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 âblebâ 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.

Opuntia ficus-indica, the prickly pear cactus, is well adapted for cultivation in arid and semi-arid regions, with a yield of 10 to 40 tonnes (dry wt) cladode biomass per ha. The cladodes (the âleavesâ, which in fact are the stems) might serve as lignocellulosic biomass feedstock for second generation bioethanol production, without competing for agricultural land or replacing significant natural vegetation. The main objective of this study was to investigate the feasibility of bioethanol production from an enzymatic hydrolysate of O. ficus-indica cladodes. The potential of a Kluyveromyces marxianus isolate UOFS Y-2791, a yeast capable of utilising a wider range of carbon substrates and of ethanol production at higher temperatures than Saccharomyces cerevisiae, was investigated for bioethanol production using an O. ficus-indica cladode enzymatic hydrolysate as feedstock. S. cerevisiae UOFS Y-0528, a wine yeast strain, was used as benchmark. Compositional analysis of the cladode biomass indicated that it had a low lignin content of 8% (dry wt). The content of readily fermentable carbohydrates in the cladode, which was 34.3 g per 100 g dry biomass of which 23 g was glucose, was comparable to other conventional biomass feedstocks such as sugar cane bagasse and corn stover, whereas it had a low xylose content. By applying a statistical design experimental approach where acid concentration and contact time were varied, optimum conditions for dilute acid pretreatment of the dried and milled cladode were determined to be 1.5% (w/w) sulphuric acid for 50 min at a temperature of 120oC and a dry biomass loading of 30% (w/v). Enzymatic hydrolysis experiments were performed with varied enzyme loadings of cellulase and β-glucosidase with or without the addition of pectinase, and the enzyme loadings chosen were 15 FPU cellulase, 15 IU β-glucosidase and 100 IU pectinase per gram of dry biomass. These parameters yielded an O. ficus-indica hydrolysate containing (per litre) 45.5 g glucose, 6.3 g xylose, 9.1 g galactose, 10.8 g arabinose and 9.6 g fructose. Using a chemically-defined medium with a sugar composition similar to the hydrolysate as benchmark, K. marxianus and S. cerevisiae were grown in the O. ficus-indica hydrolysate at 40oC and 35oC, respectively, under non-aerated conditions, whereas the performance of K. marxianus was also investigated under oxygen-limited conditions where the DOT was controlled at less than 1% saturation. The fermentation profiles of both yeasts were compared using separate hydrolysis and fermentation (SHF) and simultaneous hydrolysis and fermentation (SSF) process configurations, at a water-insoluble solids (WIS) content of 14%. Both yeasts achieved comparable ethanol yields in SHF and SSF under nonaerated conditions, although K. marxianus exhibited a lower volumetric ethanol productivity than S. cerevisiae. K. marxianus, cultivated under oxygen-limited conditions, achieved a lower ethanol yield than both yeasts cultivated without aeration. However, K. marxianus exhibited the highest volumetric ethanol productivity of 2.3 g l-1 h-1 and 1.57 g l-1 h-1 in SHF and SSF, respectively, although the ethanol produced was assimilated upon hexose depletion. K. marxianus utilised galactose poorly in the absence of aeration, but completely consumed the sugar under oxygen-limited conditions. The overall ethanol productivity of SSF was double that of SHF. An ethanol concentration of 20.6 g l-1; the highest concentration achieved in this study, was an improvement on the 14 g l-1 previously reported elsewhere. This study provided more information on the chemical composition of the O. ficus-indica cladode, particularly regarding its constituent carbohydrates, and also highlighted the feasibility of ethanol production from the cladodes, albeit at low concentrations from an industrial point of view. K. marxianus demonstrated its potential as an alternative to S. cerevisiae for bioethanol production from lignocellulosic biomass.

