Effects of various antimicrobials on in vitro Shiga toxin production and release by Shigella dysenteriae serotype 1 was investigated in this study with particular reference to the role of outer membrane vesicles in toxin release by the organism. Five antimicrobials, namely nalidixic acid, ciprofloxacin, norfloxacin, fosfomycin and mitomycin C, were chosen for the study and the toxin titre was measured by the reverse passive latex agglutination (RPLA) method using an available kit. Only mitomycin C was found to induce production of Shiga toxin in the bacteria and its release by outer membrane vesicles. The highest titre of toxin was obtained in vesicle fraction suggesting that the vesicles play an important role in the release of Shiga toxin from periplasmic space by the organism.

The fine structures of the capsules of Klebsiella pneumoniae and Escherichia coli were determined by the rapid-freezing technique. The capsular layer was seen as a densely packed accumulation of fine fibers. The thickness of the capsule was approximately 160 nm in K. pneumoniae and less than 10 nm in E. coli Kl. Two layers were observed in the Klebsiella capsule in which the arrangements of the fibers were different. The inner layer of the capsule was formed by a palisade of thick and dense bundles of the fibers standing at right angles on the surface of the outer membrane. In the outer layer these thick bundles of fibers loosened into fine fibers which spread over the bacterial surface, forming a fine network structure.The bacterial species that cause invasive infection usually have a capsule as a virulence factor (4,6,9,10,12). The capsule is said to act as an antiphagocytic factor or to give serum-resistance characteristics to the bacteria. The detailed mechanisms of this action are not known yet, though much information about the capsules is accumulating from chemical, physiological, and immunological studies (7,10,14,16). We assumed that this ambiguity about the role of the capsule in infection was due largely to insufficient knowledge about the functional structures of the capsules. The electron microscope, which has made possible great progress in our knowledge about the morphology of the bacterial cell, has been absolutely useless for examining capsular structures. This is due mostly to the very fragile nature of the bacterial capsule. During the processing for electron microscopy, major parts of the capsular structure are destroyed. Several efforts have been made to observe the fine structures of the capsules by the conventional chemical fixation method, and the structures have been demonstrated only in bacteria expressing relatively thick capsules under careful fixations (3, 9). Recently, however, a milder fixation method has been developed and applied to the observation of bacterial cell structures. This method has dramatically improved the preservation of the fine structures of bacterial cells (2,5,15). With this technique we have demonstrated the presence of a very thin capsule of Vibrio vulnificus (2). In this communication we describe the results of using this technique to reveal the fine structures of the capsules of Klebsiella pneumoniae and Escherichia coli.The bacterial strains used were a capsulated strain, K, of K. pneumoniae and a Kl-positive strain of E. coli. The strain of K. pneumoniae was obtained from our culture stock, and that of E. coli was from R. Sakazaki of The National Institute of Health, Tokyo, Japan. The presence of the capsule of K. pneumoniae was revealed by the Hiss capsular staining method. This strain is highly virulent to mice. Less than 10 CFU of bacteria injected peritoneally is sufficient to kill the animals. The strain of E. coli is of 02:K1:H2 serotype. The capsule, Kl antigen, of this strain was not visible under the light microscope after capsular staining. These...

The role of the capsule in the reaction with anti-O or anti-K serum was examined morphologically by the techniques of freeze-substitution and immunoelectron microscopy in two strains of Kkebsiella pneumoniae differing in virulence for mice. Strain Chedid (O1:K2), an encapsulated virulent strain, has a thicker capsule (150 nm) than the encapsulated avirulent strain 277 (O1:K2) (60 nm). Two morphologically recognizable domains in the capsule created by the arrangement of the capsular filaments were clearly seen in strain Chedid but were less evident in strain 277. Anti-O antibody could penetrate through the capsular layer of both strains. The capsule has no function as a barrier for the penetration of the antibody. Anti-K antibody reacted only on the surface of the capsule and induced swelling of the capsule.

Abstract:The The movement of bacteria is important for their colonization in the animal intestinal tract (5, 6, 9, 10) and the bacterial flagellum is an organelle that supports bacterial locomotion. Bacterial cells can be classified into two groups based on flagella attachments: monotrichous flagellated bacteria with a single flagellum at either one or both ends of the cell; and peritrichous flagellated bacteria with many flagella on the side of the cell. The swimming velocities of these two types of bacterial species are reported to be different (7,8). Bacterial species with polar flagella are reported to be able to move more quickly than peritrichous flagellated bacteria. However, the differences in swimming velocities among species of the monotrichous flagellated bacteria are not well known.Morphological observations of moving bacteria have been carried out by several methods. One method employed recently uses videotape (2,3,7,8). Recent advances in video, microscopy and computer-based image processing have made it possible to observe and analyze the movement of extremely small objects such as bacteria.In this paper we report the results of the measurement of swimming velocity of Vibrio cholerae and Pseudomonas aeruginosa in culture media using a computer-assisted video tracking method. In addition, we aim to show that this method is useful and simple for analysis of the details of such complicated movement. Materials and MethodsBacterial strains and cultures. The bacterial strains used in this experiment were Vibrio cholerae non-O1 strains V018, AP7, AP5 and AI1854, and Pseudomonas aeruginosa strains K, P28, P15, No. 3 and No. 6. These bacterial strains were all obtained from our own stock cultures. Bacterial strains were cultured in nutrient broth consisting of 0.5% Extract Bonito, 1.0% polypepton and 0.5% sodium chloride (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 37 C.Examination of the swimming patterns. The bacteria were cultured in nutrient broth with aeration at 37 C. Concentrations of bacterial cells were estimated from the density of the culture with a photo densitometer by light absorption at 660 nm (Fuji Kogyo Co., Ltd., Tokyo). The bacteria in log phase culture were diluted with nutrient broth up to OD 0.01 which contains approximately 106 CFU/ml of bacterial cells. Then one drop of the diluted culture was placed on a clean microscope slide and covered with a thin cover glass. To prevent evaporation of the medium which usually causes streaming of the media on the slide, the edges of the cover glass were sealed tightly with nail polish. The cover glass was gently pressed with the fingers when it was sealed with nail polish to reduce the thickness of the aqueous layer between the two glass plates.

Membrane vesicles released by E. coli 0157:H7 strains were investigated by immuno-electron microscopy using anti-0157 antibody. Anti-0157 antibody enhanced the negative-staining of vesicles and we found numerous small vesicles clearly formed around bacterial cells. An immunogold-electron microscopic examination confirmed that lipopolysaccharide (LPS) including the O-side chain is present on the surface of the vesicles. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the purified vesicles showed that the vesicles contained LPS consisting of a lipid-A and an O polysaccharide. In addition, the endotoxic activity of the vesicle was confirmed by a limulus test. These results suggest that the vesicles may play an important role in the pathogenesis of Escherichia coli 0157:H7.