Annina Zihler

In vitro inhibition activity of nisin A, nisin Z, pediocin PA-1 and antibiotics against common intestinal bacteria

Aims:  To evaluate the sensitivity of 21 common intestinal bacteria to six antibiotics and three broad‐spectrum bacteriocins (nisins Z and A and pediocin PA‐1).

In vivo study on the effectiveness of pediocin PA-1 and Pediococcus acidilactici UL5 at inhibiting Listeria monocytogenes

The anti-listerial effect of pediocin PA-1 and its producing strain, Pediococcus acidilactici UL5, was investigated in vivo using an ICR mouse model. The effect of intra-gastric administration of a single dose of P. acidilactici UL5 (4 x 10(10) CFU/animal) on the propagation of Listeria monocytogenes LSD348 in intestine, liver and spleen was negligible. P. acidilactici UL5 did not appear competitive with the mouse intestinal flora and was not detectable in fecal samples collected two days after administration. However, double-agar-layer activity assay showed the ability of P. acidilactici UL5 colonies recovered from fecal samples one day after administration to produce pediocin PA-1 and inhibit L. monocytogenes. Moreover, repeated doses (250 microg/day for three consecutive days) of purified pediocin PA-1 provided up to 2-log reductions in fecal listerial counts compared to the infected control group and slowed pathogen translocation into the liver and spleen, leading to the disappearance of L. monocytogenes infection in these two organs within six days. Neither P. acidilactici UL5 nor ingested purified pediocin PA-1 had any negative effect on feed intake or body weight development. Pediocin PA-1 did not affect the composition of the mouse intestinal flora, suggesting a potential advantage over other inhibitory agents as a prophylactic measure against L. monocytogenes.

Study of the physicochemical and biological stability of pediocin PA-1 in the upper gastrointestinal tract conditions using a dynamic in vitro model

Aims:  To evaluate the survival of Pediococcus acidilactici UL5 and its ability to produce pediocin PA‐1 during transit in an artificial gastrointestinal tract (GIT). To investigate the physicochemical and biological stability of purified pediocin PA‐1 under GIT conditions.

Unexpected consequences of administering bacteriocinogenic probiotic strains for Salmonella populations, revealed by an in vitro colonic model of the child gut.

New biological strategies for the treatment of Salmonella infection are needed in response to the increase in antibiotic-resistant strains. Escherichia coli L1000 and Bifidobacterium thermophilum RBL67 were previously shown to produce antimicrobial proteinaceous compounds (microcin B17 and thermophilicin B67, respectively) active in vitro against a panel of Salmonella strains recently isolated from clinical cases in Switzerland. In this study, two three-stage intestinal continuous fermentation models of Salmonella colonization inoculated with immobilized faeces of a two-year-old child were implemented to study the effects of the two bacteriocinogenic strains compared with a bacteriocin-negative mutant of strain L1000 on Salmonella growth, as well as gut microbiota composition and metabolic activity. Immobilized E. coli L1000 added to the proximal colon reactor showed a low colonization, and developed preferentially in the distal colon reactor independent of the presence of genetic determinants for microcin B17 production. Surprisingly, E. coli L1000 addition strongly stimulated Salmonella growth in all three reactors. In contrast, B. thermophilum RBL67 added in a second phase stabilized at high levels in all reactors, but could not inhibit Salmonella already present at a high level (>10(7) c.f.u. ml(-1)) when the probiotic was added. Inulin added at the end of fermentation induced a strong bifidogenic effect in all three colon reactors and a significant increase of Salmonella counts in the distal colon reactor. Our data show that under the simulated child colonic conditions, the microcin B17 production phenotype does not correlate with inhibition of Salmonella but leads to a better colonization of E. coli L1000 in the distal colon reactor. We conclude that in vitro models with complex and complete gut microbiota are required to accurately assess the potential and efficacy of probiotics with respect to Salmonella colonization in the gut.

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 enteropathogens.

