Title

建立高效能液相層析電化學分析法檢測胺基酸利用以鑑別多重抗藥性Salmonella Typhimurium之研究

Translated Titles

The Use of High Performance Liquid Chromatography- Electrochemical (HPLC-EC) Detection for Analysis of Amino Acid Utilization to Identify Multidrug-Resistant (MDR) Salmonella Typhimurium

Authors

吳慕澤

Key Words

多重抗藥性鼠傷寒沙門氏菌 ; Multidrug-resistant Salmonella Typhimurium

PublicationName

中興大學微生物暨公共衛生學研究所學位論文

Volume or Term/Year and Month of Publication

2014年

Academic Degree Category

碩士

Advisor

張照勤

Content Language

繁體中文

Chinese Abstract

多重抗藥性沙門氏菌的感染在人類和動物為重要的新興問題,然而傳統微生物學對於檢測多重抗藥沙門氏菌的方法相對來說比較耗時,例如以紙錠擴散法及最低抑菌濃度針對ampicillin、chloramphenicol、streptomycin、sulfonamides及tetracyclines (ACSSuT)五大類藥物做檢測。胺基酸是維持細菌生存重要的生化分子,參與DNA的複製、合成細菌毒力蛋白等。本研究目的是藉由胺基酸的利用率,透過高液相層析電化學建立一個快速診斷多重抗藥性(multidrug-resistant;MDR) S. Typhimurium之研究。本研究先使用八株S. Typhimurium(四株為MDR及四株為non-MDR)菌株,再搭配ROC曲線統計分析的評估,做為前驅試驗以篩選有潛力區辨MDR之胺基酸。結果顯示八種胺基酸,包括serine、aspartic acid、lysine、asparagine、tyrosine、tryptophan、glycine及phenylalanine,可能具有區別多重抗藥性分離株的能力。進一步使用164株MDR和27株non-MDR的S. Typhimurium進行比較,結果顯示只有serine、aspartic acid、lysine、tryptophan及phenylalanine的利用,可有效鑑別出多重抗藥性的S. Typhimurium。最後,使用多變項邏輯式迴歸模式,建構同時以此五個胺基酸利用結果組合的最佳預測方程式,計算為多重抗藥性菌株勝算之log值,並以此log值作為評估是否為多重抗藥性菌之整體指標。結果證明,此診斷方法當SI值為1.62來診斷MDR S. Typhimurium菌株,具有很高的敏感度(86%)和特異度(96%)。未來可針對其它人畜共通沙門氏菌血清型菌株進行研究,以評估該方法的廣泛使用性。

English Abstract

Multidrug-resistant (MDR) Salmonella infections become emerging issues in humans and animals. However, the traditional microbiological methods to determine MDR strains, which are time-consuming, need to apply disk diffusion or minimum inhibitory concentration methods to test resistance of ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracyclines (ACSSuT). Amino acids are major biochemical molecules to maintain bacterial survival, as they participate in DNA replication and synthesis of virulent proteins. The objective of this study was to establish a quick method to identify MDR Salmonella strains through analysis of the utilization of amino acids by high performance liquid chromatography electrochemistry (HPLC-EC). The preformance of this diagnostic method was evaluated by receiver operating characteristic curve (ROC). A total of eight S. Typhimurium strains (4 MDRs and 4 non-MDRs) were used for a pilot test. It was identified that utilization of eight amino acids, including serine, aspartic acid, lysine, asparagine, tyrosine, tryptophan, glycine and phenylalanine could be potentially useful to identify MDR isolates. Further using 164 MDR and 27 non-MDR S. Typhimurium isolates for comparison, the result indicated that utilization of serine, aspartic acid, lysine, tryptophan and phenylalanine was significantly associated with MDR isolates. Finally, using multiple logistic regression model, an equation to calculate logarithm of odds being a MDR strain as the summarized index (SI) was constructed by combinations of utilization of these five amino acids. The result showed that the established diagnostic method to identify MDR S. Typhimurium was with high sensitivity (86%) and specificity (96%) at the cut-off value of 1.62 of SI. Futher studies need to be conducted in other zoonotic Salmonella serovars to evaluate usefulness of this method.

