|Kay, Kathryn - North Carolina State University|
|Grunden, Amy - North Carolina State University|
|Oh, Deog Hwan - Kangwon National University|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 2/26/2015
Publication Date: 5/30/2015
Citation: Kay, K., Fratamico, P.M., Grunden, A., Oh, D., Breidt, F. 2015. Investigation of carbon storage regulation network (csr genes) and phenotypic differences between acid sensitive and resistant Escherichia coli O157:H7 strains. Meeting Abstract. meeting abstract.
Technical Abstract: Background: Escherichia coli O157:H7 and related serotype strains have previously been shown to vary in acid resistance, however, little is known about strain specific mechanisms of acid resistance. We examined sensitive and resistant E. coli strains to determine the effects of growth in minimal and complex media on acid stress response, including gene expression of global regulatory pathways for catabolic regulation. Methods: To determine acid resistance, E. coli O157:H7 (n=24) and a K-12 strain were grown statically in a minimal medium (M9GT) and Luria broth (LBG) with 1% glucose at 37 degrees C for 18 hours. Cells were suspended in 400 mM acetic acid at pH 3.3 and incubated at 30 degrees C. Samples were taken at T=0 and 25 min, diluted, plated on LB, incubated at 37 degrees C for 24 hours, and enumerated. Log reduction was calculated by linear regression in Excel. Transmission electron microscopy (TEM) was performed on selected strains and total RNA was extracted with Qiagen’s RNEasy Kit. The Bio-Rad One Step RT-qPCR Kit was used to target expression of carbon storage regulation, flagella, and biofilm genes. Results: E. coli O157:H7 strains grown in LBG varied significantly in acid resistance (p greater than 0.05), with log reduction values showing no apparent reduction to a 3-log reduction. Interestingly, we found that K-12 and all acid sensitive O157:H7 strains tested (less than 1.0 log reduction, n=8) flocculated and lost motility in minimal medium, but not LBG. Acid resistant strains (greater than 1.0 log reduction, n=17) showed no phenotypic differences, regardless of growth medium. TEM data showed loss of flagella and clumping for flocculating cells. Gene expression data (Ct values, see Table 1) for selected sensitive and resistant strains (B201 and B241, respectively) in M9GT showed no change in carbon storage (CsrAD), flagella (FlhCD), or biofilm (PgaABCD) mRNA transcript levels (p less than 0.05). Conclusion: The loss of motility and clumping of acid sensitive, but not acid resistant O157:H7 strains grown in a minimal medium indicates differences in global regulatory pathways. Because expression for csr, flh, and pga genes was unchanged, it is likely other regulatory pathways or physiologic changes may influence the flocculation phenotype. Interestingly, a K-12 strain (also acid sensitive) may share similar regulatory pathways as the acid sensitive O157:H7 strains.