There is a growing concern for the rise of antibiotic resistance around the world, in which bacteria change in response to people using these medicines. Resistance has become ever more prevalent within our health institutes, in which antibiotics are frequently provided to patients. One population of hospitalized patients particularly at risk are those afflicted with cystic fibrosis, a genetic disorder that results in the overproduction and/or ineffective removal of bodily fluids like sweat and mucus. Regions in the body where buildup of fluid occurs, like in the lungs, leads to high amounts of bacterial communities called biofilms that can be resistant to modern antibiotics. What makes bacterial biofilms so difficult to deal with?
We focused on the most common bacterium infecting cystic fibrosis patients, Pseudomonas aeruginosa, and investigated how it counters against a commonly prescribed antibiotic (ciprofloxacin) within a biofilm community. Previous work has demonstrated the importance of a specific DNA region for biofilm-specific antibiotic resistance, found in eleven genes of the Pseudomonas aeruginosa genetic makeup. To assess the involvement of these genes, we used strains from the Pseudomonas aeruginosa (PA14) Transposon Insertion Mutant Library that interrupted the function of these genes. We characterized the growth and biofilm formation of these strains, and assessed whether biofilm-specific antibiotic resistance was impacted through minimal bactericidal concentration (MBC) assays.
We found that the majority of transposon-insertion mutant strains had similar growth and biofilm formation to the normal wild type strain. We determined that in non-biofilm cultures (ie. planktonic), the minimal bactericidal concentration (MBC) of ciprofloxacin was similar to the wild type strain. We identified that the PA1993-tn strain had a consistently lower MBC value to the wild type strain specifically in biofilm culture.
What we know and why we care
Planet of the antibiotic-resistant bacteria
Almost 100 years since the accidental discovery of the first mass-produced antibiotic, scientists continue to use and develop antibiotic medications. Antibiotics have revolutionized medicine by treating infectious disease in humans and animals, while also enhancing our food and agriculture, allowing producers to grow large amounts of crops and livestock with greater yield. However, the widespread usage of these compounds have one great flaw: the population selection of bacteria that can counter their effects. Due to the vast number of bacteria in whichever area antibiotics are used in, there will always be some that have a genetic mutation or inherent strategy that negates any antibiotic impact, and elimination of the competition from the environment only supports their prosperity in causing disease.
The lethality of some diseases is not the disease itself
There are a number of diseases in which antibiotic resistance is a major factor in determining the mortality of afflicted individuals. One such disease is cystic fibrosis. It’s estimated that more than 70,000 people worldwide are living with cystic fibrosis and have a life expectancy of only 30 years. A genetic mutation results in thick and sticky bodily fluids like mucus in various organs. This leads to a buildup of thick mucus in places like the lungs, providing an environment for infection and inflammation that further block airways to make breathing difficult. It is the continuous inflammation and spread of infection that ultimately leads to the loss of lung function, rather than the buildup of mucus caused by the genetic mutation.
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Resistance in numbers
The most common bacterium found within respiratory regions is Pseudomonas aeruginosa, an opportunistic pathogen and leading cause of hospital-acquired infections. More specifically to cystic fibrosis, this bacterium thrives in the dense mucus environment within microbial communities called biofilms. Within this form, rather than their free-floating (planktonic) form, Pseudomonas aeruginosa can attach themselves to the mucus surface and persist, covering themselves and adjacent other cells in a slimy coating called the extracellular matrix. It’s in this configuration that this bacterium and others can hinder, or even block, the impact of commonly used antibiotics to treat Pseudomonas aeruginosa infections, either by physical barriers or cellular response pathways. The cellular response evoked by forming biofilms against antibiotics is not yet fully understood.
To learn more about the phases of biofilm formation, click any box.
Bacterium is free-floating in the environment, sensing for resources.
Bacterium attaches to a surface, due to resource collecting or resource scarcity.
Other bacteria (multiple species) can also attach to the surface and other cells, forming pillar structures that collectively form a biofilm. Microbes excrete compounds into the environment to form the extracellular matrix around the biofilm.
Some bacteria break off due to mechanical stresses or other environmental cues, restarting the process.
What we did and what we found
Do mutant strains in the study affect other things than antibiotic resistance?
In previous experiments, researchers identified a DNA region common to known genes involved in biofilm-specific antibiotic resistance. Using this region to search the entire Pseudomonas aeruginosa genetic makeup, they found it in 11 genes unknown to be involved in antibiotic resistance. To study these 11 genes, we used corresponding strains found in the Pseudomonas aeruginosa (PA14) Transposon Insertion Mutant Library. Strains in this library have been modified such that a DNA sequence (ie. transposable element) was randomly inserted into one gene, impacting its proper functioning. Seven of the total 11 were found in the library. These strains allow researchers to quickly study the effect of a gene in regards to its involvement in growth, biofilm formation and response to an antibiotic.
To learn more about cellular processes impacted by transposable elements, click their boxes below.
We first investigated whether these mutant strains affected other things than antibiotic resistance compared to the normal unmodified (wild type) strain. We focused on growth and biofilm formation, since we hypothesized that any differences in antibiotic resistance compared to the normal strain would be due to their improper response against the drug rather than growth or ability to form stable biofilms. We chose to compare growth on two common growth media, a high-nutrient medium (Luria Broth) and a low-nutrient medium (M63). Growth of the normal wild type strain (our control), along with the mutant strains (our tests), were monitored by measuring the level of how much light passes through a volume of cell culture (ie. optical density) over several hours.
