Escherichia coli O157 Cluster Evaluation

We investigated a multistate cluster of Escherichia coli O157:H7 isolates; pulsed-field gel electrophoresis subtyping, using a single enzyme, suggested an epidemiologic association. An investigation and additional subtyping, however, did not support the association. Confirmating E. coli O157 clusters with two or more restriction endonucleases is necessary before public health resources are allocated to follow-up investigations.

We investigated a multistate cluster of Escherichia coli O157:H7 isolates; pulsed-field gel electrophoresis subtyping, using a single enzyme, suggested an epidemiologic association. An investigation and additional subtyping, however, did not support the association. Confirming E. coli O157 clusters with two or more restriction endonucleases is necessary before public health resources are allocated to follow-up investigations. E scherichia coli O157:H7 is an important cause of foodborne infections estimated to cause 73,000 illnesses and 60 deaths annually in the United States (1). Implementation of pulsed-field gel electrophoresis (PFGE) molecular subtyping has greatly improved E. coli O157:H7 surveillance and detection of outbreaks (2). PFGE subtyping was initially used to identify related isolates and support epidemiologic associations during outbreak investigations. Public health laboratories in the United States now routinely subtype all E. coli O157:H7 isolates by PFGE as part of a national molecular subtyping network (PulseNet) (2) after this practice proved instrumental in identifying outbreaks not detected by traditional epidemiologic methods (3). PulseNet laboratories initially digest isolates with a single enzyme and compare the resulting PFGE patterns by using commercial software (BioNumerics, St. Martens-Latem, Belgium) to determine whether patterns are shared by multiple isolates. These patterns are then communicated electronically to the Centers for Disease Control and Prevention (CDC) (Atlanta, GA), where PFGE patterns of isolates from different states are definitively compared. PulseNet policy states that isolates with potential epidemiologic signifi-cance that have indistinguishable patterns with a primary enzyme, should be digested with a secondary enzyme before extensive epidemiologic investigations are undertaken. Indistinguishable patterns should also be confirmed by submission to a central database (2). However, time constraints and the availability of sufficient resources prevent some laboratories from adhering to this policy.

The Study
On July 5, 2000, the Michigan Department of Community Health's laboratory notified CDC of a cluster of five E. coli O157:H7 isolates, collected from May 25 to June 21, 2000, which shared an indistinguishable XbaI PFGE pattern. PulseNet staff confirmed that these isolates' patterns were indistinguishable and designated the pattern as PulseNet pattern EXHX01.0047. In 2000, this PFGE pattern represented approximately 2% of the E. coli O157 patterns in the PulseNet database. These Michigan isolates possessed genes only for Shiga toxin 2 (stx2) but not Shiga toxin 1 (stx1); approximately 30% of E. coli O157 isolates sent to CDC since 1983 expressed only stx2. From July through September 2000, six states (California, Michigan, New Jersey, New York, Ohio, and Texas) reported a total of 64 E. coli O157 isolates with PulseNet pattern EXHX01.0047, a value that exceeded expectation for this time of year and that prompted an epidemiologic investigation. Not all of these patterns were submitted to CDC's central database for confirmation. Fifty-one of these isolates were probed for Shiga toxin genes, and all possessed only stx2. Illness onsets ranged from April 1 through August 21, 2000, with a notable increase after late May 2000. The median age of case-patients was 13 years (range 1-91), and 38 (60%) were female; 36 (57%) of 64 were hospitalized, and hemolytic uremic syndrome developed in 9 (14%).
To determine the source of these E. coli O157:H7 infections, six state health departments (California, Michigan, New Jersey, New York, Ohio, and Texas) and CDC initiated an epidemiologic investigation. Informed consent was obtained from all patients or their parents or guardians and human experimentation guidelines of the U.S. Department of Health and Human Services were followed. These data were collected as part of an outbreak investigation and therefore were exempt from formal institutional review board aprovals.
Through hypothesis-generating interviews with 19 infected persons, 11 food exposures were reported by >50% of interviewees or were reported in substantial excess relative to that food's frequency of consumption in the general population (4). In a case-control study that used a survey instrument that focused on these 11 food exposures, controls were matched to case-patients by sex and age group, and were asked about exposures during the 3.65, p = 0.04); 14 (58%) of 24 case-patients, and 19 (31%) of 62 controls reported eating broccoli. Although none of the three foods that contained ground beef were individually associated with illness, consumption of "any hamburger" (a composite variable) was significant (mOR 7.30, p = 0.01), reported by 20 (87%) of 23 patients and 28 (55%) of 51 controls. In multivariate analysis, only eating "any hamburger" remained significantly associated with illness (OR = 6.13, p = 0.02).
Ground beef eaten by case-patients was recovered from three households; samples from two households (in New Jersey and California) yielded E. coli O157:H7 isolates that were indistinguishable from PulseNet pattern EXHX01.0047. One of these isolates was tested for Shiga toxin and produced only stx2. Using information from these two cases and additional information regarding likely ground beef sources for the original Michigan cases, the U.S. Department of Agriculture performed a traceback; however, an extensive investigation did not identify any common supplier for the two samples of ground beef.
We performed a retrospective review of available isolate patterns received by the PulseNet national database after the case-control study and traceback had been completed. Four additional states (Florida, Indiana, Massachusetts, and Washington) had reported E. coli O157 isolates with XbaI patterns that were indistinguishable from PulseNet pattern EXHX01.0047. Among the 46 submitted XbaI patterns from states reporting a possible match to PulseNet pattern EXHX01.0047, analysis at CDC indicated that 38 were indistinguishable and that 6 differed by one band from the PulseNet pattern EXHX01.0047 (Figure 1). Furthermore, among the 38 isolates confirmed as PulseNet pattern EXHX01.0047, digestion of 13 isolates with the restriction enzyme BlnI produced PFGE patterns that sorted into multiple distinct clusters (Figure 2).

