Age and Fecal Microbial Strain-Specific Differences in Patients With Spondyloarthritis

Matthew L. Stoll; Pamela F. Weiss; Jennifer E. Weiss; Peter A. Nigrovic; Barbara S. Edelheit; S. Lou Bridges Jr; Maria I. Danila; Charles H. Spencer; Marilynn G. Punaro; Kenneth Schikler; Andreas Reiff; Ranjit Kumar; Randy Q. Cron; Casey D. Morrow; Elliot J. Lefkowitz


Arthritis Res Ther. 2018;20(14) 

In This Article



The demographic and clinical features of the pediatric subjects are summarized in Table 1. All were naïve to immunosuppressive therapy, and none had been exposed to systemic antibiotics within 3 months prior to enrollment in the study. The patients and controls were well matched for age and BMI.

16S Sequencing

A mean of 129,226 (range 39,741–249,043) paired-end reads were obtained from the 49 subjects. Following quality filtering, merging, and chimera removal, a total of 64,016 (range 23,942–105,497) reads were used. Among the 49 subjects, a total of 1402 unique sequences (sequence variants (SVs)) were obtained; as most of these were rare and unique to specific subjects, the range was 46–236 SVs/subject.

PCoA on the 49 subjects demonstrated clustering by diagnosis (ERA versus control; Figure 1). This visually evident clustering was confirmed by the PERMANOVA test (F = 1.45, p = 0.046). Among the ERA patients, no sources of variation were observed on the basis of any single variable, including geographic location (Bray Curtis F = 1.09, p = 0.277), HLA-B27 status (Bray Curtis F = 0.902, p = 0.652), or the presence of sacroiliitis (Bray Curtis F = 0.471, p = 1). Geographic location was assessed both by looking at each site as a separate unit and by pooling into one region the four sites located in the northeast (Boston Children's Hospital, Connecticut Children's Medical Center, Hackensack University Medical Center, and Children's Hospital of Philadelphia), a geographic area that is similar in size to the catchment area of UAB.

Figure 1.

Principal coordinates analysis of the 16S sequencing results obtained from children with ERA and pediatric healthy controls demonstrating clustering by diagnosis. Blue and red dots reflect ERA patients and controls, respectively

Due to the large number of SVs as already indicated, pairwise comparisons at most of the phylogenetic levels failed to identify any groupwise differences following adjustment for multiple comparisons. The only exception was that ERA patients demonstrated decreased abundance of the Actinobacteria phylum as compared to controls (3.4% versus 9.3%, p = 0.002 uncorrected, p = 0.05 FDR-corrected). This lack of any significant pairwise differences held even after SVs within a species were collapsed into the species, thus reducing the number of unique items to 366. Consequently, based upon previous work in children with ERA,[7,8] the analysis was focused on two organisms: F. prausnitzii and the Bacteroides genus, particularly B. fragilis.

The abundance of F. prausnitzii as a whole was nominally higher in the patients (10 versus 7.8%, p = 0.192; Figure 2a), in contrast to our previous results.[8] Twenty-three unique SVs were identified within F. prausnitzii. As discussed in Methods, BLAST was used to identify strains within the F. prausnitzii SVs identified by DADA2. Of those 23 SVs, one of them was a 100% best match (253/253 bp) with strain A2-165, which has been shown previously to have regulatory effects.[39] Recent studies have demonstrated that even compared to other strains of F. prausnitzii, such as L2/6, the A2-165 strain induces increased interleukin-10 production from peripheral blood mononuclear cells[16,40] and has increased butyrate promoter activity.[41] A separate SV was a 99% best match (252/253 bp) with L2/6, the comparator strain in some of the aforementioned studies. These two strains were the two most abundant SVs matching F. prausnitzii. As a proportion of the sequences that matched F. prausnitzii, ERA patients had relatively decreased abundance of the regulatory A2-165 strain (41 ± 28% versus 54 ± 20%, p = 0.084; Figure 2b) and a relatively increased abundance of the SV that most closely matched L2/6 (28 ± 28% versus 15 ± 15%, p = 0.038; Fic 2c). Thus, the stool samples from pediatric ERA patients contained a decreased ratio of the antiinflammatory, relative to the neutral, strain of F. prausnitzii.

Figure 2.

Strain-level variation in the fecal abundance of Faecalibacterium prausnitzii in children with ERA and pediatric controls. Abundance of F. prausnitzii as a percentage of total fecal content is similar in patients and controls (a). As a percentage of total F. prausnitzii, children with ERA have a trend toward decreased fecal abundance of the anti-inflammatory A2-165 strain (b) as well as increased abundance of the L2/6 strain (c). ERA enthesitis-related arthritis

ERA patients and controls had similar levels of the Bacteroides genus (20 versus 19%). However, among the Bacteroides genus, B. fragilis is considered to have regulatory properties through its polysaccharide tail[42] and thus was investigated further. Here, consistent with our previous findings,[8] ERA patients had a four-fold increased abundance (2.0 ± 4.0% versus 0.45 0.7%, p = 0.045; Figure 3a). There were no significant differences with any of the other species of Bacteroides. In summary, children with ERA had diminished abundance of the regulatory A2-165 strain of F. prausnitzii as well as increased abundance of B. fragilis, compared to healthy control subjects.

