There are currently only two confirmed fungal vanillyl-alcohol oxidases (VAOs), one from Penicillium simplicissimum (here called PsVAO) and one from Byssochlamys fulva. Only the gene sequence of PsVAO is available. Fusarium spp. was targeted as a source of more VAOs, because they are plant pathogens known for production of lignolytic enzymes and utilization of aromatic compounds. BLAST searches of the databases of the Fungal Genome Initiative of the Broad Institute using PsVAO as query supported this choice. The predicted protein (called FvVAO) of one hit, gene number FVEG 03424 from Fusarium verticillioides, shared 63% amino acid identity with PsVAO and grouped with PsVAO in a phylogenetic analysis. Seven Fusarium strains from three species F. verticilliodes (synonym Fusarium moniliforme), Fusarium graminearum and Fusarium oxysporum were investigated for VAO activity. F. moniliforme MRC 6155 consistently displayed VAO activity in cell-free extracts with 0.036 U/mg protein obtained after veratryl alcohol induction. Primers based on the FvVAO gene were used to amplify the VAO gene (called FmVAO) from F. moniliforme MRC 6155 from both genomic DNA and mRNA. Comparison of the genomic sequences of FvVAO and FmVAO, which both have the same four introns, revealed a total of 42 nucleotide differences while the deduced amino acid sequences differed by seven amino acids. The sequences of the new FmVAO were submitted to GenBank (NCBI), accession number JQ410355. Both PsVAO and FmVAO were cloned into the pET28b(+) vector adding N-terminal His-tags and expressed in E. coli BL21(DE3)pRARE2. Using this strain to compensate for rare codons improved the expression of PsVAO but it was still not possible to detect discernable VAO bands of either PsVAO or FmVAO on SDS-PAGE gels. Comparison of substrate specificity of PsVAO and FmVAO in assays done with cell free extracts and whole cell biotransformations revealed that FmVAO preferred vanillyl alcohol as substrate and can thus be regarded as a "true" vanillyl-alcohol oxidase - possibly the first. Vanillyl-alcohol oxidase activities of PsVAO and FmVAO in cell-free extracts were respectively 0.028 and 0.018 U/mg protein, while eugenol oxidase activities were 0.030 and 0.005 U/mg protein. In whole cell biotransformations of vanillyl alcohol, specific activities of PsVAO and FmVAO were respectively 6.1 and 5.7 U/g dry weight, while with eugenol as substrate activities were 11.0 and 2.2 U/g dry weight. In whole cell biotransformations FmVAO showed higher activity with ethylphenol, again indicating its different substrate specificity. PsVAO was also cloned and expressed in the yeasts Kluyveromyces marxianus and Arxula adeninivorans while FmVAO was also cloned and expressed in A. adeninivorans. The K. marxianus vector pKM63 which gave excellent but unstable expression in K. marxianus contains 18S rDNA fragments from K. marxianus for genomic integration, a geneticin resistance marker and the native inulinase promoter of K. marxianus to drive expression of the cloned gene. The wide range vector pKM118 used for cloning into A. adeninivorans only differs from pKM63 in that it contains a hygromycin resistance marker and uses the Yarrowia lipolytica TEF promoter to drive expression of the cloned gene. Comparison of the specific activities in cell free extracts of both FmVAO and PsVAO expressed in A. adeninivorans and E. coli revealed that expression in the yeast increased the activity in cell-free extracts, with FmVAO benefiting more from expression in A. adeninivorans. The vanillyl-alcohol oxidase activity of FmVAO in A. adeninivorans was 0.045 U/mg protein and the eugenol oxidase activity, 0.015 U/mg protein. Both the vanillyl-alcohol oxidase and eugenol oxidase activities of PsVAO in A. adeninivorans were 0.04 U/mg protein. Differential centrifugation of cell free extracts showed that both PsVAO and FmVAO activity could only be detected in the soluble fraction.

