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Distinct microbiome profiles and biofilms in Leishmania donovani-driven cutaneous leishmaniasis wounds.

ABSTRACT: The endemic strain of Leishmania donovani in Sri Lanka causes cutaneous leishmaniasis (CL) rather than more common visceral form. We have visualized biofilms and profiled the microbiome of lesions and unaffected skin in thirty-nine CL patients. Twenty-four lesions (61.5%) were biofilm-positive according to fluorescence in situ hybridization. Biopsies of biofilm-positive lesions were dominated by Pseudomonas, class Bacilli and Enterobacteriaceae and distinguished by significantly lower community evenness. Higher relative abundance of a class Bacilli OTU was detected in wound swabs versus contralateral skin. Wound swabs and biopsies had significantly distinct microbiome profiles and lower diversity compared to unaffected skin. Greater abundances of potentially pathogenic organisms were observed in wet ulcers, lesions with high parasite loads and large wounds. In summary, more than half of L. donovani associated CL wounds harboured biofilms and the wounds exhibited a distinct, less diverse, microbiome than unaffected skin.

SUBMITTER: Jayasena Kaluarachchi TD

PROVIDER: S-EPMC8633208 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Distinct microbiome profiles and biofilms in Leishmania donovani-driven cutaneous leishmaniasis wounds.

Jayasena Kaluarachchi T D TD Campbell Paul M PM Wickremasinghe Rajitha R Ranasinghe Shalindra S Wickremasinghe Renu R Yasawardene Surangi S De Silva Hiromel H Menike Chandrani C Jayarathne M C K MCK Jayathilake Subodha S Dilhari Ayomi A McBain Andrew J AJ Weerasekera Manjula M MM

Scientific reports 20211130 1

The endemic strain of Leishmania donovani in Sri Lanka causes cutaneous leishmaniasis (CL) rather than more common visceral form. We have visualized biofilms and profiled the microbiome of lesions and unaffected skin in thirty-nine CL patients. Twenty-four lesions (61.5%) were biofilm-positive according to fluorescence in situ hybridization. Biopsies of biofilm-positive lesions were dominated by Pseudomonas, class Bacilli and Enterobacteriaceae and distinguished by significantly lower community ...[more]

PMID: 34848752

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Project description:BackgroundThe innate immune mediators are likely to influence the clinical phenotype of leishmaniasis by primary responses which limit or facilitate the spread of the parasite, as well as by modulating adaptive immunity. This study investigated the response of key innate immune cells in a focus which regularly reports localised cutaneous leishmaniasis (LCL) caused by Leishmania donovani, a species which typically causes visceral disease.MethodsPeripheral blood mononuclear cell (PBMC) derived macrophages and dendritic cells from patients with LCL and healthy controls from endemic and non-endemic areas, were stimulated with soluble Leishmania antigen (SLA). Inflammatory mediators produced by macrophages (TNF-α/TGF-β/IL-10, ELISA; NO, Griess method) and dendritic cells (IL-12p70, IL-10, flowcytometry) and macrophage expression of surface markers of polarization, activation and maturation (flowcytometry) were determined at 24h, 48h and 72h and compared. Study was conducted prospectively from 2015-2019.ResultsPatient derived macrophages and dendritic cells produced higher levels of both pro and anti-inflammatory mediators compared to controls (p<0.05) with the best discrimination for active disease observed at 72h. Data demonstrated an early activation of macrophages and a subsequent pro-inflammatory bias, as indicated by temporal profiles of TNF-α/TGF-β and TNF-α/IL-10 ratios and higher proportions of classical (M1) macrophages. Higher TGF-β levels were observed in cells from patients with ulcerated or persistent lesions. Immune responses by cells derived from controls in endemic and non-endemic regions did not differ significantly from each other.ConclusionsThe overall immunophenotypic profile suggests that LCL observed in the country is the result of a balancing immune response between pro-inflammatory and regulatory mediators. The mediators which showed distinct profiles in patients warrant further investigation as potential candidates for immunotherapeutic approaches. A comparison with visceral leishmaniasis caused by the same species, would provide further evidence on the differential role of these mediators in the resulting clinical phenotype.

