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Defining the complex role of the microbiome in colorectal cancer and the discovery of novel, protumorigenic microbes are areas of active investigation. In the present study, culturing and reassociation experiments revealed that toxigenic strains of Clostridioides difficile drove the tumorigenic phenotype of a subset of colorectal cancer patient–derived mucosal slurries in germ-free ApcMin/+ mice. Tumorigenesis was dependent on the C. difficile toxin TcdB and was associated with induction of Wnt signaling, reactive oxygen species, and protumorigenic mucosal immune responses marked by the infiltration of activated myeloid cells and IL17-producing lymphoid and innate lymphoid cell subsets. These findings suggest that chronic colonization with toxigenic C. difficile is a potential driver of colorectal cancer in patients. Significance: Colorectal cancer is a leading cause of cancer and cancer-related deaths worldwide, with a multifactorial etiology that likely includes procarcinogenic bacteria. Using human colon cancer specimens, culturing, and murine models, we demonstrate that chronic infection with the enteric pathogen C. difficile is a previously unrecognized contributor to colonic tumorigenesis. See related commentary by Jain and Dudeja, p. 1838. This article is highlighted in the In This Issue feature, p. 1825

Background Dyslipidemia is a feature of impaired metabolic health in conjunction with impaired glucose metabolism and central obesity. However, the contribution of factors to postprandial lipemia in healthy but metabolically at-risk adults is not well understood. We investigated the collective contribution of several physiologic and lifestyle factors to postprandial triglyceride (TG) response to a high-fat meal in healthy, overweight and obese adults. Methods Overweight and obese adults (n = 35) underwent a high-fat meal challenge with blood sampled at fasting and hourly in the 4-hour postprandial period after a breakfast containing 50 g fat. Incremental area under the curve (iAUC) and postprandial magnitude for TG were calculated and data analyzed using a linear model with physiologic and lifestyle characteristics as explanatory variables. Model reduction was used to assess which explanatory variables contributed most to the postprandial TG response. Results TG responses to a high-fat meal were variable between individuals, with approximately 57 % of participants exceeded the nonfasting threshold for hypertriglyceridemia. Visceral adiposity was the strongest predictor of TG iAUC (β = 0.53, p = 0.01), followed by aerobic exercise frequency (β = 0.31, p = 0.05), insulin resistance based on HOMA-IR (β = 0.30, p = 0.04), and relative exercise intensity at which substrate utilization crossover occurred (β = 0.05, p = 0.04). For postprandial TG magnitude, visceral adiposity was a strong predictor (β = 0.43, p p = 0.01), and exercise intensity for substrate utilization crossover (β = 0.53, p = 0.01). Conclusions Postprandial TG responses to a high-fat meal was partially explained by several physiologic and lifestyle characteristics, including visceral adiposity, insulin resistance, aerobic exercise frequency, and relative substrate utilization crossover during exercise. Trial Registration ClinicalTrials.gov, NCT04128839, Registered 16 October 2019 – Retrospectively registered.

In late December of 2019, high-throughput sequencing technologies enabled rapid identification of SARS-CoV-2 as the etiological agent of COVID-19, and global sequencing efforts are now a critical tool for monitoring the ongoing spread and evolution of this virus. Here, we provide a short retrospective analysis of SARS-CoV-2 variants by analyzing a subset (n = 97,437) of all publicly available SARS-CoV-2 genomes (n = ~11.9 million) that were randomly selected but equally distributed over the course of the pandemic. We plot the appearance of new variants of concern (VOCs) over time and show that the mutation rates in Omicron (BA.1) and Omicron sub-lineages (BA.2–BA.5) are significantly elevated compared to previously identified SARS-CoV-2 variants. Mutations in Omicron are primarily restricted to the spike and nucleocapsid proteins, while 24 other viral proteins—including those involved in SARS-CoV-2 replication—are generally conserved. Collectively, this suggests that the genetic distinction of Omicron primarily arose from selective pressures on the spike, and that the fidelity of replication of this variant has not been altered.

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.

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.

