A reference collection of 72 strains representing 37 serovars of Salmonella subspecies I has been established for use in research on genetic and phenotypic variation in natural populations. Included are isolates of the host-adapted serovars S. choleraesuis, S. dublin, S. gallinarum, S. paratyphi A , S. paratyphi B, S. paratyphi C, S. pullorum, S. sendai, S. typhi and S. typhisuis, as well as strains of S. enteritidis, S. typhimurium, and other commonly recovered serovars with broad host ranges. The isolates were characterized by enzyme electrophoresis for allelic variation in 25 chromosomal genes and represent 71 distinctive multilocus genotypes (electrophoretic types or ETs). Genetic relationships among the ETs are indicated in an evolutionary tree constructed by the neighbourjoining method from a matrix of Nei's standard genetic distance.

Variation in the chromosomal genomes of newport) in isolates of clones belonging to several evolutionary lineages, some of which are distantly related, suggests that the horizontal transfer and recombination of chromosomal genes mediating expression of cell-surface antigens has been a significant process in the evolution of the salmonellae. Two divergent clone clusters of S. derby differ in the relative frequency with which they cause disease in birds versus mammals, and two major lineages of S. newport differ in the frequency with which their clones are associated with disease in humans versus animals.

A collection of 72 reference strains of the Sufmoneffu typhimurium complex of clones recovered from a variety of hosts and environmental sources in diverse geographic locations has been established for use in studies of variation in natural populations. Included are strains of the serovars S typhimurium, S. sdntpuuf, S. heidefberg, S.purutyphi B (including varietyjatra) and S. muenchen. The strains, which have been characterized by enzyme electrophoresis for allelic variation in 24 chromosomal structural genes and represent 48 distinctive multilocus genotypes (electrophoretic types or ETs), exemplify the full range of genotypic variation in the S. typhimurium complex. Evolutionary genetic relationships among the ETs are indicated in a phylogenetic tree generated by the neighbourjoining method from a matrix of Nei's standard genetic distance.

An electrophoretic analysis of allelic variation at 24 enzyme loci among 170 isolates of the serovar Salmonella dublin (serotype 1,9,12[ViJ:g, identified three electrophoretic types (Du 1, Du 3, and Du 4), marking three closely related clones, one of which (Du 1) is globally distributed and was represented by 95% of the randomly selected isolates. All but 1 of 114 nonmotile isolates of serotype 1,9,12:-:-recovered from cattle and swine in the United States were genotypically Du 1. The virulence capsular polysaccharide (Vi antigen) is confined to clone Du 3, which apparently is limited in distribution to France and Great Britain. For all 29 isolates of Du 3, positive signals were detected when genomic DNA was hybridized with a probe specific for the ViaB region, which contains the structurally determinant genes for the Vi antigen; and 23 of these isolates had been serologically typed as Vi positive. In contrast, all 30 isolates of Du 1 tested with the ViaB probe were negative.These findings strongly suggest that the ViaB genes were recently acquired by S. dublin via horizontal transfer and additive recombination. The clones of S. dublin are closely similar to the globally predominant clone (En 1) of Salmonella enteritidis (serotype 1,9,12:g,m:-) in both multilocus enzyme genotype and nucleotide sequence of thefliC gene encoding phase 1 flagellin. Comparative sequencing offliC has revealed the molecular genetic basis for expression of the p and m flagellar epitopes by which these serovars are distinguished in the Kauffmann-White serological scheme of classification.The serovar Salmonella dublin (serotype 1,9,12 [Vi]:g, p:-) is most frequently recovered from cattle, to which it is regarded as strongly host adapted (7, 34), although it has shown a tendency to spread to swine and occasionally infects sheep and other domesticated animals (27, 48). In cattle, S. dublin causes enteric fever in both calves and adults and also induces abortion by invading the fetal blood system; and the frequent development of a carrier state, including a condition in which cows shed bacteria in their milk (40), provides the main reservoir of infection (10,46,54). Human infections with S. dublin, which are severe and may be fatal, especially in AIDS patients and other immunocompromised persons (8,19,26,53), are usually associated with the consumption of unpasteurized dairy products (13,15).An unusual feature of variation in S. dublin is that some strains express the virulence capsular antigen (Vi antigen), an acetylated polymer of galactosaminuronic acid that forms a coat on the external surface of the bacterial cell (1, 14, 25). The only other bacteria known to express this polysaccharide are the distantly related serovars Salmonella typhi and Salmonella paratyphi C (11,38,44) and a few strains of Citrobacterfreundii (12,45). For these bacteria, it has been established that the genes determining the structure of the Vi antigen are located in the ViaB region of the chromosome (20,22,44,45).Strains of S. dublin are normally motile and mono...

