Elevated Prevalence of Azole-Resistant Aspergillus fumigatus in Urban versus Rural Environments in the United Kingdom

Elevated Prevalence of Azole-Resistant Aspergillus fumigatus in Urban versus Rural Environments in the United Kingdom: Antimicrobial Agents and Chemotherapy




Elevated Prevalence of Azole-Resistant Aspergillus fumigatus in Urban versus Rural Environments in the United Kingdom.

Sewell TR1,
Zhang Y2,
Brackin AP2,
Shelton JMG2,
Rhodes J2,
Fisher MC2
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Antimicrobial Agents and Chemotherapy, 23 Aug 2019, 63(9)
DOI: 10.1128/AAC.00548-19 PMID: 31235621 PMCID: PMC6709452

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Abstract
Azole resistance in the opportunistic pathogen Aspergillus fumigatus is increasing, dominated primarily by the following two environmentally associated resistance alleles: TR34/L98H and TR46/Y121F/T289A. By sampling soils across the South of England, we assess the prevalence of azole-resistant A. fumigatus (ARAf) in samples collected in both urban and rural locations. We characterize the susceptibility profiles of the resistant isolates to three medical azoles, identify the underlying genetic basis of resistance, and investigate their genetic relationships. ARAf was detected in 6.7% of the soil samples, with a higher prevalence in urban (13.8%) than rural (1.1%) locations. Twenty isolates were confirmed to exhibit clinical breakpoints for resistance to at least one of three medical azoles, with 18 isolates exhibiting resistance to itraconazole, 6 to voriconazole, and 2 showing elevated minimum inhibitory concentrations to posaconazole. Thirteen of the resistant isolates harbored the TR34/L98H resistance allele, and six isolates carried the TR46/Y121F/T289A allele. The 20 azole-resistant isolates were spread across five csp1 genetic subtypes, t01, t02, t04B, t09, and t18 with t02 being the predominant subtype. Our study demonstrates that ARAf can be easily isolated in the South of England, especially in urban city centers, which appear to play an important role in the epidemiology of environmentally linked drug-resistant A. fumigatus.

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Antimicrob Agents Chemother. 2019 Sep; 63(9): e00548-19.
Published online 2019 Aug 23. Prepublished online 2019 Jun 24.doi: 10.1128/AAC.00548-19
PMCID: PMC6709452
PMID: 31235621
Elevated Prevalence of Azole-Resistant Aspergillus fumigatus in Urban versus Rural Environments in the United Kingdom
Thomas R. Sewell,#a Yuyi Zhang,#a Amelie P. Brackin,a Jennifer M. G. Shelton,a Johanna Rhodes,a and Matthew C. Fishera
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ABSTRACT


Azole resistance in the opportunistic pathogen Aspergillus fumigatus is increasing, dominated primarily by the following two environmentally associated resistance alleles: TR34/L98H and TR46/Y121F/T289A. By sampling soils across the South of England, we assess the prevalence of azole-resistant A. fumigatus (ARAf) in samples collected in both urban and rural locations. We characterize the susceptibility profiles of the resistant isolates to three medical azoles, identify the underlying genetic basis of resistance, and investigate their genetic relationships. ARAf was detected in 6.7% of the soil samples, with a higher prevalence in urban (13.8%) than rural (1.1%) locations. Twenty isolates were confirmed to exhibit clinical breakpoints for resistance to at least one of three medical azoles, with 18 isolates exhibiting resistance to itraconazole, 6 to voriconazole, and 2 showing elevated minimum inhibitory concentrations to posaconazole. Thirteen of the resistant isolates harbored the TR34/L98H resistance allele, and six isolates carried the TR46/Y121F/T289A allele. The 20 azole-resistant isolates were spread across five csp1 genetic subtypes, t01, t02, t04B, t09, and t18 with t02 being the predominant subtype. Our study demonstrates that ARAf can be easily isolated in the South of England, especially in urban city centers, which appear to play an important role in the epidemiology of environmentally linked drug-resistant A. fumigatus.
KEYWORDS: Aspergillus fumigatus, antifungal resistance, environment, fungal epidemiology, fungal pathogen, multidrug resistance, triazoles

