USING SHORT CHAIN FATTY ACIDS TO INACTIVATE PSEUDOMONAS AERUGINOSA BIOFILMS

Abstract Using Short Chain Fatty Acids to Disinfect Pseudomonas Aeruginosa Biofilms by Stephen Lee Background The study of bacteria typically focuses on the planktonic or free-living single-cell state that is purely cultured in a laboratory for subsequent growth with appropriate media. Although this traditional way of growing bacteria has been paramount to the understanding of bacterial behavior, physiology, and pathogenesis, bacteria rarely exist as pure cultures of planktonic growth forms in the natural habitat. Bacterial contamination of environmental surfaces, in the form of biofilms, is a prime public health problem and causes numerous infections within the general public. Biofilms are a collection of bacteria that communicate to form a protective outer layer as well as perform other metabolic processes that result in a highly resistant phenotype which can defend against numerous antimicrobial agents, including antibiotics, and survive in hostile environmental conditions. Biofilms have been found to adversely affect numerous areas that are related to human habitats. These settings include farming and food production where bacterial growth is extremely common due to the ever present amount of nutrients required for growth; urban areas and city structures such as pipes, cooling towers, or any water system associated with human contact that is subjected to stagnant water and varying temperature ranges; and most commonly in clinical or healthcare settings such as hospitals and dental offices where constant exposure to infectious agents and inadequate cleansing support growth of bacteria on surfaces. Collectively, biofilms contribute to the poor quality, functionality, and safety of all products and areas that are important to human consumption and habitation. Primary prevention of diseases is a cornerstone of public health practice and this task is significantly magnified due to the existence of bacterial infections that are now resistant to all known antibiotics. Therefore, it is even more important to focus on the basics of public health such as preventing and controlling diseases and outbreaks of infections that may not have cures. One of the ways in which biofilm infections can be prevented and controlled is through the use of biocides, which are antimicrobial agents that have broad-spectrum activity with multiple bacterial structural and metabolic targets and are designed to be used on environmental surfaces. Unfortunately, standard biocides such as sodium hypochlorite and other caustic chemicals are incapable of inactivating biofilms and are also toxic to the environment, which limits their use in public and occupational spaces. In addition, the continued use of ineffective biocides contributes to the selection of strains that are more resistant to antimicrobial agents, which subsequently become more dangerous to public health. The emergence of bacterial resistances to existing biocides and antibiotics is a public health crisis and necessitates the development of novel antimicrobial compounds and protocols that can be used for various applications in public health. Several research studies have demonstrated the antimicrobial properties of fatty acids, carboxylic acids with unbranched carbon chains that range from short to long. There were several goals of this research study. The first goal was to form a biofilm using a modified approach that has not been demonstrated prior to this study, so that it can be used for the subsequent disinfection experiments. The second goal was to evaluate the anti-biofilm potential of each fatty acid tested and whether or not the substitution of an acidic buffer, such as glycine buffer, might enhance their anti-biofilm effect. Lastly, the anti-biofilm potential of fatty acids was compared to that of sodium hypochlorite, which is typically used for disinfection purposes to help demonstrate the value of using fatty acids as not only an effective broad spectrum antimicrobial agent, but also as an economical option that can be important in the primary prevention of biofilm infections and which may also limit the development of resistant organisms. Methods To accomplish all of the aims of this research study, Pseudomonas aeruginosa was first chosen as the model organism to form a biofilm within specialized centrifugal filter devices, after which treatment experiments, using controls (sodium hypochlorite and glycine buffer) and fatty acids (butanoic, hexanoic, and octanoic), were performed at hourly time points, with a maximum time of exposure not to exceed three hours. The data was reflected by colony forming units (CFU), which indicates bacterial viability after treatment. All the data was analyzed using the IBM SPSS and Minitab®19 statistical software programs. Logarithmic transformation of the data improved the normalcy and subsequently allowed for better analysis. One-way analysis of variance (ANOVA) was performed to determine if there was CFU variation by biocidal challengers. A chi-square test was preformed to evaluate if there were any variations between replicates that were viable after 24 hours of growth. A post-hoc LSD (Least significant difference) t-test and Z-tests for the equality between two proportions were conducted to examine the relationship between biocidal challengers. Univariate GLM (General Linear Model) were also used to understand how time, and glycine buffer influences the relationship between fatty-acid and CFU. CFU was used as the dependent variable, with time, and glycine buffer used as the independent variables. Results The ineffectiveness of a 10% concentration of sodium hypochlorite at disinfecting P. aeruginosa biofilms was evidenced by the amount of CFUs that were present after treatment, indicating cell viability and survival. Of the three fatty acids tested, butanoic acid at its maximum solubility demonstrated the best results at inactivating the biofilm. Furthermore, the anti-microbial effect of hexanoic acid was greater than that of octanoic acid, thereby reflecting the trend of an inverse relationship between fatty-acid chain length and CFU or survival. When water was replaced with glycine buffer as the diluent, the data showed that all of the fatty acids had varying levels of increased disinfection efficacy, with hexanoic acid demonstrating the greatest enhancement. Conclusions This research led to the development of an experimental system that can test the disinfection efficacy of volatile fatty acids. The findings from this study are significant to the field of public health, as they relate to the primary prevention of infectious diseases and provide one of the first examples that fatty acids can function as an effective biocide for use in the environmental setting to inactivate biofilm growth, an important risk factor for human disease. The prevention of microbial diseases has been a cornerstone of public health practice, but emerging pathogens throughout the world have continuously made this effort more difficult. Clearly, microbial resistances to antimicrobial agents are of primary concern to global public health infrastructures, requiring the development of alternative broad-spectrum antimicrobial agents that can protect human and animal health. Although fatty acids have been shown to be lethal against planktonic bacterial growth, their use as disinfectants against pathogenic organisms is still not fully understood, and their potential to eradicate biofilm growth has been under-researched Therefore, the findings from this study will provide significant support for future investment and research on the anti-biofilm potential of fatty acids. Past studies have also revealed that the antimicrobial efficacy of fatty acids could be enhanced through synergism, or the combination of fatty acids. The synergistic effects of combining various fatty acids is an important area of future fatty-acid research that will not only provide more support for fatty acid use as a disinfectant but also increase the economic value to public health systems around the world