Effect of Media Modified To Mimic Cystic Fibrosis Sputum on the Susceptibility of Aspergillus fumigatus, and the Frequency of Resistance at One Center

Abstract

c Studies of cystic fibrosis (CF) patient exacerbations attributed to Pseudomonas aeruginosa infection have indicated a lack of correlation of outcome with in vitro susceptibility results. One explanation is that the media used for testing do not mimic the airway milieu, resulting in incorrect conclusions. Therefore, media have been devised to mimic CF sputum. Aspergillus fumigatus is the leading fungal pathogen in CF, and susceptibility testing is also used to decide therapeutic choices. We assessed whether media designed to mimic CF sputa would give different fungal susceptibility results than those of classical methods, assaying voriconazole, the most utilized anti-Aspergillus drug in this setting, and 30 CF Aspergillus isolates. The frequency of marked resistance (defined as an MIC of >4 g/ml) in our CF unit by classical methods is 7%. Studies performed with classical methods and with digested sputum medium, synthetic sputum medium, and artificial sputum medium revealed prominent differences in Aspergillus susceptibility results, as well as growth rate, with each medium. Clinical correlative studies are required to determine which results are most useful in predicting outcome. Comparison of MICs with non-CF isolates also indicated the CF isolates were generally more resistant. C ystic fibrosis (CF) is a genetic disorder in epithelial anion transport. One result is the development of thick respiratory secretions, which are associated with repeated pulmonary infections and inflammation. The most common bacterium associated with these infections is Pseudomonas aeruginosa. The most common fungal pathogen associated with CF airways is Aspergillus fumigatus. Infection with either has been associated with worsened or a more rapid decline in CF pulmonary function (1-11), with coinfected patients having the worst prognosis (9, 12). In addition, Aspergillus fumigatus causes an allergic bronchopulmonary disease in up to 15% of CF patients (13).An important observation made with respect to Pseudomonas aeruginosa infection in CF patients is the lack of correlation of classical in vitro susceptibility testing of isolates from patients with exacerbations with the treatment outcomes in such patients (14,15). One logical explanation for that dissociation is that classical in vitro susceptibility testing does not mimic the conditions present in CF airways, thus leading to erroneous conclusions with respect to the activity of various antibacterials. For that reason, various researchers have attempted to devise media for use in the laboratory that more closely approximate the milieu of CF airways for purposes of susceptibility testing, as well as studying growth, physiology, mutability, etc., of P. aeruginosa under such conditions. Such media have been shown to give disparate results from those obtained for P. aeruginosa using classical methods (16), such as the methods and medium of the Clinical and Laboratory Standards Institute.With standardization of susceptibility testing for fungi, we wondered whether the routine in vitro...

Azole-Resistant Aspergillus fumigatus Among Danish Cystic Fibrosis Patients: Increasing Prevalence and Dominance of TR34/L98H

“…When we compare the present study’s result to others, the current overall azole-R rate of 7.3% in the total CF population corresponded well with CF studies from other European countries [France, Germany, the Netherlands ( Bader et al, 2013 ; Seufert et al, 2018 ; Engel et al, 2019 ; Lavergne et al, 2019 ), and the United States ( Table 1 )] ( Stevens et al, 2016 ). Internationally, published azole resistance rates have varied greatly.…”

“…Azole resistance (Azole-R) in Aspergillus fumigatus is an increasing problem and complicates patient management (Lestrade et al, 2019). Azole-R A. fumigatus in patients with cystic fibrosis (CF) has been reported in several studies (Mortensen et al, 2011a;Burgel et al, 2012;Morio et al, 2012;Bader et al, 2013;Fischer et al, 2014;Stevens et al, 2016;Prigitano et al, 2017;Abdolrasouli et al, 2018;Guegan et al, 2018;Güngör et al, 2018;Seufert et al, 2018;Engel et al, 2019;Lavergne et al, 2019; Table 1). Danish CF patients are followed up monthly at the two specialized CF clinics at Copenhagen University Hospital, Rigshospitalet (RH) and at Aarhus University Hospital (AUH).…”

Parasexual recombination enablesAspergillus fumigatusto persist in cystic fibrosis

“…Furthermore, we found evidence for a role of parasexual recombination in the development of azole resistance in A. fumigatus . Several studies have reported A. fumigatus azole resistance in CF patients, both in azole-naïve patients as well as those that have received antifungal therapy [ 13 , 34 , 35 ]. Previous azole therapy was shown to be associated with increased probability of recovery of azole-resistant isolates, and resistance rates of 20% have been reported in CF patients treated with itraconazole [ 35 ].…”

“…Although this biofilm provides shielding from stressors like other microorganisms, antifungal drugs and host immune effectors [ 12 ], as a consequence the fungus cannot benefit from asexual sporulation to adapt to the lung environment. Nevertheless, antifungal drug resistance reported in azole-treated and azole-naïve CF patients indicates that A. fumigatus may employ other strategies that enable adaptation [ 13 ]. We hypothesise that during long-term hyphal colonisation A. fumigatus adapts through parasexual recombination, a process that involves fusion of compatible hyphae and nuclei leading to mitotic recombination [ 14 , 15 ].…”

Commercial porcine gastric mucin contributes to variation in production of small molecule virulence factors byPseudomonas aeruginosawhen cultured in different formulations of artificial sputum medium

“…Pulmonary mucus is a nutritionally distinct environmental niche comprised of mucin, DNA, amino acids, lipids, and micronutrients, including metals (1,2). To study the physiology of microbial pathogens and communities in the unique CF pulmonary environment, a variety of artificial sputum media (ASM) have been developed (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), and applied to investigate microbial physiology (3,5,(15)(16)(17), biofilm morphology (10,18), antibiotic susceptibility (19,20), and interspecies interactions in the context of CF (21)(22)(23). However, disparate nutrient concentration and bioavailability between ASM formulations may affect microbial growth, metabolism, and virulence (3,7,20,24).…”