Killing antibiotic-resistant bacteria in a ‘SNAPP’
Written by: Alex Sarkis
Edited by: Katherine Hill, Sienna Schaeffer, Katie Kelly
Since the development of penicillin, antibiotics have improved the quality of life for people all over the world. However, too much of a good thing can be dangerous, even indirectly. Overprescribing and overusing antibiotics have created environmental conditions to promote the evolve of antibiotic resistance bacteria. The situation has reached the point where multidrug resistant (MDR) strains of bacteria arising, to deadly effect. Methicillin Resistant Staphylococcus aureus, better known as MRSA, is a type of bacteria responsible for 11,285 deaths annually according to the CDC. Acinetobacter baumanni, a daunting health concern infecting deep wounds, bones, and the respiratory system in the Iraq war. Due to it’s ability to live on non-natural surfaces such as catheters and a variety of medical equipment, it’s been in rising concern in many hospital environments and colonization in vital organs can complicate treatment and be fatal in cases. This rise of harmful, MDR bacterium has made it important to come up with different ways to target these dangerous organisms.
In particular, a limited number of drugs are still have specificity and effectiveness against Gram negative bacteria due to the presence of their hydrophobic outer membrane protecting it’s own hydrophilic cell wall in contrast to the thicker cell wall of Gram-positive bacteria. One of the new avenues being explored that’s shown effectiveness against both types of MDR bacteria is antimicrobial peptides (AMPs). AMPs are designed and have been shown to associate to microbial membranes through charge attraction and destroy the membrane in a variety of ways. This natural attraction is also thought to make it harder for bacteria to develop resistance to them. Unfortunately, AMPs have had limited clinical success due to their high toxicity toward mammalian cells. Researchers at the University of Melbourne, including Shu J. Lam and Greg G. Qiao, have worked recently to make a star-shaped peptide polymer, shown below, they describe as “SNAPPs” (Structurally Nanaoengineered Antimicrobial Peptide Polymers), which they have shown to have exciting antimicrobial activity.
SNAPPs are comprised of either 16 or 32 arms of AMP-ike peptide chains. Previous studies of naturally occurring AMPs showed they functioned by associating with both the negatively-charged exposed part of the outer membrane and neutral, hydrophobic inner section of the outer membrane. Based on this, the researchers designed a molecule containing chains of positively-charged amino acids to interact with the negatively-charged part of the membrane and hydrophobic amino acids to act with the neutral, hydrophobic inner section.
Researchers first tested if SNAPPs had specificity toward Gram-negative bacteria. After finding that SNAPPs had preferential activity against Gram-negative bacteria, researchers tested them against a variety of MDR Gram-negative bacteria (P. aeruginosa, E.coli, K. pneumoniae, and A. baumnannii). SNAPPs had incredibly low, in some case 100 times lower, concentration needed to cause cell death (minimum bactericidal concentration, MBC) than existing AMPs across all Gram-negative species, showing that SNAPPs effect was not species specific.
Colistin is known to be the “last-resort”, hail-mary sort of antibiotic against MDR Gram-negative bacteria. To test whether SNAPPs maintained its effects against even Colistin resistant MDRs (CMDRs), SNAPPs were tested against CMDR strains of P. aeruginosa and A. baumnannii. SNAPPs was found to be equally efficient against these CMDR, which was even more surprising considering that both species have been found to be particularly resistance against antibiotics and AMPs. Researchers tested whether or not resistance could be easily gained against SNAPPs. Over 600 subsequent generations of CMDR A. baumnannii grown in the presence of SNAPPs, no resistance was found over any. The arms of lysine and valine alone required 40 times higher MBC, implying that the star shape was important. This made the researchers incredibly motivated to discern the properties and mode of action of SNAPPs.
When developing a drug for human benefit, the drug must have low toxicity to human cells. With this in mind, researchers tested SNAPPs were incubated with sheep red blood cells (RBCs), as well as human embryonic kidney and rat liver cells. Researchers found that SNAPPs effect on the sheep RBCs was negligible even at ~30 times the concentration needed for causing causing cell death (MBC), with well below 30% of red blood cells killed at even 100 times the MBC. Overall, it’s a promising sign that significantly higher than effective concentrations of SNAPPs were needed to cause toxicity than colistin.
To test SNAPPs effect in a living host, researchers introduced the A. baumnannii in mice and compared the effect of mice treated with SNAPPs to mice treated with the generally effective broad spectrum antibiotic imiprenem. They found that while SNAPPs and imiprenem were both able to reduce the cell count of A. baumnannii in the spleen, only SNAPPs reduced cell count with the CMDR variant. They also found that SNAPPs significantly increased white blood cell presence in the spleen under both variants of A. baumnannii in comparison to the antibiotic. These results illustrated the direct and indirect effectiveness of SNAPPs. This was a remarkable discovery, as SNAPPs are the first synthetic antimicrobial polymer reported to have such high efficacy!
Lastly, researchers tried to determine the mechanism(s) by which SNAPPs acted upon Gram-negative bacteria. Using advanced microscopy and visualizing techniques, they found that SNAPPs induced cell death through a multi-step mechanism summarized in the figure at the bottom. These include ripping apart the outer membrane, causing unregulated ion movement across the cytoplasmic membrane, and induction of an apoptotic-like death (ALD) cell death pathway. In layman’s terms, SNAPPs come in like a wrecking ball, tearing apart the membranes of even the hardiest Gram-negative bacteria in a nonspecific manner, which is most likely why little resistance was found against them. Compared to the ‘last resort’ drug, colistin, SNAPPs had relatively low toxicity. Additionally, SNAPPs not only directly decreased bacterial cell count, but also recruited white blood cells to the area of infection, allowing for efficient removal of the toxic parts of the bacteria from the tissue.
SNAPPs still have many drug trials to go through before they come to a hospital near you. More intensive studies into the effects a wider variety of tissue samples need to be explored, not to mention equivalent trials on human tissue. Human trials themselves might find surprising interactions with the different subjects and their unique immune systems. Still, SNAPPs have proven themselves an amazing potential treatment towards virtually every Gram-negative bacterium we may encounter.
Interaction comparison between the membrane and AMPs or SNAPPs:
Both molecules are attracted to the membrane due to charge interaction between their own positive charge and the negative charge of the outer parts of the outer membrane. However, whereas the strands of AMPs would slip through the outer membrane and cell wall and disrupt the physical integrity of the cell membrane through forming differing types of pores, SNAPPs simply kills the cell by fragmenting or destabilizing the outer membrane, slipping through or possibly going on to do the same to the cell wall and the cell membrane. This most likely causes unregulated ion movement as well as induction of a cell death pathway in the bacteria, lysing the cell.