Topic Category 醫藥衛生 > 基礎醫學
醫藥衛生 > 預防保健與衛生學
獸醫學院 > 微生物暨公共衛生學研究所
Reference
  1. 2. Uygur E, Reddy K, Ozkan FU, Soylemez S, Aydin O, et al. (2013) Salmonella enteridis Septic Arthritis: A Report of Two Cases. Case Rep Infect Dis 2013: 642805.
    連結:
  2. 3. Nakaya H, Yasuhara A, Yoshimura K, Oshihoi Y, Izumiya H, et al. (2003) Life-threatening infantile diarrhea from fluoroquinolone-resistant Salmonella enterica typhimurium with mutations in both gyrA and parC. Emerg Infect Dis 9: 255-257.
    連結:
  3. 4. Threlfall EJ (2000) Epidemic salmonella typhimurium DT 104--a truly international multiresistant clone. J Antimicrob Chemother 46: 7-10.
    連結:
  4. 6. Wiegand I, Hilpert K, Hancock RE (2008) Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3: 163-175.
    連結:
  5. 7. Andrews JM (2001) Determination of minimum inhibitory concentrations. J Antimicrob Chemother 48 Suppl 1: 5-16.
    連結:
  6. 8. French GL (2006) Bactericidal agents in the treatment of MRSA infections--the potential role of daptomycin. J Antimicrob Chemother 58: 1107-1117.
    連結:
  7. 9. Jorgensen JH, Ferraro MJ (2009) Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clin Infect Dis 49: 1749-1755.
    連結:
  8. 10. Young VR (1994) Adult amino acid requirements: the case for a major revision in current recommendations. J Nutr 124: 1517S-1523S.
    連結:
  9. 12. Jagannathan S, Forsyth TP, Kettner CA (2001) Synthesis of boronic acid analogues of alpha-amino acids by introducing side chains as electrophiles. J Org Chem 66: 6375-6380.
    連結:
  10. 13. Wu G (2009) Amino acids: metabolism, functions, and nutrition. Amino Acids 37: 1-17.
    連結:
  11. 14. Di Camillo B, Eduati F, Nair SK, Avogaro A, Toffolo GM (2014) Leucine modulates dynamic phosphorylation events in insulin signaling pathway and enhances insulin-dependent glycogen synthesis in human skeletal muscle cells. BMC Cell Biol 15: 9.
    連結:
  12. 15. Wu G, Meier SA, Knabe DA (1996) Dietary glutamine supplementation prevents jejunal atrophy in weaned pigs. J Nutr 126: 2578-2584.
    連結:
  13. 16. Wang J, Chen L, Li P, Li X, Zhou H, et al. (2008) Gene expression is altered in piglet small intestine by weaning and dietary glutamine supplementation. J Nutr 138: 1025-1032.
    連結:
  14. 17. Mohamed A, Deng X, Khuri FR, Owonikoko TK (2014) Altered glutamine metabolism and therapeutic opportunities for lung cancer. Clin Lung Cancer 15: 7-15.
    連結:
  15. 18. Wu G, Bazer FW, Davis TA, Kim SW, Li P, et al. (2009) Arginine metabolism and nutrition in growth, health and disease. Amino Acids 37: 153-168.
    連結:
  16. 19. Bronte V, Zanovello P (2005) Regulation of immune responses by L-arginine metabolism. Nat Rev Immunol 5: 641-654.
    連結:
  17. 20. Jobgen W, Meininger CJ, Jobgen SC, Li P, Lee MJ, et al. (2009) Dietary L-arginine supplementation reduces white fat gain and enhances skeletal muscle and brown fat masses in diet-induced obese rats. J Nutr 139: 230-237.
    連結:
  18. 21. Fu WJ, Haynes TE, Kohli R, Hu J, Shi W, et al. (2005) Dietary L-arginine supplementation reduces fat mass in Zucker diabetic fatty rats. J Nutr 135: 714-721.
    連結:
