臺灣博碩士論文加值系統

為了解自來水中退伍軍人菌於37℃或受熱60℃時之反應，本研究利用培養法及螢光染色法評估退伍軍人菌之可培養性、細菌活性及總細菌濃度之變化，並以此二種方法探討受熱細菌與兩種原蟲宿主- A. castellanii 和H. vermiformis共同培養3日內細菌之變化。除此短期觀察外，另以即時定量聚合酶連鎖反應法取代螢光染色法評估與原蟲共存27日之細菌總濃度變化，藉此了解退伍軍人菌的增生能力，細菌之可培養性亦同步評估。此外，在27日觀察期中，也探討以加熱（60℃,30分鐘）處理的兩種原蟲是否可成為退伍軍人菌營養源，及其對此菌可培養性與增生能力之影響。
結果顯示，退伍軍人菌（108 cells/ml）於60℃受熱5分鐘與30分鐘後，可培養濃度分別下降了4 log CFU/ml與6 log CFU/ml，而細胞膜完整性則分別下降56 %與83 %，但未造成總細菌濃度之改變，故可知受熱60℃主要影響細菌的可培養性。而37℃之退伍軍人菌與原蟲共同培養的3日內，總細菌濃度最大增生量可達2.5 log cells/ml，且以經A. castellanii之增生能力較高。當細菌在60℃受熱達30分鐘時，即使經兩種原蟲培養，72小時內仍無法觀察到具可培養性之細菌，總細菌濃度亦無增生現象；但與H. vermiformis共同培養時卻可觀察到受熱60℃的退伍軍人菌出現活性增生的情況。
27日長期觀察的結果則顯示，37℃ 5分鐘之退伍軍人菌若僅與兩種原蟲營養源共存時，營養源比例高低與營養源種類對細菌之增生能力無顯著影響，且可培養細菌濃度亦隨觀察時間而下降；但若退伍軍人菌與高濃度營養源(細菌：營養源=1:100)共存1日後再與活性A. castellanii及H. vermiformis宿主共同培養時，退伍軍人菌總濃度則出現上升現象。當將受熱60℃ 5分鐘退伍軍人菌直接與A. castellanii共同培養時，亦發現其總細菌濃度在27日內最高可上升至6 log cells/ml，且較經H. vermiformis共同培養的退伍軍人菌有較高的增生能力。但若僅有營養源而無原蟲宿主時，反而出現總細菌濃度下降之現象，且營養源濃度越高下降情形更為顯著；即使於營養源共存1或3日後亦加入活性原蟲宿主，退伍軍人菌總濃度仍趨於下降，且下降幅度亦隨營養源濃度增加而提高；至於可培養性方面，受熱於60℃之細菌僅在立即與原蟲共同培養時可偵測到具可培養性之細菌。
整體而言，受熱溫度顯著影響退伍軍人菌之可培養性與細胞膜完整性，且以可培養性所受之影響較大；受熱細菌與原蟲宿主共同培養三日之結果顯示，增加共同培養與受熱時間可分別顯著提高與降低細菌之增生性、可培養性與細胞膜完整性(p < 0.05與 < 0.001)；而在27日共同培養的觀察中發現，營養源與受熱溫度皆為顯著抑制退伍軍人菌總濃度(p < 0.001)與可培養濃度 (p < 0.001) 之主要因子。而不論觀察期間之長短，原蟲種類均顯著影響退伍軍人菌的總濃度與可培養濃度，且A. castellanii均優於H. vermiformis。

In this study, conventional standard culture method and fluorescent LIVE/DEAD BacLight staining assay were performed for evaluating the culturability, viability and total cell concentration of Legionella pneumophila after heat exposure (37℃ and 60℃) in tap water. Heated L. pneumophila in 3-day coculture with A. castellanii and H. vermiformis, respectively, were also evaluated. Real-time PCR assay was used to determine total concentration of L. pneumophila in 27-day coculture with amoebae; culturability was evaluated simultaneously. Moreover, we investigated the culturability and proliferation ability of L. pneumophila while serving the heat treated (60℃, 30 min) A. castellanii and H. vermiformis as the nutrient during this time period.
