Enzyme polymorphism and genetic population structure in Escherichia coli and Shigella

Electrophoretically demonstrable variation in 12 enzymes was studied in more than 1 600 isolates of Escherichia coli from human and animal sources and in 123 strains of the four species of Shigella. All 12 enzymes were polymorphic; and the number of allozymes (mobility variants), which were equated with alleles, averaged 9.3 per locus in E. coli. For Shigella species, the mean number of alleles was 2.9 per locus. Some 77% of the allozymes recorded in Shigella were shared with E. coli. A total of 302 unique genotypic combinations of alleles over the 12 loci (electrophoretic types, ETs) was distinguished, of which 279 represented E. coli and 23 were Shigella. Among electrophoretic types, mean allelic diversity per locus was 0.52 for E. coli and 0.29 for Shigella. It was estimated that there are, on the average, about 0.3 detectable codon differences per locus between pairs of strains of E. coli and Shigella, which is roughly equivalent to 1.2 amino acid differences per enzyme. Evidence that the enzyme loci studied are a random sample of the genome is provided by a significant positive correlation between estimates of genetic divergence between pairs of strains obtained by DNA reassociation tests and estimates of genetic distance between the same strains based on electrophoresis. A principal components analysis of allozyme profiles revealed that the 302 ETs fall into three overlapping clusters, reflecting strong non-random associations of alleles, largely at four loci. Each of the four ETs of E. coli that have been most frequently recovered from natural populations has an allozyme profile that is very similar to, or identical with, the hypothetical modal ET of one of the groups. ETs of Shigella fall into two of the groups. No biological significance can at present bbe attributed to the genetic structure revealed by Multilocus electrophoretic techniques. The electrophoretic data are fully compatible with other molecular and more conventional evidence of a close affinity between E. coli and Shigella, and they raise questions regarding the present assignments of certain strains to species. In support of evidence from DNA reassociation tests and serotyping, the present study suggests that S. sonnei is homogeneous in chromosomal genotype.     

Lipopolysaccharide core structures and their correlation with genetic groupings of Shigella strains. A novel core variant in Shigella boydii type 16

Bacteria Shigella, the cause of shigellosis, evolved from the intestinal bacteria Escherichia coli. Based on structurally diverse O-specific polysaccharide chains of the lipopolysaccharides (LPSs; O-antigens), three from four Shigella species are subdivided into multiple serotypes. The central oligosaccharide of the LPS called core is usually conserved within genus but five core types called R1-R4 and K-12 have been recognized in E. coli. Structural data on the Shigella core are limited to S. sonnei, S. flexneri and one S. dysenteriae strain, which all share E. coli core types. In this work, we elucidated the core structure in 14 reference strains of S. dysenteriae and S. boydii. Core oligosaccharides were obtained by mild acid hydrolysis of the LPSs and studied using sugar analysis, high-resolution mass spectrometry and two-dimensional NMR spectroscopy. The R1, R3 and R4 E. coli core types were identified in 8, 3 and 2 Shigella strains, respectively. A novel core variant found in S. boydii type 16 differs from the R3 core in the lack of GlcNAc and the presence of a D-glycero-D-manno-heptose disaccharide extension. In addition, the structure of an oligosaccharide consisting of the core and one O-antigen repeat was determined in S. dysenteriae type 8. A clear correlation of the core type was observed with genetic grouping of Shigella strains but not with their traditional division to four species. This finding supports a notion on the existing Shigella species as invalid taxa and a suggestion of multiple independent origins of Shigella from E. coli clones.     

Differentiation of Shigella by esterase electrophoretic polymorphism

The electrophoretic mobilities of four esterases (A, B, C, and I) of 182 strains of Shigella dysenteriae, S. flexneri, S. boydii and S. sonnei were compared to those of 636 strains of Escherichia coli from various origins, including the Alkalescens Dispar group and enteroinvasive strains. Discriminant analysis of the distribution of esterases among the strains revealed that Shigella could be distinguished from E. coli by differences in the distribution of allozymes of esterases C and I. Principal components analysis distinguished four major clusters of Shigella strains corresponding to the following: S. dysenteriae serotype 1; S. flexneri serotypes 1 to 5; S. flexneri serotype 6 and S. boydii serotypes 2 and 4; and S. sonnei. The last three were characterized by distinct electrophoretic variants of carboxylesterase B, as judged by the two-dimensional electrophoretic profile and titration curves. The distinct esterase pattern obtained for the strains of S. boydii serotype 13 substantiates the view that this serotype may constitute a new species.     