Multilocus enzyme electrophoresis was employed to measure chromosomal genotypic diversity and evolutionary relationships among 761 isolates of the serovars Salmonella typhi, S. paratyphi A, S. paratyphi B, S. paratyphi C, and S. sendai, which are human-adapted agents of enteric fever, and S. miami and S. java, which are serotypically similar to S. sendai and S. paratyphi B, respectively, but cause gastroenteritis in both humans and animals. To determine the phylogenetic positions of the clones of these forms within the context of the salmonellae of subspecies I, comparative data for 22 other common serovars were utilized. Except for S. paratyphi A and S. sendai, the analysis revealed no close phylogenetic relationships among clones of different human-adapted serovars, which implies convergence in host adaptation and virulence factors. Clones of S. miami are not allied with those of S. sendai or S. paratyphi A, being, instead, closely related to strains of S. panama. Clones of S. paratyphi B and S. java belong to a large phylogenetic complex that includes clones of S. typhimurium, S. heidelberg, S. saintpaul, and S. muenchen. Most strains of S. paratyphi B belong to a globally distributed clone that is highly polymorphic in biotype, bacteriophage type, and several other characters, whereas strains of S. java represent seven diverse lineages. The flagellar monophasic forms of S. java are genotypically more similar to clones of S. typhimurium than to other clones of S. java or S. paratyphi B. Clones of S. paratyphi C are related to those of S. choleraesuis. DNA probing with a segment of the viaB region specific for the Vi capsular antigen genes indicated that the frequent failure of isolates of S. paratyphi C to express Vi antigen is almost entirely attributable to regulatory processes rather than to an absence of the structural determinant genes themselves. Two clones of S. typhisuis are related to those of S. choleraesuis and S. paratyphi C, but a third clone is not. Although the clones of S. decatur and S. choleraesuis are serologically and biochemically similar, they are genotypically very distinct. Two clones of S. typhi were distinguished, one globally distributed and another apparently confined to Africa; both clones are distantly related to those of all other serovars studied.

To determine the evolutionary mechanisms generating serotypic diversity in Salmonella strains, we sequenced the central, antigen-determining part of the phase 1 flagellin gene (fli) in strains of several serovars for which estimates of chromosomal genomic relatedness had been obtained by multilocus enzyme electrophoresis. The nucleotide sequence of this region was identical in several chromosomally divergent strains of Salmonella heidelberg (phase 1 antigen r) but differed by 19% from the corresponding and similarly invariant sequence in strains of the closely related serovar SalmoneUa typhimurium (phase 1 antigen 'l. Mutational drift of the sequence present in the common ancestor is unlikely to have generated the difference between the phase 1 flageilins of these two serovars, which we attribute instead to a recombination event. This interpretation is supported by evidence that Salmonella strains of very diverse chromosomal backgrounds but similar phase 1 antigens may have closely similar nucleotide sequences for this highly polymorphic region. We suggest that lateral transfer and recombination of phase 1 flagellin genes is a major evolutionary mechanism generating new SalmoneUa serovars.Bacteria of the genus Salmonella are motile by means of peritrichous flagella, the filaments of which are composed of a highly antigenic protein, flagellin. There are two sources of antigenic diversity in Salmonella flagellins. First, there is a complex system of regulation causing two genes encoding structurally distinct flagellins (phase 1 and phase 2) to be alternatively expressed (23); second, the central, antigenic part of the flagellin protein is highly polymorphic in amino acid sequence (24,35,36). The phase 1 flagellin is encoded by the fliC gene, and the phase 2 flagellin is encoded by the fljB gene (15). More than 50 phase 1 flagellins and 30 phase 2 flagellins have been distinguished serologically (22), and this extensive diversity, together with antigenic variation in the somatic (0) lipopolysaccharide, has, for more than half a century, provided the basis for identification and classification of strains in the Kauffmann, White serotyping scheme (8,19,21).Comparison of the nucleotide sequences of the phase 1 flagellin locus (fliC) in strains of five Salmonella serovars has shown marked variation in the rate of evolution in different portions of the gene (30,35,36). This can be seen when the gene is divided into three segments of approximately equal length. The sequences encoding the carboxy-and aminoterminal regions of the protein, which are important for polymerization and secretion (14), are highly conserved among allelic forms (35,36), but the central region varies markedly in both sequence and length among strains of different serovars (30,35,36). The central portion of the protein has been implicated as the site of the major antigenic epitope, which suggests that this region is exposed on the surface of the flagellum (16,17), and electron micrographs of the flagellum of Salmonella typhimurium have shown that th...

