Image_2_In vitro and in vivo evaluation of antifungal combinations against azole-resistant Aspergillus fumigatus isolates.tif

Azole resistance in Aspergillus fumigatus (Af) has become a widespread threat and a major concern for optimal management of patients with invasive aspergillosis (IA). Combination of echinocandins with azoles is an attractive alternative option for the treatment of IA due to azole-resistant Af strains. The aim of this study was to evaluate the in vitro and in vivo combination of caspofungin (CAS) with either voriconazole (VRZ) or posaconazole (PSZ). In vitro interactions were assessed by two methods, and an animal model of IA in Galleria mellonella was used for in vivo evaluation. Assessment of efficacy was based on larvae mortality. Groups of 10 larvae were infected by 3 clinical strains of Af (azole susceptible, AfS; PSZ resistant, AfR1; VRZ and PSZ resistant strain, AfR2). In vitro, combination of CAS and azoles was indifferent against AfS, and AfR2, and a synergy was found for AfR1. When compared to VRZ monotherapy, the combination of VRZ at 4 µg/larva with CAS at 4 µg/larva improved survival of AfR2-infected larvae (p=0.0066). Combination of PSZ at 4µg/larva with CAS at 4 µg/larva improved survival of AfR1-infected larvae compared to CAS (p=0.0002) and PSZ (0.0024) monotherapy. Antagonism was never observed. In conclusion, the combination of caspofungin with azoles is a promising alternative for the treatment of azole resistant strains of Af.</p

Image_1_In vitro and in vivo evaluation of antifungal combinations against azole-resistant Aspergillus fumigatus isolates.tif

Azole resistance in Aspergillus fumigatus (Af) has become a widespread threat and a major concern for optimal management of patients with invasive aspergillosis (IA). Combination of echinocandins with azoles is an attractive alternative option for the treatment of IA due to azole-resistant Af strains. The aim of this study was to evaluate the in vitro and in vivo combination of caspofungin (CAS) with either voriconazole (VRZ) or posaconazole (PSZ). In vitro interactions were assessed by two methods, and an animal model of IA in Galleria mellonella was used for in vivo evaluation. Assessment of efficacy was based on larvae mortality. Groups of 10 larvae were infected by 3 clinical strains of Af (azole susceptible, AfS; PSZ resistant, AfR1; VRZ and PSZ resistant strain, AfR2). In vitro, combination of CAS and azoles was indifferent against AfS, and AfR2, and a synergy was found for AfR1. When compared to VRZ monotherapy, the combination of VRZ at 4 µg/larva with CAS at 4 µg/larva improved survival of AfR2-infected larvae (p=0.0066). Combination of PSZ at 4µg/larva with CAS at 4 µg/larva improved survival of AfR1-infected larvae compared to CAS (p=0.0002) and PSZ (0.0024) monotherapy. Antagonism was never observed. In conclusion, the combination of caspofungin with azoles is a promising alternative for the treatment of azole resistant strains of Af.</p

Table_2_In vitro and in vivo evaluation of antifungal combinations against azole-resistant Aspergillus fumigatus isolates.docx

Azole resistance in Aspergillus fumigatus (Af) has become a widespread threat and a major concern for optimal management of patients with invasive aspergillosis (IA). Combination of echinocandins with azoles is an attractive alternative option for the treatment of IA due to azole-resistant Af strains. The aim of this study was to evaluate the in vitro and in vivo combination of caspofungin (CAS) with either voriconazole (VRZ) or posaconazole (PSZ). In vitro interactions were assessed by two methods, and an animal model of IA in Galleria mellonella was used for in vivo evaluation. Assessment of efficacy was based on larvae mortality. Groups of 10 larvae were infected by 3 clinical strains of Af (azole susceptible, AfS; PSZ resistant, AfR1; VRZ and PSZ resistant strain, AfR2). In vitro, combination of CAS and azoles was indifferent against AfS, and AfR2, and a synergy was found for AfR1. When compared to VRZ monotherapy, the combination of VRZ at 4 µg/larva with CAS at 4 µg/larva improved survival of AfR2-infected larvae (p=0.0066). Combination of PSZ at 4µg/larva with CAS at 4 µg/larva improved survival of AfR1-infected larvae compared to CAS (p=0.0002) and PSZ (0.0024) monotherapy. Antagonism was never observed. In conclusion, the combination of caspofungin with azoles is a promising alternative for the treatment of azole resistant strains of Af.</p

Formation of <i>in vitro</i> biofilm of <i>A</i>. <i>fumigatus</i>.

