Investigating the effect of beta lactam antibiotic treatment on bacteria using scanning probe microscopies

Abstract

The work presented in this doctoral research thesis focuses on the physical characterisation of bacterial cells by means of scanning probe microscopies. Specifically, it aims at providing a systematic approach to physically characterise simple microbiological systems on a topographical, mechanical and electrical level. In addition to this objective, this study was also performed to validate the efficacy and the potential of a novel technique: the Scanning Microwave Microscope (SMM), used here as a tool for characterisation of samples of biological nature.

To achieve this, a Gram Positive bacterium – Rhodococcus NCIMB 13064 has been assessed by means of three different samples:

- The wild-type bacterial cell unaffected, growing in normal conditions of rich liquid medium - The cell affected by a presence of the antibiotic, ampicillin, at concentration lower than Minimum Inhibition Concentration (MIC) - A new genetically engineered strain has been created from the original one, inserting ampicillin resistance into the DNA.

Our expectation before the experiments was that the wild-type NCIMB 13064 cells and the engineered strain would bear similar findings for topographical, mechanical and electrical properties; on the contrary, the samples treated with ampicillin would show substantial differences due to the disruption of the peptidoglycan cell wall.

It has been shown, by means of AFM that wild-type and ampicillin-resistant cells are similar in length and width in a statistically significant way, although they differ in height, as the engineered strain appears overall lower in height compared to the original strain – with higher values and higher variability across cells. In addition to this, wild-type cells appear slightly more homogeneous and regular in shape, compared to uneven (measured by kurtosis factor) and spikey ampicillin-resistant cells.

Topography for the ampicillin-treated samples is much more variable and rough due to the disruption of the membrane and the leakage of inside content of the bacterium.

For the mechanical properties, the AFM has outlined several differences among the wild-type cells and those treated with ampicillin at concentration below MIC: in the latter case Young Modulus and stiffness are much more variable, following the irregularity of the dead cells themselves. For ampicillin-resistant samples, mechanical properties show a degree of variability especially due to a lower height profile for which deformation by penetration is slightly harder.

Overall, the SMM has shown potential to image electrical properties of the bacterial cells in different conditions and with uneven, highly deformed topographies, although these are still a barrier to reliability due to topography cross-talk: at higher frequencies where the signal-to-noise ratio is high, this hinders the ability to find reliable topography-free values for capacitance and conductance.

Especially in the case of ampicillin-treated cells with a disrupted wall due to the effect of the antibiotic, this has given mixed results, and as of now the results provided are still too contaminated by non-electrical information to give significant insight.

For this reason, the SMM is believed not to be a mature technique for electrical imaging of biological samples with non-flat topography.