Biernbaum, E. N. | bioRxiv Science

bioRxiv2026

Varied influence of curli on Shiga toxin-producing Escherichia coli O157, O26, O111 biofilm formation and animal cell adherence.

Shiga toxin-producing Escherichia coli (STEC) persist in cattle and can colonize the bovine recto-anal junction, making it an important reservoir for transmission. Curli are extracellular amyloid fibers associated with biofilm formation and environmental persistence, but their contribution to STEC adherence to bovine epithelial cells is unclear. This study examined the relationship between curli production, biofilm formation, and adherence of O157, O26, and O111 STEC isolates to bovine recto-anal junction (RAJ) squamous epithelial (RSE) cells. Thirty isolates were evaluated for RSE adherence, curli-associated phenotypes, and biofilm formation under environmental and host-associated growth conditions. O157 and O111 isolates predominantly exhibited strong aggregative adherence to RSE cells, whereas O26 isolates showed more heterogeneous, primarily diffuse adherence. O111 isolates produced the strongest biofilms under environmental conditions and displayed relatively stable curli phenotypes, while O157 and O26 isolates showed greater variation. However, environmental biofilm formation did not consistently correlate with RSE adherence. Biofilm formation was also markedly reduced under host-simulated conditions in DMEM-LG at 26, 37, and 39{degrees}C, including among isolates that produced strong environmental biofilms. Differences in Shiga toxin genotype and expression likewise did not account for the major adherence patterns, as O111 isolates lacking stx2 retained strong aggregative adherence. These findings indicate that curli-associated biofilm formation is primarily influenced by environmental conditions and does not directly predict STEC adherence to bovine RSE cells. The results suggest that environmental persistence and host-cell attachment represent distinct phenotypes and that STEC adherence to bovine epithelial cells is likely mediated by additional strain- and serotype-specific factors.

microbiology↗

bioRxiv2024

Growth dynamics and protein-expression of Escherichia coli serotypes O26:H11, O111:H8 and O145:NM in the bovine rumen.

To adapt to the ruminal environment, Shiga toxin-producing Escherichia coli (STEC) O157:H7 (O157) expresses proteins involved in survival rather than virulence. Additionally, STEC O157 strains exhibit distinct in vitro but shared in vivo survival patterns in rumen fluids that sets them apart from non-STEC, commensal E. coli. To determine if similar responses would be observed with other STEC, we evaluated three non-O157 serotypes, O26:H11, O111:H8 and O145:NM, along with a non-STEC E. coli, under the growth conditions used for STEC O157: (i) anaerobically, in vitro, in rumen fluid from cattle on a lactation (low fiber, high protein) or maintenance (high fiber, low protein) diet, at 39{degrees}C for 48 hr and (ii) in vivo for 48 hr within the rumen of cattle on the same diets using a non-terminal, rumen-fistulated animal model that allows for introduction of bacteria without ruminal contamination. On the lactation diet, the ruminal pH was acidic ranging from 5.2 - 6.0 and the total volatile fatty acids (VFA) concentration, 141 - 230 M/ml. On the maintenance diet, the ruminal fluid pH was close to neutral ranging from 6.0 - 7.0, with a total VFA of 87 - 190 M/ml. Unlike STEC O157, the three non-O157 serotypes demonstrated survival patterns similar to each other and the control non-STEC E. coli. A greater reduction in viable STEC counts was observed in vitro in rumen fluid from cattle fed the lactation diet than in vivo, corroborating previous reports that in vitro conditions cannot mimic those observed in vivo. Like STEC O157, the non-O157 serotypes mainly expressed proteins supporting ruminal adaptation although not all proteins matched those expressed by STEC O157, and included the virulence protein, intimin. Explorative studies such as this could provide insights into common conditions/ targets that may have application in broader STEC control in cattle. Importance of this studyThis study demonstrates that non-O157 serotypes O26:H11, O111:H8, O145:NM have similar survival and protein expression patterns in rumen fluid with variations being influenced primarily by rumen fluid composition associated with diet. Unlike STEC O157, under in vivo conditions, the growth dynamics of the non-O157 serotypes were comparable to that of non-STEC, commensal E. coli. Hence, exploring bacterial protein expression within the host is critical in discerning therapeutic targets, unique to/shared between STEC, for broad control strategies. In addition, this study further validates the value of using a non-terminal animal model for rumen studies that reduces number of animals used for an experiment.

microbiology↗