INTRODUCTION

Aspergillus fumigatus is a ubiquitous ascomycete fungus with a pan-global population distribution (1) and a primary ecological niche of decaying vegetation and soil (2). This fungus is the most prevalent species among ∼250 described aspergilli, partly due to its ability to survive and grow in a wide range of conditions but also through the large-scale dispersal of airborne conidia (2,–4). A. fumigatus is also an opportunistic pathogen and is commonly responsible for aspergillosis, a spectrum of clinical syndromes caused by Aspergillus spp. that affects millions of individuals worldwide (5). Invasive aspergillosis (IA), the most severe form of the disease, can lead to serious and even fatal illness in immunocompromised individuals, with a mortality rate of 40 to 90% (6, 7).

Triazole antifungals are used for the treatment and prophylaxis of Aspergillus spp. infections. However, resistance to these chemicals has emerged, often conferred by the presence of mutations in lanosterol 14 alpha-demethylase (erg11, syn. CYP51) which is a key component of the ergosterol biosynthetic pathway and the target for azole antifungals (8, 9). Recently, azole-resistant A. fumigatus (ARAf) has emerged environmentally, where selection is thought to be driven by the broad application of agricultural azole fungicides, structurally similar to their medical counterparts and indistinguishable in their mode of action (10, 11). Environmentally sourced ARAf is typically found with a tandem repeat mutation in the cyp51A promoter region and linked single nucleotide polymorphisms in the coding region, with common examples being TR34/L98H and TR46/Y121F/T289A (12, 13).

Studies have shown that ARAf isolates harboring resistance-associated cyp51A variants are globally distributed and are often found alongside wild-type (WT) A. fumigatus in a diverse set of environmental substrates, including agricultural soil (14, 15), flower beds (14, 16,–18), and timber mills (19). Moreover, clinical cases of aspergillosis with ARAf isolates harboring either TR34/L98H or TR46/Y121F/T289A continue to emerge (20, 21), with one study specifically linking a fatal case of aspergillosis to a genotypically indistinguishable isolate sourced from the patient’s own home (22). Retrospective studies of patients with invasive aspergillosis (IA) and infected with azole-resistant genotypes of A. fumigatus show an excess mortality of 25% at day 90 when compared with that of patients with wild-type infections, suggesting a growing clinical impact caused by the emergence of ARAf (23).

Despite a growing understanding of the global prevalence of ARAf (1), very few studies have investigated resistance in the United Kingdom (UK) or focused sampling strategies across a diverse set of substrates. Two previous studies have shown ARAf to be an emerging issue in the UK (14, 15), with one sampling program in South Wales highlighting a significant level of environmental ARAf in urban environments (14). In this study, we aim to determine the prevalence of ARAf in soil collected from a wide range of sites across the UK’s southernmost region. Sampling locations include ancient woodlands, agricultural fields, tourist attractions, and densely populated city centers. We show that the UK has similar prevalence rates to those of other countries and that both TR34/L98H and TR46/Y121F/T289A can be regularly isolated from urbanized locations, including flower beds in close proximity to city center hospitals.

RESULTS



Environmental A. fumigatus isolates.

A total of 178 soil samples were collected across Southern England, including soil and compost samples obtained from public gardens, parks, cemeteries, and flower beds outside London hospitals. Samples were collected in Central London (n=64), Bath (n=8), flower beds around Stonehenge (n=8), remote forested regions in the New Forest National Park (n=46), a lavender farm in Surry (n=13), and farmland in Cambridgeshire (n=39). Of the 178 soil samples, 131 (74%) were positive for A. fumigatus-type growth on control plates, with a varied recovery rate among different sampling sites (Table 1). The highest recovery rate was from Central London (93.8%), and the lowest was from Cambridgeshire (35.9%). Eleven A. fumigatus-positive soil samples yielded a total of 83 A. fumigatus-type isolates that were able to grow on the azole-containing plates. Nine of these soil samples were collected from urban locations in London (n=8) and Bath (n=1). Among the eight samples from London, four were collected from flower beds outside Royal Free Hospital and The Whittington Hospital, and another two samples were obtained from compost in Waterlow Park, 500 m away from The Whittington Hospital. Nineteen putatively resistant isolates were selected for further MIC testing and sequence analysis, ensuring representation from each azole-positive soil sample. Seven control isolates were randomly selected for further analyses, representing each sampling site (Fig. 1). All 26 isolates were positively identified as A. fumigatus by sequencing of the beta-tubulin gene.

