Nicola Holden

Colonization outwith the colon: plants as an alternative environmental reservoir for human pathogenic enterobacteria

Members of the Enterobacteriaceae have the capacity to adapt to a wide variety of environments and can be isolated from a range of host species across biological kingdoms. Bacteria that are pathogenic to animals, in particular humans, are increasingly found to be transmitted through the food chain by fruits and vegetables. Rather than simply contaminating plant surfaces, there is a growing body of evidence to show that these bacteria actively interact with plants and can colonize them as alternative hosts. This review draws together evidence from studies that investigate proven and potential mechanisms involved in colonization of plants by human pathogenic enterobacteria.

Transparent Soil for Imaging the Rhizosphere

Understanding of soil processes is essential for addressing the global issues of food security, disease transmission and climate change. However, techniques for observing soil biology are lacking. We present a heterogeneous, porous, transparent substrate for in situ 3D imaging of living plants and root-associated microorganisms using particles of the transparent polymer, Nafion, and a solution with matching optical properties. Minerals and fluorescent dyes were adsorbed onto the Nafion particles for nutrient supply and imaging of pore size and geometry. Plant growth in transparent soil was similar to that in soil. We imaged colonization of lettuce roots by the human bacterial pathogen Escherichia coli O157:H7 showing micro-colony development. Micro-colonies may contribute to bacterial survival in soil. Transparent soil has applications in root biology, crop genetics and soil microbiology.

Cloning, expression, and characterization of fimbrial operon F9 from enterohemorrhagic Escherichia coli O157:H7.

ABSTRACT Recent transposon mutagenesis studies with two enterohemorrhagic Escherichia coli (EHEC) strains, a sero- type O26:H- strain and a serotype O157:H7 strain, led to identification of a putative fimbrial operon that promotes colonization of young calves (1 to 2 weeks old). The distribution of the gene encoding the major fimbrial subunit present in O-island 61 of EHEC O157:H7 in a characterized set of 78 diarrheagenic E. coli strains was determined, and this gene was found in 87.2% of the strains and is therefore not an EHEC-specific region. The cluster was amplified by long-range PCR and cloned into the inducible expression vector pBAD18. Induced expression in E. coli K-12 led to production of fimbriae, as demonstrated by transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The fimbriae were purified, and sera to the purified major subunit were raised and used to demonstrate expression from wild-type E. coli O157:H7 strains. Induced expression of the fimbriae, designated F9 fimbriae, was used to characterize binding to bovine epithelial cells, bovine gastrointestinal tissue explants, and extracellular matrix components. The fimbriae promoted increases in the levels of E. coli K-12 binding only to bovine epithelial cells. In contrast, induced expression of F9 fimbriae in E. coli O157:H7 significantly reduced adherence of the bacteria to bovine gastrointestinal explant tissue. This may have been due to physical hindrance of type III secretion-dependent attachment. The main F9 subunit gene was deleted in E. coli O157:H7, and the resulting mutant was compared with the wild-type strain for colonization in weaned cattle. While the shedding levels of the mutant were reduced, the animals were still colonized at the terminal rectum, indicating that the adhesin is not responsible for the rectal tropism observed but may contribute to colonization at other sites, as demonstrated previously with very young animals.

PAPB PARALOGUES AND THEIR EFFECT ON THE PHASE VARIATION OF TYPE 1 FIMBRIAE IN ESCHERICHIA COLI

Recent work has demonstrated that expression of type 1 fimbriae is repressed by PapB, a regulator of pyelonephritis‐associated pili (P‐pili). PapB belongs to family of related adhesin regulators, for which consensus residues required for DNA binding and oligomerization have been identified. Of the regulators tested in this study, PapB, SfaB (S‐fimbriae) and PefB (Salmonella enterica serovar Typhimurium –plasmid‐encoded fimbriae) repressed FimB‐promoted off‐to‐on inversion of the fim switch, although complete repression was only demonstrated by PapB. DaaA, FaeB, FanA, FanB and ClpB had no effect on fim switching. In addition, only PapB stimulated FimE‐promoted on‐to‐off inversion. Deletion analysis demonstrated that this specificity resides in the carboxy terminal of the protein, and not the amino terminal, with the central region being homologous among the family members. Exchange of Leu82 and Ile83 of PapB for the equivalent residues from the DaaA protein (Phe and Gln) within the carboxy terminal virtually abolished cross‐talk activity. Whereas PapB can bind to a region around the left inverted repeat of the fim switch, DaaA and the PapB double mutant were effectively unable to bind this region. A previously characterized PapB DNA binding mutant also failed to bind to this region and failed to inhibit FimB activity at the fim switch. Thus, repression of fim expression appears unique to PapB and SfaB within E. coli and requires DNA binding involving amino acid residues located both within the homologous core and in the heterogeneous carboxy terminus. The variation in the carboxy terminus between the PapB family members explains their differential effects on fim. This mechanism of cross‐talk seems restricted to the P and S family adhesins with type 1 fimbriae and may ensure variable and sequential expression of adhesins during urinary tract infections.

