Species distribution and antifungal susceptibility of clinical Aspergillus isolates: A multicentre study in Taiwan, 2016-2020.

BACKGROUND: Epidemiological knowledge is important to guide antifungal therapy. OBJECTIVE: This multicentre study aimed to investigate the species distribution and antifungal susceptibility of Aspergillus isolates in Taiwan. METHOD: Four hundred and ninety-two clinical Aspergillus isolates, collected during 2016-2020, were identified by calmodulin sequencing and tested for antifungal susceptibility using CLSI M38-A3. The Cyp51A sequences of azole-resistant Aspergillus fumigatus and Aspergillus flavus isolates were analysed. RESULTS: This collection comprised 30 species from eight Aspergillus sections-Flavi (33.5%), Nigri (26.0%), Fumigati (24.2%), Terrei (10.0%), Nidulantes (5.1%), Circumdati (0.8%), Restricti (0.2%) and Aspergillus (0.2%). Sections Fumigati, Flavi and Terrei were primarily represented by A. fumigatus (99.2%), A. flavus (95.8%) and A. terreus (100%), respectively. Section Nigri comprised nine species, mostly A. welwitschiae (60.2%), A. niger (12.5%), A. brunneoviolaceus (10.9%) and A. tubingensis (10.2%). A. fumigatus (39.6%) and A. flavus (26.4%) predominated among 53 isolates from lower respiratory samples, whereas section Nigri species (46.2%) and A. terreus (29.2%) predominated among 65 isolates from ear samples. Reduced susceptibility to amphotericin B (minimal inhibitory concentration (MIC) > 1 µg/mL) was noted in A. flavus (7.0%), A. terreus (6.1%), A. nidulans and section Circumdati (A. flocculosus, A. subramanianii and A. westerdijkiae) isolates. Acquired azole resistance was observed in seven A. fumigatus (5.9%), all of which carried TR34 /L98H or TR34 /L98H/S297T/F495I mutation, and three A. flavus (1.9%), one of which carried G441S mutation. Reduced susceptibility to itraconazole (MIC >1 µg/mL) was noted in 55.5% of section Nigri isolates, mainly in A. welwitschiae, A. niger and A. tubingensis, whereas A. brunneoviolaceus, A. aculeatinus and A. japonicus were hypersusceptible to azoles. Anidulafungin was active against all isolates except for one isolate. CONCLUSIONS: This study depicted the molecular epidemiology and species-specific characteristics of Aspergillus in Taiwan, which aids in appropriate antifungal therapy and underlines the need of speciation and susceptibility testing of disease-causing Aspergillus.

Clinical and Microbiological Characteristics of Culture-Positive, Influenza-Associated Pulmonary Aspergillosis: A Single-Center Study in Southern Taiwan, 2016-2019.

This study delineated the characteristics of 24 (11.2%) culture-positive, influenza-associated pulmonary aspergillosis (IAPA) patients out of 215 patients with severe influenza during 2016-2019 in a medical center in southern Taiwan. Twenty (83.3%) patients did not have EORTC/MSG-defined host factors. The mean time from influenza diagnosis to Aspergillus growth was 4.4 days, and 20 (83.3%) developed IAPA within seven days after influenza diagnosis. All patients were treated in intensive care units and all but one (95.8%) received mechanical ventilation. Aspergillus tracheobronchitis was evident in 6 (31.6%) of 19 patients undergoing bronchoscopy. Positive galactomannan testing of either serum or bronchoalveolar lavage was noted in all patients. On computed tomography imaging, IAPA was characterized by peribronchial infiltrates, multiple nodules, and cavities superimposed on ground-glass opacities. Pure Aspergillus growth without bacterial co-isolation in culture was found in 17 (70.8%) patients. A. fumigatus (15, 62.5%), A. flavus (6, 25.0%), and A. terreus (4, 16.7%) were the major causative species. Three patients had mixed Aspergillus infections due to two species, and two had mixed azole-susceptible and azole-resistant A. fumigatus infection. All patients received voriconazole with an all-cause mortality of 41.6%. Of 14 survivors, the mean duration of antifungal use was 40.5 days. In conclusion, IAPA is an early and rapidly deteriorating complication following influenza that necessitates clinical vigilance and prompt diagnostic workup.