TABLE 1


Soil sampling areas and A. fumigatus recovery rate, with azole resistance determined by MIC and cyp51A sequencing

Sampling siteNo. of samples collectedNo. of samples with A. fumigatus growth (%)No. of samples recovered with azole-resistant A. fumigatusaPrevalence of azole-resistant A. fumigatus (%)London
    Overall 64 60 (93.8) 9 14.0
    Kensal Green Cemetery 5 5 (100) 0 0
    Queen Mary's Rose Garden and Regents Park 8 7 (87.5) 0 0
    The Whittington Hospital and Waterlow Park 5 5 (100) 3 60.0
    Hampstead Heath 3 1 (33.3) 0 0
    Royal Free Hospital 5 5 (100) 3 60.0
    Green Park & Hyde Park 9 9 (100) 1 11.1
    Charing Cross Hospital and Margravine Cemetery 8 7 (87.5) 0 0
    Brompton Cemetery 6 6 (100) 1 16.7
    Elstree Open Space 8 8 (100) 1 12.5
    Flower beds in urban city 7 7 (100) 0 0
Bath city center, Somerset
    Flower beds 8 4 (50) 1 12.5
Stonehenge, Wiltshire
    Flower beds and farm 8 6 (75) 1 12.5
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

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aResistance determined by both MIC testing and sequencing analysis.

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FIG 1


Distribution of azole-susceptible and azole-resistant Aspergillus fumigatus isolates collected in London (b) and southern UK counties (a). Isolates harboring cyp51a allele TR34/L98H are depicted in red, TR46/Y121F/T289A are in green, and wild-type isolates are in blue.



MIC measurement.

All 19 azole-tolerant isolates were confirmed to be resistant to at least one of the three medical azoles tested (Table 2). Eleven of these exhibited resistance to itraconazole (ITC) only and were susceptible to both voriconazole (VRC) and posaconazole (POS) but with elevated MICs. Six isolates were resistant to voriconazole and four of them were also resistant to itraconazole, with the remaining two having intermediate resistance against itraconazole. Isolate L2731 was cross-resistant against itraconazole and posaconazole, whereas isolate L3131 was resistant to itraconazole and had intermediate resistance toward posaconazole. Interestingly, isolates from the same soil sample yielded different susceptibility results. Among the seven azole-susceptible isolates, six of them were confirmed to be susceptible to all three medical azoles. One isolate (F311), exhibited resistance to itraconazole and elevated MIC values for voriconazole and posaconazole, 0.25g/ml and 0.125g/ml, respectively.

TABLE 2


Characteristics of azole-resistant A. fumigatus isolates and the control isolates