First, we found that after exposure to 60℃ for 5 and 30 min ,culturable L. pneumophila concentration reduced 4 and 6 log CFU/mL, and the loss of cell membrane integrity was 56 % and 83 %, respectively. However, the total concentration was not affected. These results indicate heat exposure decreases the culturability of L. pneumophila significantly.
Second, heated L. pneumophila (37℃) could proliferate to the maximum of total concentration at 2.5 log cells/mL in coculture with amoebae in 3 days. Higher proliferation ability was observed in coculture with A. castellanii. After 30 min exposure at 60℃ for 30min culturable L. pneumophila was not detected and no cellular proliferation was found in coculture with amoebae. However, increase in cellular membrane integrity of L. pneumophila was observed in H. vermiformis coculture.
Third, in the 27 observation days without coculture with viable amoebae, both nutrient contents and types of amoebae used as nutrients presented no significant effect on proliferation ability of 37℃ L. pneumophila. Also, the culturability of L. pneumophila decreased over time period. However, total concentration of L. pneumophila increased in high levels of nutrients when adding viable A. castellanii. As for 60℃ heated L. pneumophila, total concentration of L. pneumophila showed 6 log cells/ml increase when cocultured with A. castellanii, and its reproducibility is higher than that cocultured with H. vermiformis. When L. pneumophila grew with nutrient alone without viable amoebae as hosts, total concentration of L. pneumophila was decreasing, and higher nutrient contents caused more decrease. Even amoebic hosts added after L. pneumophila co-existed with nutrients for 1 day or 3 days, total concentration of L. pneumophila still decreased. And higher nutrient concentration would cause more decrease. On the other hand, the culturability of 60℃ heated L. pneumophila can be detected only when cocultured with amoebae immediately right after heating treatment.
In conclusion, the culturability and membrane integrity of L. pneumophila were affected by heating temperature. Results showed that higher temperature would significantly decrease the producibility, culturability, and membrane integrity of heated L. pneumophila cocultured with protozoa for 3 days (p<0.05). However, the longer coculture time with amoebae would significantly increase the producibility, culturability, and membrane integrity of L. pneumophila (p<0.001). And we revealed that nutrients (heated amoebae) and heating temperature are the most important factors which significantly decrease the total and culturable concentration of L. pneumophila in 27-day coculture period (p<0.001). No matter how long the period was observed, amoebic genera significantly affect the total and culturable concentration of L. pneumophila.

第一章 前言 1
第二章 文獻回顧 3
2.1 嗜肺性退伍軍人菌（Legionella pneumophila）與退伍軍人症（Legionnaires’ disease） 3
2.1.1. L. pneumophila生理特性 3
2.1.2. 環境中的L. pneumophila 3
