Staphylococcus aureus Overcomes Anaerobe-Derived Short-Chain Fatty Acid Stress via FadX and the CodY Regulon.

Chronic rhinosinusitis (CRS) is characterized by immune dysfunction, mucus hypersecretion, and persistent infection of the paranasal sinuses. While Staphylococcus aureus is a primary CRS pathogen, recent sequence-based surveys have found increased relative abundances of anaerobic bacteria, suggesting that S. aureus may experience altered metabolic landscapes in CRS relative to healthy airways. To test this possibility, we characterized the growth kinetics and transcriptome of S. aureus in supernatants of the abundant CRS anaerobe Fusobacterium nucleatum. While growth was initially delayed, S. aureus ultimately grew to similar levels as in the control medium. The transcriptome was significantly affected by F. nucleatum metabolites, with the agr quorum sensing system notably repressed. Conversely, expression of fadX, encoding a putative propionate coenzyme A (CoA)-transferase, was significantly increased, leading to our hypothesis that short-chain fatty acids (SCFAs) produced by F. nucleatum could mediate S. aureus growth behavior and gene expression. Supplementation with propionate and butyrate, but not acetate, recapitulated delayed growth phenotypes observed in F. nucleatum supernatants. A fadX mutant was found to be more sensitive than wild type to propionate, suggesting a role for FadX in the S. aureus SCFA stress response. Interestingly, spontaneous resistance to butyrate, but not propionate, was observed frequently. Whole-genome sequencing and targeted mutagenesis identified codY mutants as resistant to butyrate inhibition. Together, these data show that S. aureus physiology is dependent on its cocolonizing microbiota and metabolites they exchange and indicate that propionate and butyrate may act on different targets in S. aureus to suppress its growth. IMPORTANCE Staphylococcus aureus is an important CRS pathogen, and yet it is found in the upper airways of 30% to 50% of people without complications. The presence of strict and facultative anaerobic bacteria in CRS sinuses has recently spurred research into bacterial interactions and how they influence S. aureus physiology and pathogenesis. We show here that propionate and butyrate produced by one such CRS anaerobe, namely, Fusobacterium nucleatum, alter the growth and gene expression of S. aureus. We show that fadX is important for S. aureus to resist propionate stress and that the CodY regulon mediates growth in inhibitory concentrations of butyrate. This work highlights the possible complexity of S. aureus-anaerobe interactions and implicates membrane stress as a possible mechanism influencing S. aureus behavior in CRS sinuses.