Soil sampleSampling siteIsolateMIC (μg/ml)acyp51A
mutationcsp1 type
ITCVRCPOSL10 Waterlow Park, London L1031 2 8 0.0625 TR46/Y121F/T289A t02
L1032 4 4 0.125 TR46/Y121F/T289A t02
L1041 4 8 0.125 TR46/Y121F/T289A t02
L1043 4 0.25 0.125 TR34/L98H t02
L27 Waterlow Park, London L2731 4 1 0.5 TR34/L98H t02
L2741 4 8 0.125 TR46/Y121F/T289A t09
L28 Whittington Hospital, London L2831 4 8 0.125 TR46/Y121F/T289A t01
L2841 4 0.25 0.125 Unknownb t02
L12 Royal Free Hospital, London L1231 4 0.5 0.125 TR34/L98H t02
L1241 4 0.25 0.125 TR34/L98H t02
L29 Royal Free Hospital, London L2931 4 0.125 0.0625 TR34/L98H t01
L31 Royal Free Hospital, London L3131 4 1 0.25 TR34/L98H t02
L3141 4 0.25 0.125 TR34/L98H t02
L19 Hyde Park, London L1931 2 4 0.0625 TR46/Y121F/T289A t18
L41 Brompton Cemetery, London L4131 4 0.25 0.125 TR34/L98H t02
BS1 Bath city center BS131 4 0.25 0.125 TR34/L98H t02
BS9 Stonehenge, Wiltshire BS941 4 0.125 0.0625 TR34/L98H t02
BS942 4 0.125 0.125 TR34/L98H t02
NF63 New Forest, Hampshire NF6341 4 0.25 0.125 TR34/L98H t04B
F3 Elstree Open Space, London F311 4 0.25 0.125 TR34/L98H t04B
F44 Lavender farm, Surrey F4411 0.125 0.031 0.016 Wild type t05
F55 Wheat farm, Cambridge F5511 0.25 0.063 0.031 Wild type t04A
L11 Royal Free Hospital, London L1111 0.25 0.031 0.016 Wild type t04A
L42 Brompton Cemetery, London L4211 0.125 0.031 0.031 Wild type t03
BS10 Stonehenge, Wiltshire BS1011 0.125 0.063 0.031 Wild type t04A
NF42 New Forest, Hampshire NF4211 0.125 0.063 0.016 Wild type t08
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

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aITC, itraconazole; VRC, voriconazole; POS, posaconazole.
bIsolate L2841 amplified poorly using the primers in this study.



Molecular determination of the azole resistance mechanisms.

The molecular mechanisms of azole resistance were determined by sequencing part of the cyp51A gene and its promoter region. Of the 20 azole-resistant isolates, 13 were found to be harboring the TR34/L98H allele and six were found to be harboring the TR46/Y121F/T289A allele (Table 2). All six azole-susceptible isolates had the wild-type cyp51A allele. Both the negative and positive control isolates (Af293 and BUU09) carried the wild-type and TR34/L98H allele, respectively. One isolate did not amplify with standard cyp51a primers.



Molecular characterization of A. fumigatus.

Sequence typing of csp1 yielded 9 different csp1 types (Table 2). The six azole-susceptible wild-type environmental isolates were distributed over csp1 subtypes t03 (1/6), t04A (3/6), t05 (1/6), and t08 (1/6). The 20 azole-resistant isolates were spread across five csp1 subtypes, t01 (2/20), t02 (14/20), t04B (2/20), t09 (1/20), and t18 (1/20). All of the TR34/L98H and TR46/Y121F/T289A isolates predominantly belonged to csp1 subtype t02. The 13 isolates with the TR34/L98H allele were distributed over three csp1 subtypes, t01 (1/13), t02 (10/13), and t04B (2/13). The isolates carrying the TR46/Y121F/T289A allele were grouped into the following four csp1 subtypes: t01 (1/6), t02 (3/6), t09 (1/6), and t18 (1/6). The control isolate Af293, which was isolated from a patient with invasive aspergillosis, belonged to csp1 subtype t06, and the azole-resistant control isolate BUU09 belonged to the t04B csp1 subtype, which agreed with previously published results (24).

DISCUSSION

Given that ARAf continues to emerge globally, further environmental monitoring is required in order to fully appreciate the threat of ARAf to UK public health. Here, we present environmental sampling of London and the southeast of England, home to approximately 15 million people living both urban and rural lifestyles. We show that azole-resistant A. fumigatus was detected in 6.7% (12/178) of the soil samples collected and in 9.2% (12/131) of the soil samples that were positive for A. fumigatus growth. The prevalence of ARAf in the environment was 13.8% in urban areas (10/72), which included Greater London and Bath. In contrast, zero resistant isolates were found in soil samples collected from agricultural land, and just two resistant isolates were found in rural samples collected from nonagricultural land (1.1%). We also report the UK’s first environmental ARAf isolates confirmed to be carrying the TR46/Y121F/T289A resistance allele, alongside an expanded distribution of the TR34/L98H allele.

