Fig 1.
Survival of EHEC co-incubated with L. reuteri strains in RF samples supplemented or not with glycerol.
The strain FCH6 RifR was co-inoculated with ≈ 107 CFU/mL of L. reuteri LB1-7 (HPA producer) or 100–23 (negative control) in RF samples under anaerobiosis for 24 hours. The strain FCH6 RifR inoculated alone in RF samples was used as control. RF samples were supplemented or not with glycerol at different concentrations. Bars represent the SEM of three independent experiments. Asterisks indicate statistical significance (***: P<0.001).
Fig 2.
EHEC counts and HPA, 1,3-PD and glycerol quantification in RF-Glyc80.
The strain FCH6 RifR (≈ 104 CFU/mL) was co-incubated with L. reuteri LB1-7 (≈ 107 CFU/mL) in RF samples supplemented with 80 mM glycerol under anaerobiosis. At each time point the strain FCH6 RifR was enumerated and accumulation of HPA and 1,3-PD, and disappearance of glycerol were monitored. Bars represent the SEM of three independent experiments. Gly: glycerol.
Table 1.
Concentration of glycerol and microbial metabolites.
Fig 3.
Kinetics of EHEC growth or disappearance and HPA production in LB broth.
(A) The strain FCH6 RifR (≈ 104 CFU/mL) was co-incubated with L. reuteri LB1-7 (≈ 107 CFU/mL) in LB broth supplemented or not with different concentration of glycerol. The strain FCH6 RifR was then enumerated after 24 hours of incubation under anaerobiosis. Bacterial growth curves are expressed as a single representation of three independent experiments. (B) The strain FCH6 RifR was co-incubated with L. reuteri in LB broth supplemented with 10 mM glycerol under anaerobiosis. At each time point the strain FCH6 RifR was enumerated and accumulation of HPA was quantified. The bacterial growth curve is expressed as a single representation of three independent experiments. Bars represent the SEM of three independent experiments.
Fig 4.
Dry matter degradation (DMD) of forages and fibrolytic ruminal population.
(A) Dry matter degradation of alfalfa hay (AH) and corn silage (CS) by the rumen microbiota after 24 hours of incubation was quantified in the Daisy II incubation system containing RF in the presence or absence of L. reuteri LB1-7 and 80 mM glycerol. Non-incubated bags containing forage were used as control (see the experimental procedures section). The data are expressed as the percentage of dry matter degraded. Bars represent the SEM of three independent experiments. (B) Quantification of the rrs gene copies of the total rumen bacterial population, F. succinogenes and R. flavefaciens in DAISY II vessels. Total DNA template extracted from the Daisy II vessel containing only RF and buffer was used as control (Ctrl). The bacterial populations were quantified before and after 24 hours of incubation. pH was monitored at the start and end of incubation. Bars represent the SEM of three independent experiments. Asterisks indicate statistical significance (*: P<0.05; ***: P<0.001).
Fig 5.
Growth or survival of EHEC in rectum contents after incubation in RF.
The strain FCH6 RifR was first incubated in filter-sterilized RF (FS-RF) samples alone or inoculated with ≈ 107 CFU/mL of L. reuteri LB1-7 supplemented with 80 mM glycerol under anaerobiosis for 24 hours. The bacterial pellet was then inoculated into Rec samples and incubated under anaerobiosis. RF = 0 represents inoculation of EHEC in FS-RF samples; Rec t = 0 corresponds to RF t = 24h i.e. number of EHEC surviving the incubation in FS-RF during 24 hours; Rec t = 6h and Rec t = 24h correspond to EHEC survival in Rec samples after 6 and 24 hours of incubation respectively. Bars represent the SEM of three independent experiments. Effect of L. reuteri + Glyc80 is significant *, P<0.05; ***, P<0.001.