1EFFECT OF MEDIA MODIFIED TO MIMIC CYSTIC FIBROSIS SPUTUM ON THE 1 SUSCEPTIBILITY OF ASPERGILLUS FUMIGATUS, AND THE FREQUENCY OF 2 RESISTANCE AT ONE CENTER 3 4 David A. Stevens1,2,*, Richard B. Moss3, Cathy Hernandez3, Karl V. Clemons1,2, Marife 5 Martinez2 6 7 1Div. of Infectious Diseases and Geographic Medicine, Stanford University School of 8 Medicine, Stanford, CA; 2California Institute for Medical Research, San Jose, CA; 9 3Div. of Pulmonology, Dept. of Pediatrics, Stanford University School of Medicine, 10 Stanford, CA 11 12 *Corresponding author (California Institute for Medical Research, 2260 Clove Dr., 13 San Jose, CA 95128. Tel. 408-998-4902. Fax 408-998-2723. Email 14 stevens@stanford.edu) 15 16 17 Running head: CF sputum media and Aspergillus 18 19 Keywords: Aspergillus fumigatus, cystic fibrosis, antifungal resistance, susceptibility 20 testing21

AAC Accepted Manuscript Posted Online 25 January 2016Antimicrob. Agents Chemother. doi:10.1128/AAC.02649-15Copyright © 2016, American Society for Microbiology. All Rights Reserved.

2Abstract 22 Studies of cystic fibrosis (CF) patient exacerbations attributed to Pseudomonas 23 aeruginosa infection have indicated a lack of correlation of outcome with in vitro 24 susceptibility results. One explanation has been that media for testing do not mimic 25 the airway milieu, resulting in incorrect conclusions. Therefore media have been 26 devised to mimic CF sputum. Aspergillus fumigatus is the leading fungal pathogen in 27 CF, and susceptibility testing also is used to decide therapeutic choices. We 28 assessed whether media designed to mimic CF sputa would give different fungal 29 susceptibility results than classical methods, assaying voriconazole, the most 30 utilized anti-Aspergillus drug in this setting, and 30 CF Aspergillus isolates. The 31 frequency of marked resistance (defined as MIC >4 mcg/ml) in our CF unit by 32 classical methods is 7%. Studies with classical methods and with Digested Sputum 33 Medium, Synthetic Sputum Medium, and Artificial Sputum Medium revealed 34 prominent differences in Aspergillus susceptibility results, as well as growth rate, 35 with each medium. Clinical correlative studies are required to determine which 36 results are most useful in predicting outcome. Comparison of MICs with non-CF 37 isolates also indicated the CF isolates were generally more resistant. 38 39

3Introduction 40 41 Cystic fibrosis (CF) is a genetic disorder in epithelial anion transport. One result is 42 the development of thick respiratory secretions, associated with repeated 43 pulmonary infections and inflammation. The most common bacterium associated 44 with these infections is Pseudomonas aeruginosa. The most common fungal 45 pathogen associated with CF airways is Aspergillus fumigatus. Infection with either 46 has been associated with worsened or a more rapid decline in CF pulmonary 47 function (1-11), with the co-infected patients having the worst prognosis (9,12). In 48 addition, Aspergillus fumigatus causes an allergic bronchopulmonary disease in up 49 to 15% of CF patients (13). 50 51 An important observation made with respect to Pseudomonas aeruginosa infection 52 in CF patients has been the lack of correlation of classical in vitro susceptibility 53 testing of isolates from patients with exacerbations, with the treatment outcomes in 54 such patients (14,15). One logical explanation for that dissociation is that classical 55 in vitro susceptibility testing does not mimic the conditions present in CF airways, 56 thus leading to erroneous conclusions with respect to the activity of various 57 antibacterials. For that reason, various researchers have attempted to devise media 58 for use in the laboratory that more closely approximate the CF airways milieu, for 59 purposes of susceptibility testing, as well as studying growth, physiology, 60 mutability, etc., of P. aeruginosa in such conditions. Such media has been shown to 61 give disparate results from those obtained for P. aeruginosa using classical methods 62 (16), such as the Clinical and Laboratory Standards Institute methods & media . 63