Digital interventions for the management of chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with dyspnoea, cough or sputum production (or both) and affects quality of life and functional status. More efficient approaches to alternative management that may include patients themselves managing their condition need further exploration in order to reduce the impact on both patients and healthcare services. Digital interventions may potentially impact on health behaviours and encourage patient engagement. OBJECTIVES: To assess benefits and harms of digital interventions for managing COPD and apply Behaviour Change Technique (BCT) taxonomy to describe and explore intervention content. SEARCH METHODS: We identified randomised controlled trials (RCTs) from the Cochrane Airways Trials Register (date of last search 28 April 2020). We found other trials at web-based clinical trials registers. SELECTION CRITERIA: We included RCTs comparing digital technology interventions with or without routine supported self-management to usual care, or control treatment for self-management. Multi-component interventions (of which one component was digital self-management) compared with usual care, standard care or control treatment were included. DATA COLLECTION AND ANALYSIS: We used standard Cochrane methods. Two review authors independently selected trials for inclusion, extracted data, and assessed risk of bias. Discrepancies were resolved with a third review author. We assessed certainty of the evidence using the GRADE approach. Primary outcomes were impact on health behaviours, self-efficacy, exacerbations and quality of life, including the St George's Respiratory Questionnaire (SGRQ). The minimally important difference (MID) for the SGRQ is 4 points. Two review authors independently applied BCT taxonomy to identify mechanisms in the digital interventions that influence behaviours. MAIN RESULTS: Fourteen studies were included in the meta-analyses (1518 participants) ranging from 13 to 52 weeks duration. Participants had mild to very severe COPD. Risk of bias was high due to lack of blinding. GRADE ratings were low to very low certainty due to lack of blinding and imprecision. Common BCT clusters identified as behaviour change mechanisms in interventions were goals and planning, feedback and monitoring, social support, shaping knowledge and antecedents. Digital technology intervention with or without routine supported self-management Interventions included mobile phone (three studies), smartphone applications (one study), and web or Internet-based (five studies). Evidence is very uncertain about effects on impact on health behaviours as measured by six-minute walk distance (6MWD) at 13 weeks (mean difference (MD) 26.20, 95% confidence interval (CI) -21.70 to 74.10; participants = 122; studies = 2) or 23 to 26 weeks (MD 14.31, 95% CI -19.41 to 48.03; participants = 164; studies = 3). There may be improvement in 6MWD at 52 weeks (MD 54.33 95% CI -35.47 to 144.12; participants = 204; studies = 2) but studies were varied (very low certainty). There may be no difference in self-efficacy on managing Chronic Disease Scale (SEMCD) or pulmonary rehabilitation adapted index of self-efficacy tool (PRAISE). Evidence is very uncertain. Quality of life may be slightly improved on the chronic respiratory disease questionnaire (CRQ) at 13 weeks (MD 0.45, 95% CI 0.01 to 0.90; participants = 123; studies = 2; low certainty), but is not clinically important (MID 0.5). There may be little or no difference at 23 or 52 weeks (low to very low certainty). There may be a clinical improvement on SGRQ total at 52 weeks (MD -26.57, 95% CI -34.09 to -19.05; participants = 120; studies = 1; low certainty). Evidence for COPD assessment test (CAT) and Clinical COPD Questionnaire (CCQ) is very uncertain. There may be little or no difference in dyspnoea symptoms (CRQ dyspnoea) at 13, 23 weeks or 52 weeks (low to very low certainty evidence) or mean number of exacerbations at 26 weeks (low-certainty evidence). There was no evidence for the number of people experiencing adverse events. Multi-component interventions Digital components included mobile phone (one study), and web or internet-based (four studies). Evidence is very uncertain about effects on impact on health behaviour (6MWD) at 13 weeks (MD 99.60, 95% CI -15.23 to 214.43; participants = 20; studies = 1). No evidence was found for self-efficacy. Four studies reported effects on quality of life (SGRQ and CCQ scales). The evidence is very uncertain. There may be no difference in the number of people experiencing exacerbations or mean days to first exacerbation at 52 weeks with a multi-component intervention compared to standard care. Evidence is very uncertain about effects on the number of people experiencing adverse events at 52 weeks. AUTHORS' CONCLUSIONS: There is insufficient evidence to demonstrate a clear benefit or harm of digital technology interventions with or without supported self-management, or multi-component interventions compared to usual care in improving the 6MWD or self-efficacy. We found there may be some short-term improvement in quality of life with digital interventions, but there is no evidence about whether the effect is sustained long term. Dyspnoea symptoms may improve over a longer duration of digital intervention use. The evidence for multi-component interventions is very uncertain and as there is little or no evidence for adverse events, we cannot determine the benefit or harm of these interventions. The evidence base is predominantly of very low certainty with concerns around high risk of bias due to lack of blinding. Given that variation of interventions and blinding is likely to be a concern, future, larger studies are needed taking these limitations in consideration. Future studies are needed to determine whether the small improvements observed in this review can be applied to the general COPD population. A clear understanding of behaviour change through the BCT classification is important to gauge uptake of digital interventions and health outcomes in people with varying severity of COPD. Currently there is no guidance for interpreting BCT components of a digital intervention for changes to health outcomes. We could not interpret the BCT findings to the health outcomes we were investigating due to limited evidence that was of very low certainty. In future research, standardised approaches need to be considered when designing protocols to investigate effectiveness of digital interventions by including a standardised approach to BCT classification in addition to validated behavioural outcome measures that may reflect changes in behaviour.

Coexistence of Hidradenitis Suppurativa and Steatocystoma Multiplex: Is It a New Variant of Hidradenitis Suppurativa?

Hidradenitis suppurativa and steatocystoma multiplex may coexist in the same patient. The overlap of these 2 conditions could be suggestive of an unrecognized defect in follicular proliferation mutual in the pathogenesis of both conditions. Here we present 5 patients with both hidradenitis suppurativa and steatocystoma multiplex. Recognizing the overlap between these 2 conditions is important for accurate diagnosis, management, and identification of potential surgical candidates, as well as future basic science research.

A Systematic Review of Depression and Anxiety in Adults with Pyoderma Gangrenosum.