Pathogenic Potential of Emergent Sorbitol-Fermenting Escherichia coli O157:NM

ABSTRACT Non-sorbitol-fermenting (NSF) Escherichia coli O157:H7 is the primary Shiga toxin-producing E. coli (STEC) serotype associated with human infection. Since 1988, sorbitol-fermenting (SF) STEC O157:NM strains have emerged and have been associated with a higher incidence of progression to hemolytic-uremic syndrome (HUS) than NSF STEC O157:H7. This study investigated bacterial factors that may account for the increased pathogenic potential of SF STEC O157:NM. While no evidence of toxin or toxin expression differences between the two O157 groups was found, the SF STEC O157:NM strains adhered at significantly higher levels to a human colonic cell line. Under the conditions tested, curli were shown to be the main factor responsible for the increased adherence to Caco-2 cells. Notably, 52 of 66 (79%) European SF STEC O157:NM strains tested bound Congo red at 37οC and this correlated with curli expression. In a subset of strains, curli expression was due to increased expression from the csgBAC promoter that was not always a consequence of increased csgD expression. The capacity of SF STEC O157:NM strains to express curli at 37οC may have relevance to the epidemiology of human infections as curliated strains could promote higher levels of colonization and inflammation in the human intestine. In turn, this could lead to increased toxin exposure and an increased likelihood of progression to HUS.

Bacterial Flagella: Twist and Stick, or Dodge across the Kingdoms

The flagellum organelle is an intricate multiprotein assembly best known for its rotational propulsion of bacteria. However, recent studies have expanded our knowledge of other functions in pathogenic contexts, particularly adherence and immune modulation, e.g., for Salmonella enterica, Campylobacter jejuni, Pseudomonas aeruginosa, and Escherichia coli. Flagella-mediated adherence is important in host colonisation for several plant and animal pathogens, but the specific interactions that promote flagella binding to such diverse host tissues has remained elusive. Recent work has shown that the organelles act like probes that find favourable surface topologies to initiate binding. An emerging theme is that more general properties, such as ionic charge of repetitive binding epitopes and rotational force, allow interactions with plasma membrane components. At the same time, flagellin monomers are important inducers of plant and animal innate immunity: variation in their recognition impacts the course and outcome of infections in hosts from both kingdoms. Bacteria have evolved different strategies to evade or even promote this specific recognition, with some important differences shown for phytopathogens. These studies have provided a wider appreciation of the functions of bacterial flagella in the context of both plant and animal reservoirs.

The Endophytic Lifestyle of Escherichia coli O157:H7: Quantification and Internal Localization in Roots

The foodborne pathogen Escherichia coli O157:H7 is increasingly associated with fresh produce (fruit and vegetables). Bacterial colonization of fresh produce plants can occur to high levels on the external tissue but bacteria have also been detected within plant tissue. However, questions remain about the extent of internalization, its molecular basis, and internal location of the bacteria. We have determined the extent of internalization of E. coli O157:H7 in live spinach and lettuce plants and used high-resolution microscopy to examine colony formation in roots and pathways to internalization. E. coli O157:H7 was found within internal tissue of both produce species. Colonization occurred within the apoplast between plant cells. Furthermore, colonies were detected inside the cell wall of epidermal and cortical cells of spinach and Nicotiana benthamiana roots. Internal colonization of epidermal cells resembled that of the phytopathogen Pectobacterium atrosepticum on potato. In contrast, only sporadic cells of the laboratory strain of E. coli K-12 were found on spinach, with no internal bacteria evident. The data extend previous findings that internal colonization of plants appears to be limited to a specific group of plant-interacting bacteria, including E. coli O157:H7, and demonstrates its ability to invade the cells of living plants.

