Enterococcus faecalis’ Native CRISPR-Cas System Prevents Transfer of Antibiotic Resistance Genes In Vivo

By: Sarah Tenney ‘21 and Shannon Bennett ‘22

Enterococcus faecalis. Image Source: USDA

Antibiotic resistance is becoming an increasingly serious concern, with drug resistant microbes resulting in over 2.8 million infections, 35,000 deaths, and $4.6 billion in healthcare costs per year in the United States alone. Antibiotics have several different modes of action for disabling or killing bacteria, all of which can be evaded by changing the genome of the microbe itself. Bacteria often achieve these genomic alterations via horizontal gene transfer, in which genetic material is transferred between bacteria rather than being passed down from generation to generation. Through this mechanism bacteria acquire genes that allow for changes in expression in areas such as the peptidoglycan outer cell wall that subsequently prevent antibiotics from binding and preventing synthesis of this protective layer. Genes encoding for mutations that will serve to lessen the efficacy of antibiotics are often transferred via plasmids, small circular extrachromosomal segments of DNA present largely in prokaryotes like bacteria that replicate and segregate separately from chromosomes.

Enterococcus faecalis is a Gram-positive bacterium that is native to the mammalian intestinal tract and is not typically pathogenic unless displaced into the bloodstream or other areas of the body, a quality that categorizes E. faecalis as an opportunistic pathogen. E. faecalis is the leading cause of nosocomial (hospital-acquired) infections, and in 2017 was responsible for 54,500 infections and 5,400 deaths in the U.S. E. faecalis can horizontally transfer genes that enable the bacteria to evade the action of antibiotics through pheromone-responsive plasmids. This horizontal transfer of plasmids, referred to as conjugation, is predicated by the secretion of peptides that act as a sex pheromone allowing for communication between bacterial cells.

Half of known bacterial genomes encode a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system, which acts as an adaptive immune response to foreign DNA. CRISPR-Cas has become a household name in the past decade as research shows that this gene-editing system has vast therapeutic potential for the treatment of genetic disorders. The so-called CRISPR locus in a bacterial genome acts as a memory bank that stores genetic information as short segments of DNA called protospacers. These protospacers can be matched to viral or other foreign DNA that has been encountered in the past with the help of a segment of RNA referred to as a guide RNA, and Cas enzymes subsequently cut and remove the now-known invader DNA. A bacterium’s native CRISPR-Cas system can help prevent transmission of antibiotic resistance genes through this process of recognition and excision. In E. faecalis, about 10% of the protospacers in its CRISPR locus target pheromone-responsive plasmids, thus E. faecalis has a native immune response that codes for the removal and destruction of acquired genes that may encode for antibiotic resistance. The following infographic summarizes the basic process of CRISPR-Cas gene editing.

Image Source

The high toll on human life caused by antibiotic-resistant microbes imbues research that helps further the understanding of the acquisition of antibiotic-resistant genes with great importance, as it can lead to mediation of the spread of antibiotic resistance. In 2019, Valerie Price and her colleagues at the University of Texas at Dallas and the University of Colorado School of Medicine elucidated how crucial it is to approach such experiments from a holistic perspective; results obtained from experiments performed in vitro should not be extrapolated or assumed to perfectly mimic results from experiments performed in vivo within a microbe’s native environment. Typically, microbiological laboratory research is conducted in vitro, meaning outside of a living organism, usually in a culture medium. In the case of bacteria, this typically involves colonies of cells on a substrate such as agar. This stands in contrast to research performed in vivo, which allows for the study of bacteria within its native host or living organism. This difference might seem minute, but the differences in experimental results and implications can be vast.

The authors of this recent study utilized E. faecalis to test the horizontal gene transfer (HGT) inhibition function of its CRISPR-Cas system, specifically with conjugative pheromone-responsive plasmids that contained antibiotic resistance genes. This study stands apart because previous studies of bacteria's innate CRISPR-Cas systems have been conducted exclusively in low-complexity, in vitro contexts. Here, the researchers utilized pheromone-responsive plasmids in both in vitro and in vivo environments in order to study the efficacy of E. faecalis’ CRISPR-Cas system in preventing the horizontal transfer of plasmids, and found significantly different behaviors between the two contexts. In vivo, E. faecalis’ CRISPR-Cas system largely succeeded in preventing the HGT of antibiotic resistant genes - whereas in vitro the system was less successful in preventing the transfer.

