Evaluating the discriminating capacity of cell death (apoptotic) biomarkers in sepsis.

Background: Sepsis biomarker panels that provide diagnostic and prognostic discrimination in sepsis patients would be transformative to patient care. We assessed the mortality prediction and diagnostic discriminatory accuracy of two biomarkers reflective of cell death (apoptosis), circulating cell-free DNA (cfDNA), and nucleosomes. Methods: The cfDNA and nucleosome levels were assayed in plasma samples acquired in patients admitted from four emergency departments with suspected sepsis. Subjects with non-infectious systemic inflammatory response syndrome (SIRS) served as controls. Samples were acquired at enrollment (T0) and 24 h later (T24). We assessed diagnostic (differentiating SIRS from sepsis) and prognostic (28-day mortality) predictive power. Models incorporating procalcitonin (diagnostic prediction) and APACHE II scores (mortality prediction) were generated. Results: Two hundred three subjects were included (107 provided procalcitonin measurements). Four subjects exhibited uncomplicated sepsis, 127 severe sepsis, 35 septic shock, and 24 had non-infectious SIRS. There were 190-survivors and 13 non-survivors. Mortality prediction models using cfDNA, nucleosomes, or APACHEII yielded AUC values of 0.61, 0.75, and 0.81, respectively. A model combining nucleosomes with the APACHE II score improved the AUC to 0.84. Diagnostic models distinguishing sepsis from SIRS using procalcitonin, cfDNA(T0), or nucleosomes(T0) yielded AUC values of 0.64, 0.65, and 0.63, respectively. The three parameter model yielded an AUC of 0.74. Conclusions: To our knowledge, this is the first head-to-head comparison of cfDNA and nucleosomes in diagnosing sepsis and predicting sepsis-related mortality. Both cfDNA and nucleosome concentrations demonstrated a modest ability to distinguish sepsis survivors and non-survivors and provided additive diagnostic predictive accuracy in differentiating sepsis from non-infectious SIRS when integrated into a diagnostic prediction model including PCT and APACHE II. A sepsis biomarker strategy incorporating measures of the apoptotic pathway may serve as an important component of a sepsis diagnostic and mortality prediction tool

Separate and coordinate transcriptional control mechanisms link expression of the potentially lethal KilB spread locus to the upstream transmission operon on Streptomyces plasmid pIJ101

Efficient conjugation of the high copy plasmid pIJ101 among members of the bacterial genus Streptomyces depends on a single plasmid gene (tra) for initial inter-mycelial transfer, and involves three additional pIJ101 functions (spdA, spdB, and kilB), which may promote intra-mycelial spread of the plasmid upon its entrance into the recipient. The genes tra, spdA, and spdB are co-transcribed as part of an operon, whose expression is negatively controlled by the pIJ101 repressor KorA. Downstream of this transmission operon and in the same orientation, the kilB spread gene possesses its own promoter, which is recognized by the pIJ101 KorB repressor protein; binding of KorB appears to prevent the lethal overexpression of the KilB protein, which otherwise shows a temporally increasing pattern of production or accumulation during the streptomycete life cycle. To define better the mechanism(s) controlling the concentration of the potentially toxic KilB protein in cells, a variety of transcriptional analyses involving the kilB promoter and kilB-specific mRNA were performed. These studies demonstrated that transcription originating from the kilB promoter on pIJ101 is dramatically reduced by KorB binding under non-mating conditions; more significantly, however, as judged by evidence of readthrough transcription across the kilB promoter region and polarity effects of upstream insertion and deletion mutations, kilB was found to be expressed also as part of the transmission operon with optimal KilB production being necessarily tied to such co-transcription. Our data indicate that the genes tra, spdA, spdB, and kilB comprise an unusual operon in which separate tight control of the distal gene (kilB) by the KorB repressor is superimposed on coordinate regulation of full operon transcription by KorA. Moreover, our results imply that potential interactions between elongating RNA polymerase molecules synthesizing transmission operon transcripts and KorB repressor bound to the intercistronic kilB promoter region are important for modulating kilB expression

Growth-Regulated Expression of a Bacteriocin, Produced by the Sweet Potato Pathogen Streptomyces ipomoeae, That Exhibits Interstrain Inhibition▿

Certain strains of the bacterial sweet potato pathogen Streptomyces ipomoeae produce the bacteriocin ipomicin, which inhibits other sensitive strains of the same species. Within the signal-sequence-encoding portion of the ipomicin structural gene ipoA exists a single rare TTA codon, which is recognized in Streptomyces bacteria by the temporally accumulating bldA leucyl tRNA. In this study, ipomicin was shown to stably accumulate in culture supernatants of S. ipomoeae in a growth-regulated manner that did not coincide with the pattern of ipoA expression. Similar growth-regulated production of ipomicin in Streptomyces coelicolor containing the cloned ipoA gene was found to be directly dependent on translation of the ipoA TTA codon by the bldA leucyl tRNA. The results here suggest that bldA-dependent translation of the S. ipomoeae ipoA gene leads to growth-regulated production of the ipomicin precursor, which upon processing to the mature form and secretion stably accumulates in the extracellular environment. To our knowledge, this is the first example of bldA regulation of a bacteriocin in the streptomycetes

Oropharyngeal aspiration of Burkholderia mallei and Burkholderia pseudomallei in BALB/c mice.

