Analysis of <it>Escherichia coli </it>O157 clinical isolates by multilocus sequence typing

Abstract Background Although many strain typing methods exist for pathogenic Escherichia coli, most have drawbacks in terms of resolving power, interpretability, or scalability. For this reason, multilocus sequence typing (MLST) is an appealing alternative especially when applied to the typing of temporal and spatially separated isolates. This method relies on an unambiguous DNA sequence analysis of nucleotide polymorphisms in housekeeping genes and has shown a high degree of intraspecies discriminatory power for bacterial and fungal pathogens. Results Here we used the MLST method to study the genetic diversity among E. coli O157 isolates collected from humans from two different locations of USA over a period of several years (2000-2008). MLST analysis of 33 E. coli O157 patient isolates using the eBurst algorithm distinguished 26 different sequence types (STs), which were clustered into two clonal groups and 11 singletons. The predominant ST was ST2, which consisted of 5 isolates (14.28%) followed by ST1 (11.42%). All the isolates under clonal group I exhibited a virtually similar virulence profile except for two strains, which tested negative for the presence of stx genes. The isolates that were assigned to clonal group II in addition to the 11 singletons were found to be phylogenetically distant from clonal group I. Furthermore, we observed a positive correlation between the virulence profile of the isolates and their clonal origin. Conclusions Our data suggests the presence of genetic diversity among E. coli O157 isolates from humans shows no measurable correlation to the geographic origin of the isolates.</p

Temporal Differential Proteomes of <em>Clostridium difficile</em> in the Pig Ileal-Ligated Loop Model

<div><p>The impact of <em>Clostridium difficile</em> infection (CDI) on healthcare is becoming increasingly recognized as it represents a major cause of nosocomial diarrhea. A rising number of CDI cases and outbreaks have been reported worldwide. Here, we developed the pig ileal-ligated loop model for semi-quantitative analysis comparing temporal differential proteomes in <em>C. difficile</em> following <em>in vivo</em> incubation with <em>in vitro</em> growth using isobaric tags for relative and absolute quantification (iTRAQ). Proteins retrieved from the <em>in vitro</em> cultures and the loop contents after 4, 8, and 12 h <em>in vivo</em> incubation were subjected to in-solution digestion, iTRAQ labeling, two-dimensional liquid chromatography/tandem mass spectrometry and statistical analyses. From a total of 1152 distinct proteins identified in this study, 705 proteins were available for quantitative measures at all time points in both biological and technical replicates; 109 proteins were found to be differentially expressed. With analysis of clusters of orthologous group and protein-protein network interactions, we identified the proteins that might play roles in adaptive responses to the host environment, hence enhancing pathogenicity during CDI. This report represents the quantitative proteomic analysis of <em>C. difficile</em> that demonstrates time-dependent protein expression changes under conditions that mimic <em>in vivo</em> infection and identifies potential candidates for diagnostic or therapeutic measures.</p> </div

Evaluation of a Mycobacterium avium subsp. paratuberculosis leuD mutant as a vaccine candidate against challenge in a caprine model

