Alterações na microbiota intestinal em modelo experimental de menopausa e sua relação com a dieta rica em frutose

Abstract

Changes in gut microbiota (GM) were evaluated due to menopause and its association with high-fructose diet. Adult female Wistar rats were subjected to ovariectomy (ovx; an experimental model of menopause) or sham surgery (control) and divided into 4 groups: rats fed high-fructose diet (10% solution in drinking water; sham-F and ovx-F) and rats fed control diet (drinking water; sham-C and ovx-C). Faeces (1g) was collected immediately before surgery (T0), and at the 4th (T4), 8th (T8) and 12th (T12) subsequent weeks for processing, culture in 5% blood agar, in duplicates, and incubation (24h; 36°C). Manual counting of colony forming units (CFUs) was performed and 10 random colonies of each culture were subcultured in brain-heart-infusion agar (BHIA) for quantitative and qualitative analysis. Biochemical analysis was performed in Gram-positive cocci (GPC) and Gram-negative bacilli (GNB). Two-way ANOVA repeated measures (RM) followed by Student Newman-Keuls (SNK); One-way ANOVA RM or Friedman RM ANOVA on Ranks followed by SNK or Dunnett´s; Two-way ANOVA followed by SNK or KruskalWallis One-way ANOVA on Ranks followed by Dunn’s; and the Fisher’s exact test were used, considering the level of significance as p< 0.05. At T0, similar quantity of CFUs was observed between groups. Differently, from T4 to T12, fructose-fed groups presented increased CFUs compared to controls (p<0.05). Interestingly, sham-F had several uncountable plates (extremely high quantity of CFUs), in which odds ratio of occurring it was 16.1 times more in sham-F compared sham-C. In contrast, ovx-F had only 1 uncountable plate at T4, displaying it again only at T12 (n=6), reflecting that ovx-F had no difference in odds ratio for uncountable plates until T12 compared to ovx-C. Moreover, ovx-F odds ratio for uncountable plates was 0.09 in relation to sham-F. Of the total of 1.320 isolates (T0 to T12) that were obtained, 971 isolates were Gram-positive cocci (GPC), 222 Gram-positive bacilli (GPB), 98 Gram-negative bacilli (GNB), 22 Gram-negative cocci (GNC) and 7 yeasts (Y), in which the latter was observed only in ovx groups. Except for fructose-fed groups at T4, the majority of isolates were GPC (at least 55%) in all groups at any time. Also, all the groups, but ovx-C, had considerable increase in GPB, GNC and/or GNB at T4 (p<0.05) compared to T0. Biochemical analysis of GPC revealed that commensal microorganisms constituted at least 80% in all groups at any time, except for ovx-F at T8, in which it represented 52.1%. Sixty-seven (n=67) GNB recovered isolates, 49 isolates were fermenters, 3 isolates were non-fermenters and 15 isolates did not grow on MacConkey. The majority of GNB were obtained at T4 (n=51, wherein 48 were fermenters, of which 32 were detected in ovx group). All non-fermenters isolates (n=3) were negative on cetrimide agar, thus, non- identified Pseudomonas aeruginosa. Despite such percentual variations, no statistical differences were observed between groups in any time for biochemical analysis of GPC or GNB. Fructose-fed rats drank more solution and ate less food than their respective controls (p<0.05). In addition, as expected, ovx-F had increased body weight (BW) gain compared to sham groups (p<0.05). However, no difference was observed on BW gain due to fructose intake. In conclusion, chronic fructose intake increased CFUs in both sham and ovx females, but ovariectomy prevented the occurrence of overgrowth of CFUs. The percentual of GPB increased in fructose-fed rats from T4 onwards; while the increase of GNB and/or GNC occurred only at T4 and in all groups. Changes in GM seemed to be independently of BW gain. The data herein suggest that ovariectomy attenuated the overgrowth of CFUs, even when it is associated with fructose feeding. Despite that, general changes in GM are likely to be similar to fructose-fed sham rats.