A literature review on the role and presence of microorganisms in cheese was reviewed. The biochemical pathways involved in the cheese manufacture from the milk to the resultant cheese curd at the end of manufacturing were also reviewed. The activity of microorganisms used in cheese manufacture and microorganisms isolated from cheeses were also discussed and their role in the cheese curd formation. Yeasts, one of the microorganisms isolated from cheeses, were reviewed in detail. The use of yeasts as adjunct starter cultures in matured Cheddar cheese was investigated. The yeast cultures (Yarrowia lipolytica, Debaryomyces hansenii, Torulaspora delbrueckii and Dekkera bruxellensis) were inoculated in milk for the manufacture of matured Cheddar cheese as adjunct starter cultures. The yeast cultures supported the role of the starter culture (LAB) â lactose fermentation, and assimilated the organic acids present and inhibited spoilage microorganisms. The growth of the yeast and LAB was mutualistic in all the cheeses and no defects were detected in the cheeses as observed by the favourable sensory scores for the yeast inoculated cheeses. Co-inoculation of yeasts in the making of matured Cheddar cheese resulted in enhanced survival of the yeasts and the LAB population in the cheeses. The yeasts exhibited increased growth, without suppressing the viability and activity of LAB. Organic acids which are associated with aroma and flavour compound production were increased in the cheeses. The cheese inoculated with Dekkera bruxellensis + Yarrowia lipolytica had superior Cheddar cheese scores which were greater than 5 as well as the cheese single inoculated with Dekkera bruxellensis. The pH measurements of the cheeses indicated the deacidification abilities of the yeasts and the spoilage inhibiting acidity in the cheeses. Free amino acid accumulation in cheeses was also investigated. It was observed that the yeast inoculated cheeses had greater free amino acid accumulation compared to the control cheese. The dominant amino acids were Leu and GABA amino acids in all the cheese samples and low concentrations were observed for the other amino acids.

The epigenome represents a major regulatory interface to the eukaryotic genome. Nucleosome positions, histone variants, histone modifications and chromatin associated proteins all play a role in the epigenetic regulation of DNA function. Trypanosomes, an ancient branch of the eukaryotic evolutionary lineage, exhibit some highly unusual transcriptional features, including the arrangement of functionally unrelated genes in large, polymerase II transcribed polycistronic transcription units, often exceeding hundreds of kb in size. It is generally believed that transcription initiation plays a minor role in regulating the transcript level of genes in trypanosomes, which are mainly regulated posttranscriptionally. Recent advances have revealed that epigenetic mechanisms play an essential role in the transcriptional regulation of Trypanosoma brucei. This suggested that the regulation of gene activity is, indeed, an important control mechanism, and that the epigenome is critical in regulating gene expression programs that allow the successful migration of this parasite between hosts, as well as the continuous evasion of the immune system in mammalian hosts. A wide range of epigenetic signals, readers, writers and erasers have been identified in trypanosomes, some of which have been observed to be unique to trypanosomes. We review recent advances in our understanding of epigenetic control mechanisms in T. brucei, the causative agent of African sleeping sickness, and discuss the possible role that these mechanisms may play in the life cycle of the parasite.

