Sanja Ilic

Coliforms and Prevalence of Escherichia coli and Foodborne Pathogens on Minimally Processed Spinach in Two Packing Plants

Minimally processed spinach has been recently associated with outbreaks of foodborne illnesses. This study investigated the effect of commercial minimal processing of spinach on the coliform and Escherichia coli counts and the prevalence of E. coli O157:H7, Salmonella, Shigella spp., and Listeria monocytogenes on two types of spinach before and after minimal processing. A total of 1,356 spinach samples (baby spinach, n = 574; savoy spinach, n = 782) were collected daily in two processing plants over a period of 14 months. Raw spinach originated from nine farms in the United States and three farms in Canada. Overall, the proportion of samples positive for coliforms increased from 53% before minimal processing to 79% after minimal processing (P < 0.001). Average total coliform counts also increased significantly after processing, especially in baby spinach (mean +/- standard deviation, 1.16 +/- 0.14 log CFU/g to 2.37 +/- 0.08 log CFU/g following processing; P < 0.001). E. coli was isolated from 8.9% of the samples (mean +/- standard deviation, 1.81 +/- 0.14 log CFU/g), and no difference in prevalence or CFU counts after processing (P > 0.1) was observed. E. coli O157:H7 and Shigella spp. were not isolated from any of the samples. Salmonella and L. monocytogenes were isolated from 0.4 and 0.7% of samples, respectively. Results demonstrate that commercial minimal processing of spinach based on monitored chlorine washing and drying may not decrease microbial load on spinach leaves as expected. Further research is needed to identify the most appropriate measures to control food safety risk under commercial minimal processing of fresh vegetables.

A framework for developing research protocols for evaluation of microbial hazards and controls during production that pertain to the quality of agricultural water contacting fresh produce that may be consumed raw

Agricultural water may contact fresh produce during irrigation and/or when crop protection sprays (e.g., cooling to prevent sunburn, frost protection, and agrochemical mixtures) are applied. This document provides a framework for designing research studies that would add to our understanding of preharvest microbial food safety hazards and control measures pertaining to agricultural water. Researchers will be able to use this document to design studies, to anticipate the scope and detail of data required, and to evaluate previously published work. This document should also be useful for evaluating the strength of existing data and thus should aid in identifying future research needs. Use of this document by the research community may lead to greater consistency or comparability than currently exists among research studies, which may ultimately facilitate direct comparison of hazards and efficacy of controls among different commodities, conditions, and practices.

Clostridium difficile with Moxifloxacin/Clindamycin Resistance in Vegetables in Ohio, USA, and Prevalence Meta-Analysis

We (i) determined the prevalence of Clostridium difficile and their antimicrobial resistance to six antimicrobial classes, in a variety of fresh vegetables sold in retail in Ohio, USA, and (ii) conducted cumulative meta-analysis of reported prevalence in vegetables since the 1990s. Six antimicrobial classes were tested for their relevance as risk factors for C. difficile infections (CDIs) (clindamycin, moxifloxacin) or their clinical priority as exhaustive therapeutic options (metronidazole, vancomycin, linezolid, and tigecycline). By using an enrichment protocol we isolated C. difficile from three of 125 vegetable products (2.4%). All isolates were toxigenic, and originated from 4.6% of 65 vegetables cultivated above the ground (n = 3; outer leaves of iceberg lettuce, green pepper, and eggplant). Root vegetables yielded no C. difficile. The C. difficile isolates belonged to two PCR ribotypes, one with an unusual antimicrobial resistance for moxifloxacin and clindamycin (lettuce and pepper; 027-like, A+B+CDT+; tcdC 18 bp deletion); the other PCR ribotype (eggplant, A+B+ CDT−; classic tcdC) was susceptible to all antimicrobials. Results of the cumulative weighted meta-analysis (6 studies) indicate that the prevalence of C. difficile in vegetables is 2.1% and homogeneous (P < 0.001) since the first report in 1996 (2.4%). The present study is the first report of the isolation of C. difficile from retail vegetables in the USA. Of public health relevance, antimicrobial resistance to moxifloxacin/clindamycin (a bacterial-associated risk factor for severe CDIs) was identified on the surface of vegetables that are consumed raw.

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.

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.

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

Clinical Effects of Gamma-Radiation-Resistant Aspergillus sydowii on Germ-Free Mice Immunologically Prone to Inflammatory Bowel Disease

We report and investigated a case of inadvertent contamination of 125 mice (housed in two germ-free positive-pressurized isolators) with emerging human and coral pathogen Aspergillus sydowii. The infected mice correspond to genetic line SAMP1/YitFc, which have 100% immune predisposition to develop Crohns disease-like spontaneous pathologies, namely, inflammatory bowel disease (IBD). Pathogen update based on a scoping review of the literature and our clinical observations and experimentation are discussed. The unwanted infection of germ-free mice (immunologically prone to suffer chronic inflammation) with human pathogen A. sydowii resulted in no overt signs of clinical disease over 3-week exposure period, or during DSS-induced colitis experiments. Results and observations suggest that A. sydowii alone has limited clinical effect in immunocompromised germ-free mice or that other commensal microbial flora is required for Aspergillus-associated disease to occur.