Genetic diversity in Campylobacter jejuni is associated with differential colonization of broiler chickens and C57BL/6J IL10-deficient mice.

Previous studies have demonstrated that Campylobacter jejuni, the leading causative agent of bacterial food-borne disease in the USA, exhibits high-frequency genetic variation that is associated with changes in cell-surface antigens and ability to colonize chickens. To expand our understanding of the role of genetic diversity in the disease process, we analysed the ability of three C. jejuni human disease isolates (strains 11168, 33292 and 81-176) and genetically marked derivatives to colonize Ross 308 broilers and C57BL/6J IL10-deficient mice. C. jejuni colonized broilers at much higher efficiency (all three strains, 23 of 24 broilers) than mice (11168 only, 8 of 24 mice). C. jejuni 11168 genetically marked strains colonized mice at very low efficiency (2 of 42 mice); however, C. jejuni reisolated from mice colonized both mice and broilers at high efficiency, suggesting that this pathogen can adapt genetically in the mouse. We compared the genome composition in the three wild-type C. jejuni strains and derivatives by microarray DNA/DNA hybridization analysis; the data demonstrated a high degree of genetic diversity in three gene clusters associated with synthesis and modification of the cell-surface structures capsule, flagella and lipo-oligosaccharide. Finally, we analysed the frequency of mutation in homopolymeric tracts associated with the contingency genes wlaN (GC tract) and flgR (AT tracts) in culture and after passage through broilers and mice. C. jejuni adapted genetically in culture at high frequency and the degree of genetic diversity was increased by passage through broilers but was nearly eliminated in the gastrointestinal tract of mice. The data suggest that the broiler gastrointestinal tract provides an environment which promotes outgrowth and genetic variation in C. jejuni; the enhancement of genetic diversity at this location may contribute to its importance as a human disease reservoir.

Genomic diversity of pathogenic Escherichia coli of the EHEC 2 clonal complex.

BACKGROUND: Evolutionary analyses of enterohemorrhagic Escherichia coli (EHEC) have identified two distantly related clonal groups: EHEC 1, including serotype O157:H7 and its inferred ancestor O55:H7; and EHEC 2, comprised of several serogroups (O26, O111, O118, etc.). These two clonal groups differ in their virulence and global distribution. Although several fully annotated genomic sequences exist for strains of serotype O157:H7, much less is known about the genomic composition of EHEC 2. In this study, we analyzed a set of 24 clinical EHEC 2 strains representing serotypes O26:H11, O111:H8/H11, O118:H16, O153:H11 and O15:H11 from humans and animals by comparative genomic hybridization (CGH) on an oligoarray based on the O157:H7 Sakai genome. RESULTS: Backbone genes, defined as genes shared by Sakai and K-12, were highly conserved in EHEC 2. The proportion of Sakai phage genes in EHEC 2 was substantially greater than that of Sakai-specific bacterial (non-phage) genes. This proportion was inverted in O55:H7, reiterating that a subset of Sakai bacterial genes is specific to EHEC 1. Split decomposition analysis of gene content revealed that O111:H8 was more genetically uniform and distinct from other EHEC 2 strains, with respect to the Sakai O157:H7 gene distribution. Serotype O26:H11 was the most heterogeneous EHEC 2 subpopulation, comprised of strains with the highest as well as the lowest levels of Sakai gene content conservation. Of the 979 parsimoniously informative genes, 15% were found to be compatible and their distribution in EHEC 2 clustered O111:H8 and O118:H16 strains by serotype. CGH data suggested divergence of the LEE island from the LEE1 to the LEE4 operon, and also between animal and human isolates irrespective of serotype. No correlation was found between gene contents and geographic locations of EHEC 2 strains. CONCLUSION: The gene content variation of phage-related genes in EHEC 2 strains supports the hypothesis that extensive modular shuffling of mobile DNA elements has occurred among EHEC strains. These results suggest that EHEC 2 is a multiform pathogenic clonal complex, characterized by substantial intra-serotype genetic variation. The heterogeneous distribution of mobile elements has impacted the diversification of O26:H11 more than other EHEC 2 serotypes.

