# Recent Advances in Bacterial Persistence Mechanisms
## 6 Remaining Challenges
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Technique** & **Principle** & **Advantages** & **Disadvantages** & **Application** \\ \hline BacDrop & Droplet-based technique & Enables the massively parallel transcriptional & Prior knowledge of the genomes of interest & Evaluate the heterogeneity in _K. pneumoniae_[96] \\  & RNA-seq & Proffing of millions of single bacterial cells & & \\ \hline Single-cell Raman & Based on the D\({}_{2}\)O & A powerful tool to identify & It is challenging to directly & Evaluate the biochemical \\  & absorption rate and & persisters and normal & detect Raman spectra with & properties of _E. coli_ and \\  & carbon-deuterium Raman & & level to allow further & the presence of & persisters [88] \\  & metabolic activity of a single cell & downstream analyses & & \\ \hline  & A micron-sized femtoliter & (i) Individual droplets can & & \\  & droplet array is fixed on a & be collected with a & It is challenging to form a & Collect the single cells of _P. aeruginosa_ and \\  & hydrophilic-in- & micropipette. (ii) This & thin and uniform CYTOP & microscopically observe \\  & hydrophobic & array can be used for gene & layer as the surface & them [94] \\  & micropatterned surface & and protein analyses & & \\ \hline  & Uses a susceptible strain & & \\  & harboring a plasmid that & Directly visualize and & The bacterial strain is & Monitor the lysis of \\  & encodes a dose-dependent & measure bacterial & inserted with a reporter & intracellular _S. aureus_[7] \\  & tetracycline-inducible & replication & & \\  & _sfp_ gene & & & \\ \hline  & A liquid culture is & & & \\  & sandwiched in a glass & Provides a long-term & The thickness of & Characterize the lag phase \\  & bottom dish beneath the & single-cell microscopy & glass-bottom dishes needs & and persistence of \\  & glass-bottom dishes and a & nutrient agarose pad & observation to capture & to be adjusted to different & individual _E. coli_ \\  & nutrient agarose pad & which can maintain a & high-quality quantitative & microscopes & cells [100] \\  & consistent environment & information & & \\  & around the cells & & & \\ \hline  & Microfabricated chamber & & & \\  & holds cells against the & & & \\  & glass imaging surface to & & & \\  & maintain a single focal & & & \\  & plane during & & & \\  & perfusion-based imaging & & & \\  & experiments, and & individual cells & & \\  & _psulA:sfp_ plasmid is used & & & \\  & as a fluorescent reporter & & & \\ \hline  & Useful information from & It is an open-source & Data source should be & Investigate the \\  & complex time-lapse & software tool and provides & based on the time-lapse & contribution of single-cell \\  & bacterial single-cell & multiple ways to visualize & microscopy live-cell & heterogeneity to emerging \\  & movies is transformed into & cell attribute trends & imaging & cellular phenotypes at \\  & a digital representation & & & different scales [103] \\ \hline  & Silicon nitride cantilevers & & & \\  & are selected to perform & Studying persistent & & \\  & are selected to perform & phenotypic means from a & characterizing changes & \\  & force measurements on the & morphological level & that occur on the bacterial & properties of MDR-_E. coli_ \\  & bacterial surface & & & strains [24] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Overview of the novel techniques at the single-cell level to study persisters. Despite the progress in persister formation, the mechanism of its resuscitation is still unclear [93]. Resuscitation has long been considered a stochastic event [11]. It was also reported that the resuscitation of persisters in the presence of nutrients is not synchronized [9]. It has been found that during and after a 4 h resuscitation, the metabolic activity of _E. coli_ persisters is significantly lower than that of normal cells [88]. An innovative method based on flow cytometry and ampicillin-mediated cell lysis can help to monitor persister resuscitation at the single-cell level [106].