Co-occurrence of Campylobacter Species in Children From Eastern Ethiopia, and Their Association With Environmental Enteric Dysfunction, Diarrhea, and Host Microbiome

Abstract

High Campylobacter prevalence during early childhood has been associated with stunting and environmental enteric dysfunction (EED), especially in low resource settings. This study assessed the prevalence, diversity, abundance, and co-occurrence of Campylobacter spp. in stools from children in a rural area of eastern Ethiopia and their association with microbiome, diarrhea, and EED in children. Stool samples (n = 100) were collected from randomly selected children (age range: 360-498 days) in five kebeles in Haramaya District, Ethiopia. Diarrhea, compromised gut permeability, and gut inflammation were observed in 48, 45, and 57% of children, respectively. Campylobacter prevalence and species diversity were assessed using PCR and meta-total RNA sequencing (MeTRS). The prevalence of Campylobacter spp. in the children's stools was 50% (41-60%) by PCR and 88% (80-93.6%) by MeTRS (P < 0.01). Further, seven Campylobacter species (Campylobacter jejuni, Campylobacter upsaliensis, Campylobacter hyointestinalis, Campylobacter coli, Campylobacter sp. RM6137, uncultured Campylobacter sp., and Campylobacter sp. RM12175) were detected by MeTRS in at least 40% of children stools in high abundance (>1.76-log read per million per positive stool sample). Four clusters of Campylobacter species (5-12 species per cluster) co-occurred in the stool samples, suggesting that Campylobacter colonization of children may have occurred through multiple reservoirs or from a reservoir in which several Campylobacter species may co-inhabit. No associations between Campylobacter spp., EED, and diarrhea were detected in this cross-sectional study; however, characteristic microbiome profiles were identified based on the prevalence of Campylobacter spp., EED severity, and diarrhea. Forty-seven bacterial species were correlated with Campylobacter, and 13 of them also correlated with gut permeability, gut inflammation and/or EED severity. Forty-nine species not correlated with Campylobacter were correlated with gut permeability, gut inflammation, EED severity and/or diarrhea. This study demonstrated that (1) in addition to C. jejuni and C. coli, multiple non-thermophilic Campylobacter spp. (i.e., Campylobacter hyointestinalis, Campylobacter fetus, and Campylobacter concisus) were frequently detected in the children's stools and (2) the Campylobacter, gut permeability, gut inflammation, EED severity, and diarrhea were associated with characteristic microbiome composition. Additional spatial and longitudinal studies are needed to identify environmental reservoirs and sources of infection of children with disparate Campylobacter species and to better define their associations with EED in low-income countries.

Keywords: Campylobacter; EED; MeTRS; diarrhea; livestock reservoirs; malnutrition; non-thermotolerant Campylobacter; stunting.

Figure 1

Prevalence of Campylobacter, environmental enteric dysfunction (EED) and diarrhea in children in the five kebeles from Haramaya District (East Ethiopia). Pie graphs in red (positive for Campylobacter) and blue (negative for Campylobacter) represent the Campylobacter prevalence in stool samples (n = 100) collected from the designated kebele. Prevalence was determined using conventional PCR (c-PCR) or MeTRS (meta-total RNA sequencing). Pie graphs in green (“normal”), yellow (“moderate” EED), and orange (“severe” EED) represent the prevalence and severity of the EED for the designated kebele. Additional details concerning the EED severity determination are presented in Table S1. Pie graphs in brown (child currently having or had diarrhea in the past 15 days), gray (child had no diarrhea in the past 15 days) represent the diarrheal status of the children. GC, Gobe Chala; NG, Negaya; FK, Finkle; DM, Damota; BG: Biftu Geda.

Figure 2

Campylobacter spp. prevalence, diversity, and abundance in children stools. A total of 27 classified Campylobacter spp. and 12 unclassified Campylobacter spp. were detected in the 100 child stool samples collected from children in the five kebeles using MeTRS. (A) Campylobacter spp. prevalence and diversity in the stool samples. Blue and red cells represent the absence or presence of Campylobacter spp. in the designated stool samples (cut-off; contigs number ≥ 10; Read length ≥ 50; Z-score ≥ 1). Kebeles with the same color code belong to the same cluster and therefore harbored equivalent Campylobacter sp. diversity. (B) Abundance of Campylobacter spp. in the positive stools. White bar represents Campylobacter spp. with a prevalence lower than 40% and an abundance lower than 0.95-log rpm per stool sample. Gray bar represents Campylobacter spp. with a prevalence higher than 40% and an abundance lower than 1.76-log rpm per stool sample. Black bar represents Campylobacter spp. with a prevalence higher than 40% and an abundance higher than 1.76-log rpm per stool sample. rpm: read per million.

Figure 3

Co-occurrence of Campylobacter spp. in the children stool samples. Co-occurrence profiles were created using K-means clustering data extracted from the multivariate analysis data (r²) based on the prevalence of Campylobacter spp. in the stool samples (see Figure 2). Additional details regarding the multivariate data are displayed in Figure S3.

Figure 4

Associations between Campylobacter, gut permeability, gut inflammation, environmental enteric dysfunction (EED), diarrhea, and child stool microbiome. A total of 47 bacterial species positively or negatively correlated (red or blue cells, respectively) with Campylobacter prevalence and abundance (reads per million, rpm; r² > 0.20 or r² < −0.20; P < 0.05), gut permeability (lactulose %), [MPO] in ng/ml, EED status (“normal,” “moderate” EED, “severe” EED), and/or the diarrhea prevalence data. Additional details concerning the EED severity determination are presented in Table S1. The phylogenetic tree was built using NCBI website (https://www.ncbi.nlm.nih.gov/Taxonomy/CommonTree/wwwcmt.cgi). N: number of stools positive for the designated bacterial species. The bar graph represents the average abundance (log rpm per positive stool sample) for the selected bacterial species. rpm, read per million; MPO, myeloperoxidase.