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The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that plays an integral role in homeostatic maintenance by regulating cellular functions such as cellular differentiation, metabolism, barrier function, and immune response. An important but poorly understood class of AHR activators are compounds derived from host and bacterial metabolism of tryptophan. The commensal bacteria of the gut microbiome are major producers of tryptophan metabolites known to activate the AHR, while the host also produces AHR activators through tryptophan metabolism. We used targeted mass spectrometry-based metabolite profiling to determine the presence and metabolic source of these metabolites in the sera of conventional mice, germ-free mice, and humans. Surprisingly, sera concentrations of many tryptophan metabolites are comparable between germ-free and conventional mice. Therefore, many major AHR-activating tryptophan metabolites in mouse sera are produced by the host, despite their presence in feces and mouse cecal contents. AHR activation is rarely studied in the context of a mixture at relevant concentrations, as we present here. The AHR activation potentials of individual and pooled metabolites were explored using cell-based assays, while ligand binding competition assays and ligand docking simulations were used to assess the detected metabolites as AHR agonists. The physiological and biomedical relevance of the identified metabolites was investigated in the context of cell-based models for cancer and rheumatoid arthritis. We present data here that reframe AHR biology to include the presence of ubiquitous tryptophan metabolites, improving our understanding of homeostatic AHR activity and models of AHR-linked diseases.

Immunosurveillance of the gastrointestinal epithelium by mononuclear phagocytes (MNPs) is essential for maintaining gut health. However, studying the complex interplay between the human gastrointestinal epithelium and MNPs such as dendritic cells (DCs) is difficult, since traditional cell culture systems lack complexity, and animal models may not adequately represent human tissues. Microphysiological systems, or tissue chips, are an attractive alternative for these investigations, because they model functional features of specific tissues or organs using microscale culture platforms that recreate physiological tissue microenvironments. However, successful integration of multiple of tissue types on a tissue chip platform to reproduce physiological cell-cell interactions remains a challenge. We previously developed a tissue chip system, the gut organoid flow chip (GOFlowChip), for long term culture of 3-D pluripotent stem cell-derived human intestinal organoids. Here, we optimized the GOFlowChip platform to build a complex microphysiological immune-cell-epithelial cell co-culture model in order to study DC-epithelial interactions in human stomach. We first tested different tubing materials and chip configurations to optimize DC loading onto the GOFlowChip and demonstrated that DC culture on the GOFlowChip for up to 20 h did not impact DC activation status or viability. However, Transwell chemotaxis assays and live confocal imaging revealed that Matrigel, the extracellular matrix (ECM) material commonly used for organoid culture, prevented DC migration towards the organoids and the establishment of direct MNP-epithelial contacts. Therefore, we next evaluated DC chemotaxis through alternative ECM materials including Matrigel-collagen mixtures and synthetic hydrogels. A polysaccharide-based synthetic hydrogel, VitroGel®-ORGANOID-3 (V-ORG-3), enabled significantly increased DC chemotaxis through the matrix, supported organoid survival and growth, and did not significantly alter DC activation or viability. On the GOFlowChip, DCs that were flowed into the chip migrated rapidly through the V-ORG matrix and reached organoids embedded deep within the chip, with increased interactions between DCs and gastric organoids. The successful integration of DCs and V-ORG-3 embedded gastric organoids into the GOFlowChip platform now permits real-time imaging of MNP-epithelial interactions and other investigations of the complex interplay between gastrointestinal MNPs and epithelial cells in their response to pathogens, candidate drugs and mucosal vaccines.

Summary Chronic low-grade inflammation is a subclinical condition directly and indirectly linked to the development of a wide range of diseases responsible for the vast majority of morbidity. To examine mechanisms coupled to chronic disease, a group of overweight and obese human subjects without known inflammatory diseases participated in a high-fat meal challenge as an acute inflammation stimulus. Analysis of serum metabolites grouped by baseline cytokine levels revealed that single samples had little power in differentiating groups. However, an analysis that incorporated temporal response separated inflammatory response phenotypes and allowed us to create a metabolic signature of inflammation which revealed metabolic components that are crucial to a cytokine-mediated inflammation response. The use of temporal response, rather than a single time point, improved metabolomic prediction of high postprandial inflammation responses and led to the development of a dynamic biosignature as a potential tool for stratifying risk to a wide range of diseases., Graphical abstract, Highlights • Dynamic responses often provide insight into disease pathology • Temporal metabolic responses to acute inflammation were explored in obese people • Temporal metabolite levels differentiated low, mid, and high inflammation groups • Inflammation-linked metabolites were shown to be predictors of cytokine responses, Pathophysiology; Systems biology; Metabolomics

Longitudinal human gut microbiome datasets generated using community-level, sequence-based approaches often report a sub-set of long-lived “resident” taxa that rarely, if ever, are lost. This result contrasts with population-level turnover of resident clones on the order of months to years. We hypothesized that the disconnect between these results is due to a relative lack of simultaneous discrimination of the human gut microbiome at both the community and population-levels. Here, we present results of a small, longitudinal cohort study (n = 8 participants) of healthy human adults that identifies static and dynamic members of the gut microbiome at the clone level based on cultivation/genetic discrimination and at the operational taxonomic unit/amplified sequence variant levels based on 16S rRNA sequencing. We provide evidence that there is little “stability” within resident clonal populations of the common gut microbiome bacterial family, Enterobacteriaceae. Given that clones can vary substantially in genome content and that evolutionary processes operate on the population level, these results question the biological relevance of apparent stability at higher taxonomic levels.