The objectives of this study were to determine the effects of a commercial dietary CLA feed supplement on the production and meat quality parameters of pigs under commercial production conditions. It included the study of the chemical and sensory stability of processed meat products manufactured from the meat of such animals. One hundred and forty four Landrace x Large White crossbred pigs, weighing ± 30 kg, were randomly divided into two groups of seventy two pigs each, that were assigned to one of two dietary treatments. Diets consisted of a control diet supplemented with 1% SFO and the experimental diet where 0.5% SFO was replaced with 0.5% CLA. Each dietary group was further divided into three gender groups (boars, barrows and gilts) that consisted of twenty four pigs each. Each gender group was further divided into two slaughter weight groups (70 kg and 90 kg) consisting of twelve pigs each. Pigs were fed until the average live weight of the pigs was ± 70 kg for the porkers and ± 90 kg for the baconers. Growth performance (weight increase, ADG and FCR) and carcass characteristics (warm and cold carcass mass, dressing percentage, carcass length, shoulder and buttock circumference, pH, backfat thickness, eye muscle thickness and LMC) were assessed. Animals receiving the CLA diet had improved FCR and carcasses with thinner backfat and higher LMC, compared to animals on the SFO diets. This resulted in a higher frequency of P and O classification of carcasses from CLA supplemented pigs. Backfat, belly fat and M. longissimus thoracis quality of the dietary treatment and slaughter weight groups were compared. Baconers had improved technological properties compared to porkers. Dietary CLA supplementation resulted in improved technological properties of backfat and belly fat, demonstrated by decreased IV; RI; DBI; UFA; MUFA; PUFA; MUFA/SFA ratio; PUFA/SFA ratio; 9 desaturase index; C16:1 + C18:1/C16:0 + C18:0 ratio and increased C18:0; cis-9, trans-11; trans-10, cis-12; SFA; AI; C16:0 + C18 and ratios of C18:0/C18:2; C18:2/C18:1; C16:0/C18:2. M. longissimus thoracis from CLA supplemented pigs had higher a*-values, drip loss and WHC. Dietary CLA supplementation resulted in a decrease of health and nutritional properties of M. longissimus thoracis, demonstrated by increased SFA content and AI, while UFA, MUFA, PUFA, n-6, n-3 and ratios of MUFA/SFA and PUFA/SFA decreased. Technological and health properties were inversely related. The decreased health properties must be weighed against the numerous health benefits, ranging from improved immune function to prevention of cancer that can be attributed to CLA supplementation. Conjugated linoleic acid isomers were deposited into the neutral- and glycolipid fraction of subcutaneous adipose tissue and into the phospholipid fraction of IMF. Processed products (patties, bacon and salami) were manufactured from meat from the experimental treatment groups. The chemical stability and sensory properties of fresh meat and processed products manufactured from the experimental treatment groups were compared. Conjugated linoleic acid also demonstrated antioxidant properties in animal feed. Sensory analysis indicated the small effect of dietary CLA supplementation on the sensory properties of fresh and processed pork products. In the case of fresh pork chops and pork patties, dietary CLA supplementation had a stabilizing effect on the a*-value of the products. The lipid stability of pork patties was improved by dietary CLA supplementation as indicated by TBARS values. Salami from the CLA groups was firmer. That could be ascribed to the fat hardening effect of CLA. Pork and pork products enriched with CLA can be considered functional foods and even ânutraceuticalsâ with positive effects on human health. South African pig producers may therefore consider marketing CLA enriched pork products as a health food. The potential advantages and the premium that can be earned on such meat has to be balanced against the reality of increased feed cost.

South Africaâs economy is primarily driven by the utilization of coal to provide electricity, which results in more fossil fuels to be burnt that contributes towards global warming. The average daily temperature is estimated to rise between 1.1 to 6.4ËC by 2100. Carbon sequestration is a technology that can limit CO2 emission into the atmosphere by storing the CO2 away in oceans or the terrestrial subsurface. South Africa is focusing on geological storage at depths of 1 000 m. Limited scientific knowledge is available on the direct impact when large amounts of supercritical CO2 is injected into the subsurface. This includes the diversity of the deep subsurface microbial communities as well as their ecosystems and biogeochemical processes. The main aim of this project was to use selected deep subsurface micro-organisms (T. scotoductus, Geobacillus sp. GE-7 and Geobacillus sp. A12) and an organism that was known to grow under pressure (E. limosum) and introduce them to CCS conditions using a high pressure syringe incubator system. The identities of the selected micro-organisms were verified using molecular techniques, the genomes of these micro-organisms were retrieved and information regarding possible CO2 fixation pathways was verified using the Metacyc database collection. The CO2 fixation pathways of interest were the Calvin cycle, the reductive acetyl Co-enzyme A and the reductive citric acid cycles. Surprisingly, T. scotoductus and E. limosum were able to remain viable and metabolically active even at 100 bar and 100% CO2. This has never been previously reported in literature. However Geobacillus sp. GE-7 and Geobacillus sp. A12 could not remain viable when the pressure was increased from 2 bar to 20 bar or higher. The outcomes of this study indicate that the interactions between supercritical CO2 and the subsurface organisms should be considered as biogeochemical cycling. However, these interactions in the subsurface are still relatively unknown and the availability of interactive metabolic pathways indicate that the subsurface communities could survive and interact with this introduced substrate.

