Alexander Rodriguez-Palacios

Clostridium difficile PCR Ribotypes in Calves, Canada

C. difficile, including epidemic PCR ribotypes 017 and 027, were isolated from dairy calves in Canada.

Transmission of Clostridium difficile in foods.

Clostridium difficile is a human intestinal pathogen most frequently involved in diarrheal illnesses following the administration of antibiotics. There is growing concern that some C difficile infections (CDI) may be acquired from ingestion of C difficile spores in contaminated foods. The number of CDI cases is increasing with a heightening in the severity of disease symptoms and an increasing number of community-associated infections not connected to health care-associated risk. This article provides an overview of information related to assessing the risk of foodborne transmission of CDI, highlighting studies on C difficile relevant to food safety in health care settings.

Prevalence, enumeration, and antimicrobial agent resistance of Clostridium difficile in cattle at harvest in the United States.

To assess the potential for food contamination with Clostridium difficile from food animals, we conducted a cross-sectional fecal prevalence study in 944 randomly selected cattle harvested at seven commercial meat processing plants, representing four distant regions (median distance of 1,500 km) of the United States. In all, 944 animals were sampled in the summer of 2008. C. difficile was isolated from 1.8% (17 of 944) of cattle, with median fecal shedding concentration of 2.2 log CFU/g (range = 1.6 to 4.8, 95% confidence interval = 1.6, 4.3). Toxigenic C. difficile isolates were recovered from only four (0.4%) cattle. One of these isolates was emerging PCR ribotype 078/toxinotype V. The remaining toxigenic isolates were toxinotype 0, one of which was an isolate with resistance to linezolid, clindamycin, and moxifloxacin (by the E-test). All isolates were susceptible to vancomycin, metronidazole, and tigecycline, but the MICs against linezolid were as high as the highest reported values for human-derived isolates. The source of the linezolid-clindamycin-moxifloxacin resistance in a toxigenic C. difficile isolate from cattle is uncertain. However, since the use of these three antimicrobial agents in cattle is not allowed in North America, it is possible that resistance originated from an environmental source, from other species where those antimicrobial agents are used, or transferred from other intestinal bacteria. This study confirms that commercial cattle can carry epidemiologically relevant C. difficile strains at the time of harvest, but the prevalence at the time they enter the food chain is low.

Dysregulated NOD2 predisposes SAMP1/YitFc mice to chronic intestinal inflammation.

Significance We discovered that SAMP1/YitFc (SAMP) mice, which develop spontaneous Crohn’s disease (CD)-like ileitis in the absence of nucleotide-binding oligomerization domain-containing 2 (NOD2) genetic mutations, fail to respond to muramyl dipeptide and display impaired bacterial clearance. These results support the concept that a dysregulated NOD2 in SAMP mice predisposes them to chronic intestinal inflammation. We believe that our study provides a paradigm shift by demonstrating that CD-like ileitis is caused by an innate immune defect, rather than an overly aggressive adaptive immune response. Therefore, preventive and curative treatments for CD should be directed to boost, rather than suppress, mucosal innate immune responses. Nucleotide-binding oligomerization domain-containing 2 (NOD2) is an intracellular receptor that plays an essential role in innate immunity as a sensor of a component of the bacterial cell wall, muramyl dipeptide (MDP). Crohn’s disease (CD)-associated NOD2 variants lead to defective innate immune responses, including decreased NF-κB activation and cytokine production. We report herein that SAMP1/YitFc (SAMP) mice, which develop spontaneous CD-like ileitis in the absence of NOD2 genetic mutations, fail to respond to MDP administration by displaying decreased innate cytokine production and dysregulated NOD2 signaling compared with parental AKR control mice. We show that, unlike in other mouse strains, in vivo administration of MDP does not prevent dextran sodium sulfate-induced colitis in SAMP mice and that the abnormal NOD2 response is specific to the hematopoietic cellular component. Moreover, we demonstrate that MDP fails to enhance intracellular bacterial killing in SAMP mice. These findings shed important light on the initiating molecular events underlying CD-like ileitis.