information indicate that CYP450s are prevalent in members of the bacterial phylum Deinococcus-Thermus as well as the archaeal family Halobacteriaceae that belong to the phyulm Euryarchaeota. A property shared by these phylogenetically distant extremophiles is the production of carotenoid pigments. It became the purpose of this study to use genome sequence information to clone and study new CYP450s from the genera Thermus and Halobacterium and to explore the role of these CYP450s in pigment production. The non-pigmented thermophilic bacterium Thermus scotoductus SA-01 was screened by PCR for the presence of a cytochrome P450 monooxygenase (CYP450). No CYP450 could be found and subsequent genome sequencing confirmed this finding. However, a CYP450 gene (CYP175A) was isolated from the closely related yellow pigmented strain Thermus sp. NMX2.A1 using oligonucleotides based on the DNA sequence of the β-carotene gene cluster from three Thermus strains. The genome sequence of T. scotoductus SA-01, revealed a ferredoxin (Fdx) and ferredoxin reductase (FNR) that were almost identical to those of Thermus thermophilus HB27. In T. thermophilus HB27 the Fdx and FNR are the native redox partners for CYP175A1, a β- carotene hydroxylase. After heterologous expression in Escherichia coli, we attempted to hydroxylate β-carotene with the CYP450 from Thermus sp. NMX2.A1 and the redox partners of T. scotoductus SA-01 using cell free extracts, but no products were detected. Thirty two CYP450s have been identified in the sequenced genomes of thirteen extremely halophilic archaea. Initial attempts to clone and heterologously express a CYP174A2- homologue from a Haloarcula LK-1 strain in E. coli and Pseudomonas fluorescens were unsuccessful. In order to study the physiological role of CYP450s in halophilic archaea and to create a strain that can be used for heterologous expression of CYP450s from halophiles CYP174A1 was deleted from H. salinarum R1. CYP174A1 is the only CYP450 in H. salinarum R1 and H. salinarum R1 is a genetically tractable strain. Upon culturing the wildtype and deletion strains, a difference in red pigmentation of stationary phase cultures was observed; implying that CYP174A1 might play a role in carotenoid synthesis. Microarray analyses revealed that the bop gene, which codes for bacterioopsin (BO) was severely repressed in stationary phase cultures of the deletion strain and sucrose gradient experiments showed a consequent loss of purple membrane (PM) in the deletion strain. The classical causes of bop repression e.g. insertion elements in the bop open reading frame as well as in the brz gene was ruled out by PCR screening. In addition to bop repression, the neighboring vng1459 and vng1468 genes (both part of the bopregulon) were also down regulated, but the genes normally involved in regulation of the bop gene were not affected. Currently the functions of vng1459 and vng1468 are unknown. Retinal, together with BO, is a key component of bacteriorhodopsin (BR) and essential for PM synthesis. Retinal is formed by the central cleavage of β-carotene which can be achieved by monooxygenases or dioxygenases.The Blh and Brp proteins in H. bacterium salinarum are very closely related to a confirmed bacterial 15,15â²-β-carotene dioxygenase and studies have shown that deletion of both brp and blh results in complete abolishment of retinal and BR. It is therefore unlikely that CYP174A1 plays a role in retinal biosynthesis. Another possible function for CYP174A1 might be the hydroxylation of β-carotene, since it is known that H. salinarum strains produce hydroxylated carotenoids such as transastaxanthin, but no genes encoding typical β-carotene hydroxylases or ketolases have been identified in the genomes of H. salinarum strains. This will imply that hydroxylated carotenoids play a role in the regulation of bop.

Candida albicans and C. dubliniensis are commensals of the gastrointestinal and genitourinary tract in healthy individuals. However, in diseased individuals they can cause superficial infections to deep seated mycoses. Both species form mycelial networks called biofilms, and formation of biofilms results in increased resistance towards antifungal compounds currently in use. Therefore, there is a need for alternative antifungal compounds such as fatty acids. Research has shown that supplementation of growth medium with polyunsaturated fatty acids (PUFAs), increased the unsaturation index and made cells susceptible to lipid peroxidation and cell death. During this study this phenomenon was evaluated on biofilms of C. albicans and C. dubliniensis using selected PUFAs. Due to differences in the carbon chain length and saturation of fatty acids, they interact differently with the cell membrane and will have different peroxidisability values. The results from the study showed C18:4 n-3 and C20:5 n-3 were taken in by the cell and resulted in increased unsaturation index. The results further indicated oxidative stress-induced apoptosis following supplementation with C18:4 n-3 and C20:5 n-3 in biofilms of both C. albicans and C. dubliniensis. The induction of apoptosis following supplementation by C18:4 n-3 and C20:5 n-3 was confirmed by mitochondrial membrane potential assay, Annexin V-FITC staining, TUNEL assay and DAPI staining. The use of C18:4 n-3 in synergism with amphotericin B resulted in decreased dosage of the antifungal compound needed to inhibit biofilms of C. albicans and C. dubliniensis.

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.

The screening, biomass production of lipase-producing microorganisms from several sources, as well as the purification, characterization and utilization of the enzymatic extracts for the interesterification of butter fat were investigated. Pseudomonas fragi CRDA 323 and Aspergillus niger CBS 131.52 were considered to be good lipase producers, whereas, those from Pseudomonas putida ATCC 795 and Rhizopus oryzae ATCC 34612 as weak ones; all four microorganisms produced maximal amount of extracellular lipases by batch fermentation after three-four days of incubation in a continuously stirred tank reactor. The lipases were partially purified by ammonium sulfate precipitation and characterized with respect to pH, kinetic parameters and molecular size. The lipases from P. fragi and P. putida were optimal at pH 8.5 and 8.0, respectively, whereas those from A. niger and R. oryzae were optimal at pH 7.5. The A. niger lipase had the lowest V$ sb{max}$ value $ rm(0.51 times 10 sp3 U min sp{-1});$ R. oryzae the highest $ rm (1.86 times 10 sp3 U min sp{-1}).$ The K$ sb{m}$ values for P. fragi, P. putida, A. niger and R. oryzae lipases were 0.70, 1.18, 0.97 and 0.98 mg ml$ sp{-1},$ respectively. Interesterification of butter fat by the partially purified enzymatic extracts in a microemulsion free co-surfactant system containing sorbitol monostearate and polyoxyethylene sorbitan monostearate in the ratio 48:52 (V/V) decreased the water activity as well as the hydrolytic activity. The P. fragi lipase had the highest interesterification yield value (43%) and the R. oryzae lipase the lowest (4%). In addition, P. fragi lipase exhibited the highest decrease (18%) in long-chain hypercholesterolemic fatty acids (C12:0, C14:0 and C16:0) at the sn-2-position; the P. putida lipase demonstrated the least favorable changes in specificity at the same position. Continuous cultivation technique was developed to investigate the screening for lipase-producing microorganisms from four commercial

