Elevated prevalence of azole resistant Aspergillus fumigatus in urban versus rural environments in the 1 United Kingdom 2 Thomas R Sewell 1 , Yuyi Zhang 1 , Amelie P Brackin, Jennifer MG Shelton, Johanna Rhodes and Matthew C 3 Fisher 4 MRC Centre for Global Infectious Disease Analysis, Department of Infectious Disease Epidemiology, 5 Imperial College London, London, UK 6 1 These authors contributed equally to this work 7 . CC-BY-NC-ND 4.0 International license certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was not this version posted April 5, 2019. . https://doi.org/10.1101/598961 doi: bioRxiv preprint
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Elevated prevalence of azole resistant Aspergillus fumigatus · 27 Introduction 28 Aspergillus fumigatus is a ubiquitous ascomycete fungus with a pan-global population distribution
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Elevated prevalence of azole resistant Aspergillus fumigatus in urban versus rural environments in the 1
United Kingdom 2
Thomas R Sewell1, Yuyi Zhang1, Amelie P Brackin, Jennifer MG Shelton, Johanna Rhodes and Matthew C 3
Fisher 4
MRC Centre for Global Infectious Disease Analysis, Department of Infectious Disease Epidemiology, 5
Imperial College London, London, UK 6
1These authors contributed equally to this work 7
.CC-BY-NC-ND 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted April 5, 2019. . https://doi.org/10.1101/598961doi: bioRxiv preprint
.CC-BY-NC-ND 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted April 5, 2019. . https://doi.org/10.1101/598961doi: bioRxiv preprint
clinical cases of aspergillosis with ARAf isolates harbouring either TR34/L98H or TR46/Y121F/T289A 50
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continue to emerge (19, 20), with one study specifically linking a fatal case of aspergillosis to a 51
genotypically indistinguishable isolate sourced from the patients own home (21). Retrospective studies 52
of patients with invasive aspergillosis (IA) and infected with azole-resistant genotypes of A. fumigatus 53
show an excess mortality of 25% at day 90 when compared against patients with wild-type-infections 54
(22). 55
56
Despite a generally good understanding of ARAf global prevalence, very few studies have investigated 57
resistance in the United Kingdom or focused sampling strategies across a diverse set of substrates. In 58
this study, we aim to determine the prevalence of ARAf in soil collected from a wide range of sites 59
across the UK's southernmost region. Sampling locations include ancient woodlands, agricultural fields, 60
tourist attractions and densely populated city centres. We show that the UK has similar prevalence rates 61
to other countries and that both TR34/L98H and TR46/Y121F/T289A can be regularly isolated from 62
urbanised locations, especially flower beds in close proximity to city centre hospitals. 63
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Seven wild-type isolates and 19 suspected azole-resistant isolates were tested for antifungal drug 86
susceptibility against three medical azoles, itraconazole (ITC), voriconazole (VRC) and posaconazole 87
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(PSC) using the European Committee Antimicrobial Susceptibility Testing (EUCAST) microdilution 88
method using MICRONAUT-AM EUCAST MIC (Minimum Inhibitory Concentration) plates (Merlin 89
Diagnostika GmbH, Bornheim, Germany). The reference wild-type clinical A. fumigatus isolates Af293 90
and an environmental azole-resistant isolate BUU09 (TR34/L98H) were used as control strains. All isolates 91
were grown for 72 hours at 37 OC before washing with 8 to 10 ml of 0.01% Tween 20. The resulting 92
conidial suspension was filtered through Whatman 1 filter paper (Whatman plc, GE Healthcare Life 93
Sciences, UK) to remove hyphae and debris. The filtered conidial suspension was adjusted to 0.5 94
McFarland with phosphate-buffered saline (PBS pH7.4 (1X), gibco, Life Technologies Limited, Paisely, UK) 95
using a BioTek ELx808 spectrophotometer (BioTek Instruments Ltd, Winooski, USA). The adjusted 96
conidial suspension (0.5 ml) was added to 9.5 ml of the MICRONAUT-RPMI medium (MICRONAUT-RPMI 97
Medium + MOPS + Glucose, Merlin Diagnostika GmbH, Bornheim, Germany) and 100 µl of the mixture 98
was then used to populate the MICRONAUT-AM test plate. All plates were examined after 48-hour 99
incubation at 37OC in a humid chamber. The MIC endpoint was read visually and determined as the 100
