Study of Transitional Dynamics Towards the Non-Culturable State of Antibiotic-Resistant Escherichia coli Cells

Abstract

Background. It is known that bacteria develop resistance to antibiotics in response to their presence and tend to maintain it for a long time. In addition, microbes can remain viable by passing into an uncultivated state that is not detected by microbiological methods. This state of the population is characterized by cell resistance to various stresses, including the effects of antibiotics.Aim. To study the parameters of transition into an uncultivated state in E.coli M-17 cells resistant to an antibiotic.Material and methods. Resistant E.coli M-17 cells were obtained by culturing the initial population in media with increasing concentrations of ampicillin (up to 50 µg/mL). Seed cultures were prepared from sensitive and resistant substrains, from which the cells were transferred to a hyperosmotic, «starvation» medium — artificial sea water. The resulting populations were incubated for a long time, samples were periodically taken to conduct the assessment of viability parameters.Results. The experiments on long-term incubation of sensitive and resistant populations of E.coli M-17 showed that ampicillin-resistant substrain of the studied culture transitioned into an uncultivated state significantly faster and quantitatively higher than its sensitive variant. The presence of the antibiotic in the inoculum increased the time for the transition of resistant R2 bacteria to a viable uncultivated state to a level of >90% compared to R1. The R1 population at the beginning of the stress exposure massively (up to 77.7%) transitioned into viable but non-culturable cells (VBNCs) in the medium without an antibiotic in a similar seed culture. Periods of cell death were noted during the observation, with those cells becoming a substrate for the living part of the population and could lead to secondary growth of bacteria or partial restoration of dormant cells.Conclusion. Cells resistant to the antibiotic quickly transitioned into the non-culturable state compared to sensitive cells of the studied strain. The addition of an antibiotic to the medium for obtaining a seed culture slowed down the transition of cells to an uncultivated state.