An investigation into the efflux mechanism against quaternary ammonium compounds as a contribution of bacterial resistance
Bacterial infections, a major problem in the poultry industry, are controlled through the use of antibiotics. Due to the increase in antibiotic resistance and the restrictions placed on the use of antibiotics in animals, the poultry industry is slowly heading for a post-antibiotic era. The use of disinfectants, like quaternary ammonium compounds (QACs), could possibly be the last resort in the fight against bacterial infections. Resistance against QACs has been observed but needs to be investigated in order to prevent similar resistance problems. The overall aim of this study was to understand bacterial resistance to QACs, using the following objectives: To examine the presence of qac resistance genes in field isolates and determining if the number of genes present confer higher resistance. To study the expression of one of these qac resistance genes in the presence of increasing QAC concentration. To study the efflux system, to determine the uptake and efflux of disinfectant, and to determine if the bacteria causes structural changes of the disinfectant. Bacterial resistance is conferred through acquisition of resistance genes; therefore qac resistance genes were studied to understand resistance to QACs. Screening of field isolates using conventional PCR for qac resistance genes (smr, qacJ, qacH and qacG) showed that one strain could contain more than one resistance gene. This could not be correlated with the minimum inhibitory concentration (MIC) of the three QACs tested and possession of more genes did not necessarily make the strain more resistant. Staphylococcus aureus strains known to contain at least one of the resistance genes were also screened for these qac genes. Conventional PCR showed that all the genes could only be detected in the strain when it was exposed to QAC. Conversely, real-time PCR showed that the genes could be detected even in the absence of QAC, and was detected in susceptible strains as well. Therefore containing the genes does not necessarily confer resistance. Relative quantitative real-time PCR was used to determine the expression of the qacJ gene in the S. aureus strains VB3_qacJ and ATCC 25923. It was hypothesised that the expression of the qacJ gene would increase with increasing didecyldimethylammonium chloride (DDAC) concentration. However, in the VB3_qacJ strain there was no significant difference in expression when induced with different DDAC concentrations. Expression of known qac resistance genes needs to be investigated simultaneously to determine whether a relationship exists between the qac genes conferring resistance. The mechanism of resistance is mainly efflux of the disinfectants; thereby reducing the disinfectant concentration inside the cell. The efflux mechanism of S. aureus was also studied using liquid chromatography-mass spectrometry (LC-MS). The hypothesis was that the bacteria are able to alter the structure of QAC before extruding it from the cell. Samples were prepared for LC-MS using solid phase extraction (SPE). The SPE protocol was optimised for extraction DDAC from growth media. It was shown that the DDAC concentration in the growth media decreased after 18 hrs of growth. It was not determined if structural changes of DDAC occurred. Resistance to disinfectants is much more complex than originally thought, and therefore resistance is still not fully understood. Further research is required to understand the full mechanism of resistance against QACs, so that similar problems associated with antibiotic resistance can be prevented.