lowest concentrations of the azoles yielding no visible growth. The results of all susceptibility tests were 101
interpreted using EUCAST clinical breakpoints [91]. The isolates were regarded as susceptible when the 102
MIC was ≤ 1 mg/l for ITC and VRC, and ≤ 0.125 mg/l for POS; and resistant when the MIC was > 2mg/l for 103
ITC and VRC, and > 0.25 mg/l for POS. Isolates with MIC values in between the susceptible and resistant 104
breakpoint were considered to be intermediately susceptible. 105
106
DNA extraction for sequence analysis 107
Genomic DNA was extracted using a modified MasterPure Yeast DNA Purification (Lucigen Corporation, 108
Cambridge, UK) protocol which included an additional bead-beating treatment to enhance DNA yield. 109
110
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Cultured A. fumigatus isolates were washed in 10 ml of 0.05% Tween 20; 1.8 ml of the conidial 111
suspension was transferred into a 2 ml Eppendorf tube and centrifuged at 8000 rpm for 5 mins. The 112
condial pellet was mixed with 300 µl of Yeast Cell Lysis Solution and 1 µl of 5 g/l RNase A and transferred 113
into a new 2 ml Eppendorf tube containing 1.0 mm diameter Zirconia/Silica Bead (Thistle Scientific, 114
Glasgow, UK). The tube was subjected to bead beating for 3 x 45 seconds at 30 m/s in a TissueLyser II 115
(Qiagen, Hilden, Germany) and placed on ice for 2 mins before repeating. The lysate was transferred to a 116
new 1.5 Eppendorf tube and was twice centrifuged at 13000 rpm for 2 mins to pellet debris. The 117
supernatant was transferred to a 1.5 Eppendorf tube and incubated at 65OC for 15 mins before placing 118
on ice for another 30 mins. After incubation, 150 µl of MPC Protein Precipitation Reagent was added to 119
the sample and mixed by pulse vortexing for 10 seconds. For further pelleting of cellular debris, the 120
mixture was centrifuged at 13000 rpm for 2 mins and the supernatant was transferred to a clean 1.5 121
Eppendorf tube. DNA was precipitated by adding 500 µl of isopropanol (SIGMA, Sigma-Aldrich Chemie 122
Gmbh, Steinheim, Germany) to the supernatant before 4OC centrifugation at 14000 rpm for 10 mins. The 123
supernatant was removed, and the remaining DNA pellet washed with 500 µl of 70% ethanol (SIGMA, 124
Sigma-Aldrich Chemie Gmbh, Steinheim, Germany) before centrifugation at 13000 rpm for 2 mins. After 125
removing the ethanol, the DNA pellet was air-dried for approximately 10-15 mins in a biosafety cabinet. 126
Finally, 40 µl of nuclease-free water was added to the DNA pellet and left to resuspend at 4OC for 24 127
hours. The DNA was either used immediately or stored at -20OC until required. DNA yield was quantified 128
using a Qubit 2.0 Fluorometer (Invitrogen by Thermo Fisher Scientific corporation, Massachusetts, USA) 129
and the Qubit dsDNA BR (Broad-Range) Assay Kit (Invitrogen) according to the products guidelines. 130
131
PCR amplification 132
PCR was used to amplify part of the cyp51A gene and promoter, the csp1 region and beta-tubulin as 133
previously described [45], [80]. Amplification of cyp51A was performed using the L98HR primer (5′-134
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and 1 µl of DNA template. Amplification conditions were: a 5 mins denaturation step at 95OC, followed 150
by 35 cycles of 30 seconds at 95OC, 30 seconds at 55OC, and 1 min at 72OC. A final elongation step for 5 151
mins at 72OC was followed after the last cycle. 152
153
PCR product visualisation, purification and sequencing 154
Both cyp51A gene and csp1 gene PCR products were visualised on a 2% agarose gel using agarose gel 155
electrophoresis. 100 ml of 2% agarose solution was prepared by mixing 2 g of agarose powder with 100 156
ml of TBE buffer. 10 µl of SafeView Nucleic Acid Stain was added for visualisation of the PCR product in 157
the gel. Before loading the samples and molecular weight ladder (Quick-load Purple 50 bp DNA Ladder 158
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and Quick-load Purple 1 kb DNA Ladder, New England Biolabs Inc, UK) to the gel, 1 µl of Gel Loading Dye 159
was mixed with 5 µl of each sample. Finally, the gel was run at 120 to 130 V for approximately 45 to 60 160
mins. The DNA fragments were visualised using a G:Box Gel Image Analysis System (Syngene UK, 161
Cambridge, UK). The PCR products were then purified using ExoSAP-IT PCR Product Cleanup Reagent 162
(ThermoFisher Scientific, Massachusetts, USA) following the product guidelines. Briefly, 7.5 µl of each 163
