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 when harsh environmental conditions are introduced [21]. The VBNC state is seen as a survival strategy and once the stress factor disappears, the bacteria resuscitate and become cultivable again [21]. This was also the case for B. pullicaecorum as the cultivability was restored during small intestinal simulation when the pH conditions were more optimal. The concentration of cultivable B. pullicaecorum at low gastric pH (2 and 3) was below the detection limit and subse- quently increased in 2 h of small intestinal conditions to 3.1 log CFU/mL (gastric pH 2) and 3.8 log CFU/mL (gastric pH 3). If this increase was due to regrowing of a few cultivable cells under the detection limit, this would indicate a generation time of ca. 34 min in case of gastric pH 2 and ca. 20 min in case of gastric pH 3. As the generation time of B. pullicaecorum is typically 216 ± 14 min (Fig. 1), we conclude that there was a resuscitation of VBNC cells during small intestine incubation.
Bile salts and pancreatic enzymes are factors to which B. pullicaecorum is subjected during small intestinal conditions. This environment had no inhibitory effect on B. pullicaecorum because the concentration of cells did not decrease during small intestinal simulation. The final concentration of bile extract used in this study was 0.3% (w/v) oxgall and corresponds with a concen- tration of 3.7 mM bile salts which is at the lower limit of the in vivo concentrations found in the small intestine (varies from 2 to 15 mM) [22,23]. However this concentration is used in several other tolerance studies with probiotics [14,24,25] as it is considered a critical concentration to screen for bile resistant strains [26]. Bac- terial bile acid tolerance is strain-specific and may not be gener- alized between species [27]. Butyrate-producing bacteria also showed a difference in bile tolerance: isolates of Faecalibacterium prausnitzii showed an average growth inhibition of 76% and 95% to bile salt concentrations of 0.1% respectively 0.25% (w/v) and an Enterococcus durans isolate was only able to grow in 0.05% (w/v) bile salts [28,29]. From the presented results it can be concluded that B. pullicaecorum is able to withstand conditions that prevail in the small intestine.
B. pullicaecorum was still metabolically active upon upper gastrointestinal conditions but there was a lower butyrate pro- duction and a shift to acetate production. This indicates that certain stress factors, affecting bacterial metabolism and SCFA production,
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 when harsh environmental conditions are introduced [21]. The VBNC state is seen as a survival strategy and once the stress factor disappears, the bacteria resuscitate and become cultivable again [21]. This was also the case for B. pullicaecorum as the cultivability was restored during small intestinal simulation when the pH conditions were more optimal. The concentration of cultivable B. pullicaecorum at low gastric pH (2 and 3) was below the detection limit and subse- quently increased in 2 h of small intestinal conditions to 3.1 log CFU/mL (gastric pH 2) and 3.8 log CFU/mL (gastric pH 3). If this increase was due to regrowing of a few cultivable cells under the detection limit, this would indicate a generation time of ca. 34 min in case of gastric pH 2 and ca. 20 min in case of gastric pH 3. As the generation time of B. pullicaecorum is typically 216 ± 14 min (Fig. 1), we conclude that there was a resuscitation of VBNC cells during small intestine incubation.Bile salts and pancreatic enzymes are factors to which B. pullicaecorum is subjected during small intestinal conditions. This environment had no inhibitory effect on B. pullicaecorum because the concentration of cells did not decrease during small intestinal simulation. The final concentration of bile extract used in this study was 0.3% (w/v) oxgall and corresponds with a concen- tration of 3.7 mM bile salts which is at the lower limit of the in vivo concentrations found in the small intestine (varies from 2 to 15 mM) [22,23]. However this concentration is used in several other tolerance studies with probiotics [14,24,25] as it is considered a critical concentration to screen for bile resistant strains [26]. Bac- terial bile acid tolerance is strain-specific and may not be gener- alized between species [27]. Butyrate-producing bacteria also showed a difference in bile tolerance: isolates of Faecalibacterium prausnitzii showed an average growth inhibition of 76% and 95% to bile salt concentrations of 0.1% respectively 0.25% (w/v) and an Enterococcus durans isolate was only able to grow in 0.05% (w/v) bile salts [28,29]. From the presented results it can be concluded that B. pullicaecorum is able to withstand conditions that prevail in the small intestine.B. pullicaecorum was still metabolically active upon upper gastrointestinal conditions but there was a lower butyrate pro- duction and a shift to acetate production. This indicates that certain stress factors, affecting bacterial metabolism and SCFA production,
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