1 in 5 deaths worldwide are the result of sepsis1. Severe sepsis cases require time in an intensive care unit, a location where as much as 54% of patients have an infection at some point during their time there. During one study, 30% of patients with a suspected or proven infection died2
Sepsis is an Overreaction to an Infection
Infections can occur anywhere in the body. The natural immune response is equipped to handle most microbial infections, whether that be in the skin, urinary tract, or somewhere else. However, if the infection spreads into the blood, the immune system can easily become overwhelmed and overreact. Blood infections are particularly hard to overcome because they are not localized which results in excessive inflammation and often a prolonged or extreme immune response. The increase in inflammation and invigorated immune response can lead to dilated blood vessels which would decrease the overall blood pressure and subsequently limit blood supply to vital organs. Lack of blood supply results in tissue necrosis, organ failure, and death. Sepsis encompasses this extreme immune response.
Intensive Care Units are for individuals who are very sick, they often have weakened immune defenses and are more susceptible to infections. Because of this, healthcare professionals are trained to watch for signs of sepsis. Sepsis is best treated early, each hour of delayed treatment allows the infection to further weaken the body to the point where it might not be strong enough to ever overcome it. Unfortunately, the signs of sepsis are ambiguous, with common ailments such as fever, rapid breathing, slurred speech, extreme shivering, and discolored skin 3. This makes it difficult to pinpoint what medication, if any, to administer. Bacterial associated sepsis can be treated with broad spectrum antibiotics. A blood culture test is taken to determine if any bacteria are found and if so, what bacterial types can be identified as the culprit. Two or more blood samples are taken from different veins to ensure the sample was not contaminated and to confirm it is a blood infection even if the culture is small 4. This test can take days to return a conclusive result and still further tests need to be conducted to determine the bacterial type. Because of the slow speed, and non-specificity, this detection can come too late to help the patient.
An Idea that Could Change the World
This year’s project idea was inspired by the 2021 OhioState iGEM team which sought to tackle the issue of treating sepsis knowing the growing ineffectiveness of antibiotics. The team hypothesized that neutralizing the invading bacteria so the immune system can no longer react to it would help allow the body to overcome the infection without resulting in a severe overreaction.
Gram-negative bacteria have a unique outer membrane that contains lipopolysaccharide (LPS) structures. The hydrophobic region of LPS, lipid A, is the endotoxin that activates the immune system when it binds to an immune receptor, TLR4. This endotoxin is what the 2021 OhioState iGEM team aimed to neutralize by delivering engineered sequences, such as pFraB-LpxR, to the bacteria. The added DNA would then manufacture a molecule that would prevent lipid A from binding to TLR4.
Since sepsis infections are generally located in the blood, localized delivery of the engineered sequences was not feasible. The team developed an engineered phage therapy to allow for an efficient delivery system that would inject the DNA into specific bacteria. This is possible because bacteriophages or phages, are viruses that can only infect bacteria. There are many types of phages that have matching receptors to the matching surface receptors on various bacterial strains. A phage infects by injecting genetic material into a bacterial cell where it becomes a plasmid. As a plasmid, the bacteria reads it like DNA, but never incorporates it into its own genetic material. Repeated transcriptions of this plasmid result in growing numbers of phage inside the bacterial cell until the cell bursts, releasing all the new phage and killing the host bacterial cell.
The phage life cycle was advantageous to the 2021 OhioState iGEM team in multiple ways. Phages naturally kill the bacterial host through lysing and phages can be engineered to include the desired lipid A neutralizing proteins to be delivered to the bacterial host. As the plasmid is transcripted, lipid A can be neutralized so that even the lysed bacteria cell parts would not be able to activate the immune system. A LAL assay showed that the major result from this iGEM project was the demonstration that the engineered genes successfully decreased the amount of endotoxin units.
Engineered Phages can be Used to Optimize Bacterial Detection in Sepsis Cases
The results of the 2021 OhioState iGEM team were promising and there are numerous areas to expand to make this idea a reality. Initially, the team thought to continue the treatment-centered work from the 2021 OhioState iGEM team by further supporting the efficacy of the engineered phage therapy. This would be done by engineering a lambda bacteriophage to infect Escherichia coli and then using a HEK assay, a more robust and accurate assay, to investigate the amount of endotoxin units present.
However, discussing with professionals in the field, the project became focused on the optimization of bacterial detection. This could be done by using an engineered phage to more easily detect bacteria strains on a blood culture taken from the patient with sepsis. The engineered phage would carry a fluorescent gene into the bacteria, where it will be transcribed in the host and produce fluorophores; molecules that fluoresce. After a couple of hours, instead of the original days that a typical blood culture takes, a UV light could be shined on the sample and the fluorescence measured. Any fluorescence will be indicative of bacteria present. The designed promoter associated with the fluorophore production would be highly expressive. In this way even if there are low numbers of bacteria in the sample, fluorescence can still be observed and detected because of the large number of fluorophores produced.
Since phages are specific, the use of a single phage type does not show the absence of bacteria if there is no glow. It could be that the type of phage simply cannot infect the type of bacteria present. To that end, when these engineered phages are used, they will be in cocktail form. However, instead of the cocktail being composed of phages that all target the same bacteria, but different receptors, the cocktail will feature up to four phages that target different bacteria. In this way a few cocktails can be applied to different samples and fluorescence observed. When one of the cocktails yields a fluorescing sample then based on the phage in that cocktail, the type of bacteria can be narrowed down. Rapid testing can be further conducted by removing different phage from that one cocktail until the bacteria type is largely identified.
The engineered phage that detects the bacteria in the sample (Phinder) can then be used in the method described by the 2021 OhioState iGEM team, to administer it as a phage therapy and reverse the onset of sepsis (Phighter). This combination of phage therapy to combat sepsis makes the therapeutic one step closer to becoming a usable solution.
References
(1) - World Health Organization. “WHO calls for global action on sepsis - cause of 1 in 5 deaths worldwide.” WHO, 2020, https://www.who.int/news/item/08-09-2020-who-calls-for-global-action-on-sepsis---cause-of-1-in-5-deaths-worldwide.
(2) - Vincent, Jean-Louis, et al. "Prevalence and outcomes of infection among patients in intensive care units in 2017." Jama 323.15 (2020): 1478-1487, https://www-ncbi-nlm-nih-gov.proxy.lib.ohio-state.edu/pmc/articles/PMC7093816/.
(3) - NHS England. “Improving outcomes for patients with sepsis.” NHS England, London, 2015, https://www.england.nhs.uk/wp-content/uploads/2015/08/Sepsis-Action-Plan-23.12.15-v1.pdf.
(4) - labcorp. “Patient Test Information - Blood Culture.” labcorp, 2022, https://www.labcorp.com/help/patient-test-info/blood-culture#:~:text=Blood%20cultures%20are%20used%20to,serious%20and%20life%2Dthreatening%20complication.