A Randomized, Controlled Trial of the Analytic and Diagnostic Performance of Singleton and Trio, Rapid Genome and Exome Sequencing in Ill Infants.

The second Newborn Sequencing in Genomic Medicine and Public Health study was a randomized, controlled trial of the effectiveness of rapid whole-genome or -exome sequencing (rWGS or rWES, respectively) in seriously ill infants with diseases of unknown etiology. Here we report comparisons of analytic and diagnostic performance. Of 1,248 ill inpatient infants, 578 (46%) had diseases of unknown etiology. 213 infants (37% of those eligible) were enrolled within 96 h of admission. 24 infants (11%) were very ill and received ultra-rapid whole-genome sequencing (urWGS). The remaining infants were randomized, 95 to rWES and 94 to rWGS. The analytic performance of rWGS was superior to rWES, including variants likely to affect protein function, and ClinVar pathogenic/likely pathogenic variants (p < 0.0001). The diagnostic performance of rWGS and rWES were similar (18 diagnoses in 94 infants [19%] versus 19 diagnoses in 95 infants [20%], respectively), as was time to result (median 11.0 versus 11.2 days, respectively). However, the proportion diagnosed by urWGS (11 of 24 [46%]) was higher than rWES/rWGS (p = 0.004) and time to result was less (median 4.6 days, p < 0.0001). The incremental diagnostic yield of reflexing to trio after negative proband analysis was 0.7% (1 of 147). In conclusion, rapid genomic sequencing can be performed as a first-tier diagnostic test in inpatient infants. urWGS had the shortest time to result, which was important in unstable infants, and those in whom a genetic diagnosis was likely to impact immediate management. Further comparison of urWGS and rWES is warranted because genomic technologies and knowledge of variant pathogenicity are evolving rapidly.

Diagnosis of genetic diseases in seriously ill children by rapid whole-genome sequencing and automated phenotyping and interpretation.

By informing timely targeted treatments, rapid whole-genome sequencing can improve the outcomes of seriously ill children with genetic diseases, particularly infants in neonatal and pediatric intensive care units (ICUs). The need for highly qualified professionals to decipher results, however, precludes widespread implementation. We describe a platform for population-scale, provisional diagnosis of genetic diseases with automated phenotyping and interpretation. Genome sequencing was expedited by bead-based genome library preparation directly from blood samples and sequencing of paired 100-nt reads in 15.5 hours. Clinical natural language processing (CNLP) automatically extracted children's deep phenomes from electronic health records with 80% precision and 93% recall. In 101 children with 105 genetic diseases, a mean of 4.3 CNLP-extracted phenotypic features matched the expected phenotypic features of those diseases, compared with a match of 0.9 phenotypic features used in manual interpretation. We automated provisional diagnosis by combining the ranking of the similarity of a patient's CNLP phenome with respect to the expected phenotypic features of all genetic diseases, together with the ranking of the pathogenicity of all of the patient's genomic variants. Automated, retrospective diagnoses concurred well with expert manual interpretation (97% recall and 99% precision in 95 children with 97 genetic diseases). Prospectively, our platform correctly diagnosed three of seven seriously ill ICU infants (100% precision and recall) with a mean time saving of 22:19 hours. In each case, the diagnosis affected treatment. Genome sequencing with automated phenotyping and interpretation in a median of 20:10 hours may increase adoption in ICUs and, thereby, timely implementation of precise treatments.

New-found fundamentals of bacterial persistence.

Persister cells display tolerance to high doses of bactericidal antibiotics and typically comprise a small fraction of a bacterial population. Recently, evidence was provided for a causal link between therapy failure and the presence of persister cells in chronic infections, underscoring the need for research on bacterial persistence. A series of recent breakthroughs have shed light on the multiplicity of persister genes, the contribution of gene expression noise to persister formation, the importance of active responses to antibiotic tolerance and heterogeneity among persister cells. Moreover, the development of in vivo model systems has highlighted the clinical relevance of persistence. This review discusses these recent advances and how this knowledge fundamentally changes the way in which we will perceive the problem of antibiotic tolerance in years to come.

