PCR product was subsequently purified using magnetic beads (AMPure beads, selleck chemicals llc Beckman Coulter Genomics, Danvers, USA). Concentration was measured on Qubit fluorometer (Invitrogen, Carlsbad, CA, USA). Equimolar amounts of PCR product from each sample were used for unidirectional 454 FLX amplicon pyrosequencing using LIB-L emPCR kits following the manufacturer’s protocols (Roche Diagnostics, Basel, Switzerland). Metagenomic data processing Flowgrams were processed using amplicon analysis option in data processing software from Roche. The sequencing resulted in 161551 overall number of reads. Quality trimmed sequences obtained from the FLX sequencing run were processed using RDP pyrosequencing pipeline. Beforehand, data files were depleted of chimeras by Black Box chimera Checker [24] using default settings.

Processing involved aligning of sequences with fast, secondary-structure aware Infernal aligner, subsequent clustering with max distance of 3% based on complete-linkage clustering method and classifying of incurred clusters by na?ve Bayesian rDNA classifier [25]. Bootstrap cutoff was set to 50%, which was sufficient for accurate classification at the genus level. Generated designations of clustered sequences together with their relative abundances within the given samples were used for comparing bacterial diversity. Statistical analysis One-way analysis of variance (ANOVA) with Dennett’s multiple comparison test was used to compare multiple experimental groups with the control group.

Differences between two groups were evaluated using an unpaired two-tailed Student’s t-test and deviation of values from hypothetical mean were calculated by one sample t-test. The data is presented as the mean �� standard deviation (SD) unless stated otherwise and differences were considered statistically significant at P��0.05. GraphPad Prism statistical software (version 5.0, GraphPad Software, Inc., La Jolla, CA,USA) was used for analyses. Results Oral administration of lysate L. casei attenuate the acute colitis in BALB/c mice but not in SCID mice In our previous study we showed that oral treatment with L. casei DN-114 001 attenuates the severity of acute experimental colitis [21]. To test if its lysate have similar activity, we pretreated mice with four weekly oral doses of Lc and induced colitis by DSS in BALB/c and SCID mice.

Oral (Table 1A) but not parenteral (data not shown) administration of Lc is effective in preventing the acute DSS colitis in BALB/c mice, improving clinical and morphological markers of colitis. In contrast, when colitis was induced in SCID mice (Table 1A) pretreatment with Lc failed to improve acute Carfilzomib colitis in all tested parameters. Also no significant effects of Lc were found when the model of chronic colitis was used (data not shown). Table 1 Lc improves the severity of DSS-induced colitis in BALB/c, but not in SCID mice. Lysate of L.