Interrogating the spectrum of bacterial pathogens in ulcers of cutaneous leishmaniasis

Abstract

Purpose: Secondary bacterial infections are commonly identified in ulcers of cutaneous leishmaniasis (CL), thus, antibiotics are often empirically prescribed prior to anti-Leishmania therapy. Here, we aim to identify the spectrum of bacterial pathogens present in CL ulcers to better enhance antimicrobial stewardship and targeted antimicrobial therapy. Methods & Materials: DNA was extracted from filter paper lesion impressions (FPLIs) of 6 CL ulcers and amplified by real-time PCR (qPCR) and end-point PCR targeting organisms implicated in skin- and soft-tissue infections. Targeted species included: Staphylococcus aureus, Enterobacter cloacae, Enterococcus spp., Citrobacter freundii, Klebsiella pneumoniae, and 16S rDNA. Amplified products were Sanger sequenced for bacterial species confirmation. Samples were then sent for Whole-Genome Sequencing (WGS). The severe CL phenotype was defined as: intercurrent mucosal involvement; multifocal disease (ulcers ≥4 in number in ≥2 anatomic locations); purulent/exudative ulcers with associated erythema and pain (“secondarily infected” appearance); or ulcers with lymphatic involvement. Results: Of 6 FPLIs, 4 (68%) were identified as Leishmania Viannia braziliensis, 1 (16%) was L. V. peruviana, and 1 (16%) was L. tropica. Based on single assay detection, the following common potentially pathogenic flora were detected in all 6 FPLIs: S. aureus (100%), C. freundii (100%), Enterobacter spp. (67%), and Klebsiella pneumoniae (67%). Whole Genome Sequencing yielded identification of many environmental and skin contaminants; however, Brevundimonas nasdae was detected in 3 out of the 6 FPLIs (50%), in addition to all of the aforementioned flora using the single assay detection protocol. Overall, the distribution of flora in ulcers of severe (n = 2) and non-severe phenotypes (n = 4) did not differ (p = 1.000). Conclusion: CL ulcers with a secondarily-infected phenotype are universally treated with broad-spectrum antibiotics, often without microbiological confirmation of presence of bacterial pathogens. Therefore, understanding the presence and complement of bacterial potential pathogens in ulcers of CL has implications for antimicrobial stewardship and evidence-based management strategies. WGS enables detection and identification of the full range of organisms present in the microbiome of the CL ulcer. Further prospective analysis, including additional WGS studies of CL ulcers, is necessary to determine the role of empiric antibiotic therapy in CL ulcers with an inflammatory phenotype.