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.     

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.     

Comparative electrophoretic polymorphism of esterases and other enzymes in Escherichia coli

The electrophoretic polymorphism of esterases was compared with that of other enzymes in Escherichia coli populations by investigating allozyme distribution of four esterases (A, B, C and I) within both the subspecific groups I, II and III and the new groups A, B1, B2, C, D and E, which have been distinguished by electrophoretic analysis of 11 and 35 enzymes respectively in the 72 reference strains of the ECOR collection. Electrophoretic distribution of esterases was distinct for each of the three subspecific groups as indicated by distributions of allozymes and electrophoretic types (distinctive combination of allozyme for the four esterases). Esterase polymorphisms of the three subspecific groups appeared to have similar features to those of three previously studied natural populations of strains obtained from human and animal gastro-intestinal tracts and extra-intestinal infections in humans. Multiple correspondence analyses using data obtained from the four esterases and the 11 other enzymes also distinguished the groups A, B1, B2, C, D and E. All strains of group B2 showed the B2 electrophoretic pattern of esterase B, which appeared to be a marker of a distinct cluster of strains frequently implicated in extra-intestinal infections.     

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.     

Establishment of three categories of P-fimbriated Escherichia coli strains that show different toxic phenotypes and belong to particular O serogroups

Eight hundred and nineteen strains of Escherichia coli isolated in Spain between 1986 and 1991 from extraintestinal infections and feces of healthy controls were investigated for expression of P-fimbriae using a particle agglutination test. Among strains causing urinary tract infections, sepsis and other extraintestinal infections, P-fimbriae were found in 31% (130/420) (P < 0.001), 25% (30/118) (P < 0.001) and 12% (11/92) (P < 0.5) respectively. In contrast, only 7% (14/189) of faecal isolates from healthy individuals carried P-fimbriae. According to two more common toxic markers detected in this study (alpha-haemolysin and cytotoxic necrotizing factor type 1), P-fimbriated E. coli strains were grouped into three categories: haemolysin+cytotoxic necrosing factor+ (Hly+CNF1+) (68/185; 37%), haemolysin+cytotoxic necrosing factor- (Hly+CNF1-) (61/185; 33%) and Hly-CNF1- (56/185; 30%). The 185 P-fimbriated strains belonged to 17 different O serogroups. However, 148 (80%) were of one of six serogroups (O1, O2, O4, O6, O7 and O18). The most frequent serogroups determined in the Hly+CNF1+ strains were the O4 and O6 (53/68; 78%), in the Hly+CNF1- strains it was the O18 (27/61; 44%) and in the Hly-CNF1- strains the O1, O2 and O7 (41/56; 73%). The majority (160/185; 86%) of P-fimbriated E. coli expressed the mannose-resistant haemagglutinin type IVa.     

Infectious Drug Resistance Among Clinically Isolated Escherichia coli

Of 398 strains of clinically isolated Escherichia coli from three Birmingham, Alabama, hospitals, 38% were found to be resistant to one or more drugs tested. Fifty-seven per cent of the resistant strains transferred all or a part of their resistance pattern to sensitive cells during mixed cultivation. Of the 152 resistant strains, 29.1% were singly resistant, and 70.5% were resistant to more than one drug. Of the multiply resistant strains, 61% transferred all or a part of their pattern. Strains isolated from Veterans Hospital patients demonstrated higher percentages of resistance than strains isolated from Children's Hospital patients. An extremely low incidence of infective drug resistance was noted among E. coli isolated from the stools of healthy hospital employees.     

Release of the type I secreted alpha-haemolysin via outer membrane vesicles from Escherichia coli

The alpha-haemolysin is an important virulence factor commonly expressed by extraintestinal pathogenic Escherichia coli. The secretion of the alpha-haemolysin is mediated by the type I secretion system and the toxin reaches the extracellular space without the formation of periplasmic intermediates presumably in a soluble form. Surprisingly, we found that a fraction of this type I secreted protein is located within outer membrane vesicles (OMVs) that are released by the bacteria. The alpha-haemolysin appeared very tightly associated with the OMVs as judged by dissociation assays and proteinase susceptibility tests. The alpha-haemolysin in OMVs was cytotoxically active and caused lysis of red blood cells. The OMVs containing the alpha-haemolysin were distinct from the OMVs not containing alpha-haemolysin, showing a lower density. Furthermore, they differed in protein composition and one component of the type I secretion system, the TolC protein, was found in the lower density vesicles. Studies of natural isolates of E. coli demonstrated that the localization of alpha-haemolysin in OMVs is a common feature among haemolytic strains. We propose an alternative pathway for the transport of the type I secreted alpha-haemolysin from the bacteria to the host cells during bacterial infections.     

