Analyses of the red-dry-rough phenotype of an Escherichia coli O157:H7 strain and its role in biofilm formation and resistance to antibacterial agents

Abstract

In a previous study, we identified Congo red-binding and -nonbinding phase variants of Escherichia coli serotype O157:H7 strain ATCC 43895. The Congo red-binding variant, strain 43895OR, produced a dry, aggregative colony that was similar to the red, dry, and rough (rdar) phenotype characteristic of certain strains of Salmonella. In contrast, variant 43895OW produced a smooth and white colony morphology. In this study, we show that, similar to rdar strains of Salmonella enterica serovar Typhimurium, strain 43895OR forms large aggregates in broth cultures, firm pellicles at the air-medium interface on glass, and dense biofilms on glass and polystyrene. However, unlike S. enterica serovar Typhimurium, strain 43895OR does not stain positive for cellulose production. When strain 43895OR was fixed on agar, scanning electron microscopy showed cells expressing extracellular matrix (ECM) containing curli fibers. Strain 43895OW was devoid of any ECM or curli fibers on agar but showed expression of curli fibers during attachment to glass. Strain 43895OR produced >4-fold-larger amounts of biofilm than strain 43895OW on polystyrene, glass, stainless steel, and Teflon; formation was >3-fold higher in rich medium than in nutrient-limited medium. Biofilm-associated cells of both strains showed statistically greater resistance (P < 0.05) to hydrogen peroxide and quaternary ammonium sanitizer than their respective planktonic cells. This study shows that the rdar phenotype of E. coli O157:H7 strain 43895OR is important in multicellular growth, biofilm formation, and resistance to sanitizers. However, the lack of cellulose production by strain 43895OR indicates important differences in the ECM composition compared to that of Salmonella.

FIG. 1.

Topographical images of colonies of E. coli O157:H7 strains 43895OW (saw) (A) and 43895OR (rdar) (B) grown and fixed on YESCA agar for 48 h at 28°C. (C) Negatively stained TEM of dispersed colonies of strain 3895OR (rdar) harvested from a YESCA plate grown for 48 h at 28°C and stained with uranyl acetate. (D) Immunoblot of formic acid-treated cells collected from YESCA plates after 48 h at 28°C. Separated proteins of strains 43895OR (lane 1) and 43895OW (lane 2) reacted with anti-CsgA antibody. The arrow indicates the position of the 15-kDa marker. The predicted monomeric form of CsgA is 15.1 kDa.

FIG. 2.

Fluorescence (366 nm) of bacterial strains grown on LB agar containing 200 mg/liter calcofluor at 28°C for 72 h. (A) S. enterica serovar Enteritidis strain 3934 (curli positive; cellulose positive); (B) S. enterica serovar Enteritidis strain 942 (curli positive; cellulose negative); (C) S. enterica serovar Enteritidis strain 1170/97 (curli negative; cellulose negative); (D) E. coli 0157:H7 strain 43895OR; (E) E. coli 0157:H7 strain 43895OW.

FIG. 3.

Crystal violet-stained glass coupons from biofilm assays with strains 43895OW (A) and 43895OR (B). Coupons were submerged in 50-ml tubes containing 20 ml LB-NS broth for 48 h at 28°C. The arrowhead indicates the medium fill line.

FIG. 4.

Scanning electron micrographs of glass coupons with biofilms generated in LB-NS medium for 48 h at 28°C. (A) E. coli O157:H7 strain 43895OW (saw) from the region of the slide immediately below the medium-air interface. (B) E. coli O157:H7 strain 43895OR (rdar) from the biofilm pellicle located below the medium-air interface.

FIG. 5.

Biofilm formation of E. coli O157:H7 strains 43895OR and 43895OW on polystyrene in TSB, 1/20 tryptic soy broth, and LB-NS. Each bar represents the mean optical density measured at 590 nm ± standard deviation of eight replicates from a single sample taken from each of three independent trials for strains 43895OR and 43895OW or two independent trials from the medium controls.

FIG. 6.

Biofilm formation of E. coli O157:H7 strains 43895OR and 43895OW on glass, Teflon, and stainless steel in LB broth (without salt). Each bar represents the mean optical density measured at 590 nm ± standard deviation of a single sample taken from each of three independent trials for strains 43895OR and 43895OW or two independent trials from the medium controls.