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Biomedical subjects

B Picard

Publications and source records attributed to B Picard.

At least 109 records · Page 6Linked to original sources
PubMed1989

Biochemical, immunological and physicochemical comparisons between OHIO-1 and four SHV-type beta-lactamases.

The biochemical, immunological and physicochemical properties of the beta-lactamase OHIO-1 were compared to those of four beta-lactamases commonly found in Klebsiella pneumoniae: SHV-1, SHV-3 and the beta-lactamases of strains GN 11-03 and GN 422. The substrate profile of SHV-1, OHIO-1 and of the beta-lactamases GN 11-03 and GN 422 were similar, while that of SHV-3 appeared comparable to that of the extended spectrum SHV-2. Moreover, anti-TEM-1 serum inactivated OHIO-1 as well as SHV-1 and the beta-lactamases of strains GN 11-03 and GN 422. Analysis of the electrophoretic mobilities, isoelectric points and titration curves demonstrated that OHIO-1 and the 4 other beta-lactamases examined were closely related variants. From these findings it appears that OHIO-1 could be classified among the SHV-type beta-lactamases.

Electrophoresis↗
PubMed1989

The analysis of five carbenicillin-hydrolysing enzymes by electrophoretic methods.

Five carbenicillin-hydrolysing enzymes (carbenicillinases, or CARB), PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3, CARB-4 and CARB-5, and the beta-lactamase PSE-2 were compared by analysing their isoelectric points (pI), electrophoretic mobilities (mR) and titration curves (pH gradient electrophoresis). The pI determined by isoelectric focusing were 4.3 (CARB-4), 5.3 (PSE-4/CARB-1), 5.7 (PSE-1/CARB-2), 5.75 (CARB-3), 6.1 (PSE-2) and 6.35 (CARB-5). Their mR were estimated by zone electrophoresis as congruent to 26 for PSE-1 (CARB-2), CARB-3 and CARB-5, congruent to 30 for PSE-2, congruent to 33 for PSE-4 (CARB-1) and congruent to 61 for CARB-4. Titration curve analyses indicated that (1) PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3 and CARB-5 are closely related variants differing by a few amino acid substitutions; (2) the qualitative titration curve of CARB-4 is different from those of PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3 and CARB-5, although their patterns are somewhat similar; and (3) PSE-2 has no structural relationship with any of the other carbenicillin-hydrolysing enzymes or carbenicillinases (CARB) studied. Electrophoretic methods, and in particular titration curve determination combined with other physicochemical and enzymatic data, allowed a rapid comparison of the molecular structures of the beta-lactamases, and hence their classification.

Acinetobacter↗
PubMed1989

Correlation between electrophoretic types B1 and B2 of carboxylesterase B and sex of patients in Escherichia coli urinary tract infections.

One hundred and sixty-eight strains of Escherichia coli isolated from 84 men and 84 women who had urinary tract infections (134 cases) or bacteremia of urinary tract origin (34 cases) were assessed for their carboxylesterase B electrophoretic types B1 and B2, alpha-haemolysin production, the presence of mannose resistant haemagglutinin (MRHA) and antibiotic susceptibility. Electrophoretic type B2 was phenotypically linked with alpha-haemolysin and MRHA productions. The strains isolated from males were more frequently of type B2, haemolytic and both haemolytic and haemagglutinating than those isolated from females. The strains isolated during bacteremia were more frequently haemolytic and haemagglutinating than those obtained from urinary tract infections. Type B1 strains were more frequently resistant to antimicrobial agents than type B2 strains. The results reinforced the distinction, in terms of virulence and antibiotic sensitivity, between B1 and B2 strains and demonstrated the influence of the sex of patients on the host-parasite interaction during urinary tract infections.

Bacterial Proteins↗
PubMed1989

Esterase electrophoresis: a molecular tool for studying the epidemiology of Branhamella catarrhalis nosocomial infection.

