Molecular typing of clinical and environmental Aspergillus fumigatus isolates from Iran using microsatellites

Background and Purpose: Because of the growing incidence of Aspergillus infection, typing methods of Aspergillus species are increasingly being used. Accordingly, studying the spread and population dynamics of strains isolating from clinical and environment, from a single host to large-scale ecosystems is definitely needed. In the current study, we carried out a genetic analysis of nine microsatellite loci in isolates from different regions of Iran to compare and explore the genetic diversity between environmental and clinical A. fumigatus strains. Materials and Methods: Sixty-six clinical (n=43) and environmental (n= 23) isolates of A. fumigatus, have collected from six cities of Iran. All A. fumigatus isolates identified based on macroscopic and microscopic characters, the ability to grow at above 45°C, and confirmed using DNA sequencing of the partial b-tubulin gene. Sixty-six A. fumigatus isolates were subjected by microsatellite typing using three separate multiplex PCRs with a panel of nine short tandem repeats (STR) to evaluate the genetic relatedness. Results: The STR typing of 66 A. fumigatus isolates revealed 38 distinct genotypes distributed among environmental and clinical isolates. We identified 12 clones including 40 different isolates representing 60% of all isolates tested, which each clone included 2–7 isolates. Conclusion: The STR typing is considered as a valuable tool with excellent discriminatory power to study the molecular epidemiology and genotypic diversity of A. fumigatus isolates. These findings show that the high genetic diversity observed of Iranian A. fumigatus isolates with those outside Iran and formed a separate cluster.


Introduction
spergillus fumigatus is an everywhere saprophytic mold with a global distribution frequently occurred in human fungal infections ranging from slight allergic reactions, colonization to serious systemic infections. Invasive aspergillosis (IA) is a main reason of infections among immunocompromised individuals with over 200,000 life-threatening fungal infections yearly [1][2][3][4]. Remarkably, A. fumigatus is the predominant species to be found from cultures of respiratory samples in cystic fibrosis patients with frequency approximately from 6% to 60% [5,6]. The incidence of IA due to A. fumigatus infections in susceptible patients has dramatically increased in recent years due to construction, renovation, demolition and excavation activities in hospitals and clinical centers [7,8]. In addition, failure in treatment of infections caused by acquired triazoles resistant A. fumigatus isolates have been increasingly reported, recently. [9][10][11]. Aspergillus flavus is another species of Aspergillus that is more common in Iran due to tropical and subtropical climatic conditions. Various studies in Iran from clinical and environmental isolates of A. flavus have identified using molecular and genotyping techniques [12][13][14]. Antifungal resistance is a major threat for treatment and prophylaxis of fungal infections in both immunocompetent and immunocompromised host. Resistance to azole can occur in patients who are used azole for long-term treatment for the management of invasive aspergillosis or may acquire from the environment as a consequence of exposure to azole fungicides applied in agriculture [2,[15][16][17]. Therefore, the international surveillance network of A. fumigatus azole resistance designed a global project to realize how resistance is created in the environment. This project conducts various studies with the aim of maintaining the use of azoles for the production of food and human medicines [18]. Regarding the upward trend in incidence of Aspergillus infection, typing methods of Aspergillus species are extremely being used. Accordingly, studying the spread and population dynamics of strains isolating from clinical and environment, from a single host to large-scale ecosystems is definitely needed [19,20]. Nowadays, molecular typing creates new methods for better infection control. Also, it's useful for infection or colonization of a single patient and determine the population structure of a species and study of the epidemiological association between environmental and clinical isolates. The disadvantages of pervious molecular typing of A. fumigatus methods such as (AFLP), multilocus sequence typing (MLST), pulsedfield gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), RNA-induced silencing complex (RISC), repetitive element sequence-based PCR (rep-PCR), single-stranded DNA binding protein (SSDP), and variable number tandem repeat (VNTR) is lack of discriminatory power and poor interlaboratory reproducibility. However, microsatellites were used to determine and analyzed the genetic distances and have demonstrated to be powerful instruments for molecular typing [20][21][22]. Microsatellites are repeated motifs of about 1-9 non-coding nucleotides fragments. Due to high rates of mutation and variability, microsatellite sequence modifications is particularly useful for studying differences between closely related species [19,23]. Nevertheless, genetic variation and molecular epidemiology of A. fumigatus from different sources in Iran are underestimated. Hence, the main object of this study was to investigate the molecular epidemiology of clinical and environmental A. fumigatus isolates from Iran using microsatellite typing.

