Forms of Bacterial Survival in Model Biofilms

Abstract

Bacterial survival upon sharp fluctuations of environmental parameters and exposure to lethal doses of stressors (antibiotics, disinfectants, heat shock, and others) is ensured by the use of different strategies of resistance, an important place among which is occupied by the forms with reduced or stopped metabolism, antibiotic tolerant (AT) persister (P) cells, anabiotic dormant forms (DFs), and viable but non-culturable (VBNC) cells. Elucidating the role of these forms of bacterial resistance to an impact of chemical and biological toxicants and physical stressors is of great fundamental and practical interest. The aim of this research was to study the dynamics of the resistance forms in bacteria developing in biofilms and, for comparison in liquid media, upon exposure to lethal doses of antibiotics and heat shock (80 °C, 15 min). In the trials, the experimental model of the development of monospecies and binary forms of bacterial biofilms including contaminants of meat products (eight strains of genera Pseudomonas, Escherichia, Salmonella, Staphylococcus, and Kocuria) on the fiberglass filters was used. It was established that survival of populations in the presence of lethal doses of antibiotics and upon heating was ensured by persister cells forming in bacterial populations and, at the late stages of the development of biofilm or planktonic cultures (28 days of incubation), by anabiotic DFs. With that, the number of thermoresistant (TR) DFs (103–104 CFU/mL) in dying biofilms (28 days) developing in the standard conditions (composition and volume of a medium, pH, growth temperature) weakly depended on the bacterial taxonomic status. This study demonstrates the heterogeneity of DF populations of biofilm bacterial cultures in terms of the depth of dormancy, as a result of which the number of thermoresistant DFs after heating can exceed their total number before heating (due to the effect of DF revival, resuscitation). When studying the dynamics of TR cells and P cells in bacterial biofilm and planktonic cultures, it was found that their number (CFU/mL) in populations decreased up to the absence of TR cells and P cells on the 21st day of growth and was restored on the 28th day of growth. The revealed phenomenon can be explained only by cardinal changes in the ultrastructural organization of cells, namely, cytoplasm vitrification due to a sharp decrease in an amount of free water in a cell, which, according to the results obtained, occurs in the period between the 21st and 28th days of incubation. A high degree of correlation between the number of AT cells and TR cells (0.5–0.92) confirms the hypothesis that regards P cells as precursors of DFs.