Johne's disease (JD) is prevalent worldwide and has a significant impact on the global agricultural economy. In the present study, we evaluated the protective efficacy of a leuD (Δleud) mutant and gained insight into differential immune responses after challenge with virulent M. avium subsp. paratuberculosis in a caprine colonization model. The immune response and protective efficacy were compared with those of the killed vaccine Mycopar. In vitro stimulation of peripheral blood mononuclear cells with johnin purified protein derivative showed that Mycopar and ΔleuD generated similar levels of gamma interferon (IFN-γ) but significantly higher levels than unvaccinated and challenged phosphate-buffered saline controls. However, only with ΔleuD was the IFN-γ response maintained. Flow cytometric analysis showed that the increase in IFN-γ correlated with proliferation and activation (increased expression of CD25) of CD4, CD8, and γδT cells, but this response was significantly higher in ΔleuD-vaccinated animals at some time points after challenge. Both Mycopar and ΔleuD vaccines upregulated Th1/proinflammatory and Th17 cytokines and downregulated Th2/anti-inflammatory and regulatory cytokines at similar levels at almost all time points. However, significantly higher levels of IFN-γ (at weeks 26 and 30), interleukin-2 (IL-2; week 18), IL-1b (weeks 14 and 22), IL-17 (weeks 18 and 22), and IL-23 (week 18) and a significantly lower level of IL-10 (weeks 14 and 18) and transforming growth factor β (week 18) were detected in the ΔleuD-vaccinated group. Most importantly, ΔleuD elicited an immune response that significantly limited colonization of tissues compared to Mycopar upon challenge with wild-type M. avium subsp. paratuberculosis. In conclusion, the ΔleuD mutant is a promising vaccine candidate for development of a live attenuated vaccine for JD in ruminants

Overview of the experimental workflow.

<p><i>C. difficile</i> retrieved from pig ileal-ligated loops following the <i>in vivo</i> incubation for a period of 4, 8 and 12 h were used for biological replicates (dotted line; A and B). The <i>in vitro</i> cultures of <i>C. difficile</i> were used as a control and subjected to the identical workflow. Proteins extracted from the control and the loop contents after 4, 8 and 12 h incubation were subjected to trypsin digestion and were labeled with the tags 114, 115, 116, and 117, respectively. Mass spectrometric results were analyzed for protein identification and quantification. Technical replicates were performed from the trypsin digestion step (solid line; A1 versus A2 and B1 versus B2).</p

Distribution of differentially expressed proteins in <i>C. difficile</i> following <i>in vivo</i> incubation according to COGs functional categories.

<p>Of 3,753 proteins in <i>C. difficile</i> strain 630, there are 3,000 proteins found in COGs database. The stacked bar chart shows the percentage of differentially expressed proteins (blue) in each COGs functional category. The number of differentially expressed proteins and the total number of proteins in each COGs are also shown on the right panel. Of 109 differentially expressed proteins, 8 are not annotated in any COGs.</p

Microscopic sections of the ileal loops representing pseudomembrane formation due to the infection of <i>C. difficile</i> under the experimental conditions.

<p>(A) The mucosal surface is covered by a thick layer of fibrinopurulent exudate (‘pseudomembrane’). Note the extensive submucosal edema. (B) Focal erosion of the surface enterocytes is accompanied by exudation of fibrin and neutrophils into the lumen of the intestine.</p

Overview of the iTRAQ results.

<p>(A) Number of distinct peptides identified in a protein. The peptides were identified using Mascot Daemon software with a 95% confidence level. About 17–19% of proteins were identified by single peptides. More than 80% of proteins were identified by at least two peptides. Approximately 47% of proteins were identified by more than 5 peptides. (B) Comparison of protein identification in iTRAQ experiments. Venn diagram shows the comparison of protein identification between biological replicates (A versus B) as well as technical replicates (A1 versus A2 and B1 versus B2). There were 207 proteins identified in a single experiment and 217 proteins identified in two or three of the experiments. A total of 728 proteins were identified in all four experiments. (C) Protein expression pattern in <i>C. difficile</i> following the <i>in vivo</i> incubation. Protein expression after 4, 8, and 12 h <i>in vivo</i> incubation is compared with that of <i>ex vivo</i> growth. Rank normalized data from biological and technical replicates were used to create a heatmap for <i>C. difficile</i>. The proteins were arranged according to the numbering of the <i>C. difficile</i> 630 coding sequences, with CD0001 at the top and CD3680 at the bottom, followed by proteins from the plasmid pCD630 (CDP01 to CDP11). Each column represents a particular time point in one iTRAQ experiment, and each row corresponds to a specific protein. The status of each protein is indicated by color as follows: red, induced; green, repressed; black, unchanged; and gray, undetected in our conditions.</p