I-CeuI fragment analysis of the Shigella species: evidence for large-scale chromosome rearrangement in S. dysenteriae and S. flexneri

I-CeuI fragments of four Shigella species were analyzed to investigate their taxonomic distance from Escherichia coli and to collect substantiated evidence of their genetic relatedness because their ribosomal RNA sequences and similarity values of their chromosomal DNA/DNA hybridization had proved their taxonomic identity. I-CeuI digestion of genomic DNAs yielded seven fragments in every species, indicating that all the Shigella species contained seven sets of ribosome RNA operons. To determine the fragment identities, seven genes were selected from each I-CeuI fragment of E. coli strain K-12 and used as hybridization probes. Among the four Shigella species, S. boydii and S. sonnei showed hybridization patterns similar to those observed for E. coli strains; each gene probe hybridized to the I-CeuI fragments with sizes similar to that of the corresponding E. coli fragment. In contrast, S. dysenteriae and S. flexneri showed distinct patterns; rcsF and rbsR genes that located on different I-CeuI fragments in E. coli, fragments D and E, were found to co-locate on a fragment. Further analysis using an additional three genes that located on fragment D in K-12 revealed that some chromosome rearrangements involving the fragments corresponding to fragments D and E of K-12 took place in S. dysenteriae and S. flexneri.     

Evolutionary genetics of a new pathogenic Escherichia species: Escherichia albertii and related Shigella boydii strains

A bacterium originally described as Hafnia alvei induces diarrhea in rabbits and causes epithelial damage similar to the attachment and effacement associated with enteropathogenic Escherichia coli. Subsequent studies identified similar H. alvei-like strains that are positive for an intimin gene (eae) probe and, based on DNA relatedness, are classified as a distinct Escherichia species, Escherichia albertii. We determined sequences for multiple housekeeping genes in five E. albertii strains and compared these sequences to those of strains representing the major groups of pathogenic E. coli and Shigella. A comparison of 2,484 codon positions in 14 genes revealed that E. albertii strains differ, on average, at approximately 7.4% of the nucleotide sites from pathogenic E. coli strains and at 15.7% from Salmonella enterica serotype Typhimurium. Interestingly, E. albertii strains were found to be closely related to strains of Shigella boydii serotype 13 (Shigella B13), a distant relative of E. coli representing a divergent lineage in the genus Escherichia. Analysis of homologues of intimin (eae) revealed that the central conserved domains are similar in E. albertii and Shigella B13 and distinct from those of eae variants found in pathogenic E. coli. Sequence analysis of the cytolethal distending toxin gene cluster (cdt) also disclosed three allelic groups corresponding to E. albertii, Shigella B13, and a nontypeable isolate serologically related to S. boydii serotype 7. Based on the synonymous substitution rate, the E. albertii-Shigella B13 lineage is estimated to have split from an E. coli-like ancestor approximately 28 million years ago and formed a distinct evolutionary branch of enteric pathogens that has radiated into groups with distinct virulence properties.     

Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase.

Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the alpha-decarboxylation of L-glutamic acid to yield gamma-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-beta-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65 degrees C but not at 68 degrees C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.     

