Therefore, in this study, the ability of PAM-5 to inhibit biofilm formation as well as eradicate mature biofilm formed by a clinical isolate of multidrug-resistant (MDR) P . On the other hand, only 8 μg/ml PAM-5 was required to eradicate >50% of mature biofilm, as well as metabolic activity of biofilm-embedded bacteria, in which PAM-5 was able to reduce viable bacteria embedded in the biofilm in a dose-dependent manner. This project report titled “SCREENING FOR ANTI-BIOFILM EFFECT OF PAM-5 ON CLINICAL STAIN OF MULTIDRUG RESISTANT Pseudomonas aeruginosa” was prepared by WONG LI CHIK and submitted.
In particular, P aeruginosa is also reported as one of the biofilm-forming bacteria (Thi et al., 2020; Tuon et al., 2022), which enables the bacterium to establish many chronic infections in its hosts, such as cystic fibrosis (CF) , malignant external otitis, endophthalmitis and pneumonia (Bodey et al., 1983; . Gellatly and Hancock, 2013; Bush, 2022). Antibacterial peptides (ABPs) are short peptides with bacteriostatic and/or bactericidal effects (Li et al., 2017). However, these antibacterial findings were based on the action of PAM-5 against planktonic bacteria, while its effect on sessile bacteria in the biofilm community remains to be elucidated.
To screen for the ability of PAM-5 to inhibit biofilm formation by clinical strain of multidrug-resistant (MDR) Pseudomonas aeruginosa via crystal violet assay. To screen for the ability of PAM-5 to eradicate matured biofilm by clinical strain of multidrug-resistant (MDR) Pseudomonas aeruginosa via crystal violet and MTT assays.
Biofilm 4
- Overview of Biofilm 4
- Biofilm Formation 5
- Adverse Impacts of Biofilm Towards Human 7
- Overview of Antibacterial Peptides 13
- Advantages of Antibacterial Peptides 15
EPS provides an ideal reservoir for cellular exchange of resistant plasmids between the bacterial community via horizontal gene transfer (Flemming et al., 2016). Most importantly, in the clinical context, these ECM layers can limit or block the penetration of antibiotics to reach the bacteria in deeper layers (Dincer et al., 2020). Certain components of the EPS were found to be able to bind to certain antibiotics, reducing the concentrations of the latter to reach the bacteria embedded in the biofilm (Singh et al., 2021).
Worse, the sublethal antibiotic concentrations can impose induction pressures that promote mutation-mediated resistance among the bacterial community (Knudsen et al., 2016). A thorough review of the documented ABPs shows that these peptides exhibit remarkable structural and functional diversity (Yasir et al., 2018). First, most of the ABPs are cationic in nature with a net positive charge ranging from +2 to +11 ( Pasupuleti et al., 2011 ).
This cationicity is attributed to the presence of a large proportion of positively charged amino acids in the peptide sequence, such as lysine (Lys, K), histidine (His, H) and arginine (Arg, R) (Kumar et al., 2018). 15 consists of both hydrophobic and hydrophilic regions in different proportions or ratios (Lei et al., 2019).
General Experimental Design 22
Materials 22
Labware and Equipment 22
The bacterium was isolated from a contaminated granular setting on the patient's hand after two weeks of hospitalization for bacteremia. With a laboratory number as 1894170, this bacterium was a multidrug-resistant strain that was found to be insensitive to levofloxacin, moxifloxacin, doripenem, ertapenem, meropenem, ceftazidime, ceftriaxone and cefepime. The bacterium was initially subcultured from the clinical isolation culture agar supplied by the hospital laboratory onto a Muller Hilton (MH) agar.
For long-term storage, the bacteria was cultured in broth from Luria Bertani (LB), then preserved in 25% (v/v) glycerol solution and stored at -80°C. Before performing the anti-biofilm test, the bacteria was removed from the frozen glycerol stock and inoculated onto MH agar as the master culture plate via single colony streaking. After overnight incubation at 37°C, the master culture plate was stored at 4°C for up to seven days to ensure freshness of the bacteria.
In order to prepare 500 µL of peptide stock solution at the concentration of 1.024 µg/ml, 1.024 µg of PAM-5 was weighed and dissolved in 100 µL of degassed, filter-sterilized distilled water. Next, the peptide stock solution at the concentration of 1.024 µg/mL was subjected to twofold serial dilution to yield a range of peptide concentrations from 1.024 μg/mL to 8 μg/mL.
