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Geno-phenotypic characterization of genes envolved in expression of curli fimbriae in VTEC (Verocytotoxigenic Escherichia coli) isolated from dairy farms environment and cattle.
ROSANA POLIFRONI; DANIEL FERNÁNDEZ; ANALÍA I. ETCHEVERRÍA; NORA L. PADOLA; ALBERTO E. PARMA
Centro Cultural Borges - C.A.B.A.
Congreso; VTEC2009 -7th. International Symposium on Shiga Toxin (verocytotoxin)- Producing Escherichia coli Infections Buenos Aires; 2009
Asociación Argentina de Microbiología
When a bacteria is expose to stress conditions during the exponential growth phase, it enters the stationary phase. This response is controlled at molecular level by sigma unit of RNA polymerase, sigmas (RpoS). The RpoS accumulates in the cell during the exponential growth phase, then it associates with the core enzyme and directs the transcription of genes essentials for the general stress response and for stationary phase survival. Recent studies have shown that the crl gene product is a regulator of sigmas activity in Escherichia coli. Crl modulates the expression of sigmas- regulated genes (poxB, katE, bolA and csgB) and binds sigmas enhancing its activity. CsgD depends on RpoS and Crl and controls the expression of csgBAC operon. This indicates that at least two sigmas regulated genes are implicated in the sintesis of curli fimbriae. This capacity could be related with the docking phase of the biofilm formation. In this study, our aim was to recognize the capacity to express the curli operon in strains isolated from dairy farm´s environment and cattle. We used monoplex PCR for csgD, csgA y crl genes and Congo red plates to determinate the expression of phenotype saw (soft and white) for not expression of curli operon in the colony, and rdar (red, dry and rough) for expression of the operon. The plates were incubated for 48 h at 37° C. The strains were previously characterized as VTEC using multipex PCR for ehlyA, eae, vt2, vt1 and saa genes then checked for curli fimbriae genes and phenotype in Congo red plate. The stains used in the experiment were collected from soil from corrals (O116:NM, vt2), feed cattle (O116:NM, vt2), water from animal-drinking troughs (ND*, vt2) and swab from solid surfaces: swab from feeder (O26:H21, ehlyA, eae, vt1) and teatcup (ND, ehlyA, vt2, saa) , and their results were compared with those obtained from cattle (O157:H7, ehlyA, eae, vt2; O26:H21, ehlyA, vt1; O91:H21,ehlyA, vt2, saa; O113:H21, ehlyA,vt2, vt1, saa). The results of monoplex PCR showed that all strains had the necessary genes for the expression of the curli fimbriae, but when these strains were cultivated in Congo red plates not all expressed that fimbriae. We observed that strains isolated from solid surfaces showed phenotype rdar, as swab from feeder and teatcup and calf´s feed, whiles the others strains were saw. Our conclusion is that VTEC could be expressing the curli fimbriae in response to environmental stressing conditions. This adaptation could enhance the Escherichia coli capacity to start biofilm formation and enhance their survival time in the environment. The persistence in environmental conditions might be increase cattles infection and may be an important source of infection for human illness. * ND: Not determined Key words: VTEC, Biofilm, curli, PCR.