Restriction Endonuclease-Based Modification-Dependent Enrichment (REMoDE) of DNA for Metagenomic Sequencing.

Metagenomic sequencing is a swift and powerful tool to ascertain the presence of an organism of interest in a sample. However, sequencing coverage of the organism of interest can be insufficient due to an inundation of reads from irrelevant organisms in the sample. Here, we report a nuclease-based approach to rapidly enrich for DNA from certain organisms, including enterobacteria, based on their differential endogenous modification patterns. We exploit the ability of taxon-specific methylated motifs to resist the action of cognate methylation-sensitive restriction endonucleases that thereby digest unwanted, unmethylated DNA. Subsequently, we use a distributive exonuclease or electrophoretic separation to deplete or exclude the digested fragments, thus enriching for undigested DNA from the organism of interest. As a proof of concept, we apply this method to enrich for the enterobacteria Escherichia coli and Salmonella enterica by 11- to 142-fold from mock metagenomic samples and validate this approach as a versatile means to enrich for genomes of interest in metagenomic samples. IMPORTANCE Pathogens that contaminate the food supply or spread through other means can cause outbreaks that bring devastating repercussions to the health of a populace. Investigations to trace the source of these outbreaks are initiated rapidly but can be drawn out due to the labored methods of pathogen isolation. Metagenomic sequencing can alleviate this hurdle but is often insufficiently sensitive. The approach and implementations detailed here provide a rapid means to enrich for many pathogens involved in foodborne outbreaks, thereby improving the utility of metagenomic sequencing as a tool in outbreak investigations. Additionally, this approach provides a means to broadly enrich for otherwise minute levels of modified DNA, which may escape unnoticed in metagenomic samples.

Weather factors, soil microbiome, and bacteria-fungi interactions as drivers of the epiphytic phyllosphere communities of romaine lettuce.

Lettuce is associated with seasonal outbreaks of Shiga toxin-producing Escherichia coli (STEC) infections. Little is known about how various biotic and abiotic factors affect the lettuce microbiome, which in turn impacts STEC colonization. We characterized the lettuce phyllosphere and surface soil bacterial, fungal, and oomycete communities at harvest in late-spring and -fall in California using metagenomics. Harvest season and field type, but not cultivar, significantly influenced the microbiome composition of leaves and surface soil near plants. Phyllosphere and soil microbiome compositions were correlated with specific weather factors. The relative abundance of Enterobacteriaceae, but not E. coli, was enriched on leaves (5.2%) compared to soil (0.4%) and correlated positively with minimum air temperature and wind speed. Co-occurrence networks revealed seasonal trends in fungi-bacteria interactions on leaves. These associations represented 39%-44% of the correlations between species. All significant E. coli co-occurrences with fungi were positive, while all negative associations were with bacteria. A large proportion of the leaf bacterial species was shared with those in soil, indicating microbiome transmission from the soil surface to the canopy. Our findings provide new insight into factors that shape lettuce microbial communities and the microbial context of foodborne pathogen immigration events in the lettuce phyllosphere.

Breathing new life into death certificates: Extracting handwritten cause of death in the LIFE-M project.

The demographic and epidemiological transitions of the past 200 years are well documented at an aggregate level. Understanding differences in individual and group risks for mortality during these transitions requires linkage between demographic data and detailed individual cause of death information. This paper describes the digitization of almost 185,000 causes of death for Ohio to supplement demographic information in the Longitudinal, Intergenerational Family Electronic Micro-database (LIFE-M). To extract causes of death, our methodology combines handwriting recognition, extensive data cleaning algorithms, and the semi-automated classification of causes of death into International Classification of Diseases (ICD) codes. Our procedures are adaptable to other collections of handwritten data, which require both handwriting recognition and semi-automated coding of the information extracted.

Tobacco smoking and risk of thyroid cancer according to BRAFV600E mutational subtypes.