Clostridioides difficile infection (CDI) is the most prevalent healthcare-associated infection in the United States and carries a significant healthcare system burden. As part of an ongoing, active surveillance system of C. difficile throughout Texas, the objective of this study was to assess changes in C. difficile ribotypes of clinical isolates obtained from hospitalized patients in Texas over the past seven years. Fifty hospitals located in Texas, USA sent C. difficile positive stool specimens to a centralized laboratory for PCR ribotyping and toxin characterization between 2011 and 2018. Data collected included specimen collection date, patient age, and sex. Strain genotypes were compiled, and changes in ribotype distribution over time were assessed. Overall, 7796 samples were ribotyped from predominately female patients (58.4%) aged 62 ± 19 years. Samples were obtained from all geographic regions of Texas including Houston/Southwest region (n = 5129; 85%), Dallas/North Texas (n = 579, 9.6%), Central Texas (n = 164; 2.7%), and South Texas (n = 162; 2.6%). The 10 most common ribotypes comprised 73% of all isolates tested during the study period. The most common ribotypes were 027 (17.5%), followed by 014–020 (16.1%), 106 (11.6%), and 002 (9.1%). The prevalence of ribotypes 027, 001, and 078–126 declined significantly over time, while ribotypes 106 and 054 increased in prevalence (P

Extraintestinal infection with Clostridium difficile has been reported but remains uncommon. Treatment of this unusual complication is complex given the limitations of current therapeutic options. Here we report a novel case of recurrent extraintestinal C. difficile infection that occurred following fecal microbiota transplantation. Using whole genome sequencing, we confirmed recrudescence rather than reinfection was responsible. The patient ultimately responded to prolonged, targeted antimicrobial therapy informed by susceptibility testing.

Purpose. This study aimed to describe the correlation between Clostridium difficile spore and toxin levels within the human host. In addition, we assessed whether overgrowth of Candida albicans modified this association. Methodology. We measured toxin, spore and Candida albicans levels among 200 successively collected stool samples that tested positive for C. difficile, and PCR ribotyped these C. difficile isolates. Analysis of variance and linear regression were used to test the association between spore and toxin levels. Kruskal–Wallis tests and t-tests were used to compare the association between spore or toxin levels and host, specimen, or pathogen characteristics. Results. C. difficile toxin and spore levels were positively associated (P

Aims analysis of the stability and safety of Sulfolobus monocaudavirus 1 (SMV1) during passage through the mammalian GI tract. Methods and Results A major challenge of using nano-vectors to target gut microbiome is their survival during passage through the extremely acidic and proteolytic environment of the mammalian GI tract. Here we investigated the thermo-acidophilic archaeal virus SMV1 as a candidate therapeutic nano-vector for the distal mammalian GI tract microbiome. We investigated the anatomical distribution, vector stability, and immunogenicity of this virus following oral ingestion in mice and compared these traits to the more classically used Inovirus vector M13KE. We found that SMV1 particles were highly stable under both simulated GI tract conditions (in vitro) and in mice (in vivo). Moreover, SMV1 could not be detected in tissues outside the GI tract and it elicited a nearly undetectable inflammatory response. Finally, we used human intestinal organoids (HIOs) to show that labeled SMV1 did not invade or otherwise perturb the human GI tract epithelium. Conclusion SMV1 appeared stable and safe during passage though the mammalian GI tract. Significance and Impact this is the first study evaluating an archaeal virus as a potential therapeutic nanoparticle delivery system and it opens new possibilities for future development of novel nanoplatforms. This article is protected by copyright. All rights reserved.

Human intestinal organoids (HIOs) are millimeter-scale models of the human intestinal epithelium and hold tremendous potential for advancing fundamental and applied biomedical research. HIOs resemble the native gut in that they consist of a fluid-filled lumen surrounded by a polarized epithelium and associated mesenchyme; however, their topologically-closed, spherical shape prevents flow through the interior luminal space, making the system less physiological and leading to the buildup of cellular and metabolic waste. These factors ultimately limit experimentation inside the HIOs. Here, we present a millifluidic device called the gut organoid flow chip (GOFlowChip), which we use to "port" HIOs and establish steady-state liquid flow through the lumen for multiple days. This long-term flow is enabled by the use of laser-cut silicone gaskets, which allow liquid in the device to be slightly pressurized, suppressing bubble formation. To demonstrate the utility of the device, we establish separate luminal and extraluminal flow and use luminal flow to remove accumulated waste. This represents the first demonstration of established liquid flow through the luminal space of a gastrointestinal organoid over physiologically relevant time scales. Flow cytometry results reveal that HIO cell viability is unaffected by long-term porting and luminal flow. We expect the real-time, long-term control over luminal and extraluminal contents provided by the GOFlowChip will enable a wide variety of studies including intestinal secretion, absorption, transport, and co-culture with intestinal microorganisms.

