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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.

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.

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.

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.

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

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.

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.