scholarly journals Heterogeneity among Isolates Reveals that Fitness in Low Oxygen Correlates withAspergillus fumigatusVirulence

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Caitlin H. Kowalski ◽  
Sarah R. Beattie ◽  
Kevin K. Fuller ◽  
Elizabeth A. McGurk ◽  
Yi-Wei Tang ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Experimental Evolution ◽  
Invasive Pulmonary Aspergillosis ◽  
Clinical Isolates ◽  
Low Oxygen ◽  
Single Strain ◽  
Hypoxia Exposure ◽  
Significant Difference

ABSTRACTPrevious work has shown that environmental and clinical isolates ofAspergillus fumigatusrepresent a diverse population that occupies a variety of niches, has extensive genetic diversity, and exhibits virulence heterogeneity in a number of animal models of invasive pulmonary aspergillosis (IPA). However, mechanisms explaining differences in virulence amongA. fumigatusisolates remain enigmatic. Here, we report a significant difference in virulence of two common lab strains, CEA10 and AF293, in the murine triamcinolone immunosuppression model of IPA, in which we previously identified severe low oxygen microenvironments surrounding fungal lesions. Therefore, we hypothesize that the ability to thrive within these lesions of low oxygen promotes virulence ofA. fumigatusin this model. To test this hypothesis, we performedin vitrofitness andin vivovirulence analyses in the triamcinolone murine model of IPA with 14 environmental and clinical isolates ofA. fumigatus. Among these isolates, we observed a strong correlation between fitness in low oxygenin vitroand virulence. In further support of our hypothesis, experimental evolution of AF293, a strain that exhibits reduced fitness in low oxygen and reduced virulence in the triamcinolone model of IPA, results in a strain (EVOL20) that has increased hypoxia fitness and a corresponding increase in virulence. Thus, the ability to thrive in low oxygen correlates with virulence ofA. fumigatusisolates in the context of steroid-mediated murine immunosuppression.IMPORTANCEAspergillus fumigatusoccupies multiple environmental niches, likely contributing to the genotypic and phenotypic heterogeneity among isolates. Despite reports of virulence heterogeneity, pathogenesis studies often utilize a single strain for the identification and characterization of virulence and immunity factors. Here, we describe significant variation betweenA. fumigatusisolates in hypoxia fitness and virulence, highlighting the advantage of including multiple strains in future studies. We also illustrate that hypoxia fitness correlates strongly with increased virulence exclusively in the nonleukopenic murine triamcinolone immunosuppression model of IPA. Through an experimental evolution experiment, we observe that chronic hypoxia exposure results in increased virulence ofA. fumigatus. We describe here the first observation of a model-specific virulence phenotype correlative within vitrofitness in hypoxia and pave the way for identification of hypoxia-mediated mechanisms of virulence in the fungal pathogenA. fumigatus.

Azole-resistant and -susceptible Aspergillus fumigatus isolates show comparable fitness and azole treatment outcome in immunocompetent mice

2017 ◽  
Vol 56 (6) ◽  
pp. 703-710
Author(s):  
Michaela Lackner ◽  
Günter Rambach ◽  
Emina Jukic ◽  
Bettina Sartori ◽  
Josef Fritz ◽  
...  
Keyword(s):  
Body Weight ◽  
Aspergillus Fumigatus ◽  
Severe Disease ◽  
Weight Ratio ◽  
Clinical Parameters ◽  
Fitness Advantage ◽  
Significant Difference ◽  
Immunocompetent Mice

Abstract No data are available on the in vivo impact of infections with in vitro azole-resistant Aspergillus fumigatus in immunocompetent hosts. Here, the aim was to investigate fungal fitness and treatment response in immunocompetent mice infected with A. fumigatus (parental strain [ps]) and isogenic mutants carrying either the mutation M220K or G54W (cyp51A). The efficacy of itraconazole (ITC) and posaconazole (PSC) was investigated in mice, intravenously challenged either with a single or a combination of ps and mutants (6 × 105 conidia/mouse). Organ fungal burden and clinical parameters were measured. In coinfection models, no fitness advantage was observed for the ps strain when compared to the mutants (M220K and G54W) independent of the presence or absence of azole-treatment. For G54W, M220K, and the ps, no statistically significant difference in ITC and PSC treatment was observed in respect to fungal kidney burden. However, clinical parameters suggest that in particular the azole-resistant strain carrying the mutation G54W caused a more severe disease than the ps strain. Mice infected with G54W showed a significant decline in body weight and lymphocyte counts, while spleen/body weight ratio and granulocyte counts were increased. In immunocompetent mice, in vitro azole-resistance did not translate into therapeutic failure by either ITC or PSC; the immune system appears to play the key role in clearing the infection.

