Food industry members are often ideally placed on the front lines of manufacturing and processing to ensure best practices for food safety. This responsibility is heightened when dealing with products like powdered infant formula (PIF), where food-borne disease (FBD) pathogen contamination with microbes such as Cronobacter and Salmonella species can be extremely harmful for these vulnerable populations. Many producers use microbial detection workflows such as the Thermo Scientific™ SureTect™ PCR range of assays to provide quick and efficient screening. These screenings increase consumer confidence and enhance industry reputation, and research innovation in assay development also helps emphasize good laboratory practice (GLP) to reduce hazardous waste output.
Cronobacter species, including the former Enterobacter sakazakii (now C. sakazakii), are found naturally in the environment. Cronobacter can thrive in dried foods such as PIF, herbal teas, powdered milk and starches. People carrying this microbe often do not show symptoms, but in vulnerable populations such as infants (particularly neonates), immunocompromised patients and the elderly, infection causes meningitis, necrotizing enterocolitis, bacteremias and septicemia. In these groups, infection can be fatal or leave lasting damage.
Premature babies and neonates are particularly vulnerable to Cronobacter. Since these babies are often looked after in baby-care units and tube-fed formula, it is vital that PIF manufacturers test for FBD pathogens.
Testing protocols for FBD pathogens often involve molecular workflows such as PCR (polymerase chain reaction) assays that identify microbial DNA to detect contamination. Following a positive DNA result, safety testing requires positive identification by an established culture-based method to determine pathogen viability. It is therefore important that sample preparation in whatever microbial workflow manufacturers use in food safety testing does not compromise viability and the results can be read with confidence.
When testing for Cronobacter species in PIF, it is important to make sure that results reflect the presence of viable organisms, even at low levels, that could cause disease. Since PCR assays report microbial DNA regardless of viability, the culture-based test is important in establishing product safety.
Reference methods that test for Cronobacter include an enrichment step to boost microbial numbers to detection levels prior to testing. An added complication for PIF safety protocols is that manufacturers often include probiotic organisms in their formulations to promote gut health and reduce FBD pathogens. During sample preparation and culture in enrichment medium, these probiotic organisms also multiply. This can lower the pH of the enrichment during incubation, which may kill off the target organisms. PCR still detects DNA from these non-viable organisms, but standard culture to confirm the positive result fails to identify pathogenic colonies.
In order to overcome this masking effect during the enrichment step, the standard reference method for detecting Cronobacter species in PIF, ISO 22964:2017, requires the addition of vancomycin to inhibit the growth of probiotic strains and maintain a suitable pH for Cronobacter species viability. However, for GLP it is advisable to neutralize antibiotic-containing culture media prior to disposal or to place it only in the hazardous waste stream.
However, a recent study by Thermo Fisher Scientific scientists has shown that vancomycin is not required during enrichment if the PIF product does not contain probiotics. The scientists compared the Thermo Scientific™ SureTect™ Cronobacter PCR Assay workflow for the detection of Cronobacter species with the standard reference method, omitting vancomycin from the enrichment medium prior to PCR analysis.
Taking 300 g samples (27 PIF; 3 powdered milk) spiked with low levels of Cronobacter species injured by desiccation, the team found good agreement between the two methods. Since the results show that the performance of the SureTect Cronobacter PCR Assay method was comparable to the reference method, food safety labs do not need to add vancomycin to the enrichment medium when testing for Cronobacter in PIF and milk powders that do not contain probiotics.
Read more about testing infant formula in this Application Note: Detection of Cronobacter from Non-Probiotic Powdered Infant Formula and Milk Powder.
Cahill, S.M., et al. (2008). “Powdered infant formula as a source of Salmonella infection in infants,” Clinical Infectious Diseases 15, 46, pp. 268-273, doi: 10.1086/524737, https://www.ncbi.nlm.nih.gov/pubmed/18171262
CDC, Cronobacter Infection in Infants, https://www.cdc.gov/features/cronobacter/index.html
ISO 22964:2017 Horizontal method for the detection of Cronobacter spp. https://www.iso.org/standard/64708.html
Meyer, E.L., et al. (2017). “Is Your Institution Disposing of Culture Media Containing Antibiotics?” Applied Biosafety: Journal of ABSA International, 22, pp. 164-167, https://journals.sagepub.com/doi/pdf/10.1177/1535676017735521
Post Author: Alyssa Gonzalez.