Comparative esterase electrophoretic polymorphism of Escherichia coli isolates obtained from animal and human sources

To determine whether enzyme electrophoretic polymorphism in Escherichia coli populations was influenced by environmental background, the mobilities of four electrophoretically variable esterases (A, B, C and I) were examined. The distinction between isolates was established by significant differences in the electrophoretic distribution and the genetic diversity coefficient of individual esterases. Principal components analysis on each population and on all strains revealed three groups of allozymes. The first, characterized by slow electrophoretic mobilities of esterase B, was frequently observed in strains obtained from human extra-intestinal infections and rarely in commensal organisms. The second, characterized by fast mobilities of esterases A and B, was frequently found in animal isolates. The third, characterized by prominence of the most common mobilities of esterases B and A, was recovered in all populations. These results were confirmed by discriminant analysis. Among the 610 strains investigated, 316 electrophoretic types (distinctive combinations of allozymes of the four varieties of esterases) were distinguished, illustrating high esterase polymorphism.     

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.     

Highly pathogenic strains of Escherichia coli revealed by the distinct electrophoretic patterns of carboxylesterase B

One hundred and ninety one strains of Escherichia coli isolated from extra-intestinal infections and 85 strains isolated from the stools of healthy human beings were compared for electrophoretic mobility and isoelectric point of carboxylesterase B, and for production of alpha-haemolysin and the presence of mannose resistant haemagglutinin. Fast and slow electrophoretic mobilities were distinguished among the strains. The frequency of strains showing slow mobilities was considerably higher when they originated from extra-intestinal infections (40%) than when they were obtained from the stools of healthy individuals (7%). In a two-dimensional electrophoretic profile, the fast and slow mobility variants of carboxylesterase B were resolved into two patterns, B1 and B2, respectively. The frequency of pathogenic strains that concomitantly produced alpha-haemolysin and mannose resistant haemagglutinin was 48.7% for strains of pattern B2 but only 2.8% for strains of pattern B1. Thus, the electrophoretic pattern B2 of carboxylesterase B appears to be a molecular marker for a group of highly pathogenic E. coli strains which are frequently implicated in extra-intestinal infections.     

Esterase electrophoresis compared with biotyping for epidemiological typing of Acinetobacter baumannii strains

Forty-nine Acinetobacter baumannii strains belonging to three biotypes and isolated from four hospitals were differentiated by electrophoretic typing of their esterases. Six main kinds of esterases were distinguished by their spectra of hydrolytic activity toward seven synthetic substrates. The electrophoretic variations of these enzymes were used to define ten zymotypes among the three biotypes. Esterase electrophoresis appeared to be more sensitive than biotyping, and could represent an additional marker for epidemiological analysis.     

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.     

Characterization of Enterobacter cloacae and E. sakazakii by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases

Acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases of 34 strains of Enterobacter cloacae and 22 strains of Enterobacter sakazakii were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. The two species could be separated on the basis of distinct electrophoretic patterns of all enzymes analysed. Glutamate dehydrogenase and acid phosphatase were detected exclusively in E. cloacae, whereas esterase bands were more intensively stained in E. sakazakii. For each species, two zymotypes could be distinguished, on the basis of electrophoretic mobilities of malate dehydrogenase and banding patterns of esterase for E. cloacae, and by both isoelectric point and electrophoretic mobilities of an esterase and of lactate and malate dehydrogenases for E. sakazakii. The high degree of enzyme polymorphism within the two species permitted precise identification of strains. The variations in electrophoretic patterns might therefore provide useful epidemiological markers.     

Conservation of components of the Escherichia coli export machinery in prokaryotes

Esterase electrophoretic typing was used to classify clinical isolates of Pseudomonas aeruginosa. One hundred and twenty-seven P. aeruginosa strains belonging to 16 serotypes (including 16 non-typeable strains) and isolated from diverse human infections in three hospitals, and the type strain ATCC 10 145, were tested. Four main kinds of esterase and 4 additional esterases were distinguished by their spectra of hydrolytic activity toward synthetic substrates and by their sensitivity or resistance to di-isopropyl fluorophosphate. The electrophoretic variations of these enzymes were used to define 42 zymotypes. Electrophoretic typing of esterase appeared to be more sensitive than serotyping and the results of the two methods did not correlate. When the two typing methods were used in parallel, 78 different combinations of serotype and zymotype were obtained.     

