Four types of beneficial bacteria were investigated, including bifidobacteria, Lactobacillus spp. (all strains, since all are beneficial), E. Coli, and Enteroccus - see Table . The children with autism had much lower levels of Bifidobacterium (-45%, p = 0.002), slightly lower levels of Enterococcus (-16%, p = 0.05 per Wilcox), and much higher levels of Lactobacillus (+100%, p = 0.00003).
Beneficial bacteria from stool analysis.
Table lists the dysbiotic bacteria, which were observed during aerobic culture growth. Since these bacteria were only observed rarely, the table lists how many individuals had measurable levels of these bacteria, and the average levels of the bacteria. There were no significant differences between the children with autism and the typical children.
Dysbiotic bacteria found in stool analysis.
Some bacteria are not believed to be especially beneficial or detrimental, and we term those commensal bacteria, and list the results for them in Table . Since these bacteria were only observed rarely, the table lists how many individuals had measurable levels of these bacteria, as well as the average level. The only possibly significant differences were that the autism group was more likely to have Bacillus spp (21% vs. 2.6%, p = 0.05) and less likely to have Klebsiella oxytoca (1.7% vs. 12.8%, p = 0.04).
Commensal bacteria found in stool analysis.
The presence of yeast was determined by both culture and by microscopic observation. Yeast was only rarely observed by culture in the autism or typical groups, and the difference between the two groups was not significant, as shown in Table . Yeast was more commonly observed microscopically, but again the difference between the two groups was not significant.
Cultured and microscopic yeast.
Elastase, a digestive enzyme, was measured and found to be similar in both the autism and typical groups, shown in Table . The presence of fat, muscle fibers, vegetable fibers, and monosaccharides were measured in both groups, and again no significant differences were found.
Possible markers of inflammation, including lysozyme, lactoferrin, white blood cells, and mucus, were investigated and shown in Table . The autism group had a lower level of lysozyme (-27%, p = 0.03 by Wilcox analysis), but no other significant differences.
Summary of inflammatory markers found in the stool analysis.
Levels of secretory IgA were measured, and no significant difference between the two groups was observed as shown on Table . Secretory IgA was highly correlated with lysozyme for the autism group (R = 0.69, p < 0.001), and moderately correlated for the controls (R = 0.35, p < 0.01).
Secretory IgA (sIgA) in stool (mg/dL)
Short Chain Fatty Acids
The presence of several short chain fatty acids (SCFA), including acetate, proprionate, butyrate, and valerate were measured. The total amount of SCFA was significantly lower in children with autism (-27%, p = 0.00003). Similarly, the levels of acetate, proprionate, and valerate were also lower in the autism group, as shown in Table . The lower level of SCFA's appears to be partly due to probiotic usage (see Effect of Probiotics section below). The low level of SCFA's is also partly due to lower sacchrolytic fermentation by beneficial bacteria, lower intake of soluble fiber, prolonged transit time due to constipation, and/or possibly increased absorption by the gut due to increased permeability. However, from this study we cannot determine among those possibilities.
Summary of short chain fatty acids (SCFA's) in stool.
The presence of RBC or occult blood was very rare in both groups, and not significantly different between the two groups. The fecal pH, a reflection of colonic pH, was very similar in both groups, although the autism group had a somewhat larger standard deviation, as listed in Table . (F-test for equal value p-value = 0.0004).
Gender differences were investigated in the typical group for all of the measurements, and there were no statistically significant differences between males and females, expect for a possibly significant higher level of yeast in the females (p = 0.04). For the autism group, there were too few females to justify comparing the males vs. females.
Effect of Probiotics
The autism group was split into two groups, A-Probiotic (those that used any type of probiotic daily) and those that did not use any probiotics, A-No-Probiotic. The only significant differences (p < 0.01) were that, compared to the A-No Probiotic group, the A-Probiotic group had a lower level of total SCFA's and each individual SCFA - see Table and Figure . Probiotics did not have a significant effect on most of the beneficial bacteria, except for a marginally higher level of the level of lactobacillus in the A-Probiotic group compared to the A-No-Probiotic group (+30%, p = 0.08).