scholarly journals Combination Therapy in Treatment of Experimental Pulmonary Aspergillosis: In Vitro and In Vivo Correlations of the Concentration- and Dose- Dependent Interactions between Anidulafungin and Voriconazole by Bliss Independence Drug Interaction Analysis

2009 ◽  
Vol 53 (6) ◽  
pp. 2382-2391 ◽  
Author(s):  
Vidmantas Petraitis ◽  
Ruta Petraitiene ◽  
William W. Hope ◽  
Joseph Meletiadis ◽  
Diana Mickiene ◽  
...  
Keyword(s):  
Drug Interaction ◽  
Interaction Analysis ◽  
Invasive Pulmonary Aspergillosis ◽  
Pulmonary Aspergillosis ◽  
Treatment Groups ◽  
Synergistic Interactions ◽  
Significant Difference ◽  
Bliss Independence

ABSTRACT We studied the antifungal activity of anidulafungin (AFG) in combination with voriconazole (VRC) against experimental invasive pulmonary aspergillosis (IPA) in persistently neutropenic rabbits and further explored the in vitro and in vivo correlations by using Bliss independence drug interaction analysis. Treatment groups consisted of those receiving AFG at 5 (AFG5 group) and 10 (AFG10 group) mg/kg of body weight/day, VRC at 10 mg/kg every 8 h (VRC group), AFG5 plus VRC (AFG5+VRC group), and AFG10 plus VRC (AFG10+VRC group) and untreated controls. Survival throughout the study was 60% for the AFG5+VRC group, 50% for the VRC group, 27% for the AFG10+VRC group, 22% for the AFG5 group, 18% for the AFG10 group, and 0% for control rabbits (P < 0.001). There was a significant reduction of organism-mediated pulmonary injury, measured by infarct scores, lung weights, residual fungal burdens, and galactomannan indexes, in AFG5+VRC-treated rabbits versus those treated with AFG5 and VRC alone (P < 0.05). In comparison, AFG10+VRC significantly lowered only infarct scores and lung weights in comparison to those of AFG10-treated animals (P < 0.05). AFG10+VRC showed no significant difference in other outcome variables. Significant Bliss synergy was found in vivo between AFG5 and VRC, with observed effects being 24 to 30% higher than expected levels if the drugs were acting independently. These synergistic interactions were also found between AFG and VRC in vitro. However, for AFG10+VRC, only independence and antagonism were observed among the outcome variables. We concluded that the combination of AFG with VRC in treatment of experimental IPA in persistently neutropenic rabbits was independent to synergistic at a dosage of 5 mg/kg/day but independent to antagonistic at 10 mg/kg/day, as assessed by Bliss independence analysis, suggesting that higher dosages of an echinocandin may be deleterious to the combination.

scholarly journals Antifungal effect of all-trans retinoic acid against Aspergillus fumigatus in vitro and in a pulmonary aspergillosis in vivo model

2020 ◽  
Author(s):  
Elena Campione ◽  
Roberta Gaziano ◽  
Elena Doldo ◽  
Daniele Marino ◽  
Mattia Falconi ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Aspergillus Fumigatus ◽  
Rat Model ◽  
Fungal Infections ◽  
Invasive Pulmonary Aspergillosis ◽  
Antifungal Drugs ◽  
Pulmonary Aspergillosis ◽  
Fungistatic Activity

