Lactobacillus reuteri suppresses E. coli O157:H7 in bovine ruminal fluid: Toward a pre-slaughter strategy to improve food safety?

Abstract

The bovine gastrointestinal tract (GIT) is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Therefore, it is crucial to develop strategies, such as EHEC suppression by antagonistic microorganisms, to reduce EHEC survival in the GIT of cattle and to limit shedding and food contamination. Most human-derived Lactobacillus reuteri strains produce hydroxypropionaldehyde (HPA), an antimicrobial compound, during anaerobic reduction of glycerol. The capacity of L. reuteri LB1-7, a strain isolated from raw bovine milk, to produce HPA and its antimicrobial activity against an O157:H7 EHEC strain (FCH6) were evaluated in bovine rumen fluid (RF) under strict anaerobiosis. EHEC was totally suppressed when incubated in RF inoculated with L. reuteri LB1-7 and supplemented with 80 mM glycerol (RF-Glyc80). The addition of LB1-7 or glycerol alone did not modify EHEC survival in RF. Glycerol was converted to HPA (up to 14 mM) by LB1-7 during incubation in RF-Glyc80, and HPA production appeared to be responsible for EHEC suppression. The bactericidal activity of L. reuteri LB1-7, the concentration of glycerol required and the level of HPA produced depended on physiological and ecological environments. In vitro experiments also showed that EHEC inoculated in rumen fluid and exposed to L. reuteri and glycerol had a very limited growth in rectal contents. However, L. reuteri exerted an antimicrobial activity against the rumen endogenous microbiota and perturbed feedstuff degradation in the presence of glycerol. The potential administration of L. reuteri and glycerol in view of application to finishing beef cattle at the time of slaughter is discussed. Further in vivo studies will be important to confirm the efficiency of L. reuteri and glycerol supplementation against EHEC shedding in ruminants.

Fig 1. Survival of EHEC co-incubated with L. reuteri strains in RF samples supplemented or not with glycerol.

The strain FCH6 Rif^R 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 Rif^R 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 Rif^R (≈ 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 Rif^R 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.

Fig 3. Kinetics of EHEC growth or disappearance and HPA production in LB broth.

(A) The strain FCH6 Rif^R (≈ 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 Rif^R 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 Rif^R was co-incubated with L. reuteri in LB broth supplemented with 10 mM glycerol under anaerobiosis. At each time point the strain FCH6 Rif^R 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 Rif^R 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.