New methods to analyse microarray data that partially lack a reference signal

BACKGROUND: Microarray-based Comparative Genomic Hybridisation (CGH) has been used to assess genetic variability between bacterial strains. Crucial for interpretation of microarray data is the availability of a reference to compare signal intensities to reliably determine presence or divergence each DNA fragment. However, the production of a good reference becomes unfeasible when microarrays are based on pan-genomes.When only a single strain is used as a reference for a multistrain array, the accessory gene pool will be partially represented by reference DNA, although these genes represent the genomic repertoire that can explain differences in virulence, pathogenicity or transmissibility between strains. The lack of a reference makes interpretation of the data for these genes difficult and, if the test signal is low, they are often deleted from the analysis. We aimed to develop novel methods to determine the presence or divergence of genes in a Staphylococcus aureus multistrain PCR product microarray-based CGH approach for which reference DNA was not available for some probes. RESULTS: In this study we have developed 6 new methods to predict divergence and presence of all genes spotted on a multistrain Staphylococcus aureus DNA microarray, published previously, including those gene spots that lack reference signals. When considering specificity and PPV (i.e. the false-positive rate) as the most important criteria for evaluating these methods, the method that defined gene presence based on a signal at least twice as high as the background and higher than the reference signal (method 4) had the best test characteristics. For this method specificity was 100% and 82% for MRSA252 (compared to the GACK method) and all spots (compared to sequence data), respectively, and PPV were 100% and 76% for MRSA252 (compared to the GACK method) and all spots (compared to sequence data), respectively. CONCLUSION: A definition of gene presence based on signal at least twice as high as the background and higher than the reference signal (method 4) had the best test characteristics, allowing the analysis of 6-17% more of the genes not present in the reference strain. This method is recommended to analyse microarray data that partially lack a reference signal

Enterococcal surface protein Esp is not essential for cell adhesion and intestinal colonization of Enterococcus faecium in mice

<p>Abstract</p> <p>Background</p> <p><it>Enterococcus faecium </it>has globally emerged as a cause of hospital-acquired infections with high colonization rates in hospitalized patients. The enterococcal surface protein Esp, identified as a potential virulence factor, is specifically linked to nosocomial clonal lineages that are genetically distinct from indigenous <it>E. faecium </it>strains. To investigate whether Esp facilitates bacterial adherence and intestinal colonization of <it>E. faecium</it>, we used human colorectal adenocarcinoma cells (Caco-2 cells) and an experimental colonization model in mice.</p> <p>Results</p> <p>No differences in adherence to Caco-2 cells were found between an Esp expressing strain of <it>E. faecium </it>(E1162) and its isogenic Esp-deficient mutant (E1162Δ<it>esp</it>). Mice, kept under ceftriaxone treatment, were inoculated orally with either E1162, E1162Δ<it>esp </it>or both strains simultaneously. Both E1162 and E1162Δ<it>esp </it>were able to colonize the murine intestines with high and comparable numbers. No differences were found in the contents of cecum and colon. Both E1162 and E1162Δ<it>esp </it>were able to translocate to the mesenteric lymph nodes.</p> <p>Conclusion</p> <p>These results suggest that Esp is not essential for Caco-2 cell adherence and intestinal colonization or translocation of <it>E. faecium </it>in mice.</p

Dynamics of ampicillin-resistant Enterococcus faecium clones colonizing hospitalized patients: data from a prospective observational study

<p>Abstract</p> <p>Background</p> <p>Little is known about the dynamics of colonizing <it>Enterococcus faecium </it>clones during hospitalization, invasive infection and after discharge.</p> <p>Methods</p> <p>In a prospective observational study we compared intestinal <it>E. faecium </it>colonization in three patient cohorts: 1) Patients from the Hematology Unit at the University Hospital Basel (UHBS), Switzerland, were investigated by weekly rectal swabs (RS) during hospitalization (group 1a, n = 33) and monthly after discharge (group 1b, n = 21). 2) Patients from the Intensive Care Unit (ICU) at the University Medical Center Utrecht, the Netherlands (group 2, n = 25) were swabbed weekly. 3) Patients with invasive <it>E. faecium </it>infection at UHBS were swabbed at the time of infection (group 3, n = 22). From each RS five colonies with typical <it>E</it>. <it>faecium </it>morphology were picked. Species identification was confirmed by PCR and ampicillin-resistant <it>E. faecium </it>(ARE) isolates were typed using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). The Simpson's Index of Diversity (SID) was calculated.</p> <p>Results</p> <p>Out of 558 ARE isolates from 354 RS, MT159 was the most prevalent clone (54%, 100%, 52% and 83% of ARE in groups 1a, 1b, 2 and 3, respectively). Among hematological inpatients 13 (40%) had ARE. During hospitalization, the SID of MLVA-typed ARE decreased from 0.745 [95%CI 0.657-0.833] in week 1 to 0.513 [95%CI 0.388-0.637] in week 3. After discharge the only detected ARE was MT159 in 3 patients. In the ICU (group 2) almost all patients (84%) were colonized with ARE. The SID increased significantly from 0.373 [95%CI 0.175-0.572] at week 1 to a maximum of 0.808 [95%CI 0.768-0.849] at week 3 due to acquisition of multiple ARE clones. All 16 patients with invasive ARE were colonized with the same MLVA clone (<it>p </it>< 0.001).</p> <p>Conclusions</p> <p>In hospitalized high-risk patients MT159 is the most frequent colonizer and cause of invasive <it>E. faecium </it>infections. During hospitalization, ASE are quickly replaced by ARE. Diversity of ARE increases on units with possible cross-transmission such as ICUs. After hospitalization ARE are lost with the exception of MT159. In invasive infections, the invasive clone is the predominant gut colonizer.</p