Arsenic poisons an estimated 200 million people worldwide through contaminated food and drinking water. Confusingly, the gut microbiome has been suggested to both mitigate and exacerbate arsenic toxicity. Here, we show that the microbiome protects mice from arsenic-induced mortality. Both antibiotic-treated and germ-free mice excrete less arsenic in stool and accumulate more arsenic in organs compared to control mice. Mice lacking the primary arsenic detoxification enzyme (As3mt) are hypersensitive to arsenic after antibiotic treatment or when derived germ-free, compared to wild-type and/or conventional counterparts. Human microbiome (stool) transplants protect germ-free As3mt-KO mice from arsenic-induced mortality, but protection depends on microbiome stability and the presence of specific bacteria, including Faecalibacterium. Our results demonstrate that both a functional As3mt and specific microbiome members are required for protection against acute arsenic toxicity in mouse models. We anticipate that the gut microbiome will become an important explanatory factor of disease (arsenicosis) penetrance in humans, and a novel target for prevention and treatment strategies., It is unclear whether the gut microbiome can mitigate or exacerbate arsenic toxicity. Here, Coryell et al. show that the human gut microbiome protects mice from arsenic-induced mortality, with protection levels correlating with the relative abundance of the human commensal Faecalibacterium.

The microbial ars operon encodes the primary bacterial defense response to the environmental toxicant, arsenic. An important component of this operon is the arsR gene, which encodes ArsR, a member of the family of proteins categorized as DNA-binding transcriptional repressors. As currently documented, ArsR regulates its own expression as well as other genes in the same ars operon. This study examined the roles of four ArsR proteins in the well-developed model Gram-negative bacterium Agrobacterium tumefaciens 5A. RNASeq was used to compare and characterize gene expression profiles in ± arsenite-treated cells of the wild-type strain and in four different arsR mutants. We report that ArsR-controlled transcription regulation is truly global, extending well beyond the current ars operon model, and includes both repression as well as apparent activation effects. Many cellular functions are significantly influenced, including arsenic resistance, phosphate acquisition/metabolism, sugar transport, chemotaxis, copper tolerance, iron homeostasis, and many others. While there is evidence of some regulatory overlap, each ArsR exhibits its own regulatory profile. Furthermore, evidence of a regulatory hierarchy was observed; i.e. ArsR1 represses arsR4, ArsR4 activates arsR2, and ArsR2 represses arsR3. Additionally and unexpectedly, aioB (arsenite oxidase small subunit) expression was shown to be under partial positive control by ArsR2 and ArsR4. Summarizing, this study demonstrates the regulatory portfolio of arsenite-activated ArsR proteins and includes essentially all major cellular functions. The broad bandwidth of arsenic effects on microbial metabolism assists in explaining and understanding the full impact of arsenic in natural ecosystems, including the mammalian gut.

Environmental toxicant exposure contributes to morbidity and mortality of many human diseases. With respect to arsenic, microbially driven chemical transformations dictate its toxicity and mobility in virtually every environment yet studied, so a general hypothesis is that the human gut microbiome determines disease outcome following exposure. However, the complex nature of the gut microbiome and the myriad of potential interactions with human cells/tissues make it challenging to quantify the influence of specific arsenic-active functions—a requisite step in developing effective disease prevention and/or clinical intervention strategies. To control both mammalian and microbial function during toxicant exposure, we genetically defined the gut microbiome of mice using only Escherichia coli strain, AW3110 (▵arsRBC), or the same strain carrying a single genome copy of the Fucus vesiculosus metallothionein gene (AW3110::fmt); a cysteine-rich peptide that complexes with arsenite, facilitating bioaccumulation and reducing its toxic effects. AW3110::fmt bioaccumulated significantly more arsenic and gnotobiotic mice colonized by this strain excreted significantly more arsenic in stool and accumulated significantly less arsenic in organs. Moreover, AW3110::fmt gnotobiotic mice were protected from acute toxicity exposure (20 ppm AsIII) relative to controls. This study demonstrates—in a highly controlled fashion—that a single microbiome function (arsenic bioaccumulation) encoded by a single gene in a single human gut microbiome bacterium significantly alters mammalian host arsenic exposure. The experimental model described herein allows for a highly controlled and directed assessment of microbiome functions, and is useful to quantify the influence of specific microbiome-arsenic interactions that help mitigate human disease.

