Impact of Enterobius vermicularis infection and mebendazole treatment on intestinal microbiota and host immune response (2024)

Chin-An Yang, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Visualization, Writing – original draft, Writing – review & editing,^1,2,3 Chao Liang, Data curation, Formal analysis, Methodology, Software, Validation, Visualization, Writing – original draft,⁴ Chia-Li Lin, Investigation, Project administration, Validation,⁵ Chiung-Tzu Hsiao, Methodology, Project administration, Resources,¹ Ching-Tien Peng, Project administration, Resources, Supervision,^6,7 Hung-Chih Lin, Funding acquisition, Resources, Supervision,^#8 and Jan-Gowth Chang, Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review & editing^#1,3,9,*

Ethics statement

The study cohort consisted of 109 primary school children (1^st and 4^th grades) who had undergone pinworm screening in 2015, Taichung, Taiwan. The study was approved by the Research Ethics Committee of China Medical University Hospital (CMUH104-REC1-115). Written informed consent was obtained from parents.

Study design

A total of 30 children were tested negative for enterobiasis, while 79 were tested positive by anal tape screening. In children with positive pinworm anal tape results, additional stool samples were collected in tipped ova concentration tubes and were stained and fixed with freshly prepared merthiolate-iodine formaldehyde (MIF). After further mixing with ethyl acetate and centrifugation at 1500 rpm for 5 minutes, the sediments were examined carefully under light microscope to detect the presence of co-infected helminth eggs or protozoans as described previously [20]. Stool specimens were collected again from 65 pinworm-infected individuals 2 weeks following 100 mg mebendazole treatment, which underwent MIF-microscopic examination and 16s rRNA gene sequencing. We did not detect co-infection with other helminths or protozoans by MIF-concentration-sedimentation method in pinworm (+) samples before and after mebendazole treatment (Table 1). As shown in the flow diagram (S1 Fig), metagenomics analysis was performed on 30 pinworm (-), 65 paired pinworm (+) mebendazole (-) and pinworm (+) mebendazole (+) samples.

DNA extraction and gene sequencing

Stool specimens were collected at home and transported to our laboratory within 3 hours in ice, and were fixed in Transwab tubes (Sigma, Dorset, UK). DNA extraction was performed using the QIAamp DNA Stool Mini Kit (Qiagen, California, USA). PCR primers F515 (5’-GTGCCAGCMGCCGCGGTAA-3’) and R806 (5’-GGACTACHVGGGTWTCTAAT-3’), were designed to amplify the V4 domain of bacterial 16S ribosomal RNA gene as described previously [21]. The Nextera adapter sequence (Illumina, California, USA) was added to the 5’-end of the primer set for library preparation. PCR using 50~150 ng DNA was performed with 1 cycle of 98°C for 30 sec, 30 cycles of 98°C for 10 sec, 60°C for 30 sec, 72°C for 30 sec, and a final extension of 72°C for 5 min. Amplicons were purified using the AMPure XP Beads (Beckman Coulter, Indianapolis, USA), and quantified using Nanophotometer (IMPLEN, München, Germany). The Illumina Nextera Index Primer kit was used to create the library. The qualities and quantities of purified libraries were checked by 2% agarose gel electrophoresis, Qubit (Thermo Fisher Scientific, Massachusetts, USA) and qPCR methods. Finally, libraries were normalized to the same concentration and sequenced by Illumina Miseq sequencer.

Bioinformatic analysis of 16S rRNA gene sequencing data

FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit) was used to process the raw read data files. Sequence qualities were checked in 3 steps: (i) The minimal acceptable Phred quality score of sequences was 20 (having over 70% of the sequence bases ≥ 20). (ii) Following quality trimming from the sequence tail, the sequences over 100 bp and those with an acceptable Phred quality score of 20 were retained. (iii) Both forward and reverse sequencing reads which met the first and second requirements were retained for subsequent analyses.

