| Aspergillus fumigatus is an opportunistic fungal pathogen,which is ubiquitous in the natural environment and is mainly spread by abundantly produced asexual spores.It could cause allergic,chronic,and acute aspergillus diseases in humans and animals,with invasive pulmonary aspergillosis(IPA)as the most serious illness,for which the mortality was always more than 50%.In recent years,with the increase of susceptible patients such as malignant tumor and hematopoietic stem cell transplantation,the incidence of IPA exhibited an increasing trend,and A.fumigatus has now become one of the main infectious pathogens associated with death of hospitalized patients.Azoles are the main drug class used in the management of aspergillus diseases;however,since the emergence of itraconazole(ITC)resistant A.fumigatus in 1997,the report of azole-resistant A.fumigatus from clinical and environmental sources has been increasing,the prevalence of azole resistance in A.fumigatus from some clinical care centers was as high as 10%,which poses a great challenge for clinical treatment of aspergillus disease.The most common resistance mechanism is characterized by combinations of a tandem repeat in the sterol 14α-demethylase gene cyp51 A promoter and a concomitant mutation in the cyp51 A gene itself,including TR34/L98 H and TR46/Y121F/T289 A mutations.The wide use of agricultural azole fungicides was supposed to be associated with the emergence and prevalence of these mutations.As the diagnosis rates of aspergillus disease in Chinese clinical settings was very low,the microbiological laboratory does not commonly conduct antifungal susceptibility testing for A.fumigatus,and the study of association between agricultural fungicides use and antifungal resistance was rare,the epidemiology and mechanisms of azole resistance in clinical and environmental A.fumigatus from China was largely unknown.The main purpose of this study include:(1)To investigate the occurrence and resistance mechanisms of azole resistance in clinical and environmental through conducting antifungal resistance surveillance in different parts of China;(2)To analyze the genetic variation and evolutionary relationship of azole resistant and susceptible A.fumigatus isolates using molecular typing data of representative isolates from this study and published literatures,in combination with whole genome sequencing analyses;(3)To evaluate the impact of agricultural azole fungicides use on emergence of azole resistance in A.fumigatus through comparing the characterizations of azole resistance between plant pathogenic fungi and A.fumigatus,as well as conducting azole fungicides susceptibility testing and in vitro azole resistance induction experiments.A surveillance of clinical A.fumigatus was conducted in 11 hospitals located in different parts of China between 2014 and 2015,317 A.fumigatus isolates were identified.A total of 392 soil samples and leaves were collected from the hospital gardens,city parks and farmland located in 13 cities,144 A.fumigatus isolates were cultured and identified according to microscopic and macroscopic morphologies.All the 461 A.fumigatus isolates were screened for azole resistance by assessing growth in RPMI 1640+2% glucose agar plates containing 4 mg/L ITC and 1 mg/L voriconazole(VRC).There were ten A.fumigatus isolates showing the ability to grow on both ITC-and VRC-containing agars.These isolates were further identified as azole-resistant(MIC>4mg/L for ITC or VRC)after in vitro susceptibility testing and detection of cyp51 A mutations,including 8 clinical isolates and 2 environmental isolates.The prevalence of azole resistance in clinical and environmental A.fumigatus isolates were 2.5%(8/317)and 1.4%(2/144),respectively.These isolates originated from five different cities,including Fuzhou,Shanghai,Chengdu,Beijing and Shenyang.Two clinical isolates and two environmental isolates harboring TR34/L98H/S297T/F495 I mutation,as well as five clinical isolates harboring TR34/L98 H mutation exhibited high-level resistance to ITC(MIC≥16 mg/L).The emergence of a new environmental mechanism(TR46/Y121F/T289A)in A.fumigatus from a patient admitted into a hospital in Beijing was firstly identified in this study,this strain was isolated from the sputum sample of the patient.This patient was suspected of having IPA,no azole was used before isolation of this strain,the patient died two weeks after the date of acquiring suspected IPA.Among the 153 randomly selected azole-susceptible A.fumigatus isolates,36 isolates(23.5%)harboured at least one loci of amino acid substitution in cyp51 A.The most common cyp51 A mutation was N248 K both in clinical and environmental isolates.A9 T,M172V/A330 P and N248K/M499 K mutations were only detected in environmental isolates,while D343 N,I242V,K314 T,D262Y,F46Y/M172V/E427 K were only detected from clinical isolates.F46Y/M172V/N248T/D255E/E427 K mutation,which has been reported to be associated with azole resistance,was detected in one clinical isolate from Shanghai and one environmental isolate from Xinjiang,respectively.CSP,STR and Mating types were determined for the 10 azole-resistant and 153 susceptible control isolates.The CSP typing results showed that the five TR34/L98 H isolates and four TR34/L98H/S297T/F495 I isolates corresponded to t02 and t04 A,respectively,while the TR46/Y121F/T289 A isolates corresponded