4 64 With standardization of susceptibility testing for fungi, we wondered whether the 65 routine in vitro conditions for testing A. fumigatus, and on which therapeutic 66 antifungal decisions in CF are based, might also give divergent results from media 67 constituted to more closely approximate CF airway contents and sputum. We also 68 compared, in classical in vitro susceptibility testing, the MIC results with CF and 69 non-CF isolates. 70 71 72 73 Materials and Methods 74 75 76 Isolates. CF isolates from patient respiratory cultures were obtained after written 77 informed consent, for biobanking of the patients’ specimens and subsequent use, 78 approved by the Stanford University Institutional Review Board. Thirty A. 79 fumigatus isolates from 28 different CF patients were studied, one patient provided 80 3 different isolates at different times in the patient’s course. All were identified as A. 81 fumigatus sensu stricto (17). 82 83 Non-CF isolate susceptibility data was from requested testing in our laboratory of 84 clinical isolates of patients with invasive or suspected invasive aspergillosis. 85 86 Media. The three media described in the literature were prepared. All were 87 prepared under sterile conditions, and stored in the dark at 40C when not in use. 88 The pH of all three media was 6.8-7.0. 89

5 90 a. Digested Sputum Medium. This was originally described by Davis and Bruns (18) 91 as an equal mixture of pooled, ultrasonically homogenized, autoclaved CF sputum 92 and broth. A later modification (16) used dithiothreitol-digested sputum. We 93 obtained 10 fungus culture-negative sputa from CF outpatients in 0.3-0.5 ml 94 amounts, and pooled them. The sputa were obtained after written informed 95 consent, for biobanking of the patients’ specimens and subsequent use, approved 96 by the Stanford Institutional Review Board. Concordant with methods previously 97 described (19,20), the pool was mixed with an equal volume of 0.1% dithiothreitol 98 (Calbiochem, San Diego, CA) in normal saline, filtered through a 0.2 micron filter, 99 vortex-mixed for 3 seconds, and incubated 5 min. in a 370C water bath with 100 vigorous shaking. Further mixing occurred by aspirating up and down 20 times in a 101 pipette. The incubations and pipetting were repeated 2 more times. Any residual 102 unsolubilized material was removed by centrifugation at 800 x g for 5 min. at 40C. 103 The supernatant was then tested as 50:50, 25:75 and 10:90 mixtures with RPMI 104 1640 medium and a reference A. fumigatus isolate, strain 10AF (21), to determine 105 which mixture gave optimal A. fumigatus growth at 370C. The 10:90 mixture with 106 RPMI 1640 was then selected for further studies. A control medium was prepared 107 with equal volumes of RPMI 1640 (in lieu of sputum) and 0.1% dithiothreitol, then 108 subjected to all the steps previously described. This dithiothreitol control medium 109 thus had a final concentration of 0.005% dithiothreitol, the same as the test 110 medium. 111 112

6b. Synthetic Sputum Medium. In contrast to “a”, Palmer et al. (22) developed a 113 defined medium, based on CF sputum analyses, which nutritionally mimics CF 114 sputum. The amino acids and carbon sources are supplemented with 3-N-115 morpholino)propanesulfonic acid (MOPS) buffer to mimic the native buffering 116 system of the lung, and we followed the recipe detailed elsewhere (22). MOPS is the 117 same buffer as in the standard RPMI test medium used. 118 119 c. Artificial Sputum Medium. Sriramulu et al. developed a partially defined medium 120 with a composition that closely resembles CF sputum (23). Notable in the contents, 121 aside from the amino acids, are mucin, DNA, Tris buffer, and egg yolk emulsion 122 (source of lecithin). We prepared a filtered (0.2 micron) version of this medium as 123 detailed by Kirchner et al. (24), using herring sperm as the source of the DNA 124 (Sigma, St. Louis, MO) and egg yolk emulsion (Fisher Scientific, Hampton, NH) as 125 the source of the lecithin. 126 127 Testing. For testing with each of the three media described, MICs of three different 128 sets of 10 randomly selected CF sputum A. fumigatus isolates were determined by a 129 classical broth macrodilution method as detailed (25). Each of the 3 test media was 130 tested with a different set of the 10 isolates concurrently with the standard RPMI 131 1640 media described (25). The 3 isolates from a patient had been randomized to 2 132 different sets of 10. Minimum fungicidal concentrations (MFCs) were determined as 133 previously described (26), with the cutoff representing ≥96% killing of the initial 134 inoculum. Voriconazole was selected as the test drug, as the most recommended in 135