Genetic deletion of the bacterial sensor NOD2 improves murine Crohn’s disease-like ileitis independent of functional dysbiosis

Although genetic polymorphisms in NOD2 (nucleotide-binding oligomerization domain-containing 2) have been associated with the pathogenesis of Crohn’s disease (CD), little is known regarding the role of wild-type (WT) NOD2 in the gut. To date, most murine studies addressing the role of WT Nod2 have been conducted using healthy (ileitis/colitis-free) mouse strains. Here, we evaluated the effects of Nod2 deletion in a murine model of spontaneous ileitis, i.e., the SAMP1Yit/Fc (SAMP) strain, which closely resembles CD. Remarkably, Nod2 deletion improved both chronic cobblestone ileitis (by 50% assessed, as the % of abnormal mucosa at 24 wks of age), as well as acute dextran sodium sulfate (DSS) colitis. Mechanistically, Th2 cytokine production and Th2-transcription factor activation (i.e., STAT6 phosphorylation) were reduced. Microbiologically, the effects of Nod2 deletion appeared independent of fecal microbiota composition and function, assessed by 16S rRNA and metatranscriptomics. Our findings indicate that pharmacological blockade of NOD2 signaling in humans could improve health in Th2-driven chronic intestinal inflammation.

The Artificial Sweetener Splenda Promotes Gut Proteobacteria, Dysbiosis, and Myeloperoxidase Reactivity in Crohn’s Disease–Like Ileitis

Abstract Background Epidemiological studies indicate that the use of artificial sweeteners doubles the risk for Crohn’s disease (CD). Herein, we experimentally quantified the impact of 6-week supplementation with a commercial sweetener (Splenda; ingredients sucralose maltodextrin, 1:99, w/w) on both the severity of CD-like ileitis and the intestinal microbiome alterations using SAMP1/YitFc (SAMP) mice. Methods Metagenomic shotgun DNA sequencing was first used to characterize the microbiome of ileitis-prone SAMP mice. Then, 16S rRNA microbiome sequencing, quantitative polymerase chain reaction, fluorescent in situ hybridization (FISH), bacterial culture, stereomicroscopy, histology, and myeloperoxidase (MPO) activity analyses were then implemented to compare the microbiome and ileitis phenotype in SAMP with that of control ileitis-free AKR/J mice after Splenda supplementation. Results Metagenomics indicated that SAMP mice have a gut microbial phenotype rich in Bacteroidetes, and experiments showed that Helicobacteraceae did not have an exacerbating effect on ileitis. Splenda did not increase the severity of (stereomicroscopic/histological) ileitis; however, biochemically, ileal MPO activity was increased in SAMP treated with Splenda compared with nonsupplemented mice (P < 0.022) and healthy AKR mice. Splenda promoted dysbiosis with expansion of Proteobacteria in all mice, and E. coli overgrowth with increased bacterial infiltration into the ileal lamina propria of SAMP mice. FISH showed increase malX gene–carrying bacterial clusters in the ilea of supplemented SAMP (but not AKR) mice. Conclusions Splenda promoted gut Proteobacteria, dysbiosis, and biochemical MPO reactivity in a spontaneous model of (Bacteroidetes-rich) ileal CD. Our results indicate that although Splenda may promote parallel microbiome alterations in CD-prone and healthy hosts, this did not result in elevated MPO levels in healthy mice, only CD-prone mice. The consumption of sucralose/maltodextrin-containing foods might exacerbate MPO intestinal reactivity only in individuals with a pro-inflammatory predisposition, such as CD.

Uncovering Pathogenic Mechanisms of Inflammatory Bowel Disease Using Mouse Models of Crohn’s Disease–Like Ileitis: What is the Right Model?

Crohn’s disease and ulcerative colitis, together known as inflammatory bowel disease, are debilitating chronic disorders of unknown cause and cure. Our evolving understanding of these pathologies is enhanced greatly by the use of animal models of intestinal inflammation that allow in vivo mechanistic studies, preclinical evaluation of new therapies, and investigation into the causative factors that underlie disease pathogenesis. Several animal models, most commonly generated in mice, exist for the study of colitis. The appropriateness of their use often can be determined by their mode of generation (ie, chemical induction, T-cell transfer, targeted genetic manipulation, spontaneously occurring, and so forth), the type of investigation (mechanistic studies, pathogenic experiments, preclinical evaluations, and so forth), and the type of inflammation that occurs in the model (acute vs chronic colitis, tissue injury/repair, and so forth). Although most murine models of inflammatory bowel disease develop inflammation in the colon, Crohn’s disease most commonly occurs in the terminal ileum, where a very limited number of mouse models manifest disease. This review discusses appropriate experimental applications for different mouse models of colitis, and highlights the particular utility of 2 highly relevant models of Crohn’s-like ileitis—the spontaneous SAMP1/YitFc inbred mouse strain and the genetically engineered TnfΔAU-rich element/+ mouse model of tumor necrosis factor overexpression, both of which bear strong resemblance to the human condition. Similar to patients with Crohn’s disease, SAMP1/YitFc ileitis develops spontaneously, without chemical, genetic, or immunologic manipulation, making this model particularly relevant for studies aimed at identifying the primary defect underlying the occurrence of Crohn’s ileitis, as well as preclinical testing of novel treatment modalities.