UPARSE [22] was used to create operational taxonomic unit (OTU) clustering. Bowtie2 [23] was then used to align OTUs with 16S rRNA gene sequences of bacteria. These sequences were taken from the SILVA ribosomal RNA sequence database (version 115). Following sequence data collection, sequences were extracted by V4 forward primer and reverse primer. To prevent repetitive sequence assignments, V4 sequences from SILVA were then grouped into several clusters by 97% similarity using UCLUST. A standard of 97% similarity with the database was applied.

Intestinal SIgA and cytokine detection

Fecal samples were weighed before adding equal amounts of sterile PBS together with Pierce proteinase inhibitor (Thermo Fisher Scientific). After thorough mixing and centrifugation at 10000 g for 10 minutes, the fecal supernatants were stored at -80°C until analysis. Stool secretory IgA (SIgA) was analyzed using Immundiagnostik ELISA kit (Bensheim, Germany), IL-1ß, and IL-4 levels were measured using Quantikine ELISA kits (R&D Systems, Minneapolis, USA) according to manufacturer’s instructions.

We further grouped the samples according to levels of SIgA, IL-1ß, and IL-4. Based on median levels detected, SIgA was considered high at >150 μg/ml, and low at <80 μg/ml; IL-1ß was high at >10 pg/ml, and low at <0.5 pg/ml; and IL-4 was high at >10 pg/ml, and low at <5 pg/ml. In paired samples (collected before and after mebendazole treatment), SIgA level was considered to be elevated given a greater than 1.1-fold increase.

Statistical analysis

A rarefaction process was applied to normalize the operational taxonomic unit (OTU) table following taxonomy assignment in the bioinformatic analyses. Alpha diversity (Shannon index, inverse Simpson index and richness) was calculated. Beta diversity using weighted UniFrac distance metrics [24], principal coordinate analysis (PCoA) and unsupervised clustering were performed. Multiple response permutation procedure (MRPP) in an R package “vegan” (https://cran.r-project.org/web/package=vegan) was performed to assess community difference in PCoA. Wilcoxon rank sum test were used to compare non-paired variables (e.g., P- v.s. P+M-), and Wilcoxon signed rank test were used to compare paired variables (e.g., P+M- v.s. P+M+). The differentially expressed bacteria were filtered by the following criteria: (i) P value < 0.05 (ii) Fold change > 1.40 or < 0.71 (iii) At least one group achieved an average relative abundance of 0.5%. Sex and pair factors were adjusted. For multiple group comparisons the false discovery rate (FDR) was controlled by using Benjamini-Hochberg (BH) FDR multiple test correction. Pathway enrichment analysis was performed using an R package 'Tax4Fun' [25]. ANOVA test was used to calculate the enrichment difference. Mann-Whitney U tests using GraphPad Prism version 5 were performed to compare fecal cytokine and SIgA levels between groups. Wilcoxon signed rank test was used to analyse the paired stool SIgA data before and after mebendazole treatment.

Study population and characteristics of intestinal microbiota

We analyzed the influence of pinworm infection on gut microbiome in a cohort of 109 children in the first or fourth grade of primary school. Grade and sex effects were both insignificant among the groups (p = 0.650, p = 0.403, respectively, Table 1). In the pinworm (+) group, additional stool specimens were collected to perform MIF concentration sedimentation procedure on to detect co-infection of other parasites. As shown in Table 1, no co-infection was detected. In the pinworm (-) group, possible confounding factors including a mostly-meat diet, recent (within these 2 months) gastroenteritis, recent respiratory tract infection with oral medication, and recent confirmed use of antibiotics were recorded, and diversity analysis of the gut microbiota showed that recent respiratory tract infection with oral medication might decrease the intestinal microbial diversity (Table 1). Further differential abundance analysis at the phylum level showed that children with recent respiratory tract infection and oral medication (8 of 10 had possible antibiotics usage) had a trend to correlate with relatively less abundance of Fusobacteria (0.01%± 0.18% vs. 1.65%±4.62%, p = 0.03, FDR = 0.17). Information about the above confounding factors was not available in the pinworm (+) group.

