Introduction
Autism spectrum disorders (ASD) are pervasive developmental disorders that depend on triadic presentation of social abnormalities, communication impairments, and stereotyped and repetitive behaviors for diagnosis (DSM-IV-TR criteria, American Psychiatric Association, 2000). Gastrointestinal (GI) symptoms are commonly reported in children with autism and may correlate with autism severity (
1,
2). Intestinal disturbances in autism have been associated with macroscopic and histological abnormalities, altered inflammatory parameters, and various functional disturbances (
3–9).
In a previous study, we showed that a complex interplay exists between human intestinal gene expression for disaccharidases and hexose transporters and compositional differences in the mucoepithelial microbiota of children with autism and gastrointestinal disease (AUT-GI children) compared to children with GI disease but typical neurological status (Control-GI children). Significant compositional changes in
Bacteroidetes,
Firmicutes/
Bacteroidetes ratios, and
Betaproteobacteria in AUT-GI intestinal biopsy samples have been reported (
10). Although others have demonstrated changes in fecal bacteria of children with autism (
2,
11–15), our study differed from these by investigating mucoepithelial microbiota (
10). The GI microbiota plays an essential role in physiological homeostasis in the intestine and periphery, including maintaining resistance to infection, stimulating immunological development, and perhaps even influencing brain development and behavior (
16–19). Thus, disruption of the balanced communication between the microbiota and the human host could have profound effects on human health.
In our previous metagenomic study, we found sequences corresponding to members of the family
Alcaligenaceae in the class
Betaproteobacteria that were present in ileal and cecal biopsy samples from 46.7% (7/15) of AUT-GI children.
Alcaligenaceae sequences were completely absent from biopsy samples from Control-GI children (
10). Members of the family
Alcaligenaceae inhabit diverse habitats, ranging from humans and animals to soil (
20). Several members of
Alcaligenaceae cause clinically relevant infections or are suspected opportunistic pathogens in humans and animals, including members of the genus
Bordetella (including the human respiratory pathogens
B. pertussis and
B. parapertussis, the mammalian respiratory pathogen
B. bronchiseptica, and the poultry respiratory pathogen
B. avium), a member of the genus
Alcaligenes (the human opportunistic pathogen
A. faecalis), members of the genus
Achromobacter (the human opportunistic pathogens
A. xylosoxidans and
A. piechaudii), members of the genus
Oligella (the potential opportunistic genitourinary species
O. urethralis and
O. ureolytica), a member of the genus
Taylorella (the equine urogenital pathogen,
T. equigenitalis), and a member of the genus
Pelistega (the pigeon respiratory pathogen
P. europaea) (
20).
In some cases, the pathogenic potential of
Alcaligenaceae members is unclear. The genus
Sutterella represents one such member. Members of the genus
Sutterella are anaerobic or microaerophilic, bile-resistant, asaccharolytic, Gram-negative, short rods (
21). Members of the genus
Sutterella have been isolated from human infections below the diaphragm (
22,
23).
Sutterella 16S rRNA gene sequences have also been identified in intestinal biopsy and fecal samples from individuals with Crohn’s disease and ulcerative colitis (
24,
25). Whether the presence of
Sutterella species at sites of human infection and inflammation represents cause or consequence or whether
Sutterella is a normal part of the microbiota in some individuals remains unclear. The dearth of knowledge concerning the epidemiology and pathogenic potential of
Sutterella derives in part from the lack of specific, culture-independent assays to detect and characterize members of this genus.
Here we further characterize Alcaligenaceae sequences identified in AUT-GI children and describe PCR assays for detection, quantitation, and genotyping of Sutterella as well as serological assays for detection of immunological responses to Sutterella.
DISCUSSION
We previously reported detection by pyrosequencing of
Alcaligenaceae sequences in AUT-GI children (
10). More focused analysis revealed that this finding reflects the presence of
Sutterella species. Whereas 12 of 23 AUT-GI patients (52%) were PCR positive in both ileum and cecum, 0 of 9 Control-GI children were PCR positive for
Sutterella.
Sutterella abundance in the seven
Sutterella-positive AUT-GI patients, assessed by pyrosequencing, ranged from 1 to 7% of total bacterial sequences. Novel real-time PCR assays confirmed high copy numbers of
Sutterella species in DNA from ileal and cecal biopsy samples from
Sutterella-positive patients, with averages ranging from 10
3 to 10
5 Sutterella 16S rRNA gene copies amplified from only 25 ng of total genomic biopsy DNA.
OTU analysis of V2 region pyrosequencing reads indicated that only two OTUs accounted for the majority of Sutterella sequences in the seven AUT-GI patients that were Sutterella positive by pyrosequencing. Sequencing of PCR products from V6–V8 and C4–V8 Sutterella-specific PCR assays corroborated this finding. Our analysis also suggests that C4–V8 Sutterella products can be accurately classified at the species level. Classification with RDP and phylogenetic analysis of Sutterella sequences obtained from C4–V8 Sutterella-specific PCR indicated that the predominant sequences obtained from patients 1, 3, 10, 11, 12, 24a, 27a, and 29a were most closely related to the isolate S. stercoricanis, supported by a sequence similarity of over 98%. The predominant C4–V8 sequences obtained from patients 5, 7, and 25a were most closely related to the isolate S. wadsworthensis, supported by a sequence similarity of 100%. Our results suggest that these two species of Sutterella are the dominant phylotypes present at high levels in the intestines of AUT-GI children in this cohort. Of the known isolates, the predominant C4–V8 sequence obtained from patient 28a was most closely related to Sutterella sp. strain YIT 12072. However, the low sequence similarity (95.3%) between sequences from patient 28a and Sutterella sp. strain YIT 12072 suggests that these are not likely to be the same species. Sequences from patient 28a did have 100% sequence similarity to uncultured Sutterella sequences in GenBank, suggesting that this undefined species has been detected previously in human samples by nonspecific techniques.
