Background. The clinical application of Manuka honey, particularly for the treatment of chronic wound infections, has recently gained momentum. With the widespread use of honey-impregnated wound dressings, however, concerns have been raised regarding the potential for prolonged honey exposure to drive changes in bacterial antimicrobial sensitivity and virulence. This doctoral thesis aims to evaluate the bacteriological effects of bacterial passaging in planktonic and biofilm growth modes in the presence of a commercially available Manuka honey wound gel. Methods. Eight bacteria, including chronic wound isolates, were repeatedly exposed to sub-therapeutic concentrations of Manuka honey over ten passages (P10) and again following ten additional passages in honey-free media (X10) using an agar-based diffusion system. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) of honey and antibiotic susceptibilities were determined before and after long-term exposure to Manuka honey. Biofilm passaging was achieved using an MBEC device comprising sub-therapeutic concentrations of wound gel prepared in Mueller Hinton media. Passaged bacteria were subjected to further bacteriological analysis. Biofilm-formation was quantified using a crystal violet assay, and bacterial pathogenicity was assessed via a Galleria mellonella waxworm model. Passaged planktonic isolates exhibiting significant changes in virulence were further investigated for changes (versus parent strain) in haemolysin, coagulase, DNase, pyocyanin and motility. Where significant changes in virulence or antimicrobial sensitivity profiles were observed, passaging experiments were repeated through the repeated exposure of biofilm growth modes to subtherapeutic concentrations of honey wound gel using an MBEC assayTM. Here, changes in colony morphology, antimicrobial susceptibilities, biofilm-formation ability, dynamic growth rate and exotoxin production were assessed in biofilm derived isolates and compared to passage controls. Phenotypic analyses were conducted in conjunction with whole-genome sequencing of differential colony morphotypes Results. Compared to parent strains, moderate changes (⤠1-fold) in susceptibility to Manuka honey wound gel were observed following passaging. Staphylococcal strains exhibited a â¥4-fold increase in susceptibility to vancomycin. Additionally, transient phenotypic resistance to erythromycin following exposure to Manuka honey was noted in S. epidermidis. Increased MBECs for gentamicin were documented in both strains of P. aeruginosa, with strain WIBG 2.2 representing a 7-fold reduction in susceptibility. Relative-pathogenicity significantly increased after honey exposure in 4/8 bacterial strains, including Staphylococcus aureus WIBG 1.2, S. epidermidis, P. aeruginosa WIBG 1.3 and Escherichia coli. The enhanced virulence in pseudomonads and S. epidermidis occurred in association with enhanced biofilm formation and haemolysis, in addition to increased pyocyanin, swimming and swarming motility in P. aeruginosa. Following passaging with Manuka honey, one strain of S. aureus displayed non-pigmented colonies with reduced virulence, haemolysin, DNase, and coagulase. When passaged in sessile form, both P. aeruginosa and S. epidermidis exhibited colonies with reduced diameter and reduced sensitivity to gentamicin and vancomycin, respectively. Genome analysis of P. aeruginosa variants identified point mutations in fbcH, cheB, mcpB, hudA and lasR genes, while point mutations in cdaR, sdrG, scrK and lipA genes were observed in biofilm-derived colony variants of S. epidermidis when compared to passage controls. Enhanced virulence in vivo was observed in both P. aeruginosa and S. epidermidis biofilm variants in conjunction with increased biofilm formation, whilst P. aeruginosa also demonstrated overproduction of extracellular protease, elastase and pyocyanin. Conclusion. These
Modulating the Susceptibilities of Bacteria to Antibiotics using Manuka Honey.
Mokhtar, J. (Author). 31 Dec 2021
Student thesis: Phd