Phenotypic and genotypic characterization of biofilm forming capabilities in non-O157 Shiga toxin-producing Escherichia coli strains

Abstract

The biofilm life style helps bacteria resist oxidative stress, desiccation, antibiotic treatment, and starvation. Biofilm formation involves a complex regulatory gene network controlled by various environmental signals. It was previously shown that prophage insertions in mlrA and heterogeneous mutations in rpoS constituted major obstacles limiting biofilm formation and the expression of extracellular curli fibers in strains of Escherichia coli serotype O157:H7. The purpose of this study was to test strains from other important serotypes of Shiga toxin-producing E. coli (STEC) (O26, O45, O103, O111, O113, O121, and O145) for similar regulatory restrictions. In a small but diverse collection of biofilm-forming and non-forming strains, mlrA prophage insertions were identified in only 4 of the 19 strains (serotypes O103, O113, and O145). Only the STEC O103 and O113 strains could be complemented by a trans-copy of mlrA to restore curli production and Congo red (CR) dye affinity. RpoS mutations were found in 5 strains (4 serotypes), each with low CR affinity, and the defects were moderately restored by a wild-type copy of rpoS in 2 of the 3 strains attempted. Fourteen strains in this study showed no or weak biofilm formation, of which 9 could be explained by prophage insertions or rpoS mutations. However, each of the remaining five biofilm-deficient strains, as well as the two O145 strains that could not be complemented by mlrA, showed complete or nearly complete lack of motility. This study indicates that mlrA prophage insertions and rpoS mutations do limit biofilm and curli expression in the non-serotype O157:H7 STEC but prophage insertions may not be as common as in serotype O157:H7 strains. The results also suggest that lack of motility provides a third major factor limiting biofilm formation in the non-O157:H7 STEC. Understanding biofilm regulatory mechanisms will prove beneficial in reducing pathogen survival and enhancing food safety.

Figure 1. Congo red affinity of STEC strains with or without complementing plasmids pUC19::mlrA or pTOPO::rpoS.

Strains carrying mutant mlrA or rpoS genes were marked with black boxes. AmpR/KanR: antibiotic resistance preventing the complementation experiments. WT: strain carries wild-type RpoS protein thus not complemented. NA: stable transformant not achievable.

Figure 2. Protein alignment of RpoS.

Mutant RpoS proteins from 5 STEC strains compared with the archetype strain ATCC 43895. Amino acids are listed by one-letter symbols.

Figure 3. SDS-PAGE of CsgA (curli) extractions.

(A) from parent strains and (B) from mlrA mutant strains complemented with pUC19::mlrA plasmid (+). Strain 43894OR was used as positive control. MW: Precision Plus dual color protein standards (Bio-Rad).

Figure 4. Protein alignments of CsgA (A) and CsgB (B) from selected STEC strains.

Figure 5. CsgD promoter sequence and activity.

(A) Alignment of csgD promoters and (B) β-galactosidase activities (Miller Units) over time. A representative time course was presented.