2.2. L. pneumophila 加熱消毒法 4
2.3. 阿米巴原蟲 (amoebae) 5
2.3.1. Acanthamoeba castellanii 5
2.3.2. Hartmannella vermiformis 6
2.3.3. 原蟲對熱處理之耐受溫度 9
2.3.4. L. pneumophila與原蟲互動機制 11
2.4. 細菌生理觀察指標 12
2.4.1. 可培養性 13
2.4.2. 細胞膜完整性 13
2.4.3. 宿主互動下的細菌增生 14
第三章 研究目的 15
第四章 研究架構 17
4.1加熱處理對L.pneumophila之影響 17
4.2 加熱處理對L. pneumophila與不同原蟲共同培養之短期影響 18
4.3 加熱處理對L. pneumophila與不同原蟲共同培養之長期影響 19
第五章 材料與方法 21
5.1. 實驗菌種 21
5.1.1 Legionella 21
5.1.2 Amoebae 21
5.2. 培養基製備 21
5.2.1 BCYE α agar 21
5.2.2 ATCC medium 712 22
5.2.3 ATCC medium 1034 23
5.3. L. pnuemophila加熱實驗 23
5.3.1 無菌自來水之製備 23
5.3.2 加熱實驗 23
5.4. L. pneumophila與原蟲共同培養 24
5.4.1 Page’s Amoeba Saline (PAS)緩衝液製備 25
5.5. 加熱處理之原蟲 25
5.6 加熱時對L. pneumophila之影響評估 26
5.6.1 可培養濃度與可培養性 26
5.6.2 總濃度、活性濃度與細胞膜完整性 26
5.6.3 細菌增生性 27
5.7 QA/QC 28
5.7.1 加熱殺菌時水浴槽溫度之調控 28
5.7.2 L. pneumophila加熱實驗中的起始濃度 29
5.7.3 以60℃加熱30分鐘後原蟲之觀察 29
5.7.4 qPCR檢量線與複製效率 30
5.7.5 原蟲種類與濃度對以qPCR分析退伍軍人菌之影響 30
5.8. 統計分析 30
第六章 結果 31
6.1 加熱處理對L. pneumophila之影響 31
6.1.1 總細菌濃度、可培養細菌濃度與可培養性 31
6.1.2 活性細菌濃度與細胞膜完整性 31
6.2 加熱處理對退伍軍人菌與原蟲共同培養之短期影響 37
6.2.1 L. pneumophila 在 A. castellanii 之表現 37
6.2.2 L. pneumophila在H. vermiformis之表現 44
6.3 以qPCR及BacLight-Microscopy定量總細菌數之比較 50
6.3.1 與A. castellanii共同培養 50
6.3.2 經H. vermiformis共同培養 52
6.4 加熱處理對退伍軍人菌與原蟲共同培養之長期影響 53
6.4.1 37℃暴露之L. pneumophila與A. castellanii 53
6.4.2 暴露於60℃之L. pneumophila與A. castellanii 59
6.4.3 與A. castellanii共同培養之統計檢定結果 62
6.4.4 37℃與60℃結果之比較(A. castellanii) 65
6.4.5 37℃暴露之L. pneumophila 與 H. vermiformis 74
6.4.6 60℃暴露之L. pneumophila與H. vermiformis 80
6.4.7 與H. vermiformis共同培養之統計檢定結果 82
6.4.8 37℃與60℃結果之比較(H. vermiformis) 85
6.4.9 L. pneumophila 在 A. castellanii與H. vermiformis之比較 91
6.5 QA/QC 94
6.5.1 L. pneumophila與原蟲共同培養之感染濃度比與原蟲外L. pneumophila之移除率 94
6.5.2 加熱殺菌時水浴槽溫度之調控 94
6.5.3 L. pneumophila於加熱實驗中之起始濃度 94
6.5.4 以60℃加熱30分鐘後原蟲之觀察 96
6.5.5 60℃30分鐘加熱原蟲對L. pneumophila體內增生之影響 100
6.5.6 qPCR檢量線與複製效率 102
6.5.7 原蟲種類與濃度對以qPCR分析退伍軍人菌之影響 102
第七章 討論 105
7.1 加熱處理對L. pneumophila可培養性、總濃度與活性細菌濃度之影響 105
7.2 加熱處理對L. pneumophila與原蟲共同培養之短期影響 106
7.3 加熱處理對L. pneumophila與原蟲共同培養之長期影響 107
7.4 加熱處理之原蟲做為L. pneumophila之營養源時，其對L. pneumophila及與原蟲宿主互動之影響 108
7.5 60℃加熱殺菌之成效 110
7.5.1 無原蟲存在 110
7.5.2 有原蟲存在 110
7.6 QA/QC 111
7.6.1 L. pneumophila與原蟲共同培養之感染濃度比與原蟲外L. pneumophila之移除 111
7.6.2 原蟲受熱60℃ 30分鐘後之表現 112
第八章 結論與建議 113
第九章 參考文獻 115
第十章 附錄 123

1.Abu Kwaik, Y. 1996. The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum. Appl Environ Microbiol 62: 2022- 2028.
2.Abu Kwaik Y., Fields, B. S., and Engleberg, N.C. 1994. Protein expression by the protozoan Hartmannella vermiformis upon contact with its bacterial parasite Legionella pneumophila. Infect. Immune. 62:1860-1866.
3.Bender, L., Ott, M., Marre, R., and Hacker, J. 1990. Genome analysis of Legionella spp. by orthogonal field alternation gel electrophoresis (OFAGE). FEMS Microbiol. Let. 60: 253-257.
4.Boulos L., Prévost M., Barbeau B., Coallier J., and Desjardins R. 1999. Live/Dead® BacLightTM : application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water.