A new epidemiologic typing method based on electrophoresis of esterases had been developed for differentiating between clinical isolates of Branhamella catarrhalis. Twenty-two epidemiologically significant strains obtained from three Chest Units, a Paediatric Intensive Care Unit and a Paediatric Unit were compared with 54 randomly selected strains and 4 reference strains, including the species type strain, ATCC 25238. Thirty-four distinct zymotypes were characterized by polyacrylamide-agarose gel electrophoresis of the 80 strains. One infrequent zymotype was found in 2 neonates and another in 2 adults with nosocomial bronchopulmonary infections, suggesting the nosocomial spread of 2 outbreak strains of B. catarrhalis. A more frequent zymotype was isolated from 3 neonates with nosocomial bronchopulmonary infection and from 2 children with nosocomial rhinopharyngitis. The remaining 12 epidemiologically significant strains were of varied zymotypes. This work demonstrates that esterase electrophoresis is a suitable, readily reproducible, stable typing system applicable to the wide range of strains found in B. catarrhalis nosocomial infections.

Adult↗
PubMed1989

Analysis of the molecular relatedness of four extended spectrum beta-lactamases (SHV-2, SHV-3, SHV-4 and SHV-5) by comparative protein titration curves.

Six beta-lactamases from Klebsiella pneumoniae, five of which (SHV-1, SHV-2, SHV-3, SHV-4 and SHV-5) were plasmid-encoded and one which (beta 1a GN 11-03) was chromosomally-encoded, were compared by analysis of their isoelectric points (pI), electrophoresis mobilities (MF) and titration curves or pH gradient electrophoresis. Four groups were defined by their pI and MF, namely SHV-1 and SHV-2 (pI = 7.6, MF approximately 14), SHV-3 and beta 1a GN 11-03 (pI = 7.0, MF approximately 20), SHV-4 (pI = 7.8 MF approximately 12) and SHV-5 (pI = 8.2, MF approximately 5). The titration curves of SHV-1 and SHV-2 enzymes on the one hand, and SHV-3 and beta 1a GN 11-03 on the other were completely superimposable for the whole of the pH gradient (3.5-10), indicating strongly similarity. Conservative amino-acid substitutions could account for the differences in the substrate spectra of the purified enzymes. The differences observed between the titration curves of the enzymes SHV-1/SHV-3, SHV-1/beta 1a GN 11-03, SHV-2/SHV-3 and SHV-5/SHV-4 pairs were consistent with the replacement of a basic amino-acid residue in the former enzyme of each pair by an acidic residue in the latter. Similarly, the titration curves of SHV-1/SHV-4 and of SHV-2/SHV-4 pairs may suggest the replacement of an acidic amino-acid in the former beta-lactamases by a neutral amino-acid in the latter of each pair. However, the presence of several self-cancelling or neutral substitutions is also possible. In contrast, when SHV-1 and TEM-1 (pI = 5.4 MF approximately 45) were titrated together, no structural relationship could be inferred.

Chromosomes, Bacterial↗
PubMed1989

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.

Electrophoresis, Agar Gel↗
PubMed1988

Electrophoretic data in designing strategies for purification and identification of a highly polymorphic bacterial esterase.

We have purified a bacterial enzyme, designated esterase M, by tailoring an efficient and rapid strategy with information derived from titration curves of proteins in crude extract. The pH-dependent stability of the enzyme activity observed by titration pattern allowed an acidic pH treatment of extract and a cationic exchange chromatography at pH 4.1. These two steps were followed by an anionic exchange chromatography and a preparative electrophoresis. Thus, the enzyme was purified about 2000-fold within two days with a recovery of 13.3%. The electrophoretic variants of esterase M were investigated for their molecular relationship through the specific effect of antibodies on esterase electrophoretic pattern (immunosubtractive electrophoresis) which is applicable to large series of samples. By this process, we have demonstrated the presence of common antigenic determinants among the electromorphs of esterase M produced by the three species of motile Aeromonas.

Aeromonas↗
PubMed1988

Double-sandwich enzyme-linked immunosorbent assay for determination of Escherichia coli heat-labile porcine enterotoxins.

A "double-sandwich" ELISA for the detection and measurement of a heat-labile enterotoxin produced by porcine enterotoxigenic strains of Escherichia coli (LTp) is described. In contrast with other heat-labile toxins, LTp did not bind to agarose gels and exhibited a very low affinity for GM1 in the classical GM1-ELISA technique. The similarity of LTp with cholera toxin was confirmed by immunoblotting. This property allowed the binding of LTp to rabbit IgG anti-cholera toxin antibodies (covalently linked to polystyrene plates) and sheep anti-cholera toxin serum. The immunocomplex was revealed by anti-sheep immunoglobulin antibodies conjugated with peroxidase. Application of the "double-sandwich" ELISA to the quantitation of toxin production by two strains, which differ only in the presence or the absence of the K88ab antigen, showed that the Ent+, K88+ strain produced significantly less toxin than the Ent+, K88- derivative.