All cells need iron. Both too much and too little iron lead to diseases and unwanted outcomes., Iron is an essential micronutrient for life. In mammals, dietary iron is absorbed primarily in the small intestine. Currently, the impacts of dietary iron on the taxonomic structure and function of the gut microbiome and reciprocal effects on the animal host are not well understood. Here, we establish a mouse model of low-iron challenge in which intestinal biomarkers and reduced fecal iron reveal iron stress while serum iron and mouse behavioral markers indicate maintenance of iron homeostasis. We show that the diversity of the gut microbiome in conventional C57BL/6 mice changes dramatically during 2 weeks on a low-iron diet. We also show the effects of a low-iron diet on microbiome diversity are long lasting and not easily recovered when iron is returned to the diet. Finally, after optimizing taxon association methods, we show that some bacteria are unable to fully recover after the low-iron challenge and appear to be extirpated from the gut entirely. In particular, operational taxonomic units (OTUs) from the Prevotellaceae and Porphyromonadaceae families and Bacteroidales order are highly sensitive to low-iron conditions, while other seemingly insensitive OTUs recover. These results provide new insights into the iron requirements of gut microbiome members and add to the growing understanding of mammalian iron cycling. IMPORTANCE All cells need iron. Both too much and too little iron lead to diseases and unwanted outcomes. Although the impact of dietary iron on human cells and tissues has been well studied, there is currently a lack of understanding about how different levels of iron influence the abundant and diverse members of the human microbiome. This study develops a well-characterized mouse model for studying low-iron levels and identifies key groups of bacteria that are most affected. We found that the microbiome undergoes large changes when iron is removed from the diet but that many individual bacteria are able to rebound when iron levels are changed back to normal. That said, a select few members, referred to as iron-sensitive bacteria, seem to be lost. This study begins to identify individual members of the mammalian microbiome most affected by changes in dietary iron levels.

Clostridioides difficile is an enteric bacterial pathogen that can a cause nosocomial infection leading to debilitating colitis. The development of a murine model of C. difficile infection has led to fundamental discoveries in disease pathogenesis and the host immune response to infection. Recently, C. difficile endogenously present in the microbiota of mice has been reported and was found to complicate interpretation of mouse studies. Here, we report a novel C. difficile strain, named NTCD-035, isolated from the microbiota of our mouse colony. The presence of NTCD-035 in mice prior to challenge with a highly pathogenic C. difficile strain (VPI10463) led to significantly reduced disease severity. Phylogenetic characterization derived from whole genome sequencing and PCR ribotyping identified the isolate as a novel clade 1, ribotype 035 strain that lacks the pathogenicity locus required to produce toxins. Deficiency in toxin production along with sporulation capacity and secondary bile acid sensitivity was confirmed using in vitro assays. Inoculation of germ-free mice with NTCD-035 did not cause morbidity despite the strain readily colonizing the large intestine. Implementation of a culture-based screening procedure enabled the identification of mice harboring C. difficile in their microbiota, the establishment of a C. difficile-free mouse colony, and a monitoring system to prevent future contamination. Taken together, these data provide a framework for screening mice for endogenously harbored C. difficile and support clinical findings that demonstrate the therapeutic potential of non-toxigenic strains in preventing C. difficile associated disease. Abbreviations: PaLoc - Pathogenicity locus, CFUs - Colony forming units, TcdA - toxin-A, TcdB - toxin-B, CdtA - binary toxin A, CdtB - binary toxin B, CdtR - binary toxin R, NTCD - non-toxigenic C. difficile.

Background The epidemic Clostridioides difficile ribotype 027 strain resulted from the dissemination of 2 separate fluoroquinolone-resistant lineages: FQR1 and FQR2. Both lineages were reported to originate in North America; however, confirmatory large-scale investigations of C difficile ribotype 027 epidemiology using whole genome sequencing has not been undertaken in the United States. Methods Whole genome sequencing and single-nucleotide polymorphism (SNP) analysis was performed on 76 clinical ribotype 027 isolates obtained from hospitalized patients in Texas with C difficile infection and compared with 32 previously sequenced worldwide strains. Maximum-likelihood phylogeny based on a set of core genome SNPs was used to construct phylogenetic trees investigating strain macro- and microevolution. Bayesian phylogenetic and phylogeographic analyses were used to incorporate temporal and geographic variables with the SNP strain analysis. Results Whole genome sequence analysis identified 2841 SNPs including 900 nonsynonymous mutations, 1404 synonymous substitutions, and 537 intergenic changes. Phylogenetic analysis separated the strains into 2 prominent groups, which grossly differed by 28 SNPs: the FQR1 and FQR2 lineages. Five isolates were identified as pre-epidemic strains. Phylogeny demonstrated unique clustering and resistance genes in Texas strains indicating that spatiotemporal bias has defined the microevolution of ribotype 027 genetics. Conclusions Clostridioides difficile ribotype 027 lineages emerged earlier than previously reported, coinciding with increased use of fluoroquinolones. Both FQR1 and FQR2 ribotype 027 epidemic lineages are present in Texas, but they have evolved geographically to represent region-specific public health threats.

Interactions between the gut microbiota and the host are important for health, where dysbiosis has emerged as a likely component of mucosal disease. The specific constituents of the microbiota that contribute to mucosal disease are not well defined. The authors sought to define microbial components that regulate homeostasis within the intestinal mucosa. Using an unbiased, metabolomic profiling approach, a selective depletion of indole and indole-derived metabolites was identified in murine and human colitis. Indole-3-propionic acid (IPA) was selectively diminished in circulating serum from human subjects with active colitis, and IPA served as a biomarker of disease remission. Administration of indole metabolites showed prominent induction of IL-10R1 on cultured intestinal epithelia that was explained by activation of the aryl hydrocarbon receptor. Colonization of germ-free mice with wild-type Escherichia coli, but not E. coli mutants unable to generate indole, induced colonic epithelial IL-10R1. Moreover, oral administration of IPA significantly ameliorated disease in a chemically induced murine colitis model. This work defines a novel role of indole metabolites in anti-inflammatory pathways mediated by epithelial IL-10 signaling and identifies possible avenues for utilizing indoles as novel therapeutics in mucosal disease.

