Polysaccharide-based liquid storage and transport media for non-refrigerated preservation of bacterial pathogens

Abstract

The preservation of biological samples for an extended time period of days to weeks after initial collection is important for the identification, screening, and characterization of bacterial pathogens. Traditionally, preservation relies on cold-chain infrastructure; however, in many situations this is impractical or not possible. Thus, our goal was to develop alternative bacterial sample preservation and transport media that are effective without refrigeration or external instrumentation. The viability, nucleic acid stability, and protein stability of Bacillus anthracis Sterne 34F2, Francisella novicida U112, Staphylococcus aureus ATCC 43300, and Yersinia pestis KIM D27 (pgm-) was assessed for up to 28 days. Xanthan gum (XG) prepared in PBS with L-cysteine maintained more viable F. novicida U112 cells at elevated temperature (40°C) compared to commercial reagents and buffers. Viability was maintained for all four bacteria in XG with 0.9 mM L-cysteine across a temperature range of 22-40°C. Interestingly, increasing the concentration to 9 mM L-cysteine resulted in the rapid death of S. aureus. This could be advantageous when collecting samples in the built environment where there is the potential for Staphylococcus collection and stabilization rather than other organisms of interest. F. novicida and S. aureus DNA were stable for up to 45 days upon storage at 22°C or 40°C, and direct analysis by real-time qPCR, without DNA extraction, was possible in the XG formulations. XG was not compatible with proteomic analysis via LC-MS/MS due to the high amount of residual Xanthomonas campestris proteins present in XG. Our results demonstrate that polysaccharide-based formulations, specifically XG with L-cysteine, maintain bacterial viability and nucleic acid integrity for an array of both Gram-negative and Gram-positive bacteria across ambient and elevated temperatures.

Fig 1. Structure of xanthan gum (XG) with sub-structural components denoted.

For XG, a majority of R1 groups on the inner mannose residue are 6-acetylated and an estimated 40% of the terminal mannose residues are 4,6-pyruvated (R2, R3).

Fig 2. Screening sulfur sources at varying concentration to maintain F. novicida viability.

XG and reference buffers were supplemented with two sulfur sources at varying concentrations. Viability of F. novicida in test matrices were spiked with ~10⁴ CFU/mL and stored at 22 and 40°C for 28 days. Data are the average of one biological replicate and two sample replicates plated in triplicate (n = 6 measurements).

Fig 3. Stabilization of B. anthracis, F. novicida, S. aureus, and Y. pestis over time.

Test matrices were spiked with ~10⁵ CFU/mL of Gram-negative F. novicida U112 or Y. pestis KIM D27 (pgm-) (left) or Gram-positive B. anthracis Sterne 34F2 or S. aureus ATCC 43300 (right). All were incubated at 22°C for 14 days. Viable plate counts were conducted at various time points to determine cell stability. Data are the average of three biological replicates and three sample replicates plated in triplicate (n = 9 measurements).

Fig 4. Role of temperature on S. aureus and F. novicida viability over time.

Test matrices were spiked with ~10⁵ CFU/mL of S. aureus or F. novicida and incubated at 22–40°C for 28 days. Viable plate counts were conducted at various time points to determine cell stability. Data are the average of three biological replicates and three sample replicates plated in triplicate (n = 9 measurements).