Regulation of P-fimbrial phase variation frequencies in Escherichia coli CFT073

ABSTRACT Adherence of uropathogenic Escherichia coli to host tissue is required for infection and is mediated by fimbriae, such as pyelonephritis-associated pili (Pap). Expression of P fimbriae is regulated by phase variation, and to date, phase transition frequencies have been measured only for pap regulatory region constructs integrated into the E. coli K-12 chromosome. The aim of this work was to measure P phase transition frequencies in clinical isolates for the first time, including frequencies for the sequenced strain E. coli CFT073. P fimbriation and associated phase transition frequencies were measured for two E. coli clinical isolates and compared with levels for homologous pap constructs in E. coli K-12. Fimbriation and off-to-on transition frequencies were always higher in the clinical isolate. It was concluded that the regulatory inputs controlling papI expression are likely to be different in E. coli CFT073 and E. coli K-12 as (i) phase variation could be stimulated in E. coli K-12 by induction of papI and (ii) the level of expression of a papI::gfp+ fusion was higher in E. coli CFT073 than in E. coli K-12. Furthermore, phase transition frequencies for the two E. coli CFT073 pap clusters were shown to be different depending on the culture conditions, indicating that there is a hierarchy of expression depending on signal inputs.

Regulatory interplay between pap operons in uropathogenic Escherichia coli

Pathogenic bacteria have a large repertoire of surface organelles involved in adherence, motility and protein export, but how individual bacteria co‐ordinate surface organelle expression to prevent interference and excessive immune stimulation is unclear. Phase variation is a mechanism by which expression of surface factors is limited to a fraction of the bacterial population; however, the presence of multiple homologous surface structures controlled by related mechanisms and regulators antagonizes the independent expression achieved by phase variation. To investigate whether other mechanisms have evolved to sort out the bacterial cell surface, we examined regulatory cross‐talk between multiple phase‐variable pyelonephritis‐associated pili (pap) operons in Escherichia coli isolates associated with urinary tract infections. Allelic variation identified in the regulatory regions and regulators acts synergistically to limit coexpression of homologous fimbrial operons. In particular, there is evidence that papI is under positive selection and PapI variants displayed differences in their capacity to activate related pap operons. Alleles of the high‐affinity binding site for PapB were shown to contain a variable number of (T/A)3 repeats occurring every 9 bp that altered the sensitivity of pap operon activation. Taken together with other examples of surface organelle cross‐talk, we illustrate how this regulation could promote sequential expression.

Alignment-Free Design of Highly Discriminatory Diagnostic Primer Sets for Escherichia coli O104:H4 Outbreak Strains

Background An Escherichia coli O104:H4 outbreak in Germany in summer 2011 caused 53 deaths, over 4000 individual infections across Europe, and considerable economic, social and political impact. This outbreak was the first in a position to exploit rapid, benchtop high-throughput sequencing (HTS) technologies and crowdsourced data analysis early in its investigation, establishing a new paradigm for rapid response to disease threats. We describe a novel strategy for design of diagnostic PCR primers that exploited this rapid draft bacterial genome sequencing to distinguish between E. coli O104:H4 outbreak isolates and other pathogenic E. coli isolates, including the historical hæmolytic uræmic syndrome (HUSEC) E. coli HUSEC041 O104:H4 strain, which possesses the same serotype as the outbreak isolates. Methodology/Principal Findings Primers were designed using a novel alignment-free strategy against eleven draft whole genome assemblies of E. coli O104:H4 German outbreak isolates from the E. coli O104:H4 Genome Analysis Crowd-Sourcing Consortium website, and a negative sequence set containing 69 E. coli chromosome and plasmid sequences from public databases. Validation in vitro against 21 ‘positive’ E. coli O104:H4 outbreak and 32 ‘negative’ non-outbreak EHEC isolates indicated that individual primer sets exhibited 100% sensitivity for outbreak isolates, with false positive rates of between 9% and 22%. A minimal combination of two primers discriminated between outbreak and non-outbreak E. coli isolates with 100% sensitivity and 100% specificity. Conclusions/Significance Draft genomes of isolates of disease outbreak bacteria enable high throughput primer design and enhanced diagnostic performance in comparison to traditional molecular assays. Future outbreak investigations will be able to harness HTS rapidly to generate draft genome sequences and diagnostic primer sets, greatly facilitating epidemiology and clinical diagnostics. We expect that high throughput primer design strategies will enable faster, more precise responses to future disease outbreaks of bacterial origin, and help to mitigate their societal impact.