To test the potency of PAM-5 on selected Gram-positive bacteria using the microbroth dilution assay. To verify the effects of surface disrupting PAM-5 on Gram-positive bacteria by scanning electron microscope (SEM) analysis.
The Antibiotic Resistance Crisis
Overview
As the name implies, these peptides possess antibacterial effects against bacteria either selectively or in a broad spectrum manner (Hancock and Falla, 1996; Nusslein et al., 2006). Structural studies revealed that ABPs are generally present in various configurations such as α-helix, β-sheet, loop and extended structures (Brogden, 2005; Pushpanathan et al., 2013).
Advantages of ABPs
Furthermore, many ABPs have been shown to exert rapid killing effects against their target bacteria, with complete bactericidal effect being achieved within minutes of treatment (Mohamed et al., 2016). According to the results of Le et al. 2017), the majority of membrane-active ABPs exert their bactericidal actions via non-receptor-mediated binding to bacterial membrane, followed by membrane disruption and lysis of the bacteria.
Previous Findings on the Spectrum of ABPs
13 A natural ABP isolated from human neutrophils, namely LL-37, was found to have a broad spectrum of antibacterial activity against both gram-positive and gram-negative pathogenic bacteria (Turner et al., 1998; Shurko et al. , 2018). The 14 amino acid residues have a stronger and broader spectrum of antibacterial activity against both Gram-positive and Gram-negative bacteria (Park et al., 2000).
General Experimental Design
- Glassware, Consumable and Equipment Refer to Appendix A
- Preparation of Buffer and Media Refer to Appendix B
- Bacterial Strains
- Preparation of Bacterial Glycerol Stock and Master Culture Plate The bacteria were first enriched on enrichment media and/or selective media
- Preparation of PAM-5 Solution
- Preparation of Polymyxin B Solution
- Preparation of Melittin Solution
The peptide was stored at -20°C in a dry, tightly closed, screw-capped vial supplemented with silica gel. The peptide was then dissolved in 100 µL of degassed, filter-sterilized distilled water, followed by the addition of 900 µL of degassed, filter-sterilized phosphate buffered saline (PBS) to a final concentration of 1.024 µg/mL. After that, the peptide was serially diluted twice with degassed, filter-sterilized PBS from the stock concentration of 1,024 µg/mL to 4 µg/mL in silica vials.
The peptide solution was prepared using the similar procedure as described in the preparation of PAM-5 in section 3.2.6. The diluted peptides were stored at 4°C for a maximum period of seven days to ensure peptide efficacy.
Methodology
Preparation of Streptococcus pyogenes Suspension for Antibacterial Assay
The following day, the absorbance of the overnight culture was measured to obtain the log phase of bacterial growth. After removing the supernatant, the bacterial pellet was washed by resuspending it with 2 ml of phosphate buffer saline (PBS, pH 7.4), followed by another round of centrifugation. After the final wash, the bacterial pellet was resuspended in 1 ml of PBS to become the crude bacteria.
After overnight incubation, 200 μL of the overnight culture was added to 20 mL of fresh MH broth and allowed for further incubation in the same condition until it reached its average bacterial growth phase, which was equivalent to the absorbance value that falls within range at OD600. The bacterial pellet was washed twice by resuspending it in 2 mL of PBS (pH 7.4), followed by another round of centrifugation.
Preparation of Streptococcus anginosus Suspension for Antibacterial Assay
Antibacterial Assay
Then 10 µl of the bacterial suspension from each well was inoculated onto the agar for general inspection of the antibacterial effect of PAM-5. Subsequently, the turbid content in the wells of the microtiter plate indicating bacterial growth was serially diluted with PBS and inoculated onto semi-solid media as described above. The agar plates were incubated overnight at 37°C followed by colony counting to determine the titer of viable bacteria.
Briefly, the preparation of the bacterial sample and the treatment of the bacteria was performed according to the protocols described in section 3.3.2. After the serial dehydration with ethanol, further dehydration of the samples was performed using a freeze dryer (Scanvac COOLSAFE™) for approximately 18 hours.
Antibacterial Effect of PAM-5 towards Selected Gram-positive Bacteria
Similarly, bacteria treated with PAM-5 at concentrations ranging from 2 µg/mL to 128 µg/mL (Panel A to Panel G) were able to grow to a similar extent as the negative control, indicating that the bacteria could survive PAM -5 pressure. even at these high concentrations. The peptide was able to inhibit bacterial growth at 32 µg/mL, so this concentration of the peptide was defined as its MBC against this bacterium. At this MBC (16 µg/mL), melittin was able to eliminate bacteria by causing a decrease in titer of approximately 7 Log10 CFU/mL.
Plates A to H were inoculated with PAM-5-treated bacteria, Plates I to P were inoculated with polymyxin B-treated bacteria, while Plates Q to V were inoculated with melittin-treated bacteria. The viability of the treated bacteria was determined by the bacterial titer in Log10 CFU/mL.
Antibacterial potency of PAM-5 against Staphylococcus aureus ATCC 25923
As shown in Figure 4.2 (a), despite treatment with PAM-5 at a concentration of up to 256 µg/mL, the bacteria were able to survive and grow strongly on the inoculation medium (panel A to panel H). According to the degree of inhibition, as shown in Figure 4.2 (b), no sign of antibacterial effect was observed. This was indicated by a relatively similar titer between bacteria treated with PAM-5 and the negative control.
