Geogenomic segregation and temporal trends of human pathogenic Escherichia coli o157:H7, Washington, USA, 2005-2014

The often-noted and persistent increased incidence of Escherichia coli O157:H7 infections in rural areas is not well understood. We used a cohort of E. coli O157:H7 cases reported in Washington, USA, during 2005–2014, along with phylogenomic characterization of the infecting isolates, to identify geographic segregation of and temporal trends in specific phylogenetic lineages of E. coli O157:H7. Kernel estimation and generalized additive models demonstrated that pathogen lineages were spatially segregated during the period of analysis and identified a focus of segregation spanning multiple, predominantly rural, counties for each of the main clinical lineages, Ib, IIa, and IIb. These results suggest the existence of local reservoirs from which humans are infected. We also noted a secular increase in the proportion of lineage IIa and IIb isolates. Spatial segregation by phylogenetic lineage offers the potential to identify local reservoirs and intervene to prevent continued transmission

Microbial pathogens in ticks, rodents and a shrew in northern Gyeonggi-do near the DMZ, Korea

A total of 1,618 ticks [420 individual (adults) and pooled (larvae and nymphs) samples], 369 rodents (Apodemus agrarius, Rattus norvegicus, Tscherskia triton, Mus musculus, and Myodes regulus), and 34 shrews (Crocidura lasiura) that were collected in northern Gyeonggi-do near the Demilitarized Zone (DMZ) of Korea during 2004-2005, were assayed by PCR for selected zoonotic pathogens. From a total of 420 individual and pooled tick DNA samples, Anaplasma (A.) phagocytophilum (16), A. platys (16), Ehrlichia (E.) chaffeensis (63), Borrelia burgdorferi (16), and Rickettsia spp. (198) were detected using species-specific PCR assays. Out of 403 spleens from rodents and shrews, A. phagocytophilum (20), A. platys (34), E. chaffeensis (127), and Bartonella spp. (24) were detected with species-specific PCR assays. These results suggest that fevers of unknown causes in humans and animals in Korea should be evaluated for infections by these vector-borne microbial pathogens.Funding for portions of this work was provided by the US Department of Defense Global Emerging Infections Surveillance and Response System, Silver Spring, MD, the Armed Forces Medical Intelligence Center, Ft Detrick, MD. Dr. Joon-seok Chae received funding from the LG Yeonam Foundation

STX-ENCODING BACTERIOPHAGE CONTENT OF ESCHERICHIA COLI O157:H7 IS ASSOCIATED WITH VIRULENCE IN A PIGLET MODEL AND WITH FREQUENCY OF ISOLATION FROM HUMAN DISEASE

Enterohemorrhagic E. coli O157:H7 (EHEC O157) is an important cause of food and waterborne illness in the developed countries. Cattle are a reservoir host and a major source of human exposure via contaminated food. Shiga toxins (Stx) are an important cardinal virulence factors. EHEC O157 strains can be differentiated into several genotypes based on stx content and Stx-encoding bacteriophage insertion sites. Bovine-biased genotypes (BBG) are over-represented among cattle isolates, while clinical genotypes (CG) are over-represented among human disease-associated isolates. Since both BBG and CG are carried by cattle as similar prevalence and both typically express one or more Stx, we hypothesized that the difference in representation in human disease is due to reduced virulence potential of BBG compared to CG resulting from different Stx-encoding bacteriophages and/or different Stx gene variant content. We compared the virulence potential of human and bovine isolates of CG and BBG in a newborn conventional pig model. CG-challenged piglets experienced more severe disease accompanied by earlier and higher mortality than BBG-challenged piglets. The CG strains used in this study carried stx2a and produced significantly higher amounts of Stx, whereas the BBG strains carried the stx2c gene variant only. Genomes of four BBG and three CG strains were compared using optical mapping technology to identify bacteriophage mediated differences. BBG strains were found to consistently differ from CG strains in the insertion sites of the Stx2 encoding phages in their chromosome, and specific bacteriophage / insertion site combinations correlated with the stx2 gene variant content of both BBG and CG strains. Multiplex-PCR based testing of 419 additional EHEC O157 strains confirmed these differences in the predominant stx2 variant content and their respective encoding bacteriophages in BBG strains and CG strains: BBG strains typically carry a Stx2c-encoding bacteriophage in sbcB and stx2c is typically their sole Stx2 gene, while CG strains typically carry a Stx2a-encoding bacteriophage in wrbA or argW and stx2a. These results suggest that BBG are less virulent than CG and their differential virulence may simply result from the presence of different stx2 variants, the encoding-bacteriophages and/or the amount of Stx produced

Protozoan Predation of Escherichia coli O157:H7 Is Unaffected by the Carriage of Shiga Toxin-Encoding Bacteriophages.