The alpha diversity of the pinworm (+) mebendazole (+) group was significantly higher than the pinworm (-) group (inverse Simpson index, p = 0.002), and the alpha diversity was only marginally higher when comparing the microbial composition between the pinworm (+) mebendazole (-) group and the pinworm (-) group (p = 0.061, Fig 1a). The principle coordinate analysis also showed a significant beta diversity difference among the 3 groups (pinworm (+) mebendazole (+) group vs. pinworm (-) group, p = 0.001, Fig 1b).

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Fig 1

Intestinal microbiota community comparison of pinworm infection and mebendazole treatment groups.

a. Boxplot of alpha diversity (Inverse Simpson index) is shown in genus level. **: p<0.01 by ANOVA test. P-: pinworm negative, n = 30; P+M-: pinworm positive before mebendazole treatment, n = 65; P+M+: pinworm positive after mebendazole treatment, n = 65. b. Principal coordinate analysis of weighted UniFrac beta diversity between subjects, colored by community subgroups. c. Bacterial relative abundance distribution of the community subgroups and differentially expressed phyla. *: p < 0.05 by Kruskal-Wallis test.

Analysis of the intestinal microbiome operational taxonomic units (OTUs) of our cohort revealed the following major bacterial phyla: Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, Verrucobacteria, and Fusobacteria. The phylum microbial distribution pattern differed significantly in the proportion of Actinobacteria (pinworm (+) mebendazole (+) vs. pinworm (-), 1.08% ± 1.15% vs. 4.23% ± 5.52%, fold = 2.56, P = 5.19 x 10⁻⁴, FDR = 0.012, Fig 1c) and Fusobacteria (pinworm (+) mebendazole (+) vs. pinworm (-), 0.04% ± 0.17% vs. 1.10% ± 3.82%, fold = 0.04, P = 3.30 x 10⁻³, FDR = 0.012, Fig 1c).

Impact of pinworm infection and mebendazole deworming treatment on intestinal microbial composition

At the genus level, a trend of higher relative abundance of Alistipes (fold = 2.56, p = 0.008) and Faecalibacterium (fold = 1.64, p = 0.004), and a decreased proportion of Fusobacterium (fold = 0.18, P = 0.050), Veilonella (fold = 0.25, p = 0.042), Megasphaera (fold = 0.28, p = 0.021), and Acidaminococcus (fold = 0.56, p = 0.030) were found in the pinworm (+) mebendazole (-) group as compared with the pinworm (-) group (Fig 2a). However, the corrected p values (FDRs) for all differentially distributed taxa were all > 0.05. In the 65 pinworm (+) mebendazole (+) subjects, the intestinal bacterial diversity further increased and was correlated with significantly more abundant Collinsella (fold = 3.04, p = 1.58 x 10⁻⁴, FDR = 0.028), Streptococcus (fold = 2.94, p = 1.32 x 10⁻³, FDR = 0.043), Blautia (fold = 1.71, p = 1.22 x 10⁻³, FDR = 0.043), as well as a lower proportion of Suterrella (fold = 0.30, p = 1.32 x 10⁻³, FDR = 0.043), as compared with the microbial composition of pinworm (+) mebendazole (-) group, Fig 2b. The relative abundance of the probiotic Bifidobacterium increased after pinworm infection, and it became even higher in the mebendazole treated group (pinworm (+) mebendazole (+) vs. pinworm (-), 7.32% ± 9.28% vs. 2.86% ± 3.67%, p = 1.97 x 10–3, FDR = 0.100, Fig 2a and 2b).

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Fig 2

Visualization of differentially expressed genus-level bacteria by volcano plots.