In situ-synthesized virulence and marker gene biochip for detection of bacterial pathogens in water.

Pathogen detection tools with high reliability are needed for various applications, including food and water safety and clinical diagnostics. In this study, we designed and validated an in situ-synthesized biochip for detection of 12 microbial pathogens, including a suite of pathogens relevant to water safety. To enhance the reliability of presence/absence calls, probes were designed for multiple virulence and marker genes (VMGs) of each pathogen, and each VMG was targeted by an average of 17 probes. Hybridization of the biochip with amplicon mixtures demonstrated that 95% of the initially designed probes behaved as predicted in terms of positive/negative signals. The probes were further validated using DNA obtained from three different types of water samples and spiked with pathogen genomic DNA at decreasing relative abundance. Excellent specificity for making presence/absence calls was observed by using a cutoff of 0.5 for the positive fraction (i.e., the fraction of probes yielding a positive signal for a given VMG). A split multiplex PCR design for simultaneous amplification of the VMGs resulted in a detection limit of between 0.1 and 0.01% relative abundance, depending on the type of pathogen and the VMG. Thermodynamic analysis of the hybridization patterns obtained with DNA from the different water samples demonstrated that probes with a hybridization Gibbs free energy of approximately -19.3 kcal/mol provided the best trade-off between sensitivity and specificity. The developed biochip may be used to detect the described bacterial pathogens in water samples when parallel and specific detection is required.

On-chip non-equilibrium dissociation curves and dissociation rate constants as methods to assess specificity of oligonucleotide probes.

Nucleic acid hybridization serves as backbone for many high-throughput systems for detection, expression analysis, comparative genomics and re-sequencing. Specificity of hybridization between probes and intended targets is always critical. Approaches to ensure and evaluate specificity include use of mismatch probes, obtaining dissociation curves rather than single temperature hybridizations, and comparative hybridizations. In this study, we quantify effects of mismatch type and position on intensity of hybridization signals and provide a new approach based on dissociation rate constants to evaluate specificity of hybridized signals in complex target mixtures. Using an extensive set of 18mer oligonucleotide probes on an in situ synthesized biochip platform, we demonstrate that mismatches in the center of the probe are more discriminating than mismatches toward the extremities of the probe and mismatches toward the attached end are less discriminating than those toward the loose end. The observed destabilizing effect of a mismatch type agreed in general with predictions using the nearest neighbor model. Use of a new parameter, specific dissociation temperature (T(d-w), temperature of maximum specific dissociation rate constant), obtained from probe-target duplex dissociation profiles considerably improved the evaluation of specificity. These results have broad implications for hybridization data obtained from complex mixtures of nucleic acids.

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 approximately 4-fold. Most ORFs of the LEE pathogenicity island also decreased approximately 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 (stx1AB and stx2B) 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.

Virulence factor activity relationships: challenges and development approaches.

Virulence factor activity relationships (VFAR) is a predictive approach proposed by the National Research Council's Committee on Drinking Water Contaminants (Washington, D.C.) to classify and rank waterborne pathogens. It is based on the presumption that health threats of waterborne pathogens can be predicted from descriptors at different levels of cellular organization. This paper summarizes challenges that need to be addressed while developing VFAR, with a focus on genomics, such as genomic variability among related pathogens and the need to incorporate genetic descriptors for persistence and host susceptibility. Three key components of VFAR development and validation are also presented, including (1) compilation of a comprehensive VFAR database, (2) development of predictive mathematical models relating descriptors to health effects and other microbial responses, and (3) high-throughput molecular monitoring of drinking water supplies and sources. Bayesian approach and on-chip polymerase chain reaction are discussed as examples of mathematical models and molecular monitoring.

Potential of DNA microarrays for developing parallel detection tools (PDTs) for microorganisms relevant to biodefense and related research needs.