OBJECTIVES: We evaluated the antimicrobial susceptibility and ribotypes of Clostridium difficile isolates from participants in a Phase 2 study of ridinilazole, a novel targeted-spectrum agent for treatment of C. difficile infection. METHODS: Participants received ridinilazole (200 mg twice daily) or vancomycin (125 mg four times daily) for 10 days (ClinicalTrials.gov: NCT02092935). The MICs of ridinilazole and comparators for C. difficile isolates from stool samples were determined by agar dilution. Toxin gene profiling was performed by multiplex PCR and ribotype identification by capillary electrophoresis. RESULTS: Eighty-nine isolates were recovered from 88/100 participants (one participant had two strains at baseline). The median colony count (cfu/g stool) was 1.9 × 10(4) (range: 2.5 × 10(2)–7.0 × 10(6)). Twelve participants (three received ridinilazole and nine received vancomycin) experienced recurrence, confirmed by immunoassays for free toxin in stool samples. The ribotype of eight out of nine isolates obtained at recurrence matched those of the initial isolates. All isolates, including those obtained at recurrence, were susceptible to ridinilazole within the expected range [median (range) MIC: 0.12 (0.06–0.5) mg/L]. The median (range) vancomycin MIC was 1 (0.5–4.0) mg/L. At baseline, 13.6% and 13.3% of samples in the ridinilazole and vancomycin groups were positive for VRE, increasing to 23.7% and 29.7% by day 40, respectively. Common ribotypes included 014-20 (14 isolates), 027 (13), 106 (7), 002 (7), 078-126 (4), 001 (4), 087 (3) and 198 (3). Toxin gene profiling of nearly all baseline isolates (98.9%) revealed a binary toxin gene (cdtA/cdtB) prevalence of 35%. CONCLUSIONS: Ridinilazole potently inhibited recovered C. difficile isolates. Recurrence was not associated with altered susceptibility.

The human gastrointestinal tract is immature at birth, yet must adapt to dramatic changes such as oral nutrition and microbial colonization. The confluence of these factors can lead to severe inflammatory disease in premature infants; however, investigating complex environment-host interactions is difficult due to limited access to immature human tissue. Here, we demonstrate that the epithelium of human pluripotent stem-cell-derived human intestinal organoids is globally similar to the immature human epithelium and we utilize HIOs to investigate complex host-microbe interactions in this naive epithelium. Our findings demonstrate that the immature epithelium is intrinsically capable of establishing a stable host-microbe symbiosis. Microbial colonization leads to complex contact and hypoxia driven responses resulting in increased antimicrobial peptide production, maturation of the mucus layer, and improved barrier function. These studies lay the groundwork for an improved mechanistic understanding of how colonization influences development of the immature human intestine., eLife digest Human newborns are exposed to large numbers of bacteria at birth. They must transition from the protective, sterile environment of the womb into the bacteria-rich world. The gut, in particular, must adapt as bacteria colonize it. Many of the first bacteria found in the newborn gut form the basis of the bacterial communities needed for a healthy intestine throughout life. In some premature infants, bacterial colonization of the intestine may trigger harmful inflammation and a serious illness called necrotizing enterocolitis. It is not known exactly how the immature intestine first responds to bacteria. It is also unclear what goes wrong that causes illness in some premature infants. Learning more about how a healthy newborn intestine becomes colonized and responds to this colonization may help medical professionals to better care for normal infants and those who are at risk of intestinal disease. But it has been difficult to study because there is not much newborn intestinal tissue available for scientific research. One solution would be to grow tissue in the laboratory that is like tissue found in the newborn intestine and see how it adapts to bacteria. Now, Hill et al. use stem cells to grow a tissue in the laboratory that is very like immature newborn intestine and show that bacterial colonization helps it to mature. In the experiments, stem cells were grown into an intestine-like tissue. Analyses showed that this laboratory-grown tissue had the same patterns of gene expression as newborn intestines. Then, a type of bacteria called Escherichia coli that is normally found in the intestines of healthy babies was introduced to the intestine-like tissue. Hill et al. show that the initial contact with bacteria and changes in oxygen levels due to bacterial activity cause shifts in gene expression. These in turn stimulate the release of mucus and other protective responses. A protein called NF-κB plays a central role in these normal bacteria-intestine interactions. Hill et al. show that using a drug to block NF-κB interferes with these processes. The experiments show that contact with bacteria encourages the immature intestine to protect itself from potential harm. More experiments like these may help scientists understand normal bacteria-intestine interactions in early life and how they may go wrong in disease. These studies might also help identify new treatments for babies with necrotizing enterocolitis.

The role of bacteriophages in influencing the structure and function of the healthy human gut microbiome is unknown. With few exceptions, previous studies have found a high level of heterogeneity in bacteriophages from healthy individuals. To better estimate and identify the shared phageome of humans, we analyzed a deep DNA sequence dataset of active bacteriophages and available metagenomic datasets of the gut bacteriophage community from healthy individuals. We found 23 shared bacteriophages in more than one-half of 64 healthy individuals from around the world. These shared bacteriophages were found in a significantly smaller percentage of individuals with gastrointestinal/irritable bowel disease. A network analysis identified 44 bacteriophage groups of which 9 (20%) were shared in more than one-half of all 64 individuals. These results provide strong evidence of a healthy gut phageome (HGP) in humans. The bacteriophage community in the human gut is a mixture of three classes: a set of core bacteriophages shared among more than one-half of all people, a common set of bacteriophages found in 20-50% of individuals, and a set of bacteriophages that are either rarely shared or unique to a person. We propose that the core and common bacteriophage communities are globally distributed and comprise the HGP, which plays an important role in maintaining gut microbiome structure/function and thereby contributes significantly to human health.