We observed no significant differences in growth for the majority of mutant strains compared to the wild type strain. Thus, this suggests that any differences we determine in antibiotic resistance of these mutant strains compared to the wild type strain are not due to their impact on general growth. There was one exception, strain PA2326-tn, which grew slower in Luria Broth and variably in M63 medium. This strain was eliminated from the list for next looking at biofilm formation. We permitted the wild type and mutant strains to adhere to the plastic surface of a culture plate in order to form biofilms, washing out any free-floating bacterial cells. We then stained cells with a dye to visualize biofilms, and solubilized biofilm cells in ethanol. Using the same method as for measuring growth, we looked at the optical density of solubilized biofilm cells.
To zoom in and learn more about measuring biofilm formation, click the magnifying glass.
We observed no significant differences in growth for the majority of mutant strains compared to the wild type strain. Thus, this also suggests that any differences we determine in antibiotic resistance compared to the wild type strain are not due to their impact on biofilm formation. There were two exceptions, strains PA1970-tn and PA1993-tn, which had approximately 10% less biofilm cells. Due to this relatively small decrease, we decided to keep these strains for further analysis.
Do the remaining strains have changes in biofilm-specific antibiotic resistance?
We next investigated whether the remaining six strains had changes in biofilm-specific antibiotic resistance. Remember, by impacting these genes via a transposon-insertion, the products of translation (ie. proteins) are dysfunctional, leading to breakdown by cellular processes or less effectively doing what they were made to do. The six genes and their protein products (represented by these strains) have very little previous research conducted about them, which is exciting for us!
To learn more about these genes and their protein products, click any box.
Probable σ70 factor. extracytoplasmic function (ECF) σ70 factor, cell-surface signalling, iron-uptake.
Hypothetical protein. Repressed by PsrA, long-chain fatty acid degradation in cystic fibrosis lungs.
Hypothetical protein. No previous work conducted on it.
Probable major facilitator superfamily (MFS) transporter. No previous work conducted on it.
We assessed the impact of these transposon-insertions via minimal bactericidal concentration (MBC) assays, first looking at their growth in the presence of an antibiotic (ciprofloxacin) in their free-floating form. The point of these assays is to determine at what concentration of antibiotic is required to kill bacteria in cell culture (hence bactericidal), after transferring cells onto solid media for visualizing. These concentrations, in comparison to the MBC of the normal wild type strain, will serve as a baseline value to determine at what extent these transposon insertions impact their general resistance to a frequently-prescribed antibiotic.
Our results... But what would you expect?
(hover mouse over to see)
Culture medium with antibiotic
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We observed no significant differences to the minimal bactericidal concentration of antibiotic for the majority of mutant strains compared to the wild type strain. Thus, this puts us in a good spot when assessing differences in antibiotic resistance of bacterial cells in their biofilm form. There was one exception, strain PA2057-tn, which due to technical errors was not able to draw any conclusions at the end of each experiment. Nonetheless, we excluded this strain and continued with the five remaining strains for further MBC assays with cells in their biofilm form.
Our results... But what would you expect?
(hover mouse over to see)
Culture medium with antibiotic
2-fold difference in concentration
We observed one mutant, PA1993-tn, demonstrated a consistent 50% reduction in the minimal bactericidal concentration of antibiotic compared to the wild type strain. Thus, this suggests that interrupting this gene (and the protein product that would be formed) increases the sensitivity of Pseudomonas aeruginosa to ciprofloxacin in their biofilm form. It is possible that the ability for this strain to form biofilms was compromised due to the transposon insertion, which may explain this reduction in its MBC in the biofilm form. Future work will look at completely removing this gene from the Pseudomonas aeruginosa genetic makeup, forming a new strain, and conducting similar assays to confirm this finding. Additionally, we will be looking at the level of how much this gene is transcribed (ie. activated) by measuring the levels of transcription products (mRNA) in the absence or presence of antibiotic, providing us a bigger picture of how significant this gene is in the context of biofilm-specific antibiotic resistance.
What this means and why you should care
Let us briefly summarize everything. We started with previous work that identified a specific DNA region common to known biofilm-specific antibiotic resistance genes in Pseudomonas aeruginosa (a bacterium commonly found in infections of cystic fibrosis patients). We used the established Pseudomonas aeruginosa PA14 transposon-insertion mutant library to study uncharacterized genes that contains this specific DNA region. We subjected mutant strains to classical growth assays and biofilm formation assays to ensure that any differences in antibiotic resistance compared to the normal (wild type) strain are likely due to direct involvement in cellular resistance processes. Of the remaining strains, we assessed the minimal bactericidal concentration (MBC) of antibiotic required to kill bacterial cells in their free-floating form, and as well as the MBC of antibiotic required to kill bacterial cells in their biofilm form, compared to the wild type strain. We identified one mutant, the PA1993-tn strain, that had no difference in its free-floating MBC, but had a 50% reduction in its biofilm MBC compared to the wild type strain. This suggests a biofilm-specific response by Pseudomonas aeruginosa that requires proper functioning of the PA1993 gene and resulting protein product.
They're now targeting our methods of disposal! Hopefully they don't notice our strategies are regulated by a specific DNA region...
Our work supports that the specific DNA region found in previous research is important for regulating biofilm-specific antibiotic resistance of Pseudomonas aeruginosa. The better we understand this region, and the activation of genes like PA1993 in biofilms, the more scientists can “counter the counter strategies” used by bacteria commonly infecting cystic fibrosis patients.
Special thanks to our funding sources! (click to learn more about them)