OBJECTIVE: To synthesize the available evidence on the prevalence and odds for anxiety and depression in adults with pyoderma gangrenosum (PG). DATA SOURCES: Observational studies examining anxiety and depression in adults with PG were systematically searched using the MEDLINE, EMBASE, PsycINFO, and CINAHL databases from the inception of each database to March 11, 2020. STUDY SELECTION: Two authors independently screened references based on predetermined eligibility criteria. DATA EXTRACTION: Of the 244 articles identified, three met the eligibility criteria. Relevant data were extracted from included studies, and methodological quality was evaluated independently by two authors using the modified Newcastle-Ottawa Scale. DATA SYNTHESIS: Three observational studies comprising 183 participants with PG met the inclusion criteria. Estimated rates of depression in adults with PG ranged from 10% to 23%. None of the studies measured rates of anxiety. CONCLUSIONS: The current systematic review suggests that depression is a common psychological comorbidity in adults with PG. Additional research is required to further assess the psychological comorbidities in this population.

Bacterial lipoproteins and other factors released by Francisella tularensis modulate human neutrophil lifespan: Effects of a TLR1 SNP on apoptosis inhibition.

Francisella tularensis infects several cell types including neutrophils, and aberrant neutrophil accumulation contributes to tissue destruction during tularaemia. We demonstrated previously that F. tularensis strains Schu S4 and live vaccine strain markedly delay human neutrophil apoptosis and thereby prolong cell lifespan, but the bacterial factors that mediate this aspect of virulence are undefined. Herein, we demonstrate that bacterial conditioned medium (CM) can delay apoptosis in the absence of direct infection. Biochemical analyses show that CM contained F. tularensis surface factors as well as outer membrane components. Our previous studies excluded roles for lipopolysaccharide and capsule in apoptosis inhibition, and current studies of [14 C] acetate-labelled bacteria argue against a role for other bacterial lipids in this process. At the same time, studies of isogenic mutants indicate that TolC and virulence factors whose expression requires FevR or MglA were also dispensable, demonstrating that apoptosis inhibition does not require Type I or Type VI secretion. Instead, we identified bacterial lipoproteins (BLPs) as active factors in CM. Additional studies of isolated BLPs demonstrated dose-dependent neutrophil apoptosis inhibition via a TLR2-dependent mechanism that is significantly influenced by a common polymorphism, rs5743618, in human TLR1. These data provide fundamental new insight into pathogen manipulation of neutrophil lifespan and BLP function.

Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies.

The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS-specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.

Inclusion of Epitopes That Expand High-Avidity CD4+ T Cells Transforms Subprotective Vaccines to Efficacious Immunogens against Virulent Francisella tularensis.

T cells are the immunological cornerstone in host defense against infections by intracellular bacterial pathogens, such as virulent Francisella tularensis spp. tularensis (Ftt). The general paucity of novel vaccines for Ftt during the past 60 y can, in part, be attributed to the poor understanding of immune parameters required to survive infection. Thus, we developed a strategy utilizing classical immunological tools to elucidate requirements for effective adaptive immune responses directed against Ftt. Following generation of various Francisella strains expressing well-characterized lymphocytic choriomeningitis virus epitopes, we found that survival correlated with persistence of Ag-specific CD4(+) T cells. Function of these cells was confirmed in their ability to more effectively control Ftt replication in vitro. The importance of understanding the Ag-specific response was underscored by our observation that inclusion of an epitope that elicits high-avidity CD4(+) T cells converted a poorly protective vaccine to one that engenders 100% protection. Taken together, these data suggest that improved efficacy of current tularemia vaccine platforms will require targeting appropriate Ag-specific CD4(+) T cell responses and that elucidation of Francisella epitopes that elicit high-avidity CD4(+) T cell responses, specifically in humans, will be required for successful vaccine development.

Antileishmanial and Cytotoxic Activity of Some Highly Oxidized Abietane Diterpenoids from the Bald Cypress, Taxodium distichum.

Two new compounds, namely, a para-benzoquinone ring-containing abietane (1) and a para-benzoquinone ring-containing 7,8-seco-abietane (2), and 14 other known highly oxidized abietane diterpenoids (3-16) were isolated from an extract prepared from the cones of Taxodium distichum, collected in central Ohio. The active subfraction from which all compounds isolated in this study were purified was tested in vivo using Leishmania donovani-infected mice and was found to dose-dependently reduce the parasite burden in the murine livers after iv administration of this crude mixture at 5.6 and 11.1 mg/kg. The structures of 1 and 2 were established by detailed 1D- and 2D-NMR experiments, HRESIMS data, and electronic circular dichroism studies. Compounds 3 and 4 were each fully characterized spectroscopically and also isolated from a natural source for the first time. Compounds 2-16 were tested in vitro against L. donovani promastigotes and L. amazonensis intracellular amastigotes. Compound 2 was the most active against L. amazonensis amastigotes (IC50 = 1.4 µM), and 10 was the most potent against L. donovani promastigotes (IC50 = 1.6 µM). These compounds may be suggested for further studies such as in vivo experimentation either alone or in combination with other Taxodium isolates.