Epidemiological typing of Acinetobacter strains by esterase electrophoresis

Fifty-three strains of Acinetobacter, belonging to the species A baumannii, A. haemolyticus and A. johnsonii, were differentiated by electrophoretic typing of their esterases, on the basis of both the enzyme specific activity profiles and their electrophoretic mobilities. Each esterase was defined by its spectrum of hydrolytic activity toward five synthetic substrates and its sensitivity to di-isopropyl fluorophosphate. Since each enzyme was not detected in all strains of a given species, several zymotypes could be defined by the patterns of combinations of esterases. Thus, 24 zymotypes were defined in the 32 A. baumannii strains, 4 were defined in the 10 A. haemolyticus strains and 6 were defined in the 11 A. johnsonii strains. When the electrophoretic mobilities of the various esterases were included, each of the 53 strains of Acinetobacter (with the exception of three A. haemolyticus strains) showed a distinct electrotype.     

The Yersinia high-pathogenicity island is highly predominant in virulence-associated phylogenetic groups of Escherichia coli

The high-pathogenicity island (HPI) present in pathogenic Yersinia and encoding the siderophore yersiniabactin, has recently been identified in the asnT tRNA region of various Escherichia coli pathotypes, especially those responsible for bacteremia and urosepsis. Most E. coli strains causing such extra-intestinal infections belong to phylogenetic groups B2 and D. In this study we investigated (i) the distribution and localization of HPI among the different E. coli phylogenetic groups, using the ECOR reference collection; and (ii) the prevalence of HPI among a set of 124 phylogenetically characterized E. coli strains responsible for neonatal meningitis. Ninety-three percent of the ECOR strains belonging to groups B2 and D harbored HPI. In contrast, the island was present in 32% and 25% of strains belonging to groups A and B1, respectively, which are considered to be non-pathogenic. HPI was found in 100% of the neonatal meningitis strains, 13 of which belonged to groups A and B1, suggesting that HPI might contain virulent factors required for the development of neonatal meningitis. Moreover, we observed for the first time that HPI can be inserted in a site different from the asnT tRNA region.     

Enhanced persistence in the colonic microbiota of Escherichia coli strains belonging to phylogenetic group B2: role of virulence factors and adherence to colonic cells

Escherichia coli segregates into four phylogenetic groups, A, B1, B2 and D. B2 and D strains usually possess virulence factors, cause most extra-intestinal infections and have superior capacity to persist in the infantile colonic microbiota. Here, we investigated 24 resident and 37 transient E. coli strains from the colonic microbiota of 13 Swedish schoolgirls sampled in the 1970s with respect to phylogenetic group identity, carriage of virulence factor genes, O and K antigens and mannose-sensitive and -resistant adherence to the colonic cell line HT-29. Resident strains more often belonged to phylogenetic group B2 than transient strains (38% vs 5% p=0.004). In contrast, transient strains more often than resident strains belonged to group A (57% vs 29%, p=0.04) or B1 (24% vs 13%, p=0.33). Most B2 strains belonged to uropathogenic O serogroups, carried genes for P fimbriae, K5 capsule and hemolysin and adhered in higher numbers to HT-29 cells via mannose-resistant mechanisms than strains from the other groups. Further, among strains carrying genes for P or S fimbriae, those belonging to group B2 adhered in highest numbers. In logistic regression, genes for P fimbriae and aerobactin predicted persistence in the colonic microbiota (p=0.050 and 0.056, respectively), while B2 origin did not reach significance as an independent variable (p=0.16). Our results indicate that virulence factors carried by group B2 strains contribute to their strong colonizing capacity. These factors may actually be regarded as fitness factors in the human gut.     

Esterases in the house fly. Polymorphisms and inheritance patterns.

Polyacrylamide gel electrophoresis was used to examine the variability and inheritance of esterases in five strains of the house fly, Musca domestica L. Individual zymograms exhibited 8 to 15 bands that could be assigned to one of five zones designated as A through E from anode to cathode. Correlations of P1-F1 banding patterns indicated the existence of at least 3 different loci in zone A. 2 each in zones B and C, and 4 in zone D; no clear inheritance patterns were discernable for the bands of zone E. Only the Es-5 locus of zone C was monomorphic in all of the strains studied. Eight loci possessed null alleles and codominant alleles were detected at six loci. The results suggest that esterases should prove useful for measuring relationships among fly populations or for various studies of population dynamics.     