7therapy of aspergillosis (27), most used in the Stanford CF clinic, and because 136 differences in susceptibility among various isolates are greater with azoles than 137 polyenes (28). Isolates appearing resistant (≥4 mcg/ml) had their tests repeated. 138 The non-CF isolates had MIC testing performed by the same method, but requests 139 for MFC testing were sporadic. In every run a known voriconazole susceptible 140 isolate (Candida kefyr, isolate SA) was tested concurrently in the standard media to 141 assure drug potency. In standard and in Synthetic Sputum Medium, the isolates 142 grew to 4+ in the absence of drug in 2 days (the time of reading for them); in 143 Digested Sputum Medium it required 5 days under the culture conditions (25) to 144 obtain 4+ growth in the absence of drug (the time of reading). 145 146 Statistical analysis. In all tests, p<0.05 was considered significant. For analysis of 147 the CF isolates studied in test medium compared to standard conditions, done in 2-148 fold dilutions, the MIC data was transformed to log base 2, with MIC and MFC ≤0.5 149 and >8 mcg/ml scored as 0.5 and 16, respectively. Comparison was then done by 150 the Wilcoxon paired signed rank test (29). For comparison of the whole CF and 151 non-CF groups, a contingency Chi square was performed to test for difference in 152 distribution of the MIC values (values of 8 and >8 were combined as ≥8 mcg/ml for 153 this analysis). After demonstration of a significant distribution difference, a Fisher 154 exact test was performed at each MIC value. 155 156 157 158 159

8Results 160 161 162 Digested Sputum Medium. The results with the Digested Sputum Medium, control 163 medium and standard medium are shown in Table 1. In the absence of drug, the 164 fungal growth was noted to clump in standard medium or dithiothreitol control 165 medium, but grow as dispersed tiny balls in Digested Sputum Medium. One of 10 166 isolates (#4) was completely resistant (MIC and MFC >8 mcg/ml) to voriconazole in 167 standard testing. Dithiothreitol alone did not appear to affect the overall 168 susceptibility profile (dithiothreitol control medium), though one isolate (#9) had a 169 minimal, 2-fold, increase in MIC and MFC. 170 171 Remarkably, and unexpectedly, the Digested Sputum Medium resulted in a shift of 172 the set toward greater susceptibility to inhibition (MIC), and toward greater 173 resistance to killing (MFC). The relative positions of the isolates in terms of 174 susceptibility within the group appeared not to change, and of note, the 2 most 175 resistant isolates (#4 and #9) were always at the resistant end of the spectrum, 176 regardless of the media. However, in the Digested Sputum Medium the otherwise 177 consistently resistant isolate (#4) would have been assigned an MIC (2 mcg/ml) 178 that could be exceeded in serum with usual dosing regimens. Three isolates had an 179 unchanged MIC in Digested Sputum Medium but the other 7 decreased 1 to 3 tube 180 dilutions (p=0.009). Two had an unchanged MFC, but the other 8 had an increase of 181 1 to 4 tube dilutions (p=0.006). 182 183

9Synthetic Sputum Medium. The results with the Synthetic Sputum Medium and 184 standard medium are shown in Table 2. No unusual features of growth in the 185 absence of drug were noted. One of 10 isolates (#19) was completely resistant (MIC 186 and MFC >8 mcg/ml) to voriconazole in standard testing. Remarkably, also, the 187 Synthetic Sputum Medium resulted in a downshift of the set toward greater 188 susceptibility (MIC and MFC) compared to standard medium. Of note, the most 189 resistant isolate (#19) was resistant in both media. 190 191 Three isolates had an unchanged MIC in Synthetic Sputum Medium but the other 7 192 decreased 1 or 2 tube dilutions (p=0.009). Three isolates had an unchanged MFC in 193 Synthetic Sputum Medium but the other 7 decreased 1 or 2 tube dilutions 194 (p=0.009). 195 196 Artificial Sputum Medium. In the standard medium , all 10 isolates had MICs and 197 MFCs ≤4 mcg/ml (Table 3, “third cohort”). However, all isolates failed to grow 198 sufficiently in Artificial Sputum Medium. 199 200 Overall susceptibility of A. fumigatus in the Stanford CF population, and comparison 201 to the non-CF population. Thus, summating the above results, 2 of 30 (7%) of 202 isolates were in the highly resistant (MIC >4 mcg/ml) category, with standard 203 media and method (Table 3, top). Neither of these 2 resistant isolates came from a 204 patient who had received antifungals previously. 205 206