Red dots represent significant taxa analyzed by Wilcoxon rank sum test (pinworm (+) mebendazole (-) vs. pinworm (-)) and Wilcoxon signed rank test (paired pinworm (+) mebendazole (+) vs. pinworm (+) mebendazole (-)). After p value corrections, those taxa remained to be significant (FDR< 0.05) are written in red, and bacterial species with FDR> 0.05 are written in blue. Boundaries of significant fold change and p value of Wilcoxon tests are shown in dashed lines. Dot size represents average relative abundance. a. Impact of pinworm infection on gut microbiome. Comparison was made between the P+M- group and the P- group. b. Impact of mebendazole administration on gut microbiome. Comparison was made between P+M+ group and P+M- group. P-: pinworm negative, n = 30; P+M-: pinworm positive before mebendazole treatment, n = 65; P+M+: pinworm positive after mebendazole treatment, n = 65.

At the species level, pinworm infection was associated with a trend of increased proportions of Faecalibacterium prausnitzii, Ruminococcus flavefaciens, Alistipes purtredinis, Bifidobacterium longum and uncultured Oscillospira sp. (Fig 3a, percentages and p values are shown in S1 Table), as well as a trend of decreased relative abundance of Acidaminococcus intestine, Megasphaera elsdenii, Veillonella dispar and Fusobacterium varium (Fig 3b and S1 Table). The relative abundance of Faecalibacterium prausnitzii and Ruminococcus flavefaciens were lower, while the proportion of Bifidobacterium longum and uncultured Oscillospira sp. were higher after mebendazole deworming (Fig 3a, S1 Table). Mebendazole deworming was not associated with an increase in the relative abundance of Acidaminococcus intestine, Megasphaera elsdenii, Veillonella dispar and Fusobacterium varium as compared with the pinworm (+) mebedazole (-) group (Fig 3b).

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Fig 3

Changes in proportions of intestinal bacterial species after pinworm infection or mebendazole deworming.

a. Bacterial species increased after pinworm infection. b. Bacterial species decreased after pinworm infection. c. Bacterial species increased after mebendazole deworming. ***: p <0.001, **: p <0.01, *: p<0.05 by Wilcoxon rank sum test (P- vs. P+M-, P- vs. P+M+) or Wilcoxon signed rank test (P+M- vs. P+M+, paired). Bars represent mean±SEM.

Furthermore, as shown in Fig 3c, the relative abundance of Collinsella aerofaciens and Streptococcus thermophilus did not change significantly after pinworm infection; however, an increase in the proportion of these 2 species was detected 2 weeks after mebendazole deworming treatment (Collinsella aerofaciens: pinworm (+) mebendazole (+) vs. pinworm (+) mebendazole (-), 3.07% ± 5.52% vs. 1.00% ± 2.00%, fold = 3.08, p = 9.18 x 10⁻⁵, FDR = 0.034; Streptococcus thermophiles: pinworm (+) mebendazole (+) vs. pinworm (+) mebendazole (-), 0.89% ± 2.04% vs. 0.31% ± 0.42%, fold = 2.93, p = 0.003, FDR = 0.158, Fig 3c).

Enrichment of microbiome by pinworm infection and mebendazole treatment

Taxonomic annotation-based enrichment analysis showed that the abundance of Gram-positive and endospore-forming bacteria was increased in the pinworm (+) mebendazole (+) group, as compared with the pinworm (-) group (p = 0.0001 and p = 0.001, respectively, S2a and S2b Fig). Further pathway enrichment analysis suggested that an enriched gut microbiome involving fat absorption and digestion pathway (ko04975) was associated with pinworm infection (pinworm (+) mebendazole (-) vs. pinworm (-), fold = 2.49, p = 0.014, S2c Fig), and the statistical significance remained when comparing the microbiota of the mebendazole treated group with the pinworm uninfected group (fold = 2.54, p = 0.007, S2c Fig). In addition, exposure to pinworm and mebendazole was found to be correlated with the enrichment of the gut microbiome involving the fatty acid elongation pathway (ko00062, pinworm (+) mebendazole (+) vs. pinworm (-), fold = 2.01, p = 0.001, S2d Fig) and the caffeine metabolism pathway (ko00232, pinworm (+) mebendazole (+) vs. pinworm (-), fold = 2.04, p = 0.002, S2e Fig).