Clostridium difficile infection (CDI) is a significant source of healthcare-associated morbidity and mortality. This study investigated whether serum 25-hydroxyvitamin D is associated with adverse outcomes from CDI. Patients with CDI were prospectively enrolled. Charts were reviewed and serum 25-hydroxyvitamin D was measured. The primary outcome was a composite definition of severe disease: fever (temperature >38°C), acute organ dysfunction, or serum white blood cell count >15,000 cells/μL within 24-48 hours of diagnosis; lack of response to therapy by day 5; and intensive care unit admission; colectomy; or death within 30 days. Sixty-seven patients were included in the final analysis. Mean (±SD) serum 25- hydroxyvitamin D was 26.1 (±18.54) ng/mL. Severe disease, which occurred in 26 (39%) participants, was not associated with serum 25-hydroxyvitamin D [odds ratio (OR) 1.00; 95% confidence interval (CI) 0.96-1.04]. In the adjusted model for severe disease only serum albumin (OR 0.12; 95%CI 0.02-0.64) and diagnosis by detection of stool toxin (OR 5.87; 95%CI 1.09-31.7) remained independent predictors. We conclude that serum 25-hydroxyvitamin D is not associated with the development of severe disease in patients with CDI.

Background. Clostridium difficile infection (CDI) can cause severe disease and death, especially in older adults. A better understanding of risk factors for adverse outcomes is needed. This study tests the hypotheses that infection with specific ribotypes and presence of stool toxins independently associate with severity and constructs predictive models of adverse outcomes. Methods. Cases of non-recurrent CDI were prospectively included after positive stool tests for toxins A and/or B by enzyme immunoassay (EIA) or tcdB by polymerase chain reaction. Outcomes included severe CDI (intensive care unit admission, colectomy, or death attributable to CDI within 30 days of diagnosis) and 30-day all-cause mortality. Adjusted models were developed to test hypotheses and predict outcomes. Results. In total, 1144 cases were included. The toxin EIA was positive in 37.2% and 35.6% of patients were of age >65 years. One of the 137 unique ribotypes was ribotype 027 (16.2%). Detectable stool toxin did not associate with outcomes. Adjusting for covariates, including age, Ribotype 027 was a significant predictor of severe CDI (90 cases; odds ratio [OR], 1.73; 95% confidence interval [CI], 1.03–2.89; P = .037) and mortality (89 cases; OR, 2.02; 95% CI, 1.19–3.43; P = .009). Concurrent antibiotic use associated with both outcomes. Both multivariable predictive models had excellent performance (area under the curve >0.8). Conclusions. Detection of stool toxin A and/or B by EIA does not predict severe CDI or mortality. Infection with ribotype 027 independently predicts severe CDI and mortality. Use of concurrent antibiotics is a potentially modifiable risk factor for severe CDI.

Clostridium difficile is the most commonly identified pathogen among health care-associated infections in the United States. There is a need for accurate and low-cost typing tools that produce comparable data across studies (i.e., portable data) to help characterize isolates during epidemiologic investigations of C. difficile outbreaks and sporadic cases of disease. The most popular C. difficile -typing technique is PCR ribotyping, and we previously developed methods using fluorescent PCR primers and amplicon sizing on a Sanger-style sequencer to generate fluorescent PCR ribotyping data. This technique has been used to characterize tens of thousands of C. difficile isolates from cases of disease. Here, we present validation of a protocol for the cost-effective generation of fluorescent PCR ribotyping data. A key component of this protocol is the ability to accurately identify PCR ribotypes against an online database ( http://walklab.rcg.montana.edu ) at no cost. We present results from a blinded multicenter study to address data portability across four different laboratories and three different sequencing centers. Our standardized protocol and centralized database for typing of C. difficile pathogens will increase comparability between studies so that important epidemiologic linkages between cases of disease and patterns of emergence can be rapidly identified.

