Individual cell lag time distributions of Cronobacter (Enterobacter sakazakii) and impact of pooling samples on its detection in powdered infant formula.

Cells of six strains of Cronobacter were subjected to dry stress and stored for 2.5 months at ambient temperature. The individual cell lag time distributions of recovered cells were characterized at 25 °C and 37 °C in non-selective broth. The individual cell lag times were deduced from the times taken by cultures from individual cells to reach an optical density threshold. In parallel, growth curves for each strain at high contamination levels were determined in the same growth conditions. In general, the extreme value type II distribution with a shape parameter fixed to 5 (EVIIb) was the most effective at describing the 12 observed distributions of individual cell lag times. Recently, a model for characterizing individual cell lag time distribution from population growth parameters was developed for other food-borne pathogenic bacteria such as Listeria monocytogenes. We confirmed this model's applicability to Cronobacter by comparing the mean and the standard deviation of individual cell lag times to populational lag times observed with high initial concentration experiments. We also validated the model in realistic conditions by studying growth in powdered infant formula decimally diluted in Buffered Peptone Water, which represents the first enrichment step of the standard detection method for Cronobacter. Individual lag times and the pooling of samples significantly affect detection performances.

Impact of pooling powdered infant formula samples on bacterial evolution and Cronobacter detection.

Enterobacter sakazakii has been identified as the causative agent of serious neonatal infections, associated with high mortality rates. In most cases, powdered infant formula (PIF) has been identified as the source of infection. Recently, strains commonly referred to as E. sakazakii were proposed for classification in a new genus, Cronobacter. The standardised method for detection of Cronobacter in PIF (ISO/TS 22964; IDF/RM 210) involves pre-enrichment in buffered peptone water (BPW), followed by selective enrichment and plating onto ESIA chromogenic agar. For greater convenience and to reduce analysis cost, the common practice in the food industry is to pool samples at a constant dilution rate, in order to perform a single pre-enrichment and subsequent analysis. The consequences on the sensitivity of Cronobacter detection are not evident. We evaluated the impact of pooling on the growth of Cronobacter and PIF background microflora in samples undergoing pre-enrichment culturing in BPW. Growth of the pathogen was monitored by direct plating onto selective agar or by using a recently developed sensitive enumeration method, based on membrane filtration followed by transfer of the filter onto the selective agar. The evolution of the total bacterial population of the PIF was monitored from a qualitative and quantitative point, using molecular or classical microbiological methods. Results showed that pooling had a negative impact on the maximum population of Cronobacter attained, whereas no clear effect was observed on the onset of growth. This observation suggests strong bacterial interactions with the PIF background microflora, confirmed by a generally higher background microflora growth potential in PIF samples from various origins. These important findings suggest that, in some cases, the practice of pooling samples may affect the performance of the detection method.