Protective effect of probiotics on Salmonella infectivity assessed with combined in vitro gut fermentation-cellular models

<p>Abstract</p> <p>Background</p> <p>Accurate assessment of probiotics with targeted anti-<it>Salmonella </it>activity requires suitable models accounting for both, microbe-microbe and host-microbe interactions in gut environments. Here we report the combination of two original <it>in vitro </it>intestinal models closely mimicking the complex <it>in vivo </it>conditions of the large intestine. Effluents from continuous <it>in vitro </it>three-stage fermentation colonic models of <it>Salmonella </it>Typhimurium infection inoculated with immobilized child microbiota and <it>Salmonella </it>were directly applied to confluent mucus-secreting HT29-MTX cell layers. The effects of <it>Salmonella</it>, addition of two bacteriocinogenic strains, <it>Bifidobacterium thermophilum </it>RBL67 (thermophilicin B67) and <it>Escherichia coli </it>L1000 (microcin B17), and inulin were tested on <it>Salmonella </it>growth and interactions with epithelial cell layers. <it>Salmonella </it>adhesion and invasion were investigated and epithelial integrity assessed by transepithelial electrical resistance (TER) measurements and confocal microscopy observation. Data from complex effluents were compared with pure <it>Salmonella </it>cultures.</p> <p>Results</p> <p><it>Salmonella </it>in effluents of all reactors of the colonic fermentation model stabilized at mean values of 5.3 ± 0.8 log₁₀cfu/ml effluent. Invasion of cell-associated <it>Salmonella </it>was up to 50-fold lower in complex reactor samples compared to pure <it>Salmonella </it>cultures. It further depended on environmental factors, with 0.2 ± 0.1% being measured with proximal, 0.6 ± 0.2% with transverse and 1.3 ± 0.7% with distal reactor effluents, accompanied by a similar high decrease of TER across cell monolayers (minus 45%) and disruption of tight junctions. Subsequent addition of <it>E. coli </it>L1000 stimulated <it>Salmonella </it>growth (6.4 ± 0.6 log₁₀cfu/ml effluent of all 3 reactors) and further decreased TER, but led to 10-fold decreased invasion efficiency when tested with distal reactor samples. In contrast, presence of <it>B. thermophilum </it>RBL67 revealed a protective effect on epithelial integrity compared to previous <it>E. coli </it>L1000 periods, as reflected by a significant mean increase of TER by 58% in all reactors. Inulin addition enhanced <it>Salmonella </it>growth and invasion when tested with distal and proximal reactor samples, respectively, but induced a limited decrease of TER (minus 18%) in all reactors.</p> <p>Conclusions</p> <p>Our results highlight the benefits of combining suitable cellular and colonic fermentation models to assess strain-specific first-level host protection properties of probiotics during <it>Salmonella </it>infection, providing an efficient system biology tool for preclinical development of new antimicrobials.</p

New in vitro colonic fermentation model for Salmonella infection in the child gut

In this study, a new in vitro continuous colonic fermentation model of Salmonella infection with immobilized child fecal microbiota and Salmonella serovar Typhimurium was developed for the proximal colon. This model was then used to test the effects of two amoxicillin concentrations (90 and 180 mg day−1) on the microbial composition and metabolism of the gut microbiota and on Salmonella serovar Typhimurium during a 43-day fermentation. Addition of gel beads (2%, v/v) colonized with Salmonella serovar Typhimurium in the reactor resulted in a high and stable Salmonella concentration (log 7.5 cell number mL−1) in effluent samples, and a concomitant increase of Enterobacteriaeceae, Clostridium coccoides-Eubacterium rectale and Atopobium populations and a decrease of bifidobacteria. During amoxicillin treatments, Salmonella concentrations decreased while microbial balance and activity were modified in agreement with in vivo data, with a marked decrease in C. coccoides-E. rectale and an increase in Enterobacteriaceae. After interruption of antibiotic addition, Salmonella concentration again increased to reach values comparable to that measured before antibiotic treatments, showing that our model can be used to simulate Salmonella shedding in children as observed in vivo. This in vitro model could be a useful tool for developing and testing new antimicrobials against enteropathogen

