Campylobacter Abundance in Breastfed Infants and Identification of a New Species in the Global Enterics Multicenter Study

Abstract

Campylobacter jejuni is a leading cause of bacterial diarrhea worldwide and is associated with high rates of mortality and growth stunting in children inhabiting low- to middle-resource countries. To better understand the impact of breastfeeding on Campylobacter infection in infants in sub-Saharan Africa and South Asia, we examined fecal microbial compositions, bacterial isolates, and their carbohydrate metabolic pathways in Campylobacter-positive infants <1 year of age from the Global Enterics Multicenter Study. Exclusively breastfed infants with diarrhea exhibited high Campylobacter abundances, and this negatively correlated with bacterial carbohydrate metabolism. Although C. jejuni and Campylobacter coli are prevalent among these infants, the second most abundant Campylobacter species was a new species, which we named "Candidatus Campylobacter infans." Asymptomatic Campylobacter carriers also possess significantly different proportions of specific gut microbes compared to diarrheal cases. These findings provide insight into Campylobacter infections in infants in sub-Saharan Africa and South Asia and help inform strategies aimed at eliminating campylobacteriosis in these areas.IMPORTANCECampylobacter is the primary cause of bacterial diarrhea in the United States and can lead to the development of the postinfectious autoimmune neuropathy known as Guillain-Barré syndrome. Also, drug-resistant campylobacters are becoming a serious concern both locally and abroad. In low- and middle-income countries (LMICs), infection with Campylobacter is linked to high rates of morbidity, growth stunting, and mortality in children, and breastfeeding is important for infant nutrition, development, and protection against infectious diseases. In this study, we examined the relationship between breastfeeding and Campylobacter infection and demonstrate the increased selection for C. jejuni and C. coli strains unable to metabolize fucose. We also identify a new Campylobacter species coinfecting these infants with a high prevalence in five of the seven countries in sub-Saharan Africa and South Asia examined. These findings indicate that more detailed studies are needed in LMICs to understand the Campylobacter infection process in order to devise a strategy for eliminating this pathogenic microbe.

Keywords: Campylobacter; GEMS; breastfeeding; l-fucose metabolism; “Candidatus Campylobacter infans,” gut microbiome.

FIG 1

Fecal microbiome in symptomatic and asymptomatic Campylobacter infections in infants <1 year of age. (A) Microbial diversities (Shannon index) in different groups. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. (B) Microbial compositions of fecal samples from cases and controls with breastfeeding (BF) or no breastfeeding (No BF) (V6-V8 of 16S rRNA). P, F, A, and B refer to phyla (P, Proteobacteria; F, Firmicutes; A, Actinobacteria; B, Bacteroidetes). (C) Microbial composition of fecal samples from cases and controls with breastfeeding or no breastfeeding (V4 of 16S rRNA). (D) Proportions of Campylobacter species at the individual-child level. Each of the bars represents one individual subject, and different Campylobacter species are shown in different colors. (E) Average Campylobacter abundance in each group. NS, not statistically significant; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

FIG 2

Investigation of the relationship between fucose metabolism and Campylobacter infection in GEMS. (A) A PCR screen for the fucose permease gene fucP was carried out for all samples, and a representative gel is shown. (B) Verification of the fucP colony PCR results to compare the abilities to metabolize fucose and show growth enhancement. Values shown are means (n = 3), and error bars represent 1 standard deviation (fucP⁺ strains are highlighted in boldface type). White, control; black, l-fucose addition. (C) Pie charts comparing the correlations between fucose metabolism and the isolated Campylobacter strains. Black, l-fucose-utilizing strains; white, non-l-fucose-utilizing strains. P values were determined by chi-squared testing compared to 50%.

FIG 3

Identification of a new Campylobacter species. (A and B) Sequences of 20 core genes from various Campylobacteraceae or their cognate proteins were concatenated and aligned using CLUSTALX. Included in the alignment were the same 20 concatenated genes or proteins extracted from the metagenomic sequences obtained from a fecal DNA sample containing 83% Campylobacter sequences (“Candidatus Campylobacter infans”). The dendrograms were constructed using the neighbor-joining algorithm and the Kimura two-parameter (gene set) (A) or Poisson (protein set) (B) distance estimation method. Bootstrap values of >75%, generated from 500 replicates, are shown at the nodes. (C) Average nucleotide identity (ANI) of “Candidatus Campylobacter infans” among known related bacteria, including Campylobacter, Sulfurimonas, Arcobacter, Sulfurospirillum, and Helicobacter species.

FIG 4

Detection of “Candidatus Campylobacter infans” and prevalences of different Campylobacter species. (A) Phylogenetic analysis of “Candidatus Campylobacter infans” in GEMS samples using the full atpA gene. Red circles, atpA from GEMS fecal DNA (the first number of the 6-digit code indicates sample site [1, The Gambia; 2, Mali; 3, Mozambique; 5, India; 7, Pakistan]); red solid circle, “Candidatus Campylobacter infans” atpA. The dendrogram was constructed using the neighbor-joining algorithm and the Tamura three-parameter method. Bootstrap values of >75%, generated from 1,000 replicates, are shown at the nodes. (B) Prevalences of different Campylobacter species in the study subjects, detected by 16S rRNA sequencing and confirmed by lpxA multiplex PCR and atpA sequencing (atpA sequencing was done only for “Candidatus Campylobacter infans”).