Authentication of Aspergillus parasiticus strains in the genome database of the National Center for Biotechnology Information

Objective The use of genome sequences from strains authenticated to correct species level is a prerequisite for confidently exploring the evolutionary relationship among related species. Aspergillus strains erroneously curated as Aspergillus oryzae and Aspergillus fumigatus have been noticed in the National Center for Biotechnology Information (NCBI) genome database. Aspergillus parasiticus is one of several aspergilli that produce aflatoxin, the most potent carcinogenic mycotoxin known up to now. To ensure that valid conclusions are drawn by researchers from their genomics-related studies, molecular analyses were carried out to authenticate identities of A. parasiticus strains in the NCBI genome database. Results Two of the nine supposedly A. parasiticus strains, E1365 and NRRL2999, were found to be misidentified. They turned out to be Aspergillus flavus based on genome-wide single nucleotide polymorphisms (SNPs) and genetic features associated with production of aflatoxin and cyclopiazonic acid. NRRL2999 lacked the additional partial aflatoxin gene cluster known to be present in its equivalent strain, designated as SU-1, and shared a very low total SNPs count specifically with A. flavus NRRL3357 but not with other A. flavus isolates. Therefore, the mislabeled NRRL2999 strain actually is a clonal strain of A. flavus NRRL3357, whose genome was first sequenced in 2005.

Molecular epidemiology of Sporothrix schenkii isolates in Malaysia

Sporothrix schenkii is a dimorphic fungus that causes infections in both humans and animals. We report on 25 S. schenkii isolates collected in 2017 from humans and cats clinically diagnosed with sporotrichosis, in Malaysia. These isolates were phenotypically identified as S. schenkii sensu lato and further defined as S. schenckii sensu stricto based on partial calmodulin gene sequence. Isolates from both humans and cats were genotypically identical but displayed phenotypic variation. Phylogenetic analyses based on partial calmodulin sequence showed that the Malaysian isolates clustered with global S. schenkii sensu stricto strains, in particular, of the AFLP type E. This analysis also revealed that partial calmodulin sequence alone was sufficient for classifying global S. schenckii sensu stricto strains into their respective AFLP types, from A to E. The genetically conserved S. schenkii sensu stricto species isolated from humans and cats is suggestive of a clonal strain present in Malaysia. To the best of our knowledge, this is the first report on molecular identification of Sporothrix schenkii strains from human infections in Malaysia. Further studies are required in order to elucidate the clonal nature of Malaysian S. schenkii isolates. Our findings indicate the presence of a predominant S. schenkii genotype in the environment, causing infections in both cats and humans in Malaysia.

232. Genomic Evidence for Dissemination of Mycobacterium marinum in an HIV Patient with Multifocal Cutaneous Disease

Background Hematogenous dissemination has been proposed to explain multifocal cutaneous disease caused by Mycobacterium marinum in certain patients. Treatment duration for disseminated disease is often months longer than for skin infection alone. However, distinguishing multiple independent inoculation events from dissemination has relied primarily on clinical judgement. Additionally, whether temperature-sensitive non-tuberculous mycobacteria such as M. marinum are indeed capable of invading the vascular space at core body temperature is debated. Here we used whole-genome sequencing (WGS) of serial isolates from a single patient with multifocal cutaneous M. marinum infection to distinguish dissemination of a clonal strain from multiple inoculation events. Methods A 35-year-old male with HIV (CD4 of 66 cells/µL) presented with a two-month history of a non-healing M. marinum wound on his left elbow (isolate MM0). This was followed a month later after initiation of antiretroviral therapy by a second M. marinum lesion on the right heel (MM1) without history of repeat inoculation, and increased swelling and erythema of the wound on the left arm (MM2) consistent with paradoxical immune reconstitution inflammatory syndrome. A PacBio genome was generated for MM0 and short read Illumina genomes were generated for MM1 and MM2. Results All isolates were found to be closely related, with MM1 and MM2 distinguished from MM0 by one and five single-nucleotide variants (SNVs), respectively. Given the substantial genetic heterogeneity among environmental M. marinum strains, such close relatedness of these isolates suggests common origin, and provides strong evidence for dissemination of a clonal strain in this patient. The SNVs included a frameshift mutation in the purT gene, which encodes a formate-dependent phosphoribosylglycinamide formyltransferase involved in de novo purine synthesis, and missense mutations in atsA and the DNA methylase hsdM. All isolates grew at 35°C, compared with the optimal growth temperature of 30°C typically observed for M. marinum, suggesting thermotolerance permissive for dissemination. Conclusion These results demonstrate the potential role of WGS for providing supportive evidence of disseminated infection with M. marinum. Disclosures All authors: No reported disclosures.

Multilocus Analysis Resolves the European Finch Epidemic Strain of Trichomonas gallinae and Suggests Introgression from Divergent Trichomonads

In Europe, Trichomonas gallinae recently emerged as a cause of epidemic disease in songbirds. A clonal strain of the parasite, first found in the United Kingdom, has become the predominant strain there and spread to continental Europe. Discriminating this epidemic strain of T. gallinae from other strains necessitated development of multilocus sequence typing (MLST). Development of the MLST was facilitated by the assembly and annotation of a 54.7 Mb draft genome of a cloned stabilate of the A1 European finch epidemic strain (isolated from Greenfinch, Chloris chloris, XT-1081/07 in 2007) containing 21,924 protein coding genes. This enabled construction of a robust 19 locus MLST based on existing typing loci for Trichomonas vaginalis and T. gallinae. Our MLST has the sensitivity to discriminate strains within existing genotypes confidently, and resolves the American finch A1 genotype from the European finch epidemic A1 genotype. Interestingly, one isolate we obtained from a captive black-naped fruit dove Ptilinopsus melanospilus, was not truly T. gallinae but a hybrid of T. gallinae with a distant trichomonad lineage. Phylogenetic analysis of the individual loci in this fruit dove provides evidence of gene flow between distant trichomonad lineages at 2 of the 19 loci examined and may provide precedence for the emergence of other hybrid trichomonad genomes including T. vaginalis.