Animals↗
PubMed1988

Correlation between electrophoretic types B1 and B2 of carboxylesterase B and host-dependent factors in Escherichia coli septicaemia.

Electrophoretic types B1 and B2 of carboxylesterase B produced by strains of Escherichia coli isolated from 100 septicaemia cases were correlated with alpha-haemolysin and mannose resistant haemagglutinin (MRHA) production and with clinical data including eventual underlying diseases, origin of septicaemia and evolution. Electrophoretic type B2 was phenotypically linked with alpha-haemolysin and MRHA production. The proportion of type B2 isolates varied significantly with occurrence of an underlying illness (45% for patients without an underlying disease and 22% for compromised patients) and with the site of origin of the septicaemia (40% for those of urinary origin and 18% for infection of digestive origin). In the former infections, type B2 isolates were obtained in the majority from male patients while type B1 isolates predominated in women. The septicaemias associated with type B1 were characterized by a lower proportion of isolates producing alpha-haemolysin and MRHA and by a greater frequency of septic shock and death than those associated with type B2. These facts emphasize the importance of host-dependent factors in E. coli septicaemia.

Adolescent↗
PubMed1988

Characterization of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate and malate dehydrogenases.

Acid phosphatase, esterases, and glutamate and malate dehydrogenases of 192 strains of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gels. The six species were clearly separated from each other by their distinct enzyme electrophoretic polymorphism. For Y. enterocolitica, the strains of biotype 5 were differentiated from the other biotypes by the mobility of glutamate dehydrogenase. For Y. frederiksenii, six zymotypes were delineated by pI and by the mobility of the enzymes. Variation in number or mobility of esterases within each species could represent a marker for epidemiological and ecological analyses. A linear relationship was obtained between the mean genetic diversity coefficient of enzymes and the mean percentage DNA-DNA relatedness of Y. intermedia, Y. aldovae, Y. enterocolitica and Y. frederiksenii.

Acid Phosphatase↗
PubMed1988

[Titration curves (ph gradient electrophoresis) of SHV-1 and SHV-2 beta-lactamases and a new type].

The molecular structures of the SHV-1 (p 453) and SHV-2 (pBP 60-1) beta-lactamases and of a new enzyme, a SHV-2 like extended broad-spectrum beta-lactamase (86-4), were compared by analysis of their titration curves (pH gradient electrophoresis). The titration curves of SHV-1 and SHV-2, which have the same isoelectric points (pI = 7.7). were completely superimposable for the whole of the pH gradient (pH 3.5-10), indicating a close homology between the two proteins, with perhaps the substitution of several amino acids by ones having the same charge. The curves of SHV-1 (pI = 7.7) and the new SHV-2-like enzyme (pI = 6.98) indicated that a basic residue in SHV-1 has been replaced by an acidic residue in the new SHV-2-like enzyme. These results show that, like SHV-2, the new beta-lactamase is a variant of SHV-1, and that the structural differences are probably limited to a very small number of amino acid residues. Nevertheless, this new beta-lactamase (SHV-3) may have arisen directly from SHV-1, indirectly via SHV-2, or even from another beta-lactamase.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

[Electrophoretic behavior of beta-lactamases in gram-negative bacteria].