Subboiling Moist Heat Favors the Selection of Enteric Pathogen Clostridium difficile PCR Ribotype 078 Spores in Food

Emerging enteric pathogens could have not only more antibiotic resistance or virulence traits; they could also have increased resistance to heat. We quantified the effects of minimum recommended cooking and higher temperatures, individually on a collection of C. difficile isolates and on the survival probability of a mixture of emerging C. difficile strains. While minimum recommended cooking time/temperature combinations (63–71°C) allowed concurrently tested strains to survive, higher subboiling temperatures reproducibly favored the selection of newly emerging C. difficile PCR ribotype 078. Survival ratios for “ribotypes 078” :  “other ribotypes” (n = 49 : 45 isolates) from the mid-2000s increased from 1 : 1 and 0.7 : 1 at 85°C (for 5 and 10 minutes, resp.) to 2.3 : 1 and 3 : 1 with heating at 96°C (for 5 and 10 minutes, resp.) indicating an interaction effect between the heating temperature and survival of C. difficile genotypes. In multistrain heating experiments, with PCR ribotypes 027 and 078 from 2004 and reference type strain ATCC 9689 banked in the 1970s, multinomial logistic regression (P < 0.01) revealed PCR ribotype 078 was the most resistant to increasing lethal heat treatments. Thermal processes (during cooking or disinfection) may contribute to the selection of emergent specific virulent strains of C. difficile. Despite growing understanding of the role of cooking on human evolution, little is known about the role of cooking temperatures on the selection and evolution of enteric pathogens, especially spore-forming bacteria.

Tu1934 Whole-Genome Sequencing and Transcriptome Analysis of Mice With Progressive Crohn's Disease-Like Ileitis

Background: Autophagy has been implicated in the pathogenesis of CD. ATG12-ATG5ATG16L1 conjugate protein complex plays a critical role in autophagy pathway. We previously reported that rs26538 in ATG12 correlated with CD at a modest level, and genetic interaction between this SNP and the focal SNP rs2241880 in ATG16L1 could contribute to CD susceptibility. Our study aimed to explore expression association of the three genes in terminal ileum biopsies in an East Anglia CD cohort. Methods: TaqMan expression & genotyping assays were all ordered from Applied Biosystems. Both expression correlation regardless of genotype and eQTL experiments for a particular SNP were undertaken in a control panel. This panel comprised 97 healthy volunteers from Addenbrookes hospital endoscopy centre. Blood DNA of normal controls was genotyped for the SNPs. mRNA was extracted from terminal ileum biopsies and then reverse-transcribed into cDNA, which was used to measure gene expression by qPCR (2-ddCt, normalised to GAPDH). A case-control study was designed to compare gene expression between control panel and case panel with non-inflamed (24) and inflamed (22) CD cases (Kruskal-Wallis test). Results: In this study ATG5, ATG12 and ATG16L1 gene expression significantly correlated one another in normal controls regardless of genotype (p 0.05). ATG12 expression was not significantly different among inflamed CD, non-inflamed CD and controls (p=0.17395); ATG12 expression also was not significantly different for any pair (all p-values >0.05). ATG16L1 expression was significantly different among inflamed CD, non-inflamed CD and controls (p=0.0003); ATG16L1 expression was significantly different for any pair comparison (all p-values <0.05). Conclusion: ATG5, ATG12 and ATG16L1 gene expression levels significantly correlated with one another in normal controls. ATG5 and ATG16L1 expression significantly differed among noninflamed, inflamed and controls. Together with our previously reported genetic findings, it can be hypothesized that the three autophagy genes may work together to contribute to CD susceptibility.

