It is increasingly believed that the intestinal microflora strongly affects human health, and the quantification of the intestinal bacterial flora quantification as an adjunctive diagnostic method has gained renewed interest in the field of Complementary and Alternative Medicine . Claims of associations between specific commensal bacterial species and health will have to be established in human feeding and intervention studies, but, to date, only a few studies have investigated the influence of diet and fasting on the bacterial flora in patients. We hypothesised that both, Mediterranean diet and fasting would differentially alter the microflora of RA patients and that the clinical course of these patients during the dietary intervention would be connected to changes in the intestinal microflora as assessed by cultural technique and stool sIgA concentration. However, the results of this study do not suggest any relationship between diet, fecal culture analysis, sIgA and disease activity in patients with RA and FM.
Some cross-sectional studies based on cultural techniques have indicated that the protein and fat content of the diet as well as the nature of carbohydrates (simple sugars vs. complex carbohydrates) do affect microflora composition and activity [7, 21]. Animal studies support the hypothesis that the intestinal microflora can be modified by diet . In contrast, food restriction has been shown in one study to have little effect on the microflora of rats as measured by conventional anaerobic culture , whereas others have observed that food restriction and diet composition both strongly affect the microflora composition as judged by newer diagnostic techniques (denaturing gel gradient electrophoresis) . Generally, data from human clinical studies are limited. Our results contrast to recent findings from a study with 43 patients with RA, which were randomised to an uncooked vegan diet or an ordinary omnivorous diet . After one month, the intervention group showed a significant, diet-induced change in the fecal flora. Furthermore, the clinical improvement was related to the changes in fecal flora, which were assessed by direct stool sample gas-liquid chromatography (GLC). It has also been noted that the GLC method to study overall changes in fecal flora due to a vegan diet might be superior than the classical quantitative culture of stool sample we used . Moreover, the studies' intervention with an uncooked vegan diet rich in lactobacilli clearly differs from the Mediterranean diet that was used in our study. However, in a recent randomised trial on RA patients a Mediterranean diet induced reductions in inflammatory activity and an improvement in physical function to a similar extent as vegan diets in the prior studies . Thus, it may be that vegan diet and Mediterranean diet share nutrients that exert beneficial effects in RA.
Of note, in a randomised trial on fasting and vegetarian diet in RA patients significant differences in the fecal flora were observed between samples obtained at times which coincided with pronounced clinical improvement compared with baseline, versus samples obtained at times of low or no improvement . Again, in this study stool samples were analysed by GLC. Thus, apart from our differing study population with a smaller sample size of RA patients, the conflicting result might be due to a potential higher sensitivity of GLC compared to the classical microbiological cultural analysis used in the present study.
However, we also could not find any effect of the dietary interventions on stool pH and sIgA concentrations (a putative marker of intestinal immune function), which supports the suggestion that Mediterranean diet and fasting do not induce relevant changes in the fecal flora in the short term. To the contrary, in an observational study on patients with chronic pain syndromes (including FM) participation in fasting therapy significantly increased sIgA levels . Yet, baseline sIgA concentrations in this study were markedly lower than in our population, and the increased concentrations at the follow-up corresponded to baseline values in the our study. Therefore, regression to the mean in that study may be a likely explanation for the differing result.
In the present study RA patients tended to show better clinical outcome with fasting than on Mediterranean Diet. However, the interpretation of differences in clinical outcome between the intervention groups is limited by potential baseline differences of nutritional habits. It is known that patients with RA frequently alter their dietary patterns for symptom relief. In fact, In the present study RA patients had a slightly higher nutritional score and a reduced intake of meat compared to FM patients and the whole population showed eating patterns with some aspects of the Mediterranean Diet already realised before study entry. Therefore, the clinical response and changes in fecal flora may have been reduced in the Mediterranean Diet group. Furthermore, due to the non-randomised study design, fasting RA patients were older and had more active disease compared to fasting Mediterranean Diet patients. Thus, we cannot rule out a selection bias in the clinical response to the two types of nutritional interventions.
Another limitation relates to the short intervention time for the Mediterranean diet. Indeed, the clinical benefit of a Mediterranean diet in patients with RA was more evident after 12 than after three or six weeks of intervention time . In contrast, change to an uncooked vegan diet and the return to a Western diet induced alterations in the fecal flora within one to two weeks . Clearly, future studies on the association between Mediterranean diet and intestinal microflora should evaluate longer study periods as minor changes could accumulate over time.
A principal limitation of the present study relates to the small sample size. This implies that no definite conclusions should be drawn from our data. However, the complete absence of effects of the interventions on the bacterial flora makes it unlikely that a larger sample size would have yielded different results. Other factors, as medication, may also have influenced the bacterial flora. Yet, neither probiotics or antibiotics were given, and, within the diagnostic groups there were no other relevant differences of prescribed medication between fasters and patients on Mediterranean Diet. Another limitation that applies to all research testing fecal samples to determine bacterial flora is the influence of the fecal water concentration. This factor may not only vary from individual to individual, but within individuals within the same day. However, drying the stool would not be possible without compromising the presence of certain bacteria.
It may be further argued whether microbiologic analysis of the fecal flora with its limitations in diagnostic accuracy is the appropriate method to assess treatment effects. However, culture is the classical approach for the identification and quantification of bacteria. Most of the data available on the gut bacteria have been generated by cultivation and enumeration . Still, this approach is limited in scope, as a majority of the bacterial species present in feces are not culturable using standard microbiologic techniques. Yet, we believe that the lack of effect of the interventions on sIgA in our study further points to the suggestion that the intestinal microflora is not connected to the dietary treatments and clinical outcome in the selected patient groups. There are now refined developments with molecular analysis tools including the availability of the completed genome sequences . Given their expense, these newer molecular techniques await their broader implementation in clinical research. Clearly, the future utilisation of molecular microflora analysis tools can help to clarify a potential interplay between diet and human microbiota.
Finally, it is important to note that labelling nonpathogenic commensal bacteria as either beneficial or detrimental remains highly speculative. Yet, as we did not found any alterations in the fecal flora of our patients, this issue remains negligible for the interpretation of our results.