Multi-subunit protein complexes are involved in many essential biochemical processes including signal transduction, protein synthesis, RNA synthesis, DNA replication and protein degradation. An accurate description of the relative structural arrangement of the constituent sub-units in such complexes is crucial for an understanding of the molecular mechanism of the complex as a whole. Many complexes, however, lie in the mega-Dalton range, and are not amenable to X-ray crystallographic or Nuclear Magnetic Resonance analysis. Techniques that are suited to structural studies of such large complexes, such as cryo-electron microscopy, do not provide the resolution required for a mechanistic insight. Mass spectrometry (MS) has increasingly been applied to identify the residues that are involved in chemical cross-links in compound protein assemblies, and have provided valuable insight into the molecular arrangement, orientation and contact surfaces of sub-units within such large complexes. This approach is known as MS3D, and involves the MS analysis of cross-linked di-peptides following the enzymatic cleavage of a chemically cross-linked complex. A major challenge of this approach is the identification of the cross-linked di-peptides in a composite mixture of peptides, as well as the identification of the residues involved in the cross-link. These analyses require bioinformatics tools with capabilities beyond that of general, MS-based proteomic analysis software. Many MS3D software tools have appeared, often designed for very specific experimental methods. We review all major MS3D bioinformatics programs currently available, considering their applicability to different workflows, specific experimental requirements, and the computational approach taken by each. We also developed AnchorMS, a new bioinformatics tool for the identification of both the sequences and cross-linked residues of di-peptides within a post-digest peptide mixture based on MS1 and MS2 data. AnchorMS is intended as a component in the workflow of an MS3D experiment where the protein sequences, cross-linking reagent and protease are known. AnchorMS is freely available as a public web service at cbio.ufs.ac.za/AnchorMS via a simple, user-friendly web interface coded in PHP/XHTML. Experimental sample preparation information and MS data may be uploaded through the web form and analysed by AnchorMS. After analysis, the web interface displays the di-peptides detected, as well as the calculated maximum inter-residue distance between crosslinked residues. This distance information can be used in the optimization of sub-unit positioning within structural models using third party software. The computational core of AnchorMS was developed as an open-source Python project. We describe in detail the overall structure and workflow of the code as well as the functionality implemented in each section of the code. AnchorMS creates a digital library of possible di-peptides and generates expected precursor and fragment mass spectra for each. In order to identify di-peptides, the observed mass spectra are matched against the library of expected mass spectra. Features that are unique to AnchorMS are highlighted, including those for the analysis of di-peptides where the sequences are identical, but the cross-linked residues differ. AnchorMS considers their possible co-fragmentation and employs a specialised second score for distinguishing between such precursors. A unique mathematical model for estimating the level of false positive matching was derived based on an in silico simulation of false positive spectrum matching using randomly generated di-peptide sequences. Subsets of the simulation data were modelled using disparate functions, which were subsequently combined to yield a composite model that described expected false matching under various conditions. The refined calibration of this model against simulation data was performed using the R programming language. AnchorMS also implemented this model as a dynamic false positive threshold, where score values greater than the threshold were considered likely to be true spectrum matches.

