Background

Cattle are a primary reservoir for pathogenic bacteria E. coli O157:H7. Although the presence of E. coli O157:H7 is asymptomatic in adult cattle, human ingestion of food products contaminated with this pathogen can lead to severe disease. The rumen is a well-studied ecosystem for bacterial growth, but little is known about the mechanisms of how E. coli O157:H7 persists and survives in this unique environment. The pathogen can apparently persist in an infected ruminant significantly longer than many other E. coli serotypes. Although E. coli O157:H7 appears to be primarily located in the lower part of the digestive system, including the cecum, colon and rectum, it is not clear what factors of the host, or the microorganism influences this localization, nor what dictates its shedding. The influence of diet, stress, antibiotics and other intervention strategies on O157 prevalence further complicates its analysis.   

Researchers in this project undertook a molecular genetics approach using DNA microarrays to identify genes that are important for growth and survival of E. coli O157:H7 in the rumen. The primary hypothesis of this project is that many of the genes that are induced by encounter with the rumen will be important for O157 survival. The researchers thought that identifying these genes and testing their function by mutagenesis might lead to novel targets for new antimicrobial agents and vaccines.   

The specific objectives of this project were: 

1. Develop an in vivo culture system for E. coli O157:H7. Since O157 is exposed to the rumen before setting up residence in the lower digestive tract, the researchers focused on studying bacterial growth in this region of the gastrointestinal tract. Bacteria were inoculated into fistulated cattle and samples were removed at various times to assess the growth and transcriptional activity of bacterial genes. 
2. Perform transcriptional profiling by DNA microarrays to provide new insights into the cellular responses of E. coli O157:H7 in its natural environment. 
3. Perform mutational analyses through reverse genetics to allow the researchers to directly determine to what extent a gene contributes to the survival and persistence of E. coli O157:H7 in the ruminant.  

High-density microarrays essentially create a “genome on a chip” and enable the transcriptional activity of specific genes when bacteria are grown under different environmental conditions. With O157 having nearly 2,000 genes not found in other E. coli strains, it is likely that several of these sequences are important for its survival in the specialized environment of the rumen.

Methodology 

Growth of E. coli O157:H7 under conditions that mimic the rumen environment 

A specific strain of E. coli O157:H7 was inoculated at low density into a variety of growth media. Cultures were recovered and stored at –80ºC so as to stabilize the RNA.   

Construction of E. coli O157:H7 microarrays 

Microarrays are experimental methods that allow the activity of each gene to be monitored simultaneously, thus the researchers incorporated this relatively new technology into their analysis of the genes that might contribute to E. coli O157:H7’s persistence in the digestive tract of animals. Microarrays containing over 5,000 of the total 5,384 genes identified in the sequenced E. coli O157:H7 strain EDL933 were constructed. The arrays were assembled using polymerase chain reaction (PCR) to amplify regions of individual genes that were identified from the published genomic sequence of strain 933. The researchers used a commercially available bacterial RNA isolation kit (Ambion) to isolate and prepare samples for analysis.

Findings 

The information collected in these studies has provided a foundation for researchers’ continuing efforts to use genomic-based approaches to understand the molecular survival and persistence of E. coli O157:H7 in cattle. The researchers did encounter some unexpected hurdles in their projects. E. coli O157:H7 grew poorly in the anaerobic lab environment that was created to be similar to the rumen. This initially limited RNA yields for subsequent analysis, but the researchers overcame that challenge by using RNA amplification. The inability of E. coli O157:H7 to proliferate under the experimental conditions created for this project may reveal a vulnerability of the bacteria that could be exploited in new intervention strategies.   

The microarrays developed through this project will enable further research to identify new genes that are important for O157 growth and survival in the rumen.

Implications   

There are nearly 2,000 genes that are found in O157 that are not present in the non-pathogenic K-12 strain. Based on that information, it is likely that new genes will be revealed that are important for the unique ecological niche inhabited by E. coli O157:H7. By understanding how E. coli O157:H7 responds to the rumen environment, there is the potential to identify weaknesses in this organism that can be targeted through new vaccines or other treatments to significantly reduce the microorganism in cattle.