sybr-green-i and Salmonella-Infections--Animal

Intestinal microbiota comprises microbial communities that reside in the gastrointestinal tract and are critical to normal host physiology. Understanding the microbiota's role in host response to invading pathogens will further advance our knowledge of host-microbe interactions. Salmonella enterica serovar Typhimurium was used as a model enteric pathogen to investigate the effect of intestinal microbiota perturbation on host susceptibility to infection. Antibiotics were used to perturb the intestinal microbiota. C57BL/6 mice were treated with clinically relevant doses of streptomycin and vancomycin in drinking water for 2 days, followed by oral infection with Salmonella enterica serovar Typhimurium. Alterations in microbiota composition and numbers were evaluated by fluorescent in situ hybridization, differential plating, and Sybr green staining. Antibiotics had a dose-dependent effect on intestinal microbiota composition. The chosen antibiotic regimen did not significantly alter the total numbers of intestinal bacteria but altered the microbiota composition. Greater preinfection perturbations in the microbiota resulted in increased mouse susceptibility to Salmonella serovar Typhimurium intestinal colonization, greater postinfection alterations in the microbiota, and more severe intestinal pathology. These results suggest that antibiotic treatment alters the balance of the microbial community, which predisposes the host to Salmonella serovar Typhimurium infection, demonstrating the importance of a healthy microbiota in host response to enteric pathogens.

Topics: Administration, Oral; Animals; Anti-Bacterial Agents; Bacteria; Benzothiazoles; Biodiversity; Colony Count, Microbial; Diamines; Disease Susceptibility; Female; In Situ Hybridization, Fluorescence; Intestines; Mice; Mice, Inbred C57BL; Organic Chemicals; Quinolines; Salmonella Infections, Animal; Streptomycin; Vancomycin

The objective of this study was to develop a rapid, reproducible, and robust method for detecting Salmonella enterica serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared. The most effective method was then combined with a real-time PCR method based on the double-stranded DNA binding dye SYBR Green I used with the ABI Prism 7700 system. The specificity of the reaction was determined by the melting temperature (T(m)) of the amplicon obtained. The experiments were conducted both on samples of chicken experimentally contaminated with serotype Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001). The results of comparisons among the four DNA extraction methods showed significant differences except for the results from the boiling and Nucleospin methods (the two methods that produced the lowest threshold cycles). Boiling was selected as the preferred extraction method because it is the simplest and most rapid. This method was then combined with SYBR Green I real-time PCR, using primers SEFA-1 and SEFA-2. The specificity of the reaction was confirmed by the T(m), which was consistently specific for the amplicon obtained; the mean peak T(m) obtained with curves specific for serotype Enteritidis was 82.56 +/- 0.22 degrees C. The standard curve constructed using the mean threshold cycle and various concentrations of serotype Enteritidis (ranging from 10(3) to 10(8) CFU/ml) showed good linearity (R(2) = 0.9767) and a sensitivity limit of less than 10(3) CFU/ml. The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting serotype Enteritidis in poultry samples.

Topics: Animals; Benzothiazoles; Chickens; Culture Media; Diamines; DNA, Bacterial; Fluorescent Dyes; Organic Chemicals; Polymerase Chain Reaction; Poultry Diseases; Quinolines; Reproducibility of Results; Salmonella enteritidis; Salmonella Infections, Animal; Serotyping; Turkeys