Sutterella species have been isolated from human and animal feces (
30–32) and have also been isolated from human infections below the diaphragm; most often from patients with appendicitis, peritonitis, or rectal or perirectal abscesses (
22,
23).
Sutterella sequences have been identified in fecal samples and intestinal biopsy samples from individuals with Crohn’s disease and ulcerative colitis but also from apparently healthy adults (
24,
25,
27,
33). Thus, based on these previous findings, it remains unclear whether
Sutterella species contribute to inflammation and infection or are simply normal inhabitants of the human microbiota in some individuals. Even if the latter is the case, our results demonstrate that
Sutterella is a major component of the mucoepithelial microbiota in some children, accounting for up to 7% of all bacteria. Relative to all other bacterial genera identified in biopsy samples,
Sutterella ranged from the third to eighth most abundant genus in the patients assessed by pyrosequencing. Only the most abundant
Bacteroidetes and
Firmicutes genera outnumbered
Sutterella sequences. This result is remarkable given that
Sutterella is not reported as a major component of the microbiota (
34).
Loss of commensals in the intestine can affect immune responses and disrupt colonization resistance to potentially pathogenic bacteria (
17,
19). In our previous study, we found a significant loss of commensals, namely, members of the phylum
Bacteroidetes, in AUT-GI biopsy samples (
10). Thus, the loss of
Bacteroidetes in AUT-GI children could facilitate the growth of opportunistic pathogens. Whether
Sutterella is pathogenic in AUT-GI children cannot be determined from current data. However, the observation that some AUT-GI children have antibodies that react with
S. wadsworthensis proteins is generally consistent with infection. We detected either IgG or IgM antibodies against
S. wadsworthensis proteins in ~48% (11/23) of AUT-GI children. Only one Control-GI child had very weak IgG immunoreactivity against
S. wadsworthensis proteins. Of the 12 patients that were positive for
Sutterella by PCR, 8 (66.7%) demonstrated plasma IgG antibodies against
S. wadsworthensis proteins. In total, 65.2% (15 out of 23) of AUT-GI children were either positive by PCR assays or had immunoglobulin reactivity to
S. wadsworthensis proteins. Three AUT-GI patients were negative by PCR but had IgG or IgM antibodies against
S. wadsworthensis proteins. As we only examined ileal and cecal biopsy samples in this cohort, we cannot exclude the possibility that
Sutterella species were present in other regions of the small or large intestine or elsewhere in the body of these three patients. This could explain the presence of
Sutterella-specific antibodies without detection of the agent by PCR. Alternatively, IgG antibodies may persist long after antigenic exposure; thus, the presence of IgG antibodies may indicate past exposure in some children. The IgM immunoreactivity of patient 26a suggests recent or current exposure to
Sutterella antigen in this patient. It is perhaps not surprising that proteins recognized by different patients’ plasma were variable. It is well recognized that the use of different strains and species as antigen leads to variations in the immunoreactive profile of immunogenic proteins (
35). Several
Sutterella-positive patients in this study had
S. stercoricanis as the dominant
Sutterella species. We recognize this limitation of our Western blot analysis, as the only antigen available to us was lysate from an
S. wadsworthensis isolate.
The nature of intestinal damage in autism has not been fully defined. Abnormalities in intestinal permeability in children with autism have been reported in two studies (
8,
9). In Crohn’s disease, a condition associated with increased intestinal permeability, a generalized enhancement of antimicrobial IgG to many members of the intestinal microbiota is reported (
36). A defective epithelial barrier could lead to enhanced contact between many members of the microbiota and antigen-presenting cells in the lamina propria. If this turns out to be the case in autism, then antibodies against
Sutterella proteins may reflect interindividual, compositional variation in the microbiota, rather than being an indication of
Sutterella infection. Additional studies are warranted in order to draw definitive conclusions from this immunological analysis.
In conclusion, we have identified Sutterella 16S rRNA gene sequences in mucoepithelial biopsy samples from AUT-GI children using nonspecific, pan-microbial pyrosequencing. We have further designed and applied novel Sutterella-specific PCR assays that confirmed high levels of Sutterella species in over half of AUT-GI children and the complete absence of Sutterella in Control-GI children tested in this study. The Sutterella-specific molecular assays reported in this study will enable more directed studies to detect, quantify, and classify this poorly understood bacterium in biological and environmental samples. With such specific techniques, we can begin to understand the epidemiology of this bacterium and its associations with human infections and inflammatory diseases, the role Sutterella plays in the microbiota, and the extent to which Sutterella may contribute to the pathogenesis of GI disturbances in children with autism.