A Multimodal Intervention to Reduce C. difficile Infections and Stool Testing.

BACKGROUND AND OBJECTIVES: The introduction of multiplex gastrointestinal panels at our institution resulted in increased Clostridioides difficile (C. difficile) detection and stool test utilization. We aimed to reduce hospital-onset C. difficile infections (HO-CDIs), C. difficile detection, and overall stool testing by 20% within 1 year. METHODS: We conducted a quality improvement project from 2018 to 2020 at a large children's hospital. Interventions included development of a C. difficile testing and treatment clinical care pathway, new options for gastrointestinal panel testing with or without C. difficile (results were suppressed if not ordered), clinical decision support tool to restrict testing, and targeted prevention efforts. Outcomes included the rate of HO-CDI (primary), C. difficile detection, and overall stool testing. All measures were evaluated monthly among hospitalized children per 10 000 patient-days (PDs) using statistical process-control charts. For balancing measures, we tracked suppressed C. difficile results that were released during real-time monitoring because of concern for true infection and C. difficile-related adverse events. RESULTS: HO-CDI decreased by 55%, from 11 to 5 per 10 000 PDs. C. difficile detection decreased by 44%, from 18 to 10 per 10 000 PDs, and overall test utilization decreased by 29%, from 99 to 70 per 10 000 PDs. The decrease in stool tests resulted in annual savings of $55 649. Only 2.3% of initially suppressed positive C. difficile results were released, and no patients had adverse events. CONCLUSIONS: Diagnostic stewardship strategies, coupled with an evidence-based clinical care pathway, can be used to decrease C. difficile and improve overall test utilization.

Dietary fat promotes antibiotic-induced Clostridioides difficile mortality in mice.

Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea, and emerging evidence has linked dietary components with CDI pathogenesis, suggesting that dietary modulation may be an effective strategy for prevention. Here, we show that mice fed a high-fat/low-fiber "Western-type" diet (WD) had dramatically increased mortality in a murine model of antibiotic-induced CDI compared to a low-fat/low-fiber (LF/LF) diet and standard mouse chow controls. We found that the WD had a pro- C. difficile bile acid composition that was driven in part by higher levels of primary bile acids that are produced to digest fat, and a lower level of secondary bile acids that are produced by the gut microbiome. This lack of secondary bile acids was associated with a greater disturbance to the gut microbiome with antibiotics in both the WD and LF/LF diet compared to mouse chow. Mice fed the WD also had the highest level of toxin TcdA just prior to the onset of mortality, but not of TcdB or increased inflammation. These findings indicate that dietary intervention to decrease fat may complement previously proposed dietary intervention strategies to prevent CDI in high-risk individuals.

Gastric injury secondary to button battery ingestions: a retrospective multicenter review.

BACKGROUND AND AIMS: Removal of gastric button batteries (BBs) remains controversial. Our aim was to better define the spectrum of injury and to characterize clinical factors associated with injury from retained gastric BBs. METHODS: In this multicenter retrospective cohort study from January 2014 through May 2018, pediatric gastroenterologists from 4 pediatric tertiary care centers identified patients, aged 0 to 18 years, who had a retained gastric BB on radiography and subsequently underwent endoscopic assessment. Demographic and clinical information were abstracted from electronic health records using a standard data collection form. RESULTS: Sixty-eight patients with a median age of 2.5 years underwent endoscopic retrieval of a gastric BB. At presentation, 17 (25%) were symptomatic. Duration from ingestion to endoscopic removal was known for 65 patients (median, 9 hours [interquartile range, 5-19]). Median time from ingestion to first radiographic evaluation was 2 hours. At endoscopic removal, 60% of cases had visual evidence of mucosal damage, which correlated with duration of BB retention (P = .0018). Time to retrieval of the BB was not statistically significant between symptomatic and asymptomatic subjects (P = .12). After adjusting for age and symptoms, the likelihood of visualizing gastric damage among patients who had BBs removed 12 hours post ingestion was 4.5 times that compared with those with BB removal within 12 hours of ingestion. CONCLUSIONS: In this study, swallowed BBs posed a risk of damage to the stomach, including a single case of impaction and perforation of the gastric wall. Clinicians may want to consider retrieval within 12 hours of ingestion of gastric BBs. Larger prospective studies to assess risk of injury are needed.

Multiple-Ascending-Dose Phase 1 Clinical Study of the Safety, Tolerability, and Pharmacokinetics of CRS3123, a Narrow-Spectrum Agent with Minimal Disruption of Normal Gut Microbiota.

CRS3123 is a novel small molecule that potently inhibits methionyl-tRNA synthetase of Clostridioides difficile, inhibiting C. difficile toxin production and spore formation. CRS3123 has been evaluated in a multiple-ascending-dose placebo-controlled phase 1 trial. Thirty healthy subjects, ages 18 to 45 years, were randomized into three cohorts of 10 subjects each, receiving either 200, 400, or 600 mg of CRS3123 (8 subjects per cohort) or placebo (2 subjects per cohort) by oral administration twice daily for 10 days. CRS3123 was generally safe and well tolerated, with no serious adverse events (SAEs) or severe treatment-emergent adverse events (TEAEs) reported. All subjects completed their assigned treatment and follow-up visits, and there were no trends in systemic, vital sign, or laboratory TEAEs. There were no QTcF interval changes or any clinically significant changes in other electrocardiogram (ECG) intervals or morphology. CRS3123 showed limited but detectable systemic uptake; although absorption increased with increasing dose, the increase was less than dose proportional. Importantly, the bulk of the oral dose was not absorbed, and fecal concentrations were substantially above the MIC90 value of 1 µg/ml at all dosages tested. Subjects receiving either of the two lower doses of CRS3123 exhibited minimal disruption of normal gut microbiota after 10 days of twice-daily dosing. CRS3123 was inactive against important commensal anaerobes, including Bacteroides, bifidobacteria, and commensal clostridia. Microbiome data showed favorable differentiation compared to other CDI therapeutics. These results support further development of CRS3123 as an oral agent for the treatment of CDI. (This study has been registered at Clinicaltrials.gov under identifier NCT02106338.).

Low diversity gut microbiota dysbiosis: drivers, functional implications and recovery.

Dysbiosis, an imbalance in microbial communities, is linked with disease when this imbalance disturbs microbiota functions essential for maintaining health or introduces processes that promote disease. Dysbiosis in disease is predicted when microbiota differ compositionally from a healthy control population, but only truly defined when these differences are mechanistically related to adverse phenotypes. For the human gut microbiota, dysbiosis varies across diseases. One common manifestation is replacement of the complex community of anaerobes typical of the healthy adult gut microbiome with a community of lower overall microbial diversity and increased facultative anaerobes. Here we review diseases in which low-diversity dysbiosis has been observed and mechanistically linked with disease, with a particular focus on liver disease, inflammatory bowel disease, and Clostridium difficile infection.

Transition state analogues of Plasmodium falciparum and human orotate phosphoribosyltransferases.

The survival and proliferation of Plasmodium falciparum parasites and human cancer cells require de novo pyrimidine synthesis to supply RNA and DNA precursors. Orotate phosphoribosyltransferase (OPRT) is an indispensible component in this metabolic pathway and is a target for antimalarials and antitumor drugs. P. falciparum (Pf) and Homo sapiens (Hs) OPRTs are characterized by highly dissociative transition states with ribocation character. On the basis of the geometrical and electrostatic features of the PfOPRT and HsOPRT transition states, analogues were designed, synthesized, and tested as inhibitors. Iminoribitol mimics of the ribocation transition state in linkage to pyrimidine mimics using methylene or ethylene linkers gave dissociation constants (Kd) as low as 80 nM. Inhibitors with pyrrolidine groups as ribocation mimics displayed slightly weaker binding affinities for OPRTs. Interestingly, p-nitrophenyl riboside 5'-phosphate bound to OPRTs with Kd values near 40 nM. Analogues designed with a C5-pyrimidine carbon-carbon bond to ribocation mimics gave Kd values in the range of 80-500 nM. Acyclic inhibitors with achiral serinol groups as the ribocation mimics also displayed nanomolar inhibition against OPRTs. In comparison with the nucleoside derivatives, inhibition constants of their corresponding 5'-phosphorylated transition state analogues are largely unchanged, an unusual property for a nucleotide-binding site. In silico docking of the best inhibitor into the HsOPRT active site supported an extensive hydrogen bond network associated with the tight binding affinity. These OPRT transition state analogues identify crucial components of potent inhibitors targeting OPRT enzymes. Despite their tight binding to the targets, the inhibitors did not kill cultured P. falciparum.

Acyclic phosph(on)ate inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase.

The pathogenic protozoa responsible for malaria lack enzymes for the de novo synthesis of purines and rely on purine salvage from the host. In Plasmodium falciparum (Pf), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) converts hypoxanthine to inosine monophosphate and is essential for purine salvage making the enzyme an anti-malarial drug target. We have synthesized a number of simple acyclic aza-C-nucleosides and shown that some are potent inhibitors of Pf HGXPRT while showing excellent selectivity for the Pf versus the human enzyme.

Acyclic immucillin phosphonates: second-generation inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase.

Plasmodium falciparum, the primary cause of deaths from malaria, is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. Here, we present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.

Plasmodium falciparum parasites are killed by a transition state analogue of purine nucleoside phosphorylase in a primate animal model.

Plasmodium falciparum causes most of the one million annual deaths from malaria. Drug resistance is widespread and novel agents against new targets are needed to support combination-therapy approaches promoted by the World Health Organization. Plasmodium species are purine auxotrophs. Blocking purine nucleoside phosphorylase (PNP) kills cultured parasites by purine starvation. DADMe-Immucillin-G (BCX4945) is a transition state analogue of human and Plasmodium PNPs, binding with picomolar affinity. Here, we test BCX4945 in Aotus primates, an animal model for Plasmodium falciparum infections. Oral administration of BCX4945 for seven days results in parasite clearance and recrudescence in otherwise lethal infections of P. falciparum in Aotus monkeys. The molecular action of BCX4945 is demonstrated in crystal structures of human and P. falciparum PNPs. Metabolite analysis demonstrates that PNP blockade inhibits purine salvage and polyamine synthesis in the parasites. The efficacy, oral availability, chemical stability, unique mechanism of action and low toxicity of BCX4945 demonstrate potential for combination therapies with this novel antimalarial agent.

Purine and pyrimidine pathways as targets in Plasmodium falciparum.

Malaria is a leading cause of morbidity and mortality in the tropics. Chemotherapeutic and vector control strategies have been applied for more than a century but have not been efficient in disease eradication. Increased resistance of malaria parasites to drug treatment and of mosquito vectors to insecticides requires the development of novel chemotherapeutic agents. Malaria parasites exhibit rapid nucleic acid synthesis during their intraerythrocytic growth phase. Plasmodium purine and pyrimidine metabolic pathways are distinct from those of their human hosts. Thus, targeting purine and pyrimidine metabolic pathways provides a promising route for novel drug development. Recent developments in enzymatic transition state analysis have provided an improved route to inhibitor design targeted to specific enzymes, including those of purine and pyrimidine metabolism. Modern transition state analogue drug discovery has resulted in transition state analogues capable of binding to target enzymes with unprecedented affinity and specificity. These agents can provide specific blocks in essential pathways. The combination of tight binding with the high specificity of these logically designed inhibitors, results in low toxicity and minor side effects. These features reduce two of the major problems with the current antimalarials. Transition state analogue design is being applied to generate new lead compounds to treat malaria by targeting purine and pyrimidine pathways.

Transport of purines and purine salvage pathway inhibitors by the Plasmodium falciparum equilibrative nucleoside transporter PfENT1.

Plasmodium falciparum is a purine auxotroph. The transport of purine nucleosides and nucleobases from the host erythrocyte to the parasite cytoplasm is essential to support parasite growth. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a major route for purine transport across the parasite plasma membrane. Malarial parasites are sensitive to inhibitors of purine salvage pathway enzymes. The immucillin class of purine nucleoside phosphorylase inhibitors and the adenosine analog, tubercidin, block growth of P. falciparum under in vitro culture conditions. We sought to determine whether these inhibitors utilize PfENT1 to gain access to the parasite cytosol. There is considerable controversy in the literature regarding the K(m) and/or K(i) for purine transport by PfENT1 in the Xenopus oocyte expression system. We show that oocytes metabolize adenosine but not hypoxanthine. For adenosine, metabolism is the rate limiting step in oocyte uptake assays, making hypoxanthine the preferred substrate for PfENT1 transport studies in oocytes. We demonstrate that the K(i) for PfENT1 transport of hypoxanthine and adenosine is in the 300-700microM range. Effects of substrate metabolism on uptake studies may explain conflicting results in the literature regarding the PfENT1 adenosine transport K(m). PfENT1 transports the tubercidin class of compounds. None of the immucillin compounds tested inhibited PfENT1 transport of [(3)H]hypoxanthine or [(3)H]adenosine. Although nucleobases are transported, modifications of the ribose ring in corresponding nucleoside analogs affect substrate recognition by PfENT1. These results provide new insights into PfENT1 and the mechanism by which purine salvage pathway inhibitors are transported into the parasite cytoplasm.

Erythrocytic adenosine monophosphate as an alternative purine source in Plasmodium falciparum.

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.

Anopheles gambiae purine nucleoside phosphorylase: catalysis, structure, and inhibition.

The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has kcat values of 54 and 41 s-1 for 2'-deoxyinosine and inosine, its preferred substrates, and 1.0 s-1 for guanosine. However, the chemical step is fast for AgPNP at 226 s-1 for guanosine in pre-steady-state studies. 5'-Deaza-1'-aza-2'-deoxy-1'-(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2'-deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable Km/Ki* of 5.4 x 10(7), and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 A to reveal the differences in substrate and inhibitor specificity. The distance from the N1' cation to the phosphate O4 anion is shorter in the AgPNP.DADMe-ImmH.PO4 complex than in HsPNP.DADMe-ImmH.SO4, offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.

Structural characterization of the fusion of two pentapeptide repeat proteins, Np275 and Np276, from Nostoc punctiforme: resurrection of an ancestral protein.

The Nostoc punctiforme genes Np275 and Np276 are two adjacently encoded proteins of 98 and 75 amino acids in length and exhibit sequences composed of tandem pentapeptide repeats. The structures of Np275 and a fusion of Np275 and Np276 were determined to 2.1 and 1.5 A, respectively. The two Nostoc proteins fold as highly symmetric right-handed quadrilateral beta-helices similar to the mycobacterial protein MfpA implicated in fluoroquinolone resistance and DNA gyrase inhibition. The sequence composition of the intervening coding region and the ability to express a fused protein by removing the stop codon for Np275 suggests Np275 and Np276 were recently part of a larger ancestral pentapeptide repeat protein.