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.     

Pathogenic characteristics of Escherichia coli strains in cases of asymptomatic bacteriuria and symptomatic urinary tract infections

The aim of this study was to examine if E. coli isolated from asymptomatic bacteriuria differed in pathogenic features from strains isolated from symptomatic infections of urinary tract. In this study 130 strains of E. coli isolated from women having asymptomatic bacteriuria and 112 strains isolated from patients with symptoms of urinary tract infection were examined. It was shown that E. coli isolated from patients with symptomatic urinary tract infection showed the more frequently ability to cause mannose-resistant haemagglutination of human erythrocytes, resistance to bactericidal activity of serum and haemolytic properties than those isolated from asymptomatic bacteriuria. These strains showed also the higher ability to adhere to Vero cells in tissue culture. Among E. coli strains isolated from persons with asymptomatic bacteriuria the pathogenic features were most frequently found in strains from healthy women and the most rarely in isolated from diabetic women.     

Polyacrylamide gel electrophoresis analysis of ribosomal protein AT-L30 as a novel approach to actinomycete taxonomy: application to the genera Actinomadura and Microtetraspora.

Actinomycete ribosomal protein AT-L30 exhibits electrophoretic mobility that is specific for each genus. On the basis of this fact, we analyzed ribosomal AT-L30 proteins from 26 type strains of species belonging to the genera Actinomadura and Microtetraspora. The electrophoretic mobilities of AT-L30 preparations from these strains, as determined by two-dimensional polyacrylamide gel electrophoresis, revealed that they could be divided into two groups, one group with relative electrophoretic mobilities of 14.0 to 41.5 and another group with relative electrophoretic mobilities of -6.5 to 0. The first group corresponded to the genus Actinomadura, and the second group corresponded to the genus Microtetraspora. Partial amino acid sequencing of AT-L30 preparations from several strains proved that we were indeed dealing with the specified protein homologous to ribosomal protein L30 of Escherichia coli. Our results strongly supported the conclusions of previous work and thus proved the efficacy of ribosomal protein analysis as a novel approach for taxonomy of actinomycetes.     

Esterase-16 (ES-16) of the rat (Rattus norvegicus): Identification and genetic characterization

Esterase-16, an esterase present in lung and other tissues of the laboratory rat, has been characterized by its biochemical properties (electrophoretic mobility, substrate pattern, sensitivity to inhibitors) and genetic variation in 107 inbred strains and substrains including 14 RI strains. It was classified as a carboxylesterase (EC 3.1.1.1). The phenotype ES-16A (BN/Han and 63 other strains) was defined as a narrow electrophoretic band migrating between ES-1A and ES-13A, ES-16B (LEW/Han and 42 other strains) exhibited the same electrophoretic mobility as ES-16A but was distinguished by its extremely weak activity. Segregation of ES-16 in RI strains and backcrosses indicated linkage to linkage group V (LGV). The Es-16 locus was tentatively placed into esterase cluster 2 and homology with Es-7 of the house mouse is proposed.     

Studies on the electrophoretic separability of B and T human lymphocytes.

Cell electrophoresis experiments were performed at physiological, ionic strength on Hypaque-Ficoll separated normal human peripheral blood lymphocytes. The initial lymphocyte preparations averaged 72% T cells and 22% B cells as estimated by resetting techniques. A bimodal electrophoretic distribution of fast T cells and slow B cells such as has been repeatedly shown to exist for experimental animals could not be demonstrated. Neither B- nor T-enriched populations showed any correlation between mobility and degree of enrichment. A strong positive correlation was found between the non-rosetting cells and a very slow electrophoretic subpopulation produced during the B enrichment procedure. It was also found that considerable variability in lymphocyte mobilities existed from person to person and even in the same individual sampled over several months; these observations imply that the pooling of electrophoretic data can be misleading. The importance of these findings lies in the growing interest in relating lymphocyte electrophoresis to disease processes and immunological rejection phenomena.     

