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Abdul Malek, Ahmad Zuhairi (2021) In silico prediction of bacteriocin gene in Weissella cibaria NM1 and in vitro enhancement of antipseudomonal activity through a combined action of antibiotics and bacteriocins from lactic acid bacteria. MPhil thesis, University of Nottingham.

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Abstract

Multi-drug resistant Pseudomonas aeruginosa is the third-leading cause of nosocomial infections and is the predominant pathogen associated with the mortality of patients with cystic fibrosis. Infections caused by this bacterium is hard to treat due to its intrinsic, acquired and biofilm-associated antibiotic resistance. Hence, there is a pressing need for discovering new antipseudomonal agent and inhibitory strategy that could effectively inhibit this pathogen. The emerging antimicrobial peptides - bacteriocins produced by lactic acid bacteria are gaining much research attention with the shift from food industry into healthcare application. Hence, this study has two key goals: first, to screen for bacteriocin genes and ascertain its present in the genome of Weissella cibaria NM1 via in silico approach; Second, to investigate the antipseudomonal potency of crude bacteriocins and when in combinations with antibiotics against planktonic cells and biofilm of P. aeruginosa ATCC 10145.

This study started with a machine learning-based prediction approach combined with a homology-based search of highly conserved bacteriocin-associated genes in the genome of W. cibaria NM1. This prediction approach discovered a bacteriocin operon with a complete set of immunity gene, transporter gene, regulator gene and modifier gene with one bacteriocin genes (WC_2064) predicted. The genome of W. cibaria NM1 was screened on probiotic related genes and the genome was found to harboured specific genes that would contribute to acid and bile tolerance, adhesion on the host cell and exhibited cholesterol-reducing ability. On top of that, the genome also shows the absence of virulence and antibiotic resistance genes that have a high chance of being transferred which signifies the safety aspect as a potential probiotic candidate.

In vitro study was then conducted to investigate the antipseudomonal effect of crude bacteriocins WC, LG and PA respectively produced by W. cibaria NM1, Lactococcus garvieae NM2 and Pediococcus acidilactici NM3 against P. aeruginosa ATCC 10145 in its planktonic cell and biofilm form. This study includes an investigation of the effect of the crude bacteriocin used alone and in combination with antibiotics Chloramphenicol (CHL), Tetracycline (TET) and Ciprofloxacin (CIP)

The Checkerboard assay confirmed that the combined treatments of the three crude bacteriocins with antibiotics CIP, CHL and TET exhibited a synergistic effect against planktonic cells of P. aeruginosa ATCC 10145 resulted in FIC indexes of 0.258 and 0.375. In a further attempt to confirm the crude bacteriocins can enhance antibiotic efficacy, time-kill assays were performed over a 24h treatment period. The antipseudomonal effect of combined treatment with WC-CHL which show synergistic effect is comparable with CIP treatment alone, and successfully inhibited P. aeruginosa ATCC 10145 after 6h of treatment. Followed by other treatment such as LG-CHL, PA-CHL, WC-TET, PA-TET which inhibited P. aeruginosa ATCC 10145 after 8h of treatment. Current findings suggested that WC is a potent antipseudomonal agent and when combined with CHL could enhance the antipseudomonal potency further against planktonic cells of P. aeruginosa ATCC 10145.

In the biofilm inhibition and eradication assays, crude bacteriocins alone successfully prevent the formation of biofilm from planktonic cells and eradicated 40-50% of preformed biofilm. When combined with antibiotics, WC exhibited the most potent eradication effect towards preformed biofilm of P. aeruginosa ATCC 10145 and with WC-TET being the most potent combination with eradication effect of 93%. In hindsight, crude bacteriocin WC in combination with either TET and CH conferred strong effect towards the P. aeruginosa’s biofilm and planktonic cells. These effects were apparent throughout the study through the reduction of MIC when in combination, the increased killing rate towards planktonic cells of P. aeruginosa and biofilm eradication capability. This output warrant for a more depth study of the crude bacteriocin in the future. Future studies can be extended into further validation of in silico prediction through an experimental approach using heterologous expression and the conventional way of bacteriocin purification. The obtainment of pure bacteriocin will allow for its mode of action to be further understood especially against the planktonic and biofilm cell of P. aeruginosa.

Item Type:	Thesis (University of Nottingham only) (MPhil)
Supervisors:	Lim, Yin Sze Tan, Boon Chin Massawe, Festo
Keywords:	pseudomonas aeruginosa, bacteriocin, Lactic acid bacteria, antibiotic resistance, bacteriocin prediction, Weissella cibaria
Subjects:	Q Science > QR Microbiology
Faculties/Schools:	University of Nottingham, Malaysia > Faculty of Science and Engineering — Science > School of Biosciences
Item ID:	64027
Depositing User:	ABDUL MALEK, AHMAD ZUHAIRI
Date Deposited:	24 Feb 2021 04:40
Last Modified:	24 Feb 2021 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/64027

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