Melanin depletion affects Aspergillus flavus conidial surface proteins, architecture, and virulence.

Melanin is an Aspergillus flavus cell wall component that provides chemical and physical protection to the organism. However, the molecular and biological mechanisms modulating melanin-mediated host-pathogen interaction in A. flavus keratitis are not well understood. This work aimed to compare the morphology, surface proteome profile, and virulence of melanized conidia (MC) and non-melanized conidia (NMC) of A. flavus. Kojic acid treatment inhibited melanin synthesis in A. flavus, and the conidial surface protein profile was significantly different in kojic acid-treated non-melanized conidia. Several cell wall-associated proteins and proteins responsible for oxidative stress, carbohydrate, and chitin metabolic pathways were found only in the formic acid extracts of NMC. Scanning electron microscopy (SEM) analysis showed the conidial surface morphology difference between the NMC and MC, indicating the role of melanin in the structural integrity of the conidial cell wall. The levels of calcofluor white staining efficiency were different, but there was no microscopic morphology difference in lactophenol cotton blue staining between MC and NMC. Evaluation of the virulence of MC and NMC in the Galleria mellonella model showed NMC was less virulent compared to MC. Our findings showed that the integrity of the conidial surface is controlled by the melanin layer. The alteration in the surface protein profile indicated that many surface proteins are masked by the melanin layer, and hence, melanin can modulate the host response by preventing the exposure of fungal proteins to the host immune defense system. The G. mellonella virulence assay also confirmed that the NMC were susceptible to host defense as in other Aspergillus pathogens. KEY POINTS: • l-DOPA melanin production was inhibited in A. flavus isolates by kojic acid, and for the first time, scanning electron microscopy (SEM) analysis revealed morphological differences between MC and NMC of A. flavus strains • Proteome profile of non-melanized conidia showed more conidial surface proteins and these proteins were mainly involved in the virulence, oxidative stress, and metabolism pathways • Non-melanized conidia of A. flavus strains were shown to be less virulent than melanised conidia in an in vivo virulence experiment with the G. melonella model.

Molecular identification of Nocardia species causing endophthalmitis using multilocus sequence analysis (MLSA): a 10-year perspective.

Introduction. Nocardia spp. can cause several ocular infections, such as keratitis, endophthalmitis and scleral abscesses. Molecular identification of Nocardia spp. by 16S rDNA sequencing is the gold standard method at present for species identification, but closely related species can only be identified by multilocus sequence analysis (MLSA) of housekeeping genes.Aim. The major objective was to profile Nocardia species, antibiotic susceptibility patterns and clinical outcomes in endophthalmitis patients.Methodology. Between January 2009 and December 2018, endophthalmitis patients who were diagnosed with Nocardia infection based on microscopic and culture characteristics were selected. Antibacterial susceptibility tests were performed and Nocardia speciation was performed using MLSA and phylogenetic tree analysis of the 16 s rRNA gene and the gyrB, hsp65 and secA1 genes.Results. A total of 43 culture-proven patients were identified during the study period. All isolates were 100 % sensitive to amikacin and 98 % resistant to ceftazidime. Fluoroquinolone sensitivity was observed in the range of 58 to 72 %. Year-wise analysis of antibiotic resistance patterns revealed there was a significant increase in resistance to fluoroquinolones. Twenty-two isolates were stored and six different species were identified. Nocardia farcinica (n=10) was found to be the most predominant, followed by Nocardia cyriacigeorgica (n=4), Nocardia otitidiscaviarum (n=3), Nocardia amikacinitolerans (n=2), Nocardia puris (n=2) and Nocardia higoensis (n=1).Conclusions. N. farcinica is the major pathogen, and this is the first report to identify N. otitidiscaviarum, N. amikacinitolerans and N. higoensis as causing endophthalmitis. Overall, visual outcomes were mostly poor even after aggressive management.

Clinical presentations, genotypic diversity and phylogenetic analysis of Acanthamoeba species causing keratitis.

Introduction. Acanthamoeba keratitis is a sight-threatening corneal infection that is commonly reported among contact lens users and those suffering from corneal trauma. The prevalence of Acanthamoeba species or genotypes in causing keratitis infection is not well known.Aim. This study was conducted to identify and genotype Acanthamoeba isolates from keratitis patients, targeting the ribosomal nuclear subunit (Rns) region, and describe the associated clinical presentation and treatment outcome.Methodology. Thirty culture-confirmed patients with Acanthamoeba keratitis, identified in a tertiary eye care centre in South India during the period from December 2016 to December 2018, were included in this study. The data collected from patient records include demographic details, history of illness, mode of trauma, treatment history and follow-up status. The genotype and species were identified based on the Rns sequence and phylogenetic tree analysis.Results. Acanthamoeba culbertsoni was the most predominant keratitis-causing species, followed by Acanthamoeba quina, Acanthamoeba castellanii, Acanthamoeba healyi, Acanthamoeba hatchetti, Acanthamoeba polyphaga and Acanthamoeba stevensoni. Three major genotypes were identified (T4, T11 and T12), with the T4 genotype being the most predominant, with four subclusters, i.e. T4A, T4B, T4D and T4E. This is the first report on corneal infection by the A. stevensoni T11 genotype and the A. healyi T12 genotype. No significant correlation was observed between the clinical outcomes of corneal disease and the genotypes or species.Conclusion. Rns genotyping is very effective in identifying the Acanthamoeba species and genotype in keratitis. Genotyping of Acanthamoeba spp. will help to advance our understanding of genotype-specific pathogenesis and geographical distribution.