In vitro evaluation of potential bitterness-masking terpenoids from the Canada goldenrod (Solidago canadensis).

In a screening of extracts of selected plants native to Ohio against the human bitterness receptor hTAS2R31, a chloroform-soluble extract of the aerial parts of Solidago canadensis (Canada goldenrod) was determined to have hTAS2R31 antagonistic activity and, thus, was fractionated for isolation of potential bitterness-masking agents. One new labdane diterpenoid, solidagol (1), and six known terpenoids, including two labdane diterpenoids (2 and 3), three clerodane diterpenoids (6ß-angeloyloxykolavenic acid, 6ß-tigloyloxykolavenic acid, and crotonic acid), and a triterpenoid (longispinogenin), were isolated. Among these compounds, 3ß-acetoxycopalic acid (2) was found to be the first member of the labdane diterpene class shown to have inhibitory activity against hTAS2R31 activation (IC50 8 µM). A homology model of hTAS2R31 was constructed, and the molecular docking of 2 to this model indicated that this diterpenoid binds well to the active site of hTAS2R31, whereas this was not the case for the closely structurally related compound 3 (sempervirenic acid). The content of 2 in the chloroform-soluble portion of the methanolic extract of S. canadensis was up to 2.24 g/100 g dry weight, as determined by HPLC.

Commensal-derived short-chain fatty acids disrupt lipid membrane homeostasis in Staphylococcus aureus.

The role of commensal anaerobic bacteria in chronic respiratory infections is unclear, yet they can exist in abundances comparable to canonical pathogens in vivo. Their contributions to the metabolic landscape of the host environment may influence pathogen behavior by competing for nutrients and creating inhospitable conditions via toxic metabolites. Here, we reveal a mechanism by which the anaerobe-derived short chain fatty acids (SCFAs) propionate and butyrate negatively affect Staphylococcus aureus physiology by disrupting branched chain fatty acid (BCFA) metabolism. In turn, BCFA impairment results in impaired growth, diminished expression of the agr quorum sensing system, as well as increased sensitivity to membrane-targeting antimicrobials. Altered BCFA metabolism also reduces S. aureus fitness in competition with Pseudomonas aeruginosa, suggesting that airway microbiome composition and the metabolites they produce and exchange directly impact pathogen succession over time. The pleiotropic effects of these SCFAs on S. aureus fitness and their ubiquity as metabolites in animals also suggests that they may be effective as sensitizers to traditional antimicrobial agents when used in combination.

The Francisella tularensis migR, trmE, and cphA genes contribute to F. tularensis pathogenicity island gene regulation and intracellular growth by modulation of the stress alarmone ppGpp.

The Francisella tularensis pathogenicity island (FPI) encodes many proteins that are required for virulence. Expression of these genes depends upon the FevR (PigR) regulator and its interactions with the MglA/SspA and RNA polymerase transcriptional complex. Experiments to identify how transcription of the FPI genes is activated have led to identification of mutations within the migR, trmE, and cphA genes that decrease FPI expression. Recent data demonstrated that the small alarmone ppGpp, produced by RelA and SpoT, is important for stabilizing MglA/SspA and FevR (PigR) interactions in Francisella. Production of ppGpp is commonly known to be activated by cellular and nutritional stress in bacteria, which indicates that cellular and nutritional stresses act as important signals for FPI activation. In this work, we demonstrate that mutations in migR, trmE, or cphA significantly reduce ppGpp accumulation. The reduction in ppGpp levels was similar for each of the mutants and correlated with a corresponding reduction in iglA reporter expression. In addition, we observed that there were differences in the ability of each of these mutants to replicate within various mammalian cells, indicating that the migR, trmE, and cphA genes are likely parts of different cellular stress response pathways in Francisella. These results also indicate that different nutritional and cellular stresses exist in different mammalian cells. This work provides new information to help understand how Francisella regulates its virulence genes in response to host cell environments, and it contributes to our growing knowledge of this highly successful bacterial pathogen.

High-throughput quantification of microbial-derived organic acids in mucin-rich samples via reverse phase high performance liquid chromatography.