Association of fecal SIgA and cytokine levels with pinworm infection and intestinal bacterial abundance

To analyze the impact of Enterobius exposure on a host’s intestinal immune response, stool samples from pinworm-uninfected, pinworm-infected and untreated, and pinworm-infected and treated groups were collected and measured for their SIgA, IL-1ß and IL-4 levels. Pinworm infection was found to be associated with a significant decrease in gut SIgA levels (median level of uninfected vs. pinworm (+) mebendazole (-) group: 125.59 μg/ml vs. 109.56 μg/ml, p<0.01, Fig 4a). The amount of fecal IL-1ß and IL-4 were similar before and after pinworm infection (Fig 4b and 4c).

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Fig 4

Associations of SIgA/ cytokine levels with pinworm infection and with relative abundance of gut bacterial species.

a. Fecal SIgA, b. fecal IL-1ß, and c. fecal IL-4 levels in P-, P+M-, and P+M+ groups. **: p<0.01 by Mann-Whitney U test. Lines represent medians. d. Correlation of the relative abundance of Prevotella with gut SIgA levels, and correlation of the relative abundance of Collinsella in the P- group (low SIgA: <80000 ng/ml, n = 5; medium-high SIgA: ≥ 150000 ng/ml, n = 22; low IL-4: < 5 pg/ml, n = 12, medium-high: ≥5, n = 11). Bars represent mean±SEM. *: p<0.05, **: p<0.01 by Wilcoxon rank sum test, corrected for the following factors: recent gastroenteritis and recent respiratory tract infection with oral medication.

Furthermore, the fecal levels of SIgA and cytokines were grouped into low, medium and high as described in the Methods Section. In the pinworm (-) group, possible confounding factors such as recent gastroenteritis and respiratory tract infection with oral medication were collected; and in this group, correlation studies of SIgA and cytokine levels with intestinal microbial taxa revealed an association of a higher Prevotella proportion with a decreased amount of gut SIgA (relative abundance of Prevotella in SIgA medium-high vs. SIgA low, 6.22% ± 2.97% vs. 26.46% ± 11.52%, p = 0.006 (corrected for respiratory and gastrointestinal infection factors), Fig 4d), and association of a higher Collinsella abundance with a decreased amount of gut IL-4 (relative abundance of Collinsella in IL-4 medium-high vs. IL-4 low, 0.36% ± 0.21% vs. 1.69% ± 0.84%, p = 0.043 (corrected for respiratory and gastrointestinal infection factors), Fig 4d).

Microbiota associated with increased SIgA level after mebendazole treatment

After mebendazole deworming, the amount of intestinal SIgA only increased in half of the treated subjects (Fig 5a). We then investigated the fecal microbial composition of the mebendazole-treated samples with and without SIgA-restoration. Before mebendazole deworming, the SIgA-non-increased group was associated with a higher proportion of the gut pathogen Salmonella (SIgA-non-increased vs. SIgA-increased group, 1.40% ± 3.21% vs. 0.18% ± 0.45%, p = 0.012, Fig 5b), and a lower abundance of the commensal Klebsiella, as compared with the SIgA-increased group (0.00% ± 0.00% vs. 0.74% ± 2.84%, p = 0.010, Fig 5b). Furthermore, mebendazole deworming was associated with increased percentages of Bifidobacterium and Streptococcus in the SIgA-increased specimens (P+M+ vs. P+M-, Bifidobacterium: 9.96% ± 12.53% vs. 5.83% ± 8.47%, p = 0.037; Streptococcus: 1.58% ± 3.23% vs. 0.27% ± 0.35%, p = 0.004, Fig 6a), and a decreased relative abundance of Salmonella in the SIgA non-increased subjects (P+M+ vs. P+M-, 0.12% ± 0.48% vs. 1.40% ± 3.21%, p = 0.010, Fig 6b). A mebendazole-deworming associated increase in the proportions of Collinsella was observed in both the SIgA-increased and the SIgA-non-increased groups (P+M+ vs. P+M-, SIgA-increased group: 2.08% ± 3.43% vs. 0.43% ± 0.69%, p = 0.002; SIgA-non-increased group: 3.71% ± 5.95% vs. 1.38% ± 2.43%, p = 0.018, Fig 6a and 6b).

