ABSTRACT
Nitric oxide (NO), a reactive free radical, acts as a broad-spectrum antimicrobial agent via mechanisms of nitrosative and oxidative stress that negatively impact the integrity of the bacterial membrane. These multiple bactericidal mechanisms prevent the development of resistance, which makes NO particularly promising as a therapeutic for combating a variety of bacterial infections. This study investigates the impact of bacterial physiology (i.e. growth phase and gram type) on the efficacy of NO. To determine the role of bacterial growth phase, the concentrations of NO-releasing G1-PO dendrimers that were bactericidal for mid-log and stationary phase bacteria were compared. The role of gram type on NO efficacy was examined by comparing minimum bactericidal concentration assays using gram-positive (Staphylococcus aureus) and gram-negative (Pseudomonas aeruginosa) species. For both species, bacteria in mid-log phase were most susceptible to NO-treatment. P. aeruginosa was more susceptible to NO than S. aureus. To determine if these susceptibility differences were primarily caused by membrane accessibility and stability, assays were performed using G1-hexyl dendrimers, alternative membrane-disrupting agents. Similar trends were observed, which suggests that membrane integrity is a key factor in the antibacterial efficacy of NO.
INTRODUCTION
Bacterial infections are known to cause a number of human health complications, both in community and hospital settings. These infections range in severity from dental plaque to
traditional antibiotics, but resistance can render these treatments ineffective (Levy & Marshall, 2004). For this reason, there is an urgent need to develop alternative effective therapeutics.
Nitric oxide (NO), a reactive free radical used naturally by microphages in the body’s immune system, has proven effective as a broad-spectrum antibacterial agent (De Groote & Fang, 1995). Antibacterial activity occurs because reactive byproducts of NO (e.g. peroxynitrite and dinitrogen trioxide) induce both nitrosative and oxidative stress on bacteria, which result in several toxic effects, including direct modification of membrane proteins, lipid peroxidation, and DNA cleavage (Privett, Broadnax, Bauman, Riccio, & Schoenfisch, 2012; Wink & Mitchell, 1998). Previous studies have suggested that bacteria do not develop resistance to NO, as they can with traditional antibiotics (Privett et al., 2012). Thus, in recent years, NO has been explored as a potential alternative to traditional antibiotics in the treatment of a number of bacterial infections, including respiratory infections in cystic fibrosis, skin infections in wounds and medical
implants, and dental plaque (Barraud et al., 2006; Hetrick, Shin, Paul, & Schoenfisch, 2009; Nablo, Prichard, Butler, Klitzman, & Schoenfisch, 2005; Allaker, 2010).
Nitric oxide is a gas, and so in order to effectively target sites of infection in vivo, it must be stored and released from a macromolecular scaffold. To date, the Schoenfisch lab has
Lu, & Schoenfisch, 2012). This has been confirmed by fluorescence microscopy studies (Sun et al., 2012). Additionally, the high secondary amine concentration allows dendrimers to effectively store large NO payloads (Stasko & Schoenfisch, 2006). However, some studies have found that dendrimers can be toxic to mammalian cells, which presents an obstacle to their application in clinical infections (Leroueil, et al., 2008). For this reason, members of the Schoenfisch lab are exploring ways to alter surface properties in order to decrease the cytotoxicity of the dendrimer scaffold.
Bacteria populations exhibit two important physiological characteristics: gram type and growth phase. Gram type refers to the two broad categories of bacteria cell walls, which are distinguished based on their retention of crystal-violet dye (Cabeen & Jacobs-Wagner, 2005). Gram-positive cell walls are characterized by a thick (20-80 nm) multilayered peptidoglycan sheath located directly outside the cytoplasmic membrane (Cabeen & Jacobs-Wagner, 2005). In contrast, the gram-negative bacterial cell wall consists of an outer membrane surrounding a thin (1-7 nm), largely single-layered peptidoglycan sheath, with a periplasmic space located between the inner and outer membranes (Cabeen & Jacobs-Wagner, 2005) (Fig. 1).
continue growing at this rate if not for constraints imposed by space and resource availability (Al-Qadiri et al., 2008). Next, the culture undergoes a much longer late or stationary phase. At this point the growth and death rates are equal, so there is no increase in bacterial concentration within the culture (Al-Qadiri et al., 2008). This occurs due to limitations in the supply of nutrients and space and the accumulation of toxic waste products (Al-Qadiri et al., 2008). If the culture is grown long enough, eventually bacteria will enter the death phase. In this phase the death rate exceeds the growth rate and so the culture undergoes a decrease in the concentration of viable cells (Al-Qadiri et al., 2008). Thus, the logarithmic bacteria concentration vs. time plot assumes a sigmoidal shape, which is referred to as a growth curve (Zwietering, Jongenburger, Rombouts, & Van’t Riet, 1990) (Fig. 2).