Geographic and temporal trends in the distribution of PCR ribotypes for Clostridioides difficile associated diarrheal isolates obtained in the United States (US) are changing. As part of a US national surveillance program of C. difficile susceptibility to fidaxomicin, we quantified the distribution of PCR ribotypes of stool isolates collected from 2011 to 2016.C. difficile isolates or C. difficile toxin + stools from patients with C. difficile infection (CDI) were submitted for testing to Tufts Medical Center from 6 geographically distinct medical centers. Following isolation and confirmation as C. difficile, approximately 35% of the isolates were randomly sampled, stratified by center, for PCR ribotyping by capillary gel electrophoresis. Toxin gene profiling was performed on all isolates.939 isolates from a total of 2814 (33.4%) isolated over the 6 years were analyzed. Seventy unique ribotypes were observed, including 19 ribotypes observed 10 or more times. Sixteen ribotypes were not previously observed in our data base. Ribotype 027 declined by more than 60% over the 6 years of the survey from 35.3% to 13.1% (p 0.001). Ribotype 106 was the most common in 2016, followed by 027 and 014-020. There were strong correlations between 027 and binary toxin with the 18 base pair deletion of tcdC and ribotype 078-126 had 100% concordance with the previously described tcdC 39 base pair deletion.The frequency of ribotypes in the US has changed with a marked decline in 027. Each of the geographical areas had variations which differed from each other, but collectively, these results suggest that the changing epidemiology of C. difficile in the US is consistent with what is being seen in Europe. Continued surveillance and monitoring of changes in ribotype distributions of C. difficile are warranted.

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.

BACKGROUNDConflicting reports have been published on the association between Clostridium difficile ribotypes and severe disease outcomes in patients with C. difficile infection (CDI); several so-called hypervirulent ribotypes have been described. We performed a multicenter study to assess severe disease presentation and severe outcomes among CDI patients infected with different ribotypes.METHODSStool samples that tested positive for C. difficile toxin were collected and cultured from patients who presented to any of 7 different hospitals in Houston, Texas (2011–2013). C. difficile was characterized using a fluorescent PCR ribotyping method. Medical records were reviewed to determine clinical characteristics and ribotype association with severe CDI presentation (ie, leukocytosis and/or hypoalbuminemia) and severe CDI outcomes (ie, ICU admission, ileus, toxic megacolon, colectomy, and/or in-hospital death).RESULTSOur study included 715 patients aged 61±18 years (female: 63%; median Charlson comorbidity index: 2.5±2.4; hospital-onset CDI: 45%; severe CDI: 36.7%; severe CDI outcomes: 12.3%). The most common ribotypes were 027, 014-020, FP311, 002, 078-126, and 001. Ribotype 027 was a significant independent predictor of severe disease (adjusted odds ratio [aOR], 2.24; 95% confidence interval [CI], 1.53–3.29; PP=.041) compared with all other ribotypes in aggregate. However, in an analysis using all common ribotypes as individual variables, ribotype 027 was not associated with severe CDI outcomes more often than other ribotypes.CONCLUSIONRibotype 027 showed virulence equal to that of other ribotypes identified in this endemic setting. Clinical severity markers of CDI may be more predictive of severe CDI outcomes than a particular ribotype.Infect. Control Hosp. Epidemiol. 2015;36(11):1318–1323

