Phylogenetic Analysis of Enterohemorrhagic Escherichia coli O157, Germany, 1987–2008

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Volume 16, Number 4–April 2010
Research
Phylogenetic Analysis of Enterohemorrhagic Escherichia coli O157, Germany, 1987–2008
Christian Jenke, Dag Harmsen, Thomas Weniger, Jörg Rothgänger, Eija Hyytiä-Trees, Martina Bielaszewska, Helge Karch, and Alexander Mellmann
Author affiliations: Institute for Hygiene, Münster, Germany (C. Jenke, M. Bielaszewska, H. Karch, A. Mellmann); Department of Periodontology, Münster (D. Harmsen, T. Weniger); Ridom GmbH, Würzburg, Germany (J. Rothgänger); and Centers for Disease Control and Prevention, Atlanta, Georgia, USA (E. Hyytiä-Trees)

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Abstract
Multilocus variable number tandem repeat analysis (MLVA) is a subtyping technique for characterizing human pathogenic bacteria such as enterohemorrhagic Escherichia coli (EHEC) O157. We determined the phylogeny of 202 epidemiologically unrelated EHEC O157:H7/H– clinical isolates through 8 MLVA loci obtained in Germany during 1987–2008. Biodiversity in the loci ranged from 0.66 to 0.90. Four of 8 loci showed null alleles and a frequency <44.1%. These loci were distributed among 48.5% of all strains. Overall, 141 MLVA profiles were identified. Phylogenetic analysis assigned 67.3% of the strains to 19 MLVA clusters. Specific MLVA profiles with an evolutionary persistence were identified, particularly within sorbitol-fermenting EHEC O157:H–.These pathogens belonged to the same MLVA cluster. Our findings indicate successful persistence of this clone.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infections have substantial medical, public health, and economic effects (1,2). Most symptomatically infected patients have painful bloody diarrhea (2,3). Hemolytic uremic syndrome (HUS) develops in ≈15% of infected children ≈1 week after the first loose stool. HUS is a thrombotic microangiopathy and consists of nonimmune hemolytic anemia, thrombocytopenia, and renal failure (1). Currently, HUS is the main cause of acute renal failure in children (4). In Germany, E. coli O157:H7, which is the most frequent EHEC serotype implicated in HUS, is not the only relevant EHEC O157 involved. Sorbitol-fermenting (SF) E. coli O157:H– (nonmotile) strains cause ≈20% of all cases of HUS (5). Unlike E. coli O157:H7, organisms within this clone can ferment sorbitol after overnight incubation on sorbitol MacConkey agar. Although EHEC O157:H7 causes a zoonotic disease mainly associated with cattle, efforts to determine the animal reservoir of SF EHEC O157:H– have been unsuccessful (5).

To identify reservoirs of EHEC O157:H7 infections and of other foodborne pathogens and to elucidate the molecular epidemiology of these pathogens in the United States, PulseNet was established in 1996 (6). This US national molecular subtyping network for foodborne disease surveillance facilitates subtyping of bacterial foodborne pathogens for epidemiologic purposes. This network is based on characterization of whole bacterial genomes by using macrorestriction digestion patterns that are separated by pulsed-field gel electrophoresis (PFGE), a technique that has emerged as a common standard for subtyping EHEC O157 isolates (6). Despite its high discriminatory power, PFGE can be problematic because it requires great efforts to ensure intralaboratory and interlaboratory reproducibility (7–10). Furthermore, its application is labor-intensive and difficult to automate. Thus, this technique can be biased by subjective interpretation of band patterns (7,8). In addition, band patterns can be altered by the presence of mobile genetic elements.

To overcome these drawbacks, other molecular methods were developed, among them multilocus variable number tandem repeat (VNTR) analysis (MLVA). MLVA is based on the characterization of different VNTR regions throughout the bacterial genome. Repeat regions are amplified by using PCRs, and resulting fragments are sized to determine the number of repeats. The combination of numbers of repeats of different VNTR loci results in an allelic profile known as the typing result. First developed in 1995 for Mycobacterium tuberculosis (11), MLVA is now a common typing method for an increasing number of pathogens (12,13). For EHEC O157, different MLVA schemes with some overlaps of VNTR regions have been published and have demonstrated a capability to detect outbreaks and differentiate closely related EHEC O157 isolates not discriminated by PFGE (8,14,15). These findings qualify MLVA as the second-generation subtyping method for PulseNet (8).

In addition to its use in infectious disease surveillance, MLVA also can be used to study phylogeny of pathogens, especially recently evolved clonal pathogens such as M. tuberculosis (16,17) or Bacillus anthracis (18). However, because of limited diversity in their housekeeping genes, which are the genomic targets for phylogenetic investigations based on multilocus sequence typing (MLST), the common technique for phylogenetic studies (19,20), certain monomorphic organisms could not be sufficiently differentiated by MLST (16,18). Similarly, EHEC O157 lacks diversity in its housekeeping genes (21,22), which hampers phylogenetic analysis of EHEC O157 by MLST.

We investigated the phylogeny of EHEC O157:H7 and SF EHEC O157:H– strains isolated during 1987–2008 in Germany by applying the current PulseNet MLVA protocol for E. coli O157 (23). The purpose of our study was to gain a deeper insight into the evolution and spread of this pathogen since 1987, when the first cases of EHEC O157 infections were detected (24,25).

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Suggested Citation for this Article
Jenke C, Harmsen D, Weniger T, Rothgänger J, Hyytiä-Trees E, Bielaszewska M, et al. Phylogenetic analysis of enterohemorrhagic Escherichia coli O157, Germany, 1987–2008. Emerg Infect Dis [serial on the Internet]. 2010 Apr [date cited]. http://www.cdc.gov/EID/content/16/4/610.htm

DOI: 10.3201/eid1604.091361