A New <i>Aspergillus fumigatus</i> Typing Method Based on Hypervariable Tandem Repeats Located within Exons of Surface Protein Coding Genes (TRESP)

<div><p><i>Aspergillus fumigatus</i> is a saprotrophic mold fungus ubiquitously found in the environment and is the most common species causing invasive aspergillosis in immunocompromised individuals. For <i>A</i>. <i>fumigatus</i> genotyping, the short tandem repeat method (STR<i>Af</i>) is widely accepted as the first choice. However, difficulties associated with PCR product size and required technology have encouraged the development of novel typing techniques. In this study, a new genotyping method based on hypervariable tandem repeats within exons of surface protein coding genes (TRESP) was designed. <i>A</i>. <i>fumigatus</i> isolates were characterized by PCR amplification and sequencing with a panel of three TRESP encoding genes: cell surface protein A; MP-2 antigenic galactomannan protein; and hypothetical protein with a CFEM domain. The allele sequence repeats of each of the three targets were combined to assign a specific genotype. For the evaluation of this method, 126 unrelated <i>A</i>. <i>fumigatus</i> strains were analyzed and 96 different genotypes were identified, showing a high level of discrimination [Simpson’s index of diversity (D) 0.994]. In addition, 49 azole resistant strains were analyzed identifying 26 genotypes and showing a lower D value (0.890) among them. This value could indicate that these resistant strains are closely related and share a common origin, although more studies are needed to confirm this hypothesis. In summary, a novel genotyping method for <i>A</i>. <i>fumigatus</i> has been developed which is reproducible, easy to perform, highly discriminatory and could be especially useful for studying outbreaks.</p></div

Minimum spanning tree (MST) showing the genotypic relationship between the azole-resistant and azole-susceptible <i>A</i>. <i>fumigatus</i> isolates.

<p>Each circle corresponds to a unique genotype, and the size of the circle proportionally represents the number of isolates with that genotype (1 to 21). Connecting lines correspond to the number of differences between the genotypes. Short bold line, 1 difference; black line, 2 differences; long grey line, 3 differences; dotted line, 4 or more differences. Grey circles, azole resistant TR₃₄/L98H, (n = 27); black circles, azole resistant non TR₃₄/L98H, (n = 22); white circles, azole susceptible strains, (n = 126). Four clusters were found (A-D).</p

Tandem repeats and flanking sequence for CSP types identified among 175 <i>A</i>. <i>fumigatus</i> isolates.

<p>Tandem repeats and flanking sequence for CSP types identified among 175 <i>A</i>. <i>fumigatus</i> isolates.</p

MP2 alleles identified among 175 <i>A</i>. <i>fumigatus</i> isolates.

<p>MP2 alleles identified among 175 <i>A</i>. <i>fumigatus</i> isolates.</p

Summary data for TRESP typing method using unrelated strains.

<p>Summary data for TRESP typing method using unrelated strains.</p

Assessing the effects of adsorptive polymeric resin additions on fungal secondary metabolite chemical diversity

<div><p>Adsorptive polymeric resins have been occasionally described to enhance the production of specific secondary metabolites (SMs) of interest. Methods that induce the expression of new chemical entities in fungal fermentations may lead to the discovery of new bioactive molecules and should be addressed as possible tools for the creation of new microbial chemical libraries for drug lead discovery. Herein, we apply both biological activity and chemical evaluations to assess the use of adsorptive resins as tools for the differential expression of SMs in fungal strain sets. Data automation approaches were applied to ultra high performance liquid chromatography analysis of extracts to evaluate the general influence in generating new chemical entities or in changing the production of specific SMs by fungi grown in the presence of resins and different base media.</p></div