There are currently no standardized phenotypic methods for the screening and detection of AmpC enzymes. This study aimed to evaluate different methods to detect AmpC enzymes in Escherichia coli, Klebsiella spp., and Proteus spp., comparing the results from two disk-based methods and an agar dilution method. AmpC activity was determined for 255 clinical isolates by use of a three-dimensional enzyme assay combined with a multiplex PCR assay for plasmid-borne ampC genes. These results were compared against a disk-based inhibitor assay using various combinations of cefpodoxime and cefoxitin as antibiotic substrates and boronic acid or cloxacillin as an AmpC inhibitor. The presence of enzyme induction by disk approximation was evaluated using imipenem, cefoxitin, and amoxicillin-clavulanate as inducing agents against ceftazidime. Finally, an agar dilution assay was performed, using cefoxitin with and without added cloxacillin. AmpC activity was present in 49.8% of test isolates, 93.7% of which were positive for plasmid-borne ampC genes. CIT-like enzymes were predominant in E. coli, and DHA-like enzymes were predominant in Klebsiella spp. The disk-based inhibitor tests performed better than the agar dilution assay, while detection of AmpC by disk induction had a poor sensitivity. The cefoxitin-cloxacillin disk combination provided the best overall performance, with a sensitivity and specificity of 95%. This study confirmed the accuracy of disk-based inhibitor screening for AmpC enzymes, which proved reliable at detecting CIT-and DHA-like plasmid-borne ampC genes. The methods are simple enough for introduction into clinical microbiology laboratories.

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Rupture of vulnerable plaques is the main trigger of acute cardio-cerebral vascular events, but mechanisms responsible for transforming a stable atherosclerotic into a vulnerable plaque remain largely unknown. Melatonin, an indoleamine hormone secreted by the pineal gland, plays pleiotropic roles in the cardiovascular system; however, the effect of melatonin on vulnerable plaque rupture and its underlying mechanisms remains unknown. Here, we generated a rupture-prone vulnerable carotid plaque model induced by endogenous renovascular hypertension combined with low shear stress in hypercholesterolemic ApoE −/− mice. Melatonin (10 mg/kg/d by oral administration for 9 weeks) significantly prevented vulnerable plaque rupture, with lower incidence of intraplaque hemorrhage (42.9% vs. 9.5%, P = 0.014) and of spontaneous plaque rupture with intraluminal thrombus formation (38.1% vs. 9.5%, P = 0.029). Mechanistic studies indicated that melatonin ameliorated intraplaque inflammation by suppressing the differentiation of intraplaque macrophages toward the proinflammatory M1 phenotype, and circadian nuclear receptor retinoid acid receptor-related orphan receptor-α (RORα) mediated melatonin-exerted vasoprotection against vulnerable plaque instability and intraplaque macrophage polarization. Further analysis in human monocyte-derived macrophages confirmed the role of melatonin in regulating macrophage polarization by regulating the AMPKα-STATs pathway in a RORα-dependent manner. In summary, our data provided the first evidence that melatonin-RORα axis acts as a novel endogenous protective signaling pathway in the vasculature, regulates intraplaque inflammation, and stabilizes rupture-prone vulnerable plaques. K E Y W O R D S atherosclerosis, macrophage polarization, melatonin, nuclear receptor, RAR-related orphan receptor, vulnerable plaques 2 of 15 | DING et al. 12 of 15 | DING et al. F I G U R E 7 Melatonin regulates macrophage polarization through the signal transducer and activator of transcription (STAT) pathway.A, Flow cytometry analysis of cell surface markers for M1 (CD80 and CD197) and M2 (CD163 and CD206) macrophages during M1 and M2 polarization with vehicle or melatonin/SR3335 treatment. The results showed that co-culture with melatonin significantly reduced IFN-γ and LPSinduced CD80 and CD197 expression, whereas RORα agonism with SR3335 markedly promoted M1 polarization (n = 6 per group). *P < 0.05 or **P < 0.01 versus monocyte; # P < 0.05 versus M0. B and C, Western blotting was conducted on cell lysates with antibodies against P-STAT1, P-STAT3, and P-STAT6; total STAT1, STAT3, and STAT6; and GAPDH. Relative densities of phosphorylated STAT compared with total STAT are shown as histograms (n = 6 per group). *P < 0.05 or **P < 0.01 versus control; ##P < 0.01 versus M1 + siRORα in C. D and E, Western blotting was conducted on cell lysates with antibodies against RORα, P-AMPKα, total AMPKα, and GAPDH. *P < 0.05 versus Mel (0 mmol/L); ##P < 0.01 versus control siRNA

