, Clindamycin shows good penetration into tissues and is often used to treat skin or soft tissue infections. Pseudomembranous colitis (PMC) caused by overgrowth of Clostridium difficile
is a potentially life-threatening complication of antibiotic therapy. The probiotic product VSL#3 is a dietary supplement often used for treatment of various gastrointestinal complaints directly associated with microbial dysbiosis such as chronic constipation, diarrhea, flatulence, ulcerative colitis and pouchitis [16
The in vitro
model used in this study provides standardized and reliable conditions to study the effects of pro- and antibiotics on the human intestinal microbiota [17
] and is has an advantage over living system in continuous sampling over a defined period of time. Moreover, the system is hardly biased by environmental factors, e.g. temperature, humidity or oxygen, which can be controlled to a high extent.
The TIM-2 experiments were performed using a standardized microbiota from healthy individuals. In the control unit the standard ileal efflux meal (SIEM) was fed to the system. In one experiment the antibiotic was administered together with a probiotic mixture (VSL#3) and in the other experiment the probiotic was administered after the antibiotic treatment.
Production of beneficial microbial metabolites
Short chain fatty acids (SCFA) and lactate are beneficial microbial metabolites. SCFA and lactate acidify the intestinal lumen, causing growth arrest or even death of (opportunistic pathogens). In addition, the SCFA are an energy source for the host: butyrate for colonic epithelial cells, acetate and propionate, in amongst others liver and muscle cells [28
]. Figure presents cumulative total production of the short chain fatty acids, e.g acetate, propionate and n-butyrate during the different experiments in TIM-2, and represents metabolites present in lumen and dialysate. The amount of SCFA present at the start of the experiment has been artificially set to zero so the graphs only reflect the production of metabolites after start of addition of the test products.
Figure 3 Cumulative production of the short chain fatty acids (SCFA) acetate, propionate and n-butyrate during the different experiments in TIM-2: (A) Clindamycin for 7 days (d 1-7 a) followed by VSL#3 (d 8-14 p); (B) Clindamycin + VSL#3 for 7 days (d 1-7 a + (more ...)
The total SCFA production was not affected by the use of Clindamycin or Clindamycin plus probiotics. When probiotics were administered after the administration of Clindamycin for one week, the SCFA production increased since the slope of the total SCFA production increased in the second week, compared with the first week of the experiment. The production of n-butyrate and propionate was increased when probiotics were added. The acetate concentration was unaffected by the addition of Clindamycin or probiotics. When Clindamycin and probiotics were administered together the propionate production was decreased. These differences are likely to be caused by changes in the microbiota composition.
Figure presents the cumulative total production of lactate. Lactate was produced in all variations, but when probiotics were added the lactate production was increased, independent of the presence of Clindamycin. The probiotics were lactic acid bacteria and the extra production of lactate proved the probiotics were active in the microbiota. Lactate is only accumulating when there is a fast fermentation. If substrates are fermented slowly, lactate is converted into the other SCFA (primarily propionate and butyrate) and does not accumulate.
Figure 4 Cumulative production of lactate (D- and L-lactate) during the different experiments in TIM-2: (A) Clindamycin for 7 days (d 1-7 a) followed by VSL#3 (d 8-14 p); (B) Clindamycin + VSL#3 for 7 days (d 1-7 a + p); (C) no therapy group for 7 days (controls) (more ...)
The total SCFA production was not affected by the use of antibiotics or antibiotics plus probiotics. When probiotics were added after using antibiotics, the SCFA production increased. Propionate production was decreased when antibiotics and probiotics were used together. Enhanced production of lactate was observed both when probiotics were administrated together with Clindamycin or when they were administered after seven days of clindamycin administration.
Production of putrefactive microbial metabolites
Branched chain fatty acids (BCFA; iso-butyrate and iso-valerate) and ammonia are metabolites produced from protein fermentation, a process which is generally believed to be putrefactive and leading to production of toxic metabolites. These products are deleterious for host health [22
Figure presents the cumulative total production of BCFA. BCFA are produced in small amounts for every test variation compared to the SCFA (about 20 to 40 fold lower). Total BCFA production was highest when probiotic was administered after clindamycin. However, when Clindamycin and probiotics were administered at the same time, the BCFA production was decreased. In the experiments in which Clindamycin was administered (the first 7 days), the BCFA production was comparable to the control. Therefore the decreasing effect probably was induced by the use of probiotics. When probiotics were administered after a week treatment with Clindamycin, this decreasing effect in BCFA production was not observed.
Figure 5 Cumulative production for the branched chain fatty acids (BCFA) iso-butyrate and iso-valerate during the different experiments in TIM-2: (A) Clindamycin for 7 days (d 1-7 a) followed by VSL#3 (d 8-14 p); (B) Clindamycin + VSL#3 for 7 days (d 1-7 a + p); (more ...)
Figure shows the cumulative total production of ammonia. For ammonia the production was decreased between day 3 and 7 in the test experiments compared to the control. In the experiments in which Clindamycin was administered, as well as in which Clindamycin was administered together with probiotics, the ammonia production was reduced just as observed for the BCFA.
Figure 6 Cumulative production for ammonia during the different experiments in TIM-2 (A) (Clindamycin for 7 days (d 1-7 a) followed by VSL#3 (d 8-14 p); Clindamycin + VSL#3 for 7 days (d 1-7 a + p); no therapy group for 7 days (controls). Figure 6B shows the comparison (more ...)