Figure 3.

Fecal abundance of Bacteroides fragilis in pediatric and adult subjects with SpA. Increased fecal abundance is observed in ERA patients compared to pediatric controls (a), while a trend toward decreased fecal abundance was observed in adult SpA patients compared to adult controls (b). ERA enthesitis-related arthritis, SpA spondyloarthritis

To evaluate whether these strain-level and species-level differences between arthritis subjects and controls are unique to pediatric subjects or are potentially a marker of SpA as a whole, 16S sequencing was run on a cohort of 11 adults with longstanding SpA and 10 healthy adult controls. Characteristics of the adult subjects are presented in Table 2; most had longstanding disease, with a mean disease duration of 11 years, and most were on immunosuppressive therapy. As with the pediatric subjects, none of the adult SpA patient or controls had been exposed to systemic antibiotics within 3 months of enrollment.

Among the adults, the opposite trend with respect to Bacteroides abundance was seen. Specifically, Bacteroides abundance as a proportion of total sequencing depth was 11 ± 9% among the patients, compared to 26 ± 18% in the controls (p = 0.036), although there were no significant differences in the abundance of B. fragilis (0.2 ± 0.6% in patients versus 1 ± 1.8% in controls, p = 0.106) (Figure 3b). In contrast, the trends involving F. prausnitzii strains were similar in adults as compared to children (total F. prausnitzii 6.9 ± 6.9% in controls versus 10 ± 10% in patients (p = 0.427); A2-165 as percentage of F. prausnitzii 41 ± 30% in controls versus 25 ± 13% in patients (p = 0.175)), although the differences were not statistically significant, possibly due to a smaller sample size. Similar trends for both organisms were observed if the three subjects taking sulfasalazine were excluded (data not shown.)

Finally, the abundance of additional organisms that have emerged as being of interest in SpA was evaluated. Specifically, the Lachnospiraceae family was reduced in children with ERA in our previous work,[8] yet was elevated in both biopsy[43] and fecal[44] specimens in prior studies of adults with SpA. There were, however, no differences in the abundance of this family in either the pediatric or the adult subjects (data not shown). A study of ileal biopsies demonstrated that the abundance of the Dialister genus correlated with disease activity in treatment-naïve SpA patients;[6] this organism was virtually undetectable (<1%) in the fecal specimens of all subjects in both age groups, likely indicating specificity to mucosal specimens. Ruminococcus gnavus was elevated in the feces of adult subjects with SpA in a prior report,[44] but the differences of this organism herein were not statistically significant in either age group. Finally, both Stebbings et al.[5] and our group[8] have shown increased abundance of Bifidobacterium in the feces of SpA patients; surprisingly, the same genus was depleted among pediatric ERA subjects compared to healthy controls (2.6% versus 7.2%, p = 0.008), with no differences among the adult subjects.

Whole Genome Sequencing

To assess the potential functional consequences of the presented findings, whole genome sequencing of fecal DNA with the HiSeq device was performed on a subset of the aforementioned pediatric patients, consisting of 14 patients each (ERA and healthy controls), supplemented by 12 additional subjects (five controls and seven ERA patients) who were sequenced with the MiSeq device. These subjects were similar with respect to demographic characteristics and BMI (Table 3). A mean of 23 million (range 14.7–38.7 million) high-quality sequences from the Illumina HiSeq sequencer was analyzed, as was a mean of 5.3 million (range 3.5–7.8 million) high-quality sequences from the MiSeq device. There was no obvious clustering based upon diagnosis at either the level of individual genes or whole pathways (data not shown), consistent with a core microbiome not affected by the disease state.[45] In light of our findings in the present study and our previous report[8] showing alterations in the fecal abundance of F. prausnitzii, we specifically examined the butanoate pathway (MetaCyc title CENTFERM-PWY pyruvate fermentation to butanoate), which results in synthesis of butyrate from precursors such as pyruvate and acetyl-CoA. With both devices, there was a modest difference in the abundance of this pathway. For both the MiSeq and HiSeq devices, each value was normalized to the mean value for the controls for the respective run, and the two sets were pooled, revealing a statistically significant decreased representation of the butanoate pathway in ERA patients compared to controls (1 ± 0.48 versus 0.72 ± 0.33, p = 0.037; Figure 4.) Thus, our data indicate that the fecal microbiota of ERA patients compared to controls had decreased capacity to synthesize a compound that is generally considered to have anti-inflammatory properties within the intestinal environment.[46]

Figure 4.

Fecal abundance of bacterial genes comprising a pathway involved in butyrate synthesis. Results of two separate studies shown. For each, each subject was normalized to the mean of the healthy controls for that study, and the combined results are shown. ERA enthesitis-related arthritis