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Application of Novel PCR-based Methods for Detection, Quantitation, and Phylogenetic Characterization of Sutterella Species in Intestinal Biopsy Samples From Children With Autism and Gastrointestinal Disturbances

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Application of Novel PCR-based Methods for Detection, Quantitation, and Phylogenetic Characterization of Sutterella Species in Intestinal Biopsy Samples From Children With Autism and Gastrointestinal Disturbances

Brent L Williams et al. mBio.

Abstract

Gastrointestinal disturbances are commonly reported in children with autism and may be associated with compositional changes in intestinal bacteria. In a previous report, we surveyed intestinal microbiota in ileal and cecal biopsy samples from children with autism and gastrointestinal dysfunction (AUT-GI) and children with only gastrointestinal dysfunction (Control-GI). Our results demonstrated the presence of members of the family Alcaligenaceae in some AUT-GI children, while no Control-GI children had Alcaligenaceae sequences. Here we demonstrate that increased levels of Alcaligenaceae in intestinal biopsy samples from AUT-GI children result from the presence of high levels of members of the genus Sutterella. We also report the first Sutterella-specific PCR assays for detecting, quantitating, and genotyping Sutterella species in biological and environmental samples. Sutterella 16S rRNA gene sequences were found in 12 of 23 AUT-GI children but in none of 9 Control-GI children. Phylogenetic analysis revealed a predominance of either Sutterella wadsworthensis or Sutterella stercoricanis in 11 of the individual Sutterella-positive AUT-GI patients; in one AUT-GI patient, Sutterella sequences were obtained that could not be given a species-level classification based on the 16S rRNA gene sequences of known Sutterella isolates. Western immunoblots revealed plasma IgG or IgM antibody reactivity to Sutterella wadsworthensis antigens in 11 AUT-GI patients, 8 of whom were also PCR positive, indicating the presence of an immune response to Sutterella in some children.

Importance: Autism spectrum disorders affect ~1% of the population. Many children with autism have gastrointestinal (GI) disturbances that can complicate clinical management and contribute to behavioral problems. Understanding the molecular and microbial underpinnings of these GI issues is of paramount importance for elucidating pathogenesis, rendering diagnosis, and administering informed treatment. Here we describe an association between high levels of intestinal, mucoepithelial-associated Sutterella species and GI disturbances in children with autism. These findings elevate this little-recognized bacterium to the forefront by demonstrating that Sutterella is a major component of the microbiota in over half of children with autism and gastrointestinal dysfunction (AUT-GI) and is absent in children with only gastrointestinal dysfunction (Control-GI) evaluated in this study. Furthermore, these findings bring into question the role Sutterella plays in the human microbiota in health and disease. With the Sutterella-specific molecular assays described here, some of these questions can begin to be addressed.

Figures

FIG 1
FIG 1
Presence of Sutterella sequences in a subset of AUT-GI patients. Shown are the results from detection by pyrosequencing of the V2 region of the 16S rRNA gene. (A and B) Distribution of Sutterella sequences as a percentage of total bacterial 16S rRNA gene reads from ileal (A; Mann-Whitney, tied P = 0.022) and cecal (B; Mann-Whitney, tied P = 0.037) biopsy samples from AUT-GI and Control-GI patients. (C to F) Distribution of Sutterella sequences by individual patient as a percentage of total bacteria (C and D) or total Betaproteobacteria (E and F) 16S rRNA reads from ileal (C and E) and cecal (D and F) biopsy samples from AUT-GI (patients 1 to 15) and Control-GI (patients 16 to 22) patients. *, P < 0.05.
FIG 2
FIG 2
Pie chart indicating the percentage of Sutterella sequences in the dominant OTU (either OTU 1 or OTU 2) relative to sequences from subdominant Sutterella OTUs in ileum and cecum of the seven Sutterella-positive patients. The percentage of the dominant OTU is shown per patient.
FIG 3
FIG 3
Sutterella-specific PCR assays. (A) Schematic representation showing the location of PCR primers and products evaluated in this study. (B) Sutterella-specific 16S rRNA gene (V6–V8) PCR amplification of 10-fold dilutions of Sutterella plasmid DNA standards spiked into ileal DNA from a Sutterella-negative Control-GI patient. Note the linear amplification down to 5 × 102 copies and the endpoint detection limit of 5 × 101 copies. (C) Real-time PCR amplification plot of 10-fold serial dilutions of Sutterella plasmid DNA standards. ΔRn, magnitude of the signal generated by the PCR conditions. (D) Standard curve generated from our Sutterella-specific quantitative real-time PCR assay.
FIG 4
FIG 4
PCR-based detection of Sutterella 16S rRNA gene sequences (V6–V8 region and C4–V8 region) in biopsies from AUT-GI and Control-GI patients. (A) Agarose gel detection of 260-bp Sutterella products in ileal (4 biopsy samples/patient) and cecal (4 biopsy samples/patient) biopsy DNA using SuttFor and SuttRev primers (V6–V8 region) in conventional PCR assays. (B) Agarose gel detection of 715-bp Sutterella products in ileal and cecal biopsy DNA using pan-bacterial primer 515For and SuttRev primer (C4–V8) in conventional PCR assays. The negative control is PCR reagents with water substituted for DNA. The positive control is DNA isolated from cultured S. wadsworthensis (ATCC 51579).
FIG 5
FIG 5
Quantitation of Sutterella sequences in ileal and cecal biopsy samples from AUT-GI and Control-GI patients using a novel Sutterella-specific real-time PCR assay. Bars in the graph show mean copy number in 4 biopsy samples from ileum (blue) and 4 biopsy samples from cecum (red) + the standard error of the mean (SEM) for each patient.
FIG 6
FIG 6
Distribution of Sutterella species in ileal and cecal biopsy samples from AUT-GI patients based on C4–V8 products. The closest sequence match to known Sutterella isolates was determined using the RDP seqmatch tool. The frequency of Sutterella species matches in ileal and cecal clone libraries are shown as pie charts for patients 1 (A), 3 (B), 5 (C), 7 (D), 10 (E), 11 (F), 12 (G), 24a (H), 25a (I), 27a (J), 28a (K), and 29a (L). *, Sutterella 16S sequences obtained from patient 28a were less than 97% similar to the 16S sequence of all known isolates of Sutterella species.
FIG 7
FIG 7
Phylogenetic tree based on predominant 16S rRNA gene sequences obtained by C4–V8 Sutterella PCR from AUT-GI patients, Sutterella species isolates, and related species. The tree was constructed by the neighbor-joining method. Bootstrap values (>60%) based on 1,000 replicates are shown next to the branches. There were a total of 653 positions in the final data set. The evolutionary distances were computed using the Jukes-Cantor method and are in units representing the number of base substitutions per site. The optimal tree with the sum of branch length of 0.66371685 is shown. The tree is rooted to the outgroup Escherichia coli. Accession numbers are shown in parentheses. AUT-GI patient sequences are boxed in red.
FIG 8
FIG 8
Western immunoblot analysis of AUT-GI and Control-GI patients’ plasma antibody immunoreactivity against S. wadsworthensis antigens. (A) Patients’ plasma IgG antibody immunoreactivity against S. wadsworthensis antigens. (B) Patients’ IgM antibody immunoreactivity against S. wadsworthensis antigens. 2°, secondary antibody control.

Comment in

  • A Microbial Association With Autism
    JL Benach et al. mBio 3 (1). PMID 22334515.
    Autism is a heterogeneous group of complex developmental disabilities that result from a number of possible etiologies. There are a well-known number of comorbidities ass …

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