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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.

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

Clostridium difficile infection (CDI) is increasingly prevalent in individuals in health care settings and in the community. C. difficile is a gram positive, anaerobic bacteria that forms spores resistant to common disinfectants. Upon infection, C. difficile spores germinate, divide and produce damaging toxins resulting in intestinal inflammation. While inflammatory mediators are known to affect disease severity, the role of type I IFN signaling in CDI has not been characterized. We hypothesized that type I IFN signaling was important for innate protection in CDI. IFNAR-deficient mice succumbed to CDI more rapidly than wild type mice. Since type I IFNs can reduce inflammasome activity, we assessed IL-1β expression in multiple tissues, and discovered that IFNAR mice expressed more IL-1β protein, and less insulin-like growth factor (IGF) transcripts in response to CDI. To investigate a potential treatment, we treated mice with the type I IFN-inducing dsRNA mimetic, polyIC. PolyIC treatment delayed onset of symptoms in survival studies. Surprisingly, the protective effect of polyIC was not a result of IFNAR signaling; treatment increased IGF and decreased IL-1β in both wild type and IFNAR-deficient mice. Finally, treating wild type mice with recombinant type I IFN also reduced disease severity. These findings suggest direct type I IFN signaling protects from CDI and treatment with polyIC may protect mice via a pathway independent of IFNAR signaling. Considering that recombinant type I IFN is already used clinically and novel treatment options for CDI are needed, further investigation of innate immune pathways may present new treatment options for this emerging infection.

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 widespread agricultural use of antimicrobials has long been considered a crucial influence on the prevalence of resistant genes and bacterial strains. It has been suggested that antibiotic applications in agricultural settings are a driving force for the development of antimicrobial resistance, and epidemiologic evidence supports the view that there is a direct link between resistant human pathogens, retail produce, farm animals, and farm environments. Despite such concerns, little is understood about the population processes underlying the emergence and spread of antibiotic resistance and the reversibility of resistance when antibiotic selective pressure is removed. In this study, hierarchical log-linear modeling was used to assess the association between farm type (conventional versus organic), age of cattle (calf versus cow), bacterial phenotype (resistant versus susceptible), and the genetic composition of Escherichia coli populations ( E. coli Reference Collection [ECOR] phylogroup A, B1, B2, or D) among 678 susceptible and resistant strains from a previously published study of 60 matched dairy farms (30 conventional and 30 organic) in Wisconsin. The analysis provides evidence for clonal resistance (ampicillin resistance) and genetic hitchhiking (tetracycline resistance [Tet r ]), estimated the rate of compositional change from conventional farming to organic farming (mean, 8 years; range, 3 to 15 years), and discovered a significant association between low multidrug resistance, organic farms, and strains of the numerically dominant phylogroup B1. These data suggest that organic farming practices not only change the frequency of resistant strains but also impact the overall population genetic composition of the resident E. coli flora. In addition, the results support the hypothesis that the current prevalence of Tet r loci on dairy farms has little to do with the use of this antibiotic.

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.