Background Trends in the distribution of ribotypes for C. difficile associated diarrheal isolates obtained over time in the United States are lacking. As part of surveillance program for C. difficile susceptibility, we analyzed stool isolates for ribotype distribution from a phase 2 trial of surotomycin (2010–2011) (North America sites) as well as a national surveillance study from 2011–2014. Isolates for the surveillance study were referred from 6 geographically distinct medical centers. Methods C. difficile isolates or C. difficile toxin + stools from patients with C. difficile associated diarrhea (CDAD) were submitted for testing to Tufts Medical Center. Following isolation and confirmation as C. difficile, a random sample of isolates were ribotyped by PCR capillary gel electrophoresis. Results 673 isolates over the 5 years of the analysis have been ribotyped to date. There were 49 unique ribotype designations, and 16 ribotypes had more than 10 isolates. The ribotype distribution by year is shown in the table. Conclusion There has been a change in the frequency of ribotypes over time in the US. Of the most common ribotypes seen, 027 has decreased by over 50% while there has been an increase of 014-020, 002, and 106. 014-020 is now the most common ribotype seen in the US. These data suggest that there is a changing epidemiology of C. difficile in the US and continuous monitoring of the ribotype distributions and clinical implications is warranted. Disclosures D. R. Snydman, Merck: Consultant and Grant Investigator, Consulting fee and Research grant; Shire: Consultant, Consulting fee; Summit PLC: Consultant and Grant Investigator, Consulting fee and Research grant; BioK+: Consultant, Consulting fee; Actelion: Grant Investigator, Research grant; S. G. Jenkins, Cormedix: Consultant, Consulting fee; Bayer: Consultant, Consulting fee; Merck: Grant Investigator and Scientific Advisor, Research grant; E. J. C. Goldstein, Merck: Grant Investigator, Scientific Advisor and Speaker’s Bureau, Consulting fee and Grant recipient; Cubist: Grant Investigator, Scientific Advisor and Speaker’s Bureau, Consulting fee; Actelion: Grant Investigator, Grant recipient; Summit PLC: Grant Investigator and Scientific Advisor, Grant recipient; R. Patel, Curetis: Consultant, Grant Investigator and Speaker’s Bureau, Consulting fee and Research grant; Pocared: Grant Investigator, Research grant; nanoMR: Grant Investigator, Research grant; BioFire: Grant Investigator, Research grant; Check-Points: Grant Investigator, Research grant; 3M: Grant Investigator, Research grant; Cubist: Grant Investigator, Research grant; Merck: Grant Investigator, Research grant; S. Johnson, Bio-K+: Consultant, Consulting fee; D. N. Gerding, Merck, Shire, Cubist, Rebiotix, sanofi pasteur, Summit, DaVoltera, Actelion: Consultant, Consulting fee; CDC, US Dept of Veterans Affairs Research Service: Grant Investigator, Research grant

Chondrocyte mechanotransduction is the process by which cartilage cells transduce mechanical loads into biochemical and biological signals. Previous studies have identified several pathways by which chondrocytes transduce mechanical loads, yet a general understanding of which signals are activated and in what order remains elusive. This study was performed to identify candidate mediators of chondrocyte mechanotransduction using SW1353 chondrocytes embedded in physiologically stiff agarose. Dynamic compression was applied to cell-seeded constructs for 0–30 minutes, followed immediately by whole-cell metabolite extraction. Metabolites were detected via LC-MS, and compounds of interest were identified via database searches. We found several metabolites which were statistically different between the experimental groups, and we report the detection of 5 molecules which are not found in metabolite databases of known compounds indicating potential novel molecules. Targeted studies to quantify the response of central energy metabolites to compression found a transient increase in the ratio of NADP+ to NADPH and a continual decrease in the ratio of GDP to GTP, suggesting a flux of energy into the TCA cycle. These data are consistent with the remodeling of cytoskeletal components by mechanically induced signaling, and add substantial new data to a complex picture of how chondrocytes transduce mechanical loads.

Since 2000, Clostridium difficile isolates of ribotype 027 have been linked to outbreaks in North America and Europe and also an increased rate of colectomy and death among infected individuals. It has been proposed that enhanced sporulation and toxin production were associated with this apparent increase in virulence of 027 isolates. Since only a limited number of isolates have been examined, the relationship of these phenotypes to a specific ribotype, and as well as to clinical disease severity, remains controversial. 106 recent clinical isolates from the University of Michigan Health System were characterized for the ability to sporulate, produce viable spores, grow in rich media, and produce toxins in vitro. Significant variation was observed between isolates for each of these phenotypes. Isolates of ribotype 027 produced higher levels of toxin and exhibited slower growth compared to other ribotypes. Importantly, increased spore production did appear to be relevant to severe C. difficile infection, as determined by available clinical meta-data. These data provide the first significant difference between isolates from severe vs. less severe disease based on an in vitro C. difficile phenotype and suggest that clinical outcome is better predicted by bacterial attributes other than ribotype.

To the Editor—We thank Dr McDonald for his interest in our work and concur that the role of particular ribotypes in the clinical outcome of Clostridium difficile infection (CDI) is complex [1]. Our study identified several factors that predicted severe CDI in unadjusted analysis, including ribotype 027/078 (Table 2 in our article) [2]. We agree that including intermediate white blood cell (WBC) counts in our final model (Table 4 in our article) may have potentially attenuated direct effects of ribotype on outcome. In Table 3 of our article, we show, however, that adjustment for any of the significant predictors rendered ribotype 027/078 effect on CDI severity nonsignificant [2], indicating that WBC count was not required for the conclusions drawn in our study. To directly address the possibility that WBC count was influenced by ribotype-specific host responses requires temporal patient data, including treatment, which we did not collect. To indirectly address this issue, we performed additional analyses for our combined dataset (N = 412 cases with complete data) in the following 4 contexts: We added abnormal WBC count ( 12 000 cells/mL) to the definition of severe CDI. We determined whether ribotype 027/078 predicted an abnormal WBC count. We excluded both WBC count and albumin levels from the analysis, as is appropriate if they are potentially confounded. We again added abnormal WBC to the severe CDI definition and excluded albumin level from the analysis. Ribotype 027/078 was not significant in any of these contexts (adjusted analyses). Although these results do not exclude the possibility raised by McDonald, they do add support to our original conclusion that predictors other than ribotype may be more important in explaining the variability of CDI severity. We did not explore the influence of treatment on outcome, so the influence of ribotype was either not strong enough to overcome these effects or they were relatively minor. Why the incidence and severity of CDI increased in the early 2000s remains unclear. However, epidemic spread of clonal pathogens need not result from advantageous phenotypes (toxin production, sporulation, antibiotic resistance, etc). Such population expansions may result simply by chance, as transmission rates overcome clonal interference. Temporal fluctuations in ribotype prevalence support a “drift” scenario [3]. Such fluctuations suggest that many ribotypes reach a high local prevalence, which would increase transmission and potentiate a high global prevalence. This phenomenon does not necessitate strong selection, although increased antibiotic resistance suggests that strong selective pressure from fluoroquinolones and other antimicrobials has been important [4, 5]. As noted by McDonald, the prevalences of both 027 CDI cases and severe outcome are declining in apparent synchrony. However, both declines have occurred in light of heightened surveillance, improved diagnostics, more effective/aggressive infection control practices and therapies, and the popularization of antimicrobial stewardship. Correlations among these shifting practices, ribotype prevalence, and clinical outcome are understudied and require more data from properly designed studies that recognize the potential role of chance (null hypothesis). As 027 prevalence wanes in Europe [6], it appears that the C. difficile population is changing back to a more even distribution. These are exciting times in C. difficile epidemiology, but the complexity of ribotype-specific influences on disease progression and/or severity remains mottled.