Methodology 26
Optimization of Biofilm Growth 26
The absorbance of solubilized CV was measured with a microplate reader (BMG Labtech FLUOstar Omega) at 595 nm to quantify and compare the amount of biofilm formed in different culture media. The culture medium that produced the highest mean CV absorbance reading was selected as the medium for the subsequent biofilm inhibition and biofilm eradication assays, which will be described later. Wells B2 to B8, D2 to D8 and F2 to F8 were filled with diluted culture in MHB, TSB and BHIB respectively.
Wells A1 to A4, C1 to C4 and E1 to E4 were filled with the respective fresh medium which served as sterility control.
Biofilm Inhibition Assay 29
The absorbance of solubilized crystal violet in the wells was then measured with a microplate reader (BMG Labtech FLUOstar Omega) at the wavelength of 595 nm. Wells A1 to A4 (yellow color), which served as the sterility test, were filled with fresh BHIB. 32 3.3.3 Determination of planktonic viability after peptide treatment For the biofilm inhibition assay as described in section 3.3.2, after overnight.
Determination of Planktonic Viability After
The absorbance of the dissolved CV stain in the wells was then measured by a microplate reader (BMG Labtech FLUOstar Omega) at a wavelength of 595 nm. The setup for this analysis was similar to the protocols as described in Section 3.3.4 and the setup layout is shown in Figure 3.6. To quantify the antibiofilm effect of PAM-5, the biofilm inhibition percentage, biofilm eradication percentage, and biofilm metabolic activity reduction percentage were evaluated.
48 The absorbances of the CV spots in the wells were measured with a microtiter plate reader and the average absorbances from triplicate assays are shown in Figure 4.4. In contrast, absorbance readings from all wells filled with bacteria treated with PAM-5 were significantly lower than the negative control (p <. 0.05), regardless of peptide concentrations. At the concentration of 8 μg/mL, the peptide was able to inhibit almost 50% of biofilm formation compared to untreated bacteria.
After treatment with PAM-5, the ability of the peptide to disperse mature biofilm was assessed by two different methods. Using one of the microtiter plates pre-grown with the biofilm, the amount of biofilm still adhering to the wells after peptide treatment was quantified by CV staining and absorbance measurement. 63 Figure 4.11: Gross view of formazan formation for one of the triplicated biofilm eradication tests on mature biofilm produced by MDR P. A) Well A1 to A7 mature biofilms were treated with PAM-5 at increasing concentrations from 8 µg/mL to 512 µg /ml, respectively; while (B) Well B1 to B4 are mature biofilms without peptide treatment which served as the negative control.
The total absorption of the formazan from the biofilms treated with all concentrations of PAM-5 (8 µg/ml to 512 µg/ml) was significantly lower than the untreated biofilm (N) (p < 0.05). As can be seen in the figure, as the concentrations of the peptide treatment increased, the amount of formazan formed by the treated biofilms became smaller. At a concentration of 512 µg/ml, PAM-5 was able to eliminate 90.24% of viable bacteria in the biofilm.
69 bacteria, while the peptide's potency against biofilm-embedded bacteria has yet to be elucidated. Therefore, in this study, PAM-5 was pre-coated on the well surfaces of the microtiter plate before the addition of the P. 72 membrane (Phoon, 2016, unpublished), which could explain its ability to inhibit bacterial adherence and biofilm formation on the well surfaces of the microtiter plate, such as was found in this study.
As mentioned in section 4.3.1, surface coating of the wells with PAM-5 was able to reduce biofilm formation compared to the uncoated wells (negative control). Interestingly, the anti-biofilm effect of PAM-5 is not limited to inhibition and eradication of biofilm alone, as it was followed by killing the biofilm-embedded bacteria. 77 the range of PAM-5 concentrations used in this study (as indicated in Section 4.4.2), but more than 90% of sessile bacteria were killed by the peptide at the highest concentration tested (512 µg/ml).
According to the trend of decreasing metabolic activity as shown in Figure 4.13, complete destruction of sessile bacteria can be achieved if a higher concentration of PAM-5 is tested.
