Lindsey Ouellette

Variation in virulence among clades of Escherichia coli O157:H7 associated with disease outbreaks

Escherichia coli O157:H7, a toxin-producing food and waterborne bacterial pathogen, has been linked to large outbreaks of gastrointestinal illness for more than two decades. E. coli O157 causes a wide range of clinical illness that varies by outbreak, although factors that contribute to variation in disease severity are poorly understood. Several recent outbreaks involving O157 contamination of fresh produce (e.g., spinach) were associated with more severe disease, as defined by higher hemolytic uremic syndrome and hospitalization frequencies, suggesting that increased virulence has evolved. To test this hypothesis, we developed a system that detects SNPs in 96 loci and applied it to >500 E. coli O157 clinical strains. Phylogenetic analyses identified 39 SNP genotypes that differ at 20% of SNP loci and are separated into nine distinct clades. Differences were observed between clades in the frequency and distribution of Shiga toxin genes and in the type of clinical disease reported. Patients with hemolytic uremic syndrome were significantly more likely to be infected with clade 8 strains, which have increased in frequency over the past 5 years. Genome sequencing of a spinach outbreak strain, a member of clade 8, also revealed substantial genomic differences. These findings suggest that an emergent subpopulation of the clade 8 lineage has acquired critical factors that contribute to more severe disease. The ability to detect and rapidly genotype O157 strains belonging to such lineages is important and will have a significant impact on both disease diagnosis and treatment guidelines.

Global transcriptional response of Escherichia coli O157:H7 to growth transitions in glucose minimal medium

Background:Global patterns of gene expression of Escherichia coli K-12 during growth transitions have been deeply investigated, however, comparable studies of E. coli O157:H7 have not been explored, particularly with respect to factors regulating virulence genes and genomic islands specific to this pathogen. To examine the impact of growth phase on the dynamics of the transcriptome, O157:H7 Sakai strain was cultured in MOPS minimal media (0.1% glucose), RNA harvested at 10 time points from early exponential to full stationary phase, and relative gene expression was measured by co-hybridization on high-density DNA microarrays. Expression levels of 14 genes, including those encoding Shiga toxins and other virulence factors associated with the locus of enterocyte effacement (LEE), were confirmed by Q-PCR.Results:Analysis of variance (R/MAANOVA, Fs test) identified 442 (36%) of 1239 O157-specific ORFs and 2110 (59%) of 3647 backbone ORFs that changed in expression significantly over time. QT cluster analysis placed 2468 of the 2552 significant ORFs into 12 groups; each group representing a distinct expression pattern. ORFs from the largest cluster (n = 1078) decreased in expression from late exponential to early stationary phase: most of these ORFs are involved in functions associated with steady state growth. Also represented in this cluster are ORFs of the TAI island, encoding tellurite resistance and urease activity, which decreased ~4-fold. Most ORFs of the LEE pathogenicity island also decreased ~2-fold by early stationary phase. The ORFs encoding proteins secreted via the LEE encoded type III secretion system, such as tccP and espJ, also decreased in expression from exponential to stationary phase. Three of the clusters (n = 154) comprised genes that are transiently upregulated at the transition into stationary phase and included genes involved in nutrient scavenging. Upregulated genes with an increase in mRNA levels from late exponential to early stationary phase belonged to one cluster (n = 923) which includes genes involved in stress responses (e.g. gadAB, osmBC, and dps). These transcript levels remained relatively high for > 3 h in stationary phase. The Shiga toxin genes (stx 1AB and stx 2B) were significantly induced after transition into stationary phase.Conclusion:Expression of more than 300 O157-specific ORFs, many implicated in virulence of the O157 pathogen, was modulated in a growth dependent manner. These results provide a baseline transcriptional profile that can be compared to patterns of gene expression of this important foodborne pathogen under adverse environmental conditions.