AIM: Aspergillus fumigatus is the most common opportunistic fungal pathogen and causes invasive pulmonary aspergillosis (IPA), with high mortality among immunosuppressed patients. Fungistatic activity of all-trans retinoic acid (ATRA) has been recently described in vitro. We evaluated the efficacy of ATRA in vivo and its potential synergistic interaction with other antifungal drugs. MATERIALS AND METHODS: A rat model of IPA and in vitro experiments were performed to assess the efficacy of ATRA against Aspergillus in association with classical antifungal drugs and in silico studies used to clarify its mechanism of action. RESULTS: ATRA (0.5 and 1 mM) displayed a strong fungistatic activity in Aspergillus cultures, while at lower concentrations, synergistically potentiated fungistatic efficacy of sub-inhibitory concentration of Amphotericin B (AmB) and Posaconazole (POS). ATRA also enhanced macrophagic phagocytosis of conidia. In a rat model of IPA, ATRA reduced mortality similarly to Posaconazole. CONCLUSION: Fungistatic efficacy of ATRA alone and synergistically with other antifungal drugs was documented in vitro, likely by inhibiting fungal Hsp90 expression and Hsp90-related genes. ATRA reduced mortality in a model of IPA in vivo. Those findings suggest ATRA as suitable fungistatic agent, also to reduce dosage and adverse reaction of classical antifungal drugs, and new therapeutic strategies against IPA and systemic fungal infections.

scholarly journals In Vivo Analysis of Aspergillus fumigatus Developmental Gene Expression Determined by Real-Time Reverse Transcription-PCR

2008 ◽  
Vol 76 (8) ◽  
pp. 3632-3639 ◽  
Author(s):  
Fabrice N. Gravelat ◽  
Thomas Doedt ◽  
Lisa Y. Chiang ◽  
Hong Liu ◽  
Scott G. Filler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Aspergillus Fumigatus ◽  
Real Time ◽  
Reverse Transcription ◽  
Invasive Pulmonary Aspergillosis ◽  
Pcr Analysis ◽  
Invasive Infection ◽  
Reverse Transcription Pcr

ABSTRACT Very little is known about the developmental stages of Aspergillus fumigatus during invasive aspergillosis. We performed real-time reverse transcription-PCR analysis on lung samples from mice with invasive pulmonary aspergillosis to determine the expression of A. fumigatus genes that are expressed at specific stages of development. In established infection, A. fumigatus exhibited mRNA expression of genes specific to developmentally competent hyphae, such as stuA. In contrast, mRNA of genes expressed by conidia and precompetent hyphae was not detected. Many genes required for mycotoxin synthesis, including aspHS, gliP, mitF, and metAP, are known to be expressed by developmentally competent hyphae in vitro. Interestingly, each of these genes was expressed at significantly higher levels during invasive infection than in vitro. The expression of gliP mRNA in vitro was found to be highly dependent on culture conditions. Furthermore, gliP expression was found to be dependent on the transcription factor StuA both in vitro and in vivo. Therefore, developmentally competent hyphae predominate during established invasive infection, and many mycotoxin genes are expressed at high levels in vivo. These results highlight the importance of the evaluation of putative virulence factors expressed by competent hyphae and analysis of gene expression levels during invasive infection rather than in vitro alone.

scholarly journals Establishing In Vitro-In Vivo Correlations for Aspergillus fumigatus: the Challenge of Azoles versus Echinocandins

2008 ◽  
Vol 52 (10) ◽  
pp. 3504-3511 ◽  
Author(s):  
Maiken Cavling Arendrup ◽  
Susanne Perkhofer ◽  
Susan J. Howard ◽  
Guillermo Garcia-Effron ◽  
Aimanianda Vishukumar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Aspergillus Fumigatus ◽  
Expression Profiling ◽  
Susceptibility Testing ◽  
Northern Blotting ◽  
Clinical Isolates ◽  
Susceptible Control ◽  
Polysaccharide Composition