<p>(<b>A</b>) Kinetics of biofilm formation visualized in CLSM: 8 h (1), 12 h (2) and 24 h (3) after inoculation. (<b>B</b>) 24 h–old biofilm in SEM: general aspect of Af biofilm (1), hyphae embedded in ECM and presence of conidia (2), ECM with holes (3). ECM = extracellular matrix, C = conidia.</p

Formation of <i>in vitro</i> mixed biofilm of <i>S</i>. <i>maltophilia</i> and <i>A</i>. <i>fumigatus</i>.

<p>(<b>A</b>) Kinetics of mixed biofilm formation visualized in CLSM: 8 h (1), 12 h (2) and 24 h (3) after inoculation. (<b>B</b>) 24 h–old biofilm in SEM: general aspect of mixed biofilm (1), bacteria covering <i>A</i>. <i>fumigatus</i> hyphae and embedded in ECM (2), bacteria between hyphae and embedded in ECM (3). ECM = extracellular matrix.</p

Growth of <i>S</i>. <i>maltophilia</i> and <i>A</i>. <i>fumigatus</i> in the single and mixed biofilms.

<p>Data are expressed in log of CE or BE/mL as measured by qPCR over 24h and presented in the form of mean±SE. Sm = <i>S</i>. <i>maltophilia</i>, Af = <i>A</i>. <i>fumigatus</i>. The experiment was repeated 3 times, using 3 wells per biofilm. Results are expressed in mean±SE, * p < 0.05 compared with the single biofilms.</p

<i>A</i>. <i>fumigatus</i> and mixed biofilms thicknesses.

<p>(<b>A</b>) Means of <i>A</i>. <i>fumigatus</i> and mixed biofilms thicknesses after 24 h of culture (<b>B</b>) CLSM observations of 24 h-old biofilms thicknesses inoculated on Lab-Tek^TM slides. Sm = <i>S</i>. <i>maltophilia</i>, Af = <i>A</i>. <i>fumigatus</i>. For each biofilm, 50 measurements were taken. Results are expressed in mean±SE, * <i>p</i> < 0.0001.</p

Cell wall thickness of <i>A</i>. <i>fumigatus</i> in the single and mixed biofilms.

<p>(<b>A</b>) Observation on 24 h–old single <i>A</i>. <i>fumigatus</i> biofilm (1–2) and mixed biofilm (3–4) by TEM (<b>B</b>) Cell wall thickness of <i>A</i>. <i>fumigatus</i> measured on TEM images of the single and mixed biofilms. H = hyphae, B = bacteria, CW = cell wall, ECM = extracellular matrix, Sm = <i>S</i>. <i>maltophilia</i>, Af = <i>A</i>. <i>fumigatus</i>. For each biofilm, approximately 15 measurements on 27 hyphae were taken. Results are expressed in mean±SE, * <i>p</i> < 0.0001.</p

Formation of <i>in vitro</i> biofilm of <i>S</i>. <i>maltophilia</i> observed by SEM.

<p>(<b>A-B</b>) Single biofilm of <i>S</i>. <i>maltophilia</i> after 24 h of culture. ECM = extracellular matrix.</p

Characteristics of <i>Aspergillus fumigatus</i> in Association with <i>Stenotrophomonas maltophilia</i> in an <i>In Vitro</i> Model of Mixed Biofilm - Fig 5

<p><b>Conidiation and phenotype of <i>A</i>. <i>fumigatus</i> in the mixed biofilm visualized on SEM (A, B) and CLSM (C, D).</b> (<b>A, C</b>) 24 h-old single <i>A</i>. <i>fumigatus</i> biofilm (A’) zoom on the presence of conidial head (<b>B, D</b>) 24 h-old mixed biofilm of <i>A</i>. <i>fumigatus</i> and <i>S</i>. <i>maltophilia</i>. Grey circle represents conidial head of <i>A. fumigatus</i> which is only present in the single biofilm.</p