Intra- and Interspecies Genomic Transfer of the Enterococcus faecalis Pathogenicity Island

Enterococci are the third leading cause of hospital associated infections and have gained increased importance due to their fast adaptation to the clinical environment by acquisition of antibiotic resistance and pathogenicity traits. Enterococcus faecalis harbours a pathogenicity island (PAI) of 153 kb containing several virulence factors including the enterococcal surface protein (esp). Until now only internal fragments of the PAI or larger chromosomal regions containing it have been transfered. Here we demonstrate precise excision, circularization and horizontal transfer of the entire PAI element from the chromosome of E. faecalis strain UW3114. This PAI (ca. 200 kb) contained some deletions and insertions as compared to the PAI of the reference strain MMH594, transferred precisely and integrated site-specifically into the chromosome of E. faecalis (intergenic region) and Enterococcus faecium (tRNAlys). The internal PAI structure was maintained after transfer. We assessed phenotypic changes accompanying acquisition of the PAI and expression of some of its determinants. The esp gene is expressed on the surface of donor and both transconjugants. Biofilm formation and cytolytic activity were enhanced in E. faecalis transconjugants after acquisition of the PAI. No differences in pathogenicity of E. faecalis were detected using a mouse bacteraemia and a mouse peritonitis models (tail vein and intraperitoneal injection). A 66 kb conjugative pheromone-responsive plasmid encoding erm(B) (pLG2) that was transferred in parallel with the PAI was sequenced. pLG2 is a pheromone responsive plasmid that probably promotes the PAI horizontal transfer, encodes antibiotic resistance features and contains complete replication and conjugation modules of enterococcal origin in a mosaic-like composition. The E. faecalis PAI can undergo precise intra- and interspecies transfer probably with the help of conjugative elements like conjugative resistance plasmids, supporting the role of horizontal gene transfer and antibiotic selective pressure in the successful establishment of certain enterococci as nosocomial pathogens

A combined linkage, microarray and exome analysis suggests MAP3K11 as a candidate gene for left ventricular hypertrophy

Background: Electrocardiographic measures of left ventricular hypertrophy (LVH) are used as predictors of cardiovascular risk. We combined linkage and association analyses to discover novel rare genetic variants involved in three such measures and two principal components derived from them. Methods: The study was conducted among participants from the Erasmus Rucphen Family Study (ERF), a Dutch family-based sample from the southwestern Netherlands. Variance components linkage analyses were performed using Merlin. Regions of interest (LOD > 1.9) were fine-mapped using microarray and exome sequence data. Results: We observed one significant LOD score for the second principal component on chromosome 15 (LOD score = 3.01) and 12 suggestive LOD scores. Several loci contained variants identified in GWAS for these traits; however, these did not explain the linkage peaks, nor did other common variants. Exome sequence data identified two associated variants after multiple testing corrections were applied. Conclusions: We did not find common SNPs explaining these linkage signals. Exome sequencing uncovered a relatively rare variant in MAPK3K11 on chromosome 11 (MAF = 0.01) that helped account for the suggestive linkage peak observed for the first principal component. Conditional analysis revealed a drop in LOD from 2.01 to 0.88 for MAP3K11, suggesting that this variant may partially explain the linkage signal at this chromosomal location. MAP3K11 is related to the JNK pathway and is a pro-apoptotic kinase that plays an important role in the induction of cardiomyocyte apoptosis in various pathologies, including LVH