ABSTRACT Two clinical isolates of Aspergillus fumigatus, designated AT and DK, were recently obtained from patients failing caspofungin and itraconazole therapy, respectively. The isolates were tested by microdilution for susceptibility to itraconazole, voriconazole, posaconazole, ravuconazole, and caspofungin and by Etest for susceptibility to amphotericin B and caspofungin. Susceptibility testing documented that the DK isolate was azole resistant (itraconazole and posaconazole MICs, >4 μg/ml; voriconazole MIC, 2 μg/ml; ravuconazole MIC, 4 μg/ml), and the resistance was confirmed in a hematogenous mouse model, with mortality and the galactomannan index as the primary and secondary end points. Sequencing of the cyp51A gene revealed the M220K mutation, conferring multiazole resistance. The Etest, but not microdilution, suggested that the AT isolate was resistant to caspofungin (MIC, >32 μg/ml). In the animal model, this isolate showed reduced susceptibility to caspofungin. Sequencing of the FKS1 gene revealed no mutations; the enzyme retained full sensitivity in vitro; and investigation of the polysaccharide composition showed that the β-(1,3)-glucan proportion was unchanged. However, gene expression profiling by Northern blotting and real-time PCR demonstrated that the FKS gene was expressed at a higher level in the AT isolate than in the susceptible control isolate. To our knowledge, this is the first report to document the presence of multiazole-resistant clinical isolates in Denmark and to demonstrate reduced susceptibility to caspofungin in a clinical A. fumigatus isolate with increased expression of the FKS gene. Further research to determine the prevalence of resistance in A. fumigatus worldwide, and to develop easier and reliable tools for the identification of such isolates in routine laboratories, is warranted.

scholarly journals 2599. Studying the Effects of Altering Histone Modification on Aspergillus fumigatus Virulence

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S903-S903
Author(s):  
Pam Lee ◽  
Hong Liu ◽  
Scott Filler
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Histone Modification ◽  
Histone Deacetylase ◽  
Cell Damage ◽  
Invasive Pulmonary Aspergillosis ◽  
Epithelial Cell Line ◽  
Saga Complex

Abstract Background As there are few drugs for treating invasive aspergillosis, there is an urgent need for new antifungal agents. Enzymes involved in histone modification are possible antifungal drug targets. We set out to investigate whether genes whose products are involved in histone modifications influence the virulence of Aspergillus fumigatus (Af). Methods Genes whose products were likely involved in histone modification were deleted in strain Af293 using CRISPR-Cas9. Virulence was assessed in a triamcinolone-treated mouse model of invasive pulmonary aspergillosis. The extent of Af-induced damage to the A549 pulmonary epithelial cell line was determined by Cr51 release assay. Results Af genes were selected for investigation based on their homology to genes encoding known histone modifying proteins and their high expression level in vivo. The genes were predicted to encode members of the COMPASS histone methyltransferase complex (cclA/bre2, set2/Afu5g06000), the SAGA histone acetyltransferase complex (spt3, spt8), and the RPDL histone deacetylase complex (hosA). The ΔcclA and Δset2 mutants had significant growth defects on rich media and were not tested further. The Δspt3 and Δspt8 mutants grew normally and had mild conidiation defects. The ΔhosA mutant had wild-type (WT) growth and conidiation in vitro. Mice infected with the WT strain had 100% mortality within 9 days whereas mice infected the Δspt3, Δspt8, and ΔhosA mutants had only 40% mortality by 21 days. The ΔhosA mutant also had impaired capacity to damage pulmonary epithelial cells in vitro. Conclusion Ccla and Set2, components of the COMPASS complex, are required for normal growth in vitro. Spt3 and Spt8, members of the SAGA complex, are required for normal conidiation and virulence. HosA, part of the RPD3L complex, is necessary for maximal virulence and induction of host cell damage. Our results suggest that the HosA histone deacetylase may be a promising drug target for treating invasive aspergillosis. Disclosures All authors: No reported disclosures.

scholarly journals TLR7 expression aggravates invasive pulmonary Aspergillosis by suppressing anti-Aspergillus immunity of macrophages

2021 ◽  
Author(s):  
Banglao Xu ◽  
Qin Luo ◽  
Yi Gong ◽  
Jiaxi Li ◽  
Ju Cao
Keyword(s):  
Aspergillus Fumigatus ◽  
Knockout Mice ◽  
Tissue Injury ◽  
Critical Role ◽  
Invasive Pulmonary Aspergillosis ◽  
Negative Regulator ◽  
Immune Recognition ◽  
Wild Type

