Chapter 5: Prospective development and applications
5.2 Gelbead-based AMR evolution kinetics study
5.2.2 Provisional study workflow
During co-incubation of bacteria and target antibiotic, the proposed study will investigate the emergence of resistant subpopulation and corresponding temporal profiles of significant SNPs. Salmonella Typhimurium can be employed as a model strain, with ciprofloxacin as a model fluoroquinolone antibiotic. The study involves the following milestones: a) determine single nucleotide polymorphisms (SNPs) significant in antibiotic resistance evolution by end point single cell sequencing, b) design and test PCR primers specifically targeting the SNPs, c) develop the fluorescence activated beads sorter and validate Gelbead multiplex PCR after sorting, and d) antibiotic resistance evolution experiments. The schematic of the study is shown in Figure 5.2.
Target SNPs will be identified using methods similar to literature (Zhang et al., 2011). Bacteria and antibiotic will be co-incubated, and a small portion of the cells will be Figure 5.2 Schematic of the proposed study on antibiotic resistance evolution kinetics.
tested for viability. After acquired resistance is observed, the whole genome
sequence will be examined to understand what mutations occurred and spread within the population. The sequences of single cells phenotyped in Gelbeads will also be examined to exclude mutations induced by the Gelbead phenotyping process. SNP will be identified by comparing the sequencing data with wild-type bacteria sequences. The significant SNPs (ideally one seems directly related to resistance and one seems irrelevant) will be selected as the targets of PCR assays. The primers amplifying the genes containing the SNP will be either found in literature or designed using BLAST. Molecular beacon specifically targeting the SNPs will be designed (Mhlanga & Malmberg, 2001). The assays will be optimized with qPCR and verified in gdPCR. Multiplexing will be attempted to include 16s rDNA detection as a reference for cell presence.
For the experiments, during the co-incubation of bacteria and the antibiotic, cell samples will be extracted and washed prior to entering the single cell analysis workflow. The sampling time points will be determined based on the timeframe observed in 2.1. The cell samples will be resuspended in the mixture for phenotyping and compartmentalized into Gelbeads, which then are sorted into positive and negative beads. Each population of beads is then analyzed by PCR for SNPs. The results will be collectively analyzed for temporal profiles of quantitative resistance phenotype emergence and the presence of SNPs in each phenotypic population. The analysis will likely generate information as shown in Figure 5.3.
The PCR profiles would inform which SNP appears first. The timing of emergence SNPs relative to the temporal profile of the resistant subpopulation may inform the role of SNP in antibiotic resistance and single cell fitness. Collectively, the above information may suggest the role of these molecular variations in antibiotic resistance evolution as a community.
The completion of this study will hopefully be able to answer the following questions about the crucial SNPs during antibiotic heteroresistance evolution: a) What is the order of their occurrence during the evolution, and are their kinetics interdependent? b) what are their roles in cell fitness? and c) what are their roles in the evolving community? The answers to these questions will have implications in modulation of antibiotic treatments for enhanced efficacy and resistance prevention. Once the workflow is established, its application can be extended to studies of complicated heterogeneous cell systems such as in microbial ecology and oncology.
Figure 5.3 Provisional obtainable information from the designed experiments.
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C h a p t e r 6
CHAPTER 6: CONCLUSIONS AND OUTLOOKS