Development of parallel detection tools using microarrays is critically reviewed in view of the need for screening multiple microorganisms in a single test. Potential research needs with respect to probe design and specificity, validation, sample concentration, selective target enrichment and amplification, and data analysis are discussed. Data illustrating selected probe design issues for detecting multiple targets in mixed microbial systems is presented. Challenges with respect to cost, time, and ease of use compared to other methods are also summarized.

Identification of non-specific hybridization using an empirical equation fitted to non-equilibrium dissociation curves.

Non-equilibrium dissociation curves (NEDCs) have the potential to identify non-specific hybridizations on high throughput, diagnostic microarrays. We report a simple method for the identification of non-specific signals by using a new parameter that does not rely on comparison of perfect match and mismatch dissociations. The parameter is the ratio of specific dissociation temperature (T(d-w)) to theoretical melting temperature (T(m)) and can be obtained by automated fitting of a four-parameter, sigmoid, empirical equation to the thousands of curves generated in a typical experiment. The curves fit perfect match NEDCs from an initial experiment with an R(2) of 0.998±0.006 and root mean square of 108±91 fluorescent units. Receiver operating characteristic curve analysis showed low temperature hybridization signals (20-48°C) to be as effective as area under the curve as primary data filters. Evaluation of three datasets that target 16S rRNA and functional genes with varying degrees of target sequence similarity showed that filtering out hybridizations with T(d-w)/T(m)<0.78 greatly reduced false positive results. In conclusion, T(d-w)/T(m) successfully screened many non-specific hybridizations that could not be identified using single temperature signal intensities alone, while the empirical modeling allowed a simplified approach to the high throughput analysis of thousands of NEDCs.

Influence of dangling ends and surface-proximal tails of targets on probe-target duplex formation in 16S rRNA gene-based diagnostic arrays.

Dangling ends and surface-proximal tails of gene targets influence probe-target duplex formation and affect the signal intensity of probes on diagnostic microarrays. This phenomenon was evaluated using an oligonucleotide microarray containing 18-mer probes corresponding to the 16S rRNA genes of 10 waterborne pathogens and a number of synthetic and PCR-amplified gene targets. Signal intensities for Klenow/random primer-labeled 16S rRNA gene targets were dissimilar from those for 45-mer synthetic targets for nearly 73% of the probes tested. Klenow/random primer-labeled targets resulted in an interaction with a complex mixture of 16S rRNA genes (used as the background) 3.7 times higher than the interaction of 45-mer targets with the same mixture. A 7-base-long dangling end sequence with perfect homology to another single-stranded background DNA sequence was sufficient to produce a cross-hybridization signal that was as strong as the signal obtained by the probe-target duplex itself. Gibbs free energy between the target and a well-defined background was found to be a better indicator of hybridization signal intensity than the sequence or length of the dangling end alone. The dangling end (Gibbs free energy of -7.6 kcal/mol) was found to be significantly more prone to target-background interaction than the surface-proximal tail (Gibbs free energy of -64.5 kcal/mol). This study underlines the need for careful target preparation and evaluation of signal intensities for diagnostic arrays using 16S rRNA and other gene targets due to the potential for target interaction with a complex background.

Evolution of genomic content in the stepwise emergence of Escherichia coli O157:H7.

Genome comparisons have demonstrated that dramatic genetic change often underlies the emergence of new bacterial pathogens. Evolutionary analysis of Escherichia coli O157:H7, a pathogen that has emerged as a worldwide public health threat in the past two decades, has posited that this toxin-producing pathogen evolved in a series of steps from O55:H7, a recent ancestor of a nontoxigenic pathogenic clone associated with infantile diarrhea. We used comparative genomic hybridization with 50-mer oligonucleotide microarrays containing probes from both pathogenic and nonpathogenic genomes to infer when genes were acquired and lost. Many ancillary virulence genes identified in the O157 genome were already present in an O55:H7-like progenitor, with 27 of 33 genomic islands of >5 kb and specific for O157:H7 (O islands) that were acquired intact before the split from this immediate ancestor. Most (85%) of variably absent or present genes are part of prophages or phage-like elements. Divergence in gene content among these closely related strains was approximately 140 times greater than divergence at the nucleotide sequence level. A >100-kb region around the O-antigen gene cluster contained highly divergent sequences and also appears to be duplicated in its entirety in one lineage, suggesting that the whole region was cotransferred in the antigenic shift from O55 to O157. The beta-glucuronidase-positive O157 variants, although phylogenetically closest to the Sakai strain, were divergent for multiple adherence factors. These observations suggest that, in addition to gains and losses of phage elements, O157:H7 genomes are rapidly diverging and radiating into new niches as the pathogen disseminates.