Increased Adherence and Expression of Virulence Genes in a Lineage of Escherichia coli O157:H7 Commonly Associated with Human Infections

Background Enterohemorrhagic Escherichia coli (EHEC) O157:H7, a food and waterborne pathogen, can be classified into nine phylogenetically distinct lineages, as determined by single nucleotide polymorphism genotyping. One lineage (clade 8) was found to be associated with hemolytic uremic syndrome (HUS), which can lead to kidney failure and death in some cases, particularly young children. Another lineage (clade 2) differs considerably in gene content and is phylogenetically distinct from clade 8, but caused significantly fewer cases of HUS in a prior study. Little is known, however, about how these two lineages vary with regard to phenotypic traits important for disease pathogenesis and in the expression of shared virulence genes. Methodology/Principal Findings Here, we quantified the level of adherence to and invasion of MAC-T bovine epithelial cells, and examined the transcriptomes of 24 EHEC O157:H7 strains with varying Shiga toxin profiles from two common lineages. Adherence to epithelial cells was >2-fold higher for EHEC O157:H7 strains belonging to clade 8 versus clade 2, while no difference in invasiveness was observed between the two lineages. Whole-genome 70-mer oligo microarrays, which probe for 6088 genes from O157:H7 Sakai, O157:H7 EDL 933, pO157, and K12 MG1655, detected significant differential expression between clades in 604 genes following co-incubation with epithelial cells for 30 min; 186 of the 604 genes had a >1.5 fold change difference. Relative to clade 2, clade 8 strains showed upregulation of major virulence genes, including 29 of the 41 locus of enterocyte effacement (LEE) pathogenicity island genes, which are critical for adherence, as well as Shiga toxin genes and pO157 plasmid-encoded virulence genes. Differences in expression of 16 genes that encode colonization factors, toxins, and regulators were confirmed by qRT-PCR, which revealed a greater magnitude of change than microarrays. Conclusions/Significance These findings demonstrate that the EHEC O157:H7 lineage associated with HUS expresses higher levels of virulence genes and has an enhanced ability to attach to epithelial cells relative to another common lineage.

Differences in adherence and virulence gene expression between two outbreak strains of enterohaemorrhagic Escherichia coli O157:H7.

The Escherichia coli O157 : H7 TW14359 strain was implicated in a multi-state outbreak in North America in 2006, which resulted in high rates of severe disease. Similarly, the O157 : H7 RIMD0509952 (Sakai) strain caused the largest O157 : H7 outbreak to date. Both strains were shown to represent divergent phylogenetic lineages. Here we compared global gene expression patterns before and after epithelial cell exposure, as well as the ability to adhere to and invade epithelial cells, between the two outbreak strains. Epithelial cell assays demonstrated a 2.5-fold greater adherence of the TW14359 strain relative to Sakai, while whole-genome microarrays detected significant differential expression of 914 genes, 206 of which had a fold change >/=1.5. Interestingly, most locus of enterocyte effacement (LEE) genes were upregulated in TW14359, whereas flagellar and chemotaxis genes were primarily upregulated in Sakai, suggesting discordant expression of these genes between the two strains. The Shiga toxin 2 genes were also upregulated in the TW14359 strain, as were several pO157-encoded genes that promote adherence, including type II secretion genes and their effectors stcE and adfO. Quantitative RT-PCR confirmed the expression differences detected in the microarray analysis, and expression levels were lower for a subset of LEE genes before versus after exposure to epithelial cells. In all, this study demonstrated the upregulation of major and ancillary virulence genes in TW14359 and of flagellar and chemotaxis genes in Sakai, under conditions that precede intimate bacterial attachment to epithelial cells. Differences in the level of adherence to epithelial cells were also observed, implying that these two phylogenetically divergent O157 : H7 outbreak strains vary in their ability to colonize, or initiate the disease process.

Escherichia albertii in wild and domestic birds.

The isolates were similar to those that cause disease in humans.

