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United States Department of Agriculture

Agricultural Research Service

Research Project: MICROBIAL ECOLOGY AND SAFETY OF FRESH ON-FARM ORGANICALLY GROWN PRODUCE Title: Adherence of curli producing Shiga-toxigenic Escherichia coli to baby spinach leaves

Authors
item Macarisin, Dumitru
item Bauchan, Gary
item Giron, Jorge -
item Patel, Jitu

Submitted to: BARC Poster Day
Publication Type: Abstract Only
Publication Acceptance Date: March 22, 2011
Publication Date: April 27, 2011
Citation: Macarisin, D., Bauchan, G.R., Giron, J., Patel, J.R. 2011. Adherence of curli producing Shiga-toxigenic Escherichia coli to baby spinach leaves. BARC Poster Day.

Technical Abstract: Cellular appendages, such as curli fibers have been suggested to be involved in STEC persistence in fresh produce as these curli are critical in biofilm formation and adherence to animal cells. We determined the role of curli in attachment of STEC on spinach leaves. The curli expression by wild-type STEC strain, EDL933, and deriving curli-deficient (csgA and csgD) and curli-restored (csgA(pCsgA)) mutants grown on TSB agar at 20, 26 and 37ºC was determined by Congo red (CR) binding assay and Scanning Electron Microscopy (SEM). Spinach leaves were spot inoculated with 106 CFU of bacteria/g of leaf and incubated at 12, 26 and 37ºC for 18h. After incubation, leaves were washed three times in PBS and then homogenized by Polytron® to determine populations of strongly attached bacteria. Further, attachment was confirmed by Laser Scanning Confocal Microscopy (LSCM). STEC 'csgA and 'cgsD mutants didn’t express curli at 20, 26, and 37ºC, whereas EDL933 and csgA(pCsgA) developed a clear red phenotype on CR medium at 20 and 27ºC. At 37 ºC curli expression by EDL933 on CR medium was less pronounced compared to 'csgA(pCsgA). SEM and LSMS analysis confirmed EDL933 and 'csgA(pCsgA) cells expressing extracellular matrix containing curli fibres. When incubated at 12 and 26ºC, 'csgA and 'cgsD attached to spinach leaves at significantly (P=0.01) lower numbers compared to EDL933 and 'csgA(pCsgA). However, attached bacterial populations among strains weren’t significant when leaves were incubated at 37°C. Understanding the role of curli in STEC attachment and persistence in vegetal matrices will help developing intervention strategies to remove pathogens from fresh produce and thereby reduce future recalls.

Last Modified: 12/22/2014
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