Mechanistics of secretome proteins from Paenibacillus polymyxa Kp10 and Lactococcus lactis Gh1 against methicillin-resistant Staphylococcus aureus and Vancomycin-resistant enterococcus
Resistance in pathogenic bacteria has emerged as a major global public health concern. Antibiotic-resistant bacterial infections are a major cause of patient mortality and morbidity, and rising antibiotic resistance is seriously compromising the vast medical advances made possible by antibiotics...
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| Format: | Thesis |
| Language: | English |
| Published: |
2023
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/114154/1/114154.pdf |
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| Summary: | Resistance in pathogenic bacteria has emerged as a major global public health concern.
Antibiotic-resistant bacterial infections are a major cause of patient mortality and
morbidity, and rising antibiotic resistance is seriously compromising the vast medical
advances made possible by antibiotics over the last decade. Hence, alternative
approaches in controlling the bacterial infections are urgently needed. Paenibacillus
polymyxa Kp10 (Kp10) and Lactococcus lactis Gh1 (Gh1) both are bacterial isolates that
were believed to exhibit antimicrobial activity. Therefore, the effectiveness of secretome
protein extracts isolated from Kp10 and Gh1 as the therapeutic agent in the treatment
against antibiotic-resistant bacterial strains, namely, vancomycin-resistant Enterococcus
(VRE) and methicillin-resistant Staphylococcus aureus (MRSA) were investigated. The
main objective of this study is to determine the inhibition mechanisms of secretome
protein extracted from Kp10 and Gh1 against MRSA and VRE bacteria. Minimal
Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and timeto-
kill assays were used to determine the sensitivity and viability of MRSA and VRE
bacterial cells following treatment with the secretome proteins of Kp10 and Gh1. Next,
to determine the morphological changes of MRSA and VRE after treated with Kp10 and
Gh1, the microscopic analysis using scanning electron microscopy (SEM) and
transmission electron microscopy (TEM) were observed. Then, to elucidate the
antimicrobial mechanism of secretome protein of Kp10 and Gh1 against MRSA and
VRE, 2D gel and sonication proteomic analysis based on time dependent manner by
using liquid chromatography-mass spectrometry (LCMS) were run by comparing
upregulated and downregulated proteins. Subsequently, the proton motive force study
included the efflux of ATP; the pH gradient and the membrane potential study in treated
MRSA and VRE were conducted. The differential proteins expression in MRSA and
VRE treated with secretome proteins Kp10 and Gh1 in time dependent manner were
analyzed. Protein extracts were obtained from treated MRSA and VRE cells by
sonication and the protein profiling were identified by using liquid chromatography-mass
spectrometry (LCMS). The safety of both secretome proteins in human cell also were
evaluated by the characterization of serum stability towards secretome proteins and their
potential toxicity towards Medical Research Council cell strain 5 cell (MRC5), a kind of
human lung cells. MRSA and VRE bacteria that were found to be sensitive to secretome
proteins of Kp10 and Gh1 were treated with the respective secretome protein extract and
were found to display several distinguished and apparent signs of morphological and
internal composition changes, based on the microscopic analysis. Several proteins that
were found to be important in cell wall functions and cell division, cell wall biosynthesis/
protein synthesis and the stress response were identified to be down-regulated or upregulated
in both treated cells without changing the membrane potential gradient. Next,
the cytotoxicity test suggested that there were no cytotoxic effects have been observed
on both secretome proteins when treated on MRC5 cells. Hence, there is no IC50 was
determined. Finally, the preliminary test on the effect of secretome proteins in human
serum was done using the agar well diffusion method. From this study, there are no
significant changes in the inhibition zone of secretome proteins in serum when treated
on the bacterial strain, thus giving the initial impression that peptide is safe to use in the
human body. In conclusion, secretome proteins of Kp10 and Gh1 were demonstrated to
reduce the growth number of VRE and MRSA by damaging the cell membrane,
suggesting that both secretome proteins may serve as a potential therapeutic agent for
antibiotic-resistant pathogen. |
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