Factors Associated with Shiga Toxin-Producing Escherichia coli Shedding by Dairy and Beef Cattle

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that can cause hemorrhagic colitis and hemolytic-uremic syndrome. Cattle are the primary reservoir for STEC, and food or water contaminated with cattle feces is the most common source of infections in humans. Consequently, we conducted a cross-sectional study of 1,096 cattle in six dairy herds (n = 718 animals) and five beef herds (n = 378 animals) in the summers of 2011 and 2012 to identify epidemiological factors associated with shedding. Fecal samples were obtained from each animal and cultured for STEC. Multivariate analyses were performed to identify risk factors associated with STEC positivity. The prevalence of STEC was higher in beef cattle (21%) than dairy cattle (13%) (odds ratio [OR], 1.76; 95% confidence interval [CI], 1.25, 2.47), with considerable variation occurring across herds (range, 6% to 54%). Dairy cattle were significantly more likely to shed STEC when the average temperature was >28.9°C 1 to 5 days prior to sampling (OR, 2.5; 95% CI, 1.25, 4.91), during their first lactation (OR, 1.8; 95% CI, 1.1, 2.8), and when they were <30 days in milk (OR, 3.9; 95% CI, 2.1, 7.2). These data suggest that the stress or the negative energy balance associated with lactation may result in increased STEC shedding frequencies in Michigan during the warm summer months. Future prevention strategies aimed at reducing stress during lactation or isolating high-risk animals could be implemented to reduce herd-level shedding levels and avoid transmission of STEC to susceptible animals and people. IMPORTANCE STEC shedding frequencies vary considerably across cattle herds in Michigan, and the shedding frequency of strains belonging to non-O157 serotypes far exceeds the shedding frequency of O157 strains, which is congruent with human infections in the state. Dairy cattle sampled at higher temperatures, in their first lactation, and early in the milk production stage were significantly more likely to shed STEC, which could be due to stress or a negative energy balance. Future studies should focus on the isolation of high-risk animals to decrease herd shedding levels and the potential for contamination of the food supply.

Genetic Diversity and Antimicrobial Resistance in Group B Streptococcus Colonizing Young, Nonpregnant Women

The genetic diversity of group B Streptococcus in young, nonpregnant women is not well studied. Application of multilocus sequence analysis to 85 group B Streptococcus strains recovered from college students revealed similarities and differences in distribution of group B Streptococcus lineages, compared with that of previously studied pregnant populations, and revealed that strains of 1 clone were associated with antibiotic resistance.

Comparative analysis of five methods of emergency zipper release by experienced versus novice clinicians.

The entrapment of penile tissue (foreskin, shaft, or glans) within the actuator or teeth of a zipper accounts for one of the most common genital injuries in young boys [1]. Literature suggests that zipper injuries are relatively uncommon, and that localized edema and pain are the most common outcomes, with significant injury such as skin loss and necrosis occurring rarely [1,2]. The purpose of our studywas to compare five common techniques for releasing zipper-entrapped skin using an animal model. This was a prospective, randomized trial using an animal model consisting of chicken skin firmly entrapped by a metal zipper on a pair of denim jeans. Volunteers consisted of 12 Emergency Medicine (EM) physician faculty and 18 medical students (novice clinicians). During the simulation lab, participants were taught the five common techniques for releasing zipper-entrapped skin: 1) cutting the median bar, 2) using a screwdriver to separate faceplates, 3) manipulation of the zipper using mineral oil lubricant, 4) lateral compression of the zip fastener using pliers, and 5) removal of teeth of the zip mechanism using trauma scissors [2-6]. The order in which the techniques were performed by each volunteer was chosen by a random number generator. Subjects were timed by evaluators using a digital stopwatch from the time they began until the successful release of the entrapped skin, or for five minutes, whichever came first. Success was defined as the release of the entrapped skinwhileminimizing trauma to the skin. Failure to successfully release the skinwithin fiveminutes, or causing full thickness laceration to the skin, were logged as failures.