Investigation of Biofilm Development by Antibiotic Resistant Urinary Tract Infection Pathogens and Evaluation of the Antimicrobial Activity of Natural Products on Carbapenem-Resistant Escherichia coli IMP and Klebsiella pneumoniae NDM-1.

Extended-spectrum β-lactamase (ESβL) producing bacteria, including Carbapenem-Resistant Enterobacteriaceae (CRE), are becoming increasingly prevalent worldwide. Understanding the biology behind the ability of these pathogens to form biofilms is crucial to the treatment of the infections caused by these pathogenic bacterial strains. Resistance to carbapenems is of major concern because they are typically used as a treatment of last resort for infections associated with UTI pathogens. The ability of these pathogens to develop resistance to carbapenems has been linked to the formation of biofilms. However, there is limited information available about molecular entities that can disrupt biofilm formation that could potentially be used as therapeutic agents. Hence, it is vital to identify therapeutic modalities with novel mechanisms of action that target the bacterial ability to form biofilms. Moreover, understanding the efficacy of known antimicrobials against drug-resistant bacteria associated with such common infections as UTIs may result in the discovery of existing therapeutics that are able to impact, at least partially, the bacterial biofilm. Aims The aim of the study is to understand the different stages in the formation and development of biofilms in Escherichia coli IMP and Klebsiella pneumoniae NMD-1 in different growth media and under different growth conditions; to estimate quantify the gene expression patterns of these pathogens using real-time PCR assay to measure the expression of genes that are responsible for biofilm formation; and to assess the anti-virulence effect of Manuka honey and garlic extract on the expression of eight virulence genes in each strain. To that end, structural changes during biofilm development and the impact of antimicrobial before and after treatment were assessed via confocal laser scanning microscopy (CLSM). Methods This study investigates the effects of the biofilm characteristics of CRE pathogens under different treatment conditions. First, the amount of biofilm formed was evaluated by measurement of TCP at different incubation time points during (6, 12, 24, and 48 hours), which was performed under static and shaking conditions using two different types of media (nutrient-poor AB broth and nutrient-rich LB broth) by the carbapenem-resistant Escherichia coli IMP and Klebsiella pneumoniae NDM-1 compared to non-resistant control E. coli (12241) and K. Pneumoniae (9633). Second, structural changes during biofilm development were assessed via Confocal Laser Scanning Microscopy (CLSM). In parallel, to assess quantitively their biofilm-related gene expression, these pathogens were profiled using real-time PCR assay. This was designed using amplicon melting analysis to detect the relative expression of the eight virulence genes in each strain. The effect of Manuka honey and garlic extract treatment on the gene expression profiles of these pathogens was determined against the biofilm formation of E. coli IMP, and K. pneumoniae NDM-1 after the antimicrobial Minimum Inhibitory Concentrations (MICs) were found. Finally, CLSM was used to compare the effects of Manuka honey and garlic extract on the phenotype structure of both bacterial strains before and after treatment. Results The biofilm formation of CRE is correlated with the biofilm development stages, with the maximum effect observed at the 24-hour time point. Our data analysis shows that there was a statistically significant difference in biofilm production (p < 0.05). A high amount of biofilm formation was observed for carbapenem antibiotic-resistant using different types of media and different incubation times. Additionally, these results indicate that the CLSM analysis shows greater biofilm formation after 24 h for the different growth stages of incubation. Also, the relative changes in the expression levels of the selected genes were observed at all the categories of genes that were highly upregulated in the nutritionally nutrient-rich LB media at 24 h compared to the nutrient-poor AB media. Evidence from the statistical analysis shows that there was a statistically significant difference (p < 0.05) during most of the development of growth biofilm formation capacity, which correlated with gene expression. The antimicrobial activities of Manuka honey and garlic extract were determined against biofilm formation of E. coli IMP, and K. pneumoniae NDM-1. The results show that both strains are sensitive to antimicrobial treatment after 24 h growth incubation. However, the significant reduction in biofilm was confirmed by an independent T-test, as the p-values of the strains were reported to be lower than 0.001. Results from the qPCR assay showed that Manuka honey and garlic extract were able to significantly reduce the expression of all the genes investigated. The CLSM analysis showed that both test strains of carbapenem-resistant E. coli IMP and K. pneumoniae NDM-1, after treatment with Manuka honey and garlic extract suspension, demonstrated red dense mats of cell aggregates after the incubation period compared to the untreated strains. Conclusions This study demonstrated that biofilm formation and gene expression profiles are affected by incubation conditions, strains’ stage of growth and media type indicated that these conditions may play a role inadaptability of the carbapenem-resistant Escherichia coli IMP and Klebsiella pneumoniae NDM-1 bacteria on different environmental conditions and their increased prevalence in biofilm associated-infections. Our analysis also demonstrates that Manuka honey and garlic extract can kill the bacteria, potentially through inhibiting the biofilm formation process. The ability to decrease the expression of critical virulence factors and the capacity to rapidly and exactly detect MβL genes is an important step in developing high-efficiency antimicrobial therapeutics against drug-resistant bacteria that target the urinary tract. Antimicrobials that act through this mechanism may help reduce the morbidity caused by MβL-producing pathogenic infections, and indeed could lead to the development of alternatives to antibiotics. Due to their antibacterial activity and anti-biofilm efficacy, Manuka honey and garlic extract could represent safe alternatives to combatting the infections caused by these bacteria.