Multicenter Study of Azole-Resistant Aspergillus fumigatus Clinical Isolates, Taiwan1.

In a multicenter study, we determined a prevalence rate of 4% for azole-resistant Aspergillus fumigatus in Taiwan. Resistance emerged mainly from the environment (TR34/L98H, TR34/L98H/S297T/F495I, and TR46/Y121F/T289A mutations) but occasionally during azole treatment. A high mortality rate observed for azole-resistant aspergillosis necessitates diagnostic stewardship in healthcare and antifungal stewardship in the environment.

Comparison of the Sensititre YeastOne and CLSI M38-A2 Microdilution Methods in Determining the Activity of Amphotericin B, Itraconazole, Voriconazole, and Posaconazole against Aspergillus Species.

This study compared the YeastOne and reference CLSI M38-A2 broth microdilution methods for antifungal susceptibility testing of Aspergillus species. The MICs of antifungal agents were determined for 100 Aspergillus isolates, including 54 Aspergillus fumigatus (24 TR34/L98H isolates), 23 A. flavus, 13 A. terreus, and 10 A. niger isolates. The overall agreement (within 2 2-fold dilutions) between the two methods was 100%, 95%, 92%, and 90% for voriconazole, posaconazole, itraconazole, and amphotericin B, respectively. The voriconazole geometric mean (GM) MICs were nearly identical for all isolates using both methods, whereas the itraconazole and posaconazole GM MICs obtained using the YeastOne method were approximately 1 dilution lower than those obtained using the reference method. In contrast, the amphotericin B GM MIC obtained using the YeastOne method was 3.3-fold higher than that observed using the reference method. For the 24 A. fumigatus TR34/L98H isolates assayed, the categorical agreement (classified according to the CLSI epidemiological cutoff values) was 100%, 87.5%, and 83.3% for itraconazole, voriconazole, and posaconazole, respectively. For four A. niger isolates, the itraconazole MICs were >8 µg/ml using the M38-A2 method due to trailing growth, whereas the corresponding itraconazole MICs obtained using the YeastOne method were all ≤0.25 µg/ml without trailing growth. These data suggest that the YeastOne method can be used as an alternative for azole susceptibility testing of Aspergillus species and for detecting the A. fumigatus TR34/L98H isolates but that this method fails to detect A. niger isolates exhibiting trailing growth with itraconazole. Additionally, for isolates with azole MICs that approach or that are at susceptibility breakpoints or with high amphotericin B MICs detected using the YeastOne method, further MIC confirmation using the reference CLSI method is needed.

Prevalence, mechanisms and genetic relatedness of the human pathogenic fungus Aspergillus fumigatus exhibiting resistance to medical azoles in the environment of Taiwan.

Emerging azole resistance in Aspergillus fumigatus poses a serious threat to human health. This nationwide surveillance study investigated the prevalence and molecular characteristics of azole-resistant A. fumigatus environmental isolates in Taiwan, an island country with increasing use of azole fungicides. Of the 2760 air and soil samples screened from 2014 to 2016, 451 A. fumigatus isolates were recovered from 266 samples and 34 isolates from 29 samples displayed resistance to medical azoles (itraconazole, voriconazole or posaconazole). The resistance prevalence was 10.9% and 7.5% in A. fumigatus-positive samples and isolates respectively. Most (29, 85.3%) azole-resistant isolates harboured TR34 /L98H mutations, which were widely distributed, clustered genetically with clinical isolates, and had growth rates that were similar to those of the wild-type isolates. Microsatellite genotyping revealed both the global spread of the TR34 /L98H isolates and the occurrence of TR34 /L98H/S297T/F495I isolates belonging to local microsatellite genotypes. AfuMDR3 and atrF, two efflux transporter genes, were constitutively upregulated in two individual resistant isolates without cyp51A mutations, highlighting their potential roles in azole resistance. These results emphasize the need for periodic environmental surveillance at the molecular level in regions in which azole fungicides are applied, and agricultural fungicide management strategies that generate less selective pressure should be investigated.

