SUPPLEMENTARY MATERIALS
Supplementary results:
Burkholderia and Bradyrhizobium were represented by Burkholderia fungorum and
Bradyrhizobium pachyrhizi respectively. In the original analysis using QIIME, 6,689
sequence reads were classified to the genus Burkholderia. For species identification, we
analyzed their relationship to 16S rRNA genes of 59 Burkholderia species on a phylogenetic
tree and found all except 4 of the reads were clustered to Burkholderia fungorum
(Supplementary Figure 1A). To confirm their relationship to B. fungorum, we amplified full
length 16S rRNA genes from a duodenal sample from one of the HIV-infected patients using
universal primers 8F and 1510R. The PCR products were ligated with the pGEM T Easy
(Promega) and cloned in E. coli DH5α competent cells as previously described[1]. One hundred
and three clones were initially sequenced using primer 8F and classified using SEQMATCH in
RDP II[2]. Clones that were classified as either Burkholderia or Bradyrhizobium were bidirectionally sequenced with the vector-based primers T7 and SP6. Sixteen of the 103 clones
were found to belong to Burkholderia, of which 14 were identical to and 2 had only one
mismatch with a 16S rRNA gene of the B. fungorum type strain LMG 16225 (GenBank
accession number AF215705). QIIME assigned 432 reads in the sequence dataset to
Bradyrhizobium. Initial phylogenetic analysis with the V3/4 (366-784) regions of the type strains
of 14 Bradyrhizobium species indicated that nearly all reads (n=411) formed a single cluster
with 4 species, i.e. B. elkanii, B. jicamae, B. lablabi, and B. pachyrhizi (Supplementary Figure
1B). Similarity search using BLAST showed that the reads could not be further classified
because the four species are identical in this region. To distinguish these species, we found
three of the 103 clones aforementioned belong to Bradyrhizobium and their full length 16S rRNA
gene sequences were closely matched with a 16S rRNA gene of the Bradyrhizobium pachyrhizi
type strain PAC48 (GenBank accession number AY624135) with only one, two, and four
mismatches over the 1501-nucleotide sequence, respectively (Supplementary Table 2). We
classified this taxon as Bradyrhizobium pachyrhizi because this degree of mismatch (0.070.13%) is comparable with the average diversity (0.55%) among 16S rRNA gene copies within
individual prokaryotic genomes[3] but far below either the popular threshold of 3% or the more
strict threshold of 1% used to define species[4].
Supplementary Figure 1. Phylogenetic relationship between 16S rRNA gene sequence
reads of Burkholderia and Bradyrhizobium and 16S rRNA genes of type strains. Phylogeny
was estimated by neighbor-joining of nucleic acid pairwise distance based on the analysis of
the 419-bp region of 16S rRNA genes corresponding to the positions 366 to 784 of the E. coli
16S rRNA gene. Numbers represent percentage bootstrap support (1,000 replicates). Scale
bar indicates nucleic acid substitutions per site. For Burkholderia, a phylogenetic tree
constructed using 16S rRNA genes of type strains representing 57 Burkholderia species and
6,785 sequence reads oriiginally classified to Burkholderia by QIIME (A). For
Bradyrhizobium, a phylogenetic tree constructed with type strains of 14 Bradyrhizobium
species and 432 sequence reads originally classified to Bradyrhizobium by QIIME (B).
Supplementary Figure 2. Difference between HIV-infected patients and controls in the
duodenal microbiome by Gram-stain property and major phyla. Taxa were grouped based on
tinctorial properties of bacteria as Gram-positive or Gram-negative, or unclassified (not shown)
and compared between HIV-infected patients and controls (A). Phylum level compositions
between HIV-infected patients and controls were focused on the five major phyla that account
for more than 95% (B) of the duodenal bacteria population (C). P values were calculated using
Mann–Whitney U test.
Supplementary Figure 3. Correlation between the relative abundance values of B. fungorum
and B. pachyrhizi and blood CD4+ T cell counts. Taxa informative of HIV infection were further
analyzed at each of the three specific sites of the proximal gut including the duodenum (A),
stomach (B), and esophagus (C) as well as the mouth (D) as a reference. X axis denotes the
CD4+ T cell counts/mm3. The dashed line demarcates the threshold of normal CD4+ T cell
counts (500/mm3). The assessment was done with Spearman's rank order correlation.
Supplementary Table 1. Subject characteristics
Category
HIV-1-infected
Subjects (range)
8
36.5 (24-50)
5/8 (62.5%)
327.5 (12-708)
835.4 (267-1462)
0.4 (0.1-0.8)
23,729 (9,090-49,700)
2/8
3/8
2.1 (0.16-7)
0/8
8/8
1/8
Uninfected
subjects
8
47.6 (25-60)
4/8 (50%)
n/a
n/a
n/a
n/a
0/8
0/8
n/a
n/a
0/8e
0/8
Number of subjects
Age (years)
Gender: male (%)
CD4 count (cells/mm3)a
CD8 count (cells/mm3)b
CD4:CD8 ratioc
Plasma viral load (HIV-1 RNA copies/ml)
Upper GI symptoms (nausea, vomiting)
Lower GI symptoms (watery diarrhea)
Years since first HIV-seropositive test
HAARTd
HIV positive test
Antibiotics treatmentf
Race:
White
3 (37.5%)
1 (12.5%)
African American
5 (62.5%)
6 (75.0%)
Asian
0
1 (12.5%)
3
a. Normal range CD4+ T cells: 500-1000 cells/mm .
b. Normal range for CD8+ T cells: 150-1000 cells/mm3
c. Normal range for CD4:CD8 ratio: 0.9 to 3.7 in adults.
d. No highly active antiretroviral therapy for ≥6 month prior to specimen collection.
e. OraQuick ADVANCE Rapid HIV-1/2 antibody test.
f. Of the 8 HIV+ subjects, seven had no antibiotics treatment for at least 8 weeks before
specimen collection while one was on antibiotics for syphilis.
Supplementary Table 2. Number of positions differed between cloned near full length 16S rRNA
genes and those of closely related Bradyrhizobium species over a 1,356-bp overlapping region.
B. pachyrhizi
B. elkanni
B. jicamae
B. lablabi
Clone 3
Clone 42
Clone 79
Dissimilarity (%)
2
1
4
0.07-0.29
5
4
7
0.37-0.52
12
11
6
0.44-0.88
10
9
6
0.44-74
Supplementary Table 3. Correlation between CD4/CD8 ratio and bacterial taxa in HIV-infected
patients.
CD4/CD8 ratio
Taxa
Mouth
Esophagus
Stomach
Duodenum
p value
p value
p value
p value
r2
r2
r2
r2
<0.0001 0.693 0.0003
B. fungorum
absent absent
0.091
0.066
0.722
B. pachyrhizi
0.846
<0.000
0.091
0.222
0.146
0.244 0.0003
0.721
Ralstonia
absent absent
0.091
0.148
0.139
0.045 0.0007
0.729
Fusobacterium
0.493
0.041
0.665
0.003
0.823
0.010
0.058
0.479
Lactobacillus
0.078
0.041 <0.0001
0.322
0.352
0.322 absent
absent
Prevotella
0.693
0.041
0.243
0.384
0.779
0.064
0.102
0.376
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4.
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