The ongoing discoveries of new beta-lactamases, mainly penicillinases, in Gram-negative bacteria has emphasized the problem of their precise identification, and thus their phylogeny. Crude extracts, prepared by sonication, of 14 plasmid beta-lactamases, types TEM, carbenicillinases (CARB or PSE) and oxacillinases (OXA) were analysed by a simple, rapid (3.5 to 4 hours) method of electrophoresis on polyacrylamide (7%) agarose (1.4%) gels, using Tris-glycine buffer at pH 8.7. Preliminary serial dilutions were made to determine enzymic activity levels. Enzymes were then characterized by their relative electrophoretic mobilities. These mobilities had coefficients of variability between 2% and 10%, ranged from 5 to 61, and were correlated with their isoelectric points (pI). Thus, the lower the pI is, the greater the mobility is. Despite the high resolving power of the polyacrylamide-agarose gel system, enzymes with similar pI's and of similar types (PSE-1 and CARB-3, or OXA-1 and OXA-4) or different types (SHV-1 and OXA-6) could not be distinguished on the basis of their mobilities. However, this technique provides for rapid and easy identification of the major penicillinases in Gram-negative bacteria. A combination of polyacrylamide-agarose gel electrophoresis and pH gradient electrophoresis (titration curve) could provide a powerful approach to the study of the molecular structure of these enzymes.

Electrophoresis, Agar Gel↗
PubMed1987

A novel approach to study of the structural basis of enzyme polymorphism. Analysis of carboxylesterase B of Escherichia coli as model.

In order to understand the structural basis of charge differences among enzyme variants without undertaking purification and sequencing of the protein, an original approach was developed. The approach is applicable to any enzyme or protein provided that there is a specific staining procedure. This consists, as a first step, in the projection of electrophoretically obtained mobility values versus pI of all variants into a two-dimensional profile. In a second step, starting from the most common variant, various theoretical possibilities of substitutions are envisaged, taking into consideration the pH of the electrophoretic conditions, pI of the variants and range of variations of the pK values of several amino acid side chains. In a third step, verification of the theoretical data is obtained through comparative protein titration curves by combined isoelectrofocusing-electrophoresis of several pairs of relevant variants. The validity of this approach is tested on the highly polymorphic carboxylesterase B enzyme of Escherichia coli and is found to provide valuable information.

Carboxylesterase↗
PubMed1987

Seasonal prevalence of nosocomial Aeromonas hydrophila infection related to aeromonas in hospital water.

A seasonal variation in nosocomial Aeromonas hydrophila infection was correlated with the number of aeromonas in the hospital water supply. The high summer prevalence of A. hydrophila infection coincided with periods when water counts from storage tanks were highest. The waterborne origin of these infections highlights the importance of maintaining clean water supplies, especially where storage tanks are used. Monitoring A. hydrophila in hospital water, particularly during the summer months, may prove helpful.

Aeromonas↗
PubMed1987

Epidemiological complexity of hospital aeromonas infections revealed by electrophoretic typing of esterases.

An epidemiology analysis of a series of 12 Aeromonas hydrophila infections, including six of septicaemia, which occurred on several wards of one hospital during the summer of 1982 is presented. The hypothesis that the hospital water could be the source of these infections was supported by the isolation of 1-10 motile aeromonads per ml in most of the water samples collected from various points on the hospital water system. Electrophoretic esterase typing was used as an epidemiological screening method to determine the relationship between bacterial strains isolated from the patients and those from water samples. The epidemiology of A. hydrophila infection in the hospital was found to be complex. Amongst the 15 strains of A. hydrophila isolated from patients were 8 zymotypes, while amongst the 126 strains from the water samples there were 37. In some cases, several zymotypes were isolated simultaneously from the same tap water. On one ward, the same zymotype was found in 2 patients and in 2 water samples. The prophylactic measures taken in 1982-5 to avoid oral contamination of immuno-compromised patients with infected hospital water have significantly reduced the number of cases of septicaemia. This success has constituted additional retrospective evidence for the water-borne origin of these infections.

Aeromonas↗
PubMed1987

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.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

[Titration curves of beta-lactamases using pH gradient electrophoresis].

The molecular relationships of two types of plasmid-mediated beta-lactamases, TEM-1 (R 111), TEM-2 (RP 4) and OXA-1 (RGN 238), OXA-4 (pMG 90) were analysed by combined isoelectrofocusing-electrophoresis. Titration curves of TEM-1 (pI 5.4) and TEM-2 (pI 5.6) together were consistent with the known substitution of a glutamic acid in the former by a lysin in the latter. When OXA-1 (pI 7.4) and OXA-4 (pI 7.45) were titrated, one single mobility curve was obtained reflecting their structural homogeneity. The titration curve technique will be usefull for the study of structure of beta-lactamases.

Electrophoresis, Polyacrylamide Gel↗