Detection of reactive oxygen species during the cell cycle under normal culture conditions using a modified fixed-sample staining method.

We developed an alternative method of simultaneously monitoring the generation of reactive oxygen species (ROS) and cellular oxidative responses using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF) in fixed samples. In this study, we evaluated the ability of this method to detect ROS generation during the cell cycle under normal culture conditions using flow cytometric analyses. Among the fixatives tested, only acetone and paraformaldehyde did not alter the endogenous oxidation of the responsive dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), which is a chloromethyl derivative of H2DCFDA. Only acetone fixation followed by staining with propidium iodide was able to detect ROS generation during the cell cycle without altering DCF oxidation. Further thymidine treatment led to cell cycle arrest at the G1 phase followed by the downregulation of total intracellular ROS. Paraformaldehyde-based fixation enabled the evaluation of ROS generation by immunostaining at a different phase of the cell cycle, whereas MPM2 co-staining enabled identification of the specific mitotic phase. This study demonstrates a modified fixed-sample method that can be used to measure intracellular ROS production during the cell cycle using standard immunostaining techniques.

Autophagy facilitates cytokine-induced ICAM-1 expression.

ICAM-1 can be induced by inflammatory cytokines such as IFN-γ and TNF-α. This study investigated whether autophagy regulates ICAM-1 given that autophagy facilitates signaling of these two cytokines. Exogenous IFN-γ induced ICAM-1 in human lung epithelial A549 cells carrying wild type p53, a transcription factor reported for ICAM-1, but not in PC14PE6/AS2 (AS2) cells carrying mutated p53. However, IFN-γ also induced ICAM-1 in A549 cells with short hairpin RNA-silenced p53. No changes in IFN-γ receptor expression were observed in AS2 cells, but IFN-γ-activated Jak2/STAT1/IFN regulatory factor 1 was markedly decreased. In AS2 cells, increased levels of reactive oxygen species induced the activation of Src homology domain-containing phosphatase 2 (SHP2), while SHP2 was essential for IFN-γ resistance. AS2 cells showed autophagy resistance, and the manipulation of the autophagy pathway altered IFN-γ resistance. Aberrant Bcl-2 expression and mammalian target of rapamycin activation contributed to both autophagy resistance and IFN-γ resistance. Autophagy, but not p53, also modulated TNF-α-induced NF-κB activation and ICAM-1 expression. Inhibiting autophagy decreased the adhesion of human monocytic U937 cells to IFN-γ-treated A549 cells. These results demonstrated that IFN-γ and TNF-α induced ICAM-1 expression through a common pathway that was regulated by autophagy, but not p53.

A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses.

The generation of reactive oxygen species (ROS) in a live-cell system is routinely measured using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF). However, it is difficult to simultaneously monitor cellular oxidative responses and ROS generation in cells, and analyses of cellular oxidative responses are typically performed after ROS generation has been evaluated. In this study, we developed a modified fixed staining method that allows the simultaneous analysis of ROS generation and oxidative responses using standard immunostaining techniques. A microplate reader-based assay showed that of the fixatives tested, only methanol did not alter the hydrogen peroxide (H(2)O(2))-mediated oxidation of the responsive dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), a chloromethyl derivative of H(2)DCFDA, or the fluorescence of oxidized DCF in vitro. Further in vivo assays using flow cytometry showed that both methanol and acetic acid maintained the fluorescence of oxidized DCF in H(2)O(2)-, antimycin A-, and serum starvation-treated human lung adenocarcinoma A549 cells and human microvascular endothelial HMEC-1 cells. Following acetic acid-based fixation, the ROS generation in starved HMEC-1 cells could be evaluated by flow cytometric analysis while simultaneously monitoring the phosphorylation status of p38 mitogen-activated protein kinase. Immunostaining also revealed the synchronization of ROS generation and the H(2)O(2)-induced phosphorylation of Src homology-2 domain-containing phosphatase2. This study describes a modified method that may be used in future biomedical investigations to simultaneously measure intracellular ROS production and cellular oxidative responses.