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Fig 5

Gut microbial composition differences in SIgA-increased and SIgA-non-increased groups.

a. 48 pinworm (+) mebendazole (-) and pinworm (+) mebendazole (+) paired fecal samples were evaluated for SIgA levels. 24 subjects showed >1.1 fold increase of SIgA after mebendazole deworming (SIgA-increased group, marked in red), and the other 24 subjects fell in the SIgA-non-increased group (marked in black). b. Volcano plot showing differentially expressed genus-level bacteria comparing the untreated, pinworm-infected P+M- samples of SIgA non-increased group and the P+M- samples of SIgA-increased group. Significant microbes analyzed by Wilcoxon rank sum tests are shown in red dots. Boundaries of significant fold change and p value are shown in dashed lines. Dot size represents average relative abundance.

See Also

Uses of Mebendazole 500mg and Mebendazole 100mg

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Fig 6

The shifts of intestinal microbiota after mebendazole deworming in the SIgA-increased (a) and SIgA non-increased groups (b).

Differentially expressed bacterial genera comparing P+M+ samples vs. P+M- samples are shown in volcano plots.

At the species level, a higher proportion of Salmonella enterica was noted in pre-treatment samples of the SIgA-non-increased group (fold = 7.81, p = 0.012), and it decreased after mebendazole deworming (fold = 0.08, p = 0.010) (S3a and S3b Fig). The association of the increased relative abundance of the probiotic bacteria Streptococcus thermophilus and Bifidobacterium longum with mebendazole deworming was detected only in the SIgA-increased group (Streptococcus thermophilus: fold = 5.84, p = 0.006; Bifidobacterium longum: fold = 1.67, p = 0.046, S3c Fig).

S1 Table

(DOCX)

S1 Fig

Flow diagram.

P(-): pinworm-negative; P(+)M(-): pinworm-infected, before mebendazole treatment; P(+)M(+): pinworm-infected, 2 weeks after mebendazole treatment.

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S2 Fig

Microbiology characteristics and pathway enrichment analysis.

A-B. Box plots showing microbiology characteristic enrichment analyses based on Gram staining (a) and endospore-forming features (b). ***: p <0.001 by ANOVA test. c-e. Pathway enrichment analyses showing significant increase in fat metabolism (c), fatty acid elongation (d) and caffeine metabolism (e) pathways after pinworm infection plus mebendazole treatment. R package “Tax4Fun” was used to transform the OTU table into pathway activity value. ***: p <0.001, **: P<0.01, *: P<0.05 by ANOVA tests.

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S3 Fig

Gut bacterial species composition differences in SIgA-increased and SIgA-non-increased groups.

Volcano plots showing differentially expressed species-level bacteria comparing the untreated, pinworm-infected P+M- samples of SIgA non-increased group vs. the P+M- samples of SIgA-increased group (a), P+M+ samples vs. P+M- samples in SIgA non-increased group (b), and P+M+ samples vs. P+M- samples in SIgA-increased group (c). Red dots represent significant taxa analyzed by Wilcoxon tests. After p value corrections, those taxa remained to be significant (FDR< 0.05) are written in red, bacterial species with FDR> 0.05 are written in blue. Boundaries of significant fold change and p value are shown in dashed lines. Dot size represents average relative abundance.

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