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

BackgroundAccurate assessment of probiotics with targeted anti-Salmonella activity requires suitable models accounting for both, microbe-microbe and host-microbe interactions in gut environments. Here we report the combination of two original in vitro intestinal models closely mimicking the complex in vivo conditions of the large intestine. Effluents from continuous in vitro three-stage fermentation colonic models of Salmonella Typhimurium infection inoculated with immobilized child microbiota and Salmonella were directly applied to confluent mucus-secreting HT29-MTX cell layers. The effects of Salmonella, addition of two bacteriocinogenic strains, Bifidobacterium thermophilum RBL67 (thermophilicin B67) and Escherichia coli L1000 (microcin B17), and inulin were tested on Salmonella growth and interactions with epithelial cell layers. Salmonella 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 Salmonella cultures.ResultsSalmonella in effluents of all reactors of the colonic fermentation model stabilized at mean values of 5.3 ± 0.8 log10 cfu/ml effluent. Invasion of cell-associated Salmonella was up to 50-fold lower in complex reactor samples compared to pure Salmonella 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 E. coli L1000 stimulated Salmonella growth (6.4 ± 0.6 log10 cfu/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 B. thermophilum RBL67 revealed a protective effect on epithelial integrity compared to previous E. coli L1000 periods, as reflected by a significant mean increase of TER by 58% in all reactors. Inulin addition enhanced Salmonella growth and invasion when tested with distal and proximal reactor samples, respectively, but induced a limited decrease of TER (minus 18%) in all reactors.ConclusionsOur results highlight the benefits of combining suitable cellular and colonic fermentation models to assess strain-specific first-level host protection properties of probiotics during Salmonella infection, providing an efficient system biology tool for preclinical development of new antimicrobials.

In vitro inhibition activity of different bacteriocin-producing Escherichia coli against Salmonella strains isolated from clinical cases

Aims:  To compare in vitro the inhibitory activity of four bacteriocin‐producing Escherichia coli to a well‐characterized panel of Salmonella strains, recently isolated from clinical cases in Switzerland.

Ecology of Probiotics and Enteric Protection

Insight into the diversity and function of the human gut microbiota has been gained through recent studies with probiotic bacteria that exhibit specific functions positively affecting our health, such as immunomodulation. Initial efforts have been centered on establishing scientific support for the efficacy of some probiotic bacteria, mainly Lactobacillus and Bifidobacterium species, for prevention of enteric infection. Following these evidence-based functional approaches, considerable research is focused on elucidation of mechanisms of action of particular probiotic bacteria. Different probiotic strains vary in their ability to protect the host from enteric pathogen interactions and, the efficient dose, frequency and duration of the probiotic consumption required in different population groups remain to be determined. The taxonomy of probiotics, in relation to their functional physiology and their potential for enteric protection will be discussed in this chapter. A special focus is placed on recent in vitro and in vivo assessments of particular probiotics for inhibition of enteric pathogens using gut fermentation models, cellular tests, animal and human trials. Finally, mechanisms that contribute to the inhibition of pathogens in the gut environment will be addressed through intestinal epithelial cell models.

Bifidobacterium thermophilum RBL67 Inhibits Salmonella enterica Serovar Typhimurium in an In vitro Intestinal Fermentation Model

Infectious gastroenteritis caused by non-typhoidal serovars of Salmonella is usually self-limiting but life-threatening complications may require appropriate antibiotic therapies. Probiotics have evolved as potential alternatives to antibiotics in response to an increasing prevalence of resistant strains, but there are no studies comparing the effectiveness of both treatment strategies on the same intestinal microbiota. We used an in vitro intestinal fermentation system with immobilized fecal microbiota to model enteric Salmonella infection in children. The effects of antibiotics, amoxicillin/clavulanic acid at two dosages, and a probiotic candidate, Bifidobacterium thermophilum RBL67, on Salmonella counts, intestinal microbiota composition, and metabolic activity were compared. Antibiotic therapy, in agreement with current clinical reports, produced only a transient decrease of Salmonella concentrations but strongly modified bacterial population profiles and metabolic activity, confirming the suitability of the model. B. thermophilum RBL67 grew in the intestinal environment and exerted a strong inhibition on Salmonella when added before or after infection and was able to rebalance the metabolic activity of gut microbiota after antibiotic treatments. Our study revealed a high potential of B. thermophilum RBL67 for prevention and treatment of Salmonella infection. The exact mechanism has to be further elucidated.