D from aerial sampling of patient rooms of the VPCI hospital, and 2 from soil containing bird droppings in Tamil Nadu (Table 1). Overall, 5 (24/486) of the samples tested harbored itraconazole resistant A. fumigatus. Among the positive samples, 11.9 (24/201) showed at least one colony of resistant A. fumigatus. The isolation rate of itraconazole resistant A. fumigatus was highest 33 (9/27) from the soil of tea gardens followed by soil from flower pots of the hospital garden 20 (15/Origin(s) of the Azole-resistant A. fumigatus Genotype in IndiaThe widespread occurrence of a single azole-resistant genotype across India contrasts with those found in several other regions outside of India. In our analyses, a diversity of genotypes has been found for clinical TR34/L98H azole-resistant A. fumigatus 4-IBP cost strains in China, France, Germany and in both clinical and environmental sources in the Netherlands (Figs. 2 and 3). To examine the origin(s) of the azole – resistant genotype in India, we first attempted to isolate azole – susceptible strains from the 24 soil samples that contained the 44 azole-resistant strains. Among these 24 soil samples, we successfully obtained and analyzed eight azolesusceptible isolates from seven of the 24 samples through dilution plating, single colony purification, and screening using itraconazole-containing and non-containing media. Our genotype analyses using the 9 microsatellite markers revealed that none of the eight strains had a genotype identical to the azole-resistant genotype in India. These eight azole-susceptible strains belonged to four different genotypes. Interestingly, three of the genotypes shared no allele with the azole-resistant genotype at any of the nine microsatellite loci while the remaining genotype shared an allele with the azole-resistant genotype at only one of the nine loci. To further explore the potential origin(s) of 16574785 the azole-resistant genotype in India, we further analyzed the genotypes of all the azole-susceptible strains from within India. Among the nine microsatellite loci, we were able to find allele-sharing at only sixAzole Resistant A. fumigatus from IndiaFigure 1. An outline map of India showing state-wise isolation of multiple-triazole resistant Aspergillus fumigatus isolates from variety of environmental samples. doi:10.1371/journal.pone.0052871.gloci between the Indian azole-resistant genotype and the 35 azolesusceptible clinical and soil/air isolates in India. The highest number of loci with shared alleles between any of the 35 azole susceptible strains and the resistant genotype was at only two of the nine loci. Therefore, even with free recombination among the genotypes represented by the 35 azole susceptible strains in India, the azole-resistant genotype could not be generated due to the lack of corresponding alleles at three of the nine loci (loci 2A, 3A, and 4C, Fig. 2) found only in the azole-resistant strains.Interestingly, though not identical, several strains from outside of India were found to have genotypes more similar to the Indian azole-resistant strains than the Indian azole-susceptible strains (Fig. 2). For example, ten of the 51 strains from outside of India shared alleles in at least four of the nine loci with the Indian azole esistant genotype, with four of the 10 strains sharing alleles at five loci. These 10 strains were all similarly resistant to azoles as the Indian azole-resistant genotype and all 10 strains JI 101 carried the same TR34/L98H mutation. The.D from aerial sampling of patient rooms of the VPCI hospital, and 2 from soil containing bird droppings in Tamil Nadu (Table 1). Overall, 5 (24/486) of the samples tested harbored itraconazole resistant A. fumigatus. Among the positive samples, 11.9 (24/201) showed at least one colony of resistant A. fumigatus. The isolation rate of itraconazole resistant A. fumigatus was highest 33 (9/27) from the soil of tea gardens followed by soil from flower pots of the hospital garden 20 (15/Origin(s) of the Azole-resistant A. fumigatus Genotype in IndiaThe widespread occurrence of a single azole-resistant genotype across India contrasts with those found in several other regions outside of India. In our analyses, a diversity of genotypes has been found for clinical TR34/L98H azole-resistant A. fumigatus strains in China, France, Germany and in both clinical and environmental sources in the Netherlands (Figs. 2 and 3). To examine the origin(s) of the azole – resistant genotype in India, we first attempted to isolate azole – susceptible strains from the 24 soil samples that contained the 44 azole-resistant strains. Among these 24 soil samples, we successfully obtained and analyzed eight azolesusceptible isolates from seven of the 24 samples through dilution plating, single colony purification, and screening using itraconazole-containing and non-containing media. Our genotype analyses using the 9 microsatellite markers revealed that none of the eight strains had a genotype identical to the azole-resistant genotype in India. These eight azole-susceptible strains belonged to four different genotypes. Interestingly, three of the genotypes shared no allele with the azole-resistant genotype at any of the nine microsatellite loci while the remaining genotype shared an allele with the azole-resistant genotype at only one of the nine loci. To further explore the potential origin(s) of 16574785 the azole-resistant genotype in India, we further analyzed the genotypes of all the azole-susceptible strains from within India. Among the nine microsatellite loci, we were able to find allele-sharing at only sixAzole Resistant A. fumigatus from IndiaFigure 1. An outline map of India showing state-wise isolation of multiple-triazole resistant Aspergillus fumigatus isolates from variety of environmental samples. doi:10.1371/journal.pone.0052871.gloci between the Indian azole-resistant genotype and the 35 azolesusceptible clinical and soil/air isolates in India. The highest number of loci with shared alleles between any of the 35 azole susceptible strains and the resistant genotype was at only two of the nine loci. Therefore, even with free recombination among the genotypes represented by the 35 azole susceptible strains in India, the azole-resistant genotype could not be generated due to the lack of corresponding alleles at three of the nine loci (loci 2A, 3A, and 4C, Fig. 2) found only in the azole-resistant strains.Interestingly, though not identical, several strains from outside of India were found to have genotypes more similar to the Indian azole-resistant strains than the Indian azole-susceptible strains (Fig. 2). For example, ten of the 51 strains from outside of India shared alleles in at least four of the nine loci with the Indian azole esistant genotype, with four of the 10 strains sharing alleles at five loci. These 10 strains were all similarly resistant to azoles as the Indian azole-resistant genotype and all 10 strains carried the same TR34/L98H mutation. The.