Escherichia coli O157:H7 is a food-borne bacterium that causes hemorrhagic diarrhea and hemolytic uremic syndrome in humans. While cattle are a known source of E. coli O157:H7 exposure resulting in human infection, environmental reservoirs may also be important sources of infection for both cattle and humans. Bacteriophage-encoded Shiga toxins (Stx) carried by E. coli O157:H7 may provide a selective advantage for survival of these bacteria in the environment, possibly through their toxic effects on grazing protozoa. To determine Stx effects on protozoan grazing, we co-cultured Paramecium caudatum, a common ciliate protozoon in cattle water sources, with multiple strains of Shiga-toxigenic E. coli O157:H7 and non-Shiga toxigenic cattle commensal E. coli. Over three days at ambient laboratory temperature, P. caudatum consistently reduced both E. coli O157:H7 and non-Shiga toxigenic E. coli populations by 1-3 log cfu. Furthermore, a wild-type strain of Shiga-toxigenic E. coli O157:H7 (EDL933) and isogenic mutants lacking the A subunit of Stx 2a, the entire Stx 2a-encoding bacteriophage, and/or the entire Stx 1-encoding bacteriophage were grazed with similar efficacy by both P. caudatum and Tetrahymena pyriformis (another ciliate protozoon). Therefore, our data provided no evidence of a protective effect of either Stx or the products of other bacteriophage genes on protozoan predation of E. coli. Further research is necessary to determine if the grazing activity of naturally-occurring protozoa in cattle water troughs can serve to decrease cattle exposure to E. coli O157:H7 and other Shiga-toxigenic E. coli

Density (mean ± s. e.) of <i>P</i>. <i>caudatum</i> when co-cultured with matched MOI of <i>E</i>. <i>coli</i> O157:H7 EDL933 and cattle commensal <i>E</i>. <i>coli</i> (186) for three days at ambient laboratory temperature.

<p>Squares, <i>P</i>. <i>caudatum</i> with added <i>E</i>. <i>coli</i>; no marker, <i>P</i>. <i>caudatum</i> in trough water without added bacteria.</p

Sequence of primers for PCR amplification used throughout these studies.

<p>Sequence of primers for PCR amplification used throughout these studies.</p

<i>E</i>. <i>coli</i> strains used in these studies.

<p><i>E</i>. <i>coli</i> strains used in these studies.</p

CFU (mean ± s. e.) of <i>E</i>. <i>coli</i> O157:H7 EDL933 and isogenic mutants inoculated at matched MOI when co-cultured with <i>T</i>. <i>pyriformis</i> (solid lines) and <i>P</i>. <i>caudatum</i> (dashed lines) at ambient laboratory temperature.

<p>(A) <i>E</i>. <i>coli</i> O157:H7 EDL933 (squares) and EDL933ΔStx 2a subunit A (triangles). (B) <i>E</i>. <i>coli</i> O157:H7 EDL933 (squares) and EDL933ΔStx 1 phage (diamonds). (C) <i>E</i>. <i>coli</i> O157:H7 EDL933 (squares) and EDL933ΔStx 2a phage (circles). (D) <i>E</i>. <i>coli</i> O157:H7 EDL933 (squares) and EDL933ΔStx 1 & Stx 2a phages (asterisks).</p

<i>P</i>. <i>caudatum</i> grazing of <i>E</i>. <i>coli</i> O157:H7 EDL933 and cattle commensal <i>E</i>. <i>coli</i> (186) over three days at ambient laboratory temperature.

<p>CFU (mean ± s. e.) for three grazing trials each containing a matched MOI of <i>E</i>. <i>coli</i> O157:H7 EDL933 (squares) and cattle commensal <i>E</i>. <i>coli</i> (triangles).</p