5.Bozue, J.A., and Johnson, W. 1996. Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome±lysosome fusion. Infect Immun 64: 668- 673.
6.Breeuwer, P. and Abee, T. 2000. Assessment of viability of microorganisms employing fluorescence techniques. Inter. J. Food. Microbiol. 55:193-200.
7.Chang, C. W., Hwang, Y. H., Cheng, W. Y., and Chang, C. P. 2007. Effects of chlorination and heat disinfection on long-term starved Legionella pneumophila in warm water. J. Appl. Microbiol. 102:1636-1644.
8.Clarke, D. W., Alizadeb, H., and Niederkorn, J. Y. 2005. Failure of Acanthamoeba castellanii to prosuce intraocular infection. Invest. Ophthalmol. Vis. Sci. 46:2472-2478.
9.Colbourne, J. S., Pratt, D. J., Smith, M. G., Fischer-Hock, S. P., and Harper, D. 1984. Water fittings as sources of Legionella pneumophila in a hospital plumbing system. Lancet i :210-213.
10.De Jonckheere JF. 2003. Epidemiological typing of Acanthamoeba strains isolated from keratitis cases in Belgium. Bull. Soc. Belge. Ophtalmol. 287: 27-33.
11.Dennis, P. J., Green, D., and Jones, B. P. C. 1984. A note on the temperature tolerance of Legionella. J. Appl. Bacteriol. 56:349-350.
12.Donlan, R. M., Forster, T., Murga, R., Brown, E., Lucas, C., Carpenter, J., and Fields, B. 2005. Legionella pneumophila associated with the protozoa Hartmanella vermiformis in a model multi-species biofilm has reduced susceptibility to disinfectants. Biofouling. 21:1-7.
13.Dowsett, R. J., Dowsett, A. B, Dennis, P. J., Lee, J. V., and Keevil C.W. 1994. Influence of temperature and plumbing material selection on biofilm formation and growth of Legionella pneumophila in a model potable water system containing complex microbial flora. Appl. Environ. Environ. 60:1585-1592.
14.Gant, V. A., Warnes, G., Philips, I., and Savidge, G. F. 1993. The application of flow cytometry to the study of bacterial responses to antibiotics. J. Med. Microbiol. 39: 147-154.
15.Gao, L.-Y., and Abu Kwaik, Y. 2000. The mechanism of killing and exiting the protozoan host Acanthamoeba polyphaga by Legionella pneumophila. Environ Microbiol. 2 :79-90.
16.Gao, L. Y., Susa, M., Ticac, B., and Abu Kwaik, Y. 1999. Heterogeneity in intracellular replication and cytopathogenicity of Legionella pneumophila and Legionella micdadei in mammalian and protozoan cells. Microbial Pathogenesis. 27: 273-287.
17.Gast RK, and Holt PS. 2001. Multiplication in egg yolk and survival in egg albumen of Salmonella enterica serotype Enteritidis strains of phage types 4, 8, 13a, and 14b. J Food Prot. 64:865-868.
18.Gunasekera, T. S., SØrensen, A., Attfield, P. V., SØrensen, S. J., and Veal, D. A. 2002. Inducible gene expression by nonculturable bacteria in milk after pasteurization. Appl. Environ. Microbiol. 68: 1988-1993.
19.Gupte, A. R., Rezende, C. L. E., and Joseph, S. W. 2003. Induction and resuscitation of viable but nonculturable Salmonella enterica serovar Typhimurium DT104. Appl. Environ. Microbiol. 69:6669-6675.
20.Harb, O.S., Venkataraman, C., Haack, B.J., Gao, L.Y., and Abu Kwaik, Y. (1998) Heterogeneity in the attachment and uptake mechanisms of the Legionnaires'' disease bacterium, Legionella pneumophila, by protozoan hosts. Appl Environ Microbiol. 64: 126-132.
21.Hoffmann, R. and Michel, R. 2001. Distribution of free-living amoebae (FLA) during preparation and supply of drinking water. Int. J. Hyg. Environ. Health. 203: 215-219.
22.Jacquemin, L. J., Simitzis-Le Flohic, A. M., Chauveau, N. 1981. Free-living Amoebae in fresh water. Study of the water supply of the town of Poitiers. Bull Soc Pathol Exot Filiales. 74 :521-524.
