Publications by Year: 1986

The ability to bind Congo red (Crb+) is associated with virulence of Shigella flexneri and is encoded by a large, 220-kilobase plasmid. We cloned fragments of this plasmid to isolate the sequences encoding Congo red binding, to determine the degree of conservation of these sequences among S. flexneri strains, and to study the molecular basis for loss of the Crb+ phenotype. At least two separate BamHI fragments cloned into plasmid vectors encode Congo red binding in E. coli or S. flexneri. One Crb+ clone, pTKS2, contains a copy of IS1 adjacent to the crb sequences. IS1 appears to be responsible for deletions leading to loss of Congo red binding in this clone. In addition, this clone was found to integrate into the chromosome at relatively high frequency. Integration resulted in loss of the Crb+ phenotype. A second clone, pTKS15, which has only limited homology to pTKS2, also encodes Congo red binding. The Crb+ phenotype of transformants carrying pTKS15 was detected at 37 degrees C but not at 30 degrees C, and thus it resembles Congo red binding in wild-type S. flexneri. HindIII digests of plasmid DNA from 10 different S. flexneri strains were hybridized to both of these Crb+ clones and to an IS1 probe. More than one fragment hybridized to pTKS2 or pTKS15. In general, the sizes of these fragments were the same in S. flexneri strains of different serotypes, indicating conservation of these sequences. Three of five copies of IS1 were also found on the large S. flexneri plasmids. Two of the copies were on fragments of the same size in each strain. Analysis of Crb- derivatives of the 10 strains indicated that, although IS1 may be closely linked to crb sequences on the 220-kilobase plasmid, it is not responsible for the majority of deletions of this plasmid associated with loss of Congo red binding.

Eleven-day-old chicken embryos were used to compare the relative virulence of minimally passaged human isolates of Campylobacter jejuni and Campylobacter coli. Graded doses of bacteria were inoculated onto the chorioallantoic membrane, and 50% lethal doses were calculated at 72 h postinfection. Strains varied markedly in their ability to invade the chorioallantoic membrane and kill the embryos. The 50% lethal doses varied by about 6 logs for 25 strains of C. jejuni, and by 2 logs for 5 strains of C. coli. Although both outbred and inbred embryos were employed in the study, the latter were found to be more susceptible to infection with most strains. All isolates were screened for plasmid DNA, but there was no apparent relationship between plasmid content and virulence of strains for the embryos. Neither could virulence be associated with the production of siderophores by the strains. The ability of selected strains of C. jejuni to invade the liver of embryos was also studied. The number of campylobacters culturable from the liver was found to be inversely related to the 50% lethal dose of the strain. By inoculating 11-day-old embryos intravenously, it was possible to demonstrate that a strain of C. jejuni which was poorly virulent after chorioallantoic inoculation was relatively noninvasive. Invasiveness alone, however, could not fully account for the lethality of two highly virulent strains of C. jejuni administered by the intravenous route. Finally, there was no correlation between motility and virulence in this model system.

Three human isolates of Campylobacter jejuni were grown in a biphasic culture medium with and without the addition of a synthetic chelator to induce iron limitation. Cells grown in low-iron medium exhibited slower growth rates and altered cellular morphology. Increased numbers of longer, more filamentous forms were seen in Gram-stained smears. Three proteins, with apparent Mrs of 82,000, 76,000, and 74,000, were consistently present in the outer membrane of cells grown in low-iron medium. At least one of these proteins (76,000 to 74,000) was exposed on the cell surface. A bioassay was used to look for the production of siderophores by these and other strains of C. jejuni. Seven of 26 strains tested produced detectable amounts of siderophores. Growing strains at 42 degrees C failed to suppress siderophore synthesis or to alter the outer membrane protein profiles of iron-starved cells. The ability of three strains to utilize exogenously supplied siderophores for growth in low-iron medium was also examined. All three strains were able to utilize enterochelin and ferrichrome, but none utilized aerobactin, rhodotorulic acid, or desferrioxamine B. The effect of iron on the virulence of C. jejuni for 11-day-old chicken embryos inoculated via the chorioallantoic membrane was also determined.