10Compared to the non-CF isolates, with the same standard media and method, the CF 207 isolates were more resistant (MIC) (p=0.001) (Table 3, bottom). This was because 208 of differences at the ≤0.5 mcg/ml MIC (p<0.0001) and the 4 mcg/ml MIC (p=0.02). 209 The frequency of highly resistant isolates, though almost twice as common in the CF 210 group (2/30) than the non-CF (2/55), was not statistically significant. 211 212 The 3 isolates from one patient produced 2 different susceptibility patterns. Four of 213 the 28 CF patients had allergic bronchopulmonary aspergillosis, but only one had 214 received any azole prior to the isolate we obtained. Of the other 24 CF patients, only 215 2 had received an azole (voriconazole) prior to the isolate we studied. 216 217 218 219 220 221 Discussion 222 223 To our knowledge, this is the first study of the effect of media mimicking CF sputa 224 on A. fumigatus susceptibility results. We had the opportunity to test this with A. 225 fumigatus isolates from CF patients. It may prove to be the case that tests in such 226 designed media prove more relevant to clinical outcome than standard methods, 227 but this will require correlations of in vitro results, clinical courses, and post-228 treatment cultures. This has not yet even been done with P. aeruginosa infections, 229 although studies of growth and metabolism of that bacterium in these media have 230 been performed [30,31]. Of note, the two media tested in which A. fumigatus could 231 grow appeared to make the isolates generally more susceptible to drug inhibition, 232 although Digested Sputum Medium made the isolates more resistant to killing. It 233

11will be important to discover what components of the sputum or artificial media 234 result in altered growth characteristics for the fungus, and altered drug 235 susceptibility. The explanation does not lie in pH, nor dithiothreitol (our studies 236 comparing medium with and without dithiothreitol), nor any effect from saliva that 237 may contaminate sputum samples, as Davis et al. (18) did not find saliva to affect 238 test results. It is also of interest to know why one media in which P. aeruginosa 239 grows adequately (31), inhibits growth of A. fumigatus. It is tempting to speculate 240 the principal inhibitor of fungi in Artificial Sputum Medium is mucin (32), given 241 that P. aeruginosa can use mucin as an energy source (23), and mucin would be at 242 least partially digested in Digested Sputum Medium (in which A. fumigatus grows, 243 albeit more slowly) by dithiothreitol, but present in Artificial Sputum Medium. 244 Finally, it should also be realized that even the most artfully constructed medium to 245 mimic CF sputum does not completely reconstruct the in vivo condition, where, e.g., 246 pO2, ionic composition, consistency, effects from other resident microbes, etc. may 247 be different than in in vitro conditions. 248 249 Of note, media using digested CF sputa have been tested for their effect on P. 250 aeruginosa susceptibility in vitro. Serisier et al. (16) found such medium to increase 251 the susceptibility of P. aeruginosa to antibacterial inhibition, the same effect that we 252 found with an antifungal. Davis et al. (18) found the minimum bactericidal 253 concentration to increase for some antibacterials, remarkably also the same effect 254 that we found with an antifungal, whereas there was no change in killing with some 255 other antibacterials (18). Synthetic Sputum Medium has not apparently been tested 256

12in P. aeruginosa susceptibility. Artificial Sputum Medium is reported to decrease 257 the susceptibility of P. aeruginosa to antibacterials, but we cannot compare our 258 results because A. fumigatus fails to grow sufficiently in that medium. Waters et al. 259 (15) concluded from the literature that results of susceptibility testing P. 260 aeruginosa as a biofilm, regardless of medium, did not correlate any better with 261 clinical outcome than did testing planktonic P. aeruginosa; whether this would 262 apply to A. fumigatus biofilm (33) with these media is unknown . 263 264 With the frequency of A. fumigatus infections in CF patients, and use of antifungals 265 in some in treatment, one might expect A. fumigatus drug resistance to possibly be a 266 problem. In addition to the development of resistance on therapy, there is also the 267 possibility of cross-infection with resistant A. fumigatus in the clinic setting (34). At 268 this time, there is rising azole resistance of A. fumigatus in areas of Europe, with 269 unique mutations in the azole fungal target, attributed to concomitant use of azole 270 antifungals in agricultural practice (35). Thus far, North America appears to have 271 rarely shown this pattern (35a). There are few studies of resistance of A. fumigatus 272 specifically in CF patients, all from Europe. Some have reported CF azole resistance 273 with a frequency similar to ours (34,36,37), whereas others have reported a very 274 low frequency (38-40); this will vary with the definition of resistance and the azole 275 used to define resistance. Our resistance appears unrelated to prior antifungal use, 276 as our 2 resistant isolates came from patients who had no prior antifungal therapy. 277 This finding of resistance in the absence of drug exposure, and the different 278 patterns in one patient with multiple isolates in the absence of drug exposure, are 279