Avibacterium paragallinarum is an avian pathogen that causes the upper respiratory disease Infectious coryza (IC) in chickens. This disease has the ability to cause vast economic losses due to a decrease in egg production. To date the factors contributing to pathogenicity, immunogenicity and serotyping are still not clearly understood. Vaccine failures are a major problem that occurs due to no or poor cross-protection occurring especially between the C-serovars of A. paragallinarum. This problem will be overcome by having a more accurate serotyping technique available for the diagnosis of IC. Therefore one of the aims of this study was the development of a molecular serotyping technique, where a serotyping PCR was developed which distinguished between the Modesto (C-2) and SA-3 (C-3) A. paragallinarum isolates which is the major cause of IC in South Africa. Reported in a recently published article was the presence of a HP2-like and Mu-like phage within the genome of the Modesto (C-2) strain of A. paragallinarum. Therefore another major question addressed during this study was whether there are prophage genes present in all of the reference isolates as well as in field isolates of A. paragallinarum and what the effect of these phages might be on the virulence and pathogenicity of these isolates. Phage genes that are important during lysogeny were selected and screened for by means of PCR. From the results it was able to determine that some of these genes are present in some of these isolates but no discernible patterns were detected in terms of the effect on pathogenicity. Therefore future studies will be conducted to mainly focus on the effect these phages might have on the virulence and pathogenicity as well as whether these phages are responsible for the occurrence of different A. paragallinarum serovars.

Fermentation has been used for over three thousand years as an effective and inexpensive means to preserve the quality and safety of foods. In Africa most of the traditional foods are fermented at household level before consumption. The cereals are fermented, and then cooked prior to consumption and this offers added safety advantages because pathogenic microorganisms are inactivated thereby increasing shelf life of product. However, some cereals are cooked first and then other sources of fermentable carbohydrates and amylase such as other cereals (malts) or roots from Rhynchosia spp. (generally called munkoyo roots) are added and then fermented prior to consumption. Chibwantu and munkoyo beverages, Zambiaâs important cereal based fermented foods are prepared in such a way. The substrates added after cooking of cereals are a major concern in terms of product quality and safety. In literature there is limited information on munkoyo roots. Knowledge on quality of these roots, their effect on quality and microbiological safety of the fermented food products prepared by their use is essential for the development of improved products for increased consumption, commercial production and marketing. The present research work aimed at contributing to closing up this knowledge gap by evaluating the microbiological quality and safety of chibwantu. This was done by first gathering information on the production and utilisation of indigenous cereal based fermented food products in Lusaka and Chongwe districts of Lusaka province, Zambia with special emphasis on chibwantu and munkoyo beverages prepared using munkoyo roots. Second to isolate and characterize the microorganisms associated with the munkoyo roots and maize grit used during the preparation of chibwantu and to establish whether the same microorganisms are involved during the fermentation process of the beverage. Third to study the survival of selected food borne enteropathogenic microorganisms during fermentation of chibwantu beverage and finally to investigate the effect of munkoyo roots on growth of selected microorganisms. From the information gathered during this study, it was found that different types of indigenous cereal based fermented foods especially the beverages are prepared in Zambian households. The foods contribute to the nutritional, economical and medical needs of the consumers, thereby making them relevant to the consumers and the country at large. The raw materials; maize grit and munkoyo roots, used in the preparation of the beverage chibwantu are associated with coliforms, lactic acid bacteria (LAB), yeasts and moulds, particularly the munkoyo roots. The groups of microorganisms; coliforms, LAB, Yeasts and moulds, associated with the raw materials used in the preparation of the beverage chibwantu, particularly from the roots, were also responsible for the fermentation of the beverage. Some microorganisms that could not be identified from the raw materials were also encountered during the fermentation process of chibwantu. Staphylococcus aureus, E. coli, Salmonella and Shigella species were not encountered during natural fermentation of chibwantu and growth of selected enteropathogenic microorganisms was significantly suppressed during the fermentation process and storage. Both the yellow and white munkoyo roots possess some antimicrobial activity against some enteropathogenic microorganisms as well as microbial growth stimulatory activity. This study forms basis for further studies on the active components of the roots for possible use in preservation for improved food quality and safety. The quality of the beverage is varied due to different methods of preparation and the microorganisms involved during fermentation which are dependent on the quality of the munkoyo roots. There is still a lot of work needed on the munkoyo roots and the fermented products prepared from them to be able to establish their safety convincingly. However, fermented foods generally have a very good safety record and chibwantu should be relatively safe to consume.