OBJECTIVE: Smoking has been associated with a reduced risk of thyroid cancer, but whether the association varies between higher- and lower-risk cancers remains unclear. We aimed to assess the association between smoking and risk of thyroid cancer overall as well as by tumour BRAF mutational status as a marker of potentially higher-risk cancer. DESIGN AND PATIENTS: We recruited 1013 people diagnosed with thyroid cancer and 1057 population controls frequency-matched on age and sex. METHODS: Multivariable logistic regression was used to assess the association overall and in analyses stratified by tumour characteristics. We used sensitivity analysis to assess the potential for selection bias. RESULTS: We found little evidence of an association with current smoking (odds ratio [OR] = 0.93; 95% confidence interval [CI]: 0.69-1.26; current vs. never smoking), but a higher number of pack-years of smoking was associated with a lower risk of thyroid cancer (OR = 0.75; 95% CI: 0.57-0.99; ≥20 pack-years vs. never). However, after correcting for potential selection bias, we observed a statistically significant inverse association between current smoking and risk of thyroid cancer (bias-corrected OR = 0.65; 95% CI: 0.51-0.83). Those with BRAF-positive cancers were less likely to be current smokers than those with BRAF-negative cancers (prevalence ratio: 0.79; 95% CI: 0.62-0.99). CONCLUSION: We found smoking was inversely related to thyroid cancer risk and, in particular, current smoking was associated with a reduced risk of potentially more aggressive BRAF-positive than the likely more indolent BRAF-negative papillary thyroid cancers.

Investigating the Relatedness of Enteroinvasive Escherichia coli to Other E. coli and Shigella Isolates by Using Comparative Genomics.

Enteroinvasive Escherichia coli (EIEC) is a unique pathovar that has a pathogenic mechanism nearly indistinguishable from that of Shigella species. In contrast to isolates of the four Shigella species, which are widespread and can be frequent causes of human illness, EIEC causes far fewer reported illnesses each year. In this study, we analyzed the genome sequences of 20 EIEC isolates, including 14 first described in this study. Phylogenomic analysis of the EIEC genomes demonstrated that 17 of the isolates are present in three distinct lineages that contained only EIEC genomes, compared to reference genomes from each of the E. coli pathovars and Shigella species. Comparative genomic analysis identified genes that were unique to each of the three identified EIEC lineages. While many of the EIEC lineage-specific genes have unknown functions, those with predicted functions included a colicin and putative proteins involved in transcriptional regulation or carbohydrate metabolism. In silico detection of the Shigella virulence plasmid (pINV), which is essential for the invasion of host cells, demonstrated that a form of pINV was present in nearly all EIEC genomes, but the Mxi-Spa-Ipa region of the plasmid that encodes the invasion-associated proteins was absent from several of the EIEC isolates. The comparative genomic findings in this study support the hypothesis that multiple EIEC lineages have evolved independently from multiple distinct lineages of E. coli via the acquisition of the Shigella virulence plasmid and, in some cases, the Shigella pathogenicity islands.

Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia sp. Cryptic Lineage 1 Strain 7v Harbors a Hybrid Plasmid.

UNLABELLED: Hybrid isolates of Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) encoding heat-stable enterotoxin (ST) are being reported with increasing frequency from a variety of sources. However, information regarding the plasmids that these strains harbor is scarce. In this study, we sequence and characterize a plasmid, p7v, from the STEC/ETEC hybrid strain 7v. Whole-genome phylogenetic analyses of STEC/ETEC hybrid strains and prototype E. coli isolates of other pathotypes placed 7v in the Escherichia sp. cryptic lineage 1 (CL1) clade. The complete plasmid, p7v, was determined to be 229,275 bp and encodes putative virulence factors that are typically carried on STEC plasmids as well as those often carried on ETEC plasmids, indicating that the hybrid nature of the strain extends beyond merely encoding the two toxins. Plasmid p7v carries two copies of sta with identical sequences, which were discovered to be divergent from the sta sequences found in the prototype human ETEC strains. Using a nomenclature scheme based on a phylogeny constructed from sta and stb sequences, the sta encoded on p7v is designated STa4. In silico analysis determined that p7v also encodes the K88 fimbria, a colonization factor usually associated with porcine ETEC plasmids. The p7v sequence and the presence of plasmid-encoded virulence factors are compared to those of other STEC/ETEC CL1 hybrid genomes and reveal gene acquisition/loss at the strain level. In addition, the interrogation of 24 STEC/ETEC hybrid genomes for identification of plasmid replicons, colonization factors, Stx and ST subtypes, and other plasmid-encoded virulence genes highlights the diversity of these hybrid strains. IMPORTANCE: Hybrid Shiga toxin-producing Escherichia coli/enterotoxigenic Escherichia coli (STEC/ETEC) strains, which have been isolated from environmental, animal, and human clinical samples, may represent an emerging threat as food-borne pathogens. Characterization of these strains is important for assessing virulence potential, aiding in the development of pathogen detection methods, and understanding how the hybrid strains evolve to potentially have a greater impact on public health. This study represents, to our knowledge, both the first characterization of a closed plasmid sequence from a STEC/ETEC hybrid strain and the most comprehensive phylogenetic analysis of available STEC/ETEC hybrid genomes to date. The results demonstrate how the mobility of plasmid-associated virulence genes has resulted in the creation of a diverse plasmid repertoire within the STEC/ETEC hybrid strains.