to t01.There were 15 CSP types in 92 clinical azole-susceptible A.fumigatus and 7 CSP types in 61 environmental azole-susceptible A.fumigatus,with t01,t04 A and t03 as the three most common CSP types.The mating type of all the four TR34/L98H/S297T/F495 I A.fumigatus from this study was MAT1-2,while the other six resistant isolates was MAT1-1.There were 87(56.9%)MAT1-1 and 66(43.1%)MAT1-2 isolates among 153 azole-susceptible A.fumigatus.STR typing analysis of the 163 azole-resistant and-susceptible isolates showed that the distribution of clinical and environmental isolates were scattered,eight azole-resistant isolates belonged to one major cluster,which consisted of 49.1% of all the surveyed isolates.The clinical TR34/L98 H isolate from Chengdu represented one separate clade far away from other strains,while the TR46/Y121F/T289 A belonged to another clade,which mainly consisted of azole-susceptible environmental isolates.STR typing results of 836 isolates from previous studies were included for comparisons,which found that most of TR34/L98 H and TR46/Y121F/T289 A isolates belonged to one predominant clone.Many of the TR34/L98 H isolates from India harboured the same STR types,which might be caused by clonal expansion.The TR46/Y121F/T289 A isolate in this study haboured the similar genetic background with clinical and environmental isolates from the Netherlands.The STR types of TR34/L98H/S297T/F495 I isolates from China were quite different from all the other azole resistant isolates.Whole genome sequencing(WGS)was conducted in 18 A.fumigatus isolates use Miseq 600 sequencer from Illumina company,and analyzed in combination with WGS data from the Netherlands,United Kingdom and India,the phylogenetic analysis based on genomic SNP showed that four of the five TR34/L98 H isolates from this study were divided into one major clade which consisted of all the azole resistant isolates from the Netherlands,United Kingdom and India,while four TR34/L98H/S297T/F495 I isolates were far away from the major clade,which suggested that these isolates possibly had different evolutionary source with the TR34/L98 H clone.The use of agricultural azole fungicides and characterizations of plant fungal pathogens were analyzed thorough literature review,the amino acid sequences allignment of the sterol 14α-demethylase(CYP51)of plant fungal pathogens and A.fumigatus was conducted.As a result,many triazole fungicides commonly used in agriculture had similar structure with clinical triazole drugs,the cyp51 A point mutation sites Y121,Y431,G432,G448 and F495,which have been reported to be associated with azole resistance in A.fumigatus,corresponded to the mutations sites of azole resistant plant fungal pathogens.The TR34/L98 H,TR34/L98H/S297T/F495 I and TR46/Y121F/T289 A isolates exhibited cross resistance to many agricultural azole fungicides,including epoxiconazole,bromuconazole,tebuconazole and difenoconazole.The MICs of TR34/L98H/S297T/F495 I isolates were significantly higher than TR34/L98 H and azole susceptible isolates(P<0.01).In vitro drug induction experiments of 22 A.fumigatus isolates showed that pan-azole resistant isolates could be obtained under the pressure of epoxiconazole,tebuconazole and imazalil,G54 R and M220 I isolates could be obtained under the pressure of ITC.In conclusion,this study indicated that environmental mediated TR34/L98 H and TR34/L98H/S297T/F495 I mutations were the main resistance mechanisms in A.fumigatus from China,TR46/Y121F/T289 A mutation has now emerged in clinical isolate from China and was probably associated with patient’s adverse outcome.The TR34/L98H/S297T/F495 I isolates from China had different molecular types and genetic background comparing to other resistant isolates with the same or similar mutations,possibly evolved from distinctive sources.All the environmental mediated resistant isolates exhibited cross resistance to agricultural azole fungicides,TR34/L98H/S297T/F495 I isolates exhibited higher resistance phenotype to imidazole fungicides.Under the in vitro selection pressure of agricultural azole fungicides,pan-azole resistant A.fumigatus isolates could be induced,which suggested that the wide use of agricultural azole fungicides should be the main reason for the emergence of azole-resistant isolates.Comparing to previous studies,the major innovativeness and scientific significance of this study are as followed:(1)This is the first Chinese study on the epidemiology of azole resistance in clinical and environmental A.fumigatus from different parts of China;(2)This study firstly analyzed the genetic variation and evolution of azole resistant A.fumigatus using a combinations of different molecular typing and whole genome sequencing methods;(3)This study investigated the relationship between azole fungicide use and the emergence of azole resistance in A.fumigatus through analysis of consumption and resistance of agricultural azole fungicide use,and verification of the role of azole fungicides in causing mutation in A.fumigatus under in vitro azole drug pressure.This study is very important for making up for the lack of knowledge in epidemiology and mechanisms of azole resistance in A.fumigatus in China and could help to provide the scientific basis for the control and prevention of azole resistance in A.fumigatus. |
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