Next-Generation Probiotics Targeting \u3ci\u3eClostridium difficile\u3c/i\u3e through Precursor- Directed Antimicrobial Biosynthesis

Integration of antibiotic and probiotic therapy has the potential to lessen the public health burden of antimicrobial-associated diseases. Clostridium difficile infection (CDI) represents an important example where the rational design of next-generation probiotics is being actively pursued to prevent disease recurrence. Because intrinsic resistance to clinically relevant antibiotics used to treat CDI (vancomycin, metronidazole, and fidaxomicin) is a desired trait in such probiotic species, we screened several bacteria and identified Lactobacillus reuteri to be a promising candidate for adjunct therapy. Human-derived L. reuteri bacteria convert glycerol to the broad-spectrum antimicrobial compound reuterin. When supplemented with glycerol, strains carrying the pocR gene locus were potent reuterin producers, with L. reuteri 17938 inhibiting C. difficile growth at a level on par with the level of growth inhibition by vancomycin. Targeted pocR mutations and complementation studies identified reuterin to be the precursor-induced antimicrobial agent. Pathophysiological relevance was demonstrated when the codelivery of L. reuteri with glycerol was effective against C. difficile colonization in complex human fecal microbial communities, whereas treatment with either glycerol or L. reuteri alone was ineffective. A global unbiased microbiome and metabolomics analysis independently confirmed that glycerol precursor delivery with L. reuteri elicited changes in the composition and function of the human microbial community that preferentially targets C. difficile outgrowth and toxicity, a finding consistent with glycerol fermentation and reuterin production. Antimicrobial resistance has thus been successfully exploited in the natural design of human microbiome evasion of C. difficile, and this method may provide a prototypic precursor-directed probiotic approach. Antibiotic resistance and substrate bioavailability may therefore represent critical new determinants of probiotic efficacy in clinical trials

Field trial of three different Plasmodium vivax-detecting rapid diagnostic tests with and without evaporative cool box storage in Afghanistan

<p>Abstract</p> <p>Background</p> <p>Accurate parasitological diagnosis of malaria is essential for targeting treatment where more than one species coexist. In this study, three rapid diagnostic tests (RDTs) (AccessBio CareStart (CSPfPan), CareStart PfPv (CSPfPv) and Standard Diagnostics Bioline (SDBPfPv)) were evaluated for their ability to detect natural <it>Plasmodium vivax </it>infections in a basic clinic setting. The potential for locally made evaporative cooling boxes (ECB) to protect the tests from heat damage in high summer temperatures was also investigated.</p> <p>Methods</p> <p>Venous blood was drawn from <it>P. vivax </it>positive patients in Jalalabad, Afghanistan and tested against a panel of six RDTs. The panel comprised two of each test type; one group was stored at room temperature and the other in an ECB. RDT results were evaluated against a consensus gold standard based on two double-read reference slides and PCR. The sensitivity, specificity and a measure of global performance for each test were determined and stratified by parasitaemia level and storage condition.</p> <p>Results</p> <p>In total, 306 patients were recruited, of which 284 were positive for <it>P. vivax</it>, one for <it>Plasmodium malariae </it>and none for <it>Plasmodium falciparum</it>; 21 were negative. All three RDTs were specific for malaria. The sensitivity and global performance index for each test were as follows: CSPfPan [98.6%, 95.1%], CSPfPv [91.9%, 90.5%] and SDBPfPv [96.5%, 82.9%], respectively. CSPfPv was 16% less sensitive to a parasitaemia below 5,000/μL. Room temperature storage of SDBPfPv led to a high proportion of invalid results (17%), which reduced to 10% in the ECB. Throughout the testing period, the ECB maintained ~8°C reduction over ambient temperatures and never exceeded 30°C.</p> <p>Conclusions</p> <p>Of the three RDTs, the CSPfPan test was the most consistent and reliable, rendering it appropriate for this <it>P. vivax </it>predominant region. The CSPfPv test proved unsuitable owing to its reduced sensitivity at a parasitaemia below 5,000/μL (affecting 43% of study samples). Although the SDBPfPv device was more sensitive than the CSPfPv test, its invalid rate was unacceptably high. ECB storage reduced the proportion of invalid results for the SDBPfPv test, but surprisingly had no impact on RDT sensitivity at low parasitaemia.</p

Web Messaging for Open and Scalable Distributed Sensing Applications

Abstract. Future Web applications will increasingly require real-time data from the physical world collected by a myriad of sensors and actuators. Currently, integration of such devices require customized solutions due to the lack of widely adopted protocols for devices. Because the Web architecture offers a high degree of interoperability and a low entry barrier, we propose to leverage the Web to build hybrid applications that combine the physical world with Web content. Our work builds upon recent developments in Web push techniques and extends them for embedded devices with a RESTful messaging system. Our results illustrate that fully Web-based distributed sensing applications are not only feasible- but actually desirable- because Web standards offer an ideal compromise between performance and functionality.