13consistent with findings that A. fumigatus isolation from CF sputum may be 280 transient in many patients (34, 41-43). They may be infected transiently with a 281 resistant isolate from the environment or another patient (34). It has also been 282 demonstrated that multiple A. fumigatus genotypes may at times co-exist in the 283 same patient (44,45). The CYP51A gene of our 2 resistant isolates was sequenced, 284 and none of the described nucleic acid sequence variants associated with azole 285 resistance (46) (and presumed largely related to prior azole exposure) (35,47) 286 were found (David W. Denning, personal communication). This implies a resistance 287 based on drug efflux differences or mutations in other parts of the azole 288 susceptibility pathways. 289 290 Overall, it was also notable that MICs of CF isolates in standard medium were 291 generally and significantly higher than in non-CF isolates. This has also been 292 suggested previously (48). The frequency of marked resistance (MIC ≥8 mcg/ml) 293 was however not significantly different in both groups (2/30 vs. 2/55). We were 294 unable to compare prior antifungal experience in our CF and non-CF patients, as the 295 latter was largely unknown. Some laboratories have recommended an MIC of 4 296 mcg/ml as also resistant for voriconazole (49); this would have accentuated our 297 conclusions about resistance frequency in CF. Testing with only voriconazole could 298 have caused us to miss some isolates resistant to other azoles (other laboratories 299 have used itraconazole to screen for azole resistance), as only some azole-resistant 300 isolates are cross-resistant to all azoles. 301 302

14In summary, with the shift toward resistance in our CF population, and the lack of 303 evidence that this shift is related to azole drug pressures in the population, another 304 hypothesis is suggested. Our hypothesis is that residence in the unusual milieu of 305 the CF airways may alter drug penetration, azole target, or efflux, predisposing to 306 the small, but highly significant, population shift in voriconazole susceptibility. 307 308

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21Table 1. Digested Sputum Medium 491 492 493 Number of isolates with result in mcg/ml 494 ≤0.5 1 2 4 8 >8 Standard (RPMI) MIC 1 3 2 3^ 0 1* MFC 1 3 2 3^ 0 1* Dithiothreitol control medium MIC 1 2 2 3 1^ 1* MFC 1 2 2 3 1^ 1* Digested Sputum Medium MIC 4 3 3*^ 0 0 0 MFC 1 0 0 2 2 5*^ * includes isolate #4; ^ includes isolate #9 495 496 497 498 499

22Table 2. Synthetic Sputum Medium 500 501 502 Number of isolates with result in mcg/ml 503 ≤0.5 1 2 4 8 >8 Standard (RPMI) MIC 1 4 4 0 0 1* MFC 1 4 4 0 0 1* Synthetic Sputum Medium MIC 8 1 0 0 0 1* MFC 8 1 0 0 0 1* * includes isolate #19 504 505 506 507

23Table 3. Results of testing third CF cohort, and non-CF isolates, in standard medium 508 509 Number of isolates with result in mcg/ml 510 ≤0.5 1 2 4 8 >8 Third CF cohort- standard (RPMI)a

MIC 0 0 2 8 0 0 MFC 0 0 1 9 0 0 All CF isolates-standard (RPMI)

MIC 2 7 8 11 0 2 All non- CF isolates-standard (RPMI)

MIC 23 10 16 4 1 1 511 a. Third cohort of isolates was tested concurrently with standard medium and Artificial Sputum 512 Medium, but failed to grow sufficiently in the latter to yield endpoints. 513 514