Objective: To establish the genetic characteristics of the Colombian isolates of Histoplasma capsulatum (Hc) from environmental and clinical origin by MultiLocus Sequence Typing (MLST). Methods: There were collected environmental samples from organic fertilizers, cave floors and bird droppings. Environmental samples were evaluating for Hc presence by Hc100 nested PCR. Positive environmental samples were cultivating in Mycosel to obtain the isolate. The MLST proposed by Kasuga (1999/2003), using arf, tub, antiH and ole, was selected to compare Hc The PCR protocols were standardized to our laboratory conditions. Three group of sequences were used for molecular issues: a) DNA were obtained from 3 environmental and 28 clinical Colombian isolates, then MLST was applied. The PCR products were sequenced, and compared with: b) treefile matrix #1063, and c) gene sequences from GenBank. Geneious was used for the sequences analyses: a) to obtain consensus sequences for Colombian isolates; b) to build a matrix using the 3 sources sequences and c) to concatenate the genes; d) The matrix was analyzed using IqTree to stablish the evolutionary model and the relations between isolates by Maximum Likelihood and Ultrabootstrap. The phylogenetic tree obtained was visualized using FigTree. Results: 393 environmental samples were collected from 2010 to 2017. A total of 39(9.9%) environmental samples were positive for Hc100 nested PCR. Microbiological Hc cultures were positive in 1 sample, corresponding to a not-composted chicken manure, from this, 3 colonies were isolated. The standardized conditions for the modifiedMLST PCR led us amplifying the four genes using a conventional PCR protocol. 212 isolates are represented in the molecular analyses: 31 are new Colombian isolates, 80 from treefile #1063 and 101 from GenBank. The length for each locus was: arf: 465 bp, antiH: 415 bp, tub: 263 bp and ole: 416 bp. The concatenated matrix length was 1559. The evolutionary model was stablishing as K2+G4. The phylogenetic species at the tree were marked with a bootstrap over 70%. There was identified 17 phylogenetic species similarly to the results reported by Kasuga, 2003 and Teixeira, 2016. The Colombian isolates are distributed among the tree in at least 3 clades LAm A, LAm B and Nam 2. Also, the 3 Colombian environmental isolates are grouped together and share the same group with Colombian clinical isolates. Conclusion: The conditions described for the MLST PCR according to Kasugas’s protocol were not reproducible in our laboratory that made necessary to change the conditions to obtain the amplification products. In all distributions obtained by the phylogenetic analysis, the environmental isolates were grouped with the clinical isolates, which showed a genetic homology and these results suggest that the environment, especially the non-composted chicken manure is a potential source of infection with Hc. Additionally, there was observed great genetic heterogeneity among Colombian isolates, which indicates that the Colombian Hc population are polyphyletic like had been suggested in previous studies.
Full conference title:
- ISHAM 20th (2018)