Genetic Diversity And Susceptibility To Itraconazole In Aspergillus Isolates From Avian Farms In Guangxi, China

Fungi of the genus Aspergillus are moulds, which occur most frequently in soil, water and decaying vegetation. They sporulate abundantly and the spores are easily dispersed into the environment by air. As a result of this ubiquitous presence, animals and people are constantly exposed to Aspergillus spores. Aspergillus fumigatus and A. flavus are recognized as predominant causes of fungal diseases in humans and wide range of animals. Birds are much more sensitive that mammals and in avian farms, environmental conditions are favorable to the development of many fungal species, including Aspergillus spp.The objective of the present study was to assess the genetic diversity and antifungal susceptibility of Aspergillus isolates from avian farms in Guangxi, China.The first part of the work related the dynamic of fungal contamination in3avian farms near the city of Nanning and one farm (including a hatchery) near the city of Guilin. Pharyngeal swabs and air samples were collected during several weeks and3cycles of hatching were monitored. The average contamination level with Aspergillus spp. and Mucorales was significantly different according to the farms. The survey allowed to collect a total number of188A. fumigatus and159A. flavus isolates.The second part of the work is about the genetic diversity of A. fumigatus and A. flavus. For that purpose, the Multiple Locus Variable-number tandem-repeat (VNTR) Analysis was specifically developed and used. For A. flavus,8VNTR markers were selected and a multiplex reaction was designed. A total number of91A. flavus isolates, including6reference strains were typed with the panel of8VNTRs. This analysis yielded78different genotypes, which corresponds to a combined loci index of0.993. Among all genotypes,71were only found once. The analysis of188A. fumigatus isolates using10VNTR markers led to the resolution of142distinct genotypes. Clusters of A. flavus or A. fumigatus isolates could be defined by using the graphing algorithm Minimum Spanning Tree. The typing method could be used for molecular epidemiological studies of A. flavus in many countries without the need for sophisticated equipment. Furthermore, data obtained by the present method could be easily shared in a web database. The clustering analysis was in accordance with a geographical structuring of A. flavus isolates similar to that detected in A. fumigatus.The third part of the work is about the antifungal susceptibility of177A. fumigatus isolates collected in avian farms in China and France. Most of the isolates from China were susceptible to itraconazole with a Minimum Inhibitory Concentration (MIC) comprised between0.38and0.75ug/mL Most of the isolates from birds and avian farms in France were susceptible to itraconazole with a MIC comprised between0.19and1μg/mL. MIC values of isolates collected in farms with antifungal chemoprophylaxis were not higher than those of isolates collected from birds that never received antifungal drugs before the sampling. Susceptibility testings demonstrated that4isolates should be considered as resistant to itraconazole:2isolates from avian farms in Guangxi, China and2isolates from avian farms in France. A modification of the Cyp51A sequence was identified in11isolates (3azole-resistant and8azole-susceptible isolates). Twenty-one nucleotidic mutations were detected.Twelve of these mutations is silent and9yielded to amino acid substitutions. Six of these substitutions had already been described whereas mutations A116R, E130D and Q131H were original. Continued surveillance of azole resistance should be maintained to determine the resistance rates and any possible trend of increase in the isolation frequency of resistant strains. Based on the association with agricultural azole use, environmental sampling remains significant as well.