Pathogen subtyping: methods and uses
Recently, the Food Safety and Inspection Service (FSIS) issued a white paper titled: “Current and Future Development and Use of Molecular Subtyping by USDA-FSIS.” The potential for regulatory action based on subtyping of pathogens is guaranteed to keep folks like me awake at night.
Simply put, subtyping analyzes certain portions of an organism’s DNA so as to permit one to discriminate within a common species. There are currently two primary subtyping methods.
Pulsed-field Gel Electrophoresis (PFGE) — breaks down certain large DNA molecules through use of a gel to which electricity is applied. The net result is a pattern similar to a UPC code. The laboratory can compare patterns and determine if two or more isolates have a common pattern. With a common pattern, it is possible the two organisms are related to a common source. Varying the gel will result in different patterns. For some organisms, such as E. coli O157:H7, two different gels are used, whereas for Salmonella, only one gel is used.
The Pulse-Net database (which tracks foodborne outbreaks in selected portions of the country) has numbered each pattern associated with illness — the number is a combination of the gel used followed by a number assigned to that specific UPC-type pattern. Unfortunately, there is no publicly available library of the patterns, so an outside laboratory cannot compare or identify a pattern it generates to the Pulse-Net database, even if the laboratory uses the same gel.
Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA)— This method works by examining specific DNA fragments and comparing the size. Validated MLVA methods are limited to E. coli O157:H7 and Salmonella serotypes Typhimurium and Enteritidis.
As noted above, both of these methods use only a part of the organism’s DNA, so it would be incorrect to state that these methods give the same degree of specificity as fingerprint analysis (though PFGE is often referred to as a “genetic fingerprint”).
It must be noted that FSIS and other agencies are constantly increasing their database of PFGE patterns by conducting PFGE and, when applicable, MLVA analysis on every positive Salmonella, Shiga-toxin producing E. coli (STEC) and Listeria monocytogenes regulatory sample. In addition, monitoring programs, such as the National Microbiological Resistance Monitoring System (NARMS), generate subtyping results. In all these cases, the establishment is known by the agencies.
The FSIS white paper articulates the current uses of subtyping. Since subtyping provides the ability to determine whether isolates obtained from different sources are comparable, the finding of a match provides evidence that the sources may be related.
In the context of illness investigations, if an isolate from a product “matches” an isolate from a case patient, the possibility exists that the product caused the illness. FSIS comments that a match, in and of itself, is insufficient to attribute illnesses to a particular establishment’s product. There must also be an epidemiological link — the product must have been available to the consumer and the consumer likely ate the product. Absent the epidemiological link, there would not be attribution. However, having a subtype match makes the establishment a prime suspect in the investigation, especially if the subtype is rare in the database.
In the context of regulatory sampling, repeated findings of the same subtype could indicate a continuing problem at the establishment, such as a harborage with Listeria monocytogenes or incoming raw materials with Salmonella. FSIS has already given an indication that repeated findings of the same subtype might demonstrate ongoing insanitary conditions whereby products from the establishment would be deemed adulterated.
At present, FSIS does not routinely provide subtyping results to the establishment, but the agency plans to do so shortly. In the interim, an establishment can request the information. However, the data obtained from FSIS would be limited to the data in the agency database. It would not include patterns from other databases, such as NARMS.
Moving forward, FSIS will continue to collaborate with other federal agencies to develop additional subtyping methods, such as a quick screen for STEC virulence genes. Additionally, the agency will continue to work collaboratively on facilitating data sharing.
With advances in scientific methods of identification advance, industry needs to continue to improve its food-safety systems. As I tell my clients, the best way to avoid worrying about subtyping is for your product to test negative for the pathogen.
- 77 Fed. Reg. 72,689, col. 3 (December 6, 2012)