Clostridium difficile is the most commonly isolated stool pathogen in inflammatory bowel disease (IBD). Traditional risk factors for C. difficile may not exist in patients with IBD, and no prior studies have assessed the risk factors for the isolation of C. difficile in both symptomatic and asymptomatic IBD outpatients. We prospectively recruited consecutive IBD patients presenting to our outpatient clinic between April 2015 and February 2016. We excluded patients with a diverting ostomy or ileoanal pouch. Demographics, healthcare exposures, medical therapies and disease activity were recorded from medical charts or surveys. A rectal swab was performed from which toxigenic culture and PCR analysis for the presence of toxin and fluorescent PCR ribotyping were performed. The primary outcome of interest was isolation of toxigenic C. difficile. A total of 190 patients were enrolled in this prospective study including 137 (72%) with Crohn’s disease and 53 (28%) with ulcerative colitis. At the time of enrollment, 69 (36%) had clinically active disease. Sixteen (8.4%) patients had toxigenic C. difficile isolated on rectal swab at enrollment and four (2.1%) patients had non-toxigenic C. difficile cultured. Mixed infection with more than one toxigenic isolate was present in 5/16 (33.3%) individuals. Patients with CD with a toxin positive isolate were more likely to have a history of CDI in the past 12 months (40 vs. 11.02%, p = 0.027) and an emergency department visit in the past 12 weeks (50 vs. 20.63%, p = 0.048). In UC, individuals with isolation of C. difficile were more likely to be hospitalized within the past 12 months (66.6 vs. 8.51%, p = 0.003). C. difficile isolation at the time of presentation was not associated with a subsequent disease relapse over a 6-month period in CD (p = 0.557) or UC (p = 0.131). Healthcare exposures remain a significant risk factor for C. difficile isolation in the IBD population; however, this was not associated with relapse of disease. Further studies assessing the clinical significance of C. difficile isolation is warranted in IBD.

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.

Background Clostridium difficile infection (CDI) is a significant nosocomial infection worldwide, that recurs in as many as 35% of infections. Risk of CDI recurrence varies by ribotype, which also vary in sporulation and germination rates. Whether sporulation/germination mediate risk of recurrence and effectiveness of treatment of recurring CDI remains unclear. We aim to assess the role of sporulation/germination patterns on risk of recurrence, and the relative effectiveness of the recommended tapered/pulsing regimens using an in silico model. Methods We created a compartmental in-host mathematical model of CDI, composed of vegetative cells, toxins, and spores, to explore whether sporulation and germination have an impact on recurrence rates. We also simulated the effectiveness of three tapered/pulsed vancomycin regimens by ribotype. Results Simulations underscored the importance of sporulation/germination patterns in determining pathogenicity and transmission. All recommended regimens for recurring CDI tested were effective in reducing risk of an additional recurrence. Most modified regimens were still effective even after reducing the duration or dosage of vancomycin. However, the effectiveness of treatment varied by ribotype. Conclusion Current CDI vancomycin regimen for treating recurrent cases should be studied further to better balance associated risks and benefits.

Objective.To investigate the simultaneous occurrence of more than 1Clostridium difficileribotype in patients' stool samples at the time of diagnostic testing.Methods.Stool samples submitted for diagnostic testing for the presence of toxigenicC. difficilewere obtained for 102 unique patients. A total of 95 single colonies ofC. difficileper stool sample were isolated on selective media, subcultured alongside negative (uninoculated) controls, and polymerase chain reaction (PCR) ribotyped using capillary gel electrophoresis.Results.Capillary-based PCR ribotyping was successful for 9,335C. difficileisolates, yielding a median of 93 characterized isolates per stool sample (range, 69-95). More than 1 C.difficileribotype was present in 16 of 102 (16%)C. difficileinfection (CDI) cases; 2 of the 16 mixtures were composed of at least 3 ribotypes, while the remaining 14 were composed of at least 2.Conclusions.Deep sampling of patient stool samples coupled with capillary-based PCR ribotyping identified a high rate of mixed CDI cases compared with previous estimates. Studies seeking to quantify the clinical significance of particular C.difficileribotypes should account for mixed cases of disease.

Capillary-based PCR ribotyping was used to quantify the presence/absence and relative abundance of 98 Clostridium difficile ribotypes from clinical cases of disease at health care institutions in six states of the United States. Regionally important ribotypes were identified, and institutions in close proximity did not necessarily share more ribotype diversity than institutions that were farther apart.