Project description:BackgroundLeishmaniasis is a common neglected tropical disease in Ethiopia. Visceral leishmaniasis (VL) caused by Leishmania donovani presents in the lowlands, while cutaneous leishmaniasis (CL) affects people living in the highlands. Although CL is described as being caused by Leishmania aethiopica, there is also evidence of L. tropica and L. major isolated from a patient, sand flies and potential reservoirs. Information on species causing CL in Ethiopia is patchy, and no nation-wide study has ever been done. Understanding which species are causing CL in Ethiopia can have important implications for patient management and disease prevention.MethodsWe analyzed stored routine samples and biobanked DNA isolates from previously conducted studies of CL patients from different centers in the north, center and south of Ethiopia. Species typing was performed using ITS-1 PCR with high-resolution melt (HRM) analysis, followed by HSP70 amplicon sequencing on a selection of the samples. Additionally, sociodemographic, clinical and laboratory data of patients were analyzed.ResultsOf the 226 CL samples collected, the Leishmania species could be determined for 105 (45.5%). Leishmania aethiopica was identified in 101 (96.2%) samples from across the country. In four samples originating from Amhara region, northwestern Ethiopia, L. donovani was identified by ITS-1 HRM PCR, of which two were confirmed with HSP70 sequences. While none of these four patients had symptoms of VL, two originated from known VL endemic areas.ConclusionsThe majority of CL was caused by L. aethiopica, but CL due to L. tropica and L. major cannot be ruled out. Our study is the first to our knowledge to demonstrate CL patients caused by L. donovani in Ethiopia. This should spark future research to investigate where, how and to which extent such transmission takes place, how it differs genetically from L. donovani causing VL and whether such patients can be diagnosed and treated successfully with the currently available tools and drugs.

Project description:Among the most central questions in Leishmania research is why some species remain in the skin dermis at the site of infection by the sand fly vector whereas other species migrate to visceral organs where they cause fatal visceral leishmaniasis. Although L. donovani is the species typically responsible for visceral leishmaniasis, an atypical L. donovani strain is the etiologic agent for cutaneous leishmaniasis in Sri Lanka. To identify molecular determinants for visceral disease, we have analysed the phenotype and genotype of two L. donovani clinical isolates from Sri Lanka where one isolate was derived from a cutaneous leishmaniasis patient (CL) and the other from a visceral leishmaniasis patient (VL). These isolates cause dramatically different pathology when introduced into mice; notably the CL isolate has lost the ability to survive in visceral organs while the VL isolate was highly virulent in visceral organs of BALB/c mice. Whole genome sequencing of the CL and VL isolates revealed that these genomes were very similar as there were no gene deletions and few individual gene amplifications. Indels resulting in frame shifts and loss/gain of stop codons resulted in 13 distinct pseudogenes present in each of the CL and VL isolates. There were 154 non-synonymous SNPs specific to the CL isolate and 193 non-synonymous SNPs specific to the VL isolate. Genome wide gene expression analysis revealed several transcript level differences, including the A2 virulence gene resulting in higher expression of A2 proteins in the VL isolate than in the CL isolate. Genotypic variations relevant to pathology and tropism in Leishmania can be interrogated by reverse genetics. Experimentally increasing A2 expression in the CL isolate through gene transfer significantly increased it’s ability to survive in the spleen of BALB/c mice and conversely, down-regulating A2 expression in the VL isolate abrogated attenuated its survival in BALB/c mice. These observations reveal that there are relatively few genetic differences between the CL and VL isolates apart from the A2 genes, but collectively these have profound effects on human disease and experimentally infected mice.