Prevalence of ARAf in the UK was found to be lower than that of other European countries and Colombia (16, 18, 25,–29) but higher than most Asian countries (24, 30,–33), with the exception of India (29, 34). Our findings are also in close agreement with a recent UK-based environmental prevalence study in Wales, where ARAf was detected in 4.5% (30/671) of soil samples, with resistance predominantly found in urban city locations (14). However, they also describe a notable contrast to the only other UK-based study, which found zero ARAf in urban locales but four resistant isolates from agricultural sites (1.7%) (15).

Our findings, and those of the Welsh study (14), appear to contradict the hypothesis that UK ARAf is driven by the environmental application of azoles in arable agriculture (35, 36). Indeed, of the 53 samples collected directly on or surrounding agricultural land, zero azole-tolerant isolates were identified. Rather, we found that the prevalence of resistance was higher in urban city centers, specifically flower beds and gardens, a finding that lends itself more readily to the hypothesis that the expanding range of ARAf stems more from the distribution and cultivation of horticultural crops, such as flowers, ornamentals, and vegetables (16,–18).

One particularly concerning discovery was the repeat isolation of ARAf genotypes—TR34/L98H and TR46/Y121F/T289A—from flower beds surrounding city center hospitals. Owing to the opportunistic nature of A. fumigatus, its ability to cause debilitating illnesses in immunocompromised patients, and the elevated mortality that is associated with IA caused by azole-resistant genotypes (23), the gardened areas around hospitals could be considered high-risk locations if ARAf is present. Concern over the use of azole-treated flower bulbs in hospital environments has been raised previously (16, 17, 37), and although we do not link any cases of azole-resistant aspergillosis to the isolates found in this study, our findings do add to a worrying trend of ARAf-populated soil sampled from flower beds (16, 18).

The continued emergence of ARAf in urban city centers fits with the observation that most isolates belonged to a single csp1 subtype (t02), a pattern consistent with the selective sweep of drug-resistant genotypes (38). However, despite this obvious pattern of selection, city centers still harbored greater diversity than rural locations. All csp1 subtypes identified during this study were found in Central London, suggesting human activity facilitating the introduction of diverse A. fumigatus genotypes into a densely populated metropolis. Consequently, urban city centers where A. fumigatus diversity is high could elevate the evolutionary potential of ARAf, where resistance alleles inadvertently brought into the city are given the opportunity to introgress onto novel genetic backgrounds via recombination, increasing the diversity of traits that are associated with ARAf. Indeed, in London alone, TR34/L98H was found on two csp1 subtypes and TR46/Y121F/T298A was found on four CSP subtypes.

Ultimately, this study highlights the importance of urban environments in the epidemiology of ARAf. We have shown that although ARAf appears to be environmental by origin, urban city centers that are densely populated are of high importance when mitigating strategies are considered. Further investigations using a more sensitive genotyping approach, such as whole-genome sequencing, is needed in order to fully appreciate the relationship between environmentally sourced strains and their clinically derived counterparts. The use of azole-treated plant bulbs in the environment around hospitals should be reconsidered, and wider global monitoring of ARAf is warranted due to the threat that this pathogen poses to diverse groups of susceptible patients.

MATERIALS AND METHODS



Environmental sampling.

Samples were collected from 16 sites across South England between May and July 2018. Locations were selected to incorporate a range of habitat types and included remote forested regions, urban city centers, agricultural land, and flowering fields. At each sample site, dry surface soil was loosened and collected into a 5-ml Eppendorf tube (Eppendorf AG, Hamburg, Germany).



Recovering of A. fumigatus and screening for azole resistance.