This study validated abbreviated methods for the presumptive identification of Staphylococcus lugdunensis and studied the antibiotic susceptibilities of 106 isolates. The combination of positive responses to ornithine and pyrrolidonyl arylamidase identified all S. lugdunensis isolates. Resistance to penicillin and methicillin was detected in 27 and 5% of isolates, respectively.Staphylococcus lugdunensis, a coagulase-negative staphylococcus, has clinical characteristics that resemble those of the coagulase-positive Staphylococcus aureus. Infections attributed to S. lugdunensis include infective endocarditis (19), bacteremia, meningitis (6), bone and joint infections (7), and softtissue infections (16). S. lugdunensis generally is susceptible to anti-staphylococcal antibiotics (18), but increasing penicillin resistance has been reported (9, 13). Meanwhile, oxacillin susceptibility breakpoints for S. lugdunensis were changed in 2005 (3), and oxacillin disc breakpoints were revised in 2005 and subsequently replaced by cefoxitin disc breakpoints in 2006 (2). The current reference method for the identification of coagulase-negative staphylococci (1) is labor-intensive. Screening for S. lugdunensis by detecting clumping factor (15, 21) or using a limited number of biochemical tests (5, 12, 15) has been proposed, but these methods have been evaluated only against small numbers of S. lugdunensis isolates.The aims of this study were to evaluate the use of simple screening strategies for the identification of S. lugdunensis, to comprehensively describe the microbiological characteristics of S. lugdunensis, and to evaluate the antibiotic susceptibility of clinical isolates.The accuracy of three abbreviated identification protocols and two commercial identification kits (ID 32 Staph and Vitek ID-GP; both from bioMérieux, France) was evaluated against a collection of coagulase-negative staphylococci that were isolated from clinical samples. Isolates from the collection were identified using a panel of 30 phenotypic and biochemical tests (1) and included Staphylococcus capitis (n ϭ 7), Staphylococcus caprae (n ϭ 5), Staphylococcus cohnii (n ϭ 2), Staphylococcus epidermidis (n ϭ 14), Staphylococcus haemolyticus (n ϭ 24), Staphylococcus hominis (n ϭ 3), Staphylococcus lugdunensis (n ϭ 46), Staphylococcus saprophyticus (n ϭ 2), and Staphylococcus warneri (n ϭ 3). The identification of 10 isolates (belonging to S. haemolyticus, S. cohnii, S. epidermidis, and S. lugdunensis) was further confirmed by sequencing a 457-bp sequence of the 16S rRNA gene (8). For the three abbreviated identification protocols, isolates were tested for the presence of pyrrolidonyl arylamidase (PYR) (Oxoid, United Kingdom), ornithine decarboxylase (BioMedia, Malaysia), and urease (BioMedia, Malaysia); trehalose utilization; alkaline phosphatase activity; and mannose utilization (the last three substances were from Rosco, Denmark). In the first identification protocol, isolates that were positive for both PYR and ornithine decarboxylase were identified as S...

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Superresolution microscopy enables probing of cellular ultrastructures. However, its widespread applications are limited by the need for expensive machinery, specific hardware, and sophisticated data processing. Expansion microscopy (ExM) improves the resolution of conventional microscopy by physically expanding biological specimens before imaging and currently provides ~70-nm resolution, which still lags behind that of modern superresolution microscopy (~30 nm). Here, we demonstrate a ninefold swelling (NIFS) hydrogel, that can reduce ExM resolution to 31 nm when using regular traditional microscopy. We also design a detachable chip that integrates all the experimental operations to facilitate the maximal reproducibility of this high-resolution imaging technology. We demonstrate this technique on the superimaging of nuclear pore complex and clathrin-coated pits, whose structures can hardly be resolved by conventional microscopy. The method presented here offers a universal platform with superresolution imaging to unveil cellular ultrastructural details using standard conventional laboratory microscopes.