Inhibiting glycogen synthase kinase-3 decreases 12-O-tetradecanoylphorbol-13-acetate-induced interferon-γ-mediated skin inflammation.

Glycogen synthase kinase-3 (GSK-3) facilitates interferon (IFN)-γ signaling. Because IFN-γ is involved in inflammatory skin diseases, such as psoriasis, the aim of this study was to investigate the pathogenic role of GSK-3 in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced IFN-γ-mediated ear skin inflammation. TPA (3 µg per ear) induced acute skin inflammation in the ears of C57BL/6 mice, including edema, infiltration of granulocytes but not T cells, and IFN-γ receptor 1-mediated deregulation of intercellular adhesion molecule 1 (CD54). TPA/IFN-γ induced GSK-3 activation, which in turn activated signal transducer and activator of transcription 1. Inhibiting GSK-3 pharmacologically, by administering 6-bromoindirubin-3'-oxime (1.5 µg per ear), and genetically, with lentiviral-based short-hairpin RNA, reduced TPA-induced acute skin inflammation but not T-cell infiltration. It is noteworthy that inhibiting GSK-3 decreased TPA-induced IFN-γ production and the nuclear translocation of T-box transcription factor Tbx21, a transcription factor of IFN-γ, in CD3-positive T cells. In chronic TPA-induced skin inflammation, inhibiting GSK-3 attenuated epidermis hyperproliferation and dermis angiogenesis. These results demonstrate the dual role of GSK-3 in TPA-induced skin inflammation that is not only to facilitate IFN-γ signaling but also to regulate IFN-γ production. Inhibiting GSK-3 may be a potential treatment strategy for preventing such effects.

Anesthetic propofol causes glycogen synthase kinase-3ß-regulated lysosomal/mitochondrial apoptosis in macrophages.

BACKGROUND: Overdose propofol treatment with a prolong time causes injury to multiple cell types; however, its molecular mechanisms remain unclear. Activation of glycogen synthase kinase (GSK)-3ß is proapoptotic under death stimuli. The authors therefore hypothesize that propofol overdose induces macrophage apoptosis through GSK-3ß. METHODS: Phagocytic analysis by uptake of Staphylococcus aureus showed the effects of propofol overdose on murine macrophages RAW264.7 and BV2 and primary human neutrophils in vitro. The authors further investigated cell apoptosis in vitro and in vivo, lysosomal membrane permeabilization, and the loss of mitochondrial transmembrane potential (MTP) by propidium iodide, annexin V, acridine orange, and rhodamine 123 staining, respectively. Protein analysis identified activation of apoptotic signals, and pharmacologic inhibition and genetic knockdown using lentiviral-based short hairpin RNA were further used to clarify their roles. RESULTS: A high dose of propofol caused phagocytic inhibition and apoptosis in vitro for 24 h (25 µg/ml, in triplicate) and in vivo for 6 h (10 mg/kg/h, n = 5 for each group). Propofol induced lysosomal membrane permeabilization and MTP loss while stabilizing MTP and inhibiting caspase protected cells from mitochondrial apoptosis. Lysosomal cathepsin B was required for propofol-induced lysosomal membrane permeabilization, MTP loss, and apoptosis. Propofol decreased antiapoptotic Bcl-2 family proteins and then caused proapoptotic Bcl-2-associated X protein (Bax) activation. Propofol-activated GSK-3ß and inhibiting GSK-3ß prevented Mcl-1 destabilization, MTP loss, and lysosomal/mitochondrial apoptosis. Forced expression of Mcl-1 prevented the apoptotic effects of propofol. Decreased Akt was important for GSK-3ß activation caused by propofol. CONCLUSIONS: These results suggest an essential role of GSK-3ß in propofol-induced lysosomal/mitochondrial apoptosis.

Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo.

An overdose and a prolonged treatment of propofol may cause cellular cytotoxicity in multiple organs and tissues such as brain, heart, kidney, skeletal muscle, and immune cells; however, the underlying mechanism remains undocumented, particularly in vascular endothelial cells. Our previous studies showed that the activation of glycogen synthase kinase (GSK)-3 is pro-apoptotic in phagocytes during overdose of propofol treatment. Regarding the intravascular administration of propofol, we therefore hypothesized that propofol overdose also induces endothelial cytotoxicity via GSK-3. Propofol overdose (100 µg/ml) inhibited growth in human arterial and microvascular endothelial cells. After treatment, most of the endothelial cells experienced caspase-independent necrosis-like cell death. The activation of cathepsin D following lysosomal membrane permeabilization (LMP) determined necrosis-like cell death. Furthermore, propofol overdose also induced caspase-dependent apoptosis, at least in part. Caspase-3 was activated and acted downstream of mitochondrial transmembrane potential (MTP) loss; however, lysosomal cathepsins were not required for endothelial cell apoptosis. Notably, activation of GSK-3 was essential for propofol overdose-induced mitochondrial damage and apoptosis, but not necrosis-like cell death. Intraperitoneal administration of a propofol overdose in BALB/c mice caused an increase in peritoneal vascular permeability. These results demonstrate the cytotoxic effects of propofol overdose, including cathepsin D-regulated necrosis-like cell death and GSK-3-regulated mitochondrial apoptosis, on endothelial cells in vitro and the endothelial barrier dysfunction by propofol in vivo.

Glycogen synthase kinase-3ß is critical for interferon-α-induced serotonin uptake in human Jurkat T cells.

Dysregulation of glycogen synthase kinase (GSK)-3ß contributes to the pathophysiology of mood disorders. However, how its regulation is responsible for the functioning of serotonin (5-HT) requires further investigation. Although enhancement of T-cell function may present an alternative strategy to treat depression, the precise mechanisms have yet to be established. Our previous studies have found that interferon-alpha (IFN-α) up-regulates serotonin transporter (5-HTT) expression and induces 5-HT uptake in T cells. The present study is to examine GSK-3ß regulation on IFN-α-induced 5-HTT functions. GSK-3ß short hairpin RNAs (shRNAs) or GSK-3ß inhibitors decreased IFN-α-induced 5-HT uptake and 5-HTT expression. Src activation and calcium/calcium-activated calmodulin kinase II (CaMKII) were involved in IFN-α-induced phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) (Tyr402) and GSK-3ß (Tyr216), which regulated 5-HT uptake. GSK-3ß knockdown blocked the IFN-α-induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Tyr204) and signal transducer and transactivator (STAT) 1. In addition to inhibiting ERK, a selective 5-HTT inhibitor fluoxetine blocked IFN-α-induced activations of Src, CaMKII-regulated Pyk2/GSK-3ß cascade, as well as STAT1 activation and translocation. These results indicated that calcium/CaMKII- and Src-regulated Pyk2 participated in IFN-α-induced GSK-3ß activation and GSK-3ß-regulated 5-HT uptake. GSK-3ß signaling facilitated IFN-α-activated STAT1 by regulating ERK1/2, which controlled 5-HT uptake. Fluoxetine interfered with the Pyk2/GSK-3ß cascade, thereby inhibiting IFN-α-induced 5-HT uptake.

Autophagy facilitates an IFN-γ response and signal transduction.

Autophagy, that is directly triggered by invaded pathogens and indirectly triggered by IFN-γ, acts as a defense by mediating intracellular microbial recognition and clearance. In addition, autophagy contributes to inflammation by facilitating an IFN-γ response and signal transduction. For immune escape, downregulated autophagy may be a strategy used by microbes.

Anesthetic propofol reduces endotoxic inflammation by inhibiting reactive oxygen species-regulated Akt/IKKß/NF-κB signaling.

