Cross-contamination in a laboratory was at fault for the finding of a finished product positive for Salmonella, according to a study.

In 2013, during a routine analysis of food samples, one finished chocolate product from a European factory tested positive for Salmonella Hadar. At the same time, environmental monitoring in the lab indicated a Salmonella-positive sample from the thermocouple in one incubator.

Eight months earlier, a proficiency test was completed by the unnamed lab with Salmonella Hadar spiked on a sample. This same lab analyzed the food sample for the European factory. The lab carried out regular testing for Salmonella during 2013 but had only one positive during the year.

Due to suspicion of lab cross-contamination between the Salmonella Hadar isolate used in the proficiency testing and the isolate found on the finished product by the same lab, whole genome sequencing (WGS) was used.

The analysis showed a maximum of ten single nucleotide polymorphisms (SNPs) between the isolates coming from the lab and the finished product, confirming the cross-contamination.

Results combined with the additional investigation at the factory allowed the release of finished product batches and prevented unnecessary food waste and economic losses for the factory.

As part of food safety management systems in production facilities, factories have environmental monitoring and product testing programs to detect issues in the production area and their surroundings. Samples taken can be sent to labs for analyses to control microbiological quality and safety.

During routine microbial testing in 2013, a lab found a Salmonella Hadar positive in a sample of chocolate produced at a European factory.

The plant retained the finished product batch, started cleaning and disinfection, and increased the number of analytical tests. A root cause investigation was initiated by retrieving all production and analytical data to identify the possible source of contamination.

None of the information collected indicated abnormalities at the production site. No Salmonella was detected in raw materials or environmental samples taken at the factory.

Due to the positive environmental sample at the lab and the proficiency test, the hypothesis was probable cross-contamination in the lab had resulted in a false positive result of the food product. Participation in proficiency testing demonstrates competence while performing specific microbiological examinations.

Confirmation of the lab cross-contamination was needed using a more discriminatory method than the classical phenotypic Salmonella serotyping by slide agglutination based on the Kauffmann-White-Le Minor (KW) scheme.

Salmonella serotyping analysis highlighted a possible lab cross-contamination incident that was confirmed by WGS in the research published in the International Journal of Food Microbiology.

Four Salmonella Hadar isolates from the lab cross-contamination and 12 public sequenced isolates sampled in Europe, were added to the analysis.

None of 12 public isolates collected until 2014 were genetically similar to isolates from the study. SNP analysis showed that four isolates were genetically very similar with a maximum of 11 SNPs between them suggesting they shared a common ancestor.

Researchers said it was likely contamination of the environment in the lab occurred while doing the proficiency test.

“The reason why the isolate stayed dormant for several months and was only detected in December remains unclear. Normally, the thermocouple is not placed in growth media. It is possible that this event allowed the Salmonella to enrich to high levels increase the risk of contamination to the other test samples. After the incident, GLP was revised and environmental monitoring was increased in the laboratory.”

A cross-contamination precedent

It is not the first time cross-contamination in a lab has involved chocolate and Salmonella.

In 2012, chocolate shipped from Belgium to the United States was prevented from entering the country because a Salmonella Rissen strain was isolated from one of the chocolate bars in a Belgian food lab.

However, a retrospective study of the isolates sent from the lab to the Belgian National Reference Laboratory for Salmonella revealed that seven weeks prior, a Salmonella Rissen strain was isolated from the fish meal in the same lab. It was the first time that such a strain had been isolated in the lab since September 2009.

Cross-contamination in the food lab led to a false-positive result, leading to severe economic consequences for the chocolate bar manufacturer as the container with the batch of presumptively Salmonella positive chocolate bars were destroyed.

The cause of cross-contamination was not discovered but it is possible that some fish meal dust flew into the air, where it continued to circulate in the lab environment for weeks or the strain isolated from the fish meal was stored in a slant tube which could have been improperly handled by an employee.

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