Mice lacking a microbiota are protected from diet-induced obesity. Previous studies have shown that feeding a Western diet causes hypothalamic inflammation, which in turn can lead to leptin resistance and weight gain. Here, we show that wild-type (WT) mice with depleted gut microbiota, i.e., germ-free (GF) and antibiotic-treated mice, have elevated levels of glucagon-like peptide-1 (GLP-1), are protected against diet-induced hypothalamic inflammation, and have enhanced leptin sensitivity when fed a Western diet. Using GLP-1 receptor (GLP-1R)deficient mice and pharmacological inhibition of the GLP-1R in WT mice, we demonstrate that intact GLP-1R signaling is required for preventing hypothalamic inflammation and enhancing leptin sensitivity. Furthermore, we show that astrocytes express the GLP-1R, and deletion of the receptor in glial fibrillary acidic protein (GFAP)-expressing cells diminished the antibiotic-induced protection against diet-induced hypothalamic inflammation. Collectively, our results suggest that depletion of the gut microbiota attenuates diet-induced hypothalamic inflammation and enhances leptin sensitivity via GLP-1R-dependent mechanisms.
Azole-containing agar is used in routine Aspergillus fumigatus azole resistance screening. We evaluated the impact of the type of plastic used to prepare in-house agar plates on the procedurés performance against A. fumigatus sensu stricto and cryptic species. A. fumigatus sensu stricto (n=91) and cryptic species (n=52) were classified as susceptible or resistant (EUCAST E.Def 9.3.2; clinical breakpoints v10). In-house azole-containing agar plates were prepared following EUCAST E.Def 10.1 on three types of multi-dish plates. We assessed the sensitivity, specificity, and agreement values of the agar plates to screen for azole resistance. Overall, sensitivity and specificity values of the agar screening method were 100% and 93.3%, respectively. The type of tray used did not affect these values. All isolates harbouring TR 34 -L98H substitutions were classified as resistant to itraconazole and voriconazole by the agar method; however, false susceptibility (very major error) to posaconazole was not uncommon and happened in isolates with posaconazole MICs of 0.25 mg/L. Isolates harbouring G54R and TR 46 -Y121F-T289A substitutions were correctly classified by the agar method as itraconazole/posaconazole resistant and voriconazole-resistant, respectively. False resistance (major error) occurred in isolates showing tiny fungal growth. Finally, agreements between both procedures against cryptic species were much lower. Azole-containing agar plates are a convenient and reliable tool to screen for resistance in A. fumigatus sensu stricto ; the type of plastic tray used minimally affects the method. On the contrary, the performance against cryptic species is rather poor.
The EUCAST 9.3.2 procedure recommends visual readings of azole and amphotericin B MICs against Aspergillus spp. Visual determination of MICs may be challenging. In this work, we aim to obtain and compare visual and spectrophotometric MICs readings of azoles and amphotericin B against A. fumigatus sensu lato isolates. Eight hundred and forty-seven A. fumigatus sensu lato isolates (A. fumigatus sensu stricto [n=828] and cryptic species [n=19]) were tested against amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole using the EUCAST EDef 9.3.2 procedure. Isolates were classified as susceptible or resistant/non-wild-type according to the 2020 updated breakpoints. The area of technical uncertainty for the azoles was defined in the updated breakpoints. Visual and spectrophotometric (fungal growth reduction >95% compared to control; read at 540 nm) MICs were compared. Essential (±1 twofold dilutions) and categorical agreements were calculated. Overall, high essential (97.1%) and categorical (99.6%) agreements were found. We obtained 100% categorical agreements for amphotericin B, itraconazole, and posaconazole and, consequently, no errors were found. Categorical agreements were 98.7% and 99.3% for voriconazole and isavuconazole, respectively. Most of misclassifications for voriconazole and isavuconazole were found to be associated with MIC results falling either in the area of technical uncertainty or in one two-fold dilutions above the breakpoint. Resistance rate was slightly lower when the MICs were obtained by spectrophotometric readings. However, all relevant cyp51A mutants were correctly classified as resistant. Spectrophotometric determination of azole and amphotericin B MICs against A. fumigatus sensu lato isolates may be a convenient alternative to visual endpoint readings.
We recently reported high essential (97.1%) and categorical (99.6%) agreements between azole and amphotericin B MICs against Aspergillus fumigatus sensu lato obtained by visual and spectrophotometric readings using a ≥ 95% fungal growth endpoint and following the EUCAST methodology (doi: 10.1128/AAC.01693-20). Here, we compared the aforementioned MICs against spectrophotometric MIC readings with a ≥ 90% inhibition endpoint. Spectrophotometric readings using either ≥ 90% or ≥ 95% fungal growth inhibition resulted in high categorical (>99.9%) agreements with visual MIC readings against A. fumigatus sensu stricto. In contrast, agreements with visual MICs against cryptic species were higher with a ≥ 95% fungal growth inhibition endpoint. Lay Summary Spectrophotometrically obtained MIC readings using either ≥ 90% or ≥ 95% fungal growth inhibition endpoints and following the EUCAST methodology are suitable against A. fumigatus sensu stricto. However, the ≥ 95% fungal growth inhibition endpoint is preferred against cryptic species.
Background Azole resistance screening in Aspergillus fumigatus isolates can be routinely carried out by using azole‐containing plates (E.Def 10.2 method), that requires filtering conidial suspensions prior inoculum adjustment. Objectives We evaluated whether skipping the filtration step of conidial suspensions negatively influences the performance of the E.Def 10.2. Patients/Methods A. fumigatus sensu stricto isolates (n = 92), classified as azole‐susceptible or azole‐resistant according to the EUCAST microdilution E.Def 9.4 method, were studied. Azole‐resistant isolates had either wild type cyp51A gene sequence (n = 3) or the TR34‐L98H (n = 26), G54R (n = 5), TR46‐Y121F‐T289A (n = 1), F46Y‐M172V‐N248T‐D255E‐E427K (n = 1), F165L (n = 1) or G448S (n = 1) cyp51A gene substitutions. In‐house azole‐containing agar plates were prepared according to the EUCAST E.Def 10.2 procedure. Conidial suspensions were obtained by adding distilled water (Tween 20 0.1%). Subsequently, the suspensions were either filtered or left unfiltered prior to inoculum adjustment to 0.5 McFarland. Using microdilution as the gold standard, agreement, sensitivity and specificity of the agar plates inoculated with two inoculums were assessed. Results Agreements for the agar screening method with either unfiltered or filtered conidial suspensions were high for itraconazole (100%), voriconazole (100%) and posaconazole (97.8%). Sensitivity (100%) and specificity (98.2%) of the procedure to rule in or out resistance when unfiltered suspensions were used were also high. Isolates harbouring the TR34‐L98H, G54R and TR46‐Y121F‐T289A substitutions were detected with the modified method. Conclusions Unfiltered conidial suspensions does not negatively influence the performance of the E.Def 10.2 method when screening for A. fumigatus sensu stricto.
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