Strongyloides stercoralis infection induces gut dysbiosis in chronic kidney disease patients

Background Strongyloides stercoralis infection typically causes severe symptoms in immunocompromised patients. However, the progression of infection-driven chronic kidney disease (CKD) is not understood fully. Recent studies have shown that gut dysbiosis plays an important role in the progression of CKD. Hence, this study aims to investigate the effect of S. stercoralis infection on the gut microbiome in CKD patients. Methodology/Principal Findings Among 838 volunteers from Khon Kaen Province, northeastern Thailand, 40 subjects with CKD were enrolled and divided into two groups ( S. stercoralis -infected and -uninfected) matched for age, sex and biochemical parameters. Next-generation technology was used to amplify and sequence the V3-V4 region of the 16S rRNA gene to provide a profile of the gut microbiota. Results revealed that members of the S. stercoralis -infected group had lower gut microbial diversity than was seen in the uninfected group. Interestingly, there was significantly greater representation of some pathogenic bacteria in the S. stercoralis -infected CKD group, including Escherichia-Shigella ( P = 0.013), Rothia ( P = 0.013) and Aggregatibacter ( P = 0.03). There was also a trend towards increased Actinomyces , Streptococcus and Haemophilus ( P > 0.05) in this group. On the other hand, the S. stercoralis -infected CKD group had significantly lower representation of SCFA-producing bacteria such as Anaerostipes ( P = 0.01), Coprococcus _1 (0.043) and a non-significant decrease of Akkermansia , Eubacterium rectale and Eubacterium hallii ( P > 0.05) relative to the uninfected group. Interesting, the genera Escherichia-Shigella and Anaerostipes exhibited opposing trends, which were significantly related to sex, age, infection status and CKD stages. The genus Escherichia-Shigella was significantly more abundant in CKD patients over the age of 65 years and infected with S. stercoralis . A correlation analysis showed inverse moderate correlation between the abundance of the genus of Escherichia-Shigella and the level of estimated glomerular filtration rate (eGFR). Conclusions/Significance Conclusion, the results suggest that S. stercoralis infection induced gut dysbiosis in the CKD patients, which might be involved in CKD progression.

Scientific) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an 153 Illumina platform and 250 bp paired-end reads were generated. Processing and quality control of 154 these reads used the following steps: 1) Data split (based on their unique barcode) and truncation 155 by cutting off the barcode and primer sequences; 2) Sequence assembly (Paired-end reads were 156 merged using FLASH [26] and generated the raw tags; 3) Data filtration (quality filtering on the compared with a reference database [29] using the UCHIME algorithm [30] to detect chimera sequences, which were then removed [31]. 161 Sequences sharing ≥97% similarity were assigned to the same operational taxonomic unit   CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) accumulation curves showed a saturation phase (Fig. 1). This indicates that the sample size was 210 sufficient to capture the overall microbiota structure.

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The unweighted UniFrac distances, reflecting beta diversity, showed significant 212 differences between the Ss+ and Ss groups (P = 0.00019). In terms of alpha diversity overall, 213 there were no significant differences in estimated OTU richness, Chao1, the ACE metric, the 214 Shannon diversity index and Good's coverage, (P > 0.05) ( Table 3). In contrast, the alpha 215 diversity in males in the Ss group was significantly higher than in males in the Ss+ group 216 (Shannon diversity index, P = 0.015; Simpson diversity index, P = 0.058) (Fig 2B, C).

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The principal coordinate analysis (PCoA) was used to illustrate the beta diversity based 218 on the unweighted UniFrac distances (Fig 2D). PCoA analysis revealed that the gut microbiota 219 of Ss+ subjects showed deviation from the Ss group ( Fig 2E).

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LDA score showed the significantly in abundance between the two groups. The candidate 221 biomarker for the Ss category was order Bradymonadales and for CKD with S. stercoralis 222 infection (Ss+) was species E. coli, genera Escherichia-Shigella, Dialister, family 223 Veillonellaceae and order Selenomonadales (Fig 3).

Differences in bacterial abundance between the Ss+ and Ss groups
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Proportions of sequence reads were compared between groups at the phylum and genus 226 levels using Metastats. At the phylum level, there were no significant differences (Fig 4). For  (Table 4).

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To study the similarity among different samples, clustering analysis was applied. The 233 unweighted pair group method with arithmetic mean (UPGMA), a type of hierarchical clustering 234 method widely used in ecology, Ss vs. Ss+ samples tended to cluster separately (Fig 5).  is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted March 9, 2022. ; https://doi.org/10.1101/2022.03.07.22271995 doi: medRxiv preprint  In this study, we first characterized the gut microbiota of CKD with and without S.  Our results revealed that gut microbiota was altered by S. stercoralis infection in CKD 265 patients. The alpha-diversity indices (Chao1, the ACE metric, the Shannon diversity index, 266 Good's coverage) did not significantly differ between the two groups. However, the Shannon 267 diversity index in males (n=12) in Ss+ group was significantly lower than in the Ss group. In 268 addition, the beta diversity, based on the unweighted UniFrac distances, was significantly lower 269 in the Ss+ group, suggesting a decrease in ecological diversity in CKD concurrent with S.
. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.  with low eGFR value and higher CKD stage ( Figure 6). In addition, Enterobacteriaceae and E.
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(which was not certified by peer review)
The copyright holder for this preprint this version posted March 9, 2022.   Our study has strengths and limitations. An important strength of this study is that we 327 used groups that were pair-matched for sex, age and biochemical factors. However, we did not 328 obtain data for concentrations of uremic toxins (TMAO and IS) or for amounts of SCFAs.        . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted March 9, 2022.  . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted March 9, 2022.       . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.    CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The histogram of the LDA scores presents taxa (potential biomarkers) whose abundance differed  CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 9, 2022. ; https://doi.org/10.1101/2022.03.07.22271995 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 9, 2022. ; https://doi.org/10.1101/2022.03.07.22271995 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 9, 2022. ; https://doi.org/10.1101/2022.03.07.22271995 doi: medRxiv preprint

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. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 9, 2022. ; https://doi.org/10.1101/2022.03.07.22271995 doi: medRxiv preprint