RESUMEN
Bifidobacterium breve is a common habitant of the human gut and is used as probiotic in functional foods. B. breve has to cope with multiple stress conditions encountered during processing and passage through the human gut, including high temperature, low pH and exposure to oxygen. Additionally, during industrial processing and in the gut, B. breve could encounter nutrient limitation resulting in reduced growth rates that can trigger adaptive stress responses. For this reason, it is important to develop culture methods that elicit resistance to multiple stresses (robustness) encountered by the bacteria. To investigate the impact of caloric restriction on robustness of the probiotic B. breve NRBB57, this strain was grown in lactose-limited chemostat cultures and in retentostat for 21 days, at growth rates ranging from 0.4 h-1 to 0.00081 h-1. Proteomes of cells harvested at different growth rates were correlated to acid, hydrogen peroxide and heat stress survival capacity. Comparative proteome analysis showed that retentostat-grown cells had significantly increased abundance of a variety of stress proteins involved in protein quality maintenance and DNA repair (DnaJ, Hsp90, FtsH, ClpB, ClpP1, ClpC, GroES, RuvB, RecA), as well as proteins involved in oxidative stress defence (peroxiredoxin, ferredoxin, thioredoxin peroxidase, glutaredoxin and thioredoxin reductase). Exposure to three different stress conditions, 45 °C, pH 3, and 10 mM H2O2, showed highest stress resistance of retentostat cells sampled at week 2 and week 3 grown at 0.0018 and 0.00081 h-1. Our findings show that cultivation at near-zero growth rates induces higher abundance of stress defence proteins contributing to the robustness of B. breve NRBB57, thereby offering an approach that may support its production and functionality.
Asunto(s)
Bifidobacterium breve , Probióticos , Humanos , Peróxido de Hidrógeno/metabolismo , Proteínas de Choque Térmico/metabolismo , Lactosa/metabolismoRESUMEN
It is well-established that Extended-spectrum beta-lactamase-producing (ESBL-) Escherichia coli challenge reliable detection of campylobacters during enrichment in Bolton broth (BB) following ISO 10272-1:2017. The overgrowth of Campylobacter by ESBL-E. coli in the enrichment medium BB can lead to false-negative detection outcomes, but the cause for the growth suppression is yet unknown. A plausible reason could be the competition-induced lack of certain growth substrates. Therefore, this study aimed to investigate whether campylobacters and ESBL-E. coli compete for the same medium components and whether this is the cause for the observed growth repression. The availability of possible growth substrates in BB was determined and changes in their extracellular concentration were measured over time during mono-culture enrichment of C. jejuni, C. coli or ESBL-E. coli as well as in co-culture enrichments of campylobacters and ESBL-E. coli. Comparative analysis showed lactate and fumarate utilization by C. jejuni and C. coli exclusively, whereas ESBL-E. coli rapidly consumed asparagine, glutamine/arginine, lysine, threonine, tryptophan, pyruvate, glycerol, cellobiose, and glucose. Both campylobacters and ESBL-E. coli utilized aspartate, serine, formate, a-ketoglutarate and malate. Trends in compound utilization were similar for C. jejuni and C. coli and trends in compound utilization were rather comparable during enrichment of reference and freeze-stressed campylobacters. Since final cell densities of C. jejuni and C. coli in co-cultures were not enhanced by the addition of surplus l-serine and final cell densities were similar in fresh and spent medium, growth suppression seems not to be caused by a lack of substrates or production of inhibitory compounds. We hypothesized that oxygen availability was limiting growth in co-cultures. Higher oxygen availability increased the competitive fitness of C. jejuni 81-176 in co-culture with ESBL-E. coli in duplicate experiments, as cell concentrations in stationary phase were similar to those without competition. This could indicate the critical role of oxygen availability during the growth of Campylobacter and offers potential for further improvement of Campylobacter spp. enrichment efficacy.