Blue Stilton is a blue-veined cheese manufactured in a restricted area of the UK, using lactic starter cultures plus a secondary culture of Penicillium roquefotti. The aim of this study was to determine the change in microflora during ripening of the cheese and to investigate potential microbial interactions. Additionally, the volatile compounds present in mature samples of cheeses exhibiting few blue veins were compared with those in good quality cheeses, showing ample blue veining. Experiments on cheeses from a single dairy, monitored during the ripening process, showed that the total Lactobacillus count increased from levels of around 103 cfu g-1 on day one to around 107 cfu g-1 after 8 weeks of ripening. This is comparable to values found in other cheeses including both mould-ripened and non mould-ripened varieties. Yeast counts were generally higher than those found in other cheeses and also increased to levels in the region of 107 g-1. The total viable count (TVC) decreased from around 109 g-1 initially, reflecting the presence of the starter bacteria, to 107 g.1, suggesting a decline in the starter bacteria similar to that found in other cheeses. Mature cheeses always exhibited similar numbers of microorganisms although the species varied between cheeses. High quality, mature, cheeses were compared with sub-standard cheeses from the same production site. The predominant species of lactobacilli in good quality cheeses were Lb. plantarum and Lb. curvatus, whereas in poor quality cheeses Lb. brevis predominated. This corresponded to the results of gas chromatography-olfactometry, which indicated the presence of fruity off flavours in poor quality cheeses. Several strains of these species were isolated, as indicated by differing capabilities in utilisation of a range of carbon sources. Yeast species also varied between good and poor quality cheeses with Candida sphaerlca and C. catenulata predominating in good cheeses and C. famata, C. lipolytica and C. catenulata also occurring in both good and poor quality samples. Strain differences were observed by the biochemical profiles and two strains of C. famata demonstrated inhibitory effects against P. roqueforti when incubated under anaerobiosis. It was concluded that these strains may affect the development of blue veins in Stilton cheese when maturation conditions encourage their proliferation. Comparisons were made between samples of cheeses from several Stilton producers and the results suggested that although the levels of the groups of microorganisms tested were similar, the species of lactobacilli and yeasts present were different. This suggests that the indigenous microflora may have a significant impact on the flavour of cheeses from individual production sites. It was concluded that the microflora of Blue Stilton cheese may have a significant impact on the quality of the product both in terms of flavour and the development of the blue veins.

No description available.

An understanding of the movement of bacteria in soil is of importance in many areas of microbial ecology. The study of bacterial dispersal is of fundamental importance in understanding the dissemination of soil-borne plant pathogens and symbionts as well as human pathogens introduced into soil. In addition, the increasing interest in the use of bacterial inocula for improved plant nutrition, biological control of plant pathogens and bioremediation of contaminated soils, necessitates the study of movement of such inocula, both to optimise their function and determine their fate in the environment. The fate of inocula is of particular interest when such inocula are non-indigenous (genetically-modified or otherwise), where their impact on the environment outwith the target site is uncertain. Intact soil microcosms were used in the study of factors affecting the movement of bacterial inocula through soils in the presence of percolating water. Two ecophysiologically contrasting bacterial species (<i>Pseudomonas fluorescens</i> and <i>Bacillus subtilis</i>) were used as inocula. The strains were genetically marked with <i>lux</i> genes encoding bioluminescence and with antibiotic resistance markers (chromosomal integrations), these traits were used in the selective enumeration of the introduced bacteria against the indigenous soil populations. The effect of soil type on the leaching of <i>P. fluorescens</i> inocula was investigated using intact soil microcosms sampled from contrasting soils: Craibstone (loamy sand), Insch (sandy loam) and Cruden Bay (clay loam). It was found that cells of the inoculum were leached more rapidly, in greater numbers and over a longer period from the clay loam, than the two lighter textured soils. These differences were attributed to the interaction of the inocula with percolating water, as determined by the respective soil characteristics. Leaching of <i>B. subtilis</i> differed from that of <i>P. fluorescens</i>, in that the rate of movement through soil was slower and the number of colony forming units leached was less. It was found that the colony forming units of <i>B. subtilis</i> leached were predominantly in the form of spores. The differences in leaching of <i>B. subtilis</i> and <i>P. fluorescens</i> were attributed to the greater adsorption of vegetative cells of <i>B. subtilis</i> to the soils and the requirement for formation of spores prior to leaching.

No description available.