  19. 22. Jobgen W, Fu WJ, Gao H, Li P, Meininger CJ, et al. (2009) High fat feeding and dietary L-arginine supplementation differentially regulate gene expression in rat white adipose tissue. Amino Acids 37: 187-198.
    連結:
  20. 23. Meijer AJ (2003) Amino acids as regulators and components of nonproteinogenic pathways. J Nutr 133: 2057S-2062S.
    連結:
  21. 24. Brosnan JT (2001) Amino acids, then and now--a reflection on Sir Hans Krebs' contribution to nitrogen metabolism. IUBMB Life 52: 265-270.
    連結:
  22. 25. Shi W, Meininger CJ, Haynes TE, Hatakeyama K, Wu G (2004) Regulation of tetrahydrobiopterin synthesis and bioavailability in endothelial cells. Cell Biochem Biophys 41: 415-434.
    連結:
  23. 26. Wu G, Meininger CJ (2002) Regulation of nitric oxide synthesis by dietary factors. Annu Rev Nutr 22: 61-86.
    連結:
  24. 27. Li X, Bazer FW, Gao H, Jobgen W, Johnson GA, et al. (2009) Amino acids and gaseous signaling. Amino Acids 37: 65-78.
    連結:
  25. 28. Wu G, Fang YZ, Yang S, Lupton JR, Turner ND (2004) Glutathione metabolism and its implications for health. J Nutr 134: 489-492.
    連結:
  26. 29. Nakashima K, Yakabe Y, Ishida A, Yamazaki M, Abe H (2007) Suppression of myofibrillar proteolysis in chick skeletal muscles by alpha-ketoisocaproate. Amino Acids 33: 499-503.
    連結:
  27. 31. Suryawan A, O'Connor PM, Bush JA, Nguyen HV, Davis TA (2009) Differential regulation of protein synthesis by amino acids and insulin in peripheral and visceral tissues of neonatal pigs. Amino Acids 37: 97-104.
    連結:
  28. 32. MacLennan PA, Brown RA, Rennie MJ (1987) A positive relationship between protein synthetic rate and intracellular glutamine concentration in perfused rat skeletal muscle. FEBS Lett 215: 187-191.
    連結:
  29. 33. MacLennan PA, Smith K, Weryk B, Watt PW, Rennie MJ (1988) Inhibition of protein breakdown by glutamine in perfused rat skeletal muscle. FEBS Lett 237: 133-136.
    連結:
  30. 34. Coeffier M, Claeyssens S, Hecketsweiler B, Lavoinne A, Ducrotte P, et al. (2003) Enteral glutamine stimulates protein synthesis and decreases ubiquitin mRNA level in human gut mucosa. Am J Physiol Gastrointest Liver Physiol 285: G266-273.
    連結:
  31. 35. Wu G, Thompson JR (1987) Ketone bodies inhibit leucine degradation in chick skeletal muscle. Int J Biochem 19: 937-943.
    連結:
  32. 36. Rivlin R, Asper SP (1966) Tyrosine and the thyroid hormones. Am J Med 40: 823-827.
    連結:
  33. 37. Maarsingh H, Pera T, Meurs H (2008) Arginase and pulmonary diseases. Naunyn Schmiedebergs Arch Pharmacol 378: 171-184.
    連結:
  34. 38. Elitsur Y, Moshier JA, Murthy R, Barbish A, Luk GD (1992) Polyamine levels, ornithine decarboxylase (ODC) activity, and ODC-mRNA expression in normal and cancerous human colonocytes. Life Sci 50: 1417-1424.
    連結:
  35. 39. Meijer AJ, Dubbelhuis PF (2004) Amino acid signalling and the integration of metabolism. Biochem Biophys Res Commun 313: 397-403.
    連結:
  36. 41. Griffith RS, Norins AL, Kagan C (1978) A multicentered study of lysine therapy in Herpes simplex infection. Dermatologica 156: 257-267.
    連結:
  37. 42. Flodin NW (1997) The metabolic roles, pharmacology, and toxicology of lysine. J Am Coll Nutr 16: 7-21.
    連結:
