In this study, an assay that combines the ease and simplicity of the qualitative approach for measuring catalase activity was developed. The assay reagents comprised only hydrogen peroxide and Triton X-100. The enzyme-generated oxygen bubbles trapped by Triton X-100 were visualized as foam, whose height was estimated. A calibration plot using the defined unit of catalase activity yielded the best linear fit over a range of 20–300 units (U) (y = 0.3794x − 2.0909, r2 = 0.993). The assay precision and reproducibility at 100 U were 4.6% and 4.8%, respectively. The applicability of the assay for measuring the catalase activity of various samples was assessed using laboratory strains of Escherichia coli, catalase-deficient isogenic mutants, clinically isolated Shiga toxin-producing E. coli, and human cells. The assay generated reproducible results. In conclusion, this new assay can be used to measure the catalase activity of bacterial isolates and human cells.

ATP modulates immune cell functions, and ATP derived from gut commensal bacteria promotes the differentiation of T helper 17 (Th17) cells in the intestinal lamina propria. We recently reported that Enterococcus gallinarum, isolated from mice and humans, secretes ATP. We have since found and characterized several ATP-secreting bacteria. Of the tested enterococci, Enterococcus mundtii secreted the greatest amount of ATP (>2 M/10 8 cells) after overnight culture. Glucose, not amino acids and vitamins, was essential for ATP secretion from E. mundtii. Analyses of energy-deprived cells demonstrated that glycolysis is the most important pathway for bacterial ATP secretion. Furthermore, exponential-phase E. mundtii and Enterococcus faecalis cells secrete ATP more efficiently than stationary-phase cells. Other bacteria, including Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, also secrete ATP in exponential but not stationary phase. These results suggest that various gut bacteria, including commensals and pathogens, might secrete ATP at any growth phase and modulate immune cell function. ATP is the source of energy within living cells and acts as an allosteric effector of numerous cell processes, such as active transport, nucleic acid synthesis, muscle activity, and movement (1-5).In mammalian cells, ATP is the neurotransmitter for purinergic signal transmission (6). ATP is stored in secretory vesicles and exocytosed, leading to various purinergic responses, such as central control of autonomic functions, pain and mechanosensory transduction, neural-glial interactions, control of vessel tone and angiogenesis, and platelet aggregation through purinoceptors, thus establishing its role as a chemical transmitter (7)(8)(9)(10)(11).ATP modulates immune cell function by activating the ATP receptors P2X and P2Y (11-13). Furthermore, ATP has been found in the intestinal lumen, where it promotes differentiation of T helper 17 (Th17) cells in the lamina propria (14). Germ-free (GF) mice exhibited much lower concentrations of fecal ATP, accompanied by fewer lamina propria Th17 cells, than specificpathogen-free (SPF) mice. In addition, antibiotic-treated SPF mice showed marked reductions in the number of Th17 cells and in the concentration of fecal ATP. Systemic or rectal administration of ATP markedly increases the number of lamina propria Th17 cells in GF mice. Thus, ATP driven from commensal bacteria activates the lamina propria, leading to local differentiation of Th17 cells. However, ATP-secreting bacteria have not been identified in the intestinal lumen of SPF mice.Recently, we reported that Enterococcus gallinarum, a vancomycin-resistant Gram-positive coccus isolated from SPF mice, secretes ATP; other tested bacteria, such as Enterococcus faecalis, Enterococcus faecium, Escherichia coli, and Staphylococcus aureus, did not (15). To our knowledge, this is the first report of the isolation and identification of ATP-secreting bacteria from the feces of SPF mice. However, whether other enterococcal species sec...

BackgroundCatheter-related infection (CRI) is one of the serious challenges in clinical practice. This preliminary clinical study aimed to examine whether next-generation sequencing (NGS) targeting 16S rDNA, which was PCR-amplified directly from the tip of a central venous catheter (CVC), can be used to identify causative pathogens in CRI, compared to the culture method.MethodsHospitalized patients, from whom a CVC had just been removed, were prospectively enrolled and divided into the CRI-suspected and routine removal groups. DNA was extracted from the sonication fluid of CVC specimens derived from patients. For analysis of bacterial composition by NGS, the V3–V4 fragments of bacterial 16S rDNA were PCR-amplified, followed by index PCR and paired-end sequencing on an Illumina MiSeq device. Conventional culture methods were also performed in the CRI-suspected group.ResultsOf CVCs collected from the 156 enrolled patients (114 men; mean age 65.6 years), a total of 14 specimens [nine out of 31 patients suspected with CRI and five out of 125 patients without infection symptoms (routine removal group)] were PCR-positive. In five patients with definite CRI, Staphylococcus was the most frequently detected genus by NGS (4/5 specimens), although no pathogens were detected by NGS in the one remaining specimen. The genera identified by NGS were consistent with those from conventional culture tests. There was high agreement between NGS and the culture method in the CRI-suspected group, with sensitivity and specificity values of 80.0% and 76.9%, respectively; meanwhile, the false-positive rate of NGS was as low as 4.0% in the routine removal group. Moreover, several genera, besides the genus identified by culture test, were detected in each patient with definite CRI and surgical site infection (SSI). Additionally, in one patient with SSI, Enterococcaceae were detected not only by NGS but also by abdominal abscess drainage culture.ConclusionsNGS targeting 16S rDNA was able to analyze the bacterial composition of CVC specimens and detect causative pathogens in patients with CRI and was therefore suggested as a promising diagnostic tool for CRI.