The work was commissioned by the chocolate company, which was not named, and results were confirmed by an independent third party.
In 2012, chocolate bars shipped from Belgium were prevented from entering the US because a Salmonella Rissen strain was isolated from one of them in a Belgian food laboratory using the ISO 6579 standard.
The results were communicated to the Belgian Federal Agency for the Safety of the Food Chain (AFSCA) and the US Food and Drug Administration (FDA).
Economic consequences for manufacturer
The FDA then blocked them at arrival, where 90 samples from the four containers of chocolate bars were taken for Salmonella detection and the container with the batch of presumptively positive chocolate bars was destroyed.
However, a retrospective study of the isolates from the laboratory to the Belgian National Reference Laboratory for Salmonella found that seven weeks prior, a Salmonella Rissen strain was isolated from fish meal in the same laboratory.
It was the first time a Salmonella Rissen strain had been isolated in that laboratory since September 2009.
This led to the hypothesis that the batch of chocolate bars was Salmonella-free and that cross-contamination in the laboratory had led to a false-positive result, leading to severe economic consequences for the chocolate bar manufacturer.
Chocolate bars were not expected to be contaminated because the ingredients tested negative during the production process.
As Salmonella Rissen is rarely isolated from food, it was hypothesized that the two isolates belonged to the same strain and the second isolation was caused by cross-contamination.
Only sequencing techniques can yield absolute confidence that isolates are 100% identical and next-generation sequenced based techniques for sequencing whole genomes were not routinely in use at the time of the cross-contamination.
Repetitive element palindromic PCR (rep-PCR) and random amplified polymorphic DNA PCR (RAPD) assays were not discriminatory enough but pulsed-field gel electrophoresis (PFGE) using the combination of two restriction enzymes showed sufficient discrimination to confirm the hypothesis, said the study.
They were sent to the FDA and the remaining containers were released.
The researchers said the cause of cross-contamination will probably never be entirely known, but some explanations seem plausible.
“First, fish meal is a very powdery substance. When a subsample was weighed to start the analysis, it is possible that some fish meal dust flew into the air in the laboratory, where it continued to circulate in the lab environment for several weeks.
“Second, the Salmonella Rissen strain isolated from fish meal was stored in a slant tube. It is therefore also possible that an employee handled the slant improperly, thus leading to cross-contamination of the laboratory equipment, working tables, doors, etc.”
How did cross contamination happen?
Even when precautions are taken, cross-contamination can still occur, said the study.
“Personnel working in routine laboratories should always be aware of the possibility of cross-contamination, especially when enrichment is used in the microbiological analysis.
“Therefore, results should be interpreted carefully, especially when the same rare serotypes are isolated from non-related food items in a short period of time.
“In case of doubt, additional strain typing should be used to determine if cross-contamination is a probable cause of the second isolation event.”
For Salmonella isolation according to ISO 6579, the pre-enrichment step uses Buffered Peptone Water to multiply a very small number of cells up to levels of 105–107colony forming units per ml after enrichment (depending on food type and competitive flora). The high bacterial load after enrichment theoretically results in a high chance for cross-contamination.
Reports of cross contamination events in food labs are scarce with the likely explanation being it is less likely to be detected.
“Because the target pathogen can be present in very low levels in the food, many microbiological food analyses include enrichment steps to multiply the target pathogen to detectable levels. Consequently, even a very low level of contamination in the lab environment can cause false-positive results when testing foodstuffs for microbiological food safety.”
Source: BMC Research Notes 2016 9:156
Article online, DOI: 10.1186/s13104-016-1969-7
“Case report of Salmonella cross-contamination in a food laboratory”
Authors: Geertrui Rasschaert, K. De Reu, M. Heyndrickx and L. Herman