post-PCR reaction product was mixed with 3 µl of the reagent. The mixture was incubated for 15 mins at 164
37OC in order to degrade the remaining nucleotides and primers in the PCR product, followed by 165
incubation of 15 mins at 80OC to inactivate the reagent. The treated products were then stored at -20OC 166
until required. The treated PCR products and sequencing primers, L98HR, TR34R, CSP1F and CSP1R were 167
prepared for sequencing following the DNA Sanger sequencing sample submission guidelines provided 168
by GENEWIZ (GENEWIZ UK LTD, Takeley, UK). They were then sent away for Sanger sequencing by 169
GENEWIZ UK laboratory. 170
171
Sanger sequencing of cyp51A mutations, csp1 types and beta-tubulin 172
Sanger sequencing results from all selected isolates were trimmed and assembled using CLC Main 173
Workbench 8.0.1 software (QIAGEN Bioinformatics, Hilden, Germany), with alignment stringency set to 174
medium. The forward and reverse reads were merged into a consensus sequence using the assemble 175
sequences function. The consensus cyp51A sequences of each environmental isolates, and the known 176
TR34/L98H isolate BUU09, were aligned against the referenced wild-type isolate, A. fumigatus Af293. The 177
isolates that exhibited the same 34-bp tandem repeat as the isolate BUU09 were confirmed to harbor 178
the TR34/L98H allele. The isolates that had a 46-bp tandem repeat were compared with A. fumigatus 179
isolate VRJ056 (GenBank accession number: MF070884.1), a known clinical isolate harboring the 180
TR46/Y121F/T289A mutation, to confirm their resistant mechanism. The csp1 types of each 181
environmental isolates and two control isolates were assigned according to the csp1 typing 182
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nomenclature described previously (23), which was based on manually inspecting the repeat regions in 183
each consensus csp1 sequences. 184
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A total of 178 soil samples were collected across Southern England, including soil and compost samples 187
obtained from public gardens, parks, cemeteries, and flower beds outside hospitals. Samples were 188
collected in Central London (n = 64), Bath (n = 8), flower beds around Stonehenge (n = 8), remote 189
forested regions in the New Forest National Park (n = 46), a lavender farm in Surry (n = 13) and farmland 190
in Cambridgeshire (n = 39). Of the 178 soil samples, 131 (74%) were positive for A. fumigatus type 191
growth on control plates, with a varied recovery rate amongst different sampling sites (Table 1). The 192
highest recovery rate was from Central London (93.8%) and the lowest from Cambridgeshire (35.9%). 193
Eleven A. fumigatus positive soil samples yielded a total of 83 A. fumigatus type isolates that were able 194
to grow on the azole-containing plates. Nine of these soil samples were collected from urban locations 195
in London (n=8) and Bath (n=1). Among the eight samples from London, four were collected from flower 196
beds outside Royal Free Hospital and The Whittington Hospital, and another two samples were obtained 197
from compost in Waterlow Park, 500 meters away from The Whittington Hospital. Nineteen putatively 198
resistant isolates were selected for further MIC testing and sequence analysis, ensuring representation 199
from each azole positive soil samples. Seven control isolates were randomly selected for further 200
analyses, representing each sampling site. All 26 isolates were identified as A. fumigatus by sequencing 201
of the beta-tubulin gene. 202
203
MIC measurement 204
All 19 azole-tolerant isolates were confirmed to be resistant to at least one of the three medical azoles 205
tested (Table 2). Eleven of these exhibited resistance to itraconazole only and were susceptible to both 206
voriconazole and posaconazole but with elevated MICs. Six isolates were resistant to voriconazole and 207
four of them were also resistant to itraconazole, with the remaining two having intermediate resistance 208
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against itraconazole. Isolate L2731 was cross-resistant against itraconazole and posaconazole, whereas 209
isolate L3131 was resistant to itraconazole and had intermediate resistance toward posaconazole. 210
Interestingly, isolates from the same soil sample yielded different susceptibility results. Among the 211
seven azole-susceptible isolates, six of them were confirmed to be susceptible to all three medical 212