  38. 43. Ogier de Baulny H, Saudubray JM (2002) Branched-chain organic acidurias. Semin Neonatol 7: 65-74.
    連結:
  39. 44. van Spronsen FJ, van Rijn M, Bekhof J, Koch R, Smit PG (2001) Phenylketonuria: tyrosine supplementation in phenylalanine-restricted diets. Am J Clin Nutr 73: 153-157.
    連結:
  40. 45. Wu G (1997) Synthesis of citrulline and arginine from proline in enterocytes of postnatal pigs. Am J Physiol 272: G1382-1390.
    連結:
  41. 46. Tedeschi PM, Markert EK, Gounder M, Lin H, Dvorzhinski D, et al. (2013) Contribution of serine, folate and glycine metabolism to the ATP, NADPH and purine requirements of cancer cells. Cell Death Dis 4: e877.
    連結:
  42. 47. Dai ZL, Li XL, Xi PB, Zhang J, Wu G, et al. (2013) L-Glutamine regulates amino acid utilization by intestinal bacteria. Amino Acids 45: 501-512.
    連結:
  43. 48. Pruss BM, Nelms JM, Park C, Wolfe AJ (1994) Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids. J Bacteriol 176: 2143-2150.
    連結:
  44. 49. Chaussee MS, Somerville GA, Reitzer L, Musser JM (2003) Rgg coordinates virulence factor synthesis and metabolism in Streptococcus pyogenes. J Bacteriol 185: 6016-6024.
    連結:
  45. 51. Moses S, Sinner T, Zaprasis A, Stoveken N, Hoffmann T, et al. (2012) Proline utilization by Bacillus subtilis: uptake and catabolism. J Bacteriol 194: 745-758.
    連結:
  46. 52. Wecke J, Perego M, Fischer W (1996) D-alanine deprivation of Bacillus subtilis teichoic acids is without effect on cell growth and morphology but affects the autolytic activity. Microb Drug Resist 2: 123-129.
    連結:
  47. 53. Belitsky BR, Brill J, Bremer E, Sonenshein AL (2001) Multiple genes for the last step of proline biosynthesis in Bacillus subtilis. J Bacteriol 183: 4389-4392.
    連結:
  48. 54. Burnside K, Lembo A, de Los Reyes M, Iliuk A, Binhtran NT, et al. (2010) Regulation of hemolysin expression and virulence of Staphylococcus aureus by a serine/threonine kinase and phosphatase. PLoS One 5: e11071.
    連結:
  49. 55. Stickland LH (1935) Studies in the metabolism of the strict anaerobes (genus Clostridium): The oxidation of alanine by Cl. sporogenes. IV. The reduction of glycine by Cl. sporogenes. Biochem J 29: 889-898.
    連結:
  50. 56. Stickland LH (1935) Studies in the metabolism of the strict anaerobes (Genus Clostridium): The reduction of proline by Cl. sporogenes. Biochem J 29: 288-290.
    連結:
  51. 57. Bouillaut L, Self WT, Sonenshein AL (2013) Proline-dependent regulation of Clostridium difficile Stickland metabolism. J Bacteriol 195: 844-854.
    連結:
  52. 58. Li P, Yin YL, Li D, Kim SW, Wu G (2007) Amino acids and immune function. Br J Nutr 98: 237-252.
    連結:
  53. 59. Tan B, Li XG, Kong X, Huang R, Ruan Z, et al. (2009) Dietary L-arginine supplementation enhances the immune status in early-weaned piglets. Amino Acids 37: 323-331.
    連結:
  54. 60. Van Brummelen R, du Toit D (2007) L-methionine as immune supportive supplement: a clinical evaluation. Amino Acids 33: 157-163.
    連結:
  55. 61. Grimble RF (2006) The effects of sulfur amino acid intake on immune function in humans. J Nutr 136: 1660S-1665S.
    連結:
