Stimulating antibiotic development by targeting virulence and facilitating natural product discovery

Antibiotics are a cornerstone of modern medicine and have drastically reduced the burden of infectious diseases. Unfortunately, resistance to all clinically used antibiotics has become a major challenge that is exacerbated by numerous difficulties surrounding the development of new drugs. However, inventive strategies to overcome resistance as well as discover novel antibiotics are increasingly being explored. Whereas traditional antibiotics were generally designed to directly kill as many species of bacteria as possible, several new approaches have focused on narrower spectrum agents that have significant potential benefits. Antibiotics active against only one or a small group of pathogens would spare the microbiome, which may decrease the risk of secondary infections and slow the spread of resistance. One such narrow-spectrum strategy is to target the virulence factors employed by pathogens during an infection. In chapter 2, I demonstrate that the FDA approved HIV protease inhibitor nelfinavir can be repurposed as an inhibitor of the biosynthesis of the Streptococcus pyogenes cytolytic toxin streptolysin S. Nelfinavir was utilized to explore the proteolytic processing step in streptolysin S biosynthesis and was also shown to inhibit toxin production in other pathogens known to harbor similar biosynthetic clusters. Another approach to the problem of finding new antibiotics can be found in facilitating natural product discovery. Many antibiotics are derived from natural products but continuing to find new compounds has become increasingly difficult, especially due to rediscovery of known natural products. To help circumvent this problem, I developed a probe for identifying natural products containing aldehydes and ketones from microbial extracts based on the chemical reactivity of those carbonyl functional groups (chapter 3). This method is agnostic to the activity of the product and allows for the rapid identification of low abundance compounds that may be missed through activity-based screening. I demonstrate the utility of this probe by screening a collection of bacterial extracts, leading to the discovery of an analog of the protease inhibitor antipain

Modulation of Brain β-Endorphin Concentration by the Specific Part of the Y Chromosome in Mice

International audienceBackground: Several studies in animal models suggest a possible effect of the specific part of the Y-chromosome (Y NPAR) on brain opioid, and more specifically on brain b-endorphin (BE). In humans, male prevalence is found in autistic disorder in which observation of abnormal peripheral or central BE levels are also reported. This suggests gender differences in BE associated with genetic factors and more precisely with Y NPAR. Methodology/Principal Findings: Brain BE levels and plasma testosterone concentrations were measured in two highly inbred strains of mice, NZB/BlNJ (N) and CBA/HGnc (H), and their consomic strains for the Y NPAR. An indirect effect of the Y NPAR on brain BE level via plasma testosterone was also tested by studying the correlation between brain BE concentration and plasma testosterone concentration in eleven highly inbred strains. There was a significant and major effect (P,0.0001) of the Y NPAR in interaction with the genetic background on brain BE levels. Effect size calculated using Cohen's procedure was large (56% of the total variance). The variations of BE levels were not correlated with plasma testosterone which was also dependent of the Y NPAR. Conclusions/Significance: The contribution of Y NPAR on brain BE concentration in interaction with the genetic background is the first demonstration of Y-chromosome mediated control of brain opioid. Given that none of the genes encompassed by the Y NPAR encodes for BE or its precursor, our results suggest a contribution of the sex-determining region (Sry, carried by Y NPAR) to brain BE concentration. Indeed, the transcription of the Melanocortin 2 receptor gene (Mc2R gene, identified as the proopiomelanocortin receptor gene) depends on the presence of Sry and BE is derived directly from proopiomelanocortin. The results shed light on the sex dependent differences in brain functioning and the role of Sry in the BE system might be related to the higher frequency of autistic disorder in males