The electrophoretic polymorphism of bacterial esterases

Abstract: The specific activities of esterases and certain other molecular properties including immunospecificity indicate that the electrophoretic variations of these enzymes in bacterial populations are the result of allelic variations at specific gene loci. The esterase polymorphism of Enterobacteriaceae and some other species isolated from man or animals demonstrates that esterases can distinguish between bacteria at the species or subspecies level, both by their biochemical properties and by their electrophoretic differences. The esterase data complement DNA hybridization studies and agree with ribosomal DNA polymorphism, especially for delineating a phylogenetically distinct group of highly pathogenic strains in Escherichia coli . A two-dimensional electrophoretic profile obtained by establishing a direct correspondence between homologous esterase bands resolved by independent runs of isoelectric focusing and standard electrophoresis considerably improves the detection of allelic variations, whereas protein titration curves (electrophoresis in pH gradient) can be used to demonstrate the real electrophoretic homogeneity of allozymes or evalue their molecular relationship in terms of apparent amino acid substitutions. This overview establishes that esterases, by their significant electrophoretic polymorphism, are reliable molecular markers for systematics and epidemiology, and are suitable enzyme systems for studying population genetics and phylogeny.     

Characterization of highly virulent Escherichia coli strains by ribosomal DNA restriction fragment length polymorphism

Patterns of ribosomal DNA polymorphism were examined to compare carboxylesterase B type B1 strains and B2 strains of Escherichia coli isolated from extra-intestinal infections. DNA from 14 type B2 strains showing the presence of alpha-haemolysin and mannose-resistant haemagglutinin and lethality to mice and 14 type B1 strains lacking these characteristics, was digested with HindIII, EcoRI, BamHI or BglII restriction enzymes and analysed by Southern blotting. The obtained ribotypes clearly differentiated the B2 strains from the B1 strains. These results indicate that genotypes of the highly virulent B2 strains are different from that of the less virulent B1 strains.     

Rapid and simple determination of the Escherichia coli phylogenetic group

Phylogenetic analysis has shown that Escherichia coli is composed of four main phylogenetic groups (A, B1, B2, and D) and that virulent extra-intestinal strains mainly belong to groups B2 and D. Actually, phylogenetic groups can be determined by multilocus enzyme electrophoresis or ribotyping, both of which are complex, time-consuming techniques. We describe a simple and rapid phylogenetic grouping technique based on triplex PCR. The method, which uses a combination of two genes (chuA and yjaA) and an anonymous DNA fragment, was tested with 230 strains and showed excellent correlation with reference methods.     

Allozyme Diversity and Interspecific Differentiation o the Two Diploid Bromegrass Species, Bromus tectorum L. and B. sterilis L (Poaceae)

Abstract: Genetic diversity among 50 accessions of Bromus tectorum and 43 of B. sterilis from different sites of their Eurasian ranges has been studied by electrophoretic analysis of ten enzymes encoded by 18 loci. The two species proved clearly differentiated by alternate allozymes at seven isozymes. Populations of both taxa showed differentiation into eleven (B. tectorum ) and six (B. sterilis ) multilocus allozyme lineages (MLALs). The extent of interspecific allozyme divergence estimated by Manhattan distance exceeded more than three times intraspecific differentiation between the multilocus lineages. Only two MLALs in each species have wide geographical distribution from the Near East to Europe. Other MLAL5 were found each for only one or two populations and were region-specific. Most geographically marginal European populations had widespread MLALs.     

Mitochondrial DNA variation in the hybridizing fire-bellied toads, Bombina bombina and B. variegata

Using five restriction enzymes, geographical variation of mitochondrial DNA (mtDNA) in Bombina bombina and B. variegata was studied in samples from 20 locations. Each restriction enzyme produced a species-specific fragment pattern. B. bombina haplotypes A and B were closely related to each other. In contrast, haplotypes A and B of B. variegata formed two distinct lineages. A very distinctive haplotype (C) was found in the Carpathian Mountains, whereas two other haplotypes, D and E (differing by a single AvaI site), were present in western Europe and the Balkans, respectively. Populations polymorphic for haplotypes D and E occurred in the central Balkans where the haplotypes could replace each other clinally. mtDNA sequence divergence between B. bombina and B. variegata was estimated as 6.0-8.1% and 4.7-5.2% between type C and types D/E of B. variegata. The latter divergence is contrary to allozyme and morphological data that place the western and Carpathian B. v. variegata together (Nei's D = 0.07) and separate them from the Balkan subspecies B. v. scabra (Nei's D = 0.18). Broad interspecific correlation among morphology, allozymes and mtDNA types in European fire-bellied toads argues that, despite continuous hybridization (interrupted perhaps during Pleistocene glacial maxima), little or no mtDNA introgression between the species has occurred outside the narrow hybrid zones that separate these parapatric species.     

