Investigation of biofilm development by antibiotic resistant urinary tract infection pathogens and evaluation of antimicrobial activity of Manuka honey on Escherichia coli CTX-M-15 biofilms

Abstract

Background Antibiotic resistance is one of the major healthcare issues worldwide. Over time, bacteria are becoming more resistant, making antibiotics less effective, and this in turn will lead to an increase in the morbidity rate, the mortality rate, and the healthcare system expenses as well. The rise of antibiotic resistance is reaching critical levels as new mechanisms of resistance are emerging and spreading globally, limiting the available effective treatment options. In addition, biofilms are another significant health care issue: they act as a protective cover for infectious microbes. They are also hard to remove: they require a much higher dose of antibiotics than planktonic organisms. When treating biofilm-associated infections, it is important to understand the biology of biofilm development and biofilm-related gene expression. To understand the role of biofilm in pathogenesis, biofilm formation and gene expression in Escherichia coli CTX-M-15 were investigated in this study. The antivirulence efficacy of Manuka honey as an alternative treatment was analysed for this strain. Method Biofilm formation was measured using the Tissue Culture plate method at different times: after 6, 12, 24 and 48 hours of incubation. Assuming that times correlated with the stages of biofilm development, initial attachment occurred at 6 hours, microcolonies formed at 12 hours, biofilm maturation occurred at 24 hours and biofilm dispersion occurred at 48 hours. Biofilms were quantified in both static and shaking incubators and under three different growth media: nutrient-poor (AB broth), nutrient-rich (Luria-Bertani broth) and general (nutrient broth). A quantitative Polymerase Chain Reaction was used to determine the biofilm-related gene expression at the in vitro biofilm development stages. It was also used to determine the antivirulence effect of Manuka honey on select biofilm-related gene expressions. Results The tested strains showed a variable biofilm formation ability when using different media under static and shaking conditions. Antibiotic-resistant strains were high biofilm formers. Regardless of the incubation conditions and growth media used, E. coli CTX-M-15 formed varying amounts of biofilm which did not correlate with the stages of biofilm development. Similarly, the same pattern of biofilm-related gene expression was observed, with a large variation in the growth condition tested. Manuka honey showed potent antibacterial activity: the minimum inhibitory concentration was 6 %, the minimum biocidal concentration was 8 %, and the minimum biofilm elimination concentration was 5 %. The results of the qPCR showed that Manuka honey downregulated the expression of biofilm related genes. Conclusion These findings suggested that, for both E. coli CTX-M-15 biofilm formation and biofilm-related gene expression, the profiles were not affected by the growth conditions tested in this study. This confirms the successful role of biofilm formation in pathogenesis by adapting to different environmental conditions. The findings suggest that this plays a significant role in the increased prevalence in biofilm-associated infections in third-generation cephalosporin-resistant E. coli CTX-M-15. Lastly, Manuka honey had powerful antibacterial and antibiofilm abilities and can be considered a reliable alternative therapy.The Higher Committee for Education Development in Iraq (HCED