The Escherichia coli GTPase ObgE modulates hydroxyl radical levels in response to DNA replication fork arrest.

Obg proteins are universally conserved GTP-binding proteins that are essential for viability in bacteria. Homologs in different organisms are involved in various cellular processes, including DNA replication. The goal of this study was to analyse the structure-function relationship of Escherichia coli ObgE with regard to DNA replication in general and sensitivity to stalled replication forks in particular. Defined C-terminal chromosomal deletion mutants of obgE were constructed and tested for sensitivity to the replication inhibitor hydroxyurea. The ObgE C-terminal domain was shown to be dispensable for normal growth of E.coli. However, a region within this domain is involved in the cellular response to replication fork stress. In addition, a mutant obgE over-expression library was constructed by error-prone PCR and screened for increased hydroxyurea sensitivity. ObgE proteins with substitutions L159Q, G163V, P168V, G216A or R237C, located within distinct domains of ObgE, display dominant-negative effects leading to hydroxyurea hypersensitivity when over-expressed. These effects are abolished in strains with a single deletion of the iron transporter TonB or combined deletions the toxin/antitoxin modules RelBE/MazEF, strains both of which have been shown to be involved in a pathway that stimulates hydroxyl radical formation following hydroxyurea treatment. Moreover, the observed dominant-negative effects are lost in the presence of the hydroxyl radical scavenger thiourea. Together, these results indicate involvement of hydroxyl radical toxicity in ObgE-mediated protection against replication fork stress.

Pseudomonas aeruginosa fosfomycin resistance mechanisms affect non-inherited fluoroquinolone tolerance.

Pseudomonas aeruginosa is an opportunistic pathogen that poses a threat in clinical settings due to its intrinsic and acquired resistance to a wide spectrum of antibiotics. Additionally, the presence of a subpopulation of cells surviving high concentrations of antibiotics, called persisters, makes it virtually impossible to eradicate a chronic infection. The mechanism underlying persistence is still unclear, partly due to the fact that it is a non-inherited phenotype. Based on our findings from a previously performed screening effort for P. aeruginosa persistence genes, we hypothesize that crosstalk can occur between two clinically relevant mechanisms: the persistence phenomenon and antibiotic resistance. This was tested by determining the persistence phenotype of P. aeruginosa strains that are resistant to the antibiotic fosfomycin due to either of two unrelated fosfomycin resistance mechanisms. Overexpression of fosA (PA1129) confers fosfomycin resistance by enzymic modification of the antibiotic, and in addition causes a decrease in the number of persister cells surviving ofloxacin treatment. Both phenotypes require the enzymic function of FosA, as mutation of the Arg119 residue abolishes fosfomycin resistance as well as low persistence. The role for fosfomycin resistance mechanisms in persistence is corroborated by demonstrating a similar phenotype in a strain with a mutation in glpT (PA5235), which encodes a glycerol-3-phosphate transporter essential for fosfomycin uptake. These results indicate that fosfomycin resistance, conferred by glpT mutation or by overexpression of fosA, results in a decrease in the number of persister cells after treatment with ofloxacin and additionally stress that further research into the interplay between fosfomycin resistance and persistence is warranted.

Novel persistence genes in Pseudomonas aeruginosa identified by high-throughput screening.

Persister cells are phenotypic variants that are extremely tolerant to high concentrations of antibiotics. They constitute a fraction of stationary phase cultures and biofilm populations of numerous bacterial species, such as the opportunistic pathogen Pseudomonas aeruginosa. Even though persisters are believed to be an important cause of incomplete elimination of infectious populations by antibiotics, their nature remains obscure. Most studies on persistence have focused on the model organism Escherichia coli and only a limited number of persistence genes have been identified to date. We performed the first large-scale screening of a P. aeruginosa PA14 mutant library to identify novel genes involved in persistence. A total of 5000 mutants were screened in a high-throughput manner and nine new persistence mutants were identified. Four mutants (with insertions in dinG, spuC, PA14_17880 and PA14_66140) exhibited a low persister phenotype and five mutants (in algR, pilH, ycgM, pheA and PA14_13680) displayed high persistence. These genes may serve as new candidate drug targets in the combat against P. aeruginosa infections.