Figure 1 Comparison of SCFA's for the Autism-Probiotic group, the Autism-No-Probiotic group, and the controls. Note that the Valerate data is magnified 10x so that it is visible on the chart. The stars indicate the degree of significance of the result of a t-test (more ...)
The A-Probiotic group had a lower level of lysozyme than did the A-No-Probiotic group, but the difference was not significant (277 +/- 231 vs. 371 +/- 302, n.s.). (see Lysozyme section). A t-test comparison of lysozyme in the autism groups with the Control group found a significantly lower level in the A-Probiotic group (31% lower, p = 0.03), but no significant difference for the A-No-Probiotic group (-11%, n.s.).
For the control group, only 5% used a probiotic, so they were not analyzed separately.
Effect of Seafood and Fish Oil Consumption
The autism group was split into three groups based on consumption of seafood and fish oil (see Table ):
Consumption of Fish and Fish Oil.
A- Fish - Consumption of seafood more than 2x/month - (57% also consumed fish oil daily)
A- Fish Oil - No consumption of seafood; consumes fish oil supplement daily
A-No Fish - No consumption of seafood or fish oil
The typical group was divided similarly, but none of the typicals were consuming fish oil regularly.
A t-test comparison of those groups found only two biomarkers which differed between the groups with p < 0.01, namely lactobacillus and pH. Regarding Lactobacillus, the A-Fish Oil and the A-No Fish had similar levels, but the A-Fish had dramatically lower levels than the other two groups. Regarding pH, the A-Fish Oil group had slightly higher pH values than the other two groups.
The control group was split into only two groups, C-Fish and C-No Fish, because none of the controls consumed fish oil (see Table ). There were no significant differences between the two groups.
Kruskall-Wallis Analysis on Lactobacillus
Since exploratory data analysis with t-tests found that levels of lactobacillus were associated with both fish consumption and probiotic usage, a Kruskall-Wallis rank sum test (which treat the data as the ranked values that they are) was conducted. For the autistic group, Lactobacillus levels were very significantly related to levels of fish consumption (p = 0.0008) and adding other variables (ie, fish oil consumption or probiotics) did not add to the significance of the relationship. No relationships were found for the control group.
Effect of Gut Symptoms in Autism Group
The autism group had significantly greater gut symptoms than the control group in part because the control group was chosen to be "in good health: no stomach/gut problems." To investigate the effect of gastrointestinal problems on the results, the autism group was split into two groups: Low-GI-Problems (defined as 6-GSI of 3 or less) and High-GI-Problems (defined as 6-GSI score above 3). The two groups were compared for all the biomarkers reported above, and there were no significant differences (p < 0.01) in any biomarkers.
However, the ATEC scores of the two groups were very different, and this is reported in Table and displayed in Figure . The total ATEC score was 66% higher in the High-GI-Problem group (p = 0.00002), and the four subscales were also higher (+40% to +103%, p < 0.01).
ATEC scores for the Autism-Low-GI-Problem group (6-GSI score of 3 or lower) and for the Autism-High-GI-Problem group (GSI-6 score above 3).
Comparison of ATEC subscores for the Autism-Low 6-GSI group (few GI problems) and the Autism High 6-GSI group (many GI problems); the stars indicate the significance (*** p < 0.001, ** p < 0.01).
Correlations with Gut Symptoms and Autism Severity
For the autism group, the 6-GSI was found to strongly and very significantly correlate with the total ATEC (r = 0.60, p < 0.001); see Figure . Due to this strong correlation, correlations with the ATEC subscales were also determined and are listed in Table .
6-Item Gastrointestinal Severity Index (6-GSI) vs. total ATEC score.
Correlations between total ATEC and subset divisions and total GI Severity.
Correlations of each biomarker of gut status with the 6-GSI and the total ATEC scores were investigated. (However, comparisons with individual bacteria and yeast were not done, since in most samples they were not detected, and the large number of the comparisons made the required per-comparison p-value very low). For the 6-GSI there was a small correlation with carbohydrates (R = 0.27, p = 0.04). For the total ATEC, there was a small correlation with lysozyme (R = 0.29, p = 0.03). However, since multiple correlation analyses were conducted, the cut-off for significance (p = 0.05) should be divided by the number of tests. So, these correlations are at most only possibly significant, and a larger study would be needed to assess this possible weak correlation.