Salmonella species continue to be commonly associated with cases of food-borne disease in developed countries. In the United States in 2001, the incidence per 100,000 people was highest for salmonellosis (15.1), followed by campylobacteriosis (13.8) and shigellosis (6.4). Enteric pathogens usually contaminate the surface of raw animal products during slaughter and primary processing (scalding, defeathering or dehiding, rinsing, cutting, mixing, and grinding, etc.) and can attach and/or reside in the regular and irregular surfaces of the skin, multiply and, thereafter, contaminate food preparation surfaces, hands and utensils. Trisodium phosphate (TSP) has been approved by the USDA as a sanitizer to reduce surface loads of Salmonella on chicken carcasses. A number of studies had demonstrated that TSP effectively removes surface contamination of carcasses by food-borne pathogens. However, very little scientific evidence is available which identifies the actual mechanisms of TSP antimicrobial activity and the response of food-borne pathogens exposed to TSP. This study examined both the physiological and molecular response of Salmonella enterica serovar Enteritidis to TSP treatment. The role of high pH during TSP treatment on its antimicrobial activity was examined. Adaptation of S. enterica serovar Enteritidis to TSP treatment was also examined by analyzing the proteome of serovar Enteritidis cells using two-dimensional gel electrophoresis and mass spectrometry. The role of high pH on the antimicrobial activity of TSP was examined using comparative studies involving treatment solutions containing different concentrations of TSP, treatment solutions adjusted to the equivalent pH as in each of the TSP treatments and TSP solutions pH adjusted to 7.0. Direct and indirect indices of cell survival, membrane damage, and cellular leakage were also employed to examine specific antimicrobial effects. Cell viability, loss of membrane integrity, cellular leakage, release of lipopolysaccharides and cell morphology were accordingly examined and quantified under the above treatment conditions. Exposure of serovar Enteritidis cells to TSP or equivalent alkaline pH made with NaOH resulted in the loss of cell viability and membrane integrity in a TSP concentration- or NaOH-alkaline pH-dependent manner. In contrast, cells treated with different concentrations of TSP whose pH was adjusted to 7.0 did not show any loss of cell viability or membrane integrity. These results indicate that TSP is a potent membrane-acting agent, and provide compelling evidence that high pH during TSP treatment was responsible for its antimicrobial activity. Adaptation of S. enterica serovar Enteritidis with a sublethal concentration of TSP resulted in the induction of the alkaline stress response. Alkaline stress response involves induced thermotolerance, resistance to higher concentrations of TSP, high pH and sensitivity to acid. Examination of the proteome of TSP-adapted cells revealed differential expression of a number of proteins but did not include the common heat shock proteins involved in thermotolerance. However, TSP adaptation caused a shift in the membrane fatty acid composition from unsaturated to a higher saturated and cyclic fatty acid. This shift in fatty acid composition increases the melting point of the cytoplasmic membrane so that it remains functional at high temperatures. Biofilm bacteria are more resistant to sanitizers, heat and antimicrobial agents than their planktonic counterparts. Examination of the proteome of TSP-adapted biofilm cell of S. enterica serovar Enteritidis revealed little overlap in the protein profile compared to TSP-adapted planktonic cells. Proteomic examination of planktonic and biofilm cells of S. enterica serovar Enteritidis revealed differential expression of a number of proteins involved in DNA replication, stress survival and transport of newly synthesized proteins. These results clearly indicate that changes in the expression of specific genes are involved in the biofilm mode of growth, which could play a significant role in resistance to antimicrobial agents. The results of the current study provide a better understanding of the mechanisms of antimicrobial action of TSP and also elucidate the response of S. enterica serovar Enteritidis to TSP and high pH adaptation. The study also raises new questions regarding stress tolerance of S. Enteritidis following TSP or alkaline pH adaptation with relevance to food safety.