As shown in Figure 4.2 (a), no bacterial colony was seen on Plate T inoculated with bacteria treated with 16 µg/mL melittin. Enterococcus faecalis ATCC 19433 was incubated with increasing concentrations of ABPs followed by inoculation onto media for titer determination.
Antibacterial effect of PAM-5 against Enterococcus faecalis ATCC 19433
Clearly shown in Figure 4.3 (a), the untreated bacteria, which served as the negative control, grew vigorously as a lawn of bacteria on plate W and plate X, whereby the lawn and other growing bacterial colonies were surrounded by notable zones of β-hemolysis. In comparison, bacteria treated with PAM-5 at concentrations ranging from 2 µg/ml to 32 µg/ml (plate A to plate E) showed relatively similar rates of bacterial growth to the negative control (plate W and plate X). Therefore, the MBCs of polymyxin B and melittin against this bacterium were 8 µg/ml and 16 µg/ml, respectively.
The bacteria were treated with increasing peptide concentrations from left to right (2 µg/ml to 256 µg/ml) for PAM-5 and polymyxin B. Streptococcus pyogenes ATCC 19615 was incubated with increasing concentrations of ABPs, followed by inoculation on media for titre. determination.
Antibacterial potency of PAM-5 against Streptococcus pyogenes ATCC 19615
Despite the poor potency against Gram-positive bacteria as previously described, PAM-5 was found to possess a good antibacterial effect on a clinical strain of S. Referring to the general appearance of the media inoculated with the treated bacteria [Fig. 4.4 (a )], PAM-5 was able to kill S. However, PAM-5 was found to be less effective than polymyxin B as reflected by the latter's lower MBC level against this bacterium (≤ 2 µg/mL).
As shown in Figure 4.4 (a), bacterial viability was completely inhibited even at the lowest concentration of polymyxin B tested (2 µg/mL). As shown in Figure 4.4 (a), complete killing of this bacterium could only be achieved at 32 µg/mL (panel U), which was eight times higher than the MBC of PAM-5.
Antibacterial potency of PAM-5 against Streptococcus anginosus Clinical Strain
Antibacterial Potency of PAM-5 towards Staphylococcus aureus ATCC 25923
Staphylococcus aureus (S. aureus) is one of the major human pathogens frequently isolated in clinical settings. However, in the presence of a thick cell wall layer in Gram-positive bacteria, direct access of cationic ABPs to the bacterial plasma membrane may be blocked or reduced, thereby reducing the effects of membrane disruption, as occurred in Gram-negative bacteria. Detailed analysis revealed that the bacterial cell wall can reduce the effective concentration of ABP to reach and accumulate at the plasma membrane.
Increased cationicity on the bacterial cell wall through this Dlt pathway contributes to bacterial resistance to a wide range of cationic ABPs such as vancomycin, daptomycin, polymyxin B and cathelicidins (Ruzin et al., 2003; Nishi et al., 2004). . A report has shown that the Sak protein is able to form a complex with human neutrophil peptides (HNPs) such as α-defensins and LL-37, thereby reducing their effective concentrations for antibacterial activity against core bacteria (Braff et al., 2007). .
Antibacterial Potency of PAM-5 towards Enterococcus faecalis ATCC 19433
In an in vitro study by Schmidtchen et al. 2002), this protease was found to cleave an ABP secreted by human neutrophils, namely LL-37, resulting in the loss of antimicrobial activity of the peptide. faecalis could use this protease to overcome the bactericidal effect of host ABPs, allowing it to establish an infection in its host. Assuming that the protease was able to cleave this peptide at the cleavage sites as suggested above, the peptide fragments resulting from this cleavage may retain certain amino acids or motifs that contributed to antibacterial effects of the fragments. Hence, in this study, it is speculated that the antibacterial activity of the melittin is.
Multiple studies have shown that the presence of the positively charged amino acids such as tryptophan, arginine and lysine in an ABP is essential for its antimicrobial activities, as these residues are crucial in the initial interaction between the peptides and the bacterial cell membrane (Chan ). et al., 2006; Jindal et al., 2014). Since polymyxin B carries five positive charges, it is generally believed that polymyxin B kills bacteria by membrane lysis that begins with the initial interaction of the peptide cationic side chain called α,γ-diaminobutyric acid (Dab) to the anionic components of the bacterial membrane (Yu) et al., 2015).
Antibacterial Potency of PAM-5 towards Streptococcus pyogenes ATCC 19615
This significantly reduces the initial electrostatic attraction of cationic ABPs to the bacterial membrane, thereby reducing bacterial sensitivity to polymyxin B (Bao et al., 2012). According to Frick et al., (2003), this hydrophilic compound is able to interfere and sequester a variety of substances. Consequently, a higher amount of PAM-5 may be required to overcome the barrier effect to kill the bacteria, which explains the higher MBC of the peptide against S.
Consequently, a larger amount of ABP would be required to completely kill the bacterium, resulting in moderately high MBC of the peptides against S. The absence of a capsule in this bacterium may be one of the possible explanations for the sensitivity of the bacteria to PAM-5 in this study.
Implications of This Study
Limitations of this Study and Proposed Future Studies
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