Organic acids (short chain fatty acids, amino acids, etc.) are common metabolic byproducts of commensal bacteria of the gut and oral cavity in addition to microbiota associated with chronic infections of the airways, skin, and soft tissues. A ubiquitous characteristic of these body sites in which mucus-rich secretions often accumulate in excess, is the presence of mucins; high molecular weight (HMW), glycosylated proteins that decorate the surfaces of non-keratinized epithelia. Owing to their size, mucins complicate quantification of microbial-derived metabolites as these large glycoproteins preclude use of 1D and 2D gel approaches and can obstruct analytical chromatography columns. Standard approaches for quantification of organic acids in mucin-rich samples typically rely on laborious extractions or outsourcing to laboratories specializing in targeted metabolomics. Here we report a high-throughput sample preparation process that reduces mucin abundance and an accompanying isocratic reverse phase high performance liquid chromatography (HPLC) method that enables quantification of microbial-derived organic acids. This approach allows for accurate quantification of compounds of interest (0.01 mM - 100 mM) with minimal sample preparation, a moderate HPLC method run time, and preservation of both guard and analytical column integrity. This approach paves the way for further analyses of microbial-derived metabolites in complex clinical samples.

Epigenetic regulation of the calcitonin gene-related peptide gene in trigeminal glia.

BACKGROUND: The neuropeptide calcitonin gene-related peptide (CGRP) plays a key role in migraine. CGRP gene expression involves an enhancer that is active in neurons, yet inactive in glia. In this report, we analyze epigenetic modifications that allow enhancer activation in glia. METHODS: DNA methylation and histone acetylation states were measured in rat and human- model cell lines and primary cultures of rat trigeminal ganglia glia. The functional consequence of altering the chromatin state was determined by quantitative measurements of both calcitonin (CT) and CGRP mRNAs. RESULTS: A hypermethylated CpG island flanking the enhancer was identified in glia and non-expressing cell lines. In addition, the chromatin was hypoacetylated. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine induced CT mRNA ~30-fold in glial cultures. Treatment with a histone deacetylase inhibitor alone had little effect; however, the combination of inhibitors yielded a synergistic ~80-fold increase in CT and ~threefold increase in CGRP mRNA. Treated glia contained CT precursor (pro-CT) immunoreactivity. CONCLUSIONS: Epigenetic modulation is sufficient to induce the CGRP gene in glia. Because the CGRP gene is systemically activated by inflammatory conditions, this suggests that glial pro-CT may be an unexplored biomarker during migraine.

The relevance of higher plants in lead compound discovery programs.

Along with compounds from terrestrial microorganisms, the constituents of higher plants have provided a substantial number of the natural product-derived drugs used currently in Western medicine. Interest in the elucidation of new structures of the secondary metabolite constituents of plants has remained high among the natural products community over the first decade of the 21st century, particularly of species that are used in systems of traditional medicine or are utilized as botanical dietary supplements. In this review, progress made in the senior author's laboratory in research work on naturally occurring sweeteners and other taste-modifying substances and on potential anticancer agents from tropical plants will be described.

Clostridioides difficile exploits toxin-mediated inflammation to alter the host nutritional landscape and exclude competitors from the gut microbiota.

Clostridioides difficile is a bacterial pathogen that causes a range of clinical disease from mild to moderate diarrhea, pseudomembranous colitis, and toxic megacolon. Typically, C. difficile infections (CDIs) occur after antibiotic treatment, which alters the gut microbiota, decreasing colonization resistance against C. difficile. Disease is mediated by two large toxins and the expression of their genes is induced upon nutrient depletion via the alternative sigma factor TcdR. Here, we use tcdR mutants in two strains of C. difficile and omics to investigate how toxin-induced inflammation alters C. difficile metabolism, tissue gene expression and the gut microbiota, and to determine how inflammation by the host may be beneficial to C. difficile. We show that C. difficile metabolism is significantly different in the face of inflammation, with changes in many carbohydrate and amino acid uptake and utilization pathways. Host gene expression signatures suggest that degradation of collagen and other components of the extracellular matrix by matrix metalloproteinases is a major source of peptides and amino acids that supports C. difficile growth in vivo. Lastly, the inflammation induced by C. difficile toxin activity alters the gut microbiota, excluding members from the genus Bacteroides that are able to utilize the same essential nutrients released from collagen degradation.

In Vivo Targeting of Clostridioides difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials.

Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to obligately lytic is feasible and contributes to the therapeutic suitability of intrinsic C. difficile phages, despite the specific challenges encountered in the disease phenotypes of phage-treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms.IMPORTANCEClostridioides difficile is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broad-spectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of C. difficilein vitro and in vivo. Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for C. difficile toward clinical application. These findings provide evidence into how phage can be combined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.

Human fecal metabolomic profiling could inform Clostridioides difficile infection diagnosis and treatment.

Clostridioides difficile is a significant public health threat, and diagnosis of this infection is challenging due to a lack of sensitivity in current diagnostic testing. In this issue of the JCI, Robinson et al. use a logistic regression model based on the fecal metabolome that is able to distinguish between patients with non-C. difficile diarrhea and C. difficile infection, and to some degree, patients who are asymptomatically colonized with C. difficile. The authors construct a metabolic definition of human C. difficile infection, which could improve diagnostic accuracy and aid in the development of targeted therapeutics against this pathogen.

Shifts in the Gut Metabolome and Clostridium difficile Transcriptome throughout Colonization and Infection in a Mouse Model.

Antibiotics alter the gut microbiota and decrease resistance to Clostridium difficile colonization; however, the mechanisms driving colonization resistance are not well understood. Loss of resistance to C. difficile colonization due to antibiotic treatment is associated with alterations in the gut metabolome, specifically, with increases in levels of nutrients that C. difficile can utilize for growth in vitro. To define the nutrients that C. difficile requires for colonization and pathogenesis in vivo, we used a combination of mass spectrometry and RNA sequencing (RNA Seq) to model the gut metabolome and C. difficile transcriptome throughout an acute infection in a mouse model at the following time points: 0, 12, 24, and 30 h. We also performed multivariate-based integration of the omics data to define the signatures that were most important throughout colonization and infection. Here we show that amino acids, in particular, proline and branched-chain amino acids, and carbohydrates decrease in abundance over time in the mouse cecum and that C. difficile gene expression is consistent with their utilization in vivo. This was also reinforced by the multivariate-based integration of the omics data where we were able to discriminate the metabolites and transcripts that support C. difficile physiology between the different time points throughout colonization and infection. This report illustrates how important the availability of amino acids and other nutrients is for the initial stages of C. difficile colonization and progression of disease. Future studies identifying the source of the nutrients and engineering bacteria capable of outcompeting C. difficile in the gut will be important for developing new targeted bacterial therapeutics. IMPORTANCE Clostridium difficile is a bacterial pathogen of global significance that is a major cause of antibiotic-associated diarrhea. Antibiotics deplete the indigenous gut microbiota and change the metabolic environment in the gut to one favoring C. difficile growth. Here we used metabolomics and transcriptomics to define the gut environment after antibiotics and during the initial stages of C. difficile colonization and infection. We show that amino acids, in particular, proline and branched-chain amino acids, and carbohydrates decrease in abundance over time and that C. difficile gene expression is consistent with their utilization by the bacterium in vivo. We employed an integrated approach to analyze the metabolome and transcriptome to identify associations between metabolites and transcripts. This highlighted the importance of key nutrients in the early stages of colonization, and the data provide a rationale for the development of therapies based on the use of bacteria that specifically compete for nutrients that are essential for C. difficile colonization and disease.

The Ability to Acquire Iron Is Inversely Related to Virulence and the Protective Efficacy of Francisella tularensis Live Vaccine Strain.

Francisella tularensis is a highly infectious bacterial pathogen that causes the potentially fatal disease tularemia. The Live Vaccine Strain (LVS) of F. tularensis subsp. holarctica, while no longer licensed as a vaccine, is used as a model organism for identifying correlates of immunity and bacterial factors that mediate a productive immune response against F. tularensis. Recently, it was reported that two biovars of LVS differed in their virulence and vaccine efficacy. Genetic analysis showed that they differ in ferrous iron homeostasis; lower Fe2+ levels contributed to increased resistance to hydrogen peroxide in the vaccine efficacious LVS biovar. This also correlated with resistance to the bactericidal activity of interferon γ-stimulated murine bone marrow-derived macrophages. We have extended these findings further by showing that a mutant lacking bacterioferritin stimulates poor protection against Schu S4 challenge in a mouse model of tularemia. Together these results suggest that the efficacious biovar of LVS stimulates productive immunity by a mechanism that is dependent on its ability to limit the toxic effects of oxidative stress by maintaining optimally low levels of intracellular Fe2+.