Expression of flagella and motility by Shigella

Since the discovery of Shigella as the aetiologic agent of acute dysentery almost 100 years ago, this organism has been described as a non-motile and non-flagellated organism that invades the human colonic mucosa. In this study, the production of flagella by prototypic strains of all four Shigella species and, moreover, by fresh clinical isolates was demonstrated by electron microscopy. The fla gellum of Sh igella (flash) is ∼10 µm long and 12–14 nm in diameter and is typically seen emanating from one pole of the bacterium. Flash is composed of a putative structural polypeptide subunit of 33–38 kDa that shares immunological similarities with Escherichia coli , Salmonella spp., and Proteus mirabilis flagellins, and with the recently described recombinant Shigella flagellins (FliC_SS and FliC_SF) expressed in E. coli K-12. A fliC_SS -specific oligo probe hybridized with all four Shigella species, while a fliC_SF probe hybridized with all Shigella flexneri and Shigella dysenteriae strains, but not with all Shigella sonnei or Shigella boydii strains, indicating genetic divergence among their flagellin genes. Shigella exhibits motility in low-concentration motility agar under physiological growth conditions. The expression of flash and motility appears to be strictly regulated by unidentified genetic and environmental factors. These heretofore undescribed features may allow the bacteria to circumvent the natural intestinal mucosal defences leading to bacterial colonization and disease. The motility of shigellae may represent an evolutionary adaptation important for bacterial survival.     

A simple method for semi-preparative-scale production and recovery of enterocin AS-48 derived from Enterococcus faecalis subsp. liquefaciens A-48-32

Production of enterocin AS-48 by Enterococcus faecalis A-48-32 was compared between standard and high-cell density batch fermentations. In high-cell density cultures, bacteriocin production was 2.47-fold higher, provided that the pH was controlled during the fermentation. A two-step procedure for recovery of milligram quantities of purified bacteriocin was developed, based on adsorption of the bacteriocin on Carboxymethyl Sephadex CM-25 followed by reversed-phase chromatography on a semi-preparative column. The purified bacteriocin was active on all the Gram-positive bacteria tested (for example, species of Bacillus, Paenibacillus, Staphylococcus, and Listeria). Strains E. coli U-9, E. coli CECT 102, E. coli CECT 104, E. coli CECT 432, E. coli CECT 543, E. coli CECT 877 and Shigella sonnei CECT 542 were sensitive, while seven other E. coli strains as well as Salmonella choleraesuis CECT 722, S. choleraesuis CECT 916, Enterobacter cloacae CECT 194 and Aeromonas hydrophila CECT 398 were resistant.     

Genome dynamics and diversity of Shigella species, the etiologic agents of bacillary dysentery

The Shigella bacteria cause bacillary dysentery, which remains a significant threat to public health. The genus status and species classification appear no longer valid, as compelling evidence indicates that Shigella, as well as enteroinvasive Escherichia coli, are derived from multiple origins of E.coli and form a single pathovar. Nevertheless, Shigella dysenteriae serotype 1 causes deadly epidemics but Shigella boydii is restricted to the Indian subcontinent, while Shigella flexneri and Shigella sonnei are prevalent in developing and developed countries respectively. To begin to explain these distinctive epidemiological and pathological features at the genome level, we have carried out comparative genomics on four representative strains. Each of the Shigella genomes includes a virulence plasmid that encodes conserved primary virulence determinants. The Shigella chromosomes share most of their genes with that of E.coli K12 strain MG1655, but each has over 200 pseudogenes, 300 approximately 700 copies of insertion sequence (IS) elements, and numerous deletions, insertions, translocations and inversions. There is extensive diversity of putative virulence genes, mostly acquired via bacteriophage-mediated lateral gene transfer. Hence, via convergent evolution involving gain and loss of functions, through bacteriophage-mediated gene acquisition, IS-mediated DNA rearrangements and formation of pseudogenes, the Shigella spp. became highly specific human pathogens with variable epidemiological and pathological features.     

Mobilization of phase I plasmid in Shigella sonnei and stability of its inheritance in rough strains of Shigella and Escherichia

The plasmid pSS120, determining the synthesis of species specific I phase antigen of Shigella sonnei is mobilized for genetic transfer into E. coli K12 recipient cells with the frequency 12-41%. The frequency depends on the type of mobilized plasmid and recipient strain. The I phase antigen is normally expressed in II phase recipient cells and in E. coli cells. During mobilization pSS120 forms cointegrates representing a recombinant of mobilizing and mobilized plasmids DNA. The study of pSS120 inheritance stability has shown the plasmid to be unstable during culturing of bacteria and to be partially lost from the parent Shigella sonnei strains as well as from the "hybrid" transconjugants obtained. The 60 Md plasmid present in the donor strains of Shigella sonnei is prone to structural fragmentation particularly expressed in Shigella sonnei/E. coli hybrids.     