Conclusions
Identifying outbreaks of E. coli O157:H7 infections by routinely subtyping isolates using PFGE is a relatively new phenomenon (2,3). Traditionally, PFGE has been used to support or refute the likelihood of epidemiologic related-ness among case-patients and suspect food vehicles in epidemiologic investigations. In this instance, the converse occurred; the results of routine PFGE subtyping (XbaI) of E. coli O157:H7 isolates prompted a large, multistate epidemiologic investigation. Isolates were potentially related because 1) the PFGE patterns obtained with one restriction enzyme (XbaI) were reported to be indistinguishable and a relatively uncommon pattern, and 2) the isolates shared a Shiga toxin profile that was relatively uncommon among E. coli O157 (stx2 only). A rigorous case-control study implicated a widely consumed food vehicle responsible for multiple past outbreaks of E. coli O157 infections: ground beef (5). This study and the isolation of two E. coli O157 with matching PFGE patterns from ground beef consumed by case-patients prompted two extensive traceback investigations. However, no common source could be identified. Subsequent digestion of patient isolates with a second enzyme showed that they were actually part of multiple, small clusters and that the illnesses were thus unlikely to be related to a common source.
Investigation of suspected multistate outbreaks requires substantial public health resources (6). This investigation involved more than 50 federal, state, and local staff. E. coli O157:H7 infections can cause serious and potentially lifethreatening illness that may also engender legal action. Public health authorities must ensure that linkage of illnesses to an outbreak be as complete and accurate as possible. Rapid identification of the infections' source can avert many potential illnesses. Earlier studies demonstrated the value of subtyping E. coli O157:H7 isolates with two or more restriction endonuclease digestions or using other subtyping methods, such as phage typing, to determine whether such isolates are truly related, even if these isolates have produced matching patterns using a single enzyme digestion (7)(8)(9). More recently, in the absence of epidemiologic data, single enzyme PFGE has been found to be a poor measure of genetic relatedness (10). Since 1998, the PulseNet Task Force has recommended the use of at least two enzyme digestions for optimal subtyping of E. coli O157 isolates. However, because of resource limitations, many state and local public health laboratories initially subtype E. coli O157 isolates with XbaI enzyme and perform subtyping with a second enzyme only if clusters are identified and personnel and resources are in place to do so. This investigation lends further support to the conclusion that when clusters of E. coli O157 are detected on the basis of subtyping data only (i.e., in the absence of any epidemiologic data), digestion with two or more endonucleases is warranted, even if the isolates appear to share a primary enzyme pattern or possess other microbiologic evidence of clonality (e.g., Shiga toxin profile). Furthermore, these findings underscore the importance of having a centralized database team that can rapidly verify reports of clusters from participating PulseNet laboratories and assist in determining whether isolates are likely to be part of an outbreak and whether a rapid, large-scale epidemiologic investigation and traceback would be warranted.