Microsatellite A. fumigatus typing
To perform microsatellite typing, three distinct multiplex PCRs with a panel of nine short tandem repeats (STR) were designed to evaluate the genetic relationship between the isolates as previously described [25]. To differentiate the three loci within one multiplex PCR the forward primers were labeled with FAM-, JOE-and HEX-fluorphore at the 5´side. Briefly, PCR assays were implemented in a volume of 25 µl, containing 1 µM of all amplification primers, 1 U of FastStart Taq DNA polymerase (Roche Diagnostics), 0.2 mM deoxynucleoside triphosphates and 2µl of target DNA in 1× reaction buffer. Thermocycling was done in a thermocycler )Westburg-Biometra USA ( as described before [25]. PCR products were diluted 100-fold with ddH2O; subsequently 1 µl of this diluted PCR product was added to 8.9 µl ddH2O and 0.1 µl of CC-500-ROX marker (Promega, Leiden, TheNetherlands). The samples were boiled for 1 min at 100 °C and the fragment sizes were defined using an ABI3500xL Genetic Analyzer platform, afterward (Applied Biosystems, Foster City, CA, USA) according to the manufacturer instructions. The genomic link among A. fumigatus strains was determined by comparing the profiles with BioNumerics v6.6 software (Applied Maths, Sint-Martens-Latem, Belgium). To generate the dendrogram, or the option to generate a minimum spanning tree directly from the categorical data, un-weighted paired group method was applied using arithmetic average (UPGMA) algorithm was applied. The Simpson index diversity (D) was used to measure the genetic distinction or diversity between A. fumigatus isolates. A 'D' value of 1 indicates that all isolates are different whereas a 'D' value of 0 indicates that all isolates are identical [26,27].

Results
Sixty-six A. fumigatus isolates from different sources were genotyped using the full panel of 9 short tandem repeats markers. The Simpson's index of diversity was less than 0.9 for all nine markers combined. According to the STR typing, 38 distinct genotypes were distributed among environmental and clinical isolates. Remarkably, table 1 summarized the number of clinical strains which was higher than environmental one; more different genotypes was found in the clinical isolates (n=8) rather than in the environmental one (n=2). Among all genotypes, 26 (68.4%) genotypes were only found once, 5 (13.1%) genotypes were observed 2 times, 3 (7.8%) genotypes were observed 3 times, 2 (5.2%) genotypes were observed 4 times, 1 (2.6%) genotypes were observed 6 times, and 1 (2.6%) genotypes were observed 7 times (Figure 1). In this dendrogram, two of related genotypes could be identified differing only at a single locus. Twelve clones comprising 40 different isolates and representing 60% of all isolates were identified. Each clone included 2-7 isolates. According to genotyping results two isolates from soil samples and one clinical isolates from BAL sample of hospitalized patient were identical. We identified a clonal cluster including 6 (4 environmental and 2 clinical isolates), azole resistant isolates harboring cyp51A gene mutations. The three environmental strains of this cluster were concurrently isolated from Tehran's' hospitals, one isolated from a patient hospitalized in Tehran, one isolated from a patient hospitalized in Sari, and one isolate originated from environment of hospital in Mashhad city (Figure 1). Figure 2 illustrates the genotypic diversity distribution of the environmental and clinical A. fumigatus isolates. Eight genotype clusters included only clinical isolates, two cluster only environmental isolates and two clusters contained clinical and environmental isolates. Figure  3 show the geographically diverse A. fumigatus  isolates. The STR typing depicted no genotypic correlation of Iranian A. fumigatus with isolates from other countries. In addition, the high genetic diversity observed of Iranian A. fumigatus isolates with those outside Iran and formed a separate cluster ( Figure 4).

Discussion
A. fumigatus is globally found in the hospital environments and most frequently isolated from patients with IA or aspergilloma [28][29][30][31][32][33][34]. the highest airborne Aspergillus conidia concentrations due to the construction inside or surroundings area of the hospitals as well as problems in the ventilation or conditioner systems have proposed the environment to be the potential source of infection [35,36]. Definitely, monitoring the genotype of the isolates is the only way to determine that airborne conidia have caused the infection [37]. Several molecular typing techniques have been extended in order to understand epidemiological relationships between environmental and clinical isolates obtained from various origins to more specifically demonstrate the source of disorder [38]. In several studies, various methods have been performed for Aspergillus genotyping, but [39], they lack the essential reproducibility between experiments [40,41]. de Valk et al, newly described a novel panel of 9 STRs for genotyping of A. fumigatus with highly discriminatory power, clear assignment, interlaboratory exchangeability of the results [25]. However, it has rarely been used for epidemiological study of IA outbreaks. Kidd et al. [42] and Balajee et al. [43] designed an epidemiological study of invasive aspergillosis outbreaks in hospitals wards and validated STRAf as a main genotyping tool. Unlike other Aspergillus species, there is little information about the genotypic diversity of A. fumigatus from different sources in Iran [44]. Here, it was revealed  [45,46]. Unlike Bart-Delabesse et al., we were able to two cluster isolates by their clinical or environmental origin [46]. Our study confirmed results obtained by de Valk et al. reported that majority of the patients were affected with only one genotype [23]. This observation suggesting a common environmental source in these patients and it can lead to new approaches to infection control to prevent aspergillosis in immunocompromised patient. In our pervious study, genotyping analysis identified that 41 out of 44 A. fumigatus strains with the TR34/L98H mutation, isolated from compost in 13 different Iranian cities, shared the same allele across all nine examined microsatellite loci [22]. Like our finding, Chowdhary et al. illustrated a clonal spread and emergence of environmental azole resistant A. fumigatus isolates from different parts of India. This strains shared the same genotype not found in any other evaluated samples within or outside of India [47].

Conclusion
The STR typing is considered as a valuable tool with excellent discriminatory power to study the molecular epidemiology and genotypic diversity of A. fumigatus isolates. Generally, these findings show that the high genetic diversity observed of Iranian A. fumigatus isolates with those outside Iran and formed a separate cluster.