Aflatoxins are carcinogenic mycotoxins produced by certain species of the Aspergilli, with the most prominent producers being Aspergillus flavus and A. parasiticus. Exposure to aflatoxins causes liver cancer, immune suppression, retardation in growth and in extreme cases, even death. A. parasiticus and A. flavus infect a wide range of crops, including corn, cotton, peanuts and tree nuts. Consequently, aflatoxin contamination has a severe impact on public health, as well as the agricultural sector. Several control strategies are currently in place to combat aflatoxin contamination. Although great progress has been made in developing innovative methods for aflatoxin control, no strategy is completely efficient in eliminating aflatoxin contamination. Also, the application of the strategies is often limited by practical and economic factors. This necessitates the development of further aflatoxin control strategies. A yet under-exploited resource is the aflatoxin biosynthesis pathway. Aflatoxins are synthesised in a complex polyketide-initiated pathway that requires multiple enzymatic steps. The genes encoding the aflatoxin biosynthetic enzymes are located in a cluster which contains both the regulatory and structural genes. A novel approach to aflatoxin control is the direct inhibition of a target enzyme in the aflatoxin biosynthesis pathway. An ideal candidate is the Baeyer-Villiger monooxygenase (BVMO), MoxY, that catalyses the conversion of hydroxyversicolorone to versiconal hemiacetal acetate. BVMOs are flavin-containing proteins that catalyse the oxidation of ketones or cyclic ketones to esters or lactones, respectively. BVMOs occur only in the genomes of bacteria and fungi, rendering the MoxY enzyme as a suitable candidate for targeted enzyme inhibition. In silico analysis of the moxY gene indicated that MoxY may exist with an elongated N-terminus or an alternative C-terminus, due to alternative splicing of the mRNA. In addition to cloning the moxY gene, variations of the moxY gene were created that encode a protein with both the alternative termini or either an alternative N-terminus or an alternative C-terminus. The MoxY variants were heterologously expressed in E. coli and MoxY with an elongated N-terminus (MoxYAltN) was found to be the only active recombinant form of the MoxY enzyme. MoxYAltN is promiscuous with regard to the substrate accepted, converting linear, aromatic, substituted aromatic and bicyclic ketones. MoxYAltN was purified and the kinetic parameters determined with respect to the reaction with a surrogate substrate, phenylacetone. Purified MoxYAltN was demonstrated to convert synthetic [1â-2H]hydroxyversicolorone to versiconal hemiacetal acetate, confirming the role of MoxYAltN in the aflatoxin biosynthesis pathway. Intragenomic complementation of an impaired aflatoxin biosynthetic step is a common theme that has emerged from the experimental study of the aflatoxin biosynthetic pathway. Therefore, the complementation of the activity of MoxY(AltN) by another BVMO in the A. flavus genome was investigated. The genome contains 26 putative BVMOs and phylogenetic analysis indicted that six of these are closely-related to MoxY, which were selected for heterologous expression in E. coli. Three of the homologues expressed as soluble proteins and activity was observed for two. The substrate profiles of the homologues differ significantly from that of MoxYAltN, with the only common substrate converted being (±)-cis-bicyclo[3.2.0]hept-2-en-6-one. The enantio- and regioselective conversion of (±)-cis-bicyclo[3.2.0]hept-2-en-6-one by BVMOs is a well-characterised reaction. Chiral analysis indicated divergent biocatalytic profiles of MoxYAltN and the two homologues with respect to the conversion of (±)-cis-bicyclo[3.2.0]hept-2-en-6-one, which suggests that the substrate binding pockets of the enzymes are likely to differ. The ability of the homologues to complement the activity of MoxY(AltN) by converting [1â-2H]hydroxyversicolorone remains uncertain. However, the differential substrate profiles demonstrate that phylogenetic clustering is not an absolute indication of overlapping biocatalytic abilities. Therefore, more distantly related BVMOs in A. flavus have to be considered as candidates for intragenomic complementation as well.

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: ï· To examine the presence of qac resistance genes in field isolates and determining if the number of genes present confer higher resistance. ï· To study the expression of one of these qac resistance genes in the presence of increasing QAC concentration. ï· 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.