Identifying Cattle That Are More Likely To Be E.Coli 0157 Super-Shedders
Identification of Genetic and Microbial Markers for E. Coli O157 Super-Shedders Through Longitudinal Biopsy and Monitoring
Leluo Guan Ph.D. (University of Alberta) email@example.com
Leluo Guan Ph.D. and Graham Plastow Ph.D., (University of Alberta); Tim McAllister Ph.D., (Agriculture Agri-Food Canada Lethbridge); Kim Stanford Ph.D., (Alberta Agriculture and Forestry); Tom McNeilly Ph.D., (Moredun Research Institute)
|Completed March, 2023
Packing plants have improved their control of E. coli O157:H7 and recalls due to E. coli O157:H7 in beef are becoming rare. In 2014, Canada had ten E. coli O157 related recalls and only one involved beef. Cattle are thought to be the main reservoir for E. coli O157:H7. Fewer than 10% of cattle are thought to be “super-shedders”, which are responsible for more than 90% of the shedding of this pathogen. On-farm sampling is of limited value because some super-shedders shed longer than others. Previous research has identified suppressed expression of genes related to immunity in the rectum of super shedders. This project sought to understand the factors contributing to the super-shedding phenomenon and find strategies to minimize the risk it poses to food safety.
- To determine what causes some cattle to be super-shedders and recommend improved techniques to mitigate E. coli O157 in beef cattle.
What They did
143 feedlot steers were rectally biopsied and fecal sampled on arrival at AAFC Lethbridge. DNA markers were screened to identify potential high and low shedders (at least 3% or 30 cattle per group) based on the prevalence of the Shiga toxin gene (the main virulence factor of E. coli O157).
Using gene markers identified in an earlier study, the repeated rectal samples were analyzed to monitor how gene expression changed at different stages of super-shedding and relate these changes to the bacteria found in the fecal sample.
Gene expression of both the host and the bacterial population was compared between super-and low- shedders to identify possible control mechanisms related to colonization and shedding in both the bacteria and the host. The team looked for small changes in DNA sequences that were differentially expressed between the super- and low-shedders.
The E. coli O157:H7 isolates collected were whole genome sequenced to look for genes related to heat, acid and sanitizer tolerance identified in FOS.01.17, differences in pathogenicity (virulence, toxin, and adherence genes), or differences in colonization.
What They Learned
This team successfully identified super-shedder cattle by identifying marker genes associated with Shiga toxin production. This was done using molecular techniques which cut down the cost and labour associated with the traditional culturing of STEC.
The type of stx2 did not appear to alter the microbiome but did alter microbial communities compared to those that lacked stx2 expression. Modification of the gut environment to encourage growth of microbial communities that naturally suppress stx2 expression could reduce super-shedding. Breed may also impact the prevalence and expression of stx2 genes. As a consequence, STEC could be increasing colonization by two means – by suppressing the animal’s immune system or those with a compromised intestinal immunity due to previous or current illness. The machine learning based analysis revealed that expression of key immune genes could be used to predict STEC colonization with more than 80% accuracy.
Challenging calves with STEC impacted the microbial diversity of the gut compared to those that were not challenged though the team found that this diversity was highest at times of peak shedding. The type of stx toxin did not impact microbial community composition but did alter the stability of microbial networks. The expression of genes in rectal tissue also differed depending on the type of stx toxin, and the key regulatory mechanisms in response to shedding were also identified.
The team were able to identify antimicrobial peptides with the potential to inhibit E. coli O157 colonization; -two showing the most promise – microcin H47 (MccH47) and WK2. WK2 specifically exhibited strong antimicrobial and antibiofilm activity. Studying WK2 further showed that it binds to bacterial DNA, inhibiting structures required for motility, and reducing the cells’ ability to communicate with adjacent cells which ultimately stops cells from being viable as part of a biofilm. MccH47 inhibited the growth of several E. coli O157 serogroups as well as non- E. coli STEC serotypes. The ability for Microcin H47 to impact a broad spectrum of STEC bacteria makes it a valuable peptide to reduce STEC shedding while inhibiting other foodborne pathogens. Further investigation found that MccH47 reduced STEC’s competitive advantage which means that it could be used to reduce the competitiveness of this pathogen and its ability to integrate into biofilms. The cells exposed to MccH47 stopped growing, expressed abnormal structure, and had ruptured cell walls.
What It Means
Improving testing techniques will allow researchers and producers to understand what defines a super-shedder and how to manage them appropriately. The project found that type of Shiga toxin gene presence did not have an impact on microbial community however, there was differences compared to the microbial communities of cattle that did not have the gene present.
There was evidence to say that the gut environment could be modified to stop the expression of these genes which has potential to reduce the number of super-shedders. Breed was also found to have an impact on the abundance of genes and host immunity’s ability to mitigate the gene/toxin expression.
The study identified several promising antimicrobial peptides with potent antimicrobial and antibiofilm activity including microcin H47 (MccH47) and WK2. MccH47 reduced the competitive advantage of STEC against some E. coli isolates, arrested growth, resulted in aberrant morphology and lysis. These peptides were shown to effectively interfere with biochemical pathways that reduced the viability of this pathogen.
Ultimately, understanding the interactions between cattle and bacteria helps to determine why some animals are super-shedders and the factors that can help regulate it is a crucial step in developing more effective on-farm and/or in-plant interventions against E. coli O157:H7 to further increase the safety of Canadian beef.