Rn [22]. Moreover, azole-resistant strains from the environment of Bihar and Delhi also showed the same STR pattern. Notably, genetic analysis of a collection of MTR isolates showed that all isolates with the TR34/L98H allele were all confined within a single clade and were less variable than susceptible isolates [25], consistent with a single and recent origin of the resistant genotype. Our results are consistent with the hypothesis that the azoleresistant A. fumigatus strains analyzed here from across India were due to the clonal spread of a single genotype. The lack of a single azole-susceptible strain from MedChemExpress Madrasin either clinical origin or the environment in India with the same genotype as the widespread azoleresistant genotype it may be conceivable that the resistant genotype was unlikely the result of a single mutation at the cyp51A gene in a common azole-susceptible genotype in India. In addition, our genotype analysis suggest that the azole-resistant genotype in India was likely an extremely adaptive recombinant progeny derived from a cross between an azole-resistant strain migrated from outside of India and a native azole-susceptible strain from within India, followed by mutation. The abundant phylogenetic incompatibility found in each of the sub-samples as well as in the whole sample (where 100 of the loci pairs were phylogenetically incompatible, thus consistent with recombination) supports sexual mating in natural populations of this species in India. Our inferred mechanisms have been similarly suggested for the emergence of many virulent strains of viral, bacterial and protozoan pathogens [32,33]. Once the extremely fit A. fumigatus genotype emerged in India, it could spread quickly by producing a large number of airborne asexual spores in the environment. These airborne spores can easily disperse to other geographic areas by air current or anthropogenic means. The widespread application of triazole fungicides in the environment in India in the last two decades could have contributed to its spread by reducing the azole-susceptible genotypes and selecting for this azole-resistant genotype. Whether this resistant genotype has spread to neighbouring countries remain to be determined.Materials and Methods Ethics StatementAll necessary permits were obtained for the described field studies.Collection of Environmental SamplesA total of 486 environmental samples including soil from flowerbeds of nurseries, surrounding parks of hospitals, cotton trees, tea MK 8931 web gardens, paddy fields, soil containing bird excreta, decayed wood of tree trunks and aerial samples of the indoor environment of hospital wards from the Union Territory (UT) of Delhi, Haryana, Himachal Pradesh, Uttrakhand, Bihar, West Bengal, Sikkim, Meghalaya and Tamil Nadu States were investigated during July 2011 pril 2012. The distribution of the investigated 486 samples was as follows: UT of Delhi (n = 266), Haryana (n = 21), Himachal Pradesh (n = 4), Uttrakhand (n = 21), Bihar (n = 33), West Bengal (n = 59), Sikkim (n = 6), Meghalaya (n = 11) and Tamil Nadu (n = 65).Soil and Aerial SamplingAbout two gram of soil was suspended in 8 ml of 0.85 NaCl, vortexed and allowed to settle for 30 seconds. Subsequently, theAzole Resistant A. fumigatus from Indiasuspension was diluted 1:10 and 100 ml was plated in duplicates on Sabouraud dextrose agar plates supplemented with 50 mg/L chloramphenicol and incubated at 37uC for 48 h. One gram of decayed wood was suspended in 10 ml of 0.85 NaCl an.Rn [22]. Moreover, azole-resistant strains from the environment of Bihar and Delhi also showed the same STR pattern. Notably, genetic analysis of a collection of MTR isolates showed that all isolates with the TR34/L98H allele were all confined within a single clade and were less variable than susceptible isolates [25], consistent with a single and recent origin of the resistant genotype. Our results are consistent with the hypothesis that the azoleresistant A. fumigatus strains analyzed here from across India were due to the clonal spread of a single genotype. The lack of a single azole-susceptible strain from either clinical origin or the environment in India with the same genotype as the widespread azoleresistant genotype it may be conceivable that the resistant genotype was unlikely the result of a single mutation at the cyp51A gene in a common azole-susceptible genotype in India. In addition, our genotype analysis suggest that the azole-resistant genotype in India was likely an extremely adaptive recombinant progeny derived from a cross between an azole-resistant strain migrated from outside of India and a native azole-susceptible strain from within India, followed by mutation. The abundant phylogenetic incompatibility found in each of the sub-samples as well as in the whole sample (where 100 of the loci pairs were phylogenetically incompatible, thus consistent with recombination) supports sexual mating in natural populations of this species in India. Our inferred mechanisms have been similarly suggested for the emergence of many virulent strains of viral, bacterial and protozoan pathogens [32,33]. Once the extremely fit A. fumigatus genotype emerged in India, it could spread quickly by producing a large number of airborne asexual spores in the environment. These airborne spores can easily disperse to other geographic areas by air current or anthropogenic means. The widespread application of triazole fungicides in the environment in India in the last two decades could have contributed to its spread by reducing the azole-susceptible genotypes and selecting for this azole-resistant genotype. Whether this resistant genotype has spread to neighbouring countries remain to be determined.Materials and Methods Ethics StatementAll necessary permits were obtained for the described field studies.Collection of Environmental SamplesA total of 486 environmental samples including soil from flowerbeds of nurseries, surrounding parks of hospitals, cotton trees, tea gardens, paddy fields, soil containing bird excreta, decayed wood of tree trunks and aerial samples of the indoor environment of hospital wards from the Union Territory (UT) of Delhi, Haryana, Himachal Pradesh, Uttrakhand, Bihar, West Bengal, Sikkim, Meghalaya and Tamil Nadu States were investigated during July 2011 pril 2012. The distribution of the investigated 486 samples was as follows: UT of Delhi (n = 266), Haryana (n = 21), Himachal Pradesh (n = 4), Uttrakhand (n = 21), Bihar (n = 33), West Bengal (n = 59), Sikkim (n = 6), Meghalaya (n = 11) and Tamil Nadu (n = 65).Soil and Aerial SamplingAbout two gram of soil was suspended in 8 ml of 0.85 NaCl, vortexed and allowed to settle for 30 seconds. Subsequently, theAzole Resistant A. fumigatus from Indiasuspension was diluted 1:10 and 100 ml was plated in duplicates on Sabouraud dextrose agar plates supplemented with 50 mg/L chloramphenicol and incubated at 37uC for 48 h. One gram of decayed wood was suspended in 10 ml of 0.85 NaCl an.