This experiment included in vitro conditions featuring two distinct bacterial growth modes. Planktonic and biofilm growth states are common modes of bacterial growth chosen for experimental designs. Planktonic indicates that a liquid medium in a test tube is inoculated with the bacteria for observation; in this state the free-floating bacteria are more vulnerable to antibiotics. Biofilm growth state offers the bacteria more protection and more closely represents how bacteria grow outside of a lab setting (in vivo); this growth state is achieved by inoculating agar with the bacteria on a plate. In the Price et al. (2019) study, the researchers utilized a pheromone-responsive plasmid with a known genetic variant called pAM714. This model plasmid was chosen based on its use in previous experiments and the fact that E. faecalis’ innate CRISPR system has a protospacer encoded in its CRISPR locus that exactly matches this plasmid. pAM714 was used in all three experimental conditions: in vitro in both planktonic and biofilm states and in vivo within a mouse intestine. There was no differentiation between growth states for the in vivo condition, since a planktonic growth state is not observed within the body of an animal.

The figure below shows the results of all three experimental conditions. For all three graphs the y-axis represents the frequency with which the model plasmid was successfully transferred between bacterial cells. This is measured on a negative logarithmic scale with a unit of CFU/g. CFU/g means colony-forming unit per gram, which describes the quantity of viable bacteria per gram of either substrate for in vitro models or fecal matter for in vivo. The x-axis represents the time elapsed since co-colonization. For the in vitro conditions this means the time since incubation of the bacteria with the substrates and for in vivo this means the time since delivery of the bacteria to the mouse intestine. The triangles on the graph represent E. faecalis that have a crucial gene mutation called a deletion, in which the gene for the Cas9 enzyme is removed from the bacterial genome. As previously mentioned, the Cas enzyme acts as the molecular scissors to slice foreign DNA that is recognized and matched to protospacers on the CRISPR locus. Without a functioning Cas9 enzyme, the bacteria’s CRISPR-Cas system is unable to prevent the acquisition of antibiotic resistant genes that are present on the plasmid. The squares on the graph represent the bacteria without any genomic alterations, which is referred to as the wild type. The little brackets with asterisks indicate that the researchers found a statistically significant result or significant difference between two conditions.

The frequency of conjugation is significantly lower in the in vivo condition when compared to both planktonic and biofilm in vitro conditions, even among animals that were inoculated with E. faecalis that did not have a functioning Cas enzyme, but the most remarkable difference can be seen in panel A on the lower left corner. This square represents a wild type bacteria in a mouse intestine. These bacteria in an in vivo environment with a functioning CRISPR-Cas system display a dramatically low rate of conjugation when compared to samples containing E. faecalis with a gene deletion of the Cas enzyme. This result shows two major findings from this experiment that have vast implications for future research and understanding of antibiotic resistance. First, E. faecalis’ innate CRISPR-Cas system significantly reduces the number of antibiotic resistant genes that are passed between bacteria via plasmids. Second, results from experiments performed in vitro should not be assumed to represent results when an experiment is performed within a microbes’ native environment; significant differences in results can be obtained due to variables in experimental conditions. The authors of this study provided data to support the idea that thinking outside the test tube and designing an experiment that more closely resembles real life conditions can result in novel discoveries.

This research could be expanded upon to further comprehend and eventually harness a microbe’s native CRISPR-Cas system in order to prevent the transfer of antibiotic resistant genes between organisms. Through the use of further in vivo experiments with other microorganisms or genetically altered CRISPR-Cas systems this vein of research could lead to an absolutely massive breakthrough in microbiology. Studies like this are vital for building a foundation of understanding in this realm. The clinical significance of a greater understanding of antibiotic resistance is monumental, and will help to lead towards prevention of antibiotic resistance and many human lives saved.


About the authors:

Shannon Bennett '22

Sarah Tenney '21

Sarah Tenney (‘21) and Shannon Bennett (‘22) are both Frances Perkins scholars at Mount Holyoke College. Sarah is a Neuroscience major who works in the Sabariego Lab studying time discrimination in rats with medial entorhinal cortex lesions as well as the intersection between frustration and attentional blink in humans. She plans on pursuing a PhD studying neurodegenerative disease after graduation. Shannon is a Biology major and Environmental Studies minor who is an avid gardener and looking forward to her internship with the Mount Holyoke Botanic Garden this summer. She is interested in advocating for the incorporation of native plant species into natural restoration projects to attract local populations of pollinating insects and animals, as well as preventing invasive plants from harming the life cycles of beneficial native species.