Rapid identification of pathogens from positive blood cultures by multiplex polymerase chain reaction using the FilmArray system.

Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Salt Lake City, UT, USA) Blood Culture (BC) panel can identify >25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 h. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 (91%) of 92 pathogens covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven methicillin-resistant S. aureus and vancomycin-resistant enterococci. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture.

A human polymorphism affects NEDD4L subcellular targeting by leading to two isoforms that contain or lack a C2 domain.

BACKGROUND: Ubiquitination serves multiple cellular functions, including proteasomal degradation and the control of stability, function, and intracellular localization of a wide variety of proteins. NEDD4L is a member of the HECT class of E3 ubiquitin ligases. A defining feature of NEDD4L protein isoforms is the presence or absence of an amino-terminal C2 domain, a class of subcellular, calcium-dependent targeting domains. We previously identified a common variant in human NEDD4L that generates isoforms that contain or lack a C2 domain. RESULTS: To address the potential functional significance of the NEDD4L common variant on NEDD4L subcellular localization, NEDD4L isoforms that either contained or lacked a C2 domain were tagged with enhanced green fluorescent protein, transfected into Xenopus laevis kidney epithelial cells, and imaged by performing confocal microscopy on live cells. We report that the presence or absence of this C2 domain exerts differential effects on the subcellular distribution of NEDD4L, the ability of C2 containing and lacking NEDD4L isoforms to mobilize in response to a calcium stimulus, and the intracellular transport of subunits of the NEDD4L substrate, ENaC. Furthermore, the ability of the C2-containing isoform to influence beta-ENaC mobilization from intracellular pools involves the NEDD4L active site for ubiquitination. We propose a model to account for the potential impact of this common genetic variant on protein function at the cellular level. CONCLUSION: NEDD4L isoforms that contain or lack a C2 domain target different intracellular locations. Additionally, whereas the C2-containing NEDD4L isoform is capable of shuttling between the plasma membrane and intracellular compartments in response to calcium stimulus the C2-lacking isoform can not. The C2-containing isoform differentially affects the mobilization of ENaC subunits from intracellular pools and this trafficking step requires NEDD4L ubiquitin ligase activity. This observation suggests a new mechanism for the requirement for the PY motif in cAMP-mediated exocytosis of ENaC. We have elucidated how a common genetic variant can underlie significant functional diversity in NEDD4L at the cellular level. We propose a model that describes how that functional variation may influence blood pressure. Moreover, our observations regarding differential function of the NEDD4L isoforms may impact other aspects of physiology that involve this ubiquitin ligase.