azoles. One isolate (F311), exhibited resistance to itraconazole and elevated MIC values for voriconazole 213
and posaconazole, 0.25 g/ml and 0.125 g/ml, respectively. 214
215
Molecular determination of the azole resistance mechanisms 216
The molecular mechanisms of azole resistance were determined by sequencing part of the cyp51A gene 217
and its promoter region. Of the 19 azole-resistant isolates, 13 were found to be harbouring the 218
TR34/L98H allele and six the TR46/Y121F/T289A allele (Table 2). All six azole susceptible isolates had the 219
wild-type cyp51A allele. Both the negative and positive control isolates (Af293 and BUU09) carried the 220
wild-type and TR34/L98H allele respectively. 221
222
Molecular characterisation of A. fumigatus 223
Sequence typing of csp1 yielded 9 different csp1 types (Table 2). The six azole-susceptible wild-type 224
environmental isolates were distributed over csp1 subtypes t03 (1/6), t04A (3/6), t05 (1/6) and t08 (1/6). 225
The 20 azole-resistant isolates were spread across five csp1 subtypes, t01 (2/20), t02 (14/20), t04B 226
(2/20), t09 (1/20) and t18 (1/20). All the TR34/L98H and TR46/Y121F/T289A isolates predominantly 227
belonged to csp1 subtype t02. The 13 isolates with the TR34/L98H allele were distributed over three csp1 228
subtypes t01 (1/13), t02 (10/13) and t04B (2/13). The isolates carrying TR46/Y121F/T289A allele were 229
grouped into four csp1 subtypes: t01 (1/6), t02 (3/6), t09 (1/6) and t18 (1/6). The control isolate Af293, 230
which was isolated from a patient with invasive aspergillosis belonged to csp1 subtype t06 and the azole 231
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resistant control isolate BUU09 belonged to the t04B csp1 subtype, which agreed with previously 232
published results (24). 233
234
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Given that ARAf continues to emerge globally, it is imperative that environmental monitoring, 236
particularly in previously unsampled locations, is sustained. Here we present environmental sampling of 237
London and the south east of England, home to approximately 15 million people living both urban and 238
rural lifestyles. We show that azole-resistant A. fumigatus was detected in 6.7% (12/178) of the soil 239
samples collected, and in 9.2% (12/131) of the soil samples positive for A. fumigatus growth. The 240
prevalence of ARAf in the environment was 13.8% in urban areas (10/72), which included Greater 241
London and Bath. In contrast, zero resistant isolates were found in soil samples collected from 242
agricultural land and just two resistant isolates were found in rural samples collected from non-243
agricultural land (1.1%). We also report the UK's first environmental ARAf isolates confirmed to be 244
carrying the TR46/Y121F/T239A resistance allele, alongside an expanded distribution of the TR34/L98H 245
allele. 246
247
Prevalence of ARAf in the UK was found to be lower than that of other European countries and 248
Colombia (15, 17, 25–29), but higher than most Asian countries (24, 30–33), with the exception of India 249
(29, 34). Our findings are also in close agreement with a recent UK-based environmental prevalence 250
study in Wales, where ARAf was detected in 4.5% (30/671) of soil samples, with resistance 251
predominantly found in urban city locations (13). However, they also describe a notable contrast to the 252
only other UK based study, which found zero ARAf in urban locales but four resistant isolates from 253
agricultural sites (1.7%) (14). 254
255
Our findings, and that of the Welsh study (13), appear to contradict the hypothesis that UK ARAf is 256
driven by the environmental application of azoles in arable agriculture (35, 36). Indeed, of the 53 257
samples collected directly on or surrounding agricultural land, zero azole-tolerant isolates were 258
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identified. Rather, we found that the prevalence of resistance was higher in urban city centres, 259
specifically flower beds and gardens, a finding that lends itself more readily to the hypothesis that the 260
expanding range of ARAf stems more from the distribution and cultivation of horticultural crops, such as 261
flowers, ornamentals and vegetables (15–17) 262
263
One particularly concerning discovery was the repeat isolation of ARAf genotypes – TR34/L98H and 264
TR46/Y121F/T289A – from flower beds surrounding city centre hospitals. Owing to the opportunistic 265
nature of A. fumigatus, its ability to cause debilitating illnesses in immunocompromised patients and the 266