The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli ; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli , had lipopolysaccharide profiles quite distinct from the respective strain of E. coli . It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.     

Glutamate decarboxylase genes as a prescreening marker for detection of pathogenic Escherichia coli groups.

The enzyme glutamate decarboxylase (GAD) is prevalent in Escherichia coli but few strains in the various pathogenic E. coli groups have been tested for GAD. Using PCR primers that amplify a 670-bp segment from the gadA and gadB genes encoding GAD, we examined the distribution of the gadAB genes among enteric bacteria. Analysis of 173 pathogenic E. coli strains, including 125 enterohemorrhagic E. coli isolates of the O157:H7 serotype and its phenotypic variants and 48 isolates of enteropathogenic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and other Shiga toxin-producing E. coli (STEC) serotypes, showed that gadAB genes were present in all these strains. Among the 22 non-E. coli isolates tested, only the 6 Shigella spp. carried gadAB. Analysis of naturally contaminated water and food samples using a gadAB-specific DNA probe that was labeled with digoxigenin showed that a gadAB-based assay is as reliable as standard methods that enumerate E. coli organisms on the basis of lactose fermentation. The presence of few E. coli cells initially seeded into produce rinsates could be detected by PCR to gadA/B genes after overnight enrichment. A multiplex PCR assay using the gadAB primers in combination with primers to Shiga toxin (Stx) genes stx(1) and stx(2) was effective in detecting STEC from the enrichment medium after seeding produce rinsate samples with as few as 2 CFU. The gadAB primers may be multiplexed with primers to other trait virulence markers to specifically identify other pathogenic E. coli groups.     

A study of enteropathogenic organisms isolated in the public health laboratory, Lusaka.

A total of 328 specimens of stools were examined in the Public Health Laboratory during January and February 1973. Enteropathogens were isolated from 117 of these specimens. Besides these, 12 strains of Salmonellae were isolated from blood and 8 from urine. An occasional Salmonella was isolated from the pleural fluid (S. paratyphi A) pus from the knee (S. enteritidis) and from the C.S.F. of an infant (S. paratyphi C.). Salmonella typhi and Salmonella paratyphi A are the predominant Salmonella species. No Salmonella paratyphi B has been isolated. Shigella, was isolated with slightly less frequency than Salmonella, and Shigella flexneriis was the predominant species. E. coli 0112/K66 is the most common enteropathogenic E. coli. The majority of the Shigella and Salmonella species are sensitive to the common antibiotics used. The E. coli organisms show multiple resistance to a number of antibiotics.     

Antigen sharing of Salmonella typhi non-flagellar proteins with other salmonellae and some shigellae and Escherichia coli

Cathodal moving protein components were identified in agarose gel electrophoresis of the Veronal buffer extract of a non-motile strain of S. typhi (8393, Colindale). Rabbit antiserum was raised against these cationic proteins; it had both agglutinating and precipitating activity. A total of 80 salmonella strains belonging to serogroups A, B, C1, C2, D, E1 and E2 including 26 S. typhi and 10 S. paratyphi A were tested against this antiserum in a slide agglutination test; all strains were agglutinated. Among 94 other bacterial strains tested, the antiserum agglutinated all 16 strains of Shigella flexneri, 2 of 5 Shigella sonnei, 5 of 34 E. coli and 1 of 8 Citrobacter species. These results show that there is antigenic sharing of the non-flagellar proteins of S. typhi with many other salmonellae as well as with some shigellae and E. coli.     

Genetics and regulation of heme iron transport in Shigella dysenteriae and detection of an analogous system in Escherichia coli O157:H7.

