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.
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.
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.
Current therapies for recurrent
infection do not address the disrupted microbiome, which supports
spore germination into toxin-producing bacteria. SER-109 is an investigational microbiome therapeutic ...composed of purified Firmicutes spores for the treatment of recurrent
infection.
We conducted a phase 3, double-blind, randomized, placebo-controlled trial in which patients who had had three or more episodes of
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
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.
Among the 281 patients screened, 182 were enrolled. The percentage of patients with recurrence of
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
spore germination.
In patients with symptom resolution of
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.).
Abstract
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.
In a phase 2 trial, SER-109, an investigational microbiome drug, did not reduce rates of recurrent CDI, despite a previously successful open-label study. Key contributing factors, which led to a redesign of the currently enrolling phase 3 trial, are highlighted.
The absence of the putative DNA/RNA helicase Schlafen11 (SLFN11) is thought to cause resistance to DNA-damaging agents (DDAs) and PARP inhibitors.
We developed and validated a clinically applicable ...SLFN11 immunohistochemistry assay and retrospectively correlated SLFN11 tumour levels to patient outcome to the standard of care therapies and olaparib maintenance.
High SLFN11 associated with improved prognosis to the first-line treatment with DDAs platinum-plus-etoposide in SCLC patients, but was not strongly linked to paclitaxel-platinum response in ovarian cancer patients. Multivariate analysis of patients with relapsed platinum-sensitive ovarian cancer from the randomised, placebo-controlled Phase II olaparib maintenance Study19 showed SLFN11 tumour levels associated with sensitivity to olaparib. Study19 patients with high SLFN11 had a lower progression-free survival (PFS) hazard ratio compared to patients with low SLFN11, although both groups had the benefit of olaparib over placebo. Whilst caveated by small sample size, this trend was maintained for PFS, but not overall survival, when adjusting for BRCA status across the olaparib and placebo treatment groups, a key driver of PARP inhibitor sensitivity.
We provide clinical evidence supporting the role of SLFN11 as a DDA therapy selection biomarker in SCLC and highlight the need for further clinical investigation into SLFN11 as a PARP inhibitor predictive biomarker.
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.
ObjectivesFollowing up trauma patients after discharge, to evaluate their subsequent quality of life and functional outcomes, is notoriously difficult, time consuming, and expensive. Automated ...systems are a conceptually attractive solution. We prospectively assessed the feasibility of using a series of automated phone calls administered by Emmi Patient Engagement to survey trauma patients after discharge.MethodsRecruitment into the study was incorporated into the patient discharge process by nursing staff. For this pilot, we included trauma patients discharging home and who were able to answer phone calls. A script was created to evaluate the Extended Glasgow Outcome Scale and the EuroQol EQ-5D to assess functional status and quality of life, respectively. Call attempts were made at 6 weeks, 3 months, 6 months, and 1 year after discharge.ResultsA total of 110 patients initially agreed to participate. 368 attempted patient encounters (calls or attempted calls) took place, with 104 (28.3%) patients answering a least one question in the study. 21 unique patients (19.1% of those enrolled) completed 27 surveys.ConclusionsAutomated, scripted phone calls to survey patients after discharge are not a feasible way of collecting functional and quality of life data.Level of evidenceLevel II/prospective.
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.
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.
YbeY extensively modulates Hfq-dependent and independent sRNA-mRNA interactions. YbeY-dependent sRNAs have central roles in modulating cellular response to HU stress.
PPARγ regulates multiple aspects of skin physiology, including sebocyte differentiation, keratinocyte proliferation, epithelial stem cell survival, adipocyte biology, and inflammatory skin responses. ...However, the effects of its global deletion, namely of nonredundant key functions of PPARγ signaling in mammalian skin, are yet unknown because of embryonic lethality. Here, we describe the skin and hair phenotype of a whole-body PPARγ-null mouse (PpargΔ/Δ), obtained by preserving PPARγ expression in the placenta. PpargΔ/Δ mice exhibited total lipoatrophy and complete absence of sebaceous glands. Right after birth, hair follicle (HF) morphogenesis was transiently delayed, along with reduced expression of HF differentiation markers and of transcriptional regulators necessary for HF development. Later, adult PpargΔ/Δ mice developed scarring alopecia and severe perifollicular inflammation. Skin analyses in other models of lipodystrophy, AZIPtg/+ and Adipoq-Cretg/+Ppargfl/fl mice, coupled with skin graft experiments, showed that the early defects observed in hair morphogenesis were caused by the absence of adipose tissue. In contrast, the late alteration of HF cycle and appearance of inflammation were observed only in PpargΔ/Δ mice and likely were due to the lack sebaceous glands. Our findings underscore the increasing appreciation for the importance of adipose tissue-mediated signals in HF development and function.
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