  56. 62. Witthoft T, Eckmann L, Kim JM, Kagnoff MF (1998) Enteroinvasive bacteria directly activate expression of iNOS and NO production in human colon epithelial cells. Am J Physiol 275: G564-571.
    連結:
  57. 63. Melchior D, Le Floc'h N, Seve B (2003) Effects of chronic lung inflammation on tryptophan metabolism in piglets. Adv Exp Med Biol 527: 359-362.
    連結:
  58. 64. Liu Z, Dai H, Wan N, Wang T, Bertera S, et al. (2007) Suppression of memory CD8 T cell generation and function by tryptophan catabolism. J Immunol 178: 4260-4266.
    連結:
  59. 65. Schwarcz R, Whetsell WO, Jr., Mangano RM (1983) Quinolinic acid: an endogenous metabolite that produces axon-sparing lesions in rat brain. Science 219: 316-318.
    連結:
  60. 66. Platten M, Ho PP, Youssef S, Fontoura P, Garren H, et al. (2005) Treatment of autoimmune neuroinflammation with a synthetic tryptophan metabolite. Science 310: 850-855.
    連結:
  61. 67. Ha EM, Oh CT, Bae YS, Lee WJ (2005) A direct role for dual oxidase in Drosophila gut immunity. Science 310: 847-850.
    連結:
  62. 68. Tattoli I, Sorbara MT, Vuckovic D, Ling A, Soares F, et al. (2012) Amino acid starvation induced by invasive bacterial pathogens triggers an innate host defense program. Cell Host Microbe 11: 563-575.
    連結:
  63. 70. (1950) ESSENTIAL amino acid requirements for maintenance and for rehabilitation in rats. Nutr Rev 8: 248-250.
    連結:
  64. 71. Kilberg MS, Pan YX, Chen H, Leung-Pineda V (2005) Nutritional control of gene expression: how mammalian cells respond to amino acid limitation. Annu Rev Nutr 25: 59-85.
    連結:
  65. 72. Palii SS, Kays CE, Deval C, Bruhat A, Fafournoux P, et al. (2009) Specificity of amino acid regulated gene expression: analysis of genes subjected to either complete or single amino acid deprivation. Amino Acids 37: 79-88.
    連結:
  66. 73. Kimball SR, Jefferson LS (2006) New functions for amino acids: effects on gene transcription and
    連結:
  67. translation. Am J Clin Nutr 83: 500-507.
    連結:
  68. 74. Brasse-Lagnel C, Fairand A, Lavoinne A, Husson A (2003) Glutamine stimulates argininosuccinate synthetase gene expression through cytosolic O-glycosylation of Sp1 in Caco-2 cells. J Biol Chem 278: 52504-52510.
    連結:
  69. 75. Huang YF, Wang Y, Watford M (2007) Glutamine directly downregulates glutamine synthetase protein levels in mouse C2C12 skeletal muscle myotubes. J Nutr 137: 1357-1362.
    連結:
  70. 77. Sahai A, Pan X, Paul R, Malladi P, Kohli R, et al. (2006) Roles of phosphatidylinositol 3-kinase and osteopontin in steatosis and aminotransferase release by hepatocytes treated with methionine-choline-deficient medium. Am J Physiol Gastrointest Liver Physiol 291: G55-62.
    連結:
  71. 81. Zen JM, Hsu CT, Senthil Kumar A, Lyuu HJ, Lin KY (2004) Amino acid analysis using disposable copper nanoparticle plated electrodes. Analyst 129: 841-845.
    連結:
  72. 82. Casella IG, Gatta M (2001) Determination of electroactive organic acids by anion-exchange chromatography using a copper modified electrode. J Chromatogr A 912: 223-233.
    連結:
  73. 83. Yu H, Ding YS, Mou SF, Jandik P, Cheng J (2002) Simultaneous determination of amino acids and carbohydrates by anion-exchange chromatography with integrated pulsed amperometric detection. J Chromatogr A 966: 89-97.