Shigella species can use heme as the sole source of iron. In this work, the heme utilization locus of Shigella dysenteriae was cloned and characterized. A cosmid bank of S. dysenteriae serotype 1 DNA was constructed in an Escherichia coli siderophore synthesis mutant incapable of heme transport. A recombinant clone, pSHU12, carrying the heme utilization system of S. dysenteriae was isolated by screening on iron-poor medium supplemented with hemin. Transposon insertional mutagenesis and subcloning identified the region of DNA in pSHU12 responsible for the phenotype of heme utilization. Minicell analysis indicated that a 70-kDa protein encoded by this region was sufficient to allow heme utilization in E. coli. Synthesis of this protein, designated Shu (Shigella heme uptake), was induced by iron limitation. The 70-kDa protein is located in the outer membrane and binds heme, suggesting it is the S. dysenteriae heme receptor. Heme iron uptake was found to be TonB dependent in E. coli. Transformation of an E. coli hemA mutant with the heme utilization subclone, pSHU262, showed that heme could serve as a source of porphyrin as well as iron, indicating that the entire heme molecule is transported into the bacterial cell. DNA sequences homologous to shu were detected in strains of S. dysenteriae serotype 1 and E. coli O157:H7.     

Atypical Shigella boydii 13 encodes virulence factors seen in attaching and effacing Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that causes watery diarrhea and hemorrhagic colitis. In this study, we identified StcE, a secreted zinc metalloprotease that contributes to intimate adherence of EHEC to host cells, in culture supernatants of atypical Shigella boydii 13 (Shigella B13) strains. Further examination of the Shigella B13 strains revealed that this cluster of pathogens does not invade but forms pedestals on HEp-2 cells similar to EHEC and enteropathogenic E.?coli. This study also demonstrates that atypical Shigella B13 strains are more closely related to attaching and effacing E.?coli and that their evolution recapitulates the progression from ancestral E.?coli to EHEC.     

Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid

A method was developed for the detection of the fecal coliform bacterium Escherichia coli, using the polymerase chain reaction and gene probes, based on amplifying regions of the uid gene that code for beta-glucuronidase, expression of which forms the basis for fecal coliform detection by the commercially available Colilert method. Amplification and gene probe detection of four different regions of uid specifically detected E. coli and Shigella species, including beta-glucuronidase-negative strains of E. coli; no amplification was observed for other coliform and nonenteric bacteria.     

Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid.

A method was developed for the detection of the fecal coliform bacterium Escherichia coli, using the polymerase chain reaction and gene probes, based on amplifying regions of the uid gene that code for beta-glucuronidase, expression of which forms the basis for fecal coliform detection by the commercially available Colilert method. Amplification and gene probe detection of four different regions of uid specifically detected E. coli and Shigella species, including beta-glucuronidase-negative strains of E. coli; no amplification was observed for other coliform and nonenteric bacteria.     

Escherichia coli in disguise: molecular origins of Shigella

Shigella, which still stands as a genus with four species today, in reality belongs to the extremely diverse species Escherichia coli. There are several lineages of Shigella strains derived through independent acquisition of the pINV virulence plasmid. The chromosomally determined phenotypic properties of Shigella result from convergent evolution during niche adaptation, most due to loss of function, some from negative selection pressure.     

Structure and genetics of Shigella O antigens

This review covers the O antigens of the 46 serotypes of Shigella, but those of most Shigella flexneri are variants of one basic structure, leaving 34 Shigella distinct O antigens to review, together with their gene clusters. Several of the structures and gene clusters are reported for the first time and this is the first such group for which structures and DNA sequences have been determined for all O antigens. Shigella strains are in effect Escherichia coli with a specific mode of pathogenicity, and 18 of the 34 O antigens are also found in traditional E. coli. Three are very similar to E. coli O antigens and 13 are unique to Shigella strains. The O antigen of Shigella sonnei is quite atypical for E. coli and is thought to have transferred from Plesiomonas. The other 12 O antigens unique to Shigella strains have structures that are typical of E. coli, but there are considerably more anomalies in their gene clusters, probably reflecting recent modification of the structures. Having the complete set of structures and genes opens the way for experimental studies on the role of this diversity in pathogenicity.