Advances in the treatment of prolactinomas

Prolactinomas account for approximately 40% of all pituitary adenomas and are an important cause of hypogonadism and infertility. The ultimate goal of therapy for prolactinomas is restoration or achievement of eugonadism through the normalization of hyperprolactinemia and control of tumor mass. Medical therapy with dopamine agonists is highly effective in the majority of cases and represents the mainstay of therapy. Recent data indicating successful withdrawal of these agents in a subset of patients challenge the previously held concept that medical therapy is a lifelong requirement. Complicated situations, such as those encountered in resistance to dopamine agonists, pregnancy, and giant or malignant prolactinomas, may require multimodal therapy involving surgery, radiotherapy, or both. Progress in elucidating the mechanisms underlying the pathogenesis of prolactinomas may enable future development of novel molecular therapies for treatment-resistant cases. This review provides a critical analysis of the efficacy and safety of the various modes of therapy available for the treatment of patients with prolactinomas with an emphasis on challenging situations, a discussion of the data regarding withdrawal of medical therapy, and a foreshadowing of novel approaches to therapy that may become available in the future

Assay performance with cefepime.

<p>Relative growth values from the MBT-ASTRA assay performed with cefepime are shown. A relative growth cutoff of 0.5 was utilized to classify resistance, indicated by the horizontal line. Intermediate resistance is abbreviated as Int. Strains are arranged from left to right in order of increasing MIC, with exact values given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183899#pone.0183899.t001" target="_blank">Table 1</a>. Data points colored red indicate major or very major errors. For each strain, 4–5 independent replicates were obtained on different days.</p

Assay performance on blood culture.

<p>MBT-ASTRA assay performed from spiked blood culture. Relative growth values from each replicate are shown, with data points representing major errors colored red. Strain FDAARGOS_159 represented by (●), strain ATCC 25923 represented by (■), strain BAA-44 represented by (▲), strain BR-VRSA represented by (♦). Filled symbols indicate the strain is susceptible to the antibiotic by reference standard while open symbols indicate resistance. A relative growth cutoff of 0.5 was utilized to classify resistance, indicated by the horizontal line. For each strain, 3 independent replicates were obtained on different days, with the mean and standard deviation shown for each set.</p

Isolate sources and MICs.

<p>Isolate sources and MICs.</p

Summary of MBT-ASTRA assay performance across 35 strains.

<p>Summary of MBT-ASTRA assay performance across 35 strains.</p

Assay performance with ciprofloxacin.

<p>Relative growth values for 35 <i>S</i>. <i>aureus</i> strains derived from the MBT-ASTRA assay performed with ciprofloxacin are shown. A relative growth cutoff of 0.5 was utilized to classify resistance, indicated by the horizontal line. Strains are arranged from left to right in order of increasing MIC, with exact values given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183899#pone.0183899.t001" target="_blank">Table 1</a>. Data points colored red indicate major or very major errors. For each strain, 4–6 independent replicates were obtained on different days.</p

Semi-quantitative MALDI-TOF for antimicrobial susceptibility testing in <i>Staphylococcus aureus</i>

<div><p>Antibiotic resistant bacterial infections are a significant problem in the healthcare setting, in many cases requiring the rapid administration of appropriate and effective antibiotic therapy. Diagnostic assays capable of quickly and accurately determining the pathogen resistance profile are therefore crucial to initiate or modify care. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a standard method for species identification in many clinical microbiology laboratories and is well positioned to be applied towards antimicrobial susceptibility testing. One recently reported approach utilizes semi-quantitative MALDI-TOF MS for growth rate analysis to provide a resistance profile independent of resistance mechanism. This method was previously successfully applied to Gram-negative pathogens and mycobacteria; here, we evaluated this method with the Gram-positive pathogen <i>Staphylococcus aureus</i>. Specifically, we used 35 strains of <i>S</i>. <i>aureus</i> and four antibiotics to optimize and test the assay, resulting in an overall accuracy rate of 95%. Application of the optimized assay also successfully determined susceptibility from mock blood cultures, allowing both species identification and resistance determination for all four antibiotics within 3 hours of blood culture positivity.</p></div

Assay performance with oxacillin.

<p>Relative growth values from the MBT-ASTRA assay performed with oxacillin are shown. A relative growth cutoff of 0.5 was utilized to classify resistance, indicated by the horizontal line. Strains are arranged from left to right in order of increasing MIC, with exact values given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183899#pone.0183899.t001" target="_blank">Table 1</a>. Data points colored red indicate major or very major errors. For each strain, 4–6 independent replicates were obtained on different days.</p