23.James, B. W., Mauchline, W. S., Dennis, P. J., Keevil, C. W., and Wait R. 1999. Poly-3-phdroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments. Appl. Environ. Microbiol. 65: 822-827.
24.Kilvington S, and White D. G. 1994. Acanthamoeba biology, ecology and human disease. Rev. Med. Microbiol. 5:12-20.
25.Kuchta, J. M., J. S. Navratil, M. E. Shepherd, R. M. Wadowsky, J. N. Dowling, S. J. States, and R. B. Yee. 1993. Impact of chlorine and heat on the survival of Hartmanella vermiformis and subsequent growth of Legionella pneumophila. Appl. Environ. Microbiol. 59: 4096-4100.
26.Kuiper, M. W., Wullinqs, B. A., Akkermans, A. D., Beumer, R. R., and Van Der Kooij, D. 2004. Intracellular proliferation of Legionella pneumophila in Hartmannella vermiformis in aquatic biofilm grown on plasticized polyvinyl chloride. Appl. Environ. Microbiol. 70:6826-6833.
27.Laguna, R. K., Creasey, E. A., Li, Z., Valtz, N., and Isberg, R. R. 2006. A Legionella pneumophila-translocated substrate that is required for growth within macrophages and protection from host cell death. PNAS. 103: 18745-18750.
28.Lee, J. V., and A. A. West. 1991. Survival and growth of Legionella species in the environment. Soc. Appl. Bacteriol. Symp. Ser. 20:121-129.
29.Lleò, M. D. M., Bonato, B., Benedetti, D., and Canepari, P. 2005. Survival of enterococcal species in aquatic environments. FEMS Microbiol. Ecol. 54:189-196.
30.McCulley, J. P., Alizadeh H., and Niederkorn J. Y. 1995. Acanthamoeba keratitis. CLAO J. 21: 73-76.
31.McDougald, D., Rice, S. A., Weichart, D., and Kjelleberg, S. 1998. Nonculturability: Adaptation or debilitation? FEMS Microbiol. Ecol. 25:1-9.
32.Miuetzner, S., Schwille, R. C., Farley, A., Wald, E. R., Ge, J. H., States, S. J., Libert, T., and Wadowsky, R. M. 1997. Efficacy of thermal treatment and copper-silver ionization for controlling Legionalla pneumophila in high-volume hot water plumbing systems in hospitals. AJIC. 25: 452-457.
33.Moffat, J. F. and Tompkins, L. 1992. A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii. Infection and immunity. 60 : 296-301.
34.Nu´ ria Queralt and Rosa Araujo. 2007. Analysis of the Survival of H. pylori Within a Laboratory-based Aquatic Model System Using Molecular and Classical Techniques. Microbiol. Ecol. Online first.
35.Ohno, A., Kato, N., Yamada, N., and Yamaguchi, K. 2003. Factors influencing survival of Legionella pneumophila serotype 1 in host spring water and tap water. Appl. Environ. Microbiol. 69: 2540-2547.
36.James D. Oliver. 2005. The viable but nonculturable state in bacteria. J. Microbiol. 43: 93-100.
37.Paszko-Kolva, C. M. Shahamat, and Colwell, R. R. 1992. Long term survival of Legionella pneumophila serogroup 1 under low-nutrient conditions and associated morphological changes. FEMS Microbiol. Ecol. 102:45-55.
38.Rasmussen R. 2001. Quantification on the LightCycler. In Meuer, S, Wittwer, C, Nakagawara, K, eds. Rapid Cycle Real-time PCR. Methods and Applications Springer Press, Heidelberg: 21-34.
39.Rivera, F., Lares, F., Gallegos, E., Ramirez, E., Bonilla, P., Calderon, A., Martinez, J. J., Rodriguez, S., and Alcocer, J. 1989. Pathogenic amoebae in natural thermal waters of three resorts of Hidalgo, Mexico. 50: 289-295.
40.Rivera F, Ramirez P, Vilaclara G, Robles E, Medina F. 1983. A survey of pathogenic and free-living amoebae inhabiting swimming pool water in Mexico City. Environ Res. 1983. 32: 205-211.