Studies have suggested that bacterial physiology can impact the antibacterial efficacy of traditional antibiotics. For example, Kim and Anthony (1981) found that the concentrations of penicillin and methicillin necessary to effectively eradicate Staphylococcus aureus were significantly higher for bacteria in the stationary phase compared to samples in the logarithmic phase. In addition, the characteristic differences between gram-positive and gram-negative strains have been found to impact susceptibility to many traditional antibiotics (Hoogkamp-Korstanje, 1997). Variations in cell wall integrity and/or metabolic functioning could explain why physiological differences in gram type and growth phase impact bacterial susceptibility to traditional antibiotic reagents (Cui, Murakami, Kuwahara-Arai, Hanaki, & Hiramatsu, 2000; Desai, Bühler, Weller, & Brown, 1998).
physiology, specifically gram type and growth phase, on NO efficacy. As previously discussed, dendrimers were utilized as the NO-release scaffold in this investigation. It was hypothesized that gram-positive strains would show decreased susceptibility to NO treatment compared to gram-negative strains, because the thicker peptidoglycan layer would be better equipped to resist NO-induced stress to the membrane. It was also predicted that within each strain, bacteria in the stationary phase would show greater resistance to NO than mid-log phase bacteria because a greater proportion of cells in the more mature culture would have developed a thicker, less permeable cell wall. To determine if susceptibility differences were primarily caused by membrane accessibility and stability, assays were performed using Generation 1 hexyl (G1-hexyl) dendrimers. G1-hexyl acts as an antimicrobial reagent by inserting its long alkyl chains into the cell membrane (Keifer, Peterkofsky, & Wang, 2004). If trends similar to those observed with NO are noted with this distinct type of membrane-disrupting agent, it could suggest that membrane accessibility and integrity are indeed key factors in the efficacy of NO.
METHODS Materials
= 7.4). Tryptic soy broth (TSB) and tryptic soy agar (TSA) were obtained from Becton,
Dickinson and Company (Franklin Lakes, NJ). Unless noted otherwise, these and other materials were used as received without further purification. Gram-negative Pseudomonas aeruginosa (P. aeruginosa) (ATTC #19143) and gram-positive Staphylococcus aureus (S. aureus) (ATCC #29213) were purchased from the American Tissue Culture Collection (Manassas, VA).
Dendrimer Synthesis *
Generation 1 poly(amido amine) (PAMAM) scaffolds were synthesized as described previously, by repeated alkylation/amidation steps using methyl acrylate and EDA from an EDA core (Tomalia, 2005; Tomalia et al., 1985; Lu, Slomberg, Shah, & Schoenfisch, 2013).
Secondary amine moieties were added to the dendrimers via functionalization with PO. Briefly, one molar equivalent of PO, relative to the primary amines, was added to a solution of G1-PAMAM-NH2 dendrimers in methanol (100 mg/mL) under constant stirring at room temperature for three days. After three days of reaction, solvent was removed in vacuo. The alternative membrane-disrupting dendrimers, G1-hexyl (Fig. 3b), were synthesized via a nearly identical process, but using epoxy hexane in the place of PO.
NO was measured using a Sievers 280i Chimiluminescence Nitric Oxide Analyzer (Boulder, CO). NO-releasing dendrimers (1 mg) were added to a sample containing
deoxygenated PBS (30 mL; PH = 7.4, 37°C) and nitrogen was purged through the sample vessel solution to carry liberated NO to the analyzer at a flow rate of 70 mL/min. Additional nitrogen flow was supplied to the vessel to match the collection rate of the analyzer (200 mL/min). NO release was measured in real time, allowing for the determination of NO release total, half-life, and maximum NO flux. The analysis was terminated when the NO release levels fell to below 10 ppb NO/mg dendrimer.
* The dendrimers used in this study were synthesized and analyzed for NO release by Christopher Backlund and Brittany Worley.
Minimum Bactericidal Concentration Assays
A frozen stock was incubated in TSB overnight and then 500 µL of the overnight culture were reinoculated in 50 mL of TSB. The reinoculation culture was then grown to the desired growth phase; for mid-log experiments, the culture grew until it reached the mid-log
concentration of approximately 1x108 colony-forming units per milliliter (CFU/mL) and
created in which bacteria were exposed to the PBS exposure media without added dendrimer reagent for four hours.
After the exposure period, each sample was diluted and plated on TSA plates. Once the colonies were fully-grown, they were counted and the CFU/mL values for the exposure samples were compared to the blanks to determine the minimum bactericidal concentration (MBC) of each dendrimer reagent. The MBC was defined as the concentration of dendrimer required to induce a 3-log reduction in bacterial viability. This exposure assay was repeated using G1-PO (control and NO-releasing) and G1-hexyl dendrimers against mid-log and stationary phase P. aeruginosa and S. aureus. Exposures were performed in triplicate and CFU/mL values were averaged. MBC values were reported as the mode of three trials.
RESULTS
Impact of Bacterial Growth Phase on NO Susceptibility
MBC assays using P. aeruginosa revealed that bacteria in the mid-log phase (MBC = 0.5 mg/mL) were more susceptible to NO treatment than stationary (late) phase bacteria (MBC of 1.5 mg/mL) (Fig. 4). Bactericidal NO doses for mid-log and stationary phase P. aeruginosa were 460 nmol and 1400 nmol respectively (Table 1). G1-PO control dendrimers (non-NO-releasing) displayed little antibacterial activity at concentrations that were bactericidal for NO-releasing dendrimers (Fig. 4).
as dramatic as with P. aeruginosa. Bactericidal NO doses were 6400 nmol and 7400 nmol for mid-log and stationary phase S. aureus respectively (Table 1). In addition, G1-PO control dendrimers (non-NO-releasing) displayed only minimal antibacterial activity at concentrations that were bactericidal for NO-releasing dendrimers (Fig. 5).
Impact of Gram Type on NO Susceptibility
The MBC studies presented here indicate that at both the mid-log and stationary growth stages, the gram-negative strain, P. aeruginosa, displayed much greater susceptibility to NO treatment than gram-positive S. aureus (Fig. 6). Further studies would be necessary to determine if this trend is characteristic of all gram-positive and gram-negative strains or simply with the two strains and species examined in this study.
G1-Hexyl Susceptibility Studies
DISCUSSION
The G1-PO dendrimer NO-release scaffold selected for this study was especially efficient at storing and delivering large NO-payloads. Over the 4-hour exposure period, G1-PO
dendrimers have been found to release 0.92 ± 0.13 µmol/mL of NO (Lu et al., 2013). The half-life for NO-release with G1-PO dendrimers was found to be 0.75 ± 0.04 hours, with a maximum NO release of 3800 ± 379 ppb/mg (Lu et al. 2013). Thus, the high secondary amine
concentration of the G1-PO dendrimer scaffold allows it to store and gradually release relatively large loads of NO. Additionally, this study found that non-NO-releasing G1-PO control
dendrimers exhibited only very limited antibacterial efficacy at concentrations that were bactericidal for NO-releasing dendrimers against both P. aeruginosa and S. aureus. These control studies indicate that the antibacterial effects observed with NO-releasing G1-PO dendrimers are, in large part, a direct result of NO, and not the dendrimer scaffold. Altogether, these factors make G1-PO dendrimers an especially promising scaffold for targeting NO-release to sites of bacterial infections in vivo. However, further research must be conducted to explore ways to decrease the toxicity of these dendrimers to mammalian cells before they can be used in a clinical context (Leroueil et al., 2008).
As was hypothesized, for both the gram-positive and gram-negative species, NO
(mid-log) phase into the stationary phase. During the mid-log phase, cells are dividing rapidly, and so the culture contains a greater proportion of immature bacteria cells, with cell walls that are not yet fully developed (Al-Qadiri et al., 2008; Shockman et al., 1958). By the stationary phase, the growth rate has slowed substantially and so most cells within the culture are more mature and have developed a thicker cell wall, and thus enhanced resistance to NO antibacterial activity (Al-Qadiri et al., 2008; Shockman et al., 1958).
Overall, P. aeruginosa, a gram-negative strain, was remarkably more susceptible to NO treatment than gram-positive S. aureus. Again, further studies are necessary to determine if this trend is characteristic of all gram-positive and gram-negative strains or simply the two species examined in this study. However, previous research has suggested that thickening of the cell wall plays a key role in the development of resistance to traditional antibacterial reagents (Cui et al., 2000). Specifically, gram-positive strains can be harder to eradicate, potentially because the thick cell wall prevents contact between the antibiotic and the cytoplasmic membrane (Galizzi, Cacco, Siccardi, & Mazza, 1975). Therefore, the presence of a thicker peptidoglycan layer and lack of outer membrane in gram-positive S. aureus could make it more difficult for the dendrimer reagent to penetrate the cell wall in order to release its NO load near the inner cell membrane. This could explain why S. aureus showed increased resistance to NO treatment.
From these studies, it is obvious that bacterial physiology plays a key role in determining the efficacy of NO. In theory, this could be due to any number of factors, including changes in metabolism, protein composition, or membrane integrity (Desai et al., 1998). However,
susceptibility studies with G1-hexyl dendrimers suggest that changes in cell membrane
It does this by inserting long chain alkyl groups into the bacterial membrane, which creates pores that compromise membrane integrity (Docherty & Kulpa, 2005; Keifer et al., 2004). The MBC assays conducted in this investigation found similar susceptibility trends using both NO and G1-hexyl; mid-log phase bacteria and the gram-negative species, P. aeruginosa, demonstrated increased susceptibility to both membrane-disrupting reagents. This suggests that variations in the cell membrane composition or ease of cell membrane accessibility (as a factor of cell wall permeability) could be responsible for such susceptibility trends.
Typically MBC assays with NO are conducted with mid-log phase bacteria. This is likely because bacteria are presumed to be healthiest and most metabolically active during this phase of rapid growth (Carpenter, Slomberg, Rao, & Schoenfisch, 2011). However, evidence suggests that persistent in vivo infections consist primarily of bacteria in the later portion of the mid-log phase or in the stationary phase (Costerton, Stewart, & Greenberg, 1999). Furthermore, the data presented in this study indicate that differences in bacterial physiology can dramatically impact susceptibility to NO and other membrane-disrupting reagents. For this reason, it is critical that the MBC values of potential antibacterial therapeutics against later growth phase samples are determined.
To better understand the phenomenon by which bacterial physiology impacts NO
treatment of such infections. In the future, microscopy studies would also be useful to
APPENDIX
Figure 2. Bacterial growth curve schematic. Taken from Tortora, Funke, and Case (2010). Figure 1. Visualization of gram-positive (a) and gram-negative (b) cell wall
structures. Taken from Cabeen & Jacobs-Wagner (2005).
Figure 3. Representation of G1-PO NO-releasing (a) and G1-hexyl (b) dendrimers. In (a), n denotes the number of primary amines on the periphery of the dendrimer (n = 8) and the R group is CH3 for the G1-PO dendrimers used in this study. G1-PO dendrimer image taken from Lu et al. (2013). G1-hexyl image courtesy of Brittany Worley.
a b
1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08
0 0.5 1 1.5 2 2.5
Viability
(CFU/mL
)
Dendrimer Concentration (mg/mL)
Mid-‐log NO Late NO
Mid-‐log Control Late Control
Figure 4. Bactericidal efficacy of NO-releasing and control G1-PO dendrimers against P. aeruginosa in PBS (4-hour exposure). Bacteria in the mid-log stage demonstrated increased susceptibility to NO as compared to bacteria in the stationary phase. Control dendrimers exhibited minimal bactericidal effects in comparison to their NO-releasing counterparts. Table 1. MBC values and bactericidal NO doses for G1-PO NO-releasing dendrimers against P. aeruginosa and S. aureus.
Bacteria Species Growth Phase
Minimum Bactericidal Concentration
(mg/mL)
Bactericidal NO Dose (nmol)
P. aeruginosa Mid-Log 0.5 460
Stationary 1.5 1400
S. aureus Mid-Log 7.0 6400
Figure 5. Bactericidal efficacy of NO-releasing and control G1-PO dendrimers against S. aureus in PBS supplemented with 0.25% TSB (4-hour exposure). Bacteria in the mid-log stage demonstrated increased susceptibility to NO as compared to bacteria in the stationary phase. Control dendrimers exhibited minimal bactericidal effects in comparison to their NO-releasing counterparts.
1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07
0 2 4 6 8 10
Viability
(CFU/mL
)
Dendrimer Concentration (mg/mL)
Mid-‐log NO Late NO
Mid-‐log Control Late Control
0 1 2 3 4 5 6 7 8 9
Mid-‐log Late
D en d ri m er C on ce n tr at io n ( m g/ m L)
Growth Phase
P. aeruginosa S. aureus
1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07
0 0.02 0.04 0.06 0.08 0.1
Viability
(CFU/mL
)
Dendrimer Concentration (mg/mL)
Mid-‐log
Late
Figure 7. Bactericidal efficacy of G1-hexyl dendrimers against P. aeruginosa in PBS (4-hour exposure). Mid-log bacteria were more susceptible to G1-hexyl treatment than bacteria in the stationary (late) phase.
1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07
0 0.2 0.4 0.6 0.8 1 1.2
Viability
(CFU/mL
)
Dendrimer Concentration (mg/mL)
Mid-‐log
Late
Figure 8. Bactericidal efficacy of G1-hexyl dendrimers against S. aureus in PBS
ACKNOWLEDGEMENTS
Special thanks to Katelyn Reighard and Dr. Danielle Slomberg, my graduate student mentors, Dr. Mark Schoenfisch, my Principal Investigator, and Dr. Ann Matthysse, my Biology research sponsor. I would also like to thank Brittany Worley and Christopher Backlund for providing the dendrimers used in this study.
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Mid-‐log Late
D en d ri m er C on ce n tr at io n ( m g/ m L)
Growth Phase
P. aeruginosa S. aureus
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