Microbiology

The emergence of antimicrobial resistance and the persistence of Klebsiella pneumoniae biofilms represent significant challenges to public health. Hermetia illucens (HI) larvae are considered a sustainable reservoir of novel bioactive compounds. This protocol details a method for extracting fatty acids from HI larvae fat (AWME3 fraction) and studying their effects on multidrug-resistant and hypervirulent Klebsiella pneumoniae strains. Effects are evaluated by crystal violet and ethidium bromide uptake assays, motility assays (swimming, swarming, and twitching), minimal biofilm inhibitory and eradication concentration tests (MBIC/MBEC) for single, mixed, and mature biofilms, light, fluorescence, and scanning electron microscopy imaging, and microbial adhesion to solvents (MATS). This protocol offers a reliable methodology for evaluating the anti-biofilm and anti-virulence properties of natural compounds.

Development of biofilm associated candidemia for patients with implanted biomaterials causes an urgency to develop antimicrobial and biofilm inhibitive coatings in the management of recalcitrant Candida infections. Recently, there is an increase in the number of patients with biofilm formation and resistance to antifungal therapy. Therefore, there is a growing interest to use essential oils as coating agents in order to prevent biomaterial-associated Candida infections. Often high costs, complicated and laborious technologies are used for both applying the coating and determination of the antibiofilm effects hampering a rapid screening of essential oils. In order to determine biofilm formation of Candida on essential oil coated surfaces easier, cheaper and faster, we developed an essential oil (lemongrass oil) coated surface (silicone-rubber) by using a hypromellose ointment/essential oil mixture. Furthermore, we modified the “crystal violet binding assay” to quantify the biofilm mass of Candida biofilm formed on the lemongrass oil coated silicone rubber surface. The essential oil coating and the biomass determination of biofilms on silicone rubber can be easily applied with simple and accessible equipment, and will therefore provide rapid information about whether or not a particular essential oil is antiseptic, also when it is used as a coating agent.

Biofilms are the most common lifestyle of bacteria in both natural and human environments. The organized structure of these multicellular communities generally protects bacterial cells from external challenges, thereby enhancing their ability to survive treatment with antibiotics or disinfectants. For this reason, the search for new antibiofilm strategies is an active field of study. In this context, bacteriophages (viruses that infect bacteria) and their derived proteins have been proposed as promising alternatives for eliminating biofilms. For instance, endolysins can degrade peptidoglycan and, ultimately, lyse the target bacterial cells. However, it is important to characterize the responses of bacterial cells exposed to these compounds in order to improve the design of phage-based antimicrobial strategies.

This protocol was developed to examine the transcriptional responses of Staphylococcus aureus biofilm cells exposed to endolysin treatment, as previously described in Fernández et al. (2017). However, it may be subsequently adapted to analyze the response of other microorganisms to different antimicrobials.