Drug Resistance in Strains of Escherichia Coli Isolated from the Intestinal Tract of Pigs in Norway

Faecal samples from 95 healthy pigs and samples of jejunal content from 85 piglets suffering from colienterotoxaemia were tested for the presence of drug resistant E. coli strains. Practically all pigs in both groups harboured E. coli strains resistant to one or more of the 6 antibiotics/chemotherapeutic agents tested (Oxytetracycline, streptomycin, sulphaisodimidin, neomycin, ampicillin, chloramphenicol). Almost 100% of healthy and approx. 90% of diseased pigs harboured strains resistant to Oxytetracycline, streptomycin and sulphaisodimidin. Pigs with strains resistant to neomycin, ampicillin and chloramphenicol were less frequently found. The predominant coliform flora consisted of E. coli strains” resistant to Oxytetracycline, streptomycin and sulphaisodimidin in 71% to 81% of diseased pigs and in 47% to 69% of the healthy pigs. In diseased pigs ¾ of the animals had a coliform flora dominated by neomycinresistant E. coli strains. Of the 721 resistant E. coli strains isolated from healthy pigs, 11% were single resistant while the corresponding figure for the 518 resistant strains isolated from diseased pigs was 6%. Thus 89% and 94% of strains showed simultaneous resistance to 2 or more antibiotics. E. coli strains resistant to 3 or more drugs were found in approx. 60% and 70% of the isolates from healthy and diseased animals, respectively. Oxytetracycline/streptomycin/sulphaisodimidin resistance was most commonly found, approx. 22% and 38% of the strains from healthy and diseased pigs, respectively, showing this resistance pattern. Transmission of drug resistance which was examined in E. coli strains originating from the diseased pigs was demonstrated in approx. 76% of the isolates. The incidence of drug resistance transfer in single, double, triple and quadruple resistant strains was 11%, 68%, 97% and 98%, respectively.     

Non-radioactive PCR-SSCP with a single PCR step for detection of inhibitor resistant beta-lactamases in Escherichia coli

A method based on PCR-SSCP has been developed to detect presumptive Inhibitor-Resistant TEM (IRT) beta-lactamases in Escherichia coli. The capacity of this technique to differentiate genes from 11 control strains encoding IRT beta-lactamases was evaluated with PCR products digested with RsaI. All the bla(TEM) genes studied could be distinguished by their electrophoretic mobilities. Applied to 29 epidemiologically unrelated clinical isolates of E. coli resistant to amoxicillin-clavulanate (MIC, > or =32 microg/ml), the electrophoretic mobilities of the digested bla(TEM) PCR products were identical to those of the reference bla(TEM-1A) (6 strains) and bla(TEM-1B) (18 strains) genes. The remaining five bla(TEM) PCR products displayed SSCP profiles different from those of the reference bla(TEM) genes and their nucleotide sequence identified them as bla(TEM-1C) in one strain, bla(TEM-30/IRT-2) in two strains, bla(TEM-37/IRT-8) in one strain, and bla(TEM-40/IRT-11) in one isolate. Overexpression of the wild-type bla(TEM-1) gene, as detected by high-level resistance to beta-lactams and enzyme assay, accounted for resistance in the 24 E. coli containing bla(TEM-1). We report a simple one PCR step SSCP that can be used in epidemiological studies for rapid preliminary detection of IRT beta-lactamases; identification should be confirmed by sequence data.     

Electrophoretic mobility distributions of normal human T and B lymphocytes and of peripheral blood lymphoblasts in acute lymphocytic leukemia: effects of neuraminidase and of solvent ionic strength.

Human T and B lymphocytes were found to be distinguishable on the basis of electrophoretic mobility, with the T cells having the higher mobility, in agreement with previous reports. The effects of the enzyme neuraminidase on the electrophoretic mobilities of T and B lymphocytes were determined. T lymphocytes showed a greater decrease in electrophoretic mobility after neuraminidase treatment; the relative mobilities of T and B cells were reversed by neuraminidase treatment, and the electrophoretic distinguishability was enhanced. The electrophoretic mobility distributions of lymphoblasts from patients with acute lymphocytic leukemia were found to differ from those of normal cells in their response to neuraminidase treatment and to changes in solution ionic strength. These results imply that the surface structure of the leukemic cells differs from that of either T or B lymphocytes from normal donors.     

Correlation between the presence of aggregative fibria and a type of Escherichia coli adhesion

Enteroaggregative strains of E. coli (EAEC) are an important agents possessing among many virulence factors, aggregative fimbria AAF hemagglutinating in the presence of mannose human group A or/and rats erythrocytes. The aim of the study was to determine the correlation between the presence of AAF fimbria and pattern of adherence in vitro. Tested strains of E. coli were obtained from children with diarrhea (133 strains) and healthy children (105 strains). Among strains of E. coli from children with diarrhea 81 (61%) showed the presence of AAF fimbria and 19 (23%) were adhering in aggregative pattern. In the group of strains of E. coli isolated from healthy children 31 (30%) were AAF positive and 8 (25.8%) of them presented aggregative adherence. Examination of AAF fimbria only dose not allow to distinguish EAEC strains. The data showed the participation of EAEC strains in diarrhea of children below 3 years old.     