Among the adhesin-encoding virulence operons associated with uropathogenic Escherichia coli, only pap (pyelonephritis-associated pilus)-related gene clusters typically exhibit variation in their structure and chromosomal copy number. To access further such variability, we compared pap restriction fragment length polymorphisms (RFLPs) with those detected among rRNA (rrn) operons, which encode an essential host function unrelated to virulence. To place such findings in a phylogenetic perspective, the E. coli isolates were also characterized by using multilocus enzyme electrophoresis. Variation in the rrn RFLP profiles correlated with evolutionary divergence resolved by multilocus enzyme electrophoresis; isolates with identical rrn profiles represented the same or closely related electrophoretic types. In contrast, such isolates frequently had different pap-related RFLPs, indicating that these genetic variations have developed recently relative to the changes associated with essential rrn operons or metabolic enzymes. Despite such fluctuations, two lines of evidence indicate conditions under which the pap-related RFLPs can be stably maintained. First, for each of 20 patients with urosepsis, both the primary urinary tract isolate and the concurrent blood isolate were identical. Second, although obtained from different patients, some isolates representing the same electrophoretic type also had identical pap-related RFLPs. Thus, the genotypic diversity of this virulence adhesin operon was not generated during the course of acute infection or during laboratory manipulations. Since fecal E. coli isolates frequently carry chromosomally encoded pap-related gene clusters, these findings suggest that the intraand interchromosomal recombination events generating the polymorphisms associated with the pap-related sequences likely occur among the E. coli of the commensal reservoir.Escherichia coli cause the substantial majority of urinary tract infections (33). As a group, such uropathogenic isolates exhibit properties that are not prevalent among commensal E. coli isolates found in the intestinal flora (40). Prominent among these phenotypes is the expression of adhesins that mediate bacterial attachment to the human uroepithelium and thereby confer a selective advantage in ascending colonization of the upper urinary tract by allowing the bacteria to resist the cleansing effect of urine flow in the ureters (29). pap (pyelonephritis-associated pili) and prs (pap-related sequence) are two structurally and functionally related operon clusters that have been cloned from E. coli J96 and shown to express adhesin pili (9,21,22). pap encodes a P-fimbrial adhesin that specifically recognizes the globoseries of glycolipids present on both human erythrocytes bearing the P blood group antigen and uroepithelial cells (19,21,22). prs encodes an F adhesin that preferentially binds to the Forsmann antigen, which is a major constituent of sheep erythrocyte membranes and is also present on the cells of the human renal pelvis (21,22).In a recent s...

Genetic diversity and relationships among 123 strains of Salmonella paratyphi B (serotype 1,4,[5],12:b:[1,2]) were estimated from an assessment of electrophoretically demonstrable allelic variation at 24 chromosomal enzyme gene loci. Fourteen electrophoretic types, marking clones, were distinguished, the phylogeny of the clonal lineages was reconstructed, and biotype and other phenotypic characters were mapped onto this structure. Most d-tartrate-negative strains are members of an abundant, globally distributed clone (Pb 1) that is polymorphic for many biotype characters (including d-tartrate utilization), bacteriophage type, rRNA pattern, and colicin M and phage ES18 sensitivity. This clone is largely responsible for S. paratyphi B enteric fever in humans. In contrast, d-tartrate-positive strains (formerly known as S. java) occurred in all seven of the clonal lineages identified by population genetic analysis, although most d-tartrate-positive isolates belong to only two clones (Pb 3 and Pb 4), which vary in frequency geographically. Monophasic strains represent four closely related clones forming a distinctive phylogenetic lineage. The Kauffmann hypothesis of convergence in serotype among distantly related cell lineages through recombination (via phage transduction or other means) may account for the considerable genotypic diversity among clones of S. paratyphi B. Pb 4, Pb 6, and Pb 7 are more closely allied with clones of S. typhimurium and S. saintpaul than with other clones of S. paratyphi B. Sensitivity or resistance to colicin M and phage ES18 and the electrophoretic pattern of the rRNA, which were incorporated into a recently proposed scheme for the identification of types of S. paratyphi B, individually or in combination fail to mark clones or other meaningful phylogenetic subdivisions.