochemiluminescence. RESULTS: Of the 155 participants, 144 had more than 2 throat swabs in the 2 weeks after vaccination. Of the 801 specimens evaluated, there were no positive results by culture, PCR, or electrochemiluminescence except in the control samples (n = 6), which were positive by all 3 methods. CONCLUSIONS: Based on the absence of detectable vaccinia virus in this study population, one can be 95% certain that the true rate of vaccinia virus in the oropharynx of adults during the 2 weeks after vaccination with vaccinia (smallpox) vaccine is 0% to 3.3%. These data should be reassuring to the medical community and support the Advisory Committee on Immunization Practice guidelines that respiratory precautions are not necessary after vaccinia (smallpox) vaccination in healthy adults.

Comparative analysis of the Schleicher and Schuell IsoCode Stix DNA isolation device and the Qiagen QIAamp DNA Mini Kit.

Efficient, rapid, and reproducible procedures for isolating high-quality DNA before PCR gene amplification are essential for the diagnostic and molecular identification of pathogenic bacteria. This study evaluated the Qiagen QIAamp DNA Mini Kit and the Schleicher and Schuell IsoCode Stix DNA isolation device for isolating nucleic acid. Buffer, serum, and whole-blood samples were spiked with Bacillus anthracis Sterne vegetative cells and Yersinia pestis, while water was spiked with B. anthracis Sterne spores. Although minimal variations in limit of detection occurred among matrices, both the IsoCode Stix extraction method and the Qiagen procedure have comparable detection limits.