Next-Generation Probiotics Targeting \u3ci\u3eClostridium difficile\u3c/i\u3e through Precursor- Directed Antimicrobial Biosynthesis

Integration of antibiotic and probiotic therapy has the potential to lessen the public health burden of antimicrobial-associated diseases. Clostridium difficile infection (CDI) represents an important example where the rational design of next-generation probiotics is being actively pursued to prevent disease recurrence. Because intrinsic resistance to clinically relevant antibiotics used to treat CDI (vancomycin, metronidazole, and fidaxomicin) is a desired trait in such probiotic species, we screened several bacteria and identified Lactobacillus reuteri to be a promising candidate for adjunct therapy. Human-derived L. reuteri bacteria convert glycerol to the broad-spectrum antimicrobial compound reuterin. When supplemented with glycerol, strains carrying the pocR gene locus were potent reuterin producers, with L. reuteri 17938 inhibiting C. difficile growth at a level on par with the level of growth inhibition by vancomycin. Targeted pocR mutations and complementation studies identified reuterin to be the precursor-induced antimicrobial agent. Pathophysiological relevance was demonstrated when the codelivery of L. reuteri with glycerol was effective against C. difficile colonization in complex human fecal microbial communities, whereas treatment with either glycerol or L. reuteri alone was ineffective. A global unbiased microbiome and metabolomics analysis independently confirmed that glycerol precursor delivery with L. reuteri elicited changes in the composition and function of the human microbial community that preferentially targets C. difficile outgrowth and toxicity, a finding consistent with glycerol fermentation and reuterin production. Antimicrobial resistance has thus been successfully exploited in the natural design of human microbiome evasion of C. difficile, and this method may provide a prototypic precursor-directed probiotic approach. Antibiotic resistance and substrate bioavailability may therefore represent critical new determinants of probiotic efficacy in clinical trials

New Host-Directed Therapeutics for the Treatment of Clostridioides difficile Infection

Clostridioides difficile is a spore-forming anaerobic bacterium and the leading cause of antibiotic-associated colitis. With few therapeutic options and high rates of disease recurrence, the need to develop new treatment options is urgent. Prior studies utilizing a repurposing approach identified three nonantibiotic Food and Drug Administration-approved drugs, amoxapine, doxapram, and trifluoperazine, with efficacy against a broad range of human pathogens; however, the protective mechanisms remained unknown. Here, we identified mechanisms leading to drug efficacy in a murine model of lethal C. difficile infection (CDI), advancing our understanding of the role of these drugs in infectious disease pathogenesis that center on host immune responses to C. difficile. Overall, these studies highlight the crucial involvement of innate immune responses, as well as the importance of immunomodulation as a potential therapeutic option to combat CDI.Frequent and excessive use of antibiotics primes patients to Clostridioides difficile infection (CDI), which leads to fatal pseudomembranous colitis, with limited treatment options. In earlier reports, we used a drug repurposing strategy and identified amoxapine (an antidepressant), doxapram (a breathing stimulant), and trifluoperazine (an antipsychotic), which provided significant protection to mice against lethal infections with several pathogens, including C. difficile. However, the mechanisms of action of these drugs were not known. Here, we provide evidence that all three drugs offered protection against experimental CDI by reducing bacterial burden and toxin levels, although the drugs were neither bacteriostatic nor bactericidal in nature and had minimal impact on the composition of the microbiota. Drug-mediated protection was dependent on the presence of the microbiota, implicating its role in evoking host defenses that promoted protective immunity. By utilizing transcriptome sequencing (RNA-seq), we identified that each drug increased expression of several innate immune response-related genes, including those involved in the recruitment of neutrophils, the production of interleukin 33 (IL-33), and the IL-22 signaling pathway. The RNA-seq data on selected genes were confirmed by quantitative real-time PCR (qRT-PCR) and protein assays. Focusing on amoxapine, which had the best anti-CDI outcome, we demonstrated that neutralization of IL-33 or depletion of neutrophils resulted in loss of drug efficacy. Overall, our lead drugs promote disease alleviation and survival in the murine model through activation of IL-33 and by clearing the pathogen through host defense mechanisms that critically include an early influx of neutrophils