Virulence potential and genomic mapping of the worldwide clone Escherichia coli ST131

Recently, the worldwide propagation of clonal CTX-M-15-producing Escherichia coli isolates, namely ST131 and O25b:H4, has been reported. Like the majority of extra-intestinal pathogenic E. coli isolates, the pandemic clone ST131 belongs to phylogenetic group B2, and has recently been shown to be highly virulent in a mouse model, even though it lacks several genes encoding key virulence factors (Pap, Cnf1 and HlyA). Using two animal models, Caenorhabditis elegans and zebrafish embryos, we assessed the virulence of three E. coli ST131 strains (2 CTX-M-15- producing urine and 1 non-ESBL-producing faecal isolate), comparing them with five non-ST131 B2 and a group A uropathogenic E. coli (UPEC). In C. elegans, the three ST131 strains showed intermediate virulence between the non virulent group A isolate and the virulent non-ST131 B2 strains. In zebrafish, the CTX-M-15-producing ST131 UPEC isolates were also less virulent than the non-ST131 B2 strains, suggesting that the production of CTX-M-15 is not correlated with enhanced virulence. Amongst the non-ST131 B2 group isolates, variation in pathogenic potential in zebrafish embryos was observed ranging from intermediate to highly virulent. Interestingly, the ST131 strains were equally persistent in surviving embryos as the non-ST131-group B2 strains, suggesting similar mechanisms may account for development of persistent infection. Optical maps of the genome of the ST131 strains were compared with those of 24 reference E. coli strains. Although small differences were seen within the ST131 strains, the tree built on the optical maps showed that these strains belonged to a specific cluster (86% similarity) with only 45% similarity with the other group B2 strains and 25% with strains of group A and D. Thus, the ST131 clone has a genetic composition that differs from other group B2 strains, and appears to be less virulent than previously suspected.     

Caenorhabditis elegans as a simple model to study phenotypic and genetic virulence determinants of extraintestinal pathogenic Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPEC) strains cause disease by invading normally sterile niches within the host body, e.g., urinary tract, blood and cerebrospinal fluid. Infections due to ExPEC strains, in particular urinary tract infections, cause considerable morbidity and significant health-care costs. The goal of our study is to evaluate whether Caenorhabditis elegans can be used as a model to study phenotypic and genetic virulence determinants of ExPEC strains. For this purpose, we used a collection of 31 E. coli strains isolated during acute extra-intestinal infections or from the feces of healthy individuals. For all strains, the phylogeny, the presence of ExPEC virulence factors, the resistance to biologically relevant stressors (bile, human serum and lysozyme), the motility, the growth rate, the virulence in C. elegans and in a murine septicaemia model has been established. The results show that there is a strong link between virulence in C. elegans and certain phenotypic and genetic virulence predictors of ExPEC strains determinable in vitro. Furthermore, there is a significant correlation between virulence of different ExPEC strains in C. elegans and in the murine model. Therefore, our results suggest that C. elegans can be used as a model to study virulence determinants of ExPEC strains.     

Electrophoretic heterogeneity of ribosomal protein AT-L30 among actinomycete genera.

The ribosomal proteins from 17 type strains of species belonging to various actinomycete genera were compared by two-dimensional polyacrylamide gel electrophoresis. I detected a striking variability among certain ribosomal proteins (designated AT-L30 proteins) with respect to electrophoretic mobility in the first dimension. In contrast, such variability was not observed among ribosomal L30 proteins from other bacteria, such as Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus. Although actinomycete AT-L30 proteins from different taxa exhibited considerable heterogeneity in electrophoretic mobility, within each genus the proteins had a specific mobility characteristic. On the basis of this observation, the ribosomal AT-L30 proteins from 11 type strains of species belonging to the mycolic acid-containing genera Nocardia, Rhodococcus, Gordona, and Tsukamurella were analyzed. The relative electrophoretic mobilities of AT-L30 protein preparations from these strains, as determined by two-dimensional gel electrophoresis, revealed that the genera Nocardia, Rhodococcus, Gordona, and Tsukamurella can be sharply separated from each other. My results are consistent with the previously discussed view that each of these genera merits separate genus status.     

Frequency of Escherichia coli strains producing the cytotoxic necrotizing factor (CNF1) in nosocomial urinary tract infections

The presence of cytotoxic necrotizing factor 1 (CNF1), together with various associated virulence factors (alpha-haemolysin, P-, S- and A-fimbriae), was screened in 175 uropathogenic Escherichia coli strains isolated from hospitalized adult patients. The cnf1 gene was detected in 30% of the selected strains independently of the severity of the clinical urinary infection. A significant association between CNF1, haemolytic activity and the products of the pap/sfa genes was found. However, CNF1 appeared not to play a major role in nosocomial E. coli urinary tract infections.