Longitudinal analysis of the prevalence, maintenance, and IgA response to species of the order Bacteroidales in the human gut

Abstract

Bacteroidales species are the most abundant Gram-negative bacteria of the human intestinal microbiota. These bacteria evolved to synthesize numerous capsular polysaccharides (PS) that are subject to phase variation. In Bacteroides fragilis, PS synthesis is regulated so that only one of the eight PS biosynthesis loci is transcribed at a time in each bacterium. To determine if the bacteria evolved this unusual property to evade a host IgA response, we directly studied the human fecal ecosystem. We performed a longitudinal analysis of the abundant Bacteroidales species from 15 healthy adults at four intervals over a year. For this study, we used bacterial culture to perform analyses not accurate with DNA-based methods, including quantification of total viable Bacteroidales bacteria, strain maintenance, and IgA responses. Abundant Bacteroidales isolates were identified to the species level using multiplex PCR and 16S rRNA gene sequencing. Arbitrarily primed PCR was used for strain typing. IgA responses to endogenous strains carried over the year were analyzed, and the orientations of the invertible PS locus promoters from the ecosystem were quantified. Subjects consistently harbored from 5 × 10(8) to 8 × 10(10) Bacteroidales bacteria/g of feces. Within the cohort, 20 different Bacteroidales species were detected at high concentrations. Bacteroides uniformis was the most prevalent; however, abundant Bacteroidales species varied between subjects. Strains could be maintained over the year within the ecosystem at high density. IgA responses were often not induced and did not correlate with the elimination of a strain or major changes in the orientations of the capsular PS locus promoters.

Fig. 1.

Multiplex PCR analyses. EtBr-stained agarose gels show the PCR product(s) resulting when each Bacteroidales species was analyzed by multiplex PCR I (top panel), multiplex PCR II (middle panel), and multiplex PCR III (bottom panel). The following type strains were used: B. thetaiotaomicron ATCC 29741, B. vulgatus ATCC 8482, B. fragilis NCTC 9343, B. caccae ATCC 43185, B. ovatus ATCC 8483, P. distasonis ATCC 8503, P. merdae ATCC 43184, B. uniformis ATCC 8452, and B. finegoldii DSM 17565. The B. stercoris, B. eggerthii, B. intestinalis, B. dorei, and P. johnsonii strains were all obtained from this study and confirmed by full 16S rRNA gene sequencing.

Fig. 2.

Number of stool samples from which a given Bacteroidales species was identified on the two highest-dilution plates from a total of 59 stool samples.

Fig. 3.

Strain typing by AP-PCR. (A) AP-PCR amplicons resulting from four longitudinally collected B. fragilis isolates from subjects 7 and 5 and B. fragilis type strain NCTC 9343. The top panel is AP-PCR1, and the bottom panel shows the products from the same isolates with AP-PCR3. (B) AP-PCR amplicons resulting from four longitudinally collected B. fragilis isolates from subjects 14 and 3 and B. fragilis NCTC 9343. The top panel is AP-PCR1, and the bottom panel shows the products from the same isolates with AP-PCR3. (C) AP-PCR amplicons resulting from four longitudinally collected B. uniformis isolates from subjects 14 and 3 and B. uniformis type strain ATCC 8452. The top panel is AP-PCR2, and the bottom panel shows the products from the same isolates with AP-PCR3. (D) AP-PCR products amplified from six B. fragilis isolates from the 6-month stool sample of subject 4. The top panel is AP-PCR1, the middle panel AP-PCR3, and the bottom panel shows amplification of bft.

Fig. 4.

Western immunoblot analysis of local human antibody responses to abundant endogenous Bacteroidales strains. (A) IgA reactivity in the 3-month sample from subject 1 to the Bacteroidales strains isolated from this subject at the initial collection point. (B to D) IgA reactivity of the 12-month sample from subjects 3, 7, and 14 to the endogenous B. fragilis strain that each had carried over the year of study. (E) IgA reactivity from the initial stool sample from subject 7 to the same strains of panel C.

Fig. 5.

Orientations of the B. fragilis PS locus promoters from bacteria in the fecal samples of subjects 7 and 14. (A) Schematic of the PCR digestion method used for quantitative promoter orientation analysis of the PSH locus. The inverted repeats (IR) flanking the invertible DNA region are shown with the promoter (p) indicated. The primers used for PCR and the restriction site are indicated. The sizes of the fragments resulting from digestion of the PCR product when the promoter is in both the on and off orientations are shown. (B and C) EtBr-stained agarose gels demonstrating the fragments resulting from PCR digestion of each invertible PS locus promoter region for 0-, 6-, and 12-month samples from subject 7 or 14. (D and E) Quantification of the percentages of B. fragilis bacteria with each of the PS locus promoters oriented on in the 0-, 6-, and 12-month samples from subjects 7 (D) and 14 (E).