Genomic and phenotypic analysis of shiga toxin-producing Escherichia coli O157:H7: The role of ariR in biofilm formation and acid resistance

E. coli O157:H7 was first recognized as a pathogen in an outbreak investigation study conducted in 1982 and has garnered significant attention due to its virulence potential, causing life-threatening symptoms in immunocompromised populations, and its adaptability to diverse sources, hosts, and environments. This underscores the critical necessity of advancing the understanding of E. coli O157:H7 genetic diversity and phylogenetic relationships. Whole-genome sequencing (WGS) has emerged as a potential epidemiological tool for investigating foodborne outbreaks and conducting routine surveillance, with sequenced data enhancing public health efforts in source attribution. Consequently, WGS serves as a motivation to further characterize historic O157:H7 isolates in the E. coli Reference Center (ECRC) repository at The Pennsylvania State University. Accordingly, WGS analysis was employed to directly compare the genetic characteristics retrospectively in 172 E. coli O157:H7 isolates from the ECRC, which comprised 109 strains yet-to-be sequenced and 63 strains already sequenced. WGS analysis identified phylogenetic relatedness among isolates, 315 virulence genes including key virulence factors such as Shiga toxin gene (stx) and intimin (eae), and revealed 20 multidrug resistant strains predominantly from animal sources. Additionally, the identification of IncF group plasmids and virulence genes encoded by pO157 suggested the presence of the virulence plasmid pO157 in the O157:H7 collection. Collectively, these results emphasize the necessity of utilizing WGS in integrated routine surveillance and retrospective comparisons of E. coli O157:H7 from various sources.

Interestingly, a regulator gene, ariR, responsible for biofilm regulation and acid resistance (AR) common in E. coli K-12, was flagged in 8 out of the 109 sequenced O157:H7 isolates in the Pathogen Detection database. ariR is absent in most O157:H7 strains, possibly due to a mechanism analogous to a "black hole", where non-essential genes are deleted to gain virulence genes. Accordingly, biofilm formation, curli expression, and AR assays were carried out to understand the physiological role of ariR in E. coli O157:H7. In total, 5 ariR positive, 5 ariR negative, and 2 control strains (E. coli O157:H7 EDL933 and E. coli K-12) underwent phenotypic screening. A lack of direct correlation between ariR presence and suppressed biofilm formation was observed, and no correlation was found between increased AR and the presence of ariR. Moreover, other regulatory mechanisms that might impact curli expression and biofilm formation, as well as other AR systems, might be at play in the AR assays. Collectively, environmental pressures may lead to the deletion or suppression of non-essential genes like ariR, enhancing the pathogenicity and host colonization of these strains. The observed lack of correlation between AR, biofilm formation, and ariR suggested that O157:H7 may have evolved different mechanisms compared to the domesticated K-12 strain in the lab, highlighting the need for cautious interpretation of bioinformatics results. Further loss-of-function studies are required to examine ariR and other regulatory mechanisms that might impact AR and biofilm formation in O157:H7, as gene presence alone does not necessarily imply functionality.