Elsevier

Anaerobe

Volume 30, December 2014, Pages 70-74
Anaerobe

Clinical microbiology
Butyricicoccus pullicaecorum, a butyrate producer with probiotic potential, is intrinsically tolerant to stomach and small intestine conditions

https://doi.org/10.1016/j.anaerobe.2014.08.010Get rights and content

Highlights

  • The butyrate-producing Butyricicoccus pullicaecorum has probiotic potential.

  • We evaluate its tolerance to stomach and small intestine conditions.

  • Acid conditions induce a viable and active but non-cultivable state.

  • Cultivability is restored during small intestine conditions.

  • Stomach and small intestine conditions induce a shift to acetate production.

Abstract

Butyrate has several beneficial properties that are essential to maintain gastrointestinal health. Therefore butyrate-producing bacteria are seen as the next generation of probiotics. The butyrate-producing bacterium Butyricicoccus pullicaecorum (a clostridial cluster IV strain) is such a promising probiotic candidate for people suffering from inflammatory bowel disease. To exert its beneficial properties, it is crucial that B. pullicaecorum survives the harsh conditions of the upper gastrointestinal tract to arrive in the colon in a viable and metabolically active state. Before developing a stable formulation of B. pullicaecorum for oral administration, it is important to know its intrinsic acid and bile tolerance. We monitored the survival during and short chain fatty acid production after incubation in conditions simulating the stomach and small intestine using in vitro batch experiments.

In case of acid conditions (pH 2 and pH 3), B. pullicaecorum was viable and active but not cultivable. Cultivability was restored during subsequent small intestine conditions. Importantly, bile and pancreatic juice had no lethal effect. Milk, as a suspension medium, only had a protective effect on the cultivability during the first hour at pH 2. B. pullicaecorum was still metabolically active after upper gastrointestinal conditions and produced short chain fatty acids, but a shift from butyrate to acetate production was observed. Although the butyrate-producing anaerobe B. pullicaecorum showed good intrinsic acid and bile tolerance in terms of viability and metabolic activity, colonization efficiency and butyrate production under colon conditions is needed to further evaluate its probiotic potential.

Introduction

Butyrate has several beneficial properties that help to maintain gastrointestinal health: it is the main energy source of the colonocytes, inhibits pro-inflammatory pathways, reduces the oxidative stress in the colon and maintains gut barrier function [1], [2], [3]. Several strategies are proposed to increase the concentration of butyrate in the gut. A direct strategy is the administration of pure butyrate by means of butyrate containing tablets or rectal enemas [3]. An elegant alternative could be the consumption of butyrate-producing bacteria as an indirect strategy to increase the in situ butyrate production in the gut. Therefore, butyrate-producing bacteria are seen as the next generation probiotics to improve treatments of intestinal disorders like Inflammatory Bowel Disease (IBD) [4], [5], [6]. Butyrate-producing bacteria present in the human gut are obligate anaerobic Gram-positive bacteria, originating from two important groups: the Clostridium leptum (or clostridial cluster IV) cluster and the Clostridium coccoides (or clostridial cluster XIVa) cluster [7].

A good probiotic candidate could be derived from the genus Butyricicoccus (clostridial cluster IV) [8], as fecal samples of IBD patients display lower numbers of Butyricicoccus bacteria [9]. Recently, a novel butyrate-producing species, Butyricicoccus pullicaecorum, was isolated from caecal content of broiler chickens and showed high in vitro butyrate production (18 mM overnight in its growth medium) [10]. In addition, this isolate showed promising probiotic properties in a study with rats and Caco-2 epithelial cells [9]. In the rat study a less severe colitis developed after chemically induced inflammation in rats treated with B. pullicaecorum through intra gastric gavage compared to the control group [9].

To exert its beneficial properties, it is crucial that B. pullicaecorum arrives in the ileum and colon in a viable and metabolically active state. Therefore it needs to survive the harsh conditions of the upper gastrointestinal tract (UGIT): low pH in the stomach and presence of bile salts and pancreatic enzymes in the small intestine.

There is a lack of information on the intrinsic gastric and bile salt tolerance of butyrate-producing bacteria. If these are not able to withstand the harsh conditions of the UGIT, a suitable protection strategy (e.g. encapsulation with enteric coating) will be necessary.

During this study the intrinsic tolerance of B. pullicaecorum to the conditions of stomach and small intestine was evaluated using in vitro batch experiments. The cultivability and viability during exposure to stomach conditions at different pH levels and subsequent small intestinal conditions were monitored. We also evaluated whether the tolerance was influenced by the suspension medium of B. pullicaecorum and whether milk had a protective effect. To assess the metabolic potency after UGIT conditions, B. pullicaecorum that had been subjected to gastrointestinal conditions was further incubated and short chain fatty acid production was monitored.

Section snippets

Bacterial strain and growth conditions

B. pullicaecorum (LMG 24109T) was grown in anaerobic M2GSC medium at pH 6 prepared as described by Miyazaki et al. [11] using 15% (v/v) of clarified rumen fluid instead of 30% (v/v). M2GSC (1.5% w/v) agar plates were incubated at 37 °C in an anaerobic (8% CO2, 8% H2 and 84% N2) workstation (Ruskinn Technology, Bridgend, UK) for 20 h. Before use in each experiment, a B. pullicaecorum colony was transferred into 10 mL of anaerobic M2GSC broth and incubated overnight at 37 °C. Subsequently, the

Growth rate of B. pullicaecorum in M2GSC medium

A continuous anaerobic growth curve of B. pullicaecorum in M2GSC medium was recorded to assess the growth rate and characteristics under standard cultivation conditions. A triplicate OD measurement of two biological replicates showed an average growth rate of 0.19 ± 0.01 h−1, equivalent to a generation time of 3.6 ± 0.2 h or 216 ± 14 min (Fig. 1).

Cultivability of B. pullicaecorum depends on pH during stomach conditions

The ability of a stationary culture of B. pullicaecorum (7.7 ± 0.2 log CFU/mL) to survive or grow under gastrointestinal circumstances was assessed

Discussion

In this study we assessed the intrinsic tolerance of the obligate anaerobic butyrate-producing bacterium B. pullicaecorum to the harsh conditions of the stomach and small intestine.

Our results demonstrate that during acidic conditions (pH 2 and pH 3) B. pullicaecorum lost cultivability, but still remained viable and active. These findings indicate that Butyricicoccus enters a viable but noncultivable (VBNC) state in response to acid stress. Many other bacteria are known to enter a VBNC state

Acknowledgments

We thank Jana De Bodt for technical assistance and Jessica Benner, Massimo Marzorati and Eva Van Meervenne for critical revision of this manuscript. This work was supported by a research grant of “Strategisch Basisonderzoek – SBO” of the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT-Vlaanderen, project nr. 100016) and of a GOA (BOF12/GOA/008) project from Ghent University and the Belgian Foundation for Crohn and Ulcerative Colitis Patients (CCV-vzw). JA

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