    連結:
  74. 84. Chou CC, Lin SP, Lee KM, Hsu CT, Vickroy TW, et al. (2007) Fast differentiation of meats from fifteen animal species by liquid chromatography with electrochemical detection using copper nanoparticle plated electrodes. J Chromatogr B Analyt Technol Biomed Life Sci 846: 230-239.
    連結:
  75. 86. Brooks K, Sodeman T (1974) A rapid method for determining decarboxylase and dihydrolase activity. J Clin Pathol 27: 148-152.
    連結:
  76. 87. Thibault P, Le Minor L (1957) [Simple methods of demonstration of lysine decarboxylase and tryptophan desaminase on media for rapid differentiation of Enterobacteriaceae]. Ann Inst Pasteur (Paris) 92: 551-554.
    連結:
  77. 1. Botti V, Navillod FV, Domenis L, Orusa R, Pepe E, et al. (2013) Salmonella spp. and antibiotic-resistant strains in wild mammals and birds in north-western Italy from 2002 to 2010. Vet Ital 49: 195-202.
  78. 5. Cipriani S, Masci M, Tacconi R, Marsili G (1979) [Comparative study of bacterial sensitivity to antibiotics with the "abac" automatic system and the agar diffusion disk method]. Ann Sclavo 21: 635-645.
  79. 11. Rama Rao PB, Metta VC, Norton HW, Johnson BC (1960) The amino acid composition and nutritive value of proteins. III. The total protein and the nonessential amino nitrogen requirement. J Nutr 71: 361-365.
  80. 30. Tischler ME, Desautels M, Goldberg AL (1982) Does leucine, leucyl-tRNA, or some metabolite of leucine regulate protein synthesis and degradation in skeletal and cardiac muscle? J Biol Chem 257: 1613-1621.
  81. 40. Civitelli R, Villareal DT, Agnusdei D, Nardi P, Avioli LV, et al. (1992) Dietary L-lysine and calcium metabolism in humans. Nutrition 8: 400-405.
  82. 50. Vining LC, Magasanik B (1981) Serine utilization by Klebsiella aerogenes. J Bacteriol 146: 647-655.
  83. 69. Palusiak A, Maciejewska A, Lugowski C, Rozalski A (2014) The amide of galacturonic acid with lysine as an immunodominant component of the lipopolysaccharide core region from Proteus penneri 42 strain. Acta Biochim Pol.
  84. 76. Leong HX, Simkevich C, Lesieur-Brooks A, Lau BW, Fugere C, et al. (2006) Short-term arginine deprivation results in large-scale modulation of hepatic gene expression in both normal and tumor cells: microarray bioinformatic analysis. Nutr Metab (Lond) 3: 37.
  85. 78. Levenberg B, Hartman SC, Buchanan JM (1956) Biosynthesis of the purines. X. Further studies in vitro on the metabolic origin of nitrogen atoms 1 and 3 of the purine ring. J Biol Chem 220: 379-390.
  86. 79. Ashoor SH, Monte WC, Stiles PG (1988) Liquid chromatographic identification of meats. J Assoc Off Anal Chem 71: 397-403.
  87. 80. Ashoor SH, Osman MA (1988) Liquid chromatographic quantitation of chicken and turkey in unheated chicken-turkey mixtures. J Assoc Off Anal Chem 71: 403-405.
  88. 85. Goldschmidt MC, Lockhart BM (1971) Rapid methods for determining decarboxylase activity: arginine decarboxylase. Appl Microbiol 22: 350-357.
  89. 88. Yu SY, Chang CC (2013) Epidemiology of Antimicrobial Resistant in Various Salmonella Serovars with ramR/ramA Mutation. Unpublised master’s dissertation, National Chung-Hsing University, Taichung, Taiwan.