Composition of the microbiota
To determine the effects of Clindamycin and the probiotics on the composition of the microbiota, the I-chip platform was used. The I-chip contained roughly 400 probes, some for group-level detection (e.g Bifidobacterium genus) and some for the detection of individual species (e.g. Bifidobacterium longum). Some groups and species were covered by more than one probe. In all cases the hybridization to these multiple probes correlated very well. However, not al probes gave a signal above background noise, which was expected, as not all microorganisms are present above the level of detection of the method (approximately 107 CFU/g). Due to the different nature of each probe (different sequence), hybridization intensity does not necessarily reflect abundance. Difference in GC-content results in different hybridization efficiencies. Although the I-Chip at most is semi-quantitative, comparing signals from one and the same hybridization does allow interpretation of the increase or decrease of certain probes. For the TIM-2 experiments samples from time points 0, 7 and 14 were analyzed.
Figure shows the results of the I-chip analysis. Displayed is the fold-increase in signal between the start and the end of the fermentation period compared to the control. For day 14 of the experiment with Clindamycin followed by probiotics the results at day 14 were compared with the same experiment at day 7, after Clindamycin only.
Figure 7 Graphic representation of the I-chip results showing those probes that i) give a signal above the background, and ii) differed by a factor of > 2 from the control for the first two columns. For the third column the effect of the addition of probiotics (more ...)
Different shades of green reflect more than 2, more than 3 and more than 4 times increases of microbial species, genera or groups compared to the control, while the different shades of red reflect the more than 2, 3 and 4 times decrease of microbial species, genera or groups compared to the control.
Comparing the experiments receiving Clindamycin to the control experiment, the experiments with administration of Clindamycin showed a decrease in Bifidobacerium animalis Bifidobacterium longum, Crenarchaeota, Enterobacteriaceae, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. and an increase in Bifidobacterium bifidum Eubacterium eligens, Bacteroidetes, Bactetroidales, Ruminococcus albus, Ruminococcus bromii and Fusobacterium prausnitzii.
When Clindamycin and probiotics were administered together the following species increased compared to the control: Bifidobacterium animalis, Enterobacter cloaca/Serratia marcesens/Salmonella typhi, Enterococcus species, Haloanaerobiale, Lactobacillus acidophilus, Lactobacillaceae, Lactobacillus casei and paracasei, Lactobacillus gasseri, Lactobacillus sakei, Microbacteriaceae, Nitrospirae, Parabasilidea peptostreptococcus asaccharolyticum, Streptococcus groups and Streptococcus salivarius. Bifidobacterium longum (which was in the probiotic mixture) decreased less strong than when Clindamycin was administered alone.
When Clindamycin was administered for 7 days and the probiotics were administered the week thereafter the bacteria that increased compared to the situation after antibiotic treatment alone were Bifidobacterium adolescentis/Bifidobacterium angulatum, Bifidobactrium longum, Collinsella aerofaciens, Enterococcus hirae, Eubacterium siraeum, Eubacterium xylanophilum, Euryachaeota, Moraxellaceae and Peptostreptococcus micros. The groups that decreased were Bifidobacterium catenulatum, Bifidobacteriaceae, Brevibacteriaceae, Campylobacter coli/jejuni, Clostridium coccoides/Ruminococcus productus, Clostridium sporogenes, Enterobacter cloacae/Serratia marcesens, Salmonella typhi/Klebsiella penumoniae, Eubacterium contortum, Haloanaerobiales, Lactobacillus acidophilus, Lactobacillus casei and paracasei and Phascolarcobacterium faecium.
Administration of clindamycin together with probiotics has positive effect on lactobacilli while the administration of probiotic after antibiotic has negative effect on same bacterial group. For the bifidobacteria this seemed to be divided in two groups, increase in one group (namely Bifidobacterium animalis) was observed when Clindamycin together with probiotics, but not when probiotic was administated after Clindamycin. Decrease in another group (namely Bifidobacterium catenulatum) was observed only when probiotics were administrated after clindamycin but not in other experimental setups
Statistical analyses (SAM) of the data obtained with the I-chip showed that all time point 0 samples clustered together (data not shown) and thus could be considered equal. The SAM analysis did not add new information to the other analysis performed on the I-chip data.
According to the I-chip results not all strains from the probiotic mixture increased when the mix was added to the TIM-2 system; therefore we plated the mixture to get an idea of the amount and proportions of the bacterial strains in the mixture. The amount of bifidobacteria was very low in the mixture and only Bifidobacterium longum could be identified.
After administration of clindamycin, a decrease in bifidobacteria and lactococci groups was observed, whereas in the experiment in which Clindamycin was administered together with the probiotic mix, an increase in Bifidobacterium animalis as well as several Lactobacillus strains could be observed, and decrease of Bifidobacterium longum was also less strong, decreasing from 4 fold to 2 fold.
Increase in the beneficial bacterial group Lactobacilli was observed when Clindamycin and probiotics were administered together, while if the probiotics were administered following the administration of Clindamycin the level of lactobacilli was lower. In summary, in this study we could demonstrate that the simultaneous administration of anti- and probiotics had the most significant positive effects on intestinal homeostasis by stabilizing the intestinal microbial composition, increased production of short chain fatty acids and decreasing the production of toxic microbial metabolites like ammonia and other branched chain fatty acids. We could also show that probiotics are active when applied simultaneous with antibiotics. Therefore the administration of probiotics could be of significant advantage in the prevention of AAD and CDI by surveillance of intestinal metabolic balance.