An investigation on the role of skin microbiota in the treatment response against Cutaneous Leishmaniasis in the Kingdom of Saudi Arabia

Abstract

Cutaneous Leishmaniasis (CL) is a neglected tropical disease caused by Leishmania parasites and transmitted by phlebotomine sand flies. Worldwide, CL remains a major health problem as treatment is still limited and a vaccine to either prevent or treat the disease has not yet been developed. In the Kingdom of Saudi Arabia (KSA), localised CL is mainly caused by either L. major or L. tropica species, and sodium stibogluconate (SbV) is commonly used as an anti-leishmanial drug. A recent study has highlighted the potential role that the human skin microbiota may have in modulating the outcome of CL and the response to anti-leishmanial drugs. However, not much is known about the impact that both the human and insect microbiota have in the pathogenicity and transmission of this disease. In this thesis, I carried out a field study in two regions of KSA, Al-Hasa and Asir, which are endemic for L. major and L. tropica CL, respectively. The main aim was to identify the microbiota composition of CL ulcers from infected individuals, before or after receiving anti-leishmanial treatment. In addition, in order to establish a possible correlation between sand fly microbiota and disease severity, I explored the midgut bacterial composition of Phlebotomus papatasi sand fly, which is the main vector of zoonotic CL in the Al-Hasa region. It was found that most of L. major-infected patients had a better treatment response (i.e. healing) after receiving either antifungal and/or antibiotic regimes (first line) or first line followed by a course of intralesional injections of SbV. However, the majority of L. tropica patients did not respond favorably to SbV, regardless of the presence (or not) of secondary infections. An in vitro drug susceptibility assay corroborated the natural SbV refractoriness in L. tropica strains isolated from CL patients, in agreement with the observed lack of efficacy that this drug had in most L. tropica patients. Interestingly, these isolates appeared to be more susceptible to other antileishmanial drugs in vitro, including Miltefosine, Paromomycin and Amphotericin B. These findings support the use of antifungals and antibiotics as part of the treatment regime against L. major infections, but not for L. tropica patients. Moreover, it raises the possibility for using alternative anti-leishmanial drugs against L. tropica infections, which should help to minimise development and spread of SbV-resistant parasites. 16S metagenetic analysis showed that the skin of most CL patients is composed by bacteria of the Firmicutes, Actinobacteria and Proteobacteria phyla. Furthermore, skin infected from L. major patients showed a significant increase in bacterial richness and diversity (Shannon index) with a high dissimilarity level (ANOSIM, R=0.35, p=0.001) compared to L. tropica patients. Interestingly, L. major patients who healed after receiving only first line of treatment showed a high Shannon index compared to those whose responses were delayed or required administration of SbV. On the other hand, patients with predominantly dysbiotic skin showed a significant association with a delayed treatment response, regardless of the infecting parasite species. Furthermore, patients' samples showing a high abundance of Streptococcus, Bacillus and Acinetobacter correlated with a faster healing response. Likewise, the presence of Staphylococcus, Pseudomonas and Erwinia in high abundancy was associated with a delayed healing response. In addition, the 18S metagenetic analysis showed a similar distribution of fungal genera between lesions and adjacent skin with Malassezia spp as the dominant genus. Interestingly, lesions in either type of CL infections contain a significant low abundance of fungi reads, which in the case of L. major lesions correlated with a higher parasite load (Pearson r= -0.72, p=<0.005). Overall, these findings suggest