Extraintestinal Escherichia coli (ExPEC), a heterogeneous group of pathogens, encompasses avian, neonatal meningitis, and uropathogenic E. coli strains. While several virulence factors are associated with ExPEC, there is no core set of virulence factors that can be used to definitively differentiate these pathotypes. Here we describe a multiplex of four virulence factor-encoding genes, yfcV , vat , fyuA , and chuA , highly associated with uropathogenic E. coli strains that can distinguish three groups of E. coli : diarrheagenic and animal-associated E. coli strains, human commensal and avian pathogenic E. coli strains, and uropathogenic and neonatal meningitis E. coli strains. Furthermore, human intestinal isolates that encode all four predictor genes express them during exponential growth in human urine and colonize the bladder in the mouse model of ascending urinary tract infection in higher numbers than human commensal strains that do not encode the four predictor genes ( P = 0.02), suggesting that the presence of the predictors correlates with uropathogenic potential.

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.

Toxigenic Clostridium sordellii strains are increasingly recognized to cause highly lethal infections in humans that are typified by a toxic shock syndrome (TSS). Two glucosylating toxins, lethal toxin (TcsL) and hemorrhagic toxin (TcsH) are believed to be important in the pathogenesis of TSS. While non-toxigenic strains of C. sordellii demonstrate reduced cytotoxicity in vitro and lower virulence in animal models of infection, there are few data regarding their behavior in humans. Here we report a non-TSS C. sordellii infection in the context of a polymicrobial bacterial cholangitis. The C. sordellii strain associated with this infection did not carry either the TcsL-encoding tcsL gene or the tcsH gene for TcsH. In addition, the strain was neither cytotoxic in vitro nor lethal in a murine sepsis model. These results provide additional correlative evidence that TcsL and TcsH increase the risk of mortality during C. sordellii infections.

As Clostridium difficile spores are resistant to many household cleaning products, the potential for community household contamination is high. The purpose of this study was to assess the prevalence of toxigenic C. difficile from environmental sources from a large urban area. Three to 5 household items or environmental dust was collected from 30 houses in Houston, Texas. A total of 127 environmental samples were collected from shoe bottoms (n = 63), bathroom surfaces (n = 15), house floor dusts (n = 12), or other household surfaces (n = 37). Forty one of 127 samples (32.3%) grew C. difficile. All 41 isolates were positive for toxin A and B genes and no isolate was positive for binary toxin genes. Shoe bottom swab samples had the highest percent of positive samples (25/63; 39.7%) followed by bathroom/toilet surfaces (5/15; 33.3%), house floor dust (4/12; 33.3%), and other surface swabs (7/37; 18.9%). Strains were grouped into 25 different ribotypes, the most prevalent type was 001 (5 strains). In conclusion, a high rate of environmental contamination of C. difficile was observed from community households from a large urban area.

Extended multilocus sequence typing (MLST) analysis of atypical Escherichia isolates was used to identify five novel phylogenetic clades (CI to CV) among isolates from environmental, human, and animal sources. Analysis of individual housekeeping loci showed that E. coli and its sister clade, CI, remain largely indistinguishable and represent nascent evolutionary lineages. Conversely, clades of similar age (CIII and CIV) were found to be phylogenetically distinct. When all Escherichia lineages (named and unnamed) were evaluated, we found evidence that E scherichia fergusonii has evolved at an accelerated rate compared to E. coli , CI, CIII, CIV, and CV, suggesting that this species is younger than estimated by the molecular clock method. Although the five novel clades were phylogenetically distinct, we were unable to identify a discriminating biochemical marker for all but one of them (CIII) with traditional phenotypic profiling. CIII had a statistically different phenotype from E. coli that resulted from the loss of sucrose and sorbitol fermentation and lysine utilization. The lack of phenotypic distinction has likely hindered the ability to differentiate these clades from typical E. coli , and so their ecological significance and importance for applied and clinical microbiology are yet to be determined. However, our sampling suggests that CIII, CIV, and CV represent environmentally adapted Escherichia lineages that may be more abundant outside the host gastrointestinal tract.