Burkholderia mallei and Burkholderia pseudomallei are potentially lethal pathogens categorized as biothreat agents due, in part, to their ability to be disseminated via aerosol. There are no protective vaccines against these pathogens and treatment options are limited and cumbersome. Since disease severity is greatest when these agents are inhaled, efforts to develop pre- or post-exposure prophylaxis focus largely on inhalation models of infection. Here, we demonstrate a non-invasive and technically simple method for affecting the inhalational challenge of BALB/c mice with B. pseudomallei and B. mallei. In this model, two investigators utilized common laboratory tools such as forceps and a micropipette to conduct and characterize an effective and reproducible inhalational challenge of BALB/c mice with B. mallei and B. pseudomallei. Challenge by oropharyngeal aspiration resulted in acute disease. Additionally, 50% endpoints for B. pseudomallei K96243 and B. mallei ATCC 23344 were nearly identical to published aerosol challenge methods. Furthermore, the pathogens disseminated to all major organs typically targeted by these agents where they proliferated. The pro-inflammatory cytokine production in the proximal and peripheral fluids demonstrated a rapid and robust immune response comparable to previously described murine and human studies. These observations demonstrate that OA is a viable alternative to aerosol exposure

Proinflammatory cytokine concentration in the peripheral blood following oropharyngeal aspiration challenge with <i>B. pseudomallei</i>.

<p>Serum from each of the mice (n = 5) examined above was analyzed by cytometric bead analysis for the presence of proinflammatory cytokines. Each assay was performed in triplicate. The average of the five mice is shown in pg/ml with the SEM displayed on the graph. *indicates statistical significance compared to naïve serum as determined by one-way Anova with Tukey's post-test (*<0.05, **<0.01, ***<0.001).</p

The 50% lethal dose of <i>B. pseudomallei</i> K96243 when administered by oropharyngeal aspiration.

<p>Five doses of 10-fold serial dilutions were prepared to administer 4.06×10⁴ cfu, 3.83×10³ cfu, 640 cfu, 40 cfu and 4 cfu of <i>B. pseudomallei</i> K96243 to BALB/c mice by OA as described as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115066#s2" target="_blank">materials and methods</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115066#pone-0115066-g001" target="_blank">Fig. 1</a>. Mice were observed for 14 days. The actual inhaled doses are shown in the graph and were utilized to calculate LD₅₀ (3.11 cfu).</p

Temporal lung lesions induced by <i>B. pseudomallei</i> when administered by oropharyngeal aspiration.

<p>Lungs were collected from mice at 24 h, 48 h and 72 h post infection, stained (H&E stains and Periodic Acid Schiff) and analyzed for <i>B. pseudomallei</i> induced lung damage. Focal pyogranulomatous lesions associated with bronchioles are indicated by arrows. PBS (72 h) depict sham challenged lung tissue at 72 hours following administration of PBS. The data presented here represent a consensus of five lungs examined at each timepoint.</p

Inflammatory lung scores following OA-challenge.

<p><i>B. pseudomallei</i> or PBS was delivered to BALB/c mice by OA. The lungs were collected and processed as described above and alalyzed for signs of perivascular, peribronchial and interstitial inflammation according to previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115066#s2" target="_blank">methods</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115066#pone.0115066-Norris1" target="_blank">[40]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115066#pone.0115066-McConchie1" target="_blank">[42]</a>. Controls depict lungs from uninfected naïve mice while PBS represents sham challenged tissues. The average score of the five mice is shown with the SEM displayed on the graph. *indicates statistical significance compared to the PBS controls as determined by one-way Anova with Tukey's post-test (*<0.05, ***<0.0001).</p

Challenge by oropharyngeal aspiration.

<p>Mice were lightly anesthetized as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115066#s2" target="_blank">materials and methods</a> and manually restrained in an upright position (A). Small curved forceps were applied to gently open the mouth and secure the tongue to the lower jaw (A, inset). (B) 30 µl of inoculum was administered to the back of the mouth using a pipette and sterile tip. The nares were blocked by the second investigator (C) to prevent obligate nasal breathing and compel inhalation of the inoculums.</p

Reproducibility of oropharyngeal aspiration.

<p>The results of four independent challenges are presented here. In each case, 15 BALB/c mice were challenged by OA with <i>B. pseudomallei</i> K96243 and the inhaled dose was determed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115066#s2" target="_blank">materials and methods</a> section. Asterics identify challenges performed for this work (* from model, ** from LD₅₀).</p