Summary
Background
For many years the red meat industry has been interested in E. coli O157 in relation to protecting public health and to meet the requirements of countries receiving exported beef.
However, E. coli O157 is not the only pathogenic E. coli that is present in animals used for meat production and potential exists for red meat to become contaminated with these other pathogenic strains.
There is growing awareness and importance being placed on non-0157 Shiga toxin producing E. coli (STEC) in countries that accept Australian meat exports.
Although these countries currently only require meat to be tested for E. coli O157, at some stage in the future they may require testing for non-O157 STEC's.
There is little information about the prevalence and types of non 0157 STEC found in foods of animal origin. This absence of information is primarily due to the lack of reliable methods for the detection and isolation of these organisms.
Similarly, little is known about the ecology of these organisms and how they may contaminate meat (eg. location in the animal host, survival on surfaces and transmission during processing), so the effectiveness of strategies used for controlling E. coli O157 may not be sufficient for controlling non-O157 STEC.
Purpose
The purpose of this project was to review the current methods available for the detection and isolation of pathogenic E. coli and define and evaluate the most appropriate methods for use in the red meat industry.
Once methods have been developed and evaluated, further information on the prevalence and numbers of these organisms is cattle and sheep will be investigated, to provide data for assessing the risk to the industry.
Results
A review of methods for the detection and isolation of non-O157 EHEC determined that a multi-step protocol would provide a holistic approach to the detection, isolation and characterisation of non-O157 EHEC in red meat systems.
The approach initially determines which samples harbour a combination of genes (stx and eae) commonly found in EHEC isolates before determining whether samples containing this combination of genes also contain any of the E. coli serotypes of interest.
Isolation of the target organism follows and samples from which a non-O157 EHEC isolate is recovered are enumerated using a combinatorial polymerase chain reaction - PCR - and most probable number - MPN - technique.
This approach is consistent with methods proposed by the US Food Safety and Inspection Service - FSIS- and the European Food Safety Authority - EFSA - for non-O157 EHEC.
The methods developed in this project were used to conduct an investigation to determine the prevalence of non-O157 EHEC in Australian beef cattle.
Eight EHEC serotypes encompassing the epidemiologically significant serotypes of the United States and Europe were included in the survey.
Molecular screening of enriched faecal samples determined that 128 of 300 (42.6%) contained stx and 152 of 300 (50.6%) contained eae.
A total of 78 of 300 (26%) samples had stx and eae present in the same sample.
E. coli of nearly all target serotypes were recovered, however there was a lack of EHEC virulence markers in most isolates.
The results indicated that the prevalence of EHEC serotypes other than E. coli O157 in the Australian beef cattle population is low thereby reinforcing Australia's position as a source of quality meat products.
The complexity associated with non-O157 methods was highlighted during this study.
A large proportion of samples were shown to contain eae, however this failed to translate into the isolation of EHEC isolates. Isolates harbouring eae could be isolated from beef cattle but they lacked additional EHEC virulence markers and accessory genes.
This finding, in combination with the isolation of STEC from a number of samples reinforces the need for non-O157 methods to result in the isolation of an EHEC. As the isolation phase of the method is considered the least optimised component, the need for enhanced isolation protocols is evident.
In addition to the increased attention being placed on non-O157 EHEC, there remains a need to further understand E. coli O157.
There is increasing evidence that specific genotypes of E. coli O157 are more likely to cause human disease than others.
Genetics and genomics of a collection of Australian O157 isolates from animals and humans determined that there were no obvious differences in the presence of virulence-associated genes in these isolates. However, E. coli O157 could easily be differentiated from other pathotypes of E. coli based on the presence or absence of virulence-associated genes.
As the interrogation of E. coli O157 strains that are associated with outbreaks of human disease continues, it is probable that a genetic signature(s) associated with increased pathogenicity will be identified.
Publications
The results of this research have been published in the following;Journal articles
Barlow, R.S and Mellor, G.E. (2010) Prevalence of Enterohemorrhagic Escherichia coli Serotypes in Australian Beef Cattle. Foodborne Pathogens and Disease. 7(10) 1239-1245Conference presentations
Barlow, R.S, Mellor, G.E and Fegan, N. (2009) Prevalence of non-O157 EHEC serotypes in Australian beef cattle. 7th International Symposium on Shiga Toxin (Verocytotoxin) producing Escherichia coli Infections, Buenos Aires, Argentina. May 10-13, 2009. Poster presentation.
Mellor, G.E and Barlow, R.S. (2010) Presence and characterisation of ATEC in Australian beef cattle. Australian Society for Microbiology, Sydney. July 4-8, 2010. Microbiology Australia. Poster presentation.