A. fumigatus was isolated from environmental samples using 2g of soil suspended in 8ml of sterile spore suspension buffer, as previously described (14). Supernatant was added to Sabouraud dextrose agar plates (CM0041; Oxoid Ltd, Basingstoke, Hants, UK) supplemented with 8g/ml tebuconazole (PESTANAL analysis standard; Sigma-Aldrich AG Industriestrasse, Switzerland) and control plates without tebuconazole. Plates were incubated at 42°C and examined for growth after 72h. A. fumigatus isolates were identified by observation of their macro and microscopic morphology (3, 39), with putative azole resistance assigned to isolates growing on tebuconazole-positive plates.



In vitro susceptibility testing—MIC measurement.

Seven suspected wild-type isolates and 19 suspected azole-resistant isolates were tested for antifungal drug susceptibility against three clinically used azoles, itraconazole (ITC), voriconazole (VRC), and posaconazole (POS), according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) microdilution method (40) using Micronaut-AM EUCAST MIC plates (Merlin Diagnostika GmbH, Bornheim, Germany). The reference wild-type clinical A. fumigatus isolates Af293 and an environmental azole-resistant isolate BUU09 (TR34/L98H) were used as control strains. Isolates with MIC values between the susceptible and resistant breakpoint were considered to be intermediately susceptible.



DNA extraction for sequence analysis.

Genomic DNA was extracted using a modified MasterPure yeast DNA purification (Lucigen Corporation, Cambridge, UK) protocol, which included an additional two-step bead-beating treatment to enhance DNA yield (38). Cultured A. fumigatus conidia suspended in lysis solution were subjected to bead beating for 3×45s at 30m/s in a TissueLyser II (Qiagen, Hilden, Germany) and placed on ice for 2 min before repeating. DNA was precipitated with isopropanol and quantified using a Qubit 2.0 fluorometer (Invitrogen by Thermo Fisher Scientific Corporation, MA, USA) and the Qubit dsDNA BR (broad-range) assay kit (Invitrogen) according to product guidelines.



PCR amplification, purification, and Sanger sequencing.

PCR was used to amplify part of the cyp51A gene and promoter (L98HR, 5′-TTCGGTGAATCGCGCAGATAGTCC-3′; TR34R, 5′-AGCAAGGGAGAAGGAAAGAAGCACT-3′), the csp1 region (CSP1F, 5′-TTGGGTGGCATTGTGCCAA-3′; CSP1R, 5′-GAGCATGACAACCCAGATACCA-3′), and beta-tubulin (Bt2A_F, 5′-GGTAACCAAATCGGTGCTGCTTTC-3′; Bt2A-R, 5′-ACCCTCAGTGTAGTGACCCTTGGC-3′), as previously described (39, 41, 42). PCR products were purified using ExoSAP-IT PCR product cleanup reagent (Thermo Fisher Scientific, MA, USA) following the product guidelines and Sanger sequenced by Genewiz UK (Genewiz UK Ltd, Takeley, UK). Sequencing results were trimmed and assembled using CLC Main Workbench 8.0.1 software (Qiagen Bioinformatics, Hilden, Germany).

Assembled sequences were aligned against wild-type and resistant cyp51A sequences to confirm the presence/absence of WT, TR34/L98H, or TR46/Y121F/T289 alleles. The csp1 types of each environmental isolate and two control isolates were assigned according to the csp1 typing nomenclature described previously (42).

ACKNOWLEDGMENTS


We thank Ali Abdolrasouli, Darius Armstrong-James, and Andrew Scourfield for their helpful discussions while analyzing the data. We thank all reviewers for taking the time to assess our manuscript.

We also acknowledge joint Centre funding from the UK Medical Research Council and Department for International Development. T.R.S., A.P.B., J.R., and M.C.F. were supported by the Natural Environmental Research Council (NERC; NE/P001165/1), and all authors were supported by the Medical Research Council (MRC; MR/R015600/1). M.C.F. is a CIFAR Fellow in the Fungal Kingdom program.

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. collected the data. T.R.S., Y.Z., and A.P.B. analyzed the data. T.R.S. and Y.Z. wrote the manuscript. T.R.S., Y.Z., A.P.B., J.M.G.S., J.R., and M.C.F. discussed the results and commented on the manuscript.

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