Using 8 VNTR loci of the current PulseNet MLVA O157 protocol (23
), we analyzed a large collection of 202 EHEC O157:H7/H–
strains isolated over >2 decades in Germany to determine their molecular epidemiology. Of the 141 MLVA profiles detected, 81 were clustered into 19 groups of related profiles that differed at >
2 loci. The remaining 60 profiles were not clustered. Our data demonstrate a great diversity of EHEC O157:H7 associated with human diseases in Germany over the past 2 decades. The wide distribution of strains within the MST based on MLVA typing reflects frequent occurrence of genetic events outside the EHEC O157 core genome (, ). The 19 MLVA clusters included 67.3% (136/202) of the analyzed strains. Further analysis of the clusters including >4 MLVA profiles did not show any specific clustering of strains in time. Most of the larger clusters (clusters 1, 2, 4, 5, and 6; ) contained strains widespread in the period of 10–20 years. Only cluster 3 is defined by profiles starting from 2001, which indicates a later appearance than clusters 1, 2, 4, 5, and 6 ().
The 2 most frequently identified MLVA profiles are parts of cluster 1, which indicates a consensus profile among SF EHEC O157:H–
isolates over time within this cluster. The corresponding strains include strain 493/89, which was isolated during the first documented outbreak caused by SF EHEC O157:H–
). All other isolates that exhibited the 2 most common MLVA profiles also fermented sorbitol, which identified strain 493/89 as a prototype of these strains. This finding corroborates the assumption of an epidemic bacterial population structure within a background population comprising a network between different genotypes, and that superimposed strains emerge from highly adaptive, ancestral genotypes and may be persistent for decades (35
). Nodes from cluster 1, which represent the 2 most prevalent MLVA profiles, include strains from 1988–2008 and 1995–2008. This finding indicates a persistence of these successful clones, which supports this hypothesis. Moreover, evolutionary success and uniqueness of this SF clone was recently supported by whole genome single nucleotide polymorphism analysis, in which distinct branching of these clones was determined during evolution of the O157 serotype (22
Statistical analysis demonstrated that the 2 most common profiles and the entire cluster 1 are associated with HUS, which indicates that specific MLVA profiles are associated with severe disease. Cluster 1 comprised 61 of the strains and was distributed over more than a decade. Although not statistically significant, 10 of 11 isolates in cluster 2 were also associated with HUS. Despite these similarities, they exhibited different MLVA profiles (; ). Extensive heterogeneity of EHEC O157:H7, in contrast to conservation of SF EHEC O157:H–
, could be related to observed differences in the nature of the reservoirs and vehicles for transmission. In addition, the epidemiology of SF EHEC O157:H–
infections differs markedly because these infections occur predominantly during cold (winter) months and in children <3 years of age (5
). Moreover, although EHEC O157:H7 infections have zoonotic origins, SF EHEC O157:H–
are rarely found in animals (36
). Humans are plausibly the main reservoirs, as is the case with classical enteropathogenic E
and enteroinvasive E
. This relatively stable niche may lead to the conserved genome structure and high pathogenicity for the host (37
Four strains isolated in 2007 and 2008 exhibited the same MLVA profile as the reference strain EDL933 isolated in 1982 in the United States (38
) (, ). Among the 3,200 entries in the CDC MLVA database, the EDL933 MLVA profile was detected only during an outbreak in 1982 (E. Hyytiä-Trees, pers. comm.). There are 2 possible explanations for this phenomenon. This finding is coincidental because of genetic changes in the O157 genome or EDL933 shares a common MLVA profile with other strains. The presence of such common profiles is known, especially in foodborne pathogens and other monomorphic species (39
) and frequently seen by using other typing techniques, such as PFGE.
Analysis of the number of alleles of different VNTRs produced results similar to those of a previous study (8
). Whereas the ID was high (0.74–0.90; ) in VNTR loci consisting of 6–7-bp repeats (all VNTR loci except VNTR-34), the ID was low (0.66) for the 18-bp repeat (VNTR-34). Whether a VNTR locus is located within an open reading frame did not influence the ID (). However, the frequency of null alleles differed markedly. A total of 98 (48.5%) of 202 strains exhibited null alleles in 4 of the 8 VNTR loci. Especially in VNTR-9 and VNTR-36, the frequency of null alleles was high (31.7% and 44.1%). Although null alleles were reported in other MLVA O157 studies (8
), this high frequency of null alleles determined in our study might indicate a specific feature of EHEC O157 strains from central Europe or Germany. An explanation for the frequent occurrence might be that VNTR-36 and VNTR-9 are located in noncoding or hypothetical protein encoding regions of the EHEC genome (). Nevertheless, all strains had a high ID regarding the complete MLVA profile.
Our study had some limitations. Because of the limited number of isolates obtained during 1987–1995, clustering might be biased and a more year-specific clustering might be observable. However, cluster 1 represents 30.2% (61/202) of strains widespread during 1988–2008, which contradicts this thesis, and infers a certain genetic stability of such clusters over time. In contrast to phylogenetic studies based on whole genome sequencing data (22
), we report a phylogeny based on 8 genetic loci that might be biased by larger recombinational events. However, all VNTR loci are >
50 kb from the rfb-gnd
segment, which was determined to be the only genomic region in EHEC O157 with a higher mutation rate (22
Strains (66/202, 32.7%) that were not classifiable into any MLVA cluster complement the assumption of the highly dynamic EHEC O157 genome. This finding likely indicates that genetic changes in E. coli lead to adaptation to a host-specific environment (in this case human), especially during pathogenesis and host-specific immune responses.
Applying MLVA to this highly diverse strain collection resulted in new insights into the phylogeny of EHEC O157 in Germany since their first description in 1987. In addition to its already demonstrated ability to differentiate outbreak and sporadic case strains, MLVA of O157 emerged as a major typing tool that can further characterize EHEC O157 subpopulations and associated strains. This tool can be used for studying phylogeny coherences and identifying successful clones.