The prevalence of carbapenemase-producing Enterobacteriaceae (CPE) in our hospital increased beginning in 2009. We aimed to study the clinical and molecular epidemiology of these emerging isolates. We performed a retrospective review of all adult patients with clinical cultures confirmed as CPE by positive modified Hodge test from 5/2009-5/2010 at the University of Michigan Health System (UMHS). Clinical information was obtained from electronic medical records. Available CPE isolates were analyzed by polymerase chain reaction (PCR) and sequencing of the 16S rRNA encoding gene and blaKPC locus. Multilocus sequence typing (MLST) was used to characterize Klebsiella pneumoniae isolates. Twenty six unique CPE isolates were obtained from 25 adult patients. The majority were Klebsiella pneumoniae (n=17). Other isolates included K. oxytoca (n=3), Citrobacter freundii (n=2), Enterobacter cloacae (n=2), Enterobacter aerogenes (n=1) and Escherichia coli (n=1). Molecular characterization of 19 available CPE isolates showed that 13 (68%) carried the KPC-3 allele and 6 (32%) carried the KPC-2 allele. Among 14 available K. pneumoniae strains, 12 (86%) carried the KPC-3 allele and belonged to a common lineage, sequence type (ST) 258. The other 2 (14%) K. pneumoniae isolates carried the KPC-2 allele and belonged to two unique STs. Among these ST 258 strains, 67% were isolated from patients with prior exposures to health care settings outside of our institution. In contrast, all CPE isolates carrying the KPC-2 allele and all non ST 258 CPE isolates had acquisition attributable to our hospital. Molecular epidemiology of carbapenemase producing K. pneumoniae suggests that KPC-3 producing K. pneumoniae isolates of a common lineage, sequence type (ST 258), are emerging in our hospital. While ST 258 is a dominant sequence type throughout the United States, this study is the first to report its presence in Michigan.

To the Editor—Clostridium difficile infection (CDI) is a nosocomial infection that poses a challenge to infection control procedures, often from delays in testing [1]. A nurse-driven protocol may result in earlier identification and isolation of infected patients, leading to decreased transmission. It is a common “urban legend” among nursing staff that they can identify patients with CDI by the odor of their stool alone. Based on studies that used gas chromatography, this is biologically plausible—in 2004, Probert et al demonstrated the presence of unique volatile organic compounds (VOCs) in stool from patients with CDI [2]. Similarly, Garner et al demonstrated in 2007 that derived discriminant scores from VOC data had 100% predictive accuracy in distinguishing stool from patients who were asymptomatic, had ulcerative colitis, had Campylobacter jejuni infection, or had CDI [3]. In 2002, Johansen et al found that nurses on the wards were 84% sensitive and 77% specific in diagnosing CDI by stool odor [4]. Similarly, in 2007 Burdette et al found that nurses were 55% sensitive and 83% specific in diagnosing CDI [5]. A limitation of both studies, as pointed out in an accompanying commentary [6], is that the nurses were not blinded to any patient characteristics. Thus, one could not conclude that stool odor was the sole factor informing their assessments. We hypothesized that nurses can detect the presence of C. difficile in stool by odor alone in a controlled, laboratory setting. Nurses were recruited from inpatient wards at our hospital. Our microbiology laboratory randomly set aside 5 positive and 5 negative stool samples based on results from 2-step testing (glutamate dehydrogenase/toxin enzyme immunoassay followed by polymerase chain reaction for discordant results) for C. difficile toxin (CDTOX) from patients with liquid stool. We asked nurses about their work experience and whether they believed they could detect C. difficile by odor. They were instructed to sniff each sample and record whether CDTOX positive stool was present. Fisher exact and Mann-Whitney tests were used to assess statistical significance. Eighteen nurses participated. Experience ranged from 1 to 30 years (8 with >10 years of experience). Eleven felt confident in their sniffing ability (61%). The median percentage correct was 45% (range, 40%–80%). CDTOX-positive samples elicited a lower percentage of correct answers than CDTOX-negative samples (Figure ​(Figure11A; median, 31% vs 74%; P = .0119). No single individual performed better than chance (mean sensitivity/specificity = 0.26/0.69). Those with confidence in sniffing did not perform better than others (Figure ​(Figure11B; median, 40% vs 50%; P = .2471) and more experienced nurses were no different from less experienced nurses (Figure ​(Figure11C; both 45%; P = .8887). Figure 1. The percentage of correct responses, based on smell, regarding the presence of toxigenic Clostridium difficile in stool depending on sample type (A), confidence level (B), and experience (C); lines are at the median and interquartile range. Abbreviation: ... In this controlled laboratory setting, our nurses were unable to identify stool samples with C. difficile by odor, but CDTOX-negative samples elicited more correct answers than CDTOX-positive samples. More experience with nursing and confidence in sniffing ability did not improve performance. It is likely that our findings differ from prior studies owing to their inadequate blinding of nurses to patient and stool characteristics.

