Detection of Thermal Sublethal Injury in Escherichia coli via the Selective Medium Plating Technique: Mechanisms and Improvements.

Laura Espina,Diego García-Gonzalo,Rafael Pagán

doi:10.3389/fmicb.2016.01376

Abstract

In food preservation, the synergistic combination of different technologies aims to maximize the total lethality of the process and minimize the intensity of each hurdle. This is especially the case when at least one of the treatments can cause sublethal (reparable) injury in a great proportion of the population, so that sublethally injured cells can end up being entirely inactivated by the other hurdle(s). The selective medium plating technique (SMPT) is extensively used to enumerate bacterial sublethal injury after inimical treatments, being sodium chloride added to the recovery medium to detect damaged bacterial envelopes. However, little work has been done to explain the reasons for the inability of sublethally injured cells to outgrow in selective agar media, whereas they are able to grow in non-selective agar. In the present paper, the performance of SMPT on Escherichia coli cells after heat treatments is explored by applying different selective agents in the recovery media, using mutants lacking factors involved in osmoregulation, and also by examining the integrity of the cytoplasmic membrane. In view of the results, the possibility of a specific toxic effect of Na+ as the main mechanism under SMPT was discarded, since the same level of sublethal injury was detected using KCl instead of NaCl. The synthesis of the osmoprotectant trehalose determined the maximum osmotolerance of intact cells to the selective agents, but was not crucial in the quantification of sublethal injury. Moreover, for the first time, the extent of sublethal injury detected via SMPT was directly correlated with the physical loss of integrity of the cell membrane in 99.999% of the initial population. This was achieved through statistical analysis of flow cytometry data using propidium iodide-exclusion technique when that dye was added before thermal treatments. The present work confirms the adequacy of SMPT as a tool for detecting the occurrence and quantity of sublethally injured cells after thermal treatments and thus, for efficiently designing the combination of heat with other preservation techniques. We also propose the study of statistical analysis from flow cytometry data for a more rapid quantification of bacterial sublethal injury in a broad detection range.

Highlights

In bacteriology, viability has been traditionally defined and measured as the ability of organisms to self-replicate in culture media (Bogosian and Bourneuf, 2001; Nyström, 2001)
Concentrations over 3% NaCl (MNIC) in the agar inhibited the growth of untreated cells
Cells whose osmoregulatory mechanisms or physical structures become nonfunctional after thermal treatments are unable to outgrow in osmotically challenging agar media, they can outgrow in the absence of the selective agent

Summary

Introduction

Viability has been traditionally defined and measured as the ability of organisms to self-replicate in culture media (Bogosian and Bourneuf, 2001; Nyström, 2001). Microorganisms that are metabolically active despite their inability to grow in laboratory culture media are said to be in a “viable but non-culturable” (VBNC) state, which, under harsh environmental conditions, can be triggered as a survival mechanism (Bogosian and Bourneuf, 2001). On the other hand, according to the “hurdle effect” (Leistner and Gorris, 1995), repair of sublethally injured cells after a preservation treatment can be adequately prevented by the combination of additional preservation agents (hurdles) that interfere with cellular homeostasis maintenance, thereby synergistically increasing the combined process’s global lethality (Mackey, 2000)

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Conclusion