LYSOSTAPHIN - NEW BACTERIOLYTIC AGENT FOR STAPHYLOCOCCUS
LYSOSTAPHIN - NEW BACTERIOLYTIC AGENT FOR STAPHYLOCOCCUS
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DOI：
10.1073/pnas.51.3.414
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发表时间：
1964-01-01
影响因子：
11.1
通讯作者：
SCHUHARDT, VT
中科院分区：
文献类型：
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作者：
SCHINDLER, CA;SCHUHARDT, VT
方法-媒体：除非另有说明，用于鉴定K-6-WI分离株的媒体、试剂和技术均为Shaw、Stitt和Cowan所描述的。23在胰酶-大豆(TS)肉汤(巴尔的摩生物实验室，马里兰州巴尔的摩)上，保持了生物体，并测定了表面生长特性。在含有2%(w/v)的pH 7.2-7.3时添加琼脂(Difco，Detroit，Mich.)。在18小时的TS肉汤和琼脂斜面培养上测定革兰氏染色特征。所有的琼脂表面拮抗实验都是通过在TS琼脂平板上交叉划线18小时的试验菌的肉汤培养来进行的。收集葡萄球菌滤液：从K-6-WI的肉汤培养物中回收葡萄球菌滤液，在37℃，1升芬巴赫或发酵瓶中，以76循环/分钟的速度在2.5英寸的往复摇床上操作。初步研究表明，当pH从最初的7.3下降到约6.5后，当pH增加到7.5时，溶葡萄球菌的产量最高。这通常发生在11-27小时，这取决于接种量和生长介质的数量，并被用作收获时间的决定因素。培养时间越长，pH值越高，产率越低。将培养物汇集在一起，在40℃下以13200g的重量离心20min，沉淀细胞。上清液通过纤维素膜(Schleicher and Schuell，Bac-T-Flex，0.5，科学产品公司，伊利诺伊州埃文斯顿)过滤。并保存在-20℃。溶葡萄球菌酶的定量测定：设计了一种快速、可重复性的方法来比较不同收获的溶葡萄球菌酶的活性，并对预期的纯化程序进行了评估。测定菌种为金黄色葡萄球菌FDA 209P。振荡培养18小时，13℃沉淀，用0.05M三(羟甲基)氨基甲烷-盐酸盐(Tris)缓冲液洗涤2次，缓冲液中含有0.145 M氯化钠(缓冲盐水)。在调整含有#54滤光片(500-570m)的Klett Summerson光电比色仪(Klett)，使其读数为110和0.001 M硫酸钡后，将洗净的金黄色葡萄球菌细胞重新悬浮在缓冲盐水中，Klett读数为300。将1.5毫升这种金黄色葡萄球菌悬浮液加入克利特试管，含有足够的缓冲盐水(有或没有溶葡萄球菌酶)，产生总体积为5毫升，初始克利特读数为125±5。试验和对照制剂在370水浴中孵育，克利特读数每隔5分钟进行一次。一个单位的溶葡萄球菌酶被指定为包含在该量的测试材料中，使标准金黄色葡萄球菌悬浮液在37℃下在10分钟内浊度降低50%。通过构建标准曲线，可以用最少的时间和精力来确定任何溶葡萄球菌酶制剂的单位价值。
Methods.-Media: Unless otherwise stated, the media, reagents, and techniques used in identification of the K-6-WI isolate were those described by Shaw, Stitt, and Cowan. 23 The organism was maintained, and surface growth characteristics were determined, on Trypticase-soy (Ts) broth (Baltimore Biological Laboratories, Baltimore, Md.) at pH 7.2-7.3 containing 2%(w/v) added agar (Difco, Detroit, Mich.). Gram staining characteristics were determined on 18-hr Ts broth and agar slant cultures. All agar surface antagonism experiments were performed by cross-streaking 18-hr Ts broth cultures of the test organisms on Ts agar plates. Harvesting staphylolytic filtrates: Staphylolytic filtrates were recovered from Ts broth cultures of K-6-WI after incubation at 37 in 1-liter Fernbach or fermentation flasks on a reciprocating shaker operating with a 2.5-in stroke at a rate of 76 cycles/min. Preliminary studies indicated a maximum yield of staphylolytic substance when the pH, after an initial decrease from 7.3 to ca. 6.5, increased to pH 7.5. This usually occurred at 11-27 hr, depending upon both the size of the inoculum and quantity of growth medium, and was used asa determinant of harvest time. Longer incubation resulted in higher pH and lower yields. The cultures were pooled and centrifuged at 40 for 20 min at 13,200 g to sediment the cells. The supernates were filtered through cellulose membranes (Schleicher and Schuell, Bac-T-Flex, 0.5, upore diam, Scientific Products, Evanston, Ill.) and were stored at-20. Hereafter, the staphylolytic factor will be referred to as lyso-staphin.Quantitative assay of lysostaphin: A rapid and reproducible assay was devised to compare the activity of different harvests of lysostaphin and to assess the contemplated purification procedures. The assay organism chosen was Staphylococcus aureus FDA 209P. The cells in an 18-hr, Ts broth, shake culture were sedimented at 13, DO g and washed twice with 0.05 M tris (hydroxy-methyl) aminomethane-HCl (Tris) buffer at pH 7.5, containing 0.145 M NaCl (buffered saline). After adjusting the Klett Summerson photoelectric colorimeter (Klett) containing a# 54 filter (500-570 m,) to give a reading of110 with 0.001 M barium sulfate, the washed S. aureus cells were resuspended in buffered saline to give a Klett reading of 300. The addition of 1.5 ml of this S. aureus suspension to Klett tubes, containing sufficient buffered saline (with or without lysostaphin) to yield a total volume of 5 ml, gave initial Klett readings of125±5. The test and control preparations were incubated in a 370 waterbath, and Klett readings were made at 5-min intervals. One unit of lysostaphin was designated as being containedin that amount of test material which gave a 50% reductionin turbidity of the standard S. aureus suspension in 10 min at 37. By constructing a standard curve, the unit value of any lysostaphin preparation could be established with a minimum of time and effort.