BACKGROUND: Anesthetic propofol has immunomodulatory effects, particularly in the area of anti-inflammation. Bacterial endotoxin lipopolysaccharide (LPS) induces inflammation through toll-like receptor (TLR) 4 signaling. We investigated the molecular actions of propofol against LPS/TLR4-induced inflammatory activation in murine RAW264.7 macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Non-cytotoxic levels of propofol reduced LPS-induced inducible nitric oxide synthase (iNOS) and NO as determined by western blotting and the Griess reaction, respectively. Propofol also reduced the production of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 as detected by enzyme-linked immunosorbent assays. Western blot analysis showed propofol inhibited LPS-induced activation and phosphorylation of IKKß (Ser180) and nuclear factor (NF)-κB (Ser536); the subsequent nuclear translocation of NF-κB p65 was also reduced. Additionally, propofol inhibited LPS-induced Akt activation and phosphorylation (Ser473) partly by reducing reactive oxygen species (ROS) generation; inter-regulation that ROS regulated Akt followed by NF-κB activation was found to be crucial for LPS-induced inflammatory responses in macrophages. An in vivo study using C57BL/6 mice also demonstrated the anti-inflammatory properties against LPS in peritoneal macrophages. CONCLUSIONS/SIGNIFICANCE: These results suggest that propofol reduces LPS-induced inflammatory responses in macrophages by inhibiting the interconnected ROS/Akt/IKKß/NF-κB signaling pathways.

Macrophage migration inhibitory factor regulates interleukin-6 production by facilitating nuclear factor-kappa B activation during Vibrio vulnificus infection.

BACKGROUND: Patients infected with Vibrio vulnificus (V. vulnificus) show severe inflammatory responses characterised by the upregulation of proinflammatory cytokines. Macrophage migration inhibitory factor (MIF), an upstream proinflammatory regulator, increases the inflammation caused by sepsis. Whether MIF regulates responses to V. vulnificus infection and the actual mechanism by which V. vulnificus initiates these MIF-modulated proinflammatory cytokines remain unclear. RESULTS: MIF increased inflammation during V. vulnificus infection in vivo. In V. vulnificus-infected mice, MIF was produced earlier than tumour necrosis factor (TNF)-α and interleukin (IL)-6 and was expressed in a time-dependent manner. ISO-1 ((S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester), a small-molecule inhibitor of MIF, significantly decreased IL-6, IL-8, and TNF-α production in a time- and dose-dependent manner in human peripheral blood cells infected with V. vulnificus. The induction of IL-6, IL-8, and TNF-α production by V. vulnificus infection was mediated via the NF-κB- and p38 MAPK-regulated pathways but not via the Akt pathway. ISO-1-treated human peripheral blood cells showed lower V. vulnificus-induced NF-κB activation, IL-6 mRNA expression, and IκB phosphorylation, but they did not show lower p38 MAPK activation. CONCLUSIONS: We conclude that MIF regulates V. vulnificus-induced IL-6 production via NF-κB activation and that p38 MAPK activation in V. vulnificus infection is not MIF dependent.

Autophagy facilitates IFN-gamma-induced Jak2-STAT1 activation and cellular inflammation.

Autophagy is regulated for IFN-gamma-mediated antimicrobial efficacy; however, its molecular effects for IFN-gamma signaling are largely unknown. Here, we show that autophagy facilitates IFN-gamma-activated Jak2-STAT1. IFN-gamma induces autophagy in wild-type but not in autophagy protein 5 (Atg5(-/-))-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-gamma induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5(-/-) or Atg7(-/-) MEFs are, independent of changes in IFN-gamma receptor expression, resistant to IFN-gamma-activated Jak2-STAT1, which suggests that autophagy is important for IFN-gamma signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-gamma-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-gamma-induced activation of STAT1 in Atg5(-/-) MEFs. Our study provides evidence that there is a link between autophagy and both IFN-gamma signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation.