Briefly, the minimal bactericidal concentrations (MBCs) of PAM-5, gentamicin, and polymyxin B on the two bacteria were determined using the microbroth dilution antibacterial assay. Therefore, PAM-5 is more potent compared to gentamicin and polymyxin B in terms of killing kinetics.
Overview of Antibiotic Resistance
Limitations of Conventional Antibiotics
Therefore, there is an urgent need to investigate or develop alternative antibacterial agents that kill bacteria quickly to reduce the risk of bacterial resistance.
Antibacterial Peptides (ABPs) .1 Overview
Advantages of ABPs
Compared to antibiotics, ABPs kill their target bacteria via multiple cellular targets (Teixeira et al., 2012). In addition, the action of ABPs on multiple cellular targets may further contribute to the rapid killing ( Yan et al., 2012 ).
Previous Findings on the Time-kill Kinetics of ABPs
Due to the possibility that ABPs could reduce the likelihood of drug-resistant bacteria (Mohamed et al., 2016) and the advantages mentioned above, it is highly recommended that ABPs be developed as an alternative antibacterial agent. Then, a similar study conducted by Mohamed et al. 2016) showed that a novel 12-mer synthetic peptide, namely WR12, was able to show complete killing against methicillin-resistant Staphylococcus aureus (MRSA) within 30 minutes.
Synthetic Peptide PAM-5
From a study conducted by Sainath Rao et al. 2013), a phage-displayed peptide EC5 with cationicity of +7 and hydrophobicity of 41%, was shown to reduce the growth of E. coli and Pseudomonas aeruginosa with 5 log10 reduction within 5 minutes at MIC of 8 µg/ml. Apart from that, a linear 16-mer α-helical antimicrobial peptide, T9W, showed complete killing against various strains of P. In the previous studies, PAM-5 was tested for its potency against several Gram-negative pathogenic bacteria.
In the study conducted by Chan (2016) (data not published), PAM-5 was able to kill a range of Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Shigella flexneri in different minimum bactericidal concentrations (MBCs). Another study conducted by Yong (2018) (data not published) showed that PAM-5 could exert bactericidal effects on a range of drug-resistant pathogenic bacteria, including carbapenem-resistant Enterobacteriaceae Klebsiella pneumoniae, extended-spectrum β-lactamases that kill Escherichia coli produce. , cefazolin-resistant Pseudomonas aeruginosa and clinical isolate Salmonella Typhi. Therefore, PAM-5 was studied in this study for its time-kill kinetics on selective Gram-negative bacteria.
General Overview of Experimental Design
Materials
- List of Glassware, Consumables and Laboratory Equipment Refer to Appendix A
- Preparation of Buffers and Media Refer to Appendix B
- Bacteria Strains
- Synthesis and Preparation of PAM-5
- Preparation of Gentamicin and Polymyxin B
PAM-5 was packaged in a tightly closed and dry plastic tube in lyophilized form and stored at -20°C. PAM-5 must be dissolved in sterile, degassed distilled water due to the presence of methionine residues in PAM-5, which are prone to oxidation. The peptide stock solution (1024 µg/mL) was then subjected to two-fold serial dilution to obtain a peptide concentration series from 1024 µg/mL to 4 µg/mL, as shown in Figure 3.1.
Diluted peptide solutions were stored in silica vials at 4 °C for a maximum of seven days to ensure peptide effectiveness. Gentamicin (Calbiochem®) was purchased from EMD Chemicals, Inc (Canada) and polymyxin B (Calbiochem®, Denmark) was purchased from Merck Millipore.
Methodology
Determination of Minimum Bactericidal Concentrations (MBCs) using Microbroth Dilution Assay
The bacterial suspension was then serially diluted with PBS to obtain a bacterial titer of 103 CFU/mL. To prepare the antibacterial test, 100 µL of bacterial suspension with an inoculation titer of 103 CFU/mL was loaded into the wells of a 96-well microtiter plate. The bacteria were then treated with 100 µL of PAM-5 in concentrations from 2 µg/mL to 256 µg/mL.
On the other hand, bacteria treated with 100 μL of gentamicin and polymyxin B at final concentrations ranging from 0.25 μg/mL to 8 μg/mL were also plated. The microtiter plate was then pre-incubated at 37 ˚C for one hour before loading 50 µL of MH broth into each well. This was performed to determine the minimum bactericidal concentrations (MBC) of PAM-5, gentamicin and polymyxin B.
Determination of Minimum Bactericidal Concentrations (MBCs) The antibacterial potency of PAM-5, gentamicin and polymyxin B were
Time-kill Assay
After treatment with the antibacterial agents, at every 10 minute interval, a total of 60 µL of the treated bacterial suspension from each well was inoculated onto MH agar as shown in Figure 3.4. At the same time, 60 µL of the untreated bacteria was also inoculated onto MH agar, which served as the negative control. The upper left quadrant was inoculated with PAM-5-treated bacteria, followed by the upper right quadrant inoculated with gentamicin-treated bacteria, the lower right quadrant inoculated with polymyxin B-treated bacteria, and the lower left quadrant inoculated with untreated bacteria to serve as a negative control.
The inoculated MH agar plates were incubated overnight at 37°C and the number of colonies was counted the next day.
Determination of Minimum Bactericidal Concentrations (MBCs) of PAM-5, Gentamicin and Polymyxin B Towards Escherichia coli
For polymyxin B, the absence of bacterial colony on the agar starting from plate S (0.5 µg/ml) indicated that the MBC of polymyxin B against Escherichia coli ATCC 35218 was 0.5 µg/ml. Plate A to Plate H were bacteria treated with PAM-5 at concentrations ranging from 256 µg/ml to 2 µg/ml; Plate I to Plate N were bacteria treated with gentamicin at concentrations ranging from 8 µg/ml to 0.25 µg/ml; Plate O to Plate T were bacteria treated with polymyxin B with the same concentration range of gentamicin; Plate U and Plate V served as the negative control consisting of untreated bacteria. The MBC for PAM-5 was determined to be 16 µg/ml, whereas the MBC for gentamicin and polymyxin B were 1 µg/ml and 0.5 µg/ml, respectively.
Determination of Minimum Bactericidal Concentrations (MBCs) of PAM-5, Gentamicin and Polymyxin B Towards Pseudomonas
Plate A to Plate H bacteria were treated with PAM-5 at concentrations ranging from 256 μg/ml to 2 μg/ml; Plate I to Plate N bacteria were treated with polymyxin B at concentrations ranging from 8 μg/ml to 0.25 μg/ml; Plate O in Plate T were bacteria treated with gentamicin at the same concentration range as polymyxin B; Plate U and Plate V served as negative control consisting of untreated bacterial. The MBC for PAM-5 was determined as 32 μg/ml while the MBC for polymyxin B and gentamicin were both 0.5 μg/ml.
Time-kill Kinetic Assay for PAM-5, Gentamicin and Polymyxin B Towards Escherichia coli ATCC 35218
Inoculation of Escherichia coli ATCC 35218 treated with PAM-5 (upper left quadrant), gentamicin (upper right quadrant), polymyxin B (lower right quadrant) and untreated bacteria serving as the negative control (lower left quadrant) from 10 minutes to 60 minutes (Plate A to F).
Time-kill Kinetic Assay for PAM-5, Gentamicin and Polymyxin B Towards Pseudomonas aeruginosa ATCC 27853
Uncontrolled antibiotic consumption such as incorrect dosage and dosage interval are the main culprits of antibiotic resistance. Most antibiotics only target a single site on the bacteria to exert their antibacterial action. Ala residues that bind to vancomycin and prevent vancomycin from reaching the target site of the bacteria (Lowy, 2003).
Similarly, macrolides, a class of antibiotics that inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria, become less effective when bacteria methylate the 23 rRNA ribosomal subunit. Most of these studies showed that ABPs could work better than antibiotics if they could overcome the limitations of the latter. In contrast, most antibiotics used in clinical settings target only a specific bacterial site to exert their antibacterial effect.
Time-kill Study of PAM-5 on Escherichia coli ATCC 35218 and Pseudomonas aeruginosa ATCC 27853
Since the bacterial membrane is usually the first target site of ABPs, extensive membrane damage may be associated with rapid killing of the peptides. The action of PAM-5 on bacterial DNA can be attributed to the increase in membrane permeability caused by PAM-5, thus allowing the rest of the peptides to translocate into the intracellular compartment of bacteria. Cationic PAM-5 can bind to the anionic phosphate group in the DNA backbone through electrostatic interaction.
Binding of PAM-5 to DNA can interfere with DNA replication or synthesis and subsequently inhibit the expression of proteins required for cellular processes, thereby leading to bacterial death. Therefore, it is believed that bacterial membrane disruption and inhibition of bacterial DNA metabolism contributed to the rapid killing of target bacteria by PAM-5. Since PAM-5 also possesses membrane-active mechanisms similar to the above-mentioned ABPs, this explains the rapid killing of the peptide.
Comparison of Kinetic Killing of PAM-5 and Gentamicin on Escherichia coli ATCC 35218 and Pseudomonas aeruginosa ATCC
WR12, a 12-residue peptide mainly composed of arginine and tryptophan, exhibits rapid bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA) within 30 minutes by disrupting the bacterial membrane and leaking intracellular contents (Mohamed et al., 2016) . Gentamicin is an aminoglycoside that inhibits protein synthesis only in susceptible bacteria (Hahn and Sarre, 1969). To exert its antibacterial effect, gentamicin must first bind to the lipopolysaccharide on the bacterial membrane before infusing through the outer and inner membrane to reach the bacterial cytoplasm.
Once in the cytoplasm, gentamicin irreversibly binds to the 30S subunit of the ribosome, disrupting normal protein synthesis and producing mistranslated polypeptides that damage the bacterial membrane (Poole, 2005). Since the whole process of gentamicin (aminoglycoside) bactericidal action involves several steps that are time consuming, and inhibition of protein synthesis is the only action of gentamicin, these could explain why gentamicin needs a longer duration to eliminate the two bacteria compared to PAM-5. A study by Mohamed et al. 2014) showed that six synthetic short peptides, namely RRIKA, RR, (KFF)3K, IK8, WR-12 and Penetratin, were able to rapidly kill their target bacteria within 60 minutes compared to amikacin (aminoglycosides) which only achieved that after 12 hours.
This can be explained by the increasing peptide length providing more surface area for the peptide adsorption to the anionic bacterial membrane, followed by pore formation and leakage of intracellular contents (Ringstad et al., 2006). Hence, the slower killing of polymyxin B can be explained by the shorter length of the peptide compared to PAM-5 which needs longer time to fully adsorb on the anionic membrane to cause membrane perturbation.
Implications of Studies
Limitations of Current Study and Proposed Future study
Antibiotic development challenges: the different mechanisms of action of antimicrobial peptides and of bacterial resistance. Effects of net charge and the number of positively charged residues on the biological activity of amphipathic α-helical cationic antimicrobial peptides. Evaluation of short synthetic antimicrobial peptides for treatment of drug-resistant and intracellular Staphylococcus aureus.
Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions. Potential of novel antimicrobial peptide P3 from bovine erythrocytes and its analogs to disrupt bacterial membranes in vitro and against drug-resistant bacteria in a mouse model. Evaluation of the bactericidal efficacy and modes of action of a novel T9W antimicrobial peptide against Pseudomonas aeruginosa.
