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UCD Centre for Food Safety

Ionad Sábháilteachta Bia UCD

Isolation and characterisation of Enterobacteriaceae from the infant formula food chain

Enterobacteriaceae are a family of Gram-negative, motile, non-spore forming, peritrichous, facultative anaerobic bacteria.  Some species within this family are opportunistic pathogens. This includes the genus Cronobacter, which is particularly associated with illness following ingestion of powdered infant formula (PIF).  Cronobacter can be aetiological agents of meningitis, bacteremia and necrotizing enterocolitis in neonates.  These bacteria can tolerate extreme osmotic stress and acidic conditions.  Also undefined virulence factors in these bacteria are responsible for these neonatal infections.  It is possible that other members of the Enterobacteriaceae that also contaminate infant formula could share risk factors similar to Cronobacter and cause cases of neonatal illness.  Knowledge of the prevalence and risk factors associated with Enterobacteriaceae strains present in infant formula would contribute to improved detection and control paradigms for the safety of infants.

In the first part of the study sampling (and testing) protocols will be designed and implemented to assess the efficiency of current control measures in place designed to limit the presence of Enterobacteriaceae in the environment and in final powdered product.

A surveillance network will be established across the supply chain manufacturing process, including all raw materials, by application of molecular fingerprinting. These tools will facilitate the coordinated investigation and identification
of dissemination routes of Enterobacteriaceaestrains.

Following DNA fingerprinting, it is predicted that populations of clonal or dominant strains will be identified from the above sampling events.  Phenotypic analysis of these strains to elucidate why in particular, these bacteria persist in manufacturing facilities will be conducted.  The UCD Centre for Food Safety recently commissioned a Phenotype MicroArray platform which simultaneously and in ‘real-time’, assays the expression of 2,000 strain characteristics.  The Phenotype MicroArray provides a comprehensive phenotypic profile that can provide valuable insights into strain characteristics and behavior under selected conditions. Evaluating Enterobacteriaceae recovered from the sampling points, in this way, will identify important properties of these organisms.  These features can then be targeted for analysis and control options.
An evaluation of the ability of a range of disinfectants and sanitizers to inhibit Enterobacteriaceae proliferation in planktonic and biofilm cultures will be undertaken in simulated factory environments.

Enterobacteriaceae associated with infant formula will also be evaluated for their ability to form infections. Attachment, invasion and translocation assays can initially be performed using in vitro assays on mammalian cell lines. 
These can be expanded to in vivo investigations given that ethical justification is established.  Mammalian tissue culture techniques are already established within the UCD Centre for Food Safety and Veterinary Pathology groups.

Gene micro arrays designed to detect virulence markers in pathogenic species such as Salmonella, E. coli and Cronobacter have been constructed under an existing MOU with United States Department of Agriculture (USDA).  Molecular micro array studies can be carried out in at the Agriculture Research Service laboratory at Albany, California.  Relevant Enterobacteriaceae from this study can be investigated using these tools to determine if genes of interest are present.

Investigation of the contribution of specific genes to environmental persistence or the risk of infection will be conducted using knockout mutations.  Techniques to generate mutant strains are in use within the UCD Centre for Food Safety.  The mutant strains will be assayed in comparison to isogenic parent strains using techniques described above.

Research methods will include cell & microbiological culture, biochemical and molecular characterization, bioinformatics, mutagenesis and microarray technology.

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PCR gel