New in vitro colonic fermentation model for Salmonella infection in the child gut

The definitive version is available at ww3.interscience.wiley.comInternational audienceIn this study, a new in vitro continuous colonic fermentation model of Salmonella infection with immobilized child fecal microbiota and Salmonella serovar Typhimurium was developed for the proximal colon. This model was then used to test the effects of two amoxicillin concentrations (90 and 180 mg day(-1)) on the microbial composition and metabolism of the gut microbiota and on Salmonella serovar Typhimurium during a 43-day fermentation. Addition of gel beads (2%, v/v) colonized with Salmonella serovar Typhimurium in the reactor resulted in a high and stable Salmonella concentration (log 7.5 cell number mL(-1)) in effluent samples, and a concomitant increase of Enterobacteriaeceae, Clostridium coccoides-Eubacterium rectale and Atopobium populations and a decrease of bifidobacteria. During amoxicillin treatments, Salmonella concentrations decreased while microbial balance and activity were modified in agreement with in vivo data, with a marked decrease in C. coccoides-E. rectale and an increase in Enterobacteriaceae. After interruption of antibiotic addition, Salmonella concentration again increased to reach values comparable to that measured before antibiotic treatments, showing that our model can be used to simulate Salmonella shedding in children as observed in vivo. This in vitro model could be a useful tool for developing and testing new antimicrobials against enteropathogens

Ongoing toxin-positive diphtheria outbreaks in a federal asylum centre in Switzerland, analysis July to September 2022.

Two diphtheria outbreaks occurred in a Swiss asylum center from July to October 2022, one is still ongoing. Outbreaks mainly involved minors and included six symptomatic respiratory diphtheria cases requiring antitoxin. Phylogenomic analyses showed evidence of imported and local transmissions of toxigenic strains in respiratory and skin lesion samples. Given the number of cases (n = 20) and the large genetic diversity accumulating in one centre, increased awareness and changes in public health measures are required to prevent and control diphtheria outbreaks

Novel Polyfermentor Intestinal Model (PolyFermS) for Controlled Ecological Studies: Validation and Effect of pH

Peer reviewe

Novel Polyfermentor Intestinal Model (PolyFermS) for Controlled Ecological Studies: Validation and Effect of pH

Peer reviewe

Extended-spectrum-β-lactamase-producing Enterobacteriaceae isolated from vegetables imported from the Dominican Republic, India, Thailand, and Vietnam

To examine to what extent fresh vegetables imported into Switzerland represent carriers of extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae, 169 samples of different types of fresh vegetables imported into Switzerland from the Dominican Republic, India, Thailand, and Vietnam were analyzed. Overall, 25.4% of the vegetable samples yielded one or more ESBL-producing Enterobacteriaceae, 78.3% of which were multidrug resistant. Sixty isolates were obtained: Escherichia coli, 26; Klebsiella pneumoniae, 26; Enterobacter cloacae, 6; Enterobacter aerogenes, 1; and Cronobacter sakazakii, 1. We found 29 isolates producing CTX-M-15, 8 producing CTX-M-14, 7 producing CTX-M-55, 3 producing CTX-M-65, 1 each producing CTX-M-1, CTX-M-3, CTX-M-27, and CTX-M-63, 5 producing SHV-2, 3 producing SHV-12, and 1 producing SHV-2a. Four of the E. coli isolates belonged to epidemiologically important clones: CTX-M-15-producing B2:ST131 (1 isolate), D:ST405 (1 isolate), and D:ST38 (2 isolates). One of the D:ST38 isolates belonged to the extraintestinal enteroaggregative E. coli (EAEC) D:ST38 lineage. Two of the K. pneumoniae isolates belonged to the epidemic clones sequence type 15 (ST15) and ST147. The occurrence of antibiotic-resistant pathogenic and commensal Enterobacteriaceae in imported agricultural foodstuffs constitutes a source of ESBL genes and a concern for food safety

