Multi-laboratory proficiency testing of clinical cancer genomic profiling by next-generation sequencing.

Next-generation sequencing (NGS) enables parallel analysis of multiple genomic targets. The increasing demand for NGS-based multiplexed molecular diagnostics requires standardized protocols and recommendations to ensure reproducibility and accuracy of test results for routine clinical decision making. However, the lack of clinical NGS data from multi-laboratory tests and the absence of inter-laboratory comparisons have hampered the establishment of instructive clinical NGS standards. To fill the gap, we set up Proficiency Testing (PT) for inter-laboratory comparison, in which formalin-fixed paraffin-embedded specimens from eight lung and eight colon cancers were analyzed by 15 European molecular diagnostic laboratories on three different platforms using multiple target enrichment systems. We first performed platform, test, and informatics pipeline validation and conducted sensitivity and specificity analysis by random in silico down-sampling. We then implemented a multi-level filtering strategy based on performance tests of base substitution, replicate runs, and Sanger sequencing verified variants. We finally applied the filter criteria to the NGS data from the respective PT participants and obtained high inter-laboratory agreement. We demonstrated accuracy, scalability, and robustness of NGS by means of PT, serving as a benchmark for detecting clinically actionable molecular alterations in research and diagnostic laboratories. In conclusion, this study strongly highlights the importance of establishing standards for NGS-based testing, particularly when the test results impact on clinical decisions, and systematically provides data sets from multiple different labs to infer such standards.

Comprehensive molecular, genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40.

BACKGROUND: Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type. RESULTS: We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro). CONCLUSION: Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

Complete genome sequence of the porcine isolate Enterococcus faecalis D32.

The complete and annotated genome sequence of Enterococcus faecalis D32, a commensal strain isolated from a Danish pig, suggests putative adaptation to the porcine host and absence of distinct virulence-associated traits.

Improved identification including MALDI-TOF mass spectrometry analysis of group D streptococci from bovine mastitis and subsequent molecular characterization of corresponding Enterococcus faecalis and Enterococcus faecium isolates.

We examined 199 group D streptococci isolated from clinically defined and epidemiologically unrelated cases of bovine mastitis. Samples were collected during a 5-month period from 2010 to 2011 from diseased animals in 199 herds (1 isolate per herd) raised in different counties and federal states in Germany. A classical enterococcal species identification procedure started with PYRase and catalase assays, growth on Enterococcoselagar(®) and GCG(®) agar plates and in 6.5% NaCl followed by a biochemical reaction panel. All 199 isolates were also subjected to MALDI-TOF MS diagnostics in which a simple and an extended direct transfer protocol were compared. The latter revealed a much better performance (higher log (score) values) although the same result was obtained in all but three cases. Classical and MALDI TOF MS analyses identified 64 Enterococcus faecalis and 37 Enterococcus faecium isolates which were confirmed by species-specific PCRs. These 101 enterococcal isolates did not display a specific multi-resistance phenotype and resistances to glycopeptides and antibiotics of last resort (linezolid, daptomycin, tigecycline) were absent, resistance to tetracycline was the most frequent resistance feature. Molecular typing of the 64 E. faecalis isolates revealed 3 main PFGE clusters of related strains represented by three MLST types (ST40, ST211, ST268). PFGE and MLST analysis of E. faecium isolates revealed several smaller clusters of only a few related strains and identified a number of previously unknown allele and MLST types (n=6; ST624-ST629) besides known variants (ST22, ST32). One of the 37 E. faecium strains showed properties of hospital-associated E. faecium strains (ampicillin resistance, IS16-positive; MLST CC17).

Cross-transmission rates of enterococcal isolates among newborns in a neonatal intensive care unit.

Enterococci are important pathogens causing nosocomial infections and patients at risk include also premature babies requiring intensive care treatment. Our aim was to assess occurrence and cross transmission rates of enterococci among neonatal patients of a hospital ward during a two months period. Rectal and skin samples were taken between day one and 60 of infants' age. Colonization with various potentially pathogenic bacteria was correlated with developing a subsequent infection. Enterococcal isolates were identified by colony morphology. The bacterial species was assessed and antibiotic susceptibilities were determined. A molecular analysis of 20 investigated enterococcal isolates revealed prevalence of commensal strain types; hospital-associated strain types or multi-resistant variants were absent. Cross transmission of E. faecium and E. faecalis isolates among neonatal patients attending the intensive crare unit at the same time was demonstrable. Introduction of hospital-associated, multi-resistant variants into this special setting has to be avoided to reduce the risk of subsequent infections.

Estrogen-induced upregulation of Sftpb requires transcriptional control of neuregulin receptor ErbB4 in mouse lung type II epithelial cells.

Estrogen is known for its positive stimulatory effects on surfactant proteins. ErbB4 receptor and its ligand neuregulin (NRG) positively stimulate lung development. ErbB receptors interact with nuclear receptors and ErbB4 co-regulates estrogen receptor (ER)α expression in breast cells. ERß is highly expressed in pneumocytes and its deletion leads to fewer alveoli and reduced elastic recoil. A similar picture was seen in ErbB4-deleted lungs. We hypothesized that estrogen signals its effect on surfactant protein B (Sftpb) expression through interactions of ERß and ErbB4. Estrogen and NRG treatment decreased cell numbers and stimulated Sftpb expression in type II cells. Estrogen and NRG both stimulated phosphorylation of ERß and co-localization of both receptors. Overexpression of ERß increased the cell number and Sftpb expression, which was further augmented by estrogen and NRG. Finally, estrogen and NRG stimulated ERß and ErbB4 binding to the Sftpb promoter. Overexpression of these receptors stimulated Sftpb promoter activation, which was further enhanced by estrogen and NRG. The stimulatory effect of estrogen and NRG was abolished in ErbB4 deletion and reconstituted by re-expression of full-length ErbB4 in fetal ErbB4-deleted type II cells. Estrogen-induced nuclear translocation of ErbB4 required the intact γ-secretase cleavage site but not the nuclear localization sequence of the ErbB4 receptor, suggesting that ERß might function as a nuclear chaperone for ErbB4. These studies demonstrate that estrogen effects on Sftpb expression require an interaction of ERß and ErbB4. We speculate that the stimulatory effects of estrogen on Sftpb are under transcriptional control of ErbB4.