The fish disease epidemiology is urgent to be investigated for the surveillance and prevention. The diseased fish showed splenomegaly with diffusion of white nodules and microscopical granulomatous formation. It is important to develop a method of pathogens isolated from clinical samples with serial dilution method and disc diffusion method. Representative colonies were selected from diseased cobia on BHIA plate and were inoculated onto MacConkey agar, TCBS agar, and blood agar. The cage-culture of the different bacterial groups detected in the survey of bacteria isolated from THOD, HDSB, EMD with serial dilution method. 119 from 128 isolated strains were Gram¡¦s negative (93%), including pathogenic Vibrio spp. 57% (73/128) in THOD. 54 from 90 isolated strains were Gram¡¦s negative (60%), including pathogenic Vibrio spp. 12.2% (11/90) in HDSB. 61 from 104 isolated strains were Gram¡¦s negative (59%), including pathogenic Vibrio spp. 70.2% (77/90) in EMD. In different times diseased grouper, 104 from 139 isolated strains were Gram¡¦s negative (75%), including pathogenic Vibrio spp. 88% (123/139), in 2003. While 24 from 44 isolated strains were Gram¡¦s negative (55%), including pathogenic Vibrio spp. 73% (32/44), in 2004. 66 from 75 isolated strains were Gram¡¦s negative (88%), including pathogenic Vibrio spp. 97% (73/75) by disc dilution method in EMD. 9 from 31 isolated strains were Gram¡¦s negative (30%), including pathogenic Vibrio spp. 26% (8/31), by disc dilution method of grouper in PCG. A DGGE (denaturing gradient gel electrophoresis) technique can identify six groups of bacteria from cobia, and J6, R13, T29 have similarity 100%. Quantity One Version 4.5 (Bio-Rad) can identify six groups of bacteria from diffusion methods that F group diluted the bacterial strain from serial dilution method. B group and E group diluted the bacterial strain from disc diffusion method. Higher resistance rates of the different bacterial strains isolated from cobia and were £]-lactam and susceptible were observed in quinolones.

The cyanide-degradation bacteria Klebsiella oxytoca SYSU-110 was isolated from the waste water of a metal-plating plant in southern Taiwan. K. oxytoca can utilize many nitrile compounds [including acetonitrile (100 mM), benzonitrile (1 mM), butyronitrile (100 mM), glutaronitrile (50 mM), methacrylnitrile (100 mM), phenylacetonitrile (1 mM), propionitrile (25 mM), succinonitrile (25 mM) and valeronitrile (50 mM)] as its sole nitrogen source. In this study, we found out that K. oxytoca was capable of degrading acetonitrile and propionitrile. Frome GC analysis, we recognized amide was an intermediate compound, while the carboxylic acid and ammonia were the final end-products. Therefore, we presume that K. oxytoca biodegraded nitrile compounds by two enzymes, the nitrile hydratase and amidase. We also analyzed the total cell proteins by 2-D polyacrylamide gel electrophoresis after the cells were cultured in medium containing 25mM succinonitrile. There were 23 proteins could be induced or overexpressed by nitrile and we had identified 11 by Mascot Peptide mass Fingerprint and Blast. Six proteins that can protect the cells from oxidative damage are: superoxide dismutase, glutathione s-transferase, dyp-type peroxidase, metal binding protein PsaA (that can transport metal ions into the cells), LraI, and FepA (used to transport inorganic ions into the cells). Three enzymes glutamine synthetase, methylenetetrahydrofolate reductase,¡@and dihydroxyacid dehydratase were used to synthesize amino acids. One protein was identified as ribosomal protein L9. The last identified protein is nucleoside triphosphates kinase which can convert nucleoside diphosphates to nucleoside triphosphates non-specifically. From the activity analysis, superoxide dismutase and glutathione S-transferase activities were escalated when the cells were cultured in 25mM succinonitrile, and the concentration of ROS has rise. These results suggested that succinonitrile could cause oxidative damage to the cells and induce some anti-oxidative damage proteins to protect them.

