CERELA   05438
CENTRO DE REFERENCIA PARA LACTOBACILOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Optimization of Fluorescence in situ hybridization protocol (FISH) for detection and quick identification of bifidobacteria from intestinal content samples of poultry
Autor/es:
GRANDE, SONIA MARÍA MERCEDES; PEREZ CHAIA, ADRIANA; BERTANI, MILENA SABRINA; ARGAÑARAZ MARTINEZ, ELOY
Lugar:
San Miguel de Tucumán
Reunión:
Simposio; V Simposio Internacional de Bacterias Lácticas; 2016
Institución organizadora:
CERELA CONICET
Resumen:
The fluorescence in situ hybridization (FISH) is a tool for detecting microbial populations through hybridization of 16S rRNA with fluorescent probes. It does not require prior isolation and displays viable organisms in their natural niches. FISH is useful to detect bifidobacteria, anaerobic microorganism with recognized beneficial features of interest in human and animal health, which is difficult to identify. Detection by this technique depends on the physiological state of the microorganism, fixation method, specificity of the probes, permeabilization method and conditions of temperature and time of hybridization. The probe commonly used to detect the genus Bifidobacterium is known as B164 probe, and for this there is not well standardized protocol hybridization. In this work, we proposed optimize variables of FISH to B164 probe with three reference strains of the genus Bifidobacterium, for subsequent detection and selective isolation in cecal homogenates from poultry. Strains B. animalis ssp. lactis BB12 (Chr Hansen) of milk origin; B. animalis ssp. lactis BLC 1 (Sacco), of dairy origin and B. infantis CRL 1395 (CERELA), of human origin were grown in MRS with cysteine to late exponential phase at 37 ° C in strict anaerobiosis. From these culture, cells were prepared by two methods of fixing. One included incubation for 6 and 16 h in a solution of p-formaldehyde; on the other, the cells were suspended directly a mixture 1:1 of ethanol 96°- PBS. Then the samples were washed, dried, permeabilized and fixed to slide properly prepared. Hybridization was carried out in a humid chamber with Eub338 probe for Bacteria (control +), Non338 (control -, non-specific hybridization), L158 probe for lactobacilli and enterococci (control -) and B164 for Bifidobacterium. The temperature was set at 45, 47 and 50 °C for 6 and 16h, and finally samples were observed in a fluorescence microscope. Fixed cells directly into ethanol: PBS visibly emit less fluorescence than the fixed in p-formaldehyde. With respect to the temperature-time relation, strain CRL 1395 was the only hybridized with B164 probe in 6 h at 45, 47 and 50 °C; BLC1 hybridized only to the lower temperature and BB12 not hybridized. When time rose to 16 h, all strains showed fluorescence between 45 °C and 47 °C with Eub338 and B164 probes. Subsequently to detect bifidobacteria in cecal contents of poultry, samples were fixed by 16h in p-formaldehyde and hybridized for 16 h at 47 ° C with Eub338, Non338 and B164 probes. Samples of cecal content where bifidobacteria were detected by FISH, were subjected to isolations in media containing selective agents. Two of the 59 colonies evaluated were positively identified with B164 probe. Isolation from fecal samples is very laborious because the bifidobacteria have several cultural restrictions, and when develop in artificial media, colonies have similar morphology to other genera. The protocol used in this work allowed detection of bifidobacteria from cecal homogenates of bird and quick identification of isolates. Therefore, represents a significant contribution to the study and selection of potential probiotic strains.