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United States Department of Agriculture

Agricultural Research Service

Research Project: PREVENTION AND CHARACTERIZATION OF PERSISTENT COLONIZATION BY E. COLI O157:H7 AND OTHER SHIGA TOXIN-PRODUCING E. COLI (STEC) IN CATTLE Title: Polymorphic amplified typing sequences (PATS) and pulsed-field gel electrophoresis (PFGE) yield comparable results in the strain typing of a diverse set of bovine Escherichia coli O157 isolates

Authors
item Kudva, Indira
item Davis, Margaret -
item Griffin, Robert -
item Garren, Jeonifer -
item Murray, Megan -
item John, Manohar -
item Hovde, Carolyn -
item Calderwood, Stephen -

Submitted to: International Journal of Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 25, 2012
Publication Date: September 24, 2012
Citation: Kudva, I.T., Davis, M.A., Griffin, R.W., Garren, J., Murray, M., John, M., Hovde, C.J., Calderwood, S.B. 2012. Polymorphic amplified typing sequences (PATS) and pulsed-field gel electrophoresis (PFGE) yield comparable results in the strain typing of a diverse set of bovine Escherichia coli O157:H7 isolates. International Research Journal of Microbiology. 10.1155. Available: http://www.hindawi.com/journals/ijmb/2012/140105/.

Interpretive Summary: With the current mechanization and globalization trends in food production and distribution, the need to rapidly monitor, identify and eliminate potential sources of O157 from the food chain still remains. This identification and elimination of contaminated sources is possible by matching genomic DNA patterns (fingerprints) of pathogens in the source to those in the patient/general public. Pulsed-field gel electrophoresis (PFGE) is used routinely in most laboratories to develop genomic DNA patterns of bacterial pathogens and it relies on the genomic DNA banding pattern following digestion/cleavage with restriction enzymes that have rare/few cleavage sites in the O157 genome. But inherent drawbacks in the technique are leading epidemiologists to other genomic DNA fingerprinting methodologies. Polymorphic Amplified Typing Sequences (PATS) is a PCR-based bacterial genomic DNA fingerprinting system that relies on specific DNA sequence variations (polymorphisms) at these rare restriction enzyme cleavage sites, and virulence genes in the O157 genome, to differentiate between related and unrelated O157 bacterial isolates. Previously, PATS was used to effectively fingerprint or “type” 46 different O157 isolates associated with various outbreaks and human disease. In this study, we demonstrated the ability of PATS to discriminate 25 bovine O157 isolates, from different geographic locations across Northwest United States. About 85% of these bovine O157 isolates were grouped similarly by PATS and PFGE, irrespective of the rare restriction enzyme sites being targeted. Isolates that were grouped differently were better matched by location/source using PATS. Statistical analysis showed that the PATS and PFGE profiles shared a good correlation. Overall, PATS demonstrated a potential for rapidly differentiating isolates, without any user bias/errors or the need for sophisticated analytical software, while effectively complementing PFGE. Because results can be generated in just 6-8 hrs following bacterial isolation, PATS has excellent potential as a convenient tool for early epidemiological or food safety investigations to identify sources of infection and enabling rapid notification/implementation of quarantine measures.

Technical Abstract: The PCR-based Escherichia coli O157 (O157) strain typing system, Polymorphic Amplified Typing Sequences (PATS), targets insertions-deletions (Indels) and single nucleotide polymorphisms (SNPs) at the XbaI and AvrII(BlnI) restriction enzyme sites, respectively, besides amplifying four known virulence genes (stx1, stx2, eae, hlyA) in the O157 genome. Previously, we reported the potential of PATS to effectively discriminate between 46 different O157 isolates associated with various outbreaks and human disease. Here, we demonstrate the ability of PATS to discriminate unrelated, and associate related O157 isolates from cattle. Twenty-five bovine O157 isolates, from different geographic locations across Northwest United States, were analyzed using PATS, and pulsed-field gel electrophoresis (PFGE). Comparison of the dendograms generated showed that about 85% of the bovine O157 isolates grouped similarly by PATS and PFGE, irrespective of whether it was XbaI-based, AvrII-based, or based on a combination of enzymes. Isolates that were grouped differently were better matched by location using PATS. The Pearson’s correlation coefficient, r, calculated at about 0.4, 0.3, and 0.4 for XbaI-based, AvrII-based and combined PATS and PFGE similarities, indicating that these profiles shared a good but not high correlation, an expected inference given that the two techniques discriminate differently. Overall, PATS demonstrated a potential for rapidly discriminating isolates, without inadvertent interpretive biases or the need for sophisticated analytical software, while effectively complementing but not duplicating PFGE. Because results can be generated in just 6-8 hrs following bacterial isolation, PATS has excellent potential as a convenient tool for early epidemiological or food safety investigations, enabling rapid notification/implementation of quarantine measures.

Last Modified: 7/22/2014