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Assunta Gergely
Human schistosomiasis is a highly prevalent neglected tropical disease (NTD) caused by Schistosoma species. Research on the molecular mechanisms influencing the outcomes of bladder infection by Schistosoma haematobium is urgently needed to develop new diagnostics, therapeutics and infection prevention strategies. The objective of the research study was to determine the microbiome features and changes in urine during urogenital schistosomiasis and induced bladder pathologies.
Seventy participants from Eggua, southwestern Nigeria provided morning urine samples and were screened for urogenital schistosomiasis infection and bladder pathologies in a cross-sectional study. Highthroughput NGS sequencing was carried out, targeting the 16S V3 region. Filtered reads were processed and analyzed in a bioinformatics pipeline.
The study participants (36 males and 34 females, between ages 15 and 65) were categorized into four groups according to status of schistosomiasis infection and bladder pathology. Data analytics of the next-generation sequencing reads revealed that Proteobacteria and Firmicutes dominated and had influence on microbiome structure of both non-infected persons and persons with urogenital schistosomiasis. Furthermore, gender and age influenced taxa abundance independent of infection or bladder pathology. Several taxa distinguished urogenital schistosomiasis induced bladder pathologies from urogenital schistosomiasis infection alone and from healthy persons, including known immune-stimulatory taxa such as Fusobacterium, Sphingobacterium and Enterococcus. Some of these significant taxa, especially Sphingobacterium were projected as markers of infection, while several genera including potentially beneficial taxa such as Trabulsiella and Weissella, were markers of the non-infected. Finally, expected changes in protein functional categories were observed to relate to cellular maintenance and lipid metabolism.
The human microbiome comprises bacteria (plus viruses, fungi and archeae) inhabiting different sites of the body. They do not specifically cause diseases, but their presence, absence or population influence body functions. We therefore examined such organisms found along the urinary tract, in persons living in a rural community in Nigeria who considered themselves healthy, were infected with the parasite Schistosoma haematobium or had developed bladder complications along with the parasite infection. We found that these groups shared a large portion of the microbiome, but there were microbial species unique to infected persons and those with bladder complication. Some of these were capable of inducing inflammation and could offer less protection to the host. We also predicted pathways that are affected by the difference in the microbiome.
Copyright: 2017 Adebayo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: ASA received travel support from The World Academy Academy of Sciences (TWAS)/ Department of Biotechnology (India) Postgraduate Fellowship in the course of the research. RDI acknowledges the award HRD-1435186 from the U.S. National Science Foundation. CIA acknowledges the World Health Organisation short term training grant B40394. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Research on molecular pathology influencing the outcomes of bladder infection by Schistosoma haematobium is urgently needed to develop new diagnostics, therapeutics and infection prevention strategies[3].Earlier studies have suggested that the mechanism of formation of bladder tumours will be due to formation of nitrosamines, polyaromatic hydrocarbons, free radicals, and presence of microbes [11]. More recently, studies have highlighted the role of estrogen-related molecules from the parasite in disease progression [12], based on the discovery that they could be oxidized to form adducts [13], induce infertility in females [14], and could probably induce error-prone DNA repair [15]. Molecules related to estrogen were recently detected in urine samples of persons infected with schistosomiasis [16].
The availability of volunteers with asymptomatic schistosomiasis in Eggua, in southwestern Nigeria allowed us to accomplish the research goal. Seventy volunteers were categorized into groups based on the presence of infection and induced bladder pathologies. The objectives of the study were to (1) determine the microbiome changes in urine samples during urogenital schistosomiasis and induced pathologies; and (2) identify functional biological processes that could be altered by such changes.
The approach of the research study consisted of recruiting study participants; screening for schistosomiasis infection and bladder pathologies; microbiome sequencing; pre-processing of microbiome sequences; and data analytics of microbiome sequence data collection.
