SER-109, an Oral Microbiome Therapy for Recurrent Clostridioides difficile Infection.

BACKGROUND: Current therapies for recurrent Clostridioides difficile infection do not address the disrupted microbiome, which supports C. difficile spore germination into toxin-producing bacteria. SER-109 is an investigational microbiome therapeutic composed of purified Firmicutes spores for the treatment of recurrent C. difficile infection. METHODS: We conducted a phase 3, double-blind, randomized, placebo-controlled trial in which patients who had had three or more episodes of C. difficile infection (inclusive of the qualifying acute episode) received SER-109 or placebo (four capsules daily for 3 days) after standard-of-care antibiotic treatment. The primary efficacy objective was to show superiority of SER-109 as compared with placebo in reducing the risk of C. difficile infection recurrence up to 8 weeks after treatment. Diagnosis by toxin testing was performed at trial entry, and randomization was stratified according to age and antibiotic agent received. Analyses of safety, microbiome engraftment, and metabolites were also performed. RESULTS: Among the 281 patients screened, 182 were enrolled. The percentage of patients with recurrence of C. difficile infection was 12% in the SER-109 group and 40% in the placebo group (relative risk, 0.32; 95% confidence interval [CI], 0.18 to 0.58; P<0.001 for a relative risk of <1.0; P<0.001 for a relative risk of <0.833). SER-109 led to less frequent recurrence than placebo in analyses stratified according to age stratum (relative risk, 0.24 [95% CI, 0.07 to 0.78] for patients <65 years of age and 0.36 [95% CI, 0.18 to 0.72] for those ≥65 years) and antibiotic received (relative risk, 0.41 [95% CI, 0.22 to 0.79] with vancomycin and 0.09 [95% CI, 0.01 to 0.63] with fidaxomicin). Most adverse events were mild to moderate and were gastrointestinal in nature, with similar numbers in the two groups. SER-109 dose species were detected as early as week 1 and were associated with bile-acid profiles that are known to inhibit C. difficile spore germination. CONCLUSIONS: In patients with symptom resolution of C. difficile infection after treatment with standard-of-care antibiotics, oral administration of SER-109 was superior to placebo in reducing the risk of recurrent infection. The observed safety profile of SER-109 was similar to that of placebo. (Funded by Seres Therapeutics; ECOSPOR III ClinicalTrials.gov number, NCT03183128.).

SER-109, an Investigational Microbiome Drug to Reduce Recurrence After Clostridioides difficile Infection: Lessons Learned From a Phase 2 Trial.

BACKGROUND: Recurrent Clostridioides difficile infection (rCDI) is associated with loss of microbial diversity and microbe-derived secondary bile acids, which inhibit C. difficile germination and growth. SER-109, an investigational microbiome drug of donor-derived, purified spores, reduced recurrence in a dose-ranging, phase (P) 1 study in subjects with multiple rCDIs. METHODS: In a P2 double-blind trial, subjects with clinical resolution on standard-of-care antibiotics were stratified by age (< or ≥65 years) and randomized 2:1 to single-dose SER-109 or placebo. Subjects were diagnosed at study entry by PCR or toxin testing. Safety, C. difficile-positive diarrhea through week 8, SER-109 engraftment, and bile acid changes were assessed. RESULTS: 89 subjects enrolled (67% female; 80.9% diagnosed by PCR). rCDI rates were lower in the SER-109 arm than placebo (44.1% vs 53.3%) but did not meet statistical significance. In a preplanned analysis, rates were reduced among subjects ≥65 years (45.2% vs 80%, respectively; RR, 1.77; 95% CI, 1.11-2.81), while the <65 group showed no benefit. Early engraftment of SER-109 was associated with nonrecurrence (P < .05) and increased secondary bile acid concentrations (P < .0001). Whole-metagenomic sequencing from this study and the P1 study revealed previously unappreciated dose-dependent engraftment kinetics and confirmed an association between early engraftment and nonrecurrence. Engraftment kinetics suggest that P2 dosing was suboptimal. Adverse events were generally mild to moderate in severity. CONCLUSIONS: Early SER-109 engraftment was associated with reduced CDI recurrence and favorable safety was observed. A higher dose of SER-109 and requirements for toxin testing were implemented in the current P3 trial. CLINICAL TRIALS REGISTRATION: NCT02437487, https://clinicaltrials.gov/ct2/show/NCT02437487?term=SER-109&draw= 2&rank=4.