Food Indwelling Clostridium difficile in Naturally Contaminated Household Meals: Data for Expanded Risk Mathematical Predictions

To the Editor—We read with interest the study “An Evaluation of Food as a Potential Source for Clostridium difficile Acquisition in Hospitalized Patients,” in which Kwon et al made mathematical predictions on the risk of C. difficile infection (CDI) acquisition due to consumption of C. difficile (CD)–contaminated foods in one hospital setting (presumably from a single kitchen source). Although the authors tested many small-sized, mixed-meal samples (n= 910) consumed by 149 patients (median length of hospital stay, 4 days), their mathematical predictions were based on prevalence data obtained using, apparently, nonenrichment culture methods (previously tested for fecal swabs), which are suboptimal for culturing CD and foodborne pathogens from food. Furthermore, the study used a nonstandard heat-shock treatment (80°C, 10 minutes) prior to culture, which introduces a negative bias because this heat treatment has been shown recently to kill up to 75% of CD isolates in liquid media. Hence, not surprisingly, Kwon et al reported a low prevalence of CD in the testedmeals (0.22%). This observed prevalence was then used for mathematical modeling. With low-prevalence data, their prediction regarding causal connections between food contamination and the incidence of CDI was, reasonably, that food is an unlikely risk for CDI (<1 colonization per 1,000 admissions) in their study. Although the study makes an important contribution to the controversial topic of whether CDIs are foodborne, their conclusion seems biased due to suboptimal CD culture methodology. Distinctive methodological imperfections, without critical interpretation, may set us back to the first studies in the 1980s, when CD was not found in hospital meals using nonenrichment methods. After decades of believing CDI was strictly nosocomial, there is now solid evidence based on whole-genome sequencing of CD isolates in hospitals that less than one-third of CDIs are nosocomial, whereas most sources of exposure that result in CDIs remain unknown. With such genomic hospital discoveries, and with the persistence of CDI despite immense efforts to prevent nosocomial transmission, it is not advisable to discard the most plausible source of toxigenenic CD spores (ie, food), even if some studies report negative results. Manymore unbiased reports have shown that food can be a real source of CD spores of virulent or multidrug-resistant CD strains, including studies of hospital meals showing 17% and 27% prevalence on cooked and uncooked meats, respectively. Even Kwon et al reported important CD strains in food: specifically, CD spores of toxigenic PCR-ribotypes 001 in gelatin dessert and 027 in ‘vegetable/bread/ grain.’ Food-dwelling CD became evident as a natural source of exposure to humans in 2005 when emerging hypervirulent CD strains causing severe disease in humans in Canada and United Kingdom were unexpectedly found in food animals and retail foods. To date, no studies have addressed kitchens as complex food environments where cross contamination and cooking practices may influence the prevalence of CD at the consumer level. Here, we would like to contribute to the external validity of the Kwon study on hospital-cooked meals by reporting, for the first time, CD data for household-cooked meals. Although we did not study colonization in humans, we blindly quantified CD in household meals, and we investigated the potential for environment–food cross contamination after visiting 35 rural and urban households in Ohio (2.3± 1.2 visits/each; over four months). In total, 467 samples of food (collected from 188 kitchen pots or refrigerators) and 279 samples from the household environment were processed using validated food-enrichment protocols. Meals, cooked, uncooked, or processed, were sampled, homogenized, centrifuged, and stored as sediments at −80°C until processing. Environmental swabs (8 cm× 4 cm× 1 cm) from kitchen countertops (n= 32), sinks (n= 56), refrigerator shelves (n= 59), gloves (n= 23), shoes (n= 56), and washing machines (n= 52) were taken using sponges premoistened with buffered peptone water (5mL, Hydrasponge, Biotrace, London, UK). Thawed samples were enriched anaerobically in CD broth for 15 days (37°C) and were then homogenized and ethanol shocked (not heated) prior to their inoculation and incubation onto CD agar for 5 days at 37°C. Strain identification, PCR-ribotyping, and antimicrobial resistance analyses were performed as previously described. Our methodology detected 2 CD-positive food items in 2 urban households, yielding an overall CD prevalence of 1.06% (2 of 188 meals, binomial exact 95% CI, 0.129–3.789), which is 5-fold higher than the 0.22% reported by Kwon et al (for which we computed a 95% CI, 0.026–0.792; difference> 0 was 0.84%, P= .081; probability Z> z= 0.04; binomial exact P= .06). CD positivity was not attributed to environmental cross contamination because 279 environmental swabs were CD negative (0%, one-sided 97.5% CI, 0–1.32). Control fecal samples from 54 farm animals yielded CD in 3 animals from farm households that did not contribute to contaminated foods. The CD isolates were toxigenic, moxifloxacinand clindamycin-resistant PCR-ribotype 078, representing 2.15% of uncooked and commercially processed food (10-fold greater than Kwon’s prevalence; 2 of 93 for vegetable and bologna; 95% CI, 0.26–7.55). All 88 household-cooked meals were CD negative, supporting the evidence that heat decreases the probability of recovering CD (0%; 1-sided 97.5% CI, 0–4.1; infection control & hospital epidemiology