RESUMO
A gene encoding the resuscitation-promoting factor (Rpf) from Salmonella Typhimurium LT2 was cloned and characterized. The amino acid sequence encoded by S. Typhimurium LT2 rpf gene shares 24.2% homology with Micrococcus luteus Rpf, which is secreted by growing cells, and required to resuscitate from viable but non-culturable (VNC) state. The S. Typhimurium LT2 rpf gene is 696 bp long, and shared a conserved segment with Salmonella enterica serovar Oranienburg (99.4%). Recombinant Rpf (rRpf) proteins of S. Typhimurium LT2 after expression in E. coli BL21 harboring the pET15-b plasmid was approximately 25 kDa. Since S. Oranienburg cells are relatively quick to enter the VNC state just after incubating in the presence of 7% NaCl at 37 degrees C for 3 days, we evaluated the biological effect of rRpf by using S. Oranienburg VNC cells. The rRpf not only promoted proliferation but also induced resuscitation of VNC cells to the culturable state in a dose-dependent manner. Therefore, rRpf may be useful for detection of bacterial contaminants present in the VNC form in food samples and the environment.
Assuntos
Proteínas de Bactérias/farmacologia , Citocinas/farmacologia , Microbiologia de Alimentos , Regulação Bacteriana da Expressão Gênica , Intoxicação Alimentar por Salmonella/microbiologia , Salmonella enterica/crescimento & desenvolvimento , Salmonella typhimurium/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Western Blotting , Clonagem Molecular , Contagem de Colônia Microbiana , Meios de Cultura , Citocinas/química , Citocinas/genética , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Pressão Osmótica , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Salmonella enterica/efeitos dos fármacos , Salmonella typhimurium/química , Inoculações Seriadas , Cloreto de Sódio/farmacologia , Fatores de TempoRESUMO
Shiga toxin producing-Escherichia coli (STEC) has not yet been identified as an important aetiologic agent of human disease in Thailand. To evaluate the potential for STEC to contribute to human disease in Thailand, 139 fecal samples were collected from healthy cattle from five different provinces and analysed by genotypic and phenotypic methods for STEC. Of 139 samples, 27 (19.4%) were positive for stx1 and/or stx2 by multiplex polymerase chain reaction, or for O157 lipopolysaccharide (LPS) by immunoassay. Isolates positive for stx and/or O157 were subdivided into 49 strains that varied in the presence of the virulence determinants stx1+/stx2+ (22 strains), stx2+ (22 strains), stx1+ (4 strains), and O157 LPS (1 strain). Within these 49 distinguishable strains, other virulence determinants varied as follows: hlyA+ (77.6%), eae+ and tir+ (4.1%), and katP+ (6.12%). The most predominant profile (22 isolates) was stx1+/stx2+, eae-, tir-, etpD-, hlyA+, katP-. For further characterization of the isolated strains by two molecular typing assays, plasmid profiles and ERIC PCR were performed. The results suggest that the genetic and phenotypic profiles of STEC associated with human disease are not prevalent at this time in cattle in Thailand.
