Evaluation of a rapid combination disc test (RCDT) for direct phenotypic detection of extended-spectrum ß-lactamase production in E. coli from positive blood culture bottles.

BACKGROUND: The spread of multi-resistant bacteria endangers the effectiveness of empirical antimicrobial treatment, particularly in Gram-negative bloodstream infections. Thus, rapid and reliable susceptibility testing has become a key challenge of modern microbiology. Here, we evaluated a combination disc test for rapid detection of ESBL production in Escherichia coli (rapid combination disc test, RCDT) directly from blood cultures. METHODS: RCDT with discs containing cefotaxime and ceftazidime alone or in combination with clavulanic acid was validated using a cryo-collection of 96 third-generation cephalosporin-resistant (3GCR), whole-genome sequenced E. coli isolates spiked into blood culture bottles. All isolates were subjected to RCDT and rapid antibiotic susceptibility testing (RAST). Zone diameters were assessed after 4, 6 and 8 h of incubation. All isolates also underwent conventional combination disc testing. The real-life performance of RCDT was assessed by analysis of 306 blood cultures growing E. coli. RESULTS: Eighty of 90 (88.9%) ESBL-positive E. coli validation isolates were correctly identified by RCDT after 4 h of incubation. The detection rate increased to 100% after 6 and 8 h. RCDT was negative in six 3GCR E. coli isolates expressing class B or C ß-lactamases. RCDT from routine blood cultures correctly classified all 56 ESBL producers and 245/250 ESBL-negative isolates after 4 h, resulting in 100% sensitivity and 98.8% specificity. CONCLUSIONS: RCDT is a reliable method for rapid ESBL detection in E. coli directly from positive blood cultures. RCDT might complement RAST to support antibiotic stewardship interventions and treatment decisions.

Dual NADPH oxidases DUOX1 and DUOX2 synthesize NAADP and are necessary for Ca2+ signaling during T cell activation.

The formation of Ca2+ microdomains during T cell activation is initiated by the production of nicotinic acid adenine dinucleotide phosphate (NAADP) from its reduced form NAADPH. The reverse reaction­NAADP to NAADPH­is catalyzed by glucose 6-phosphate dehydrogenase (G6PD). Here, we identified NADPH oxidases NOX and DUOX as NAADP-forming enzymes that convert NAADPH to NAADP under physiological conditions in vitro. T cells express NOX1, NOX2, and, to a minor extent, DUOX1 and DUOX2. Local and global Ca2+ signaling were decreased in mouse T cells with double knockout of Duoxa1 and Duoxa2 but not with knockout of Nox1 or Nox2. Ca2+ microdomains in the first 15 s upon T cell activation were significantly decreased in Duox2−/− but not in Duox1−/− T cells, whereas both DUOX1 and DUOX2 were required for global Ca2+ signaling between 4 and 12 min after stimulation. Our findings suggest that a DUOX2- and G6PD-catalyzed redox cycle rapidly produces and degrades NAADP through NAADPH as an inactive intermediate.

HN1L/JPT2: A signaling protein that connects NAADP generation to Ca2+ microdomain formation.

NAADP-evoked Ca2+ release through type 1 ryanodine receptors (RYR1) is a major mechanism underlying the earliest signals in T cell activation, which are the formation of Ca2+ microdomains. In our characterization of the molecular machinery underlying NAADP action, we identified an NAADP-binding protein, called hematological and neurological expressed 1-like protein (HN1L) [also known as Jupiter microtubule-associated homolog 2 (JPT2)]. Gene deletion of Hn1l/Jpt2 in human Jurkat and primary rat T cells resulted in decreased numbers of initial Ca2+ microdomains and delayed the onset and decreased the amplitude of global Ca2+ signaling. Photoaffinity labeling demonstrated direct binding of NAADP to recombinant HN1L/JPT2. T cell receptor/CD3-dependent coprecipitation of HN1L/JPT2 with RYRs and colocalization of these proteins suggest that HN1L/JPT2 connects NAADP formation with the activation of RYR channels within the first seconds of T cell activation. Thus, HN1L/JPT2 enables NAADP to activate Ca2+ release from the endoplasmic reticulum through RYR.