Heteroresistance at the Single-Cell Level: Adapting to Antibiotic Stress through a Population-Based Strategy and Growth-Controlled Interphenotypic Coordination

Abstract

Heteroresistance refers to phenotypic heterogeneity of microbial clonal populations under antibiotic stress, and it has been thought to be an allocation of a subset of “resistant” cells for surviving in higher concentrations of antibiotic. The assumption fits the so-called bet-hedging strategy, where a bacterial population “hedges” its “bet” on different phenotypes to be selected by unpredicted environment stresses. To test this hypothesis, we constructed a heteroresistance model by introducing a blaCTX-M-14 gene (coding for a cephalosporin hydrolase) into a sensitive Escherichia coli strain. We confirmed heteroresistance in this clone and that a subset of the cells expressed more hydrolase and formed more colonies in the presence of ceftriaxone (exhibited stronger “resistance”). However, subsequent single-cell-level investigation by using a microfluidic device showed that a subset of cells with a distinguishable phenotype of slowed growth and intensified hydrolase expression emerged, and they were not positively selected but increased their proportion in the population with ascending antibiotic concentrations. Therefore, heteroresistance—the gradually decreased colony-forming capability in the presence of antibiotic—was a result of a decreased growth rate rather than of selection for resistant cells. Using a mock strain without the resistance gene, we further demonstrated the existence of two nested growth-centric feedback loops that control the expression of the hydrolase and maximize population growth in various antibiotic concentrations. In conclusion, phenotypic heterogeneity is a population-based strategy beneficial for bacterial survival and propagation through task allocation and interphenotypic collaboration, and the growth rate provides a critical control for the expression of stress-related genes and an essential mechanism in responding to environmental stresses.

Metabolic variability in bioprocessing: implications of microbial phenotypic heterogeneity

“…Indeed, in the environment, diversification of fitness strategies through phenotypic heterogeneity is a common strategy used by microbial populations to thrive even in adverse conditions. In this way, diversity of fitness strategies allows a heterogeneous population to have a higher probability of survival in the face of environmental fluctuations [52,53]. Bacterial populations in the environment are far more efficient when they operate according to the division of labor strategy [54,55].…”

“…For example, phenotypic heterogeneity and microbial antibiotic resistance, a phenotype termed 'persistence', have been long interpreted as a consequence of stochasticity in gene expression [53]. More recently, new analyses have pointed out the role of metabolism in bacterial persistence [62].…”

High-Throughput Single-Cell Cultivation on Microfluidic Streak Plates

“…Full automation of the MSP method may be realized with commercially available motorized colony pickers, though these systems are currently very expensive. We believe that the MSP work flow could be used with various droplet-based microfluidic systems to facilitate many applications, such as studies of cellular heterogeneity (41), selection of mutants, and adherent-cell or microbial biofilm cultivation. In addition, the spiral droplet array generated by an automated dish drive is addressable, which facilitates serial dilution and dose-response screening.…”

Antimicrobial Heteroresistance: an Emerging Field in Need of Clarity

“…Heteroresistance was followed on a single-cell level owing to the fusion with green fluorescent protein. This study showed that cells with hydrolase overexpression formed the majority of the population upon increasing antibiotic concentrations, due to decreased growth rates rather than selection for resistant cells (136).…”

“…This permitted monitoring of heteroresistant bacteria, since a subset of the cells expressed more hydrolase and hence exhibited a higher level of resistance to ceftriaxone (136). Heteroresistance was followed on a single-cell level owing to the fusion with green fluorescent protein.…”