Duchet-Suchaux M, Bertin A, Dubray G
Institut National de la Recherche Agronomique, Station de Pathologie de la Reproduction, Centr de Tours-Nouzilly, Monnaie, France.
J Gen Microbiol. 1988 Apr;134(4):983-95. doi: 10.1099/00221287-134-4-983.
In order to describe morphologically the structures on the cell surface of bovine enterotoxigenic Escherichia coli, variants of reference strain B41 (K99+F41+) either negative for K99 and positive for F41 antigens (variants B41A, B41C), or phenotypically negative for both antigens (variants B41B1, B41B2, B41CB), and a transconjugant harbouring the K99 plasmid and expressing the K99 adhesin [transconjugant B41 x H510a:H510(2)] were examined by transmission electron microscopy using negative staining. Several negative staining procedures were tested for strain B41 and variant B41A: direct harvesting of strains into ammonium molybdate (2%, w/v), with bacitracin (50 micrograms ml-1) as wetting agent, gave the best results. Three morphologically distinct structures on the cell surface could be identified in cultures grown on Minca medium. Firstly, thin, filamentous, flexible fibrillar structures, presenting a helical structure and a mean diameter of approximately 3 nm, were recognized as K99 fimbriae, since they were present on strain B41 and on transconjugant H510(2), but not on K99-negative variants nor on the recipient strain H510a. Secondly, coil-like structures with a diameter of about 17-20 nm were observed on strain B41 and on variants B41A and B41C. These structures appeared to consist of two or more curled filaments (diameter 3 nm) joined to coil on themselves into dense spirals. They were very rare in variants B41B1 and B41B2 and were absent on variant B41CB and on a transconjugant B41* x B41CB, which had re-acquired the K99 plasmid and which again exhibited K99 fimbriae. Strains B41 and variant B41A gown at 37 degrees C for 24 h on sheep-blood agar exhibited coiled structures like those seen on Minca medium. In contrast, after growth at 18 degrees C for 48 h (which inhibits the synthesis of F41 antigen), coiled structures were no longer expressed on the cell surface of strain B41 and variants B41A and B41C. Thus the presence of coiled structures correlated with the expression of F41 antigen in strains and variants, which suggests that F41 had a coiled morphology. Finally, straight fimbriae (diameter 6.5-7 nm) were observed on the cell surface of every strain and variant. Their expression on the cell surface was enhanced by several subcultures in th e static broth, and it was inhibited by subculture on agar, but not by culture at 18 degrees C after serial subcultures in static broth. These facts indicated that the straight fimbriae could be common fimbriae, and excluded their being F41 structures.
为了从形态学上描述牛产肠毒素大肠杆菌细胞表面的结构，对参考菌株B41（K99+F41+）的变体进行了研究，这些变体要么K99抗原阴性而F41抗原阳性（变体B41A、B41C），要么两种抗原表型均为阴性（变体B41B1、B41B2、B41CB），还研究了携带K99质粒并表达K99黏附素的转接合子[转接合子B41×H510a:H510(2)]，采用负染色法通过透射电子显微镜进行观察。对菌株B41和变体B41A测试了几种负染色程序：将菌株直接接种到钼酸铵（2%，w/v）中，加入杆菌肽（50微克/毫升）作为湿润剂，效果最佳。在Minca培养基上生长的培养物中，可以在细胞表面鉴定出三种形态不同的结构。首先，细的、丝状的、柔韧的纤维状结构，呈现螺旋结构，平均直径约为3纳米，被认为是K99菌毛，因为它们存在于菌株B41和转接合子H510(2)上，但不存在于K99阴性变体和受体菌株H510a上。其次，在菌株B41以及变体B41A和B41C上观察到直径约为17 - 20纳米的盘绕状结构。这些结构似乎由两根或更多卷曲的细丝（直径3纳米）组成，它们自身盘绕成紧密的螺旋。它们在变体B41B1和B41B2中非常罕见，在变体B41CB和转接合子B41*×B41CB上不存在，转接合子B41×B41CB重新获得了K99质粒并再次表现出K99菌毛。菌株B41和变体B41A在绵羊血琼脂上于37℃培养24小时后呈现出与在Minca培养基上看到的类似的盘绕状结构。相反，在18℃培养48小时（这会抑制F41抗原的合成）后，菌株B41以及变体B41A和B41C的细胞表面不再表达盘绕状结构。因此，盘绕状结构的存在与菌株和变体中F41抗原的表达相关，这表明F41具有盘绕的形态。最后，在每个菌株和变体的细胞表面都观察到了直菌毛（直径6.5 - 7纳米）。它们在细胞表面的表达通过在静止肉汤中多次传代培养而增强，在琼脂上传代培养则受到抑制，但在静止肉汤中多次传代培养后于18℃培养不会受到抑制。这些事实表明直菌毛可能是普通菌毛，排除了它们是F41结构的可能性。
