Estado viável não cultivável em Salmonella enterica: indução, perfil de proteínas intracelulares e detecção de mRNA

• Autor(a) principal: Mendes, Renata Aparecida
• Data de Publicação: 2009
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1542
• topic: Salmonella enterica; Estado viável; Não cultivável; Proteínas; Salmonella enterica; Viable state; Nonculturable; Proteins; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA

The foodborne pathogen Salmonella spp. enters into a viable but nonculturable state (VBNC) in response to adverse environmental conditions. The presence of VBNC cells is a public health concern since it may be overlooked under standard routine laboratory procedure, resulting in improper assessment of contaminating pathogens in food. This work was done to investigate the influence of strain, cell suspension media, saline concentration, and initial population density on the induction of VBNC state in Salmonella enterica and to evaluate the intracellular protein profile and the presence of invA, rpf, katF, and 16S rDNA gene transcripts in VBNC Salmonella enterica serovar Enteritidis PT4 963. The entry into VBNC state in S. enterica varied significantly between strains and was also influenced by the saline concentration, suspension media, and initial population density. The culturability loss was lower (p<0.001) in cells maintained at 4 °C in Brain and Hearth Infusion broth (BHI) than in cells in Butterfield's Phosphate Solution (BPS). The cells maintained at 4 °C in BHI added with NaCl showed lower (p<0.05) viablility when culturability could no longer be detected. The highest rate of culturability loss was obtained in media x with 1.2 mol/L NaCl (p<0.001). The strains S. Enteritidis PT4 963 and S. Typhimurium ATCC 14028 maintained at 4 °C in BPS added with 1.2 mol/L NaCl showed the largest (p<0.001) entry index into VBNC state. The smallest culturability loss rates and also the largest reductions in viable counts were observed for an initial population density of 107 CFU/mL (p<0.05) compared to 105 and 103 CFU/mL. The intracellular protein profile of VBNC S. Enteritidis PT4 963 differed from that observed in the exponential and stationary growth phases, and under osmotic, low temperature, and starvation stresses. The absence of proteins with molecular mass superior to 63 kDa and the presence of proteins with smaller molecular masses in the VBNC state suggest the occurrence of proteolysis, probably to guarantee the maintenance of the nutrient supply and viability. Our results indicate that the VBNC state constitutes a physiologically distinct state within the life cycle of Salmonella enterica serovar Enteritidis PT4. However we could not detect any protein specific of the VBNC cells of Salmonella. The amount of total RNA decreased approximately 600-fold in VBNC cells. No katF, invA, and rpf mRNA was detected in the VBNC populations under the experimental conditions studied. However the 16S rRNA expression was detected in VBNC cells. These results suggest that, considering the 16S rRNA production, VBNC S. Enteritidis PT4 cells are metabolically active and that this housekeeping gene is a good viability marker for Salmonella enterica serovar Enteritidis PT4 in the VBNC state.