Application of metagenomic sequencing to food safety: detection of Shiga Toxin-producing Escherichia coli on fresh bagged spinach.

Culture-independent diagnostics reduce the reliance on traditional (and slower) culture-based methodologies. Here we capitalize on advances in next-generation sequencing (NGS) to apply this approach to food pathogen detection utilizing NGS as an analytical tool. In this study, spiking spinach with Shiga toxin-producing Escherichia coli (STEC) following an established FDA culture-based protocol was used in conjunction with shotgun metagenomic sequencing to determine the limits of detection, sensitivity, and specificity levels and to obtain information on the microbiology of the protocol. We show that an expected level of contamination (∼10 CFU/100 g) could be adequately detected (including key virulence determinants and strain-level specificity) within 8 h of enrichment at a sequencing depth of 10,000,000 reads. We also rationalize the relative benefit of static versus shaking culture conditions and the addition of selected antimicrobial agents, thereby validating the long-standing culture-based parameters behind such protocols. Moreover, the shotgun metagenomic approach was informative regarding the dynamics of microbial communities during the enrichment process, including initial surveys of the microbial loads associated with bagged spinach; the microbes found included key genera such as Pseudomonas, Pantoea, and Exiguobacterium. Collectively, our metagenomic study highlights and considers various parameters required for transitioning to such sequencing-based diagnostics for food safety and the potential to develop better enrichment processes in a high-throughput manner not previously possible. Future studies will investigate new species-specific DNA signature target regimens, rational design of medium components in concert with judicious use of additives, such as antibiotics, and alterations in the sample processing protocol to enhance detection.

Acquisition of the lac operon by Salmonella enterica.

BACKGROUND: Classical bacteriological characteristics of Salmonella enterica indicate that the members of this species are unable to utilize lactose as a carbon source. However, lactose-fermenting (Lac+) strains of several Salmonella serovars have been isolated from different foodborne outbreaks as well as different geographical regions worldwide. In the present study, we sequenced the genomes of 13 Lac + S. enterica isolates and characterized the lac region, comparing it to the lac region in other enteric bacterial species. RESULTS: Genetic analysis of the lac operons in the S. enterica genomes revealed that they all contain intact lacI, lacZ, and lacY genes. However, lacA was truncated in all of the S. enterica subsp. enterica isolates, encoding a 56 amino acid peptide rather than the full length 220 amino acid LacA protein. Molecular analyses of the 13 isolates revealed that the lac operon resided on a plasmid in some strains and in others was integrated into the bacterial chromosome. In most cases, an insertion sequence flanked at least one end of the operon. Interestingly, the S. enterica Montevideo and S. enterica Senftenberg isolates were found to harbor a plasmid with a high degree of sequence similarity to a plasmid from Klebsiella pneumoniae strain NK29 that also harbors the lac operon. In addition, two S. enterica Tennessee isolates carried two copies of the lac operon. Phylogenetic analysis based on lacIZY gene sequences determines distinct clusters, and reveals a greater correlation between lacIZY sequence and flanking organization than with either bacterial species or genomic location. CONCLUSIONS: Our results indicate that the lac region is highly mobile among Enterobacteriaceae and demonstrate that the Lac + S. enterica subsp. enterica serovars acquired the lac region through parallel events. The acquisition of the lac operon by several S. enterica serovars may be indicative of environmental adaptation by these bacteria.

