Pathogen Survival in a Desiccated Environment

Koyama et al. (2017) describe a statistically based method for establishing risk arising from microbial survival in desiccated food products.¹ Using an enrichment broth to revive microbes, they explore variable behavioral responses and viability characteristics in small bacterial populations, comprising 10 to 100 organisms, subjected to desiccation. Their results provide food safety officials with tools for defining contamination risks in food products, such as chocolate, with low water content.

Food processing, from raw ingredient to finished product, incorporates many different steps and protocols that ensure consumer safety. In addition to processing best practices and hygiene management, these include methods designed to inactivate or remove potential foodborne disease (FBD) pathogens. Methods such as heat treatment, irradiation, the use of additives and desiccation kill off microbial contaminants, or at least inactivate them, thus rendering them harmless. However, it is possible for cells damaged or stressed during these processes to survive as viable or infective in the final product.

Desiccation, in which producers reduce the water content (a_w) of the food to below 0.85, inhibits bacterial activity and does not support pathogen growth. However, FBD pathogen cells can survive this process, remaining viable and infective even in a damaged state. Salmonella enterica is responsible for FBD outbreaks arising from ingestion of low-a_wfoodstuffs such as chocolate, salami, and potato chips. In these cases, either a sufficient population of viable cells survived to multiply when food conditions altered to allow growth or numbers were sufficient to infect on ingestion.

To explore microbial FBD pathogen survival, the researchers prepared stocks of commercially available FBD pathogen strains, focusing on S. enterica (n = 4 serotypes), enterohemorrhagic Escherichia coli (n = 3 serotypes), and one strain of Listeria monocytogenes as representative of common microbial contaminants encountered in prepared foods. By diluting the stocks, the team created single-cell solutions of each bacterial species. They inoculated 96-well plates with single cells in 2 µl volumes, using pure water to buffer the microbes from desiccation before placing the inoculums in a drying chamber with silica gel.

The team maintained the cell preparations in 10% to 20% relative humidity at 5°C, 15°C and 25°C. At various time points (every one to two hours up to 10 hours, and then at 24 hours and 48 hours post-desiccation), they added 100 µl of enrichment medium to revive the cells. They cultured the cells for a further week at 25°C before assessing survival. From these results and from computer modeling, Koyama et al. created probability models to describe the variability in the numbers of microbes surviving desiccation.

Food safety experts find that predicting behavior and especially survival characteristics in the low population numbers of microbial cells that survive processing treatments is difficult since the responses are random. It is important to maximize bacterial recovery from these foodstuffs by using enrichment media such as Buffered Peptone Water (BPW) (Thermo Fisher Scientific; CM0509, CM1049). Food testing laboratories may, therefore, need to seek out specialist protocols for best practices in testing certain food matrices in order to achieve valid results. Enrichment broths such as BPW maximize microbial recovery by supporting damaged cells. Furthermore, they are suitable for non-selective culture where low numbers of stressed organisms are suspected in food matrices. For more challenging isolations, specialized enrichment media are available for certain foodstuffs commonly at risk of FBD pathogen contamination. For example, Cocoa Samples Recovery (CSR) Broth (Thermo Fisher Scientific; CM1155) maximizes recovery of stressed or damaged organisms for rapid identification of Salmonella in cocoa powder.

The microbiological culture model Koyama and co-authors developed in tandem with statistical analysis and modeling according to Poisson distribution assessment should help to define microbial survival characteristics for more accurate estimation. Used in conjunction with suitable enrichment media, these analytical tools could help determine contamination risks from foodstuffs subjected to desiccation or for those with reduced water content.

Learn more about enrichment and identification of food borne pathogens in our food microbiology community.

Reference
1. Koyama, K., et al. (2017) “Modeling stochastic variability in the numbers of surviving Salmonella enterica, enterohemorrhagic Escherichia coli, and Listeria monocytogenes cells at the single-cell level in a desiccated environment,” Applied and Environmental Microbiology, 83(7) (e02974-16), https://doi.org/10.1128/AEM.02974-16.