Timothy Jennings, Jason Nielsen, Brian Ratcliff, Nina Wallace, Xin Zhong Lai, Paul B. Savage, Department of Chemistry and Biochemistry
Abstract
Nosocomial infections lead to septicemia, a severe problem in the United States. The Savage group has developed a novel class of antibiotics (CSAs) that can be attached to polymers. Invasive medical equipment can be coated with CSA polymers, forming an antibiotic thin film. Testing has shown the ability of CSA polymers to kill bacteria, which will lead to further testing and future use in the medical field.
INTRODUCTION
Septicemia, or infections of the blood stream, is the 10th leading cause of death in the United States, killing over 33,000 people in 2002.1 Annually, an estimated 250,000 cases of blood stream infections are hospital derived, or nosocomial infections. Additionally, several of the important nosocomial bloodstream pathogens have developed antibiotic resistance, further aggravating the problem.2 Nosocomial infections often arise from the use of invasive equipment in medical procedures. This includes the placement of catheters and implanted screws and plates that may be contaminated.
The Savage group has developed a novel class of antibiotics, called cationic steroid antibiotics (CSAs), that mimic the properties of the cationic peptide antibiotic polymyxin (figure 1), and the steroid antibiotic squalamine (figure 2).3 The CSAs (figure 3) have proven effective as antibacterial agents. Like polymyxin, the mechanism of activity in the CSAs is the cationic nature of the amine groups after protonation. These cationic amine groups penetrate and disrupt the anionic membranes of both gram-negative and gram-positive bacteria causing cell lysis.3
To overcome the problem of nosocomial infections, the Savage group has developed a polymeric form of the CSAs that can be attached to invasive equipment. When attached, this polymeric form of the CSAs will form an antibiotic thin film covering the equipment, enabling it to inhibit infection over time.
METHODS
Polymer Preparation
In order to polymerize the cationic steroid antibiotics, a polyurethane polymer with carboxylic acid groups is used. The acid groups are converted to acyl chloride groups through treatment with oxalic chloride. Boc protected CSAs are added to the polymer to form linkages via amide bonds. These are then deprotected using trifluoroacetic acid. The CSA polymer was attached to titanium plates, catheters, and foams through dip coating and spin coating.
Activity tests
In order to test the activity of the polymerized form of the CSAs, time kills were performed for the coated catheters and titanium plates, and soaks and daily time kills were performed for the coated foams.
Coated catheters and titanium plates, activity tests
The CSA-polymer coated catheters and titanium plates were soaked in 7.5 ml of 7.0 PBS for two hours previous to performing the time kills, allowing the polymer to swell as it would in normal human physiological conditions. After the two hour soak, the soaking media containing the coated catheter or plate was inoculated with 106 CFUs/ml of MRSA (methicillin resistant Staphylococcus aureus). The amount of viable CFUs of MRSA was determined at times 0, 2, 4, and 6 hours through serial dilutions and plating onto Mueller Hinton agar plates.
After the time kill testing, the titanium plates were tested to determine if any bacteria were adhered to the surface of the plate. This was determined by washing each plate three times in 7.0 PBS. Then placing the plate in 1 ml of 7.0 PBS and sonicating it for five minutes. After sonicating, the amount of viable CFUs of MRSA that were adhered to the plates was measured by serial dilutions and plating onto MH agar plates.
Coated foams, activity tests
The CSA-polymer coated foams were soaked in 45 ml of 7.0 PBS, changing the soaking solution every 24 hours. After the foams were transferred to new soaks, the remaining solution was inoculated with 106 CFUs/ml of MRSA. The amount of viable CFUs of MRSA was then measured at times 0, 2, 4, and 6 hours through serial dilutions and plating onto MH agar plates. This was performed daily, to measure the performance of a single coated foam over time.
Bacteria Preparation
A single colony of MRSA is inoculated into 5 ml of MH broth, and incubated at 37˚C on a shaker at 100 rpm overnight. Two hours prior to performing the assay, an additional 15 ml of MH broth is added. Directly before performing the assay, the bacteria is washed 3 times in sterile 7.0 PBS, and then suspended in 7.0 PBS at a final volume of 5 ml.
RESULTS AND DISCUSSION
The CSA-polymer coated catheters and titanium plates proved to effectively kill bacteria over the six hour time period. See figure 4. In another test, they killed at bacterial concentrations of 108 CFUs/ml.
The CSA-polymer coated foams maintained their ability to kill for more than 30 days. The CSA coated foams were tested in conjunction with a commercial antibiotic coated foam to compare activity. The CSA coated foam proved to outlast and out-kill the commercial antibiotic foam. See figures 5 and 6. Also, as can be noted in figure 5, the first five days of testing showed a decrease in counts at time zero. When compared with controls, this appeared to be the polymerized form of the CSA killing the inoculants very quickly (before the solution can be plated out onto an agar plate).
CONCLUSION
Impressively, the experimental results have provided evidence that the CSAs maintain their ability to kill bacteria when in their polymeric form.
When attached to invasive equipment used in many medical procedures, the CSA-polymer rapidly killed bacteria and substantially reduced bacterial concentrations.
Due to the success of these initial tests, testing will continue to occur with polymeric CSAs. Further tests include: developing a better polymer to CSA linking method, in vivo testing with laboratory animals, and biofilm prevention testing.
In the near future, antibiotic thin films will play a major role in protecting against nosocomial infections. Initial testing suggests polymeric forms of cationic steroid antibiotics will be a key player in that field.
REFERENCES
- Anderson, R. N.; Smith, B. L. Deaths: Leading causes for 2002. National Vital Statistics Reports. 2002, 53, 17.
- Edmond, M. B.; Wallace, S. E.; McClish, D. K.; Pfaller, M. A.; Jones, R. N.; Wenzel, R. P. Nosocomial Bloodstream Infections in United States Hospitals. Clinical Infectious Diseases. 1999, 29, 239-44.
- Savage, P. B. Cationic Steroid Antibiotics. Current Medicinal Chemistry – Anti-Infective Agents. 2002, 1, 3, 293-304.