Synergistic effects of Bifidobacterium thermophilum RBL67 and selected prebiotics on inhibition of Salmonella colonization in the swine proximal colon PolyFermS model

Background Probiotics and prebiotics are promising strategies to counteract Salmonella prevalence in swine. In the present study, we investigated the effects of prebiotics (fructo- (FOS), galacto- (GOS) and mannan- (MOS) oligosaccharides) and the bacteriocinogenic Bifidobacterium thermophilum RBL67 (RBL67) on Salmonella enterica subsp. enterica serovar Typhimurium N-15 (N-15) colonization using the PolyFermS in vitro continuous fermentation model simulating the swine proximal colon. Material and methods The PolyFermS model was designed with a first-stage reactor containing immobilized fecal pig microbiota. This reactor continuously inoculated five parallel second-stage reactors, a control and four treatment reactors, all operated with proximal colon conditions. FOS and GOS (5.2g/day), and MOS (half dosage) and RBL67 (108 copy numbers/mL applied daily) were tested on the ability of N-15 to colonize reactors, inoculated with the same microbiota. Reactor effluents were collected daily and analyzed for microbial composition (quantitative PCR and 454 pyrosequencing of 16S rRNA gene pool) and main metabolites (HPLC). Results RBL67 and N-15 were shown to stably colonize the system. Colonization of N-15 was strongly inhibited by FOS and GOS, whereas addition of RBL67 alone or combined with MOS showed intermediate results. However, the effect of FOS and GOS was enhanced when prebiotics were combined with a daily addition of RBL67. FOS and GOS increased the total short chain fatty acid production, especially acetate and propionate. RBL67 combined with FOS additionally stimulated butyrate production. Conclusions Our study demonstrates the suitability of the porcine PolyFermS in vitro model to study nutritional effects of pro- and prebiotics on gut microbiota composition and activity. It can further be used to monitor Salmonella colonization. The inhibition effects of FOS and GOS on N-15 colonization are partly due to an increased acetate production, while further antimicrobial mechanisms may contribute to an enhanced inhibition with prebiotic-RBL67 combinations. A future direction of this work could be to understand the anti-Salmonella effects of Bifidobacterium thermophilum RBL67 in the presence of prebiotics to unravel the mechanism of this probiotic:pathogen interaction.ISSN:1757-474

Effect of Bifidobacterium thermophilum RBL67 and fructo-oligosaccharides on the gut microbiota in Göttingen minipigs

International audienceModulating the gut microbiota via dietary interventions is a common strategy to enhance the natural defence mechanisms of the host. Several in vitro studies have highlighted the probiotic potential of Bifidobacterium thermophilum RBL67 (RBL67) selected for its anti-Salmonella effects. The present study aimed to investigate the impact of RBL67 alone and combined with fructo-oligosaccharides (FOS) on the gut microbiota of Gottingen minipigs. Minipigs were fed a basal diet supplemented with 8 g/d probiotic powder (1x10(9) CFU/g in skim milk matrix) (probiotic diet (PRO)), 8 g/d probiotic powder plus 8 g/d FOS (synbiotic diet (SYN)) or 8 g/d skim milk powder (control), following a cross-sectional study design. Faecal and caecal microbiota compositions were analysed with pyrosequencing of 16S rRNA genes and quantitative PCR. Metabolic activity in the caecum and colon was measured by HPLC. 16S rRNA gene amplicon sequencing revealed that minipig faeces show close similarity to pig microbiota. During the treatments and at the time of killing of animals, RBL67 was consistently detected in faeces, caecum and colon at numbers of 10(5)-10(6) 16S rRNA copies/g content after feeding PRO and SYN diets. At the time of killing of animals, significantly higher Bifidobacterium numbers in the caecum and colon of SYN-fed minipigs were measured compared with PRO. Our data indicate that the Gottingen minipig may be a suitable model for gut microbiota research in pigs. Data from this first in vivo study of RBL67 colonisation suggest that the combination with FOS may represent a valuable symbiotic strategy to increase probiotic bacteria levels and survival in gastrointestinal tracts for feed and food application