Molecular components of physiological stress responses in Escherichia coli.

In order to survive under and adapt to different conditions Escherichia coli has evolved elaborate systems that are able to sense and respond to environmental stimuli. Very often, different stresses act on a bacterium simultaneously and a variety of stresses have similar effects on cellular molecules and processes. Therefore, the various stress response systems have to interact (cross talk) with each other. A complex network of global regulatory systems with a multitude of molecular components ensures a coordinated and effective answer. Such regulatory components include DNA, mRNAs, sRNAs, proteins, such as DNA-and RNA binding proteins, alternative sigma factors and two-component systems, as well as small molecular weight molecules, as for example (p)ppGpp. These regulatory systems govern the expression of a plethora of further effectors that aim at maintaining stability of the cellular equilibrium under the various conditions. Using five of the most important stress response systems, we will discuss the roles and mechanisms of such regulatory and effector molecules in more detail. The heat shock response, controlled by the sigma factor sigma32, and the envelope stress response, controlled by the sigma factor sigmaE and the Cpx two-component system, both result in an increased expression of chaperones and proteases in response to misfolded proteins. The cold shock response governs expression of RNA chaperones and ribosomal factors, ensuring accurate translation at low temperatures. The general stress response depends on the sigma factor sigmaS, which controls the expression of more than 50 genes conferring resistance to many different stresses. The (p)ppGpp-dependent stringent response reduces the cellular protein synthesis capacity and controls further global responses upon nutritional downshift. A lot has been learned in recent years about the mechanisms of action of single components. However, the main challenge for the future is to achieve an understanding of the interactions of these components under different physiological conditions.

The apparent clock-like evolution of Escherichia coli in glucose-limited chemostats is reproducible at large but not at small population sizes and can be explained with Monod kinetics.

To follow and model evolution of a microbial population in the chemostat, parameters are needed that give an indication of the absolute extent of evolution at a high resolution of time. In this study the evolution of the maximum specific growth rate ( micro (max)) and the residual glucose concentration was followed for populations of Escherichia coli K-12 under glucose-limited conditions at dilution rates of 0.1 x h(-1), 0.3 x h(-1) and 0.53 x h(-1) during 500-700 h in continuous culture. Whereas micro (max) improved only during the initial 150 h, the residual glucose concentration decreased constantly during 500 h of cultivation and therefore served as a convenient parameter to monitor the evolution of a population at a high time resolution with respect to its affinity for the growth-limiting substrate. The evolution of residual glucose concentrations was reproducible in independent chemostats with a population size of 10(11) cells, whereas no reproducibility was found in chemostats containing 10(7) cells. A model based on Monod kinetics assuming successive take-overs of mutants with improved kinetic parameters (primarily K(s)) was able to simulate the experimentally observed evolution of residual glucose concentrations. Similar values for the increase in glucose affinity of mutant phenotypes (K(s(mutant)) approximately equal 0.6 x K(s(parent))) and similar mutation rates per cell per generation leading to these mutant phenotypes (1-5 x1 0(-7)) were estimated in silico for all dilution rates. The model predicts a maximum rate of evolution at a dilution rate slightly below micro (max)/2. With increasing and decreasing dilution rates the evolution slows down, which also explains why in special cases a selection-driven evolution can exhibit apparent clock-like behaviour. The glucose affinity for WT cells was dependent on the dilution rate with highest values at dilution rates around micro (max)/2. Below 0.3 x h(-1) poorer affinity was mainly due to the effects of rpoS.