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The Progression of Antibiotic Resistance in a Spatial Gradient

by Yu Guan, Sameen Belal, and Ralph Navarro

In recent history, the prominent use of antibiotics to inhibit the growth of bacteria has become the backbone of modern medicine. Consequently, the development of antibiotic resistance in various organisms is a growing threat. Thus, the study of the progression of antibiotic resistance is crucial to countering its harmful effects in the future.

To understand the relationship between bacterial gene mutations and environmental stress, scientists created a microfluidic device that is designed to mimic ecological and physicochemical conditions in naturally occurring bacterial niches. They did so by connecting the bacteria in different wells, inducing a complex environment where both bacteria and nutrients can flow between different areas, creating a gradient. The bacteria were then introduced to ciprofloxacin, an antibiotic that stops the spread of bacteria by inhibiting DNA replication and cell division.

Two syringe pumps, one circulating a nutrient rich medium, Luria-Bertani (LB) broth, and the other circulating LB broth with ciprofloxacin, were used to create a stressor gradient where bacteria move between areas which were abundant with nutrients and areas which were abundant with both nutrients and ciprofloxacin. In the center of their device, the scientists inoculated a bacterial culture, which originally migrated to the perimeter of the device. However, rather counter-intuitively, due to greater competition for nutrients the bacteria started to migrate towards the regions of increased antibiotic concentration, where they eventually grew resistant.

The scientists then sought to test the effectiveness of the spatial gradient in the microfluidic device against other methods of inducing antibiotic resistance. First, they eliminated the gradient and spread ciprofloxacin throughout the perimeter of the device. The wild-type bacteria showed no growth when ciprofloxacin surrounded the device, suggesting that mutant bacteria were selected for as a response to high antibiotic concentrations. This proved that the bacteria evolved in response to the presence of a gradient.

Second, the scientists sought to repeat their original experiment in a smaller, unconnected environment. The bacteria demonstrated a loss of fitness with increased concentrations of ciprofloxacin in such an environment, suggesting that the device was too small to create a spatial gradient that mimics complex environmental niches.
To investigate whether the resistant bacteria were the progeny of preexisting resistant bacteria or whether the mutants evolved in response to antibiotic stress, wild-type E. Coli were inoculated in the device at several concentrations. The ability of the bacteria to develop resistance at various inoculation levels (including even a culture of only 100 bacteria) proved that the bacteria evolved over time.

In order to study the specific sites where the mutations occurred, the resistant bacteria’s genome was compared with the wild type’s. The comparison revealed four single-nucleotide polymorphisms (SNPs). These SNPs enabled the resistant bacteria to regulate the expression of antibiotic resistance genes.

This experiment demonstrated the development of antibiotic resistances in a complex environment similar to the human body. The experiment and the advent of a microfluidic device complete with spatial gradients, allows for improved research into the study of bacterial evolution.

 

Qiucen Zhang et al., Acceleration of Emergence of Bacterial Antibiotic Resistance in Connected Microenvironments, Science 333, 1764-1767 (2011)

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