Exploring agent-level calculations of risk and returns in relation to observed land-use changes in the US Great Plains, 1870-1940.

Land-use change in the U.S. Great Plains since agricultural settlement in the second half of the nineteenth century has been well documented. While aggregate historical trends are easily tracked, the decision-making of individual farmers is difficult to reconstruct. We use an agent-based model to tell the history of the settlement of the West by simulating farm-level agricultural decision making based on historical data about prices, yields, farming costs, and environmental conditions. The empirical setting for the model is the period between 1875 and 1940 in two townships in Kansas, one in the shortgrass region and the other in the mixed grass region. Annual historical data on yields and prices determine profitability of various land uses and thereby inform decision-making, in conjunction with the farmer's previous experience and randomly assigned levels of risk aversion. Results illustrating the level of agreement between model output and unique and detailed household-level records of historical land use and farm size suggest that economic behavior and natural endowments account for land change processes to some degree, but are incomplete. Discrepancies are examined to identify missing processes through model experiments, in which we adjust input and output prices, crop yields, agent memory, and risk aversion. These analyses demonstrate how agent-based modeling can be a useful laboratory for thinking about social and economic behavior in the past.

The effects of wealth, occupation, and immigration on epidemic mortality from selected infectious diseases and epidemics in Holyoke township, Massachusetts, 1850-1912.

BACKGROUND: Previous research suggests individual-level socioeconomic circumstances and resources may be especially salient influences on mortality within the broader context of social, economic, and environmental factors affecting urban 19th century mortality. OBJECTIVE: We sought to test individual-level socioeconomic effects on mortality from infectious and often epidemic diseases in the context of an emerging New England industrial mill town. METHOD: We analyze mortality data from comprehensive death records and a sample of death records linked to census data, for an emergent industrial New England town, to analyze infectious mortality and model socioeconomic effects using Poisson rate regression. RESULTS: Despite our expectations that individual resources might be especially salient in the harsh mortality setting of a crowded, rapidly growing, emergent, industrial mill town with high levels of impoverishment, infectious mortality was not significantly lowered by individual socio-economic status or resources.

Evaluation of the Increased Genetic Resolution and Utility for Source Tracking of a Recently Developed Method for Genotyping Cyclospora cayetanensis.

Cyclospora cayetanensis is a foodborne parasite that causes cyclosporiasis, an enteric illness in humans. Genotyping methods are used to genetically discriminate between specimens from cyclosporiasis cases and can complement source attribution investigations if the method is sufficiently sensitive for application to food items. A very sensitive targeted amplicon sequencing (TAS) assay for genotyping C. cayetanensis encompassing 52 loci was recently designed. In this study, we analyzed 66 genetically diverse clinical specimens to assess the change in phylogenetic resolution between the TAS assay and a currently employed eight-marker scheme. Of the 52 markers, ≥50 were successfully haplotyped for all specimens, and these results were used to generate a hierarchical cluster dendrogram. Using a previously described statistical approach to dissect hierarchical trees, the 66 specimens resolved into 24 and 27 distinct genetic clusters for the TAS and an 8-loci scheme, respectively. Although the specimen composition of 15 clusters was identical, there were substantial differences between the two dendrograms, highlighting the importance of both inclusion of additional genome coverage and choice of loci to target for genotyping. To evaluate the ability to genetically link contaminated food samples with clinical specimens, C. cayetanensis was genotyped from DNA extracted from raspberries inoculated with fecal specimens. The contaminated raspberry samples were assigned to clusters with the corresponding clinical specimen, demonstrating the utility of the TAS assay for traceback efforts.

Development of a targeted amplicon sequencing method for genotyping Cyclospora cayetanensis from fresh produce and clinical samples with enhanced genomic resolution and sensitivity.

