Murine colonization model by Campylobacter coli DSPV458.

AIMS: To generate a murine experimental model of colonization by Campylobacter coli DSPV458. METHODS AND RESULTS: Twelve adult Balb/cCmedc female mice were housed in a treated group (T-G) and a control group (C-G) for 4 weeks. Both experimental groups received antibiotics for 5 days during the first week. The T-G was administered with 6.68log10 CFU of C. coli DSPV458 by oesophageal gavage. Necropsies were performed weekly to evaluate translocation and intestinal colonization in the spleen and liver and in the ileum and cecum respectively. Samples were cultured to quantify intestinal microbiota members. Faeces were cultured weekly for a C. coli DSPV458 count. Campylobacter coli DSPV458 was isolated from all the inoculated mice. The recovered level of C. coli DSPV458 was, on average, 6.9 log10 CFUg-1 , 8.0 log10 CFUg-1 and 1.6 log10 CFUg-1 in faeces, cecum and ileum respectively. Colonization by C. coli DSPV458 does not alter the normal clinical and physiological status. CONCLUSIONS: Campylobacter coli DSPV458 does not have an invasive capacity, and the model is suitable for evaluating strategies to reduce intestinal loads. SIGNIFICANCE AND IMPACT OF STUDY: Farm animals have an important impact on thermotolerant Campylobacter transmission to humans. Extremely few colonization models by C. coli have been reported to date. In food-producing animals, infection is mild or absent and thermotolerant Campylobacter colonize the intestines of animals. Colonization models are specific models that do not cause infection as they do not generally result in diarrhoea or other signs of disease. Therefore, this model will allow to evaluate the evolution of colonization by thermotolerant Campylobacter and the alternative tools development to antibiotics that limit their colonization in food-producing animals.

Protective effect of Lactiplantibacillus plantarum LP5 in a murine model of colonisation by Campylobacter coli DSPV458.

Thermotolerant Campylobacter species are the leading cause of foodborne bacterial diarrheal disease worldwide. Campylobacter coli, abundant in pigs and pork products, have been identified as a source of human infection. In this study, we propose the use of Lactiplantibacillus plantarum LP5 as a probiotic to reduce colonisation of this intestinal pathogen in a murine colonisation model of C. coli DSPV458. Six-week-old adult female Balb/cCmedc mice were housed in groups: Control, Campy and Pro-Campy. Control and Pro-Campy groups received antibiotics for 5 days and the Campy group for 12 days. Pro-Campy group was inoculated for 7 days with 8.78 log10 cfu total of L. plantarum LP5 suspended in De Man, Rogosa and Sharpe broth. All groups were inoculated with 6.72 log10 cfu of C. coli DSPV458 suspended in brain heart infusion broth. L. plantarum LP5 was recovered only in the Pro- Campy group. C. coli DSPV458 was recovered at higher levels in the Control and Campy groups. The differences with the Pro-Campy group were significant. As regards faeces, Control and Campy groups reached 7.41 and 7.84 log10 cfu/g, respectively, and the Pro-Campy group only 4.62 log10 cfu/g. In the caecum, Control and Campy groups reached 8.01 and 9.26 log10cfu/g, respectively, and the Pro-Campy group only 4.51 log10 cfu/g. In the ileum, Control and Campy groups reached 3.43 and 3.26 log10 cfu/g, respectively, and the Pro-Campy group did not show detectable levels. The reduction of C. coli DSPV458 in the Pro-Campy group compared to the Control group in faeces, caecum and ileum was 99.55, 99.98 and 100%, respectively. Animals were maintained under normal health conditions, and haematological parameters were within the standard values for Balb/cCmedc. The incorporation of a probiotic generated a protective effect in the mice colonisation model. The protective effect would also apply to intestinal colonisation by indigenous enterobacteria. Therefore, the strategy used in this study is of great importance to understand the protection mechanisms in a murine model, as well as its application in food-producing animals.

In vitro screening and in vivo colonization pilot model of Lactobacillus plantarum LP5 and Campylobacter coli DSPV 458 in mice.

The objective of this work was to determine the antibacterial effect of Lactobacillus plantarum strains of pork origin against Campylobacter coli strains, and to conduct experimental colonization pilot models in mice for both microorganisms. Inhibition assays allowed evaluation and selection of L. plantarum LP5 as the strain with the highest antagonistic activity against C. coli and with the best potential to be used in in vivo study. Adult 6-week-old female Balb/cCmedc mice were lodged in two groups. The treated group was administered with 9.4 log10CFU/2 times/wk of L. plantarum LP5. L. plantarum LP5 was recovered from the feces and cecum of the inoculated mice. However, when bacteria stopped being administered, probiotic counts decreased. Experimental colonization with C. coli was carried out in five groups of mice. All animals were treated with antibiotics in their drinking water to weaken the indigenous microbiota and to allow colonization of C. coli. Four groups were administered once with different C. coli strains (DSPV458: 8.49 log10CFU; DSPV567: 8.09 log10CFU; DSPV570: 8.46 log10CFU; DSPV541: 8.86 log10CFU, respectively). After 8 h, mice inoculated with different C. coli strains were colonized because the pathogen was detected in their feces. L. plantarum LP5 tolerated the gastrointestinal conditions of murine model without generating adverse effects on the animals. C. coli DSPV458 colonized the mice without causing infection by lodging in their digestive tract, thus generating a reproducible colonization model. Both models combined could be used as protection murine models against pathogens to test alternative control tools to antibiotics.

Quantification of FITC-labelled probiotic Lactobacillus salivarius DSPV 001P during gastrointestinal transit in broilers.

The knowledge related to the fate of probiotics in the complex environment of the intestinal microbiota in broilers is just beginning to be elucidated; however, it is not yet well understood. A good method to investigate the mechanisms by which probiotics mediate their effects is to mark probiotic bacteria and trace them. The aim of this research was to develop a new method to estimate in vivo fluorescein isothiocyanate (FITC)-labelled Lactobacillus salivarius DSPV 001P counts during passage through the gastrointestinal tract (GIT) of broilers. Forty-five, 1 d old Cobb broilers were used in this trial. Programmed necropsies were performed 30 min, 6 h, and 12 h after the administration of the probiotic bacterium, and samples of liver, crop, duodenum, caecum, and bursa of fabricius were collected. To determine the spatial and temporal transit of L. salivarius DSPV 001P in broilers, the number of bacteria as well as its respective fluorescent signal produced by FITC were measured. In order to observe the relationship between the variables, a logistic regression analysis was applied. The amount of fluorescence could be used as an indicator of fluorescent probiotic bacteria in the crop and duodenum 30 min after probiotic bacterium supplementation. In addition, the fluorescent signal could be used to estimate bacterial counts in caecum 6 and 12 h after L. salivarius DSPV 001P administration. To the best of our knowledge, this research is the first in vivo trial to employ the bacterial FITC-labelling technique in order to enumerate probiotic bacteria during gastrointestinal transit in broilers.