Clonal relationships among bovine pathogenic Escherichia coli producing surface antigen CS31A

Abstract Forty-two Escherichia coli strains producing surface antigen CS31A isolated from bovine infections were characterized with respect to OKH serotypes, outer membrane protein (OMP) elelctrophoretic patterns, allozymes for esterases A, B, C, I and biotypes. A large majority of the strains could be clustered in a limited number of groups of clonally related strains with diverse O serogroups. CS31A producing Escherichia coli strains thus appear to have a common genetic background and are representative of an important part of bovine pathogenic Escherichia coli .     

Morphological, cytogenetic and allozymic variation within Cephalofovea (Onychophora: Peripatopsidae) with descriptions of three new species

Variation within and between populations of Cephalofovea (Peripatopsidae) has been examined by allozyme, karyological and morphological analyses. Four groups are recognized on the basis of allozyme electrophoresis. One group includes specimens from the type locality of the only described species of the genus, C. tomahmontis. While karyotypic and morphological character states show considerable inter-group variation, the distributions of these states among groups are not concordant when different characters are compared. However, each group of populations is uniquely defined by the full suite of character states it possesses. The four groups are recognized here as distinct species with three species described as new.     

Esterase Variations between the 14 Syngens of PARAMECIUM AURELIA under Axenic Growth

Under axenic growth all 14 syngens of Paramecium aurelia have 4 types of esterases. The three major types (A, B and cathodal C) vary independently in electrophoretic mobility among the syngens. Using these three esterases, stocks can be keyed to a syngen, except for the groupings 1-3-5 and 7-13. Using 5 other enzymes only syngens 1 and 5 cannot be distinguished. Most syngens differ from each other in 6 out of the 8 enzymes. An axenically-grown stock of Paramecium multimicronucleatum collected in Costa Rica has the same types of esterases as P. aurelia. Two of the types (A and C) are similar in mobility to those found in syngens 7 and 13, but its B esterase differs in mobility from all the known syngens of P. aurelia.     

GENETIC STRUCTURE OF THE BLUE RIDGE DUSKY SALAMANDER (DESMOGNATHUS ORESTES): INFERENCES FROM ALLOZYMES, MITOCHONDRIAL DNA, AND BEHAVIOR

Abstract The plethodontid salamander Desmognathus orestes, a member of the D. ochrophaeus species complex, is distributed in southwestern Virginia, eastern Tennessee, and western North Carolina. Previous allozyme analyses indicate that D. orestes consists of two distinct groups of populations (D. orestes‘B’ and D. orestes‘C’) with extensive intergradation and probable gene flow between these two groups. Spatially varying allele frequencies can reflect historical associations, current gene flow, or a combination of population‐level processes. To differentiate among these processes, we use multiple markers to further characterize divergence among populations of D. orestes and assess the degree of intergradation between D. orestes‘B’ and D. orestes‘C’, specifically investigating variation in allozymes, mitochondrial DNA (mtDNA), and reproductive behavior among populations. On a broad scale, the mtDNA genealogies reconstruct haplotype clades that correspond to the species identified from previous allozyme analyses. However, at a finer geographic scale, the distributions of the allozyme and mtDNA markers for D. orestes‘B’ and D. orestes‘C’ are discordant. MtDNA haplotypes corresponding to D. orestes‘B’ are more broadly distributed across western North Carolina than predicted by allozyme data, and the region of intergradation with D. orestes‘C’ indicates asymmetric gene flow of these markers. Asymmetric mating may contribute to observed discordance in nuclear versus cytoplasmic markers. Results support describing D. orestes as a single species and emphasize the importance of using multiple markers to examine fine‐scale patterns and elucidate evolutionary processes affecting gene flow when making species‐level taxonomic decisions.