Epidemiology of tetracycline resistance determinants in Shigella spp. and enteroinvasive Escherichia coli: characterization and dissemination of tet(A)-1.

To make a comprehensive study of tetracycline resistance determinant distribution in the genus Shigella, a collection of 577 clinical isolates of Shigella spp. and enteroinvasive Escherichia coli (EIEC) from a variety of geographical locations was screened to identify tetracycline-resistant strains. The 459 tetracycline-resistant isolates identified were then screened by PCR analysis to determine the distribution in these strains of tetracycline efflux resistance determinants belonging to classes A to E, G, and H that have been identified in gram-negative bacteria. Only classes A to D were represented in these strains. Although Tet B was the predominant determinant in all geographical locations, there were geographical and species differences in the distribution of resistance determinants. An allele of tet(A), designated tet(A)-1, was identified and sequenced, and the 8.6-kb plasmid containing determinant Tet A-1, designated pSSTA-1, was found to have homologies to portions of a Salmonella enterica cryptic plasmid and the broad-host-range resistance plasmid RSF1010. This allele and pSSTA-1 were used as epidemiological markers to monitor clonal and horizontal transmission of determinant Tet A-1. An analysis of serotype, distribution of tetracycline resistance determinants, and resistance profiles indicated that both clonal spread and horizontal transfer had contributed to the spread of specific tetracycline resistance determinants in these populations and demonstrated the use of these parameters as an epidemiological tool to follow the transmission of determinants and strains.

Construction, characterization, and animal testing of WRSd1, a Shigella dysenteriae 1 vaccine.

WRSd1 is a Shigella dysenteriae 1 vaccine containing deletions of the virG(icsA) gene required for intercellular spreading and a 20-kb chromosomal region encompassing the Shiga toxin genes (stxAB). WRSd1 was constructed from S. dysenteriae 1 strain 1617 that was originally isolated during the 1968 to 1969 epidemic of Shiga dysentery in Guatemala. The virG(icsA) deletion was constructed from a streptomycin-resistant (Str(r)) mutant of 1617 by a filter mating procedures using a virG(icsA) deletion derivative, pDeltavirG2. A colony that was invasive for HeLa cells and negative for the virG(icsA) gene by Southern blotting was grown anaerobically on plates containing chlorate for selection of resistant colonies that had lost the entire Shiga toxin gene. A virG(icsA) stxAB Str(r) mutant selected from the chlorate plates was designated WRSd1. This candidate vaccine was evaluated for safety, immunogenicity, and protective efficacy using the guinea pig keratoconjunctivitis model. WRSd1 was Sereny negative, and two applications of this strain to the cornea elicited a significant protective immune response against the S. dysenteriae 1 O antigen. Vaccination with WRSd1 conferred protection against challenge with each of three virulent S. dysenteriae 1 strains. Since a vaccine protecting against multiple Shigella species is required for most areas where Shigella is endemic, protection studies using a combination vaccine of Shigella sonnei vaccine strain WRSS1, Shigella flexneri 2a vaccine strain SC602, and WRSd1 were also performed. Guinea pigs vaccinated with a mixture of equal amounts of the three vaccine strains were protected against challenge with each of the homologous virulent strains. Unlike WRSS1 and SC602, however, the level of protection afforded by WRSd1 in a combination vaccine was lower than the protection elicited by a pure culture. A current Good Manufacturing Practice product of WRSd1 given intragastrically to rhesus monkeys proved safe and immunogenic.

Two studies evaluating the safety and immunogenicity of a live, attenuated Shigella flexneri 2a vaccine (SC602) and excretion of vaccine organisms in North American volunteers.

We report the first community-based evaluation of Shigella flexneri 2a strain SC602, a live, oral vaccine strain attenuated by deletion of the icsA (virG) plasmid virulence gene, given at 10(4) CFU. The primary objectives of this trial were to determine the safety and immunogenicity of the vaccine and to determine the duration of colonization. Four of 34 volunteers experienced transient fevers, and three reported diarrhea during the first 3 days of the study. Half of the volunteers mounted a positive serum immunoglobulin A (IgA) response to S. flexneri lipopolysaccharide. All but one of the volunteers excreted the vaccine in their stools for 1 to 33 days, and this excretion was often intermittent. Data from the community-based study were supplemented with an inpatient trial in which three volunteers received 10(3) and nine received 10(4) CFU. All volunteers who received 10(3) CFU excreted SC602 and had an IgA antibody-secreting cell response. Two of these had a serum IgA response. Six of the nine volunteers who received 10(4) CFU excreted SC602. One vaccinee had a transient fever and two met the definition of diarrhea. Six volunteers that received 10(4) CFU had an antibody-secreting cell response, and four had a serum IgA response. SC602 has now been tested at 10(4) CFU in a total of 58 volunteers. The cumulative results of these clinical trials, reported here and previously (Coster et al., Infect. Immun. 67:3437-3443, 1999), have demonstrated that SC602 is a substantially attenuated candidate vaccine that can evoke protection against the most severe symptoms of shigellosis in a stringent human challenge model of disease.