Outbreaks of cyclosporiasis, an enteric illness caused by the parasite Cyclospora cayetanensis, have been associated with consumption of various types of fresh produce. Although a method is in use for genotyping C. cayetanensis from clinical specimens, the very low abundance of C. cayetanensis in food and environmental samples presents a greater challenge. To complement epidemiological investigations, a molecular surveillance tool is needed for use in genetic linkage of food vehicles to cyclosporiasis illnesses, estimation of the scope of outbreaks or clusters of illness, and determination of geographical areas involved. We developed a targeted amplicon sequencing (TAS) assay that incorporates a further enrichment step to gain the requisite sensitivity for genotyping C. cayetanensis contaminating fresh produce samples. The TAS assay targets 52 loci, 49 of which are located in the nuclear genome, and encompasses 396 currently known SNP sites. The performance of the TAS assay was evaluated using lettuce, basil, cilantro, salad mix, and blackberries inoculated with C. cayetanensis oocysts. A minimum of 24 markers were haplotyped even at low contamination levels of 10 oocysts in 25 g leafy greens. The artificially contaminated fresh produce samples were included in a genetic distance analysis based on haplotype presence/absence with publicly available C. cayetanensis whole genome sequence assemblies. Oocysts from two different sources were used for inoculation, and samples receiving the same oocyst preparation clustered together, but separately from the other group, demonstrating the utility of the assay for genetically linking samples. Clinical fecal samples with low parasite loads were also successfully genotyped. This work represents a significant advance in the ability to genotype C. cayetanensis contaminating fresh produce along with greatly expanding the genomic diversity included for genetic clustering of clinical specimens.

Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in Produce.

Regulatory methods for detection of the foodborne protozoan parasite Cyclospora cayetanensis must be specific and sensitive. To that end, we designed and evaluated (in a single laboratory validation) a novel and improved primer/probe combination (Mit1C) for real-time PCR detection of C. cayetanensis in produce. The newly developed primer/probe combination targets a conserved region of the mitochondrial genome of C. cayetanensis that varies in other closely related organisms. The primer/probe combination was evaluated both in silico and using several real-time PCR kits and polymerases against an inclusivity/exclusivity panel comprised of a variety of C. cayetanensis oocysts, as well as DNA from other related Cyclospora spp. and closely related parasites. The new primer/probe combination amplified only C. cayetanensis, thus demonstrating specificity. Sensitivity was evaluated by artificially contaminating cilantro, raspberries, and romaine lettuce with variable numbers (200 and 5) of C. cayetanensis oocysts. As few as 5 oocysts were detected in 75%, 67.7%, and 50% of the spiked produce samples (cilantro, raspberries, and romaine lettuce), respectively, all uninoculated samples and no-template real-time PCR controls were negative. The improved primer/probe combination should prove an effective analytical tool for the specific detection of C. cayetanensis in produce.

Application of quasimetagenomics methods to define microbial diversity and subtype Listeria monocytogenes in dairy and seafood production facilities.

IMPORTANCE: In developed countries, the human diet is predominated by food commodities, which have been manufactured, processed, and stored in a food production facility. Little is known about the application of metagenomic sequencing approaches for detecting foodborne pathogens, such as L. monocytogenes, and characterizing microbial diversity in food production ecosystems. In this work, we investigated the utility of 16S rRNA amplicon and quasimetagenomic sequencing for the taxonomic and phylogenetic classification of Listeria culture enrichments of environmental swabs collected from dairy and seafood production facilities. We demonstrated that single-nucleotide polymorphism (SNP) analyses of L. monocytogenes metagenome-assembled genomes (MAGs) from quasimetagenomic data sets can achieve similar resolution as culture isolate whole-genome sequencing. To further understand the impact of genome coverage on MAG SNP cluster resolution, an in silico downsampling approach was employed to reduce the percentage of target pathogen sequence reads, providing an initial estimate of required MAG coverage for subtyping resolution of L. monocytogenes.

Determinants of tubal ligation in Puebla, Mexico.

