Investigating the physiology of viable but non-culturable bacteria by microfluidics and time-lapse microscopy

Abstract

BackgroundClonal microbial populations often harbor rare phenotypic variants that are typically hidden within the majority of the remaining cells, but are crucial for the population’s resilience to external perturbations. Persister and viable but non-culturable (VBNC) cells are two important clonal bacterial subpopulations that can survive antibiotic treatment. Both persister and VBNC cells pose a serious threat to human health. However, unlike persister cells, which quickly resume growth following drug removal, VBNC cells can remain non-growing for prolonged periods of time, thus eluding detection via traditional microbiological assays. Therefore, understanding the molecular mechanisms underlying the formation of VBNC cells requires the characterization of the clonal population with single-cell resolution. A combination of microfluidics, time-lapse microscopy, and fluorescent reporter strains offers the perfect platform for investigating individual cells while manipulating their environment.MethodsHere, we report a novel single-cell approach to investigate VBNC cells. We perform drug treatment, bacterial culturing, and live/dead staining in series by using transcriptional reporter strains and novel adaptations to the mother machine technology. Since we track each cell throughout the experiment, we are able to quantify the size, morphology and fluorescence that each VBNC cell displayed before, during and after drug treatment.ResultsWe show that VBNC cells are not dead or dying cells but share similar phenotypic features with persister cells, suggesting a link between these two subpopulations, at least in the Escherichia coli strain under investigation. We strengthen this link by demonstrating that, before drug treatment, both persister and VBNC cells can be distinguished from the remainder of the population by their lower fluorescence when using a reporter strain for tnaC, encoding the leader peptide of the tnaCAB operon responsible for tryptophan metabolism.ConclusionOur data demonstrates the suitability of our approach for studying the physiology of non-growing cells in response to external perturbations. Our approach will allow the identification of novel biomarkers for the isolation of VBNC and persister cells and will open new opportunities to map the detailed biochemical makeup of these clonal subpopulations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-017-0465-4) contains supplementary material, which is available to authorized users.

Antibiotic transport kinetics in Gram-negative bacteria revealed via single-cell uptake analysis and mathematical modelling

“…Our single-cell platform allows us to quantify heterogeneity in the cellular response to antibiotic treatment (26). However, as detailed in the Results section, quantitative estimates of systematic 15 and biological variation revealed no detectable heterogeneity in ofloxacin uptake in our experiments.…”

“…Microfluidic chip fabrication: 35 The complete protocol for the fabrication of the "mother-machine" microfluidic devices was reported previously (26). The epoxy mold used was constructed from replicas of devices kindly provided by the Jun lab (29).…”

On-chip MIC by Combining Concentration Gradient Generator and Flanged Chamber Arrays

“…Instead of batch culture, microfluidics can serve as a powerful tool for MIC because of its advances in miniaturization, integration, high throughput analysis, controllable conditions, and low sample consumption [9][10][11]. Microfluidic technology has been an effective method for bioanalysis, such as label-free cell separation, single-cell biophysics, capturing agents for specific bioseparation [12][13][14][15][16]. Moreover, microfluidics is capable of confining individual bacteria to a micrometer scale environment to enable quick bacterial growth [17,18], because the bacteria tend to accumulate in the surroundings where the space is sufficiently enclosed [19,20].…”

Microfluidic Technology for Cell Manipulation

“…The primary techniques are dielectrophoresis and electrophoresis utilizing interactions of polarity or gradients of applied electric fields and net charges of manipulated particles [27][28][29][30], optical tweezers using the scattering force and the gradient force delivered from a focused laser beam [31][32][33][34], magnetophoresis driven by application of magnetic field gradients to particles with magnetic susceptibility [35][36][37][38], and acoustophoresis wherein particle motion is controlled using acoustic waves with varying pressure profiles [39][40][41]. Additionally, there also have been other microfluidic approaches using inertial focusing and hydrodynamic stretching [42], optical stretching [43], and pressure gradients [44,45]. These techniques have been consistently applied for concentration, separation, sorting, isolation, and transport of cells [46][47][48][49].…”