An investigation of resistance to quaternary ammonium compound disinfectants in bacteria
Jansen, Arina Corli
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The widespread and unrestricted use of antibiotics in animal production has led to a surge in antibiotic resistant bacterial strains. The poultry industry is steadily headed for a post antibiotic era, thus fuelling the search for alternative treatments for bacterial infections. One of these alternative treatments is the use of quaternary ammonium compound (QAC) based disinfectants. QACs are cationic surface active detergents widely used in the poultry industry because of their low relative toxicity and good antibacterial properties. Reports on QAC resistant bacteria have been on the increase in the food industry and thus studies on bacterial resistance to QACs are on the increase. In order to try and understand disinfectant resistance, it is important to gain a better understanding of the mode of action of QAC based disinfectants of bacterial cells, particularly in the light of a pending post antibiotic era. In order to do this, bacteria treated with DDAC were examined using Scanning electron microscopy (SEM) and Nano Scanning Auger Microscopy (NanoSAM). Staphylococcus aureus strain ATCC 2357 treated with DDAC revealed protuberances or “bleb” formations on their cell walls when observed with SEM. The DDAC treated cells were further investigated using NanoSAM. NanoSAM is the combination of Scanning Auger Microscopy (SAM) and etching with an Argon (Ar+) gun. SAM has the ability to perform semi-quantitative elemental analysis on extremely small volumes while visualizing the sample with SEM. Using NanoSAM technology we were able to visualize morphological changes caused by the disinfectant that SEM could not show. Clear evidence of a disruption of the cell membrane and the leaking out of cellular content was obtained. Resistance to QAC has been attributed to the presence of the qac resistance genes, smr, qacJ, qacG, qacH. During this study the presence of the qac resistance genes could be correlated to the degree of resistance QACs. The qac resistance genes were identified using conventional PCR in strains that displayed higher tolerance to the different QACs. No qac resistance genes where identified in the susceptible strain ATCC 25923 using conventional PCR even though this strain was resistant to one of the QACs, benzalkonium chloride. An increased resistance to the different QACs could not be attributed to the presence of one specific qac resistance gene.Real time PCR was introduced in this study since it is a technique known to be more sensitive than conventional PCR. Using real time PCR, it was revealed that all the bacterial strains contained more than one qac resistance gene. Interesting results were obtained with the susceptible strain ATCC 25923, where qac resistance genes were detected with real time PCR, while these genes were not detected using conventional PCR. Similar results were obtained with the Avian pathogenic Escherichia coli (APEC) strain isolated from poultry pens. After detecting the presence of the genes, the focus of the study changed to investigate the levels of expression of one of the qac resistance genes, smr. The expression study was performed using relative quantitative real time PCR. The hypothesis was that expression is increased when QACs are present in a culture medium. During the study it was revealed that there was no significant difference in the expression of the qac genes during cultivation in the presence of different QACs. There was, however a difference in the expression of the different strains tested where the smr was only expressed in the strain VB4-smr and not in the strains VB3-qacJ and ATCC 25923 during cultivation in the QAC didecyldimethylammonium chloride (DDAC). An additional hypothesis was subsequently formed. This hypothesis postulates that there is a difference in the expression of the smr gene over a time interval. During this study it was revealed that there was a significant difference in the expression of smr cultivated in different concentrations of DDAC, but there was no significant difference in the expression over a time interval. From this study, it has been established that qac resistance genes are present in various bacteria and that using the more sensitive real time PCR test, additional qac genes were found in most of the strains. From the expression studies, it can be concluded that the levels of resistance is not merely related to the presence or absence of a particular qac resistance gene. It was also established that resistance is also not always directly related to increased levels of expression of a particular qac resistance genes. From this study, it is evident that resistance to disinfectants is multi factorial and substantial additional research is required to fully understand resistance to disinfectants.