Persistent diarrhea in CDI correlates with intestinal inflammation and not fecal pathogen burden. These findings suggest that modulation of host response, rather than adjustments to antimicrobial regimens, might be a more effective approach to patients with unremitting disease.
The potential to rapidly capture the entire microbial community structure and/or gene content makes metagenomic sequencing an attractive tool for pathogen identification and the detection of resistance/virulence genes in clinical settings. Here, we assessed the consistency between PCR from a diagnostic laboratory, quantitative PCR (qPCR) from a research laboratory, 16S rRNA gene sequencing, and metagenomic shotgun sequencing (MSS) for Clostridium difficile identification in diarrhea stool samples. Twenty-two C. difficile-positive diarrhea samples identified by PCR and qPCR and five C. difficile-negative diarrhea controls were studied. C. difficile was detected in 90.9% of C. difficile-positive samples using 16S rRNA gene sequencing, and C. difficile was detected in 86.3% of C. difficile-positive samples using MSS. CFU inferred from qPCR analysis were positively correlated with the relative abundance of C. difficile from 16S rRNA gene sequencing (r 2 ؍ ؊0.60) and MSS (r 2 ؍ ؊0.55). C. difficile was codetected with Clostridium perfringens, norovirus, sapovirus, parechovirus, and anellovirus in 3.7% to 27.3% of the samples. A high load of Candida spp. was found in a symptomatic control sample in which no causative agents for diarrhea were identified in routine clinical testing. Beta-lactamase and tetracycline resistance genes were the most prevalent (25.9%) antibiotic resistance genes in these samples. In summary, the proof-of-concept study demonstrated that next-generation sequencing (NGS) in pathogen detection is moderately correlated with laboratory testing and is advantageous in detecting pathogens without a priori knowledge. Sequencing technology has revolutionized infectious diseases research over the past decade. Whole-genome sequencing (WGS) of pure cultures has been widely used for pathogen characterization, evolutionary studies, transmission investigations, and outbreak detection (1, 2, 3). WGS of cultured isolates is now moving from the proof-of-concept phase to implementation. The two major applications of WGS of cultured strains in clinical diagnostic microbiology are molecular epidemiology and antibiotic resistance gene prediction (4). In contrast to the WGS sequencing of cultured isolates, metagenomics assesses a community of organisms but eliminates the isolation step. This can be done by focusing on a specific conserved gene, such as the 16S rRNA gene, or by the metagenomic shotgun sequencing (MSS) of total microbial nucleic acids within samples. For the purpose of this study, metagenomics sequencing refers to either 16S rRNA gene sequencing or MSS; 16S rRNA gene sequencing and MSS using next-generation sequencing (NGS) platforms produce large quantities of data in a relatively short time. Although 16S rRNA gene sequencing is less expensive than MSS, it suffers from potential PCR-related bias. Taxonomical classification based on partial 16S rRNA gene sequencing is generally limited to phylum to genus level specificity. Nevertheless, highly heterogeneous species within certain genera can be dist...
Objectives To identify specific fecal biomarkers for symptomatic Clostridium difficile infection and predictors of poor outcomes. Study design We enrolled children with positive C. difficile testing (cases) and symptomatic controls. We also analyzed stool samples from colonized and non-colonized asymptomatic children. We performed enzyme immunoassays (EIA) to determine fecal interleukin (IL)-8, lactoferrin and phosphorylated-p38 protein concentrations, and quantitative polymerase chain reactions (PCR) to determine IL-8 and CXCL-5 RNA relative transcript abundances, and C. difficile bacterial burden. Results Of 68 asymptomatic controls, 16 were colonized with C. difficile. Phosphorylated-p38 was specific for C difficile infection but lacked sensitivity. Fecal cytokines were elevated in samples from symptomatic children, whether cases or controls. In children with C difficile infection, fecal CXCL-5 and IL-8 mRNA abundances at diagnosis correlated with persistent diarrhea after five days of C difficile infection therapy and with treatment with vancomycin. When children with concomitant viral gastroenteritis were excluded, these correlations persisted. Time-to-diarrhea resolution was significantly longer in patients with elevated fecal cytokines at diagnosis. A logistic regression model identified high CXCL-5 mRNA abundance as the only predictor of persistent diarrhea. Conversely, fecal C. difficile bacterial burden was not different in symptomatic and asymptomatic children and did not correlate with any clinical outcome measure. Conclusions Fecal inflammatory cytokines may be useful in distinguishing C. difficile colonization from disease and identifying children with C difficile infection likely to have prolonged diarrhea.
Clostridium difficile infection (CDI) results in toxin-induced epithelial injury and marked intestinal inflammation. Fecal markers of intestinal inflammation correlate with CDI disease severity, but regulation of the inflammatory response is poorly understood. Previous studies demonstrated that C. difficile toxin TcdA activates p38 kinase in tissue culture cells and mouse ilium, resulting in interleukin-8 (IL-8) release. Here, we investigated the role of phosphorylated mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2 kinase, pMK2), a key mediator of p38-dependent inflammation, in CDI. Exposure of cultured intestinal epithelial cells to the C. difficile toxins TcdA and TcdB resulted in p38-dependent MK2 activation. Toxininduced IL-8 and GRO␣ release required MK2 activity. We found that p38 and MK2 are activated in response to other actin-disrupting agents, suggesting that toxin-induced cytoskeleton disruption is the trigger for kinase-dependent cytokine response. Phosphorylated MK2 was detected in the intestines of C. difficile-infected hamsters and mice, demonstrating for the first time that the pathway is activated in infected animals. Furthermore, we found that elevated pMK2 correlated with the presence of toxigenic C. difficile among 100 patient stool samples submitted for C. difficile testing. In conclusion, we find that MK2 kinase is activated by TcdA and TcdB and regulates the expression of proinflammatory cytokines. Activation of p38-MK2 in infected animals and humans suggests that this pathway is a key driver of intestinal inflammation in patients with CDI.
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