Tubal ligation provides an effective and reliable method by which women can choose to limit the number of children they will bear. However, because of the irreversibility of the procedure and other potential disadvantages, it is important to understand factors associated with women's choice of this method of birth control. Between May 1999 and August 2000, data were collected from 755 women aged 40 to 60 years from a cross-section of neighborhoods of varying socio-economic make-up in Puebla, Mexico, finding a tubal ligation rate of 42.2%. Multiple logistic regression models were utilized to examine demographic, socio-economic, and reproductive history characteristics in relation to women's choice of tubal ligation. Regression analyses were repeated with participants grouped by age to determine how the timing of availability of tubal ligation related to the decision to undergo the procedure. The results of this study suggest that younger age, more education, use of some forms of birth control, and increased parity were associated with women's decisions to undergo tubal ligation. The statistically significant difference of greater tubal ligation and lower hysterectomy rates across age groups reflect increased access to tubal ligation in Mexico from the early 1970s, supporting the idea that women's choice of tubal ligation was related to access.

Household and farm transitions in environmental context.

Recent debate in the literature on population, environment, and land use questions the applicability of theory that patterns of farm extensification and intensification correspond to the life course of farmers and to the life cycle of farm families. This paper extends the debate to the agricultural development of the United States Great Plains region, using unique data from 1875 to 1930 that link families to farms over time in 25 environmentally diverse Kansas townships. Results of multilevel statistical modeling indicate that farmer's age, household size, and household structure are simultaneously related to both the extent of farm operations and the intensity of land use, taking into account local environmental conditions and time trends as Kansas was settled and developed. These findings validate farm- and life cycle theories and offer support for intergenerational motivations for farm development that include both daughters and sons. Environmental variation in aridity was a key driver of farm structure.

Maximizing Efficiency of Telemedicine in the Skilled Nursing Facility during the Coronavirus Disease 2019 Pandemic.

Telemedicine has rapidly become a significant component of healthcare during the coronavirus disease 2019 pandemic, and is particularly beneficial in delivering care to vulnerable populations in skilled nursing facilities (SNFs). To limit coronavirus disease 2019 exposure, our team developed a quality improvement (QI) project to identify common telemedicine-related disruptions and their solutions, and created a streamlined protocol to maximize the efficiency of virtual rounding in the SNF. Through 9 Plan-Do-Study-Act cycles, we revised our protocol to decrease the percentage of rounding time spent troubleshooting telemedicine-related problems ("nonclinical care time") and were able to demonstrate repeatability at three checkpoints. Our QI project offers a framework for SNF providers and staff to deliver telemedicine-driven patient care.

Seasonality, shelf life and storage atmosphere are main drivers of the microbiome and E. coli O157:H7 colonization of post-harvest lettuce cultivated in a major production area in California.

BACKGROUND: Lettuce is linked to recurrent outbreaks of Shiga toxin-producing Escherichia coli (STEC) infections, the seasonality of which remains unresolved. Infections have occurred largely from processed lettuce, which undergoes substantial physiological changes during storage. We investigated the microbiome and STEC O157:H7 (EcO157) colonization of fresh-cut lettuce of two cultivars with long and short shelf life harvested in the spring and fall in California and stored in modified atmosphere packaging (MAP) at cold and warm temperatures. RESULTS: Inoculated EcO157 declined significantly less on the cold-stored cultivar with short shelf life, while multiplying rapidly at 24 °C independently of cultivar. Metagenomic sequencing of the lettuce microbiome revealed that the pre-storage bacterial community was variable but dominated by species in the Erwiniaceae and Pseudomonadaceae. After cold storage, the microbiome composition differed between cultivars, with a greater relative abundance (RA) of Erwiniaceae and Yersiniaceae on the cultivar with short shelf life. Storage at 24 °C shifted the microbiome to higher RAs of Erwiniaceae and Enterobacteriaceae and lower RA of Pseudomonadaceae compared with 6 °C. Fall harvest followed by lettuce deterioration were identified by recursive partitioning as important factors associated with high EcO157 survival at 6 °C, whereas elevated package CO2 levels correlated with high EcO157 multiplication at 24 °C. EcO157 population change correlated with the lettuce microbiome during 6 °C storage, with fall microbiomes supporting the greatest EcO157 survival on both cultivars. Fall and spring microbiomes differed before and during storage at both temperatures. High representation of Pantoea agglomerans was a predictor of fall microbiomes, lettuce deterioration, and enhanced EcO157 survival at 6 °C. In contrast, higher RAs of Erwinia persicina, Rahnella aquatilis, and Serratia liquefaciens were biomarkers of spring microbiomes and lower EcO157 survival. CONCLUSIONS: The microbiome of processed MAP lettuce evolves extensively during storage. Under temperature abuse, high CO2 promotes a lettuce microbiome enriched in taxa with anaerobic capability and EcO157 multiplication. In cold storage, our results strongly support a role for season and lettuce deterioration in EcO157 survival and microbiome composition, suggesting that the physiology and microbiomes of fall- and spring-harvested lettuce may contribute to the seasonality of STEC outbreaks associated with lettuce grown in coastal California.