Medical history, routine diet and demographic factors were obtained via structured questionnaire, and participants provided midstream urine samples. All samples were collected in the morning hours and immediately anonymized upon collection. The presence of analytes in the urine samples was immediately determined with Urinalysis Reagent Strips (Rapid Labs, UK), and urine microscopy was done on a 10 ml aliquot to detect S. haematobium eggs after sedimentation; and the rest was immediately frozen until further use and transported under dry ice conditions when required. Egg shedding may be infrequent in chronic infection, hence if a sample was negative for S. haematobium eggs, it was subjected to PCR detection using published Dra1 primers [25]. Briefly, 25ul PCR reaction mix containing 100ng isolated urine DNA was prepared. Cycling conditions were: initial denaturation at 95C for 5mins, 30 cycles of 95C for 30 secs, 55C for 30 secs and 72C for 1min, and final extension at 72C for 10mins. Bladder scans were carried out with Titan UltraSystem (Sonosite, WA, USA) by a trained radiologist, and all images scored according to WHO recommendations [26] and also anonymized.
(A) Mean relative abundance of various phyla in the urine microbiome of a study population in Eggua, southwestern Nigeria. (B) Bar plot of the Firmicutes/Proteobacteria log abundance ratio in different states of urogenital schistosomiasis. The correlation was not significant (p = 0.08). (C)Abundance of phyla Actinobacteria and Bacteroidetes with regard to gender in the urine microbiome of a study population in Eggua, southwestern Nigeria (p
Comparison of microbiome diversity indices (A) between urogenital schistosomiasis infection and controls, (B) in different states of urogenital schistosomiasis and controls, and (C) among age groups. Dark band represent mean diversity index, circles represent outliers. The differences in diversity were not significant (p>0.05) except in C(p = 0.038).
Beta diversity was assessed using Bray-Curtis metrics to estimate dissimilarities based on OTU level, rarefied data. Similar patterns were observed considering infection and pathology. The first two principal coordinates explained 36% of the variation among them, and a plot of the first two axes revealed little level of axes separation among the sample groups (Fig 4A). Of the ten most dominant OTUs, 4 of them, all assigned to the genus Pseudomonas, were prevalent (and in opposition to other prevalent OTUs) along the first axis (Fig 4A). They clustered close to several infected or pathology samples. Four other OTUs, all assigned to Staphylococcus were in contrast, driving the axes in the opposite direction, and clustering close to some control samples (Fig 4A).
(A) Principal Coordinates biplot of beta diversity in different states of urogenital schistosomiasis and controls. Circle and small typeset represent Greengenes ID of dominant OTUs. These dominant OTUs were mainly assigned to Pseudomonas and Staphylococcus, but had little influence on disease status. (B) Partial canonical correspondence analysis of age groups without the influence of urogenital schistosomiasis infection. Blue fonts are age categories, red typeface are Greengenes ID of the most influential OTU. Clustering together indicates no special correlation with age group. OTUs that corresponded closely with participants aged sixty years were mainly assigned to Acinetobacter.
There was a difference between sexes in the microbial community at the OTU and genus levels. There was more abundance of Actinobacteria and Bacteroidetes phyla in females than males (Fig 2C). More heterogeneity was observed in females, with 40% more OTUs present compared to males. There was a non-significant reduction in diversity in female samples. Using only control samples, unclassified Enterobacteriaceae and unclassified Pseudomonadaceae were significantly abundant in females (p = 0.035, p = 0.021, respectively).
Differentially abundant microbiome (A) between urogenital schistosomiasis induced bladder pathology (advanced) and healthy controls, and (B) between urogenital schistosomiasis (infection-only) and healthy controls (FDR
Microbiome sequence data was subjected (at OTU and genus levels) to discriminant analysis using LEfSe to identify possible biomarkers. Comparing infected and non-infected cases, all taxa that were significantly associated with each group from earlier significance analysis were also identified at the OTU level. At genus levels, only two of the significant taxa, Sphingobacterium and Aerococcus were projected as markers of infection, while several genera including Trabulsiella and Weissella, associated with non-infected (Fig 7).
Using our sequence abundance data, we obtained imputed metagenomes and the associated KEGG Orthology pathways present in the microbiome. Nearest Sequenced Taxon Index (NSTI) summary ranged from 0.021 to 0.053 (mean = 0.039). This indicated that several closely related genomes were available for inference purposes. A total of 328 functional categories from 6908 KOs were identified.
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