Project description:Sri Lanka is a recent focus having Leishmania donovani induced cutaneous leishmaniasis (CL) as the main clinical entity. A separate clinical entity within profile of CL was described in this study. Laboratory confirmed cases of CL (n= 950, 2002-2014) were analysed. Most lesions showed known classical developmental stages of CL (CCL) observed in other CL endemic settings while few cases (13%, 122/950) showed atypical skin manifestations (ACL). Clinical, geographical, and treatment response patterns of ACL were different from those of CCL. ACL was mainly found among males (68.0%), in 21-40 year age group (51.6%), and reported delayed treatment seeking (23.5% vs 16.3% in CCL), more nonclassical onset (lesions other than acne form <1cm sized papules), (12.1 vs 2.7%, P<0.05.), more head and neck lesions (41.5%. vs 27.2%), more large lesions (>4cm), (18.6 vs 9.9%), and poor laboratory positivity rates (65.6% vs 88.2%) when compared to CCL. When compared to lesions reporting a typical onset, lesions reporting nonclassical onset were more likely to develop ACL later on (50.1% vs 10.7%). As compared to lesions on limbs, those on head and neck and trunk were more likely to be ACL (7.0%, 16.3%, and 22.8%, respectively, P<0.05). ACL features were not age or gender dependent. Highest proportion within ACL category (32.8%) and small proportion of CCL (10.1%) originated from less leishmaniasis prevalent areas (other regions) (P<0.05). North reported more ACL than South (15.9% vs 7.4%). A total of 95 CL cases with a significant travel history were further analyzed. Residents of other regions when acquired infection from North or South developed more ACL than residents in North or South (60.9% vs 15.9% and 42.9% vs 7.4% respectively). Patients in other regions when travelled to North developed more ACL than when they travelled to South (60.9%, 42.9%). ACL and CCL required an average of 18 doses over 16.7 months and 10 doses over 12 weeks, respectively, to achieve a complete clinical cure. Underlying host immunological factors, parasite strain variations and regional variations of both could be underlying etiologies. Established independent transmission within less leishmaniasis prevalent regions combined with an unusual clinical picture leading to poor clinical suspicion and low laboratory confirmation rate will pose potential difficulties in early case detection in these highly populated and commercialized areas. This in turn will further facilitate silent and high disease transmission.

Project description:Among the most central questions in Leishmania research is why some species remain in the skin dermis at the site of infection by the sand fly vector whereas other species migrate to visceral organs where they cause fatal visceral leishmaniasis. Although L. donovani is the species typically responsible for visceral leishmaniasis, an atypical L. donovani strain is the etiologic agent for cutaneous leishmaniasis in Sri Lanka. To identify molecular determinants for visceral disease, we have analysed the phenotype and genotype of two L. donovani clinical isolates from Sri Lanka where one isolate was derived from a cutaneous leishmaniasis patient (CL) and the other from a visceral leishmaniasis patient (VL). These isolates cause dramatically different pathology when introduced into mice; notably the CL isolate has lost the ability to survive in visceral organs while the VL isolate was highly virulent in visceral organs of BALB/c mice. Whole genome sequencing of the CL and VL isolates revealed that these genomes were very similar as there were no gene deletions and few individual gene amplifications. Indels resulting in frame shifts and loss/gain of stop codons resulted in 13 distinct pseudogenes present in each of the CL and VL isolates. There were 154 non-synonymous SNPs specific to the CL isolate and 193 non-synonymous SNPs specific to the VL isolate. Genome wide gene expression analysis revealed several transcript level differences, including the A2 virulence gene resulting in higher expression of A2 proteins in the VL isolate than in the CL isolate. Genotypic variations relevant to pathology and tropism in Leishmania can be interrogated by reverse genetics. Experimentally increasing A2 expression in the CL isolate through gene transfer significantly increased itM-bM-^@M-^Ys ability to survive in the spleen of BALB/c mice and conversely, down-regulating A2 expression in the VL isolate abrogated attenuated its survival in BALB/c mice. These observations reveal that there are relatively few genetic differences between the CL and VL isolates apart from the A2 genes, but collectively these have profound effects on human disease and experimentally infected mice. 6 Samples in total, 3 each from VL and CL causing isolates were analyzed by Splice Leader RNASeq. These three samples from each of the isolates were grown to form one of the following three lifestages, Promastigotes, Macrophage derived Amastigotes, Axenic Amastigotes.

Dataset Information

Distinct microbiome profiles and biofilms in Leishmania donovani-driven cutaneous leishmaniasis wounds.

Publications

Distinct microbiome profiles and biofilms in Leishmania donovani-driven cutaneous leishmaniasis wounds.

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