Projects

Using phages to steer antibiotic-resistant bacteria

Bacteria, like E. coli, contain efflux pumps. These efflux pumps function like a sump pump; when water enters a house, a sump pump helps to remove the water. Likewise, when antibiotics enter a bacterial cell, an efflux pump will remove the antibiotics that may kill the bacteria.

We identify phages that bind efflux pumps. When a population of E. coli are infected with phages, most of the bacterial cells die. Some do not, and those that don't are phage-resistant. Resistance develops from mutations that don't allow the efflux pump to be made properly. So, like a broken sump pump will allow a house to fill up with water, a "broken" efflux pump will allow a bacterial cell to fill up with antibiotics. We think we can alter the genotype and phenotype of a bacterial population by infecting them with phage that binds one specific efflux pump.

We test phage-resistant mutants for antibiotic sensitivity. For example, we have isolated a bacteriophage that is able to bind the outer membrane protein, YohG. Phage-resistant clones of E. coli show greater sensitivity to the antibiotics, kanamycin and novobiocin, than the original, phage-sensitive population.

Where do we find bacteriophages? Wherever there are bacteria! For example, if you want to find a bacteriophage that infects Staphylococcus aureus, then you would search the location where S. aureus is found, such as your nose. If you wanted phages that infected E. coli, then you might search sewage water. In fact, we get most of our phages from our local sewage district!

Plaques of phages on a lawn of bacteria.

Testing for phage-resistant mutants

A broth dilution assay testing the sensitivity of phage-resistant clones to the antibiotic, novobiocin.

Using bacteriophages to treat biofilms

Biofilms are like community living for bacteria. Though we often think of bacteria as living alone (they are unicellular), they can find themselves in the comfort of a biofilm, which is made of extracellular matrix containing protein and sugars. This matrix is sticky, allowing bacteria to adhere to surfaces, and over time it may harden. In fact, the plaque on your teeth is a biofilm. Bacteria in biofilms are more resistant to drying, UV radiation, antibiotics, and other antimicrobials than are free-living bacteria (known as planktonic cells).

Bacteriophages produce lysins, which are enzymes that can break down polymeric matrices, like the cell wall of a bacteria or biofilms. Currently, we are isolating bacteriophages that target ESKAPE relatives (the ESKAPE pathogens cause the majority of hospital-acquired infections), and testing these phages to find those that best break down biofilms.

Left: A biofilm assay showing the amount of biofilm formed by Pseudomonas putida in different media.

Right: Assay with and without bacteriophages testing their ability to block biofilm formation in P. putida.

Identifying and characterizing novel antibiotics

Recently, we have begun to work with the folks at Tiny Earth to identify bacteria in our area that produce novel antibiotics that will inhibit the growth of ESKAPE pathogens (or at least their non-pathogenic relatives). These pathogens are those that cause the majority of infections in hospitals.

The Tiny Earth Chemistry Hub (TECH) identifies bacteria that produce antimicrobials on a priority list from 1-5, with a "5" representing "very high priority" isolates that likely produce novel antibiotics. We are collaborating with TECH to determine if efflux pumps encoded by E. coli have the ability to remove these novel antibiotics from bacterial cells.

WT E. coli (right) is not sensitive to the antibiotic produced by bacteria 96, but E. coli lacking tolC (left) is sensitive.

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