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Detection trace compound from human serum by matrix-assited laser desorption ionization mass spectrometry and second dimensional gel ectrophoresis

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Metaproteomics, as a proteomic approach to analyse environmental samples, is a new and expanding field of research. The field promises new ways of determining the status of the organisms present in a sample, and could provide additional information compared to metagenomics. Being a novel field of research, robust methods and protocols have not yet been established. In this thesis, we examine several methods for a reliable extraction of protein from soil and periphyton samples. The extraction should preferably be fast, compatible with downstream analysis by mass spectrometry and extract proteins in proportion to their presence in the original sample. A variety of methods and buffers were used to extract proteins from soil and periphyton samples. Concentration determinations showed that all of these methods extracted enough protein for further analysis. For purification and digestion of the samples, several methods were used. The purified samples were analysed on three different mass spectrometers, with the Orbitrap Velos Pro delivering the best results. The results were matched against four genomic and metagenomic databases for identification of proteins, of which the UniProt/SwissProt database gave the best result. A maximum of 52 proteins were identified from periphyton samples when searching against UniProt/SwissProt with strict settings, of which the majority were highly conserved proteins. The main limitation for this type of work is currently the lack of proper metagenomic databases.

In this thesis we look for potential chronic inflammation biomarkers because studies have shown that farmers with musculoskeletal disorders might be affected by the environment to develop musculoskeletal disorders. Animal farmers are highly exposed to dust, aerosols, molds and other toxins in the air and environment leading to musculoskeletal disorders, respiratory disorders, airway symptoms and febrile reactions. There is reason to believe that the farmers have a constant or chronic inflammation that develops into musculoskeletal disorders. By using a proteomic approach with Two-dimensional Gel Electrophoresis and silver staining our goal was to find biomarkers by quantifying protein spots that differ significantly from farmers with musculoskeletal disorders compared to rural controls. In our study we found 8 significant proteins, two from Alpha-2-HS-glycoprotein, one from Apolipoprotein A1, three from Haptoglobin, one from Hemopexin and 1 from Antithrombin. All 5 proteins are involved in inflammation response in some way and some proteins are linked to chronic inflammation. Out of the 5 proteins Alpha-2-HS-glycoprotein, Apolipoprotein A1 and Hemopexin seem like the most likely proteins to investigate further as potential inflammation biomarkers.

A method for the selective concentrating of DNA targets using capillary affinity gel electrophoresis is presented. Complementary ssDNA targets are retained through hybridization with oligonucleotide probes immobilized within polyacrylamide gels while non-complementary targets are removed. The captured DNA targets were concentrated by step elution, where a localized thermal zone was applied in small steps along the capillary. Evaluation of the selective capture of a 150 nt DNA target in a complicated mixture was carried out by factorial analysis. Gels with a smaller average pore size were found to retain a higher amount of complementary targets. This was thought to be due to the ssDNA target migrating through the gel by reptation, eliminating hairpin structures, making the complementary region of the target available for hybridization. This method was applied to a series of DNA targets of different lengths, 19 nt, 150 nt, 250 nt and 400 nt. The recovery of the method ranged from 0.5 to 4% for the PCR targets, and 13 to 18% for the 19 nt oligonucleotide target. The purity was calculated to be up to 44% for the PCR targets and up to 86% for the 19 nt target. This was an improvement in purity of up to 15 times and 1100 times in comparison to the original samples for the PCR targets and 19 nt oligonucleotide, respectively. The 19 nt targets were selective concentrated and delivered into a microfluidic based DNA biosensing platform. The purity of the sample improved from 0.01% to 50% while recovery decreased from 100% to 20% for a sample with 0.5 nM complementary and 1 μM non-complementary targets. An improvement in the response of the sensing platform was demonstrated on 19 nt oligonucleotide targets delivered by selective concentration versus concentration alone into the microfluidic biosensing system.