In 2009, five monophyletic Escherichia clades were described and referred to as “cryptic” based on the inability to distinguish them from representative E. coli isolates using diagnostic biochemical reactions. Since this original publication, a number of studies have explored the genomic, transcriptomic, and phenotypic diversity of cryptic clade isolates to better understand their phylogenetic, physiological, and ecological distinctiveness with respect to previously named Escherichia species. This chapter reviews the original discovery of the cryptic clades, discusses available evidence that some are environmentally adapted, and evaluates current support for taxonomic designations of these microorganisms. The importance of these clades to clinical research, epidemiology, population genetics, and microbial speciation is also discussed.

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.

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.

Studies of Clostridium difficile outbreaks suggested that certain ribotypes (eg, 027 and 078) cause more severe disease than other ribotypes. A growing number of studies challenge the validity of this hypothesis.We conducted a cross-sectional study of C. difficile infection (CDI) to test whether ribotype predicted clinical severity when adjusted for the influence of other predictors. Toxigenic C. difficile isolates were cultured from stool samples, screened for genes encoding virulence factors by polymerase chain reaction (PCR) and ribotyped using high-throughput, fluorescent PCR ribotyping. We collected data for 15 covariates (microbiologic, epidemiologic, and laboratory variables) and determined their individual and cumulative influence on the association between C. difficile ribotype and severe disease. We then validated this influence using an independent data set.A total of 34 severe CDI cases were identified among 310 independent cases of disease (11.0%). Eleven covariates, including C. difficile ribotype, were significant predictors of severe CDI in unadjusted analysis. However, the association between ribotypes 027 and 078 and severe CDI was not significant after adjustment for any of the other covariates. After full adjustment, severe cases were significantly predicted only by patients' white blood cell count and albumin level. This result was supported by analysis of a validation data set containing 433 independent CDI cases (45 severe cases; 10.4%).Ribotype is not a significant predictor of severe CDI when adjusted for the influence of any other variables separately or in combination. White blood cell count and albumin level are the most clinically relevant predictors of severe CDI cases.

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.

Clostridium difficile is a significant nosocomial threat to human health and is the most commonly identified cause of antibiotic-associated diarrhea. The development of C. difficile colitis requires production of toxins A and/or B, but some strains do not express these proteins. These non-toxigenic C. difficile (NTCD) have garnered attention for their capacity to colonize humans and potentially reduce the risk for symptomatic colitis caused by toxigenic strains. Isolates of NTCD have been obtained from the environment as well as from animal and human sources. Studies in a hamster CDI model have demonstrated a protective effect of NTCD against toxigenic infection. The extent to which this protective effect of NTCD occurs in humans remains to be defined. Evidence for a therapeutic or preventive role for NTCD is limited but clinical prophylaxis studies are ongoing. NTCD potentially represents an exciting new tool in preventing CDI and its recurrences.

Journal of Clinical Microbiology p. 728–730 February 2013 Volume 51 Number 2 also available at jcm.asm.org

The gut microbiota, the collection of all bacterial members in the intestinal tract, plays a key role in health. Disruption of the indigenous microbiota by a variety of stressors, including antibiotic therapy and intestinal infections, is associated with multiple health problems. We sought to determine if infection with Norovirus disrupts the gut microbiota. Barcoded pyrosequencing of the 16S rRNA-encoding gene was used to characterize the stool microbiota in Norovirus-infected human patients (n = 38). While the microbiota in most infected patients (n = 31) resembled that seen in uninfected healthy controls, a minority of patients (n = 7) possessed a significantly altered microbiota characterized by reduced relative numbers of Bacteriodetes and a corresponding increase in Proteobacteria. In these patients, the increase in Proteobacteria was due to a single operational taxonomic unit (OTU) of Escherichia coli. We cultured E. coli from Norovirus-infected patients and characterized them using PCR-ribotyping and virulence factor analysis. Multiple ribotypes were encountered, but none possessed typical virulence factors commonly carried by enteropathogenic E. coli strains. Microbiota disruption and elevated Proteobacteria were not significantly correlated to patient age, gender, sampling time following illness onset, or overall gut inflammation. These results demonstrate that some patients have a disrupted microbiota following Norovirus infection, and therefore may be at elevated risk for long-term health complications.

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.