Toll-like receptors (TLRs) play a critical role in early immune recognition of Aspergillus, which can regulate host defense during invasive pulmonary Aspergillosis (IPA). However, the role of TLR7 in the pathogenesis of IPA remains unknown. In this study, an in vivo model of IPA was established to investigate the contribution of TLR7 to host anti-Aspergillus immunity upon invasive pulmonary Aspergillus fumigatus infection. The effects of TLR7 on phagocytosis and killing capacities of A. fumigatus by macrophages and neutrophils were investigated in vitro. We found that TLR7 knockout mice exhibited lower lung inflammatory response and tissue injury, higher fungal clearance, and greater survival in an in vivo model of IPA as compared with wild-type mice. TLR7 activation by R837 ligand led wild-type mice more susceptible to invasive pulmonary Aspergillus fumigatus infection. Macrophages, but not neutrophils, were required for the protection against IPA observed in TLR7 knockout mice. Mechanistically, TLR7 impaired phagocytosis and killing of A. fumigatus by macrophages, but not neutrophils. Together, these data identify TLR7 as an important negative regulator of anti-Aspergillus innate immunity in IPA, and we propose that targeting TLR7 may be beneficial in treatment of IPA.

scholarly journals Analysis of Virulence of Clinical Isolates ofSalmonella enteritidis In Vivo and In Vitro

1999 ◽  
Vol 67 (11) ◽  
pp. 5651-5657 ◽  
Author(s):  
Sangwei Lu ◽  
Amee R. Manges ◽  
Yisheng Xu ◽  
Ferric C. Fang ◽  
Lee W. Riley
Keyword(s):  
Clinical Isolates ◽  
In Vitro Assays ◽  
Complex Nature ◽  
Phage Types ◽  
Food Borne ◽  
Oral Inoculation ◽  
Wide Range ◽  
Significant Difference

ABSTRACT Salmonella enterica serotype Enteritidis (S. enteritidis) is a major food-borne pathogen, and its incidence among all Salmonella serotypes has increased dramatically in the last two decades. To study the virulence characteristics of clinical isolates of S. enteritidis, we determined the 50% lethal doses (LD50) in mice of isolates of two major phage types (4 and 8). Isolates of both phage types showed a wide range of LD50 after oral inoculation, varying from under 102 organisms to over 108 organisms. No significant difference in LD50 was observed between the phage types. These observations indicated that clinical isolates ofS. enteritidis are highly heterogeneous in their ability to cause death in mice. We compared the LD50s of these isolates to the results observed from in vitro pathogenicity assays. We also analyzed these isolates for recognized Salmonellavirulence loci (spv, sodCI, sopE, and sef). The in vitro phenotypes of the isolates showed no obvious correlation with their LD50 in any given assay, and the virulence genes tested were present in all isolates. However, the isolate with the lowest LD50 (isolate 97A 2472) was resistant to acidified sodium nitrite (ASN). Moreover, the most acid-susceptible, macrophage-susceptible, and ASN-susceptible isolates were attenuated for virulence in mice. These results, based on extensive analysis of clinical isolates of S. enteritidis, demonstrate the complex nature of Salmonella pathogenesis in mice. Our results also indicate the limitation of in vitro assays in predicting in vivo virulence.

scholarly journals Defining Fractional Inhibitory Concentration Index Cutoffs for Additive Interactions Based on Self-Drug Additive Combinations, Monte Carlo Simulation Analysis, and In Vitro-In Vivo Correlation Data for Antifungal Drug Combinations against Aspergillus fumigatus

2009 ◽  
Vol 54 (2) ◽  
pp. 602-609 ◽  
Author(s):  
Joseph Meletiadis ◽  
Spyros Pournaras ◽  
Emmanuel Roilides ◽  
Thomas J. Walsh
Keyword(s):  
Monte Carlo Simulation ◽  
Monte Carlo ◽  
Aspergillus Fumigatus ◽  
Inhibitory Concentration ◽  
Simulation Analysis ◽  
Invasive Pulmonary Aspergillosis ◽  
Drug Combinations ◽  
Fractional Inhibitory Concentration

