Bacterial Infections

Longitudinal Analysis of the Prevalence, Maintenance, and IgA Response to Species of the Order Bacteroidales in the Human Gut

Naamah Levy Zitomersky, Michael J. Coyne, Laurie E. Comstock
J. N. Weiser, Editor
DOI: 10.1128/IAI.01348-10

Article Figures & Data

Figures

  • Fig. 1.
    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.
    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.
    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.
    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.
    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).

Tables

  • Table 1.

    Total Bacteroidales CFU in fecal samples from 15 human subjects over 1 year

    SubjectTotal no. of Bacteroidales CFU/g of fecal sample at:
    0 mo1 mo3 mo6 mo8/9 mo12 moa
    13 × 10101 × 10103 × 10101 × 10103 × 1010
    27 × 1092 × 1096 × 1095 × 1082 × 109
    33 × 10108 × 1091 × 10101 × 10102 × 109, 3 × 109
    46 × 1081 × 1091 × 1092 × 1091 × 109
    55 × 1097 × 1096 × 1088 × 1092 × 109, 4 × 109
    61 × 1099 × 1091 × 1093 × 1093 × 109
    72 × 1093 × 1091 × 1094 × 1092 × 1010
    83 × 10101 × 10102 × 10102 × 10102 × 109, 6 × 109
    91 × 1091 × 10103 × 1093 × 1091 × 109, 2 × 109
    102 × 10102 × 10102 × 1093 × 1010, 8 × 1010
    119 × 1096 × 1091 × 10107 × 1082 × 109, 8 × 109
    122 × 10101 × 10102 × 10102 × 10102 × 1010, 2 × 1010
    135 × 1092 × 1091 × 1095 × 1081 × 109, 4 × 109
    142 × 10102 × 10102 × 10102 × 10102 × 1010, 2 × 1010
    152 × 10103 × 10101 × 10102 × 10102 × 1010, 3 × 1010

    • ↵a Two different regions of various 12-month fecal samples were analyzed.

  • Table 2.

    Proportions of B. fragilis isolates containing bft

    SubjectProportion of B. fragilis isolates containing bft
    0 mo1 moa3 moc6 mo9 mo12 moTotal
    10/20/70/2b0/11
    20/10/1
    30/70/20/50/20/20/18
    40/180/10/21/61/27
    51/1b2/21/15/59/9
    721/213/33/33/36/636/36
    80/20/10/10/4
    90/20/2
    100/10/70/3b0/11
    110/3b0/3
    135/54/41/24/4b14/15
    1417/176/64/45/57/739/39
    152/22/2

    • ↵a The 1-month collection was screened only for B. fragilis and therefore not used in other tables and figures in this report.

    • ↵b Some of these B. fragilis isolates were present on the third-highest-dilution plate and therefore not included in the other analyses of this study.

    • ↵c The B. fragilis isolates from the 3-month sample of subject 1 were not frozen and therefore were not available for bft typing.

Additional Files

  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - Table S1. Primers used in this study.
      Table S2. Bacteroidales species detected on two highest-dilution plates.
      Fig. S1. Species detected from at least one sample on two highest-dilution plates for each subject.
      PDF file, 40K.
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