The urinary tract is one of the most frequent sites of bacterial infection in humans. Uropathogenic Escherichia coli (UPEC) strains are the leading cause of urinary tract infections (UTIs) and are responsible for greater than 80% of uncomplicated cases in adults. Infection of the urinary tract occurs in an ascending manner, with colonization of the bladder leading to possible kidney infection and bacteremia. The goal of this study was to examine the population dynamics of UPEC in vivo using a murine model of ascending UTI. To track individual UPEC lineages within a host, we constructed 10 isogenic clones of UPEC strain CFT073 by inserting unique signature tag sequences between the pstS and glmS genes at the attTn7 chromosomal site. Mice were transurethrally inoculated with a mixture containing equal numbers of unique clones. After 4 and 48 h, the tags present in the bladders, kidneys, and spleens of infected mice were enumerated using tag-specific primers and quantitative real-time PCR. The results indicated that kidney infection and bacteremia associated with UTI are most likely the result of multiple rounds of ascension and dissemination from motile UPEC subpopulations, with a distinct bottleneck existing between the kidney and bloodstream. The abundance of tagged lineages became more variable as infection progressed, especially after bacterial ascension to the upper urinary tract. Analysis of the population kinetics of UPEC during UTI revealed metapopulation dynamics, with lineages that constantly increased and decreased in abundance as they migrated from one organ to another., IMPORTANCE Urinary tract infections are some of the most common infections affecting humans, and Escherichia coli is the primary cause in most uncomplicated cases. These infections occur in an ascending manner, with bacteria traveling from the bladder to the kidneys and potentially the bloodstream. Little is known about the spatiotemporal population dynamics of uropathogenic E. coli within a host. Here we describe a novel approach for tracking lineages of isogenic tagged E. coli strains within a murine host by the use of quantitative real-time PCR. Understanding the in vivo population dynamics and the factors that shape the bacterial population may prove to be of significant value in the development of novel vaccines and drug therapies.

Escherichia coli , a cause of ∼90% of urinary tract infections (UTI), utilizes fimbrial adhesins to colonize the uroepithelium. Pyelonephritis isolate E. coli CFT073 carries 12 fimbrial operons, 5 of which have never been studied. Using multiplex PCR, the prevalence of these 12 and 3 additional fimbrial types was determined for a collection of 303 E. coli isolates (57 human commensal, 32 animal commensal, 54 asymptomatic bacteriuria, 45 complicated UTI, 38 uncomplicated cystitis, and 77 pyelonephritis). The number of fimbrial types per E. coli isolate was distributed bimodally: those with low (3.2 ± 1.1) and those with high (8.3 ± 1.3) numbers of fimbrial types (means ± standard errors of the means). The fimbrial genes ygiL , yadN , yfcV , and c2395 were significantly more prevalent among urine isolates than human commensal isolates. The effect of deletion of Ygi and Yad fimbrial operons on growth, motility, biofilm formation, adherence to immortalized human epithelial cells, and pathogenesis in the mouse model of UTI was examined. Yad fimbriae were necessary for wild-type levels of adherence to a bladder epithelial cell line and for biofilm formation. Deletion of these fimbrial genes increased motility. Ygi fimbriae were necessary for wild-type levels of adherence to a human embryonic kidney cell line, biofilm formation, and in vivo fitness in the urine and kidneys. Complementation of each fimbrial mutant restored wild-type levels of motility, biofilm formation, adherence and, for ygi , in vivo fitness. A double deletion strain, Δ ygi Δyad , was attenuated in the urine, bladder, and kidneys in the mouse model, demonstrating that these fimbriae contribute to uropathogenesis.

Defining bacterial species remains a challenging problem even for the model bacterium Escherichia coli and has major practical consequences for reliable diagnosis of infectious disease agents and regulations for transport and possession of organisms of economic importance. E. coli traditionally is thought to live within the gastrointestinal tract of humans and other warm-blooded animals and not to survive for extended periods outside its host; this understanding is the basis for its widespread use as a fecal contamination indicator. Here, we report the genome sequences of nine environmentally adapted strains that are phenotypically and taxonomically indistinguishable from typical E. coli (commensal or pathogenic). We find, however, that the commensal genomes encode for more functions that are important for fitness in the human gut, do not exchange genetic material with their environmental counterparts, and hence do not evolve according to the recently proposed fragmented speciation model. These findings are consistent with a more stringent and ecologic definition for bacterial species than the current definition and provide means to start replacing traditional approaches of defining distinctive phenotypes for new species with omics-based procedures. They also have important implications for reliable diagnosis and regulation of pathogenic E. coli and for the coliform cell-counting test.