ABSTRACT The fractional inhibitory concentration (FIC) index range of 0.5 to 4 that is commonly used to define additivity results in no interactions in most combination studies of antifungal agents. These results may differ from those of in vivo studies, where positive and negative interactions may be observed. We reassessed this in vitro FIC index range based on (i) the experimental variation of the checkerboard technique using multiple replicates, (ii) the ability to correctly determine purely additive self-drug and two-drug antagonistic combinations of amphotericin B (AMB) and voriconazole (VRC), (iii) Monte Carlo simulation analysis, and (iv) in vitro-in vivo correlation using experimental models of invasive pulmonary aspergillosis against the same Aspergillus fumigatus isolate based on visual, spectrophotometric, and colorimetric determinations of FICs after 24 and 48 h of incubation. FICs obtained after 24 h of incubation ranged from 0.5 to 1.25 for the self-drug additive combinations of AMB plus AMB and VRC plus VRC and from 2.25 to 4.25 for the antagonistic combination of AMB plus VRC. Monte Carlo simulation analysis showed that self-drug combinations were correctly classified as additive and that the combination of AMB plus VRC was correctly classified as antagonistic for >85% of the simulated FICs when deviation of the 95% confidence interval (CI) of replicate FICs from the additivity range of 1 to 1.25 was used to assess interactions after 24 h. In vitro-in vivo correlation analysis showed that the 95% CIs of the FICs of the in vivo synergistic combination anidulafungin plus VRC determined after 24 h were lower than 1 and the 95% CIs of the FICs of the in vivo antagonistic combination AMB plus ravuconazole were higher than 1.25. Adequate insight into weak pharmacodynamic interactions with in vivo relevance may be obtained by demonstrating that triplicate FICs at 24 h are outside an inclusive additivity range of 1 to 1.25.

scholarly journals Resistance to amikacin and isepamicin in rabbits with experimental endocarditis of an aac(6')-Ib-bearing strain of Klebsiella pneumoniae susceptible in vitro.

1996 ◽  
Vol 40 (12) ◽  
pp. 2848-2853 ◽  
Author(s):  
E Caulin ◽  
A Coutrot ◽  
C Carbon ◽  
E Collatz
Keyword(s):  
Klebsiella Pneumoniae ◽  
Clinical Isolates ◽  
Low Level ◽  
Susceptibility Data ◽  
Resistance Determinants ◽  
Significant Difference ◽  
Bactericidal Effects ◽  
High Level

The effect of production of the aminoglycoside 6'-N-acetyltransferase [AAC(6')-IB] in Klebsiella pneumoniae on the outcome of amikacin and isepamicin treatment of rabbits with experimental endocarditis was assessed. Isogenic high-level (Hi) and low-level (Lo) AAC(6')-Ib-producing transconjugants (T) were constructed from clinical isolates with plasmid-borne resistance determinants. The MICs of amikacin and isepamicin, their bactericidal effects, and AAC(6')-Ib production appeared to be well correlated among the clinical isolates and the transconjugants. The susceptibility data determined in vitro, with MICs (in micrograms per milliliter) of amikacin and isepamicin for LoT and HiT of 4 and 0.5 and 32 and 8, respectively, were, however, not predictive of the in vivo efficacies of the drugs. While amikacin and isepamicin caused reductions in bacterial densities (log10 CFU per gram of cardiac vegetation) of 5.1 and 4.8 of the fully susceptible recipient strain (MICs of amikacin and isepamicin, 0.5 and 0.25, respectively), the reductions in density of both LoT and HiT caused by the two drugs (2.7 and 2.4 and 2.9 and 2.2, respectively) were only marginally significant, if at all. There was no significant difference (P > 0.05) when the reductions in density of LoT and HiT by either drug were compared or when the efficacies of the two drugs in reducing the density of any strain [non-AAC(6')-producing, LoT, or HiT] were compared (P > 0.5). It is concluded that AAC(6')-Ib in K.pneumoniae, even when produced at a low level and not conferring resistance to amikacin and isepamicin in vitro, compromises the efficacies of both drugs in vivo and possibly does so beyond the experimental model studied here.

Sign in / Sign up

Export Citation Format

Share Document