Hydroxyurea induces hydroxyl radical-mediated cell death in Escherichia coli.

Hydroxyurea (HU) specifically inhibits class I ribonucleotide reductase (RNR), depleting dNTP pools and leading to replication fork arrest. Although HU inhibition of RNR is well recognized, the mechanism by which it leads to cell death remains unknown. To investigate the mechanism of HU-induced cell death, we used a systems-level approach to determine the genomic and physiological responses of E. coli to HU treatment. Our results suggest a model by which HU treatment rapidly induces a set of protective responses to manage genomic instability. Continued HU stress activates iron uptake and toxins MazF and RelE, whose activity causes the synthesis of incompletely translated proteins and stimulation of envelope stress responses. These effects alter the properties of one of the cell's terminal cytochrome oxidases, causing an increase in superoxide production. The increased superoxide production, together with the increased iron uptake, fuels the formation of hydroxyl radicals that contribute to HU-induced cell death.

A Novel Microbiome Therapeutic Increases Gut Microbial Diversity and Prevents Recurrent Clostridium difficile Infection.

BACKGROUND: Patients with recurrent Clostridium difficile infection (CDI) have a ≥60% risk of relapse, as conventional therapies do not address the underlying gastrointestinal dysbiosis. This exploratory study evaluated the safety and efficacy of bacterial spores for preventing recurrent CDI. METHODS: Stool specimens from healthy donors were treated with ethanol to eliminate pathogens. The resulting spores were fractionated and encapsulated for oral delivery as SER-109. Following their response to standard-of-care antibiotics, patients in cohort 1 were treated with SER-109 on 2 consecutive days (geometric mean dose, 1.7 × 10(9) spores), and those in cohort 2 were treated on 1 day (geometric mean dose, 1.1 × 10(8) spores). The primary efficacy end point was absence of C. difficile-positive diarrhea during an 8-week follow-up period. Microbiome alterations were assessed. RESULTS: Thirty patients (median age, 66.5 years; 67% female) were enrolled, and 26 (86.7%) met the primary efficacy end point. Three patients with early, self-limiting C. difficile-positive diarrhea did not require antibiotics and tested negative for C. difficile at 8 weeks; thus, 96.7% (29 of 30) achieved clinical resolution. In parallel, gut microbiota rapidly diversified, with durable engraftment of spores and no outgrowth of non-spore-forming bacteria found after SER-109 treatment. Adverse events included mild diarrhea, abdominal pain, and nausea. CONCLUSIONS: SER-109 successfully prevented CDI and had a favorable safety profile, supporting a novel microbiome-based intervention as a potential therapy for recurrent CDI.

Central role for RNase YbeY in Hfq-dependent and Hfq-independent small-RNA regulation in bacteria.

BACKGROUND: Conceptual parallels exist between bacterial and eukaryotic small-RNA (sRNA) pathways, yet relatively little is known about which protein may recognize and recruit bacterial sRNAs to interact with targets. In eukaryotes, Argonaute (AGO) proteins discharge such functions. The highly conserved bacterial YbeY RNase has structural similarities to the MID domain of AGOs. A limited study had indicated that in Sinorhizobium meliloti the YbeY ortholog regulates the accumulation of sRNAs as well as the target mRNAs, raising the possibility that YbeY may play a previously unrecognized role in bacterial sRNA regulation. RESULTS: We have applied a multipronged approach of loss-of-function studies, genome-wide mRNA and sRNA expression profiling, pathway analysis, target prediction, literature mining and network analysis to unravel YbeY-dependent molecular responses of E. coli exposed to hydroxyurea (HU). Loss of ybeY function, which results in a marked resistance to HU, had global affects on sRNA-mediated gene expression. Of 54 detectable E. coli sRNAs in our microarray analysis, 30 sRNAs showed a differential expression upon HU stress, of which 28 sRNAs displayed a YbeY-dependent change in expression. These included 12 Hfq-dependent and 16 Hfq-independent sRNAs. We successfully identified at least 57 experimentally inferred sRNA-mRNA relationships. Further applying a 'context likelihood of relatedness' algorithm, we reverse engineered the YbeY-dependent Hfq-dependent sRNA-mRNA network as well as YbeY-dependent Hfq-independent sRNA-mRNA network. CONCLUSION: YbeY extensively modulates Hfq-dependent and independent sRNA-mRNA interactions. YbeY-dependent sRNAs have central roles in modulating cellular response to HU stress.

Intracerebral hemorrhage with hypothyroidism.

BACKGROUND: Hypothyroidism is associated with increased ischemic stroke risk but paradoxically results in more favorable outcomes once a stroke occurs. Whether a similar pattern emerges in patients with primary intracerebral hemorrhage (ICH) is unknown. METHODS: A retrospective analysis of a prospective stroke center database was performed to analyze the clinical presentation and outcomes of hypothyroid patients with spontaneous ICH. Patients were classified into groups with no history of thyroid disease (n=491) versus those with hypothyroidism (n=72). Hypothyroid patients were further classified into patients receiving thyroid replacement on admission or those without replacement. The Glasgow Coma Scale, ICH score, and the National Institutes of Health Stroke Scale (NIHSS) were used to assess the initial severity. Outcome was assessed by admission to discharge change in the NIHSS and modified Barthel Index (mBI), in-hospital mortality, discharge disposition and mortality, and the mBI at 3 and 12 months. RESULTS: There were 563 patients in the analysis. Seventy-two patients had a history of hypothyroidism, and of these, 63% received thyroid hormone replacement. Patients receiving replacement had significantly lower NIHSS at presentation (median 4 [IQR 1, 11]) compared with either the control group (median 8 [IQR 3, 16]) or hypothyroid patients without replacement (median 9 [IQR 3.8, 15.5]; P=.004). There was no difference in in-hospital and 3-month mortality or functional outcomes at 3 and 12 months among the groups. CONCLUSIONS: This study suggests that the history of hypothyroidism does not affect clinical severity or outcome after ICH.

Long-term improvement in outcome after intracerebral hemorrhage in patients treated with statins.

BACKGROUND: Intracerebral hemorrhage (ICH) is a severe type of stroke for which there is currently no specific medical therapy. We hypothesized that statins reduce immediate inflammatory injury and improve long-term recovery from increased neurogenesis and angiogenesis. We conducted a large retrospective cohort study to assess the influence of statin therapy on patient death and disability at 12 months after ICH. METHODS: This was a retrospective analysis of a prospectively collected database at a tertiary care medical center. Patients were grouped based on statin use, and poor outcome was assessed as dead or alive with dependency (modified Barthel Index≤14). RESULTS: We compared outcomes in 190 patients exposed to statins to 236 patients who were not exposed to statins. Univariate analysis found that statin use was associated with decreased mortality in-hospital and at 12 months (P=.001). Multivariable analysis found that statin use was associated with a decreased odds of death or disability at 12 months after ICH (odds ratio 0.44; 95% confidence interval 0.21-0.95). CONCLUSIONS: Statin use is associated with improved long-term outcome at 12 months after ICH. This finding supports previous clinical studies that have shown the short-term benefits of statin therapy. In addition, this study correlates with animal studies supporting the possible long-term recovery benefits of statins.

Gemella Endocarditis Presenting as an ST-Segment-Elevation Myocardial Infarction.

Acute myocardial infarction from septic embolization is a rare initial presentation of endocarditis. We report the case of a 67-year-old man who presented with acute chest pain, in whom emergency cardiac catheterization revealed findings that suggested coronary embolism. The patient was found to have Gemella endocarditis, with its initial presentation an embolic acute ST-segment-elevation myocardial infarction. We suggest that endocarditis be considered among the potential causes of acute myocardial infarction.

Cryoglobulinemic membranoproliferative glomerulonephritis associated with mucosa-associated lymphoid tissue lymphoma treated with rituximab.

Cryoglobulinemia and mucosa-associated lymphoid tissue (MALT) lymphoma are diseases characterized by B-cell dysregulation and overproduction of antibodies. Vasculitis and cutaneous manifestations are common, but renal involvement is rare. A 65-year-old woman with type 1 cryoglobulinemia and MALT lymphomas of the right lacrimal and parotid glands successfully treated by excision and chemoradiotherapy, presented with dyspnea on exertion, edema, and hematuria. Renal biopsy findings revealed type 1 cryoglobulinemic glomerulonephritis. She underwent treatment with high-dose oral prednisone and intravenous rituximab with subsequent return of creatinine to baseline levels. To our knowledge, this is the first report of a patient in whom type 1 cryoglobulinemia, multiple MALT lymphomas, and MPGN with IgM cryoglobulin deposits coexist. Evidence for rituximab is sparse with widely varying protocols and mixed results. There is a need for high quality evidence in the treatment of these conditions.

Provider Board Certification Status and Practice Patterns in Total Knee Arthroplasty.

PURPOSE: The presumption that board certification directly affects the quality of clinical care is a topic of ongoing discussion in medical literature. Recent studies have demonstrated disparities in patient outcomes associated with type of anesthesia provided for total knee arthroplasty (TKA); improved outcomes are associated with neuraxial (or regional) versus general anesthesia. Whether board-certified (BC) and non-board-certified (nBC) anesthesiologists make different choices in the anesthetic they administer is unknown. The authors sought to study potential associations of board certification status with anesthesia practice patterns for TKA. METHOD: The authors accessed records of anesthetics provided from 2010 to 2013 from the National Anesthesia Clinical Outcomes Registry database. They identified TKA cases using Clinical Classifications Software and Current Procedural Terminology codes. The authors divided practitioners into two groups: those who were BC and those who were nBC. For each of these groups, the authors compared the following: their patient populations, the hospitals in which they worked, the nature of their practices, and the anesthetics they administered to their patients. RESULTS: BC anesthesiologists provided care for 81.7% of 97,508 patients having TKA; 18.3% were treated by nBC anesthesiologists. BC anesthesiologists administered neuraxial/regional anesthesia more frequently than nBC anesthesiologists (41.4% versus 21.2%; P < .001). CONCLUSIONS: The rates at which regional/neuraxial anesthesia were administered for TKA were relatively low, and there were significant differences in practice patterns of BC and nBC anesthesiologists providing care for patients undergoing TKA. More research is necessary to understand the causes of these disparities.

Variability in anesthetic care for total knee arthroplasty: an analysis from the anesthesia quality institute.

Anesthetic practice utilization and related characteristics of total knee arthroplasties (TKAs) are understudied. The research team sought to characterize anesthesia practice patterns by utilizing National Anesthesia Clinical Outcomes Registry data of the Anesthesia Quality Institute. The proportions of primary TKAs performed between January 2010 and June 2013 using general anesthesia (GA), neuraxial anesthesia (NA), and regional anesthesia (RA) were determined. Utilization of anesthesia types was analyzed using anesthesiologist and patient characteristics and facility type. In all, 108 625 eligible TKAs were identified; 10.9%, 31.3%, and 57.9% were performed under RA, NA, and GA, respectively. Patients receiving RA had higher median age and higher frequency of American Society of Anesthesiology score ≥3 compared with those receiving other anesthesia types under study. Relative to GA (45.0%), when NA or RA were used, the anesthesiologist was more frequently board certified (75.5% and 62.1%, respectively; P < .0001). Anesthetic technique differences for TKAs exist, with variability associated with patient and provider characteristics.

Identification and characterization of programmed cell death markers in bacterial models.