elevated mortality that is associated with IA caused by azole-resistant genotypes (22), the gardened 267
areas around hospitals could be considered high risk locations if ARAf is present. Concern over the use of 268
azole treated flower bulbs in hospital environments has been raised previously (15, 16, 37), and 269
although we do not link any cases of azole-resistant aspergillosis to the isolates found in this study, our 270
findings do add to a worrying trend of ARAf populated soil sampled from flower beds (15, 17). 271
272
The continued emergence of ARAf in urban city centres fits with the observation that most isolates 273
belonged to a single csp1 subtype (t02), a pattern consistent with the selective sweep of drug resistant 274
genotypes (38). However, despite this obvious pattern of selection, city centres still harboured greater 275
diversity than rural locations. All csp1 subtypes identified during this study were found in Central 276
London, suggesting an anthropological driver facilitating the migration of many A. fumigatus genotypes 277
into a densely populated metropolis. Consequently, urban city centres, where A. fumigatus diversity is 278
high, could facilitate a ‘melting pot’, where resistance alleles inadvertently brought into the city are 279
given the opportunity to introgress onto novel genetic backgrounds via recombination, increasing the 280
diversity potential of ARAf. Indeed, in London alone, TR34/L98H was found on two csp1 subtypes and 281
TR46/Y121F/T298A on four CSP subtypes. 282
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Ultimately, this study highlights the importance of urban environments in the epidemiology of ARAf. We 284
have shown that although ARAf appears to be environmental by origin, urban city centres, that are 285
densely populated, are of high importance when mitigating strategies are considered. The use of azole 286
treated bulbs in the environment around hospitals should be reconsidered and the continuous global 287
monitoring of ARAf is imperative. 288
289
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The authors would like to thank Ali Abdolrasouli, Darius Armstrong-James and Andrew 291
Scourfield for their helpful discussions whilst analysing the data. We also acknowledge joint 292
Centre funding from the UK Medical Research Council and Department for International 293
Development. Funding: T.R.S., A.P.B., J.R. and M.C.F. were supported by the Natural 294
Environmental Research Council (NERC; NE/P001165/1) and all authors were supported by the 295
Medical Research Council (MRC; MR/R015600/1). 296
297
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Fig 1: Distribution of azole susceptible and resistant Aspergillus fumigatus isolates collected in London 409
(b) and southern UK counties (a). Isolates haboring cyp51a allele TR34/L98H are depicted in red, 410
TR46/Y121F/T289A in blue and wild-type in green. 411
412
.CC-BY-NC-ND 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted April 5, 2019. . https://doi.org/10.1101/598961doi: bioRxiv preprint
T.R.S., Y.Z. and M.C.F. conceived and designed the study. T.R.S., Y.Z., A.P.B. and J.M.G.S. 414
collected the data. T.R.S., Y.Z. and A.P.B. analyzed the data. T.R.S. and Y.J. wrote the 415
manuscript. T.R.S., Y.Z., A.P.B., J.M.G.S., J.R., and M.C.F. discussed the results and commented 416
on the manuscript417
.CC-BY-NC-ND 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted April 5, 2019. . https://doi.org/10.1101/598961doi: bioRxiv preprint
New Forest National Park, Hampshire Remote forest 46 37 (80.4%) 1 2.2%
Surrey Lavender farm 13 10 (76.9%) 0 0%
Cambridgeshire Wheat farm 30 12 (40%) 0 0%
Open space surrounded by farm 9 2 (22.2%) 0 0%
COMBINED TOTAL 178 131 (73.6%) 12 6.7%
a. Resistance determined by both MIC testing and sequencing analysis. 422 423
.CC-BY-NC-ND 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted April 5, 2019. . https://doi.org/10.1101/598961doi: bioRxiv preprint
wild-type control Af293 0.25 0.063 0.016 wild-type t06 azole-resistant control BUU09 4 0.25 0.125 TR34/L98H t04B
a. ITC, itraconazole; VRC, voriconazole; POS, posaconazole. 425 b. Isolate L2841 amplified poorly using the primers in this study. 426
.CC-BY-NC-ND 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted April 5, 2019. . https://doi.org/10.1101/598961doi: bioRxiv preprint
.CC-BY-NC-ND 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted April 5, 2019. . https://doi.org/10.1101/598961doi: bioRxiv preprint