Risk of thyroid cancer following hysterectomy.

BACKGROUND: Hysterectomy has been associated with increased thyroid cancer risk but whether this reflects a biological link or increased diagnosis of indolent cancers due to greater medical contact remains unclear. METHODS: We recruited 730 women diagnosed with thyroid cancer and 785 age-matched population controls. Multivariable logistic regression was used to assess the association overall, and by tumour BRAF mutational status as a marker of potentially higher-risk cancers. We used causal mediation analysis to investigate potential mediation of the association by healthcare service use. RESULTS: Having had a hysterectomy was associated with an increased risk of thyroid cancer (odds ratio [OR] = 1.45, 95 % confidence interval [CI] 1.07-1.96). When stratified by indication for hysterectomy, the risk appeared stronger for those who had a hysterectomy for menstrual disorders (OR = 1.67, 95 % CI 1.17-2.37) but did not differ by tumour BRAF status. Approximately 20 % of the association between hysterectomy and thyroid cancer may be mediated by more frequent use of healthcare services. CONCLUSIONS: The observed increased risk of thyroid cancer among those with hysterectomy may be driven, at least partly, by an altered sex steroid hormone milieu. More frequent healthcare service use by women with hysterectomy accounts for only a small proportion of the association.

Comparative Transcriptomics of Shiga Toxin-Producing and Commensal Escherichia coli and Cytokine Responses in Colonic Epithelial Cell Culture Infections.

Ingestion of Shiga toxin-producing Escherichia coli (STEC) can result in a range of illness severity from asymptomatic to hemorrhagic colitis and death; thus risk assessment of STEC strains for human pathogenicity is important in the area of food safety. Illness severity depends in part on the combination of virulence genes carried in the genome, which can vary between strains even of identical serotype. To better understand how core genes are regulated differently among strains and to identify possible novel STEC virulence gene candidates that could be added to the risk assessment repertoire, we used comparative transcriptomics to investigate global gene expression differences between two STEC strains associated with severe illness and a commensal E. coli strain during in vitro intestinal epithelial cell (IEC) infections. Additionally, we compared a wide array of concomitant cytokine levels produced by the IECs. The cytokine expression levels were examined for a pattern representing STEC pathogenicity; however, while one STEC strain appeared to elicit a proinflammatory response, infection by the other strain produced a pattern comparable to the commensal E. coli. This result may be explained by the significant differences in gene content and expression observed between the STEC strains. RNA-Seq analysis revealed considerable disparity in expression of genes in the arginine and tryptophan biosynthesis/import pathways between the STEC strains and the commensal E. coli strain, highlighting the important role some amino acids play in STEC colonization and survival. Contrasting differential expression patterns were observed for genes involved in respiration among the three strains suggesting that metabolic diversity is a strategy utilized to compete with resident microflora for successful colonization. Similar temporal expression results for known and putative virulence genes were observed in the STEC strains, revealing strategies used for survival prior to and after initial adherence to IECs. Additionally, three genes encoding hypothetical proteins located in mobile genetic elements were, after interrogation of a large set of E. coli genomes, determined to likely represent novel STEC virulence factors.