In eukaryotic organisms facing terminal stress, activation of genetically encoded cell death pathways underlies fundamental changes in core cellular processes and functional modification of critical biomolecules. These physiological alterations manifest themselves as phenotypic hallmarks during programmed cell death, and are markers of the particular mode of death initiated. A growing volume of work has illustrated that prokaryotes too are capable of exhibiting hallmarks of programmed cell death, albeit without the multiple, tight regulatory layers which control these events in higher order organisms.This chapter describes how methods and materials which have been used to assay for hallmarks of programmed cell death in eukaryotic models are transferrable to prokaryotic models. In particular, we describe the applicability of these methods to the study of post-antibiotic effects on bacteria, notably the biochemical changes induced by the interaction of drug molecules and targets, including oxidative stress, that accompany and ensure cell death. Specifically we discuss techniques for detecting DNA fragmentation, chromosomal condensation, phosphatidylserine exposure, membrane depolarization, and caspase substrate peptide binding, thereby providing a launchpoint for the study of the evolution of these physiological events in bacteria.

Potentiating antibacterial activity by predictably enhancing endogenous microbial ROS production.

The ever-increasing incidence of antibiotic-resistant infections combined with a weak pipeline of new antibiotics has created a global public health crisis. Accordingly, novel strategies for enhancing our antibiotic arsenal are needed. As antibiotics kill bacteria in part by inducing reactive oxygen species (ROS), we reasoned that targeting microbial ROS production might potentiate antibiotic activity. Here we show that ROS production can be predictably enhanced in Escherichia coli, increasing the bacteria's susceptibility to oxidative attack. We developed an ensemble approach of genome-scale, metabolic models capable of predicting ROS production in E. coli. The metabolic network was systematically perturbed and its flux distribution analyzed to identify targets predicted to increase ROS production. Targets that were predicted in silico were experimentally validated and further shown to confer increased susceptibility to oxidants. Validated targets also increased susceptibility to killing by antibiotics. This work establishes a systems-based method to tune ROS production in bacteria and demonstrates that increased microbial ROS production can potentiate killing by oxidants and antibiotics.

Silver enhances antibiotic activity against gram-negative bacteria.

A declining pipeline of clinically useful antibiotics has made it imperative to develop more effective antimicrobial therapies, particularly against difficult-to-treat Gram-negative pathogens. Silver has been used as an antimicrobial since antiquity, yet its mechanism of action remains unclear. We show that silver disrupts multiple bacterial cellular processes, including disulfide bond formation, metabolism, and iron homeostasis. These changes lead to increased production of reactive oxygen species and increased membrane permeability of Gram-negative bacteria that can potentiate the activity of a broad range of antibiotics against Gram-negative bacteria in different metabolic states, as well as restore antibiotic susceptibility to a resistant bacterial strain. We show both in vitro and in a mouse model of urinary tract infection that the ability of silver to induce oxidative stress can be harnessed to potentiate antibiotic activity. Additionally, we demonstrate in vitro and in two different mouse models of peritonitis that silver sensitizes Gram-negative bacteria to the Gram-positive-specific antibiotic vancomycin, thereby expanding the antibacterial spectrum of this drug. Finally, we used silver and antibiotic combinations in vitro to eradicate bacterial persister cells, and show both in vitro and in a mouse biofilm infection model that silver can enhance antibacterial action against bacteria that produce biofilms. This work shows that silver can be used to enhance the action of existing antibiotics against Gram-negative bacteria, thus strengthening the antibiotic arsenal for fighting bacterial infections.

Oxidation of the guanine nucleotide pool underlies cell death by bactericidal antibiotics.

A detailed understanding of the mechanisms that underlie antibiotic killing is important for the derivation of new classes of antibiotics and clinically useful adjuvants for current antimicrobial therapies. Our efforts to understand why DinB (DNA polymerase IV) overproduction is cytotoxic to Escherichia coli led to the unexpected insight that oxidation of guanine to 8-oxo-guanine in the nucleotide pool underlies much of the cell death caused by both DinB overproduction and bactericidal antibiotics. We propose a model in which the cytotoxicity of beta-lactams and quinolones predominantly results from lethal double-strand DNA breaks caused by incomplete repair of closely spaced 8-oxo-deoxyguanosine lesions, whereas the cytotoxicity of aminoglycosides might additionally result from mistranslation due to the incorporation of 8-oxo-guanine into newly synthesized RNAs.