Strains phenotypically indistinguishable from Escherichia coli and belonging to at least five distinct cryptic lineages, named Escherichia clades I to V, that are genetically divergent from E. coli yet members of the genus have been recently found using multi-locus sequence typing (MLST). Very few epidemiological data are available on these strains as their detection by MLST is not suitable for large-scale studies. In this work, we developed a rapid PCR method based on aes and chuA allele-specific amplifications that assigns a strain a cryptic lineage membership. By screening more than 3500 strains with this approach, we show that the cryptic lineages of Escherichia are unlikely to be detected in human faecal samples (2-3% frequency) and even less likely to be isolated from extra-intestinal body sites (1% frequency). They are more abundant in animal faeces ranging from 3-8% in non-human mammals to 8-28% in birds. Overall, the strains from the clade V are the most abundant and from the clade II very rare. These results suggest that members of the cryptic clades are unlikely to be of significance to human and health but may influence the use of 'E. coli' as an indicator of water quality.

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.

Background The study of intestinal microbiota has been revolutionised by the use of molecular methods, including terminal restriction fragment length polymorphism (T-RFLP) analysis. Microbiota studies of Crohn9s disease patients have examined samples from stool or from the neoterminal ileum with a standard biopsy forceps, which could be contaminated by colonic bacteria when the forceps passes through the colonoscope channel. Objective To determine whether sheathed biopsy forceps are able to obtain terminal ileal microbiota samples with less colonic bacterial contamination compared with unsheathed (standard) biopsy forceps. Design Prospective randomised single-centre study. Patients and methods Four (paired) biopsy specimens were obtained from adjacent locations in the terminal ileum using the sheathed and standard forceps of 27 consecutive subjects undergoing colonoscopy and the microbiota were characterised using T-RFLP. The Bray–Curtis similarity index between samples (sheathed vs unsheathed forceps) was calculated within patients and significant differences were tested for across all patients. Results There was not a significant difference in the microbial diversity of samples obtained using sheathed versus unsheathed forceps. The difference in microbial diversity between patients was much greater than the variability within patients by proximal versus distal site or by forceps type. Limitations T-RFLP is based on PCR amplification, so it is not always sensitive to rare bacterial species. Conclusion Standard unsheathed forceps appear to be sufficient for microbiota sample collection from the terminal ileum.

We report a pseudo-outbreak of infection caused byClostridium sordellii, an uncommon human pathogen. The pseudo-outbreak involved 6 patients and was temporally associated with a change by the clinical microbiology laboratory in the protocol of handling anaerobic culture specimens. All isolates were genetically indistinguishable from a laboratory reference strain used for quality control.

The clonal analysis of a diverse collection of Salmonella Enteritidis indicates that most strains belong to a single multilocus genotype (i.e., ET-3) regardless of phage type, geographic origin, or time of isolation that spanned over 2 decades (1978 to 2004). Attachment and invasion assays, however, indicate that, among ET-3 isolates, there is a distinct invasive bacterial subpopulation that is more readily recovered from eggs and clinical cases in humans than from chicken cecal samples. These observations support the hypothesis that the specialized ability of S. Enteritidis to infect the avian reproductive tract and contaminate eggs has been critical in its emergence as a frequent cause of human illness.

Objective Clostridium difficile infection (CDI) is a major cause of morbidity and biomarkers that predict severity of illness are needed. Procalcitonin (PCT), a serum biomarker with specificity for bacterial infections, has been little studied in CDI. We hypothesized that PCT associated with CDI severity. Design Serum PCT levels were measured for 69 cases of CDI. Chart review was performed to evaluate the presence of severity markers and concurrent acute bacterial infection (CABI). We defined the binary variables clinical score as having fever (T >38°C), acute organ dysfunction (AOD), and/or WBC >15,000 cells/mm3 and expanded score, which included the clinical score plus the following: ICU admission, no response to therapy, colectomy, and/or death. Results In univariate analysis log10 PCT associated with clinical score (OR 3.13, 95% CI 1.69–5.81, P0.2 ng/mL was 81% sensitive/73% specific for a positive clinical score and had a negative predictive value of 90%. Conclusion An elevated PCT level associated with the presence of CDI severity markers and CDI was unlikely to be severe with a serum PCT level below 0.2 ng/mL. The extent to which PCT changes during CDI therapy or predicts recurrent CDI remains to be quantified.