41.Rivera F, Ramírez E, Bonilla P, Calderón A, Gallegos E, Rodríguez S, Ortiz R, Zaldívar B, Ramírez P, Durán A. 1993. Pathogenic and free-living amoebae isolated from swimming pools and physiotherapy tubs in Mexico. Environ Res. 62: 43-52.
42.Rodiguez-Zaragoza, S. 1994. Ecology of free-living amoebae. Crit. Rev. Microbiol. 20: 225-241.
43.Rogers, D. J., Dennis, P. J., Lee, J. V., and Keevil C. W. 1993. Effects of water chemistry and temperature on the survival and growth of Legionella pneumophila in potable water systerm, p248-250. In J. M. Baarbaree, R. F. Breiman, and A. P. Dufour (ed.), Legionella current status and emerging perspective. American society for Microbiology, Washington, D. C.
44.Rohr, U., Weber, S., Michel, R., Selenka, F., and Wilhelm, M. 1998. Comparison of free-living amoebae in hot water systems of hospitals with isolates from moist sanitary areas by identifying genera and determining temperature tolerance. Appl Environ Microbiol. 64:1822-1824.
45.Rowbotham, T. J. 1980. Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J. Clin. Pathol. 33:1179–1183.
46.Sansonetti, P. J., Ryter, A., Clerc, P., Maurelli, A. T., and Mounier, J. 1986. Multiplication of Shigella flexneri within HeLa Cells: Lysis of thePhagocytic Vacuole and Plasmid-Mediated Contact Hemolysis.Infect. Immun. 51:461-469.
47.Sawyer, T. K. 1989. Free-living pathogenic and non-pathogenic amoebae in Maryland soils. Appl. Environ. Microbiol. 55: 1074-1077.
48.Seno, M., Sakaki, M., Ogawa, H., Matsuda, H., and Takeda, Y. 2006. Effective proliferation of low level Legionella pneumophila serogroup 1 cells using coculture procedure with Acanthamoeba castellanii. J. Microbiol. Methods. 66:564-567.
49.Steinert, M., Ott, M., Lück, P. C., Tannich, E., and Hacker, J. 1994. Studies on the uptake and intracellular replication of Legionella pneumophila in protozoa and in macrophage-like cells. FEMS Microbiol. Ecol. 15:299-308.
50.Steinert, M., Emödy, L., Amann, R., and Hacker, J. 1997. Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii. Appl. Environ. Microbiol. 63:2047-2053.
51.Storey, M. V., Ashbolt, N. J., and Stenström, T. A. 2004. Biofilms, thermophilic amoebae and Legionella pneumophila – a quantitative risk assessment for disinfected water. Wat. Sci. Tech. 50:77-82.
52.Stout, J.E., Best, M.G., and Yu, V.L. 1986. Susceptibility of members of the family Legionellaceae to thermal stress: implications for heat eradication methods in water distribution systems. Appl Environ Microbiol 52, 396–399.
53.Temmerman, R., Vervaeren, H., Boon, N., and Verstraete, W. 2006. Necrotrophic growth of Legionella pneumophila. Appl. Environ. Microbiol. 72: 4323-4328.
54.Vatsos, I. N., Thompson, K. D., and Adams, A. 2003. Starvation of Flavobacterium psychrophilum in broth, steam water, and distilled water. Dis. Aquat. Org. 56:115-126.
55.Venkataraman, C., Gao, L.Y., Bondada, S., and Abu Kwaik, Y. (1998) Identification of putative cytoskeletal protein homologues in the protozoan host Hartmannella vermiformis as substrates for induced tyrosine phosphatase activity upon attachment to the Legionnaires'' disease bacterium, Legionella pneumophila. J Exp Med 188: 505- 514.
56.Visvesvara, G. S. 1995. Pathogenic and opportunistic free-living amoeba. In Manual of Clinical Microbiology, 6th edn, p 1196-1203. Edited by P.R. Murray. Washington, DC: American Society for Microbiology.
57.Winn, W. 1994. Legionella, p 533-543. In P. Murray, E. Baron, and M. Pfaller (eds.), Manual of Clinical Microbiology. ASM Press, Washington, D.C.