The use of plasmid profiles and nucleic acid probes in epidemiologic investigations of foodborne, diarrheal diseases.

The application of nucleic acid analyses to investigations of infectious disease outbreaks has resulted in useful molecular strain markers that distinguish the epidemic clone of a particular pathogen and help identify specific vehicles of infection. We have successfully used plasmid profile analysis, restriction endonuclease digestion of plasmid and whole-cell DNAs, and nucleic acid hybridization to investigate recent outbreaks of foodborne diarrheal illness. Plasmid analysis has been important in identifying epidemic strains of Salmonella enteritidis and Escherichia coli O157:H7. In a culture survey of S. enteritidis isolates from humans and a variety of animals, including chickens and chicken eggs, we identified 16 distinct plasmid profiles and used these to differentiate strains, especially within commonly occurring phage types (Colindale 8 and 13a). HindIII digests of plasmid DNA were useful in distinguishing plasmids of similar mass but dissimilar enzyme target sequences; they clearly distinguished S. enteritidis strains causing systemic infections in children in parts of Africa from U.S. isolates. Investigations of outbreaks of hemorrhagic colitis have also been assisted by plasmid analysis. Restriction endonuclease digests of whole-cell DNA and Southern blot analysis, hybridizing with E. coli 16S and 23S rRNA (ribotyping), have been effective subtyping techniques, especially for plasmidless isolates of Campylobacter jejuni. In five outbreaks of C. jejuni infections, ribotyping of PvuII and ClaI digests distinguished individual epidemic strains within one commonly occurring C. jejuni serotype (Penner 2, Lior 4). Preliminary data show that ribotyping of NcoI digests can also distinguish individual epidemic strains of E. coli O157:H7 and may provide a more stable marker than plasmid profiles. Specific DNA probes derived from cloned virulence genes of E. coli have been invaluable in epidemic investigations and surveys. Using colony hybridization, we found in one survey of stool specimens from 174 dairy cattle that 11% of animals were asymptomatically carrying Shiga-like toxigenic E. coli other than O157:H7. We also found that newly synthesized oligonucleotide probes for the Shiga-like toxins I and II agreed 100% with cloned gene probes in a study of 613 E. coli strains. Future studies of these organisms will include the use of additional synthetic oligonucleotides as primers to amplify the toxin genes directly in patient and animal specimens by the polymerase chain reaction. There is a continuing and expanding role for molecular approaches in epidemiological investigations. The DNA methods described above are not based on the